Cancer chemoprevention: signaling pathways and strategic approaches

Abstract

Although cancer chemopreventive agents have been confirmed to effectively protect high-risk populations from cancer invasion or recurrence, only over ten drugs have been approved by the U.S. Food and Drug Administration. Therefore, screening potent cancer chemopreventive agents is crucial to reduce the constantly increasing incidence and mortality rate of cancer. Considering the lengthy prevention process, an ideal chemopreventive agent should be nontoxic, inexpensive, and oral. Natural compounds have become a natural treasure reservoir for cancer chemoprevention because of their superior ease of availability, cost-effectiveness, and safety. The benefits of natural compounds as chemopreventive agents in cancer prevention have been confirmed in various studies. In light of this, the present review is intended to fully delineate the entire scope of cancer chemoprevention, and primarily focuses on various aspects of cancer chemoprevention based on natural compounds, specifically focusing on the mechanism of action of natural compounds in cancer prevention, and discussing in detail how they exert cancer prevention effects by affecting classical signaling pathways, immune checkpoints, and gut microbiome. We also introduce novel cancer chemoprevention strategies and summarize the role of natural compounds in improving chemotherapy regimens. Furthermore, we describe strategies for discovering anticancer compounds with low abundance and high activity, revealing the broad prospects of natural compounds in drug discovery for cancer chemoprevention. Moreover, we associate cancer chemoprevention with precision medicine, and discuss the challenges encountered in cancer chemoprevention. Finally, we emphasize the transformative potential of natural compounds in advancing the field of cancer chemoprevention and their ability to introduce more effective and less toxic preventive options for oncology.

Introduction

Cancer has become a major public health and economic issue in current society. According to GLOBOCAN 2022, there were 20 million new cases of cancer and 9.7 million deaths, and the number of new cancer cases was estimated to reach 35 million by 2050.¹ The risk of developing cancer also tends to increase as the human development index elevated. Despite the rapid progress made in research on the etiology and molecular mechanisms of cancer, no fruitful results regarding improving overall cancer survival rates have been obtained. Nonetheless, in principle, cancer epidemiology have shown that all common cancers are largely preventable.^2,3 Wherein, the concept of chemoprevention is increasingly popular, initially because it has successfully reduced the incidence rate of cardiovascular disease.^4,5 Currently, the content of chemoprevention has expanded to include the use of natural, synthetic, and biological products to block, delay, or reverse carcinogenesis.⁶ Cancer chemoprevention has always been considered an important preventive strategy for reducing the burden of cancer on healthcare systems.^7,8,9,10,11

Cancer chemoprevention refers to the use of natural, synthetic or biological products to prevent, slow down or reverse the development of cancer to reduce its incidence and mortality, and is usually divided into the following three levels (Fig. 1): (1) primary chemoprevention aims to prevent the development of diseases in the general population or especially high-risk populations; (2) secondary chemoprevention focuses on individuals diagnosed with a certain type of tumor or precancerous lesion that may develop into invasive cancer; (3) tertiary chemoprevention aims to inhibit cancer recurrence or secondary tumors.^7,12,13 However, even after decades of development, only more than ten types of available drugs have been approved by the U.S. Food and Drug Administration (FDA) for cancer chemoprevention.^14,15 Considering the constantly increasing incidence and mortality rates of cancer, it is of great significance to screen drugs for cancer chemoprevention from a large number of existing chemical substances. Moreover, given that the prevention process is lengthy, the chemoprevention process must always be safe, and the drugs used should be acceptable to both health and patients. Hence, the ideal chemopreventive agent should be nontoxic, inexpensive, and oral. Natural compounds have been a treasure reservoir for clinical use in cancer prevention and treatment for a long time.^16,17,18 From 1981 to 2019, more than 60% of the small molecules approved for cancer treatment were natural products, natural product-derived, or natural product analogs.¹⁶ Natural compounds account for a significant proportion of currently used chemotherapy drugs, and a large number of studies have confirmed their chemopreventive efficacy.^{9,17,19,20,21,22} Due to the diverse chemical components, complex chemical structures, unique biological effects, high cost-effectiveness, ease of acquisition, low toxicity, and minimal side effects of natural compounds, there has been a greater focus on the use of natural compounds for cancer chemoprevention. In addition, the development of new preventive drugs with natural compounds as the lead compound has also emerged.^23,24 Natural compounds have become a preferred approach in cancer chemoprevention.

Fig. 1

Cancer chemoprevention occurs through cancer initiation, progression, and metastasis. Cancer prevention can be effective throughout the entire process of cancer initiation and development. Primary chemoprevention can be achieved by altering diet, lifestyle habits, and increasing exercise to avoid or reduce exposure to known carcinogenic factors (chemical carcinogens such as formaldehyde, nitrosamine benzene, aflatoxin, etc.; physical carcinogenic factors, such as ultraviolet radiation, ionizing radiation, etc.; viruses such as human papillomavirus, hepatitis B virus, etc., and family genetic factors). Secondary chemoprevention helps suppress and reverse cancer. Tertiary chemoprevention aims to reduce the risk of tumor recurrence and metastasis, improve the quality of life and survival rate of patients. This figure was created with BioRender.com

In this review, we mainly discuss the concept, content, and classifications of cancer chemopreventive agents, highlight on exploring potential natural compound-derived cancer chemopreventive agents, and embark on a comprehensive investigation of multilevel regulatory mechanisms and therapeutic targets from the perspectives of targeted signaling pathways, immunotherapy, and gut microbiome regulation. We introduce various advances in drug delivery systems to assist in cancer chemoprevention. Clinical research progress of cancer chemoprevention in cancer treatment is also summarized and discussed. Moreover, we also summarize the various approaches that contribute to discovering anticancer natural compounds with low abundance and high activity to reveal the broad prospects of natural compounds as a treasure reservoir for cancer chemopreventive agent discovery. Meanwhile, we associate cancer chemoprevention with precision medicine to achieve individual precision cancer chemoprevention in the future. We also discuss the challenges encountered in cancer chemoprevention and expectations during the bench-to-bedside translation. Overall, we provide a comprehensive review of the field of cancer chemoprevention and emphasize the transformative potential of natural compounds in advancing the field of cancer chemoprevention and their ability to introduce more effective and less toxic preventive options for oncology.

Agents for cancer chemoprevention

FDA-approved medications for cancer chemoprevention

Cancer chemoprevention is a rapidly developing field with broad prospects, but the long-term benefits of intervention may take many years or remain unknown. After the proposal of cancer chemoprevention, several agents were successfully approved by FDA for cancer prevention (Table 1). Many studies are also actively underway to expand the available agents for cancer prevention, and major development history of cancer chemoprevention are depicted in Fig. 2.

Table 1 Cancer chemoprevention agents approved by FDA

Fig. 2

Historical timeline of important development in the field of cancer chemoprevention. FDA U.S. Food and Drug Administration, HPV human papillomavirus, NIH National Institutes of Health, PD-1 programmed cell death-1

Chemopreventive agents for breast cancer

Intimate relationship exists between breast cancer and estrogen, and anti-hormone drugs are the first potential chemopreventive drugs to be tested in large-scale clinical trials. Among them, tamoxifen, a selective estrogen receptor (ER) modulator (SERM), is the first specifically cancer chemopreventive drug approved by FDA, which can reduce the risk of invasive and noninvasive breast cancer. At a median follow-up of 55 months, tamoxifen reduced the risk of invasive breast cancer by 49% and noninvasive cancer by 50%, as well as a reduction in hip, radius, and spine fractures was observed, while, the risk of endometrial cancer and the incidence of thromboembolic events also increase accordingly.²⁵ In a meta-analysis, tamoxifen reduced the risk of ER-positive breast cancer by 48%.²⁶ In study of comparing tamoxifen with placebo, the incidence of adverse reactions decreased and it was equivalent to that of patients taking placebo by 10 years (or 5 years after discontinuation of tamoxifen).²⁷ In the latest study of tamoxifen and raloxifene, raloxifene has been proven to be less effective in reducing invasive breast cancer than tamoxifen, but it maintains a higher safety.²⁸ At present, raloxifene is also approved to reduce the risk of invasive breast cancer in postmenopausal women with osteoporosis and in postmenopausal women at high risk.²⁹ In addition, aromatase inhibitors such as ezetimibe and anastrozole have shown efficacy as chemopreventive agents, although they have not yet been approved.^30,31

Chemopreventive agents for skin cancer

Actinic keratosis (AK) is an abnormal proliferation of epidermal keratinocytes caused by long-term ultraviolet irradiation, and about 10% of lesions can develop into invasive squamous cell carcinoma. Compared with other chemoprevention targets, skin is easier accessibility, which can carried out the direct local treatment.⁷ Currently, fluorouracil cream, diclofenac sodium gel, imiquimod cream, ingenol mebutuate gel, and tirbanibulin have been approved by FDA for the treatment of AK.^15,32 Among them, ingenol mebutuate gel was a plant extract from Euphorbia peplus L., which provided an exciting natural compound-based chemopreventive option for skin cancer. Ingenol mebutuate gel is very effective for treating AK on face or scalp and body, exhibiting good tolerability, no systemic absorption, and excellent patient compliance in clinical settings.^33,34 The successful application of chemoprevention in skin cancer provides great confidence for the development of chemopreventive agents.

Chemopreventive agents for bladder cancer and esophageal cancer

Bacillus Calmette-Guerin (BCG) is a live attenuated strain, initially used to treat tuberculosis, and it was proved that intravesical administration of BCG can treat carcinoma in situ of the bladder and reduce its recurrence.^35,36,37 Besides, valrubicin and pembrolizumab were approved for the treatment of BCG-unresponsive carcinoma in situ of the bladder, with 21% and 41% patients achieved a complete response (CR), respectively.^38,39,40 The approval of both BCG and pembrolizumab for carcinoma in situ of the bladder manifested that immunotherapy can play a role in cancer chemoprevention.

Photodynamic therapy (PDT) in combination with the photosensitizer drug porfimer sodium (PDT/PS) is licensed for the treatment of barrett esophagus with high-grade dysplasia. It was reported PDT/PS exhibited a superior complete ablation of Barrett esophagus compared with omeprazole (77% vs. 39%), as well as fewer patients progressed to cancer (13% vs. 28%).⁴¹ This therapy indicated that treating individuals with identifiable high-risk pre-invasive lesions can elaborate the cancer chemopreventive role.

Chemopreventive agents for cervical carcinoma and hepatocellular carcinoma (HCC)

New cases of gastric cancer, liver cancer, and cervical cancer worldwide reached 2.5 million in 2022, equivalent to one-eighth of all newly diagnosed cancer patients.¹ The main carcinogenic factors (e.g., Helicobacter pylori (H. pylori), human papillomavirus (HPV), and hepatitis B virus (HBV)) that cause the above-mentioned cancers are preventable. HPV infection has been widely recognized as a necessary cause of cervical cancer.^42,43 The specificity of this virus is highly desirable as vaccination provides a highly effective chemoprevention method.⁷ The currently available HPV vaccines can protect against infection with HPV 16 and HPV 18, which account for 71% of cervical cancer cases.^44,45 These vaccines are considered to be molecularly targeted because they generate immune responses against specific proteins.⁴⁶ It was reported after 5–8 years of vaccination, the incidence of HPV 16 and 18 in girls aged 13–19 significantly decreased by 83%, and the incidence in women aged 20–24 significantly decreased by 66%.⁴⁷ Global vaccination coverage with at least one dose in girls by 15 years of age was estimated to be 20% as of 2023.⁴⁸ Moreover, the World Health Organization recommends getting the HPV vaccine before sexual activity to maximize its preventive effect.⁴⁹ Chronic hepatitis B and hepatitis C are the most important causes of HCC and account for 21–55% of HCC cases globally.^50,51 Statistical data from multiple countries and regions indicate that the popularity of HBV vaccination has significantly reduced the incidence rate of HCC.^52,53,54

Synthetic chemopreventive agents with clinical efficacy but lacking approval

Finasteride, a type of 5α-reductase inhibitor, has been shown can significantly reduce the prevalence of prostate cancer, but this protective effect is limited to lower-grade.^55,56 Although finasteride has not been approved for the prevention of prostate cancer, multiple subsequent studies have supported its ability as prostate cancer chemoprevention.^57,58,59 For example, finasteride can significantly decrease the risk of high-grade prostatic intraepithelial neoplasia.⁶⁰ In the long-term follow-up study (median follow-up time of 18.4 years) of finasteride published by Goodman et al., finasteride reduced the risk of prostate cancer death by 25%, but it was not statistically significant due to the small number of prostate cancer deaths.⁶¹

Aspirin, a non-steroidal anti-inflammatory drug (NSAID) as well as cyclooxygenase (COX) inhibitor, can prevent the incidence rate and recurrence of colorectal cancer (CRC).^62,63 Compared to a control group without aspirin, daily administration of aspirin halved the risk of colorectal metastatic adenocarcinoma and reduced distant metastases by 30–40%.⁶⁴ In addition, it was advised that individuals with an average risk of CRC should be considered to employ low-dose aspirin for CRC prevention if they are not older than 70 years with a life expectancy of at least 10 years, not at high risk for bleeding, and have a 10-year cardiovascular disease risk of at least 10%.⁶⁵ However, due to the significant gastrointestinal burden associated with long-term use of aspirin, its effectiveness as a chemopreventive agent is also limited.⁶⁶

The selective COX-2 inhibitor celecoxib can significantly reduce the burden of colon polyps,⁶⁷ and decrease the incidence of adenomas,⁶⁸ and is more effective in reducing the risk of gastrointestinal bleeding, which has been approved to reduce the number of polyps in patients with familial adenomatous polyposis (FAP). Although clinically effective, it is associated with an increased risk of cardiovascular disease, leading manufacturer withdraw the CRC prevention function from its label, highlighting the necessity of long-term safety monitoring after drug approval.¹⁵

Sulindac, a NSAID drug, was reported standard doses of sulindac did not prevent the development of adenomas in subjects with FAP,⁶⁹ however, the combination of difluoromethylornithine (DFMO) and sulindac can prevent recurrence of colorectal adenomas in patients at high risk by 70%.⁷⁰ The most notable aspect of this study is the impact of DFMO and sulindac on the number and severity of new adenomas with only one case of advanced adenoma was detected in the combination therapy group, while 11 cases were observed in the placebo group,⁷⁰ which provides impetus for further development of multi-drug combination therapy for cancer chemoprevention. In a meta-analysis of over 20,000 patients, the combination of DFMO and sulindac [relative risk, 0.24; 95% confidence interval (CI), 0.10–0.55] showed excellent protective effects compared to aspirin (relative risk, 0.77; 95% CI, 0.60–1.00), celecoxib (relative risk, 0.56; 95% CI, 0.31–1.01), and metformin (relative risk, 0.56; 95% CI, 0.22–1.39).⁷¹ Although the combination of DFMO and sulindac has a significant preventive effect, relatively cardiovascular adverse reactions are pronounced.

Diabetes is associated with an increased risk of several types of cancers.⁷² Metformin is an effective drug for treating diabetes, and because of its low cost, it is also considered as a promising cancer prevention strategy. A meta-analysis showed metformin reduced overall cancer incidence rate by 31% (summary relative risk, 0.69; 95% CI, 0.52–0.90), despite significant heterogeneity among studies (I² = 88%).⁷³ Besides, Higurashi’s study provided encouraging evidence for the cancer chemopreventive effect of metformin.⁷⁴ One hundred and fifty-one patients who underwent resection of single or multiple colorectal adenomatous polyps were enrolled, and compared with placebo after just 1 year, a low dose of metformin (250 mg/day) significantly reduced the risk of total polyps (risk ratio, 0.67; 95% CI, 0.47–0.97) and adenomas (risk ratio, 0.60; 95% CI, 0.39–0.92). This study suggested the potential role of metformin in cancer prevention, however, large-scale and long-term trials still need to provide clear conclusions.

Statins are potent competitive inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase, commonly used as lipid-lowering drugs. In recent years, an increasing number of studies have shown that statins may have potential roles in the field of cancer chemoprevention. It was reported statins have an overall positive impact on the clinical outcomes of a series of cancers, including but not limited to CRC, HCC, gastric cancer, breast cancer, lung cancer, and kidney cancer.⁷⁵ In a meta-analysis of over one million cancer patients, the use of statins was significantly associated with decreased risk of all-cause mortality [hazard ratio (HR), 0.70; 95% CI, 0.66–0.74] and cancer-specific mortality (HR, 0.60; 95% CI, 0.47–0.77) compared with non-users.⁷⁶ Other meta-analysis also found that the use of statins is significantly associated with a reduced risk of HCC development,^77,78,79 and this effect is dose-dependent, particularly evident in lipophilic statins.⁷⁸ An observation study displayed regular use of aspirin and statins can reduce the risk of cancer with systemic inflammatory diseases.⁸⁰ However, there is still controversy over the role of statins in cancer prevention, and more researches are needed to clarify their preventive effects.

There are also controversial chemopreventive agents, such as vitamin E and selenium, the results from a large-scale clinical study on the selenium and vitamin E cancer prevention trial with a follow-up period of at least 7 years exhibited the controversy surrounding the use of vitamin E and selenium as prostate cancer prevention agents.⁸¹ In another randomized clinical trial of selenium and vitamin E in patients with newly diagnosed non-muscle-invasive bladder cancer, neither selenium nor vitamin E supplementation affect disease recurrence or overall survival (OS) rate.⁸² The above researches on cancer chemoprevention agents provided valuable information for the field of cancer chemoprevention and have guiding significance for future experimental design. With the increasing cost of developing chemotherapy drugs, limited benefits, and the high economic burden, thus, as a solution, cancer chemoprevention requires the development of better and safer preventive agents.

Potential biological products as chemopreventive agents

Vaccine

In addition to traditional cancer prevention vaccines such as HPV and HBV, of which exert cancer prevention effects based on known tumor-associated antigens, currently, multiple studies have pioneered new cancer vaccines with personalized and immune system activation characteristics, expanding the potential treasure trove of cancer chemopreventive agents.

CIMAvax-EGF consists of a chemical conjugate between epidermal growth factor (EGF) and P64, targeting the immune system and inducing anti-EGF antibodies, thus leads to a decrease in circulating EGF, thereby exerting its anticancer activity.⁸³ It is worth noting that CIMAvax-EGF can also benefit patients who have not undergone genetic testing for mutations. Data shows that after treatment, the median OS of non-small cell lung cancer (NSCLC) patients is 22.46 months, with the survival rates at 6, 12, and 24 months were 97.7%, 82.7% and 45.5%, respectively. The median progression free survival (PFS) was 8.16 months, and the PFS rate at 6, 12, and 24 months were 55.4%, 36.4%, and 19.1%, respectively.⁸⁴

Autogene cevumeran (BNT122), an individualized mRNA neoantigen vaccine containing up to 20 major histocompatibility complex class I (MHC I) and MHCII restricted neoantigens in lipoplex nanoparticles intravenously delivered, was reported can delay recurrence and prolong the survival time of pancreatic ductal adenocarcinoma patients.⁸⁵ Sixteen patients were treated with atezolizumab and autogene cevumeran, then 15 patients with mFOLFIRINOX (comprising folinic acid, fuorouracil, irinotecan and oxaliplatin), the autogene cevumeran was tolerable and induced de novo high-magnitude neoantigen-specifc T cells in 8 out of 16 patients. After 18-month median follow-up, patients with vaccine-expanded T cells had a significant longer median recurrence-free survival compared with that without vaccine-expanded T cells (13.4 months, P = 0.003). Besides, the serum CA19-9 level of one patient increased and appeared a new liver lesion, while, a biopsy sample did not reveal malignant cells, indicated that the BNT122 vaccine may have the ability to eradicate micrometastases. Autogene cevumeran substantially expanded T cells that included vaccine neoantigen-specific, functional and durable CD8⁺ T cells, which can persist up to 2 years despite post-vaccination mFOLFIRINOX treatment, and the persistence of these T cells is associated with a longer median recurrence-free survival in vaccinated individuals. This is a phase I study with a small sample size, and subsequent studies has also conducted randomized phase II clinical trials on melanoma, NSCLC, CRC, etc (NCT03289962, NCT03815058, and NCT06534983).

mRNA-4157 (V940) is an mRNA-based personalized neoantigen therapy by encoding up to 34 tumor-specific antigens in a lipid nanoparticle formulation and tailored specifically for cancer patients, subsequently stimulating the immune system to produce reactive T cells targeting patient-specific tumor neoantigens, thereby achieving the goal of controlling and treating tumors. The most famous mRNA-4157 studies are KEYNOTE-603 and KEYNOTE-942. KEYNOTE-603 study mainly assessed safety, tolerability, and immunogenicity of mRNA-4157 in patients with resected NSCLC or resected cutaneous melanoma.⁸⁶ This open-label, multicenter of phase I study taking mRNA-4157 as adjuvant monotherapy or combination therapy with pembrolizumab is safe and overall tolerable, and mRNA-4157 exhibited generation of de novo and enhancement of existing neoantigen-specific T-cell responses and provided mechanistic proof to support further development of mRNA-4157 for patients with resected solid tumors.⁸⁶ Moreover, these T-cell responses are persistent and can even be detected 100 days after the last administration, indicated mRNA-4157 not only triggers strong immune responses, but these responses can be sustained for a long time. The latest research on mRNA-4157 (KEYNOTE-942) showed that combination of mRNA-4157 and pembrolizumab prolonged recurrence-free survival than monotherapy (HR, 0.561; 95% CI, 0.309–1.017), with lower recurrence or death event rate (22% vs. 40%), 18-month recurrence-free survival was 79% versus 62%.⁸⁷ A phase III trial (NCT05933577) is conducting to confirm the safety and prevention cancer from returning in people with high-risk melanoma.

GNOS-PV02 can encode up to 40 neoantigens by using DNA plasmids as vectors, these neoantigens were determined through DNA, RNA, and germ cell DNA sequencing of tumor samples from each patient. After vaccination, it can stimulate the immune system to generate tumor-infiltrating lymphocytes to kill tumor cells. Yarchoan et al. used GNOS-PV02, DNA plasmid-encoded cytokine interleukin-12 (IL-12, as an adjuvant to enhance the response to neoantigens), and pembrolizumab in combination for the treatment of advanced HCC patients.⁸⁸ The objective response rate (ORR) was 30.6% (11/36), with 8.3% (3/36) of patients achieving a CR, and the disease control rate was 55.6% (20/36). Compared to the historical value of ORR of pembrolizumab monotherapy of 18.3%,⁸⁹ the efficacy of GNOS-PV02 combination therapy is about twice that of immunotherapy alone. It is worth noting that the target lesions of two patients continued to reduction of 44% and 59%, respectively. For most HCC patients, the high-capacity plasmid used by GNOS-PV02 is sufficient to cover neoantigens in the tumor, allowing the immune system to select the most effective neoantigens on its own.

Tumor vaccine can accurately target and block key stages of cancer occurrence, prolong survival time, delay cancer metastasis or recurrence. Compared to traditional synthetic prevention agents, its side effects are mild, usually including pain, redness, and swelling at the injection site. However, due to significant individual differences, many factors can affect the immune response of vaccines, thereby reducing their effectiveness in preventing cancer, and the immune evasion mechanism of tumor cells can weaken the preventive effect. Introducing novel natural drug carriers to prepare cancer immunotherapy-targeted vaccines might improve the aforementioned shortcomings.⁹⁰ The neoantigens cancer vaccine is tailored specifically for each individual and is unique to each patient, with the continuous emergence of clinical evidence, the feasibility, safety, and promising clinical efficacy of cancer immunotherapy based on neoantigens have been demonstrated in cancer patients. With continuous exploration and research, the neoantigens cancer vaccine is expected to become a powerful solution for cancer prevention.

Immune modulator

The immune system is dedicated to protect organisms from harmful substances, and IL-2, as an important immune modulator, plays a crucial role in T-cell development and expansion, and has been approved by the FDA as an antitumor drug for patients with renal cell carcinoma (RCC) and melanoma.⁹¹ It was reported high dose (HD) IL-2 treatment yielded durable responses in metastatic RCC patients and prolonged survival.⁹² Among 356 patients receiving HD IL-2 treatment alone, 119 (33%) met the favorable risk criteria, 203 (57%) met the intermediate risk criteria, and 34 (10%) met the adverse risk criteria. The favorable and intermediate risk patients in this group demonstrated prolonged OS, and many experience years of treatment-free survival.⁹² The median OS for favorable, intermediate and poor risk groups treated with IL-2 alone is 64.5 months, 57.6 months, and 14 months, respectively, and the 2-year OS for those treated with IL-2 alone by risk category is 73.8, 63.7, and 39.8%, respectively.⁹² In radically operable CRC patients, pre-operative IL-2 immunoprophylaxis exhibited an encouraging effect.⁹³ After a median follow-up of 72 months, 6/20 recurrences (30%) were observed in IL-2 pre-operative group, with 19/40 recurrences (47.5%) in controls. Furthermore, after a 5-year follow-up, 4/20 (20%) IL-2 treated patients were dead and the control patients were 19/40 (47.5%, P = 0.05).⁹³ Interferon-α (IFN-α) is another cytokine approved by the FDA for the treatment of hematological malignancies and melanoma, it has been demonstrated to exert antitumor effect mediated by activation of immune system and antiangiogenic effect.⁹⁴ However, due to the low response rate and high toxicity associated with HD IL-2 and IFN-α, the clinical application of these cytokines has been replaced by targeted therapy.

Cancer cells typically express tumor-associated antigen (TAA) that is different from normal cells, such as human epidermal growth factor receptor-2 (HER-2). Monoclonal antibodies (mAbs) can specifically recognize and bind with these TAAs, thereby blocking the growth, proliferation, invasion, and metastasis of cancer cells. Trastuzumab, a classical anti-HER-2 mAb, inhibits the growth of HER-2 positive breast cancer cells through specific binding with HER-2 receptors,⁹⁵ and plays a leading role in adjuvant therapy and metastatic therapy of breast cancer. Researches show that trastuzumab adjuvant chemotherapy significantly improves the disease-free survival rate of HER-2 positive breast cancer patients, and reduces the risk of recurrence and death.^96,97,98 Although other anti-HER-2 drugs are currently available, such as pertuzumab and lapatinib, trastuzumab remains the gold standard for treating HER-2-positive breast cancer.⁹⁹ If TAA with abnormal expression in the body can be identified before cancer occurs and mAbs can be used for intervention in a timely manner, it may prevent the formation or development of cancer cells.

Biological products with potential cancer chemopreventive effects have highly specific and personalized preventive potential, and most of them with the advantages of strong immune regulatory effects, which may prevent cancer from occurring at the source. However, due to tumor heterogeneity and individual genetic differences, it may affect its preventive effect. In addition, as most biological products work by regulating the immune system, which might cause immune-related adverse reactions (irAEs), and long-term use may lead to changes in immune system tolerance, affecting normal immune function. In addition, biological products usually require special storage and transportation conditions, and injection is required for use. The economic burden brought by expensive prices should not be underestimated, too. Therefore, the widespread use of cancer chemopreventive agents based on biological products is limited. Considering the prevention process is lengthy, the agents used should be nontoxic, inexpensive, and take orally to both health and patients.

Natural compounds as promising cancer chemoprevention agents

Natural compounds for cancer chemoprevention at the preclinical stage

Unlike many synthetic drugs, the consumption of natural compounds extracted from food, plants, herbs, etc. has been considered as relatively safe for thousands of years, which has brought new vitality to the field. In the past few decades, natural compounds have gradually become the main source of new candidate drugs. Due to the effective tumor targeting potential and low toxicity to normal tissues, natural compounds have become the potential cancer chemoprevention drug for use alone or in combination with other chemotherapy drugs.^100,101,102 In this section, we introduced the classical and widely studied natural compounds with cancer chemoprevention characteristics, and demonstrated their potentiality in cancer chemoprevention (Fig. 3).

Fig. 3

The chemical structures and main source of presentative natural compounds exhibiting cancer chemoprevention. EGCG epigallocatechin-3-gallate, CPT camptothecin

Polyphenols

Polyphenols are a large class of compounds with over 8000 structural variants.¹⁰³ Most polyphenols have pharmacological activities, such as anti-inflammatory, immunomodulatory, and anticancer effects.^104,105

Genistein: Genistein is a multifunctional natural flavonoid mainly derived from soybeans and their products, and it is a typical example of a phytoestrogenic compound. Researches have shown a relationship between a high soy products and cancer prevention. Soy food consumption was associated with a reduced risk of prostate cancer in men,^106,107 and it was negatively associated with the risk of breast cancer in Asian women. While, this association has not been confirmed in Western women.^108,109 Moreover, soybean isoflavone intake can reduce the risk of breast cancer in both premenopausal and postmenopausal women in Asian countries.¹¹⁰ The mechanism for their cancer chemoprevention involves inducing apoptosis, cell cycle arrest, anti-proliferation, reducing angiogenesis, acting on cancer stem cells (CSCs), regulating gene and mRNA expression, and its unique estrogenic properties. Wherein, genistein induces cell apoptosis through multiple pathways, such as stimulation of the peroxisome proliferator-activated receptor gamma,¹¹¹ inactivation of the insulin-like growth factor (IGF)-1 receptor/p-Akt signaling pathway,¹¹² activation Ca²⁺-dependent pro-apoptotic proteases,¹¹³ inhibition the accumulation of lipid droplets,¹¹⁴ regulation endoplasmic reticulum stress, mitochondria-dependent or reactive oxygen species (ROS)-dependent pathways,^115,116 etc. Through inactivating DNA repair pathways, such as homologous recombination and nonhomologous end joining pathway,^117,118 regulating epigenetic proteins,^119,120,121 modulating microRNAs (miRNAs),^{122,123,124,125,126} genistein thus exhibited the cancer chemoprevention role. In addition, due to its unique estrogenic properties, genistein can disrupt estrogen binding within molecules and affect estrogen-dependent pathways in a dose-dependent manner,^127,128 It can effectively inhibit the development of breast cancer by blocking estrogen and inhibiting tumor cell proliferation, because it has a strong affinity for ERβ than ERα.¹²⁹ The above preclinical studies collectively support the role of genistein in preventing, retarding, and blocking cancer development, indicating genistein is a promising chemopreventive drug.

Quercetin: Quercetin has a wide range of sources and potential health benefits, which mainly from Allium cepa L., Crataegus pinnatifida Bge., apple, etc. It has been added as a commercial dietary supplement to functional foods, as well as playing a role in the prevention and treatment of cancer.¹³⁰ A great deal of researches have been proved the effect of quercetin in preventing the growth, proliferation and progression of cancer,^{131,132,133,134,135} In addition, quercetin as an antioxidant, can protect cells from oxidative stress and reduce DNA damage by regulating ROS signaling pathways, and maintain redox homeostasis.^136,137 Meanwhile, quercetin can also impact and specifically target oncogenic miRNA^138,139 and long non-coding RNA (lncRNA).^140,141,142 According to reports, it is safe to orally administration quercetin 1 g/day, and it can absorbed up to 60%.¹⁴³ Quercetin is mainly absorbed by intestinal cells in the form of glycosides, hydrolyzed into aglycones, and enters the intestinal cavity.¹⁴⁴ In addition, gut microbiota play important role in the production of glycosidases and enzymes, which can transfer quercetin to smaller, and more easily absorbed molecules.^145,146 Furthermore, no toxicity or side effects of quercetin have been observed in the adults within a reasonable dosage range.¹⁴⁷

Apigenin: Apigenin is a plant-derived flavonoid widely found in Apium graveolens L., Coriandrum sativum L., and Matricaria recutita L., with the health-promoting effect due to its low toxicity and significant effects on normal vs. cancer cells.^148,149 Apigenin induces various programmed cell death processes such as apoptosis, autophagy, and stimulates immune responses, inhibits cell cycle progression, migration, and invasion, suppresses CSCs by modulating signaling pathways involved in tumor development and progression.^150,151 In addition, the interplay between apigenin and miRNA,^152,153,154 lncRNA¹⁵⁵ is also a new avenue for cancer prevention. The chemopreventive potential of apigenin has been demonstrated, but further researches are needed to validate apigenin as a potential chemopreventive agent.

Epigallocatechin-3-gallate (EGCG): EGCG is the most important and abundant polyphenol in green tea, which exerts various biological effects by regulating signaling pathways, such as anti-DNA methylation, anti-apoptosis, anti-inflammatory, anti-angiogenesis, and anti-metastasis.^156,157,158 EGCG is composed of three aromatic rings and connected by a pyran ring, which is believed to be the cause for its biological activity.^159,160 Recent research progress supports the potential role of EGCG in chemoprevention of various cancers by interfering with cancer hallmarks, including sustained proliferation signaling, evasion of growth inhibitors, resistance to cell death, induction of angiogenesis, activation of invasion and metastasis, energy metabolism reprogramming and evasion.¹⁵⁸ EGCG demonstrates a multifunctional mechanism of action by targeting these features to provide possibilities for cancer prevention. In clinical trials, EGCG concentrations usually range from 100 mg to 600 mg/day.¹⁶¹ It was reported patients with Down’s syndrome received EGCG (600–800 mg/day) for 12 months have good safety and tolerability, with no significant changes in aspartate transaminase, alanine transaminase, and electrocardiogram.¹⁶² As the newly reviewed safety of EGCG by European Food Safety Agency, a daily intake of 800 mg or more of EGCG as a food supplement can induce a significant increase in serum transaminases of the human body,¹⁶³ which might be a guidance for the dosage for clinical studies.

Curcumin: Curcumin is an acidic polyphenolic compound mainly presented in Zingiberaceae plant of Curcuma longa L. Its molecular feature characterized by its unique polyphenolic framework, especially the two feruloyl groups connected by methylene, which are responsible for its anti-inflammatory and antiproliferative biological properties.^164,165 Numerous studies have shown that curcumin can inhibit cancer cell proliferation, migration, and metastasis by regulating multiple signaling pathways to induce cancer cell apoptosis, inhibit tumor angiogenesis, reverse multi-drug resistance, improve epigenetics.^166,167,168 Moreover, curcumin exhibited safe, effective, and well-tolerated characteristics, and is an encouraging drug in chemoprevention. Curcumin is recognized as generally safe by the FDA, and healthy individuals can tolerate up to 12,000 mg/day of curcumin without adverse reactions.¹⁶⁹ In a study of curcumin in the treatment of patients with advanced pancreatic cancer, oral curcumin is tolerated without toxicity at doses of 8 g/day for up to 18 months.¹⁷⁰ Moreover, it is worth noting that the safe dosage range of curcumin may vary greatly depending on the type of cancer being treated.¹⁷¹

Resveratrol: Resveratrol exists in various plants, such as grapes, blueberries, Polygonum cuspidatum Sieb. et Zucc., as well as plenty genera of fungi, including Botryosphaeria, Penicillium, Cephalosporium, etc.¹⁷² Resveratrol has been reported with plenty of health benefits, and there were compelling evidences demonstrated its anti-inflammatory, antioxidant, antiviral, anti-aging roles, as well as retarding the growth of a variety of cancer cells in vitro and in vivo models.^{173,174,175,176,177} It has shown that administrating 1.0 g of resveratrol can provide a 0.6 mM maximum plasma concentration, and it occurs within 1 h.^{178,179,180,181} Although the absorption rate of resveratrol in the systemic circulation is very low, its absorption rate can exceed 70%.^182,183 It is worth noting that the blood concentrations of glucuronide and sulfate conjugates in resveratrol are higher than those of free resveratrol, confirming the rapid metabolism of resveratrol and indicating that it may undergo enteropathic recirculation. Gut microbiota have been shown can promote the synthesis of resveratrol from its precursor, thus improving its bioavailability.^184,185 Therefore, regulating the composition of the microbiota composition is one of the mechanisms by which resveratrol exerts cancer chemoprevention.^174,186

Alkaloids

Alkaloids, as natural bioactive compounds, play an important role in the development of anticancer drugs.^187,188,189 It also constitutes a reserve for the lead compounds for drug discovery. Some of these alkaloids have been approved by the FDA as chemotherapy drugs, such as camptothecin (CPT),¹⁹⁰ a well-known inhibitor of topoisomerase I, and vinblastine,¹⁹¹ which interacts with microtubule proteins to cause mitotic disasters.

Camptothecin (CPT): The discovery of the anticancer activity of CPT is considered as a great breakthrough, it was isolated from the bark and stem of Camptotheca acuminata Decne., it has been used in clinical treatment of certain cancers in the 1970s.¹⁹² CPT exerts anticancer effects by inhibiting topoisomerase I (Top 1)-DNA complexes, referred as “Top 1 covalent complex”, the main target of CPT, rather than free TOP 1 enzyme.^193,194 Under normal physiological conditions, the equilibrium between unbound Top 1 and TOP 1 covalent complex shifts towards the free enzyme, while, under the effect of CPT, this balance strongly shifts to the formation of ternary complex, abating the amount of free Top 1 thus eventually diminishing its effect.¹⁹³ Although CPT exhibits significantly higher selectivity towards the Top 1 to cancer cells, its cytotoxic effect can also affect normal healthy cells.¹⁹⁵ Wherein, the most outstanding side effects include myelosuppression and gastrointestinal toxicity. In order to overcome the drawbacks of toxicity and poor stability of CPT, researchers have attempted to prepare it into novel drug delivery systems such as nanoformulations.¹⁹⁶

Vinblastine and vincristine: Vinca alkaloids was the first type of alkaloid applied in cancer therapy, mainly originated from Catharanthus roseus (L.) G. Don, with vinblastine, vincristine, and vinorelbine of well-known, which are the second-most widely used chemotherapy compounds for the treatment of various cancers, especially in combination chemotherapy regimens.¹⁹⁷ Among them, vinblastine and vincristine as natural compounds, have been approved for use in United States.¹⁹⁸ Vinca alkaloids are effective anti-mitotic chemotherapy drugs through inhibiting cell proliferation by binding to microtubules, which can cause mitotic arrest and cell apoptosis.¹⁹⁹ Vinca alkaloids have typical peripheral neurotoxicity, mainly manifested as peripheral, symmetric multiple sensor-motor and autonomic neuropathy.²⁰⁰ In addition to autonomic dysfunction, gastrointestinal toxicity can also be observed.

Berberine: Berberine is a natural isoquinoline alkaloid mainly distributed in Coptis chinensis Franch., Phellodendron chinense Schneid., etc. Convincing studies have shown berberine possesses diverse pharmacological activities of anti-inflammatory, antibacterial, anti-diabetic effects, as well as exhibits preventive and therapeutic effects on various types of cancer.^{201,202,203,204,205} For example, berberine plays a reliable chemopreventive role in the formation of CRC, which can alleviate intestinal ecological imbalance, increase the abundance of beneficial bacteria, regulate small-molecule metabolism of intestinal microorganisms and intestinal inflammation, thus control intestinal mucosal inflammation.^{206,207,208,209} In the stage of CRC, in addition to regulating the homeostasis of gut microbiota, berberine exerts the effect of inhibiting cell proliferation, invasion, and metastasis, blocking the cell cycle, inducing cell apoptosis, regulating cell metabolism, suppressing angiogenesis, and enhancing chemical sensitivity, similar to other active molecules.²¹⁰

Homoharringtonine: Homoharringtonine can be extracted from Cephalotaxus hainanensis H. L. Li, known as a notable bioactive compound in hematological malignancies, it has been used in the clinical treatment of leukemia in China since 1970s, and it was approved by FDA for the treatment of chronic myeloid leukemia (CML) with resistant to tyrosine kinase inhibitors in 2012.^211,212 Homoharringtonine mainly suppressing translation process by affecting the A site in the ribosome, thus effectively inhibiting the initial elongation step of protein synthesis, which is crucial for the survival and proliferation of cancer cells.^213,214 Homoharringtonine also targets the phosphorylated serine 209 residues of the eukaryotic translation initiation factor eIF4E, leading to the degradation of phosphorylated proteins and hindering the growth of leukemia cells.²¹⁵ Although it is currently used for the treatment of leukemia in clinical practice, studies have displayed its inhibitory effects on different types of cancer.^{216,217,218,219,220}

Piperine: Piperine is a widely distributed dietary phytochemical compound, mainly founded in Piperaceae family, with a broad spectrum of pharmacological activities, including anti-inflammatory, antibacterial, and antitumor properties.²²¹ Piperine as a cancer chemopreventive agent can affect cancer cells in various ways, such as influencing cell apoptosis signaling, inhibiting proliferation and survival, restraining invasion and metastasis, affecting redox homeostasis, regulating endoplasmic reticulum stress and autophagy, and blocking drug metabolism enzymes, etc.²²² In addition, piperine can enhance the biological effects of many important therapeutic nutrients and drugs.^223,224,225 Human consumption of piperine in black pepper (5 mg/kg/day) has been determined to have no adverse effect, and subacute toxicity test demonstrated a dose of 100 mg/kg pepper is nontoxic in mice.^226,227 More interestingly, piperine does not undergo any metabolic changes during absorption, as it is found in both intestinal tissue and serous fluid.²²⁶

Terpenoids

Paclitaxel: Paclitaxel is naturally produced in the bark and needles of Taxus wallichiana var. chinensis (Pilger) Florin, is one of the most successful and widely used natural anticancer drugs, and listed on the World Health Organization Essential Medicines List.^228,229 Due to the low yield and high cost of extracting paclitaxel from plants, most of the current paclitaxel is obtained from chemically synthesized or genetically modified endophytic fungi, with its trade name of Taxol.²²⁹ Paclitaxel is a mitotic inhibitor that targets microtubules and induces cell cycle arrest, and ultimately leads to cancer cell death.²³⁰ In addition, paclitaxel can also induce antitumor immunity on various types of immune cells, and the interaction with the immune system is crucial for single or combination therapy.²³¹

Artemisinin: Artemisinin, a gift from traditional Chinese medicine to the world, was extracted from Artemisia annua L., and its discovery has led to the Nobel Prize in Physiology or Medicine to Youyou Tu in 2015.²³² In addition to its well-known anti-malaria effect, it also has excellent antitumor properties. Its biological activity is mainly attributed to the internal endoperoxide trioxane moiety in its sesquiterpene lactone structure.^233,234 The cytotoxic activity of artemisinin in multiple cell lines is attributed to free iron, as it is necessary to activate artemisinin.^235,236,237 In the presence of free iron, artemisinin can convert itself into cytotoxic carbon center radicals, an efficient alkylating agent that induces direct oxidative damage to cancer cells.²³⁵ Moreover, artemisinin upregulates intracellular free ion levels, promotes the accumulation of lipid peroxides in cells, thus induce ferroptosis, and retard the development of cancer.^238,239,240 In addition, artemisinin can induce cancer cell death by disrupting cell membranes, as well as induce cell apoptosis, inhibit angiogenesis, proliferation, and migration of endothelial cells, and improve sensitivity to chemotherapy or radiotherapy.^241,242

Andrographolide: Andrographolide is the major bioactive ingredient present in Andrographis paniculata (Burm.f.) Nees, with immunosuppressive, antipyretic, analgesic, antitumor, antiviral and anti-inflammatory properties.^243,244 Andrographolide showed promising antitumor effects in preclinical studies, established the foundation for its cancer chemoprevention and cancer treatment. It was discovered that andrographolide has the highest concentration distribution in the kidneys,²⁴⁵ and the maximum concentration was 58.62 ng/mL when oral administration of 200 mg of andrographolide after 1.6 h, with the elimination half-life of 10.5 h.²⁴⁶

Ursolic acid: Ursolic acid is the most predominant representative of pentacyclic triterpenoids, obtained from various plants and fruit of Rosmarinus officinalis L., Salvia japonica Thunb., apple peel, and possesses considerable pharmacological activities.^247,248,249 In preclinical studies, ursolic acid is widely known as a cancer chemopreventive agent with the potential to manage the neoplastic progress and target caner hallmarks at various phases.²⁵⁰ Pharmacokinetics studies indicated that the plasma concentration of ursolic acid is low even though the oral administration of doses up to 300 mg/kg, and the removal half-life is relatively short.²⁵¹ Research has revealed that ursolic acid has low cytotoxicity, and even with a daily intake of up to 9.26 g/kg can be well-tolerated.²⁵² The limited bioavailability, solubility, and rapid metabolic characteristics of ursolic acid hinder its clinical application. Therefore, alternative strategies of constructing its analogs or novel delivery systems were able to improve its bioavailability.

According to a great deal of published results, it has been found that natural compounds with cancer prevention activity come from a wide range of sources. In general, the above-mentioned natural compounds have great potential as candidate cancer chemopreventive agents, and further in vivo and in vitro research is needed to explore their targets and mechanisms of action, to provide data support for the translation into clinical applications.

Therapeutic targets of natural compounds as cancer chemopreventive agents

Targeting classical signaling pathways

Testing the potential of cancer chemoprevention drugs mostly starts from in vitro research of cell lines, followed by in vivo tests, to measure the incidence rate, size and quantity of tumors. These in vitro/in vivo studies help to understand the molecular mechanism of the chemoprevention effect of natural compounds. Given that previous researches have comprehensively reviewed oxidative stress and antioxidant defense celluar signaling molecules as targets, such as nuclear factor erythroid 2-related factor 2 (Nrf-2)/antioxidant response element (ARE) pathway,^22,253,254 activator protein 1,¹⁷ matrix metalloproteinases (MMP)²⁵⁵ of the role in cancer chemoprevetion, and we will not go into much detail here. We overviewed the preclinical research results of natural compounds in the field of cancer prevention in past decade, focusing on classic cancer-related signaling pathways, as well as their main targets and pathways, which mainly including mitogen-activated protein kinase (MAPK) signaling pathway, phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway, wingless (Wnt)/β-catenin signaling pathway, nuclear factor kappa-B (NF-κB) signaling pathway, Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway, Hippo signaling pathway, Hedgehog pathway, and signaling pathway-associated crosstalk.

MAPK signaling pathway

MAPK signaling pathway is a complex and interrelated signaling cascade that frequently involved in tumor development, progression, and drug resistance.²⁵⁶ The MAPK cascade is composed of signal transduction partially regulated by phosphorylation and is a highly conserved tertiary kinase model, mainly composed of MAPK kinase kinase (MAPKKK), MAPK kinase (MEK, also known as MAPKK), and MAPK.^257,258 The relevant signals stimulate the upstream kinase MAPKK, and respond by activating the intermediate kinase MAPKK, followed by activating the downstream kinase MAPK.^259,260 The three primary MAPKs are extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 kinase.^261,262 The most extensively studied MAPK pathway is the RAS/RAF/MEK/ERK signaling, which occupies a central position in controlling cell proliferation and differentiation, as well as control various aspects of cancer cell metabolism.^263,264 JNK and p38 signaling also play essential roles in MAPK cascade, they can triggered by various damages mainly activated by MKK4/7 and MKK3/6 kinases, respectively.^265,266 Natural compounds can also exert anticancer effects by regulating the MAPK pathway.

Polyphenols

The cytoskeleton adapter protein vinculin can regulate the interaction between focal adhesion kinase and paxilin through ERK pathway,²⁶⁷ and a high concentration of genistein with 100 μM can dramatically restrain the expression levels of p-FAK, p-paxillin, tensin-2, vinculin, and α-actinin, furthermore, p-p38, p-ERK, and p-JNK levels were also significantly lessened by genistein in B16F10 melanoma cells.²⁶⁸ In HeLa cells, genistein was also reported to markedly suppressed the phosphorylations of p38 and p42/44 proteins in the MAPK pathway to inhibit the migration and invasion.²⁶⁹ Osteopontin plays significant role in determining the metastatic potential of various malignant tumors,²⁷⁰ it can be inhibited by genistein in MDA-MB-435 and MDA-MB-231 cells, meanwhile, genistein can also decreased p-ERK1/2 and p-MEK1/2, and no alteration was observed in nucleus.²⁷¹ Quercetin treatment could promote apoptosis, lead G1 cell cycle arrest and inhibit migration of PC-3 and CD44⁺/CD133⁺ cells via decreased the expression of p-ERK1/2 and p38 in MAPK pathway.²⁷² Quercetin also shows antitumor effect on melanoma cells by increasing the expression of Bcl-2 associated X protein (Bax), p-JNK, p-p38, p-ERK1/2, and cleaved poly-ADP ribose polymerase and decreasing Bcl-2 in vitro, the further in vivo study also displayed that quercetin can significantly reduce tumor volume and increase the expression of p-JNK and p-p38.²⁷³ In Tca8113 cells, apigenin restrains the p-MEKK1, p-ERK1/2, thus prevent the proliferation, invasion, and migration of tumor cells.²⁷⁴ Li et al. elucidated apigenin can inhibit HepG2 cell growth via inducing G1 arrest and activating p38 MAPK pathway by elevating the levels of phosphorylation p38 and decreasing p21.²⁷⁵ In CML cells, the proliferation inhibition and apoptosis induction caused by EGCG was associated with the increased expression of p-JNK and the decreased p38.²⁷⁶ It was reported the levels of phosphorylated ERK and p38 MAPK can be substantially enhanced when the chemo-resistant A549 sublines were treated with curcumin. Knocking down p38 MAPK can significantly reduce curcumin-induced cell apoptosis, and it is suggested activated p38 MAPK signaling was considered as a pro-apoptotic signal for curcumin-induced apoptosis of chemo-resistant human lung cancer cells.²⁷⁷ The study of Zhu et al. provided evidence to confirm the effect of curcumin in activating JNK and p38 but suppressed ERK and p-p65, which leads to SHI-1 tumor of diminished volume.²⁷⁸ Curcumin can inhibit human placental choriocarcinoma cells via activating MAPK signaling by elevating the expression of p-ERK1/2, JUN, p-p90RSK, p–c-JUN.²⁷⁹ The aberrant expression of bone morphogenetic proteins (BMPs) was related to the pathogenesis of various types of cancer, and resveratrol can effectively restrain the proliferation and promote the apoptosis of LoVo cells, which was related with the upregulation of BMP9 to activate p38 MAPK.²⁸⁰ A growing number of evidence indicate that there is a close relationship between diabetes and pancreatic cancer, and resveratrol represses hyperglycemia-driven ROS-induced invasion and migration of pancreatic cancer cells through inhibiting of ERK and p38 MAPK signaling pathways.²⁸¹ Resveratrol has also increased the relative expression of Beclin1 and LC3-II/I while decreased p62 expression, suggesting that resveratrol induced autophagy in NSCLC cells.²⁸²

Alkaloids

Piperine can activate caspase-3 and caspase-9, and cleaved PARP, as well as reduce the phosphorylation of JNK and p38 MAPK in A2780 cells, indicated piperine could induce cell death through the JNK/p38 MAPK-mediated intrinsic apoptotic pathway in ovarian cancer cells.²⁸³ IL-6 as a multifunctional inflammatory cytokine, various studies have shown that higher serum IL-6 concentrations are closely associated with advanced tumor stages and shorter survival periods.^284,285 Piperine was reported can suppress TMK-1 cell invasion via lessening IL-6 expression through inhibition of p38 MAPK signaling.²⁸⁶ Berberine can also decrease the IL-8 expression in vitro and in vivo through the inhibition of the phosphorylation levels of p38 MAPK, ERK1/2 and JNK in gastric cancer MGC-803 cell.²⁸⁷ It was shown whether MG-63 cells were treated with berberine and cisplatin alone or combination, the MAPK signaling was inhibited manifested by the downregulation of expression of p-p38, p-JNK, and p-ERK.²⁸⁸ Zheng et al. validated berberine restrained the growth and induce cell cycle arrest in G0/G1 phase, and apoptosis in NSCLC cells through p38α MAPK-mediated induction of Forkhead box O3a and p53, followed by p21 protein expression.²⁸⁹ Homoharringtonine not only has a significant effect on the treatment of leukemia related diseases, but also inhibits the proliferation of LoVo cell growth, this effect is mainly achieved through inhibition EphB4 and the downstream MAPK/EKR1/2 signaling pathway, suggested homoharringtonine might be a promising EphB4 inhibitor for CRC treatment.²⁹⁰

Terpenoids

Andrographolide inhibits growth of human T-cell acute lymphoblastic leukemia Jurkat cells by upregulation of p-p38 expression, it can also constrict Jurkat xenografts tumor growth in vivo.²⁹¹ In Huh-7 and SK-Hep-1 cells, the treatment of ursolic acid can affect the anti-apoptotic protein of Mcl-1, Bcl-xL, Bcl-2, TCTP, and apoptosis-related proteins tumor necrosis factor-α (TNF-α), Fas, FADD, Bax, cleaved caspase-3, caspase-8, caspase-9, PARP, as well as significantly upregulation the expression of p-ERK1/2 and p-JNK, and downregulation p-p38.^292,293 In addition, ursolic acid can trigger caspase-dependant and ERK1/2 MAPK associated-apoptosis in osteosarcoma MG-63 cells.²⁹⁴ Artemisinin also displays the inhibitory effects on osteosarcoma. Artemisinin could induce the phosphorylation of cAMP response element-binding protein (CREB) via the activation of p38 MAPK signaling in osteosarcoma cells, and the phosphorylation of CREB can also bond specifically to the promoter of secretion of thrombospondin-1 (TSP-1) and promote its transcriptional activation, suggest the p38 MAPK/CREB/TSP-1 signaling cascade might be a potential therapeutic target for osteosarcoma.²⁹⁵

PI3K/Akt /mTOR signaling pathway

The PI3K/Akt/mTOR signaling pathway is a highly conserved and important transduction network in all higher eukaryotic cells, involved in the processes of cell survival, growth, and proliferation, etc.²⁹⁶ It’s one of the most frequently dysregulated pathways in human cancers,^297,298,299 causing apoptosis deregulation and chemotherapeutic resistance,^300,301 and also a common target of natural compounds regulation.^{300,302,303,304}

Polyphenols

As the estrogen receptor like effects of genistein, it reduces cell cycle arrest and apoptosis with altered p-FAK, p-PI3K, p-Akt, p-GSK3β, p21 or cyclin D1 expression in ovarian cancer cells.³⁰⁵ Genistein can also play the chemopreventive potential by inducing G2/M arrest and apoptosis in T24 cells through ROS-dependent blocking of the PI3K/Akt signaling pathway.³⁰⁶ After quercetin treatment, ERα and PI3K/Akt/mTOR signaling were downregulated, concurrently with the inhibition of CD44⁺/CD24⁻ viability and clone formation, implied quercetin targeted cancer stem cells to promote tumor eradication.³⁰⁷ Quercetin might be a good candidate drug for the treatment of invasive B-cell lymphoma, as it reduced the release of IL-6 and IL-10, inhibited the PI3K/Akt/mTOR and STAT3 pathways and induce primary effusion lymphoma cells death. Quercetin also decreases the expression of latent and lytic Kaposis’ Sarcoma-associated Herpesvirus proteins, increases the human leukocyte antigen DR and calreticulin to make the dying cells more easily detected by the immune system.³⁰⁸ Apigenin can also inhibit HCC cell proliferation and induce autophagy by inhibiting the PI3K/Akt/mTOR pathway, and 3-methyladenine and Atg5 genes silencing enhanced apigenin-induced proliferation inhibition and apoptosis, evidenced that the combination of autophagy inhibitors and apigenin would be a potential chemotherapy strategy for HCC treatment.³⁰⁹ It was reported IGF2 was the target of curcumin, by suppressing IGF2, PI3K-p85, Akt, mTOR, eIF4E-bind-ing protein 1 and ribosomal protein S6 kinase beta-1 expression in bladder cancer cell to exhibit the mechanism of suppressing IGF2-mediated PI3K/Akt/mTOR signaling pathway.³¹⁰ By inhibiting the PI3K/Akt/mTOR pathway, curcumin can also exert cytotoxic effect on A549 cells with significantly increasing the expression of Beclin1 and LC3-II, and reducing p62 levels to induce autophagy.³¹¹ In the mechanisms study of curcumin in laryngeal squamous cell carcinoma (LSCC), it was found miR-145 was significantly downregulated in LSCC cells and tissues, and curcumin can dramatically upregulate miR-145 and inhibit PI3K/Akt/mTOR pathway to suppress the progression of LSCC.³¹²

Alkaloids

Han et al. exhibited piperine-induced apoptosis of oral cancer cells is associated with the inhibition of PI3K/Akt/mTOR pathway with the expression levels of the autophagy-related proteins a significant decrease in p-mTOR, Beclin1, and LC3 in vitro, and inhibiting tumor growth, inducing apoptosis in vivo.³¹³ Berberine dosed-dependently inhibited SW480 cells proliferation by inducing autophagy and cell cycle arrest under the regulation of PI3K/Akt/mTOR pathway by upregulating phosphatase and tensin homolog deleted on chromosome ten (PTEN).³¹⁴

Terpenoids

Paclitaxel increases ROS-mediated DNA damage thus triggers the activation of apoptotic signaling pathways, and inhibits the epidermal growth factor receptor (EGFR)/PI3K/Akt/mTOR signaling pathway to prevent PC9 cell proliferation to exert anticancer effect.³¹⁵ Andrographolide shows an inhibitory effect on the proliferation of MCF-7 cells through ERα-mediated transcription and undergoes crosstalk with PI3K/Akt/mTOR signaling in a concentration-dependent manner, and the effects is comparable to that of the anticancer drug fuvinsetron, indicating its potential role as a possible anti-estrogenic agent in the treatment of breast cancer.³¹⁶ Ursolic acid administration can inhibit the proliferation and induce apoptosis in LNCaP and PC-3 cell lines through PI3K/Akt/mTOR pathway, as characterized by the increased Annexin V-binding.³¹⁷ Moreover, administration of ursolic acid significantly inhibits the growth of LNCaP prostate tumor xenografts in vivo, which is confirmed to be related to the inhibition of the PI3K signaling pathway. Thus, ursolic acid appears to be an attractive natural compound for the chemoprevention of prostate cancer. The treatment of artemisinin can dramatically reduce the phosphorylation of PI3K, Akt, and mTOR in uveal melanoma cells, as well as induce mitochondrial membrane potential loss and apoptosis, demonstrating the therapeutic potential on primary intraocular malignancy.³¹⁸

Wnt/β-catenin signaling pathway

Wnt/β-catenin signaling pathway, also known as the canonical Wnt signaling pathway, involves the nuclear translation of β-catenin and activation of target genes through T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription factors.^319,320,321 Once the signaling pathway is activated, it will induce the stability of β-catenin and transfers it to the nucleus, ultimately promoting the expression of genes involved in cell proliferation, survival, differentiation, and migration.^319,322 Moreover, cytoplasmic–nuclear shuttling of β-catenin is believed to be a prominent property of Wnt/β-catenin pathway activation.³²³ Various investigations suggested its dysregulation is one of the most relevant events related to the development of cancer.

Polyphenols

Quercetin can impair the increased expression of β-catenin and cyclin D1 induced by TGF-β in prostate cancer (PC-3 cells), and it has the potency to prevent TGF-β-induced epithelial-mesenchymal transition (EMT) process by suppressing N-cadherin and vimentin while increasing E-cadherin.³²⁴ Apigenin inhibits β-catenin/TCF/LEF signaling activation induced by LiCl in a dose-dependent manner, and restrains β-catenin nuclear entry, thereby inhibiting the expression of Wnt downstream genes, thus significantly inhibits the proliferation, migration, and invasion of CRC cells (HEK293T and SW480) and the growth of intestinal organoids.³²⁵ Apigenin can also suppress in vitro and in vivo HCC (HepG2 and SMMC-7721) growth by downregulating H19 and reducing β-catenin expression, leading to deactivation of Wnt/β-catenin pathway and its downstream genes octamer-binding transcription factor 4 (OCT4), vascular endothelial growth factor (VEGF), CD44, cyclin D1, and axis inhibition protein 2 (Axin2).³²⁶ EGCG can effectively diminish the spheroid formation of lung cancer and CRC, as well as the CSC markers, along with deactivating Wnt/β-catenin pathway to suppress lung cancer and CRC cell proliferation and induce apoptosis.^327,328 In gastric cancer, EGCG decreases nuclear translocation of β-catenin, and downregulates its downstream gene, CCND1, c-Myc, and c-JUN, indicating EGCG restrained proliferation of gastric cancer cells by inhibiting activation of canonical Wnt/β-catenin signaling.³²⁹ The proliferation and invasion of NSCLC cells (95D and A549) can be retarded by curcumin via metastasis-associated protein 1-mediated inactivation of Wnt/β-catenin pathway,³³⁰ and the levels of lung CSCs markers of CD133, CD44, aldehyde dehydrogenas 1A1 (ALDH1A1), Nanog and OCT4 can be downregulated, as well as the tumorsphere formation and the number of CD133 positive cells.³³¹ In liver cancer, curcumin treatment suppresses long intergenic non-coding RNA ROR (lincROR) expression, blocks the activation of Wnt/β-catenin signaling via downregulating downstream target genes of CD44, OCT3/4, CCND1, and c-Myc,³³² sperm-associated antigen 5 expression can also be decreased resulted in the suppressed expression of β-catenin.³³³ The preventive effect of curcumin on CRC is mainly reflected in upregulating naked cuticle homolog 2, suppressing EMT and chemokine receptor 4, and inhibiting the invasion and metastasis, thus leading to the downregulation of key markers of β-catenin, axin, and TCF-4 associated with Wnt/β-catenin pathway.³³⁴ In another research, it was found that curcumin is able to retard CRC by inhibiting Wnt/β-catenin pathway via miR-130a, indicating miR-130a might serve as a novel target of curcumin for the treatment of CRC.³³⁵ In addition to exhibiting inhibitory effects on the aforementioned cancer types, curcumin can also inhibit HepG2 cell proliferation and induced apoptosis by downregulating glypican-3 expression and Wnt/β-catenin pathway,³³⁶ significantly suppressing the levels of Wnt3a, recombinant low density lipoprotein receptor related protein 6 (LRP6), p-LRP6, β-catenin, p-β-catenin, c-Myc, and surviving to induce apoptosis of gastric carcinoma cells (SNU-1, SNU-5, and AGS),³³⁷ and exerting protective effects on chronic tobacco smoke exposure mediated urocystic EMT and acquisition of bladder CSCs through inhibiting Wnt/β-catenin pathway.³³⁸ In the mechanism of treating gastric cancer with resveratrol, runt-related transcription factor 3 and caudal-related homeobox TCF 2 expression levels are upregulated to inhibit the proliferation, along with the restrain of β-catenin and TCF-4.³³⁹

Alkaloids

Berberine can also downregulate lincROR, inducing the inactivation of Wnt/β-catenin signaling in vitro and in vivo and leading to the CRC cell cycle arrest and apoptosis.³⁴⁰ Piperine inhibits the translocation of β-catenin to the nucleus and might suppress the binding of TCF/LEF to the DNA, thus suppresses Wnt/β–catenin pathway to show an anti-CRC effect.³⁴¹

Terpenoids

Tong et al. reported artemisinin notably diminishes tumor growth in A549 xenograft model via downregulated the expression of Wnt5-a/b, LRP6, disheveled, dsh homolog 2 (Dvl2), and β-catenin (including nanog, sox2, OCT3/4, and cyclin D1).³⁴² The findings of Mandal et al. indicated ursolic acid upregulates secreted frizzled related protein 4, and inhibited miR-499a-5p to inhibit Wnt/β-catenin signaling thus targeted breast CSCs.³⁴³ Li et al. explored the relationship between andrographolide and macrophage polarization in breast cancer.³⁴⁴ The in vivo studies displayed that andrographolide restrained the growth of MDA-MB-231 and HCC1806 human breast tumor xenografts and 4T-1 mammary gland tumors through tumor-associated macrophages, and this effect was closely related with the inhibition of Wnt5a/β-catenin pathway, indicated tumor-associated macrophages might be a potential novel therapeutic target for breast cancer.

NF-κB signaling pathway

There are two main pathways that regulate the transcriptional activity of NF-κB signaling proteins, with the first one was known as a canonical pathway, plays an important role in the control of innate immunity and inflammation.^345,346 Compared to canonical pathway, the activation of noncanonical pathway is slow and persistent,^347,348 and exhibits a crucial role in controlling the development, organization, and function of secondary lymphoid organs, as well as the maturation and survival of B cells.³⁴⁵ NF-κB pathway plays a vital role in inflammation-related diseases. It is suggested inflammation is a contributing factor to most solid and hematopoietic malignancies,³⁴⁹ and the activation of NF-κB has been proposed to be a major factor in linking inflammation and cancer development.³⁵⁰ The high expression level of NF-κB in cancer-related tissues leads to the aggregation of pro-inflammatory cytokines and the development of the tumorigenic microenvironment, which in turn cause the occurrence and development of tumors.^350,351 In addition, NF-κB signaling also plays a central role in the metabolic response of tumor cells by coordinating metabolic processes, affecting glycolysis, glutaminolysis, etc.³⁵²

Polyphenols

Quercetin treatment provides chemoprevention on oral squamous cell carcinoma mainly by suppressing NF-ĸB signaling pathway via downregulating NF-ĸB p50 and p65.³⁵³ Snail is a critical transcription factor in regulating EMT in cancer, and patients with higher expression of Snail usually have shorter survival.³⁵⁴ Snail also can be regulated by NF-κB, both colon cancer cells and HCC cells have been found NF-κB/Snail signaling pathway can be inhibited by apigenin treatment.^355,356 In addition, apigenin administration downregulated NF-κB transcription activity, inhibitor of NF-κB (IκB)-α phosphorylation, transcription of p65 and p50 to nucleus, and inhibitor of κB kinase (IKK)-β expression in pancreatic cancer cells and xenograft mouse model.³⁵⁷ EGCG inhibits bladder cancer SW780 cell proliferation and migration both in vitro and in vivo via downregulation of NF-κB p65 and MMP-9.³⁵⁸ Sah et al. further investigated the effect of EGCG on T24 cells, and it was found EGCG blocked the IL-1β stimulated ROS production, in turn restraining NF-κB signaling and anti-invasion effects by inhibiting the expression of urokinase-type plasminogen activator (uPA) receptor,³⁵⁹ the glycosylphosphatidylinositol (GPI)-anchored cell membrane receptors that have vital roles in cell invasion and metastasis of bladder cancer.³⁶⁰ By deactivating NF-κB p65, EGCG can repress nasopharyngeal cancer stem cell self-renewal and migration and reverses EMT.³⁶¹ Marquardt et al. displayed the growth-suppressive effects of curcumin on hepatoma cells was dependent on the extent of NF-κB inhibition, and emphasized the potential of NF-κB targeting to effectively consume CSCs in liver cancer. Meanwhile, it is pointed out that HCC patients with poor prognosis may benefit from curcumin treatment, and specific disruption of NF-κB signaling might be a potential therapeutic method for HCC patients with poor prognosis.³⁶² Curcumin can also modulate the dysregulation of miR-200c, miR-21, miR-let7c, miR-26a, and miR-125b in advanced thyroid cancer, which are associated with regulating cell differentiation and NF-κB activity, as well as decrease NF-κB p65 activity.³⁶³ Plenty of studies have shown autocrine growth hormone signaling cause EMT and trigger a metastatic profile by increasing occludin and fibronectin expression levels in breast cancer.^364,365 With the treatment of curcumin, the invasion and metastasis of breast cancer cells can be inhibited by autocrine growth hormone-mediated targeting both canonical and noncanonical NF-κB signaling.^366,367 In addition, curcumin can also play anticancer role against gastric cancer,³⁶⁸ pancreatic cancer,³⁶⁹ NSCLC,³⁷⁰ and CRC³⁷¹ via regulating NF-κB signaling. VEGF is a key regulator of angiogenesis, and IL-8 is a regulation protein involved in tumorigenic activities in cancers, resveratrol can modulate activity of VEGF and IL-8 in SKOV-3 cell aggregates via significant attenuation of the expression of NF-κB, p-NF-κB, and proliferating cell nuclear antigen.³⁷²

Alkaloids

Li et al. demonstrated berberine can constrict the growth of HepG2 cells by promoting apoptosis through the NF-κB p65 pathway.³⁷³ Homoharringtonine exhibits an anti-inflammatory activity, and can attenuate dextran sulphate sodium (DSS)-induced colitis by inhibiting macrophage-associated NF-κB activation (downregulate p-p65 and p-IκBα) and M1 polarization, which could be an option for the treatment of ulcerative colitis (UC) or prevention of CRC.³⁷⁴ A unique molecular mechanism of homoharringtonine was reported by Chen et al. that by directly binding NF-κB repressing factor (NKRF), several NF-κB target genes including MYC, MMP can be regulated.³⁷⁵ By strengthens the interaction of p65-NKRF, and interferes with p65-p50 complex formation, homoharringtonine can attenuate the transactivation activity of p65 on MYC gene in acute myeloid leukemia (AML), moreover, the expression of a frequently mutated and/or highly expressed gene, named KIT, can be significantly diminished, which indicated patients with MYC and KIT overexpression could achieve a favorable response to homoharringtonine treatment.³⁷⁵

Terpenoids

Zhang et al. found andrographolide can inhibit proliferation of SW620 cells through the toll-like receptor 4 (TLR4)/NF-κB/MMP-9 signaling pathway of decreasing the expression of TLR4, MyD88, p65, and MMP-9.³⁷⁶ In another type of CRC cell of HCT116, andrographolide attenuated TNF-α-induced IL-8 via inhibition of NADPH oxidase/ROS/NF-κB signaling and then suppresses angiogenesis in tumor microenvironment.³⁷⁷ Andrographolide remarkably constricted the expression of p65 and p-p65 in MCF-7 cells and tumor tissues of MMTV-PyMT mice to deactivate NF-κB pathway to inhibit the expression of miR-21-5p, thus stimulating programmed cell death 4 expression to abate luminal-like breast cancer growth, metastasis, and invasion.³⁷⁸ Li et al. investigated the anticancer effect of ursolic acid in four types of gastric cancer cells including BGC-823, HGC-27, AGS, and MGC-803. Different types of cells have different responses to ursolic acid, the expression of N-cadherin, vimentin, Snail, Twist p-Axl, p-IKKα/β, and p-NF-κB were downregulated in BGC-823 and MGC-803 cells, meanwhile, the expression of N-cadherin, Snail, p-Axl, and p-IKKα/β were diminished in xenograft model rats, indicating the anticancer effect of ursolic acid was conducted by the attenuation of EMT, which was associated with the regulation of Axl/NF-κB pathway.³⁷⁹ Su et al. discovered artemisinin exert preventive effects on H. pylori-induced gastric cancer both in vivo and in vitro, these effects were closely related to the inhibition of NF-κB signaling of the decreasing expression of p-p65 and p-IκB-α, as well as restraining the downstream inflammatory factors of IL-8 and TNF-α.³⁸⁰

JAK/STAT signaling pathway

The JAK/STAT signaling pathway is considered as one of the central communication nodes in cellular function, it forms a rapid membrane-nucleus signal transduction module, and induces the expression of various key mediators in cancer and inflammation.^{381,382,383,384} Blocking the JAK/STAT signaling in cancer cells can inhibit the expression of target genes that control basic cellular functions and hinder cancer cells from escaping growth control mechanisms, thus, antagonizing JAK/STAT pathway might prevent the transformation of precancerous lesions into malignant tumors.^385,386 In addition, plenty of studies have shown that activation of the JAK/STAT pathway plays significant role in the homeostasis of the immune system.^387,388 Inhibition of JAK/STAT signaling on the restrain of pro-inflammatory responses and auto-immune conditions, appears to be a promising strategy for preventing disease progress, particularly in skin diseases.³⁸⁹

Polyphenols

Igbe et al. proposed quercetin stimulated the inhibitory effect of IFN-α on HCC (HepG2 and Huh-7) cells proliferation by inhibiting Src homology domain 2 containing tyrosine phosphatase 2 activation of the JAK/STAT pathway. Interestingly, only the tyrosine phosphorylation of STAT1, but not STAT3 can be significantly raised by quercetin.³⁹⁰ In addition, quercetin can also suppress p-JAK2 and p-STAT3 to deactivate JAK/STAT signaling in LM3 cells, as well as inhibit LM3 cell tumor growth in vivo.³⁹¹ Ko’s team found apigenin can inhibit the proliferation of MDA-MB-453 cells by upregulating the levels of caspase-3 and caspase-8 to induce the cleavage of PARP, and blocking the activation of p-JAK2, p-STAT3, and the nuclear staining of STAT3.³⁹² They further studied another breast cancer cell line of BT474, and found apigenin can also effectively inhibit its proliferation in a dose- and time-dependent manner through downregulating the expression of p-JAK1, p-JAK2, and p-STAT3 and decreasing the production of STAT3 target genes such as VEGF and MMP-9 to prevent or treat HER-2-overexpressing breast cancer.³⁹³ Torres et al. demonstrated EGCG transcriptional regulated ES-2 ovarian CSC molecular signature in tumorspheres and induced a pro-apoptotic phenotype.³⁹⁴ Further mechanistic research indicated EGCG mimic as a JAK/STAT3 inhibitor to regulate the acquisition of CSC phenotype and chemotactic response of ovarian cancer tumorspheres, which supported the chemopreventive benefit of EGCG in cancer.³⁹⁴ Liu et al. evaluated the potential chemoprevention and therapeutic effect of curcumin in esophageal squamous cell carcinoma (ESCC) patient-derived xenograft models.³⁹⁵ It was indicated that curcumin inhibited JAK2 and arrested the activation of STAT3, leading to diminished expression of STAT3-regulated genes and increased apoptosis in ESCC.³⁹⁵ Moreover, prophylactic administration of curcumin was significantly more effective in inhibiting 75% of patient-derived xenograft models than giving curcumin only after the innoculation of tumors, indicating curcumin might be an effective chemoprevention agent for ESCC. Curcumin might be a chemoprevention agent for CRC, as it can suppress the activation of dendritic cells by diminishing the phosphorylation of JAK2, STAT3, and STAT6, elevating the downstream proteins of suppressor of cytokine signaling (SOCS) 1, SOCS3, and protein inhibitor of activated STAT3 to restore immunologic balance and to treat colitis in an effective manner.³⁹⁶ Curcumin can inhibit the growth and recurrence of various tumors by adjusting JAK/STAT signaling pathway. In papillary thyroid carcinoma of BCPAP, TPC-1, and SW1736 cells, curcumin blocks G2/M phase, increases apoptotic rate, and downregulates p-JAK2 and p-STAT3 to inactivate JAK/STAT signaling.^397,398 Curcumin suppresses invasiveness and vasculogenic mimicry of squamous cell carcinoma of the larynx through the inhibition the levels of JAK2, p-STAT-3, as well as endothelial nitric oxide synthase related with cell growth and apoptosis, MMP2 and VEGF associated with antiangiogenic.³⁹⁹ In osteosarcoma, the inhibition of the proliferation and migration of MG-63 cells by curcumin is through the restraining p-JAK2 and p-STAT3 to induce the arrest of the G0/G1 phase and apoptosis.⁴⁰⁰ In addition, curcumin can reduce fascin expression through inactivation JAK/STAT signaling to exhibit as a potential cancer chemoprevention agent for suppressing metastasis and recurrence of ovary cancer.⁴⁰¹ Resveratrol alleviates inflammatory bowel disease (IBD) via the reduction of O-GlcNAcylation of STAT3 in intestinal epithelial cells, thus preventing its phosphorylation and abating the activity of the JAK2/STAT3 pathway.⁴⁰² Resveratrol can also restrain the proliferation and invasive migration ability of GBM cells, as well as improve the inflammatory response of glioblastoma by inhibiting JAK2/STAT3 pathway and impairing the activation of NLRP3 inflammasomes.⁴⁰³

Alkaloids

Berberine inhibits JAK2/STAT3 pathway through reducing the expression of IL-6, thereby causing G0/G1 phase cell arrest and apoptosis in gastric cancer cell in vitro and in vivo.⁴⁰⁴ Through upregulation of miR-17-5p, berberine exhibits anti-bladder cancer effect via inactivating JAK1/STAT3 signaling. MiR-17-5p can directly bind to the 3’ UTR of JAK1 and STAT3, and block the expressions of JAK1, p-STAT3, and STAT3, thus provide supporting evidence for berberine to prevent the progression of bladder cancer.²⁰⁵ Homoharringtonine can prevent cells growth, cell viability, and induce cell apoptosis through mitochondria pathway in gefitinib-resistant NSCLC cell lines.⁴⁰⁵ Mechanistically, homoharringtonine retards IL-6-induced STAT3 tyrosine 705 phosphorylation and deactivates JAK1/STAT3 signaling.⁴⁰⁵ Wherein, cells with EGFR T790M mutation are more sensitive to homoharringtonine treatment.

Hippo signaling pathway

Hippo signaling plays a crucial role in regulating cell proliferation, differentiation, and survival, has been evidenced to contribute to the progression of various diseases, especially cancer.⁴⁰⁶ The Hippo pathway can be activated by a sequence of phosphorylation events. When cancer occurs, the Hippo signaling becomes inactive, the unphosphorylated yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ) enters the nucleus, and binds to the transcriptional enhanced associate domains (TEADs) transcription family, synergistically promoting the expression of target genes, enhancing cell proliferation, resistance to apoptosis, invasion and metastasis.^407,408 In addition, multiple studies have demonstrated the role of the Hippo pathway in regulating host immunity,^409,410,411 and it also has been proved to regulate the immune checkpoint molecule PD-L1.^412,413,414 The development of targeted Hippo pathway for cancer chemoprevention and treatment by natural compounds is still in its infancy stages with a promising future.

Polyphenols

As a highly recurrent cancer, triple-negative breast cancer (TNBC) lacks effective prevention measures.⁴¹⁵ Li et al. demonstrated the inhibitory effect of apigenin on stemness features of TNBC cells in vitro and in vivo.⁴¹⁶ It was found apigenin protected organism by reducing the activity of YAP/TAZ and the expression of target genes such as connective tissue growth factor (CTGF) and cysteine-rich protein, angiogenic inducer 61 (CYR61), as well as corrupting the YAP/TAZ-TEADs protein-protein interactions.⁴¹⁶ Apigenin reduces YAP expression to lessen migration and invasion by adjusting the expression of the EMT markers, and assisted the autophagy of HCC cells by modulating the expression of autophagy-related genes.⁴¹⁷ As a common cancer type of head and neck, tongue squamous cell carcinoma can be induced to apoptosis by EGCG via declining the Hippo-related protein levels of TAZ, large tumor suppressor homolog 1 (LATS1), MOB kinase activator 1 (MOB1).⁴¹⁸ Kruppel-like factor 5 (KLF5) has been reported to exert carcinogenic effects in various cancers.^419,420,421 Curcumin behaves an effective suppression on tumor growth and pro-proliferative YAP/TAZ/KLF5/cyclin D1 axis, thus promoting KLF5 proteasome-dependent degradation in bladder cancer.⁴²² Curcumin also play an anti-pancreatic cancer role by downregulating YAP and TAZ.⁴²³ In addition, curcumin can also inhibit autophagy in CRC and stemness in NSCLC by inhibiting nuclear cytoplasm translocation of YAP or TAZ alone, thus activating Hippo signaling.^424,425 It was known persistent liver fibrosis can progress to HCC.⁴²⁶ Li et al. found resveratrol heightened the activation of Hippo signaling and attenuated the expression of YAP and TAZ to contribute the apoptosis of hepatic stellate cells.⁴²⁷ A novel mechanism was found by Xu et al. that ST6 beta-galactosamide alpha-2,6-sialyltranferase 2 (ST6GAL2) can inactivate Hippo signaling and promote tumorigenesis of follicular thyroid cancer, and resveratrol can reverse this process by suppressing the expression of ST6GAL2 and promoting p-mammalian STE20-like protein kinase (MST) 1/2, p-LATS1, and p-YAP, which provide a preventive possibility for patients with difficult thyroid cancer diagnosis.⁴²⁸ The cell cycle arrest and apoptosis of HCT116 cells can be also abated by resveratrol via upregulation p-YAP and downregulation of total YAP protein, and reduced the mRNA expression of the YAP signaling downstream genes CTGF and CYR61, which was partially mediated by the interaction between YAP and TEAD.⁴²⁹ Resveratrol exhibits a beneficial effects on breast cancer cell invasion by suppressing RhoA, leading to the activation of LATS1 and phosphorylation of YAP.⁴³⁰ Besides, resveratrol can also inhibit EMT in gastric cancer cells through Hippo–YAP pathway.⁴³¹

Alkaloids

Homoharringtonine significantly enhances the phosphorylation levels of MST1/2, MOB1, LATS1, YAP, as well as salvador homolog 1 to activate Hippo signaling thus induce HCC cell cycle arrest at S phase and promote apoptosis.⁴³² Ursolic acid diminishes the proliferation and metastasis of gastric cancer via the modulation of upregulating MST1, MST2, LATS1, and p-YAP through ras association (RalGDS/AF-6) domain family 1, suggesting ursolic acid might be a potential chemopreventive and therapeutic agent for gastric cancer.⁴³³ Li et al. disclosed artemisinin impaired mitochondrial respiration, induced ROS production, suppressed aerobic glycolysis, as well as regulated p-YAP and cytoplasmic retention thus inhibiting HCC cell growth, migration and invasion.⁴³⁴

Hedgehog pathway

The Hedgehog signaling pathway implicated in embryonic development, tissue patterning, and wound healing.⁴³⁵ Hedgehog signaling can divided into canonical and noncanonical pathway to elicit various cellular response. The canonical signaling pathway involves binding Hedgehog ligands to patched (PTCH) receptor to relieve the suppress of Smoothened transducer (SMO) by transmembrane receptor PTCH, and allowing SMO entering the primary cilium, thus activating lima-associated oncogene (GLI) transcription factors.⁴³⁶ In noncanonical Hedgehog pathway, it is largely attributed to crosstalk with other signaling cascades, partially by affecting the activity of GLI transcription factors.⁴³⁷ The imbalance of Hedgehog signaling pathway has been emphasized in cancer, and it is currently believed that one-third of malignant tumors rely on the abnormal function of this pathway.^436,438

Polyphenols

Zhang et al. confirmed that genistein can inhibit cell proliferation, and induced apoptosis of nasopharyngeal CSCs via suppressing the tumorsphere formation capacity, decreasing the number of EpCAM⁺ cells, and downregulating the expression of nasopharyngeal CSCs markers, as well as suppressing Sonic Hedgehog (SHH) signaling mainly by downregulating the levels of SHH, SMO, and GLI 1.⁴³⁹ Genistein also shows an inhibition on renal CSCs through SHH pathway by diminishing SHH, SMO, GLI 1, and GLI2.⁴⁴⁰ In gastric CSCs, the levels of CD44, SHH, PTCH1, and GLI 1 are significantly reduced by genistein.⁴⁴¹ It was considered that genistein could be an effect cancer therapy by modulating CSCs characteristic. The CSCs markers of CD44, CD133, OCT4, ALDH1A1, and Nanog were downregulated by EGCG, and the decreased levels of the components in Hedgehog pathway mediated the inhibitory effect of EGCG on bladder CSCs.⁴⁴² This research team also studied the effect of curcumin on bladder CSCs. Curcumin can also suppress CSC properties through downregulating the expression of SHH, SMO, GLI 1, and GLI2 in SHH pathway thus diminishing the proliferation and enhancing apoptosis of bladder CSCs.⁴⁴³ Li et al. demonstrated curcumin decreased the expression of EMT and stemness in MDA-MB-231 mammospheres, which was related to the downregulation of GLI 1, GLI2, PTCH1, and SMO, especially GLI 1. Meanwhile, GLI 1 expression was significantly reduced in nucleus and cytoplasm, and it was found vimentin was interacted with GLI 1, which might be a downstream target of GLI 1 to suppress EMT and stemness.⁴⁴⁴ In addition, curcumin displays an important role in restraining hypoxia-induced pancreatic cancer metastasis via suppressing Hedgehog signaling pathway.⁴⁴⁵ It was reported resveratrol can reverse gastric cancer cell (SGC‑7901) proliferation, migration, invasive capacities, as well as EMT changes via the deactivation of Hedgehog pathway with the expression of decreased SHH, SMO, GLI 1.^446,447 In pancreatic cancer of MIA PaCa-2 cell line, resveratrol induces apoptosis of MIA PaCa-2 cells in a dose-dependent manner, and the levels of Indian Hedgehog, PTCH, and SMO were reduced;⁴⁴⁸ in BxPC-3 and Panc-1 cell lines, resveratrol also suppresses hypoxia-induced expression of metastatic-related factors, uPA and MMP2, as well as markedly inhibits hypoxia-mediated activation of the Hedgehog signaling pathway via downregulating SHH, SMO, and GLI 1.⁴⁴⁹ Therefore, the changes in the same signaling pathway may vary among different cell lines of the same type of cancer, thus, research on multi-cellular lines is necessary. Resveratrol also has a good effect on improving gynecological tumors, such as ovarian cancer and cervical cancer. Ferraresi er al. displayed the significant role of Hedgehog downstream effector polycomb complex protein BMI-1 (BMI-1) in mediating lysophosphatidic acid pro-tumorigenic activities through inhibition of autophagy, and the capability of resveratrol to prevent Hedgehog pathway and BMI-1 activation to rescue autophagy and dampen the malignant features of ovarian cancer cells.⁴⁵⁰ The Cancer Genome Atlas data pointed patients with low expression of Hedgehog pathway/EMT-related genes have a better prognosis indicating resveratrol can as an adjuvant therapeutic for ovarian cancer.⁴⁵⁰ In cervical cancer, resveratrol inhibits the expression of SHH, SMO, GLI 1 to induce apoptosis, and constricts the migration and invasion of the HeLa cells.⁴⁵¹ In addition, Hedgehog pathway can be inhibited by resveratrol via downregulating the protein of PTCH, SMO, and GLI 1 in a dose- and time- dependent manner in HCT116 cells.⁴⁵² It was found resveratrol restrained cell proliferation and induced cell apoptosis of renal CSCs, as well as the Hedgehog pathway mediated the suppressive effects of resveratrol.⁴⁵³

Alkaloids

Research by Wang et al. showed that berberine significantly inhibited Hedgehog pathway in medulloblastoma, meanwhile, berberine cannot affect transcriptional factors activities induced by TNF-α and prostaglandin E₂, indicated its selective activation of Hedgehog pathway. Furthermore, berberine inhibits the Hedgehog pathway by targeting SMO, the most successful molecular target for developing Hedgehog pathway anticancer drugs.⁴⁵⁴ It was reported berberine exerts strong potential in treating CRC with reducing the levels of SHH, PTCH1, SMO, GLI 1, enhancing the expression of suppressor of fused in vitro and in vivo, as well as retarding c-Myc downstream of Hedgehog signaling, and effectively improve the pathological profile of subcutaneous HCT116 xenograft tumor. The study also evaluates the toxicity of berberine on zebrafish, and points out that long-term and excessive consumption of berberine may cause cardiac and hepatic toxicity.⁴⁵⁵

Signaling pathway-associated crosstalk

Various studies have shown that no pathway exists independently, and direct regulation or mutual influence between signaling pathways and targets can enable cells to integrate different environmental signals and make more precise responses to meet the overall needs of the body.³⁰¹

It was reported genistein can reduce the upregulation of interferon-γ (IFN-γ)/JAK1/STAT1 and INF-γ/toll-like receptor-4/NF-κB signaling pathways and modulate interferon regulatory factor-1/calcium-independent nitric oxide synthase/ nitric oxide (NO) and IL-6/JAK2/STAT3/COX-2 pathways and consequently, reduce the levels of TNF-α and IL-1β to preserve colon function, thus treating UC.⁴⁵⁶ In this study, genistein exhibited similar therapeutic effects as sulfasalazine (the agent most widely prescribed and standard therapy for IBD treatment), and the combination of the two agents yielded a superior benefit. Quercetin has been shown to suppress chronic stress-induced TNBC cell proliferation and migration by blocking the β₂-adrenergic receptor/ERK1/2 pathway.⁴⁵⁷ Xia et al. demonstrated apigenin suppressed IL-1β-induced urokinase-type plasminogen activator receptor expression by inhibiting ERK1/2/JNK pathway and ERK1/2/JNK-dependent transcription factor AP-1 and NF-кB to exert anti-invasion effects in T24 cells.⁴⁵⁸ Kassouri et al. reported EGCG can induce transcriptomic program by increasing C-C motif chemokine ligand 5, cystatin 8, and interferon-induced transmembrane protein 3 characterized by reduced proliferative and stem-like features, which included the downregulation of Notch and CBFA2T3 pathways in macrophage-like differentiated human HL60 promyelocytic leukemia cells.⁴⁵⁹ Curcumin can effectively abolish the characteristic of lung CSC, and inhibit Wnt/β-catenin and SHH pathway to exhibit its prevention effect on lung cancer.³³¹ Kurzava Kendall et al. found the epigenetic effects of resveratrol on oncogenic signaling in breast cancer. Resveratrol led to a DNA methylation increase within GLI2 and WNT4 enhancers, and the downregulation of CCND1 and CYR61, the common targets share by both of Hedgehog and Wnt signaling.⁴⁶⁰ The regulation of resveratrol on epigenetics may be a new strategy for the prevention of breast cancer. Li et al. verified berberine markedly decreased p-AKT1 expression and disturbances ERK/MAPK as well as p38 and JNK/MAPK pathways to inhibit migration and induce mitochondrial apoptosis in diverse thyroid carcinoma cells.⁴⁶¹ In addition, berberine can also inhibit the progression of gastric cancer by regulating the crosstalk of adenosine 5‘-monophosphate (AMP)-activated protein kinase (AMPK) and Wnt signaling pathways.⁴⁶² Berberine could downregulate hepatocyte nuclear factor 4α through activating AMPK signaling, and further downregulate Wnt5 and β-catenin to attenuate the growth, invasion and metastasis of gastric cancer. Cheng et al. determined homoharringtonine as an anti-STAT3 agent by regulating the crosstalk between STAT3 and Wnt signaling to achieve preventing CRC progression and recurrence.⁴⁶³ Homoharringtonine remarkably inhibited STAT3 expression and reduced epidermal growth factor-mediated β-catenin expression, leading to the inhibition of Wnt signaling to breakdown cancer stem-like tumorspheres in selective EGFR-positive CRC. Piperine can also prevent CRC by regulating the crosstalk between Nrf-2 and NF-κB pathways. Rehman et al. investigated prophylactic treatment of piperine can activate Nrf-2 pathway which triggers antioxidant response mediators of hemeo xygenase-1, glutathione, superoxide dismutase, etc., and block NF-κB signaling and its downstream molecules of COX-2, TNF-α, IL-6, etc. to prophylactic treatment of CRC.⁴⁶⁴ Ursolic acid can not only increase the activity of glycogen synthase kinase by downregulating PI3K/Akt signaling pathway, but also inhibit inflammation and prevent the progress of breast cancer by downregulating NF-κB signaling.⁴⁶⁵

In general, partial studies are limited to exploring the mechanism of natural compounds in cancer prevention from in vitro cell models, and we found the effects of the same natural compound on different type cell lines of the same cancer are not completely consistent, which may be due to different cell phenotypes. Further researches on multiple types of cell models should be strengthened. Besides, in vitro research cannot be limited to cell lines, as more complex in vitro cancer models have been developed, such as organ-chips, which can simulate the pathophysiology of cancer and conserve organ microenvironment, allowing researchers to study the effects of drugs on cancer in a controlled environment.⁴⁶⁶ With the continuous advancement of CRISPR-Cas technology, gene manipulation can now be performed on different types of organoids, and patient-derived organoids and organoids co-culture model systems can also as excellent model with tumor microenvironment.^{467,468,469,470} In addition, further in vivo studies are needed to verify the results, and currently, in vivo research usually adopt xenograft tumor mice as subjects, but far from perfect in vivo model. Moreover, current researches usually employed inhibitors or activators of a specific target for validation, which may not be able to remove interference factors from other signaling pathways. The technologies of CRISPR-Cas,⁴⁷¹ site-specific recombinase,⁴⁷² and induced expression (small molecule and virus) can more effectively capture human genetics within mice in a time-tunable and tissue-specific manner. Besides utilizing classical techniques, combining novel technologies and models will better facilitate the study of natural compounds mechanisms and discover key targets for cancer prevention.

Targeting immune checkpoints

Natural compounds can not only act on traditional signaling pathways, but also exert cancer chemoprevention by affecting immune checkpoints (Fig. 4a). Through the years, unsatisfactory outcomes of cancer treatment are partially due to systemic immune unresponsiveness or immunosuppression to cancer. The immune system dysfunction in tumors leads to malignant cells evading immune surveillance, immune recognition, and eradication.⁴⁷³ The emergence of immunotherapy provides a revolutionary new approach for the treatment of cancer. The purpose of cancer immunotherapy is to equip patients with cancer-fighting immunity, utilize their immune system to eradicate cancer and prevent recurrence.^474,475 It was the emergence of immunotherapy that led to the 2018 Nobel Prize in Physiology or Medicine was awarded to Drs James P. Allison and Tasuku Honjo for their work in this field. Tumor immunotherapy mainly includes immune checkpoint inhibitors (ICIs), cellular immunotherapy, and tumor vaccines. Among them, ICIs are the most widely used tumor immunotherapy methods at present, and FDA has approved antibodies against programmed cell death-1 (PD-1), programmed death ligand-1 (PD-L1), and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) for various solid tumors, which proved the effectiveness and prospects of immunotherapy as an anticancer approach.

Fig. 4

Natural compounds targeting immune checkpoints and gut microbiome to serve as cancer chemopreventive agents. a Targeting immune checkpoints. T cells recognize antigens presented by the major histocompatibility complex (MHC) on the surface of cancer cells through their T-cell receptor (TCR). However, this signal is not sufficient to initiate T-cell response and requires a second signal transmitted by the costimulatory molecules of B7. The interaction between cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and costimulatory molecules mainly occurs during the initiation phase of T-cell responses. Programmed death-1 (PD-1) inhibitory receptors are expressed by T cells during long-term antigen exposure and exert negative regulation on T cells during their association with programmed death ligand-1 (PD-L1), and the PD-1 interaction occurs during the effector phase of T-cell responses. The inhibitory effect of natural compounds on tumor can be achieved by interfering with the interaction between PD-L1 or CTLA-4 with their corresponding receptors, and blocking the interaction of related signaling pathways, promoting T-cell activation, thus restoring immune cell function and promoting a strong tumor immune response. b Targeting gut microbiome. The imbalance of gut microbiota is closely related to the development of cancer, tumor growth may lead to local disruption of the barrier, resulting in microbial invasion and immune monitoring disrupted. The gut microbiome regulates various host processes, including metabolism, inflammation, and immunity. Natural compounds can improve cancer-related metabolism, inflammation, and immunity by remodeling gut microbiota homeostasis. DC dendritic cell. This figure was created with Biorender.com, and adapted from Antoni Ribas.⁴⁸²

Targeting the PD-1/PD-L1 axis

PD-1 is one of the co-inhibitory receptors expressed on T cells, B cells, and type 2 tumor-associated macrophages.⁴⁷⁶ PD-L1 is the ligand of PD-1, expressed widely on immune cells, tumor cells and other tumor microenvironment cells,⁴⁷⁷ and PD-1/PD-L1 axis plays crucial role in physiological immune homeostasis and is considered as a means for tumor cells to evade immune surveillance.^{476,478,479,480} An accumulating body of research indicates that the activation of PD-1/PD-L1 axis negatively modulates T-cell-mediated immune responses in peripheral tissues to protect tissues from immune-mediated tissue damage.^479,481,482 Currently, nivolumab, pembrolizumab, tislelizumab, atezolizumab, durvalumab, and avelumab have been approved for clinical use in the treatment of melanoma, NSCLC, etc. Evidence suggests that natural compounds have potential immunomodulatory effects and can regulate the PD-1/PD-L1 axis.

Quercetin can highly binds with PD-L1 and inhibit the binding of glycosylated PD-1/PD-L1, thus promote the activation of T cells by blocking the interaction of PD-1/PD-L1, which shows exciting potential as a cancer chemopreventive agent.¹³³ Xu et al. reported apigenin and curcumin significantly inhibited IFN-γ-induced PD-L1 upregulation, reduced p-STAT1, enhanced T-cell-mediated melanoma cell killing.⁴⁸³ In vivo studies have shown apigenin strongly suppressed tumor growth and boosted T-cell immunity via inhibiting PD-L1 expression in host dendritic cells.⁴⁸³ The dual effect of apigenin on the inhibition of PD-L1 expression provides new insights into the anticancer effect of apigenin and exhibits potential clinical significance. Lim et al. determined TNF-α is the main factor triggering cancer cell immune suppression against T-cell surveillance through stable PD-L1, and COP9 signalosome subunit 5 (CSN5) is a necessary factor in this process, with the ability to inhibit the ubiquitination and degradation of PD-L1.⁴⁸⁴ Besides, curcumin as a CSN5 inhibitor diminished PD-L1 expression and benefited anti-CTLA-4 therapy, suggesting curcumin might be a practical adjuvant to enhance immune-based therapies in breast cancer.⁴⁸⁴ Evidence suggests resveratrol act as inhibitor of glyco-PD-L1-processing enzymes to modulate N-linked glycan decoration of PD-L1, and interact with inner surface of PD-L1 to directly target PD-L1, which provide novel mean to restore T cells function.⁴⁸⁵ Resveratrol can also activate Sirtuin 1 deacetylase to deacetylate and stabilize the transcription factor Snail, which, in turn restrain the transcription of Axin2, thus lead to an enhanced binding of β-catenin/TCF and PD-L1 promoter, and display its role in inhibiting T-cell function in lung cancer.⁴⁸⁶ Liu et al. discovered berberine as a novel negative regulator of PD-L1 in NSCLC, it exerted antitumor effect through diminishing PD-L1 expression via specific binding to Glu76 of CSN5 and inhibiting its deubiquitination, as well as activating tumor-infiltrating T cells, which provided a theoretical basis for the potential application of berberine as a small-molecule inhibitor to disrupt PD-L1-mediated immunosuppression.⁴⁸⁷ Andrographolide diminishes PD-L1 expression via inducing p62-depending selective autophagy by modulating STAT3, besides, andrographolide potentiates the antitumor effect of anti-PD-1 mAb immunotherapy by stimulating the infiltration and function of CD8⁺ T cells.⁴⁸⁸ Kang et al. unveiled ursolic acid exhibited antitumor activity by repressing PD-L1 expression via EGFR/JAK2/STAT3 pathway, as well as downregulating the bindings of STAT3 to MMP2 and PD-L1 promoters in NSCLC.⁴⁸⁹

Targeting CTLA-4

CTLA-4 is also a classic immune checkpoint molecule, acting as a CD28 homolog, has a strong binding affinity to the ligands CD80 (B7-1) and CD86 (B7-2),^490,491 thus affects T cells in the priming phase of T cells activation and decreasing T-cell immune response.^492,493 In addition, CTLA-4 is also participate in other aspects of immune control, especially the regulatory T cells (Tregs) exhibit constitutive expression of CTLA-4, which is considered vital for their inhibitory effect.^493,494 It was reported the decrease of CTLA-4 expression on CD4⁺CD25⁺ Treg with curcumin was associated with the declining of Treg-mediated inhibitory activity.⁴⁹⁵ Resvertrol can elevate the reduced Treg-related genes expression of CTLA-4 and TGF-β, thus restoring the function of Tregs.⁴⁹⁶

Immunotherapy has become a promising treatment method and has made exciting progress, but there still exist several problems. For example, the patient’s responsiveness is not high and there are fewer beneficiaries. Although patients have a positive response to immunotherapy, some patients might quickly develop resistance and experience pseudo-progression, and severe irAEs. In light of natural compounds that can act on immune checkpoints and have the advantage of reducing chemotherapeutic side effects, its combination with ICIs may enhance the efficacy and reduce related irAEs, thereby assisting in cancer chemoprevention.

Gut microbiome regulation

With the expansion of research in the field of oncology, manipulating the gut microbiota to induce positive therapeutic effects in vivo has opened the door to novel technologies for cancer prevention and treatment. The gut microbiome regulates various host processes, including metabolism, inflammation, and immunity (Fig. 4b).^{497,498,499,500} Increasingly evidences indicated that the microbiome can also affect the development of cancer with the outcome may be positive or negative.^501,502,503 Some microbes have been proven to have pro-tumorigenic effects, such as H. pylori, a primary carcinogen, contribute to gastric cancer and mucosa-associated lymphoid tissue lymphomas.^504,505 Fusobacterium nucleatum and Escherichia coli are closely related to the onset and development of CRC.^506,507 In addition, tumor growth may lead to local disruption of the barrier, resulting in microbial invasion, the pro-inflammatory and immunosuppressive effects of immune monitoring can be also disrupted.^497,508

The gut microbiota plays crucial role in regulating cancer treatment outcomes, particularly in chemotherapy and immunotherapy.^509,510 Chemotherapy drugs can reduce the diversity of the microbiome in the body by affecting host immune system, leading to a decrease in its therapeutic effects.⁴⁹⁹ While, the microbiome can also enhance the efficacy of chemotherapy drugs through metabolism, enzyme degradation, and immune regulation.⁵¹¹ Moreover, chemotherapeutic drug resistance is also related to the microbiota.⁵¹² Cancer immunotherapy has emerged a promising method for treating cancer patients, and there exists significant differences in the microbial diversity and composition of fecal samples between immunotherapy responders and non-responders, indicating that changes in clinical response may be related to the gut microbiome.^513,514 Meanwhile, unremitting efforts were employed to predict immunotherapy responses through microbiome, and it helps in guiding the selection of appropriate cancer treatment methods.⁵¹⁵ Hence, regulating microbiota and improving cancer-related microecological imbalances serve new strategies for preventing and treating cancer. It is worth noting that some natural compounds possess low bioavailability, but the influence of gut microbiota cannot be ignored when exploring their mechanisms of action.

Quercetin can enrich the abundance of Akkermansia muciniphilia to enhance the anticancer effect of cyclophosphamide, as well as increase the mobilization frequency of T cells and NK cells and reduce the frequency of Tregs during cyclophosphamide treatment, which indicates that quercetin can mobilize vital antitumor immune cells to enhance the antitumor effect of cyclophosphamide, reflecting the chemoprevention potential of quercetin in TNBC.⁵¹⁶ Apigenin can effectively improve DSS-induced UC through regulating the abundance of Akkermansia, Turicibacter, Klebsiella, Romboutsia, etc., to reshape the disordered gut microbiota thus ameliorated colon injury, the expression of zonula occludens-1, claudin-1, and occludin were also upregulated to restore the integrity of the intestinal barrier.⁵¹⁷ In further azoxymethane/DSS-induced CRC model, apigenin exhibits anti-CRC effect via influencing the abundance of Firmicutes and Actinobacteria.⁵¹⁸ Furthermore, by reducing the gut microbiota and fecal transplantation, the role of apigenin in regulating gut microbiota was further confirmed. During the progression of colitis to CRC, curcumin reduces the burden of colon tumors, and increases the relative abundance of Lactobacillales and decreased Coriobacterales order.⁵¹⁹ Resveratrol can influence UC mice of intestinal microfora, improve intestinal microbiota by increasing diversity and uniformity of cecal microbiota, thus exerting a protective effect on the intestines.⁵²⁰ The prevention and treatment mechanisms of berberine for CRC are currently relatively plentiful. Evidence show berberine recovers DSS-induced UC in a microbial-dependent manner, with it maintaining the structure and function of the intestinal mucosal barrier, regulating intestinal mucosal immune homeostasis, activating Wnt/β-catenin pathway,⁵²¹ FXR/TGR5 signaling, increasing unconjugated bile acids and secondary bile acids in the gastrointestinal tract,⁵²² activating aromatic hydrocarbon receptors⁵²³ to exert its protective effect in the colon. In further CRC mice model studies, berberine can still reduce the β-diversity of gut microbiota, enrich the probiotic community and reduce pathogenic microorganisms to relieve CRC progression, and the related fecal metabolism results also support the effect of berberine on gut microbiota.^524,525 Berberine can also performance anti-breast cancer and anti-HCC role by regulating the abundance and diversity of microorganisms, as well as the production of microbiota-derived butyric acid.^526,527 The prevention effect of ursolic acid on UC is related to abate the richness of gut microbiota and avoid the inflammatory response caused by intestinal epithelial barrier disruption.⁵²⁸

The preventive effect of gut microbiota on cancer is usually influenced by the interactions between microbiota, tumor, immune system, and metabolism. The flourishing development of next-generation sequencing and bioinformatics has led to a significant increase in research on the association between gut microbiota and cancer. However, most current researches only explored the impact of microbial communities on phylum or genus level, and have not used isolation and cultivation methods for in-depth research. Another controversial issue exists whether the fecal transplantation applied for manufacture gnotobiotic mice faithfully replicate the microbiota of donors? The future development of the microbiome is expected to identify individual microbial species that lead to cancer phenotypes and reveal their potential mechanisms, and to modify dominant microbiota for personalized treatment. Emerging spatial multi-omics technologies and organoid research can help uncover the aforementioned issues from genes to phenotypes. Microbiome research is in the ascendant, there are still mountains of black boxes to be opened for preventing cancer by revealing the impact of the microbiome on cancer hosts. The multiple advantages of natural compounds are constantly being exploited in regulating gut microbiota, and more and more candidate natural compounds that can serve as microbiota regulators to prevent the development of cancer.

Drug delivery system assists in natural compounds-based cancer chemoprevention

Although these natural compounds exhibited various excellent antitumor effects in preclinical studies, most of them usually characterized as low bioavailability, poor targeting, and fast metabolic rate, which greatly hinder their further clinical application. With the introduction of novel drug delivery systems, drug delivery systems-chemoprevention therapies have established their potential with enhancing target ability, reducing side effects and drug resistance to provide better efficacy and safety, and has become a promising strategy for effective cancer treatment (Fig. 5).

Fig. 5

Drug delivery systems assist in natural compounds-based cancer chemoprevention. Diversity delivery platforms (mainly including but not limited to liposomes, polymeric nanocarriers, extracellular vesicles) enhance bioavailability and reducing toxicity of natural compounds, through active-targeting ligand decorated and passive targeting via the enhanced permeability and retention effect. This figure was created with Biorender.com, and adapted from Riya Khetan et al.⁵³²

Nanotechnology polishing up for cancer chemoprevention

Loading natural compounds into a certain size of nanocarrier can alter the biological distribution of drugs, increase in vivo stability, prolong the blood circulation time, and achieve better accumulation of drugs at the tumor site through passive targeting, due to tumor angiogenesis with leaky blood vasculature, and by active targeting via binding to proteins overexpressed on the surface of tumor cells.^529,530 By manipulating particle size and surface, controlling and sustaining release of drugs, siting specific targeting, using of multiple delivery routes of administration to improve the bioavailability, targetability, and controllability,^531,532,533 which greatly improved the shortcomings of natural compounds in clinical applications, and amplified the pharmacological advantages. Currently, the widely studied nanomaterial-delivery systems of natural compounds mainly include liposomes and polymeric nanoparticles.

Liposomes

Liposomes are spherical vesicles composed of lipid bilayers, which creating two microenvironments, thus, lipophilic or hydrophilic molecules all can be encapsulated in the lipid bilayers or aqueous cores of liposomes.⁵³⁴ Due to the similarity in composition with the cell membrane, liposomes have the ability to diffuse across the cell membrane and have certain advantages as carriers for targeted drugs.⁵³⁵ Increasingly evidence suggests liposome drug delivery system increase anticancer activity,^536,537,538 inhibit cancer cell proliferation,⁵³⁹ induce tumor cell apoptosis,^540,541 etc. In particular, liposomes are considered an ideal carrier for mediating effective cancer immunotherapy by activating immune responses.^542,543 Liposomal nanomedicine has been recognized as the most successful nanomedicine delivery system. The first liposome formulation to successfully obtain FDA approval for cancer treatment was Doxil^® (doxorubicin hydrochloride liposome), employed to treat ovarian cancer and AIDS-related Kaposi’s sarcoma.⁵⁴⁴ Doxil^® improved the pharmacokinetic properties of free doxorubicin and reduced its life-threatening toxicity.⁵⁴⁴ Marqibo^® (vincristine sulfate liposomes injection) was approved for the treatment of leukemia, which can reduce the severe neurotoxicity and gastrointestinal toxicity of vincristine.⁵⁴⁵ Jing et al. developed a folic acid-modified quercetin liposome that can further enhance the in vivo antitumor effect of quercetin.⁵⁴⁶ Zhang et al. prepared a dry powder of curcumin liposomes, which is more suitable for pulmonary delivery, and A549 cells showed significantly higher uptake of curcumin liposomes than free curcumin, and existed lower cytotoxicity to normal cells.⁵⁴⁷ Jhaveri et al. assembled transferrin moieties-modified resveratrol liposome to passive and active targeting treat glioblastoma. The modified resveratrol liposome exhibited more cytotoxic and induced higher levels of apoptosis accompanied by activation of caspases 3/7, and more effective to inhibit tumor growth and improve survival in mice than that of free resveratrol or resveratrol liposome.⁵⁴⁸

Polymeric nanocarriers

Polymeric nanoparticle is a class of polymer nanocarriers with conjugated or encapsulated drugs, exhibiting various structures. Polymer nanoparticles provide multifunctionality through the utilize of polymers with different chemical compositions, hydrophilic-lipophilic balance, charge and physical structures.⁵⁴⁹ In addition, the ability to control the degradation or decomposition of polymeric nanoparticles endows them with the ability to control drug delivery time over a wider range, which made polymeric nanoparticles attractive as therapeutic delivery carriers.⁵⁴⁹ The multiple surface functional groups of polymeric nanoparticles can be further modified by antibodies, peptides, and other targeting ligands, thereby promoting drug accumulation at the target site and mediating effective drug internalization in target cells, which can not only improve therapeutic efficacy, but also reduce potential adverse reactions of chemotherapeutics, and displayed great potential in preventing cancers.^550,551,552 Abraxane^®, also known as nab-paclitaxel, was consists of a nanosuspension of human serum albumin loaded with paclitaxel, used for the treatment of breast cancer, etc. The co-delivery polymeric nanoparticles of paclitaxel and curcumin exhibited an increasingly cellular uptake in breast cancer cells, and inducing cytotoxicity in breast cancer cells via the apoptotic pathway by blocking the G2/M phase of the cell cycle.⁵⁵³ In addition, polymeric nanoparticles can significantly inhibit tumor growth in vivo, prolong the survival time, reduce glucose metabolism in tumor tissues.⁵⁵³ Co-delivery of CPT and curcumin by cationic polymeric nanoparticles can also enhance synergistic effects to CRC.⁵⁵⁴

Polymeric micelles are composed of amphiphilic polymers, and can self-assemble in aqueous environments. These amphiphilic polymers are constructed by different polymer blocks, which can be optimized by adjusting the hydrophobic/lipophilic balance, size, drug loading capacity, micellar ability.^555,556,557 Compared to other drug delivery systems, the size of polymeric micelles allows for more effective extravasation through the leaking vascular system, and the hydrophilic polymeric coating also prevents them from being recognized by the reticuloendothelial system during circulation and can be taken to the liver and spleen for clearance.⁵⁵⁸ Genexol^®PM is the first successfully marketed polymeric micelles loading paclitaxel, it was approved for the treatment of metastatic breast cancer, etc.⁵⁵⁹ Apart from Genexol^®PM, the number of successfully marketed polymeric micelles is limited, and although there are many ongoing clinical trials, the results have not yet been announced. Gong et al. prepared curcumin polymeric micelles and studied its anti-lung cancer effects, compared with free curcumin, the cellular uptake, in vitro cytotoxicity and anti-angiogenesis effect were increased in polymeric micelles, and continuous release behavior and slow extravasation behavior were also observed.⁵⁶⁰ Moreover, curcumin polymeric micelles were more effective in suppressing tumor growth and prolonging survival in LL/2 tumor models.⁵⁶⁰ The curcumin polymeric micelles were also be proven to be effective in preventing the development of CRC in vitro and in vivo mechanismly due to the inhibition of tumor proliferation and angiogenesis and increased apoptosis of tumor cells.⁵⁶¹ The co-delivery of resveratrol and docetaxel via polymeric micelles can improve the treatment of drug-resistant tumors, and the effect is superior to the individual use of each drug.⁵⁶² Besides, co-delivery of resveratrol and quercetin polymeric micelles can alleviate cardiotoxicity induced by doxorubicin.⁵⁶³

Dendrimers is a unique class of macromolecules with highly branched three-dimensional structures, and their shape and size can be precisely controlled, they exhibit an exponential number of dendritic branches radiating out from the central nucleus.^24,564 It consists of three main components: a core or nucleus, an inner layer composed of repeating molecular units called dendrons, and the terminal functional groups on the surface.^565,566 Dendrimers can carry bioactive compounds through covalent bonds or via ion interactions or adsorption within the internal space of nanostructures, thus demonstrating great potential as carriers for anticancer drugs.^565,567 Gallien et al. encapsulated curcumin in surface-modified polyamidoamine dendrimers, and the in vitro studies have shown that the curcumin dendrimers effectively reduced viability of glioblastoma cell lines, while, unencapsulated curcumin was ineffective.⁵⁶⁸ Shen et al. developed a low-polyamidoamine dendrimers of ursolic acid conjugate modified with lactobionic acid, which could abate the migration and adhesion of SMMC-7721 cells by restraining metastasis-related protein MMP-9 expression, as well as effectively inhibit the tumor growth in H22 mice model.⁵⁶⁹ However, this study only used MTT assay as a detection method and only conducted cellular-level research, lacking in in vivo research results.

Extracellular vesicles (EVs)

Although nanotechnology-based drug delivery systems have provided advantages for therapeutic delivery and achieved encouraging results, there are still many limitations in terms of biosafety issues and low biocompatibility. With the rapid development of the field of natural drug carrier systems, one of the most prominent examples is EVs. EVs are natural nano-size lipid bilayer vesicles secreted from all cell types,^570,571 which can divide as three main subtypes based on their biogenesis: exosomes, microvesicles and apoptotic bodies.^572,573,574 Exosomes have a size ranging from 40 to 160 nm (averaging 100 nm), and formed by invagination of the cytoplasmic membrane and inward of payload sprouts to form multivesicular bodies, and then fuse with the cell membrane and eventually release into the extracellular environment.⁵⁷⁵ Microvesicles (50–1000 nm) sprout outward directly from the plasma membrane, and apoptotic bodies (1000–5000 nm) are released by cells undergoing apoptosis through membrane foaming and rupture.⁵⁷⁶ EVs are biological origin membrane structures, and exhibit low toxicity, high biocompatibility, and low immunogenicity.⁵⁷⁷ In addition, due to its targeting ability to transfer bioactive molecules to target cells, it is beneficial to increase the concentration of drugs in target cells and reduce toxic side effects. Moreover, its lipid bilayer structure provide superior modification flexibility and biofilm permeability.⁵⁷⁸ Thus, EVs offer an alternative strategy and have become a promising vehicle for drug delivery, and engineered EVs, modified by genes or chemicals can enhance their targeting ability and loading efficiency, thereby improving the anticancer activity. EVs have been applied to deliver natural compounds to enhance their cancer prevention performance.

Paclitaxel can be loaded into EVs of various cells to enhance their cytotoxicity and reduce drug resistance. In a study, paclitaxel was loaded into human normal pancreatic duct cell line hTERT-HPNE-derived EVs and enhanced its effect in pancreatic ductal adenocarcinoma (PDAC) cells through a clathrin-dependent endocytosis pathway.⁵⁷⁹ Melzer et al. demonstrated bone marrow mesenchymal stroma/stem-like cell-derived EVs loaded with paclitaxel showed stronger antitumor effects than that of human umbilical vein endothelial cells loaded with paclitaxel.⁵⁸⁰ Wang et al. indicated the exosomes from M1-polarized macrophages act as carrier to deliver paclitaxel can enhance paclitaxel antitumor activity by activating macrophages-mediated inflammation.⁵⁸¹ Chen et al. anchored ligands onto milk-derived EVs with the allowance of attachment of a substantial amount of transferrin, which significantly enhanced cellular uptake and induced pronounced cytotoxic effects when loaded with paclitaxel.⁵⁸² Zhu et al. prepared c(RGDyK)-modified and paclitaxel-loaded embryonic stem cells–exosomes of cRGD-Exo-paclitaxel, the engineered exosomes exhibited more efficiently to glioblastoma versus free drug alone and drug-loaded embryonic stem cells–exosomes.⁵⁸³ Zheng et al. engineered exosomes derived from T cells expressing the chimeric antigen receptor (CAR-Exos), and paclitaxel was encapsulated into CAR-Exos (PTX@CAR-Exos) and administered via inhalation to an orthotopic lung cancer mouse model.⁵⁸⁴ The inhaled PTX@CAR-Exos accumulated within the tumor area, and prolonged survival with increased efficacy. In addition, it reprogrammed the tumor microenvironment and reversed the immunosuppression. Yang et al. developed a patient-derived exosome CPT delivery platform as personalized therapeutic modality for cervical cancer treatment. This therapeutic approach modulated the cell cycle and enhancing the sensitivity of tumor cells to radiotherapy, thus achieved significant antitumor effects.⁵⁸⁵

When curcumin was encapsulated into milk-derived EVs, it was shown enhanced antiproliferative activity against multiple cancer cell lines, especially enhanced higher antitumor activity in cervical tumor xenograft model.⁵⁸⁶ Jia et al. loaded superparamagnetic iron oxide nanoparticles (SPIONs) and curcumin into exosomes and then conjugated the exosome membrane with neuropilin-1-targeted SC peptide to obtain glioma-targeting exosomes.⁵⁸⁷ These exosomes provided good results for the synergistic anti-glioma effect with SPION-mediated magnetic flow hyperthermia and curcumin-mediated therapy. The engineered exosomes provide a potential pathway for improving the simultaneous diagnosis and treatment of gliomas. In addition, two clinical trials aim to encapsulate curcumin into plant-derived exosomes for the prevention/treatment of CRC (NCT01294072 and NCT04879810), but no data have been released. Deng et al. engineered human placental mesenchymal stem cell-derived exosomes loaded with berberine for UC therapy, and these exosomes concurrently demonstrated anti-inflammatory and antioxidant activities, contributing to the mitigation of UC with the possible mechanism of modulating MAPK signaling pathway, and the therapeutic effect was superior than that of berberine alone.⁵⁸⁸ Salek et al. loaded berberine to immature dendritic cells-derived EVs, and evaluated the antitumor activity and antiangiogenic properties in breast cancer cells and human umbilical vein endothelial cells.⁵⁸⁹ Berberine-loaded EVs upregulated the efficacy of free berberine in inhibiting cell proliferation, cell migration, capillary-like formation, and NO release to prevent tumorigenesis and angiogenesis. In summary, as a natural medicine delivery system, although there is currently limited clinical research on natural compounds-loaded EVs, multiple studies have shown exciting and encouraging results, and the potentiality of EVs is without doubt.

The rapid development of drug delivery systems can effectively improve the low bioavailability of natural compounds, while, there are still many obstacles from laboratory research to reliable and effective clinical applications. The current evaluation of cancer drug delivery systems mainly relies on xenograft tumor models, which cannot accurately predict the therapeutic effect of formulations in advanced cancer or metastatic tumors.⁵⁹⁰ The use of nude mouse models cannot evaluate the immune response against tumor cells or drug delivery systems.⁵⁹¹ Therefore, using a spontaneous cancer model may be a better choice for evaluating drug delivery systems. In addition, studies have shown that nanotechnology-based delivery systems are more effective in small animal tumor models than in human tumors,^543,592 and organoids can be attempted for evaluation. Cancers are closely related to the imbalance of the gut microbiota, and natural compounds can exert its effects by affecting the gut microbiota, therefore, targeted drug delivery systems should not be limited to protein ligands. Researches have shown that multiple parameters such as particle size, surface charge, particle shape, porosity, and biodegradability can affect the toxicity of nanotechnology-based drug delivery systems,⁵⁹³ thus, the biocompatibility, toxicology, long-term stability, and in vivo metabolic degradation pathways should be comprehensively evaluated.⁵³⁵ At present, the preparation of most nanotechnology-based formulations are completed in laboratories with sophisticated processes and difficult to apply in practice, which means the processes and technologies for large-scale production and quality control still need breakthroughs. In addition, the large-scale purification, reproducible production, optimal storage conditions (including storage buffer and storage container), and cycling stability for biological for natural drug carrier systems remain challenges. With the continuous advancement of precision medicine, drug delivery systems also need to be combined with personalized medicine. The addition of technologies such as gene editing and artificial intelligence (AI) may provide revolutionary changes for cancer prevention.

Natural compounds improve the efficacy of chemotherapies

Increasing chemotherapeutic sensitivity

Drug resistance remains a major obstacle in cancer prevention and treatment, and seeking strategies to overcome tumor resistance has always been a focus in the field of oncology. It is encouraging that natural compounds with different chemical structures and pharmacological effects are effective substances in assisting combat drug resistance.^594,595,596 Currently, natural compounds as the fourth generation of multi-drug-resistant inhibitors have attracted the attention of researchers.⁵⁹⁴ Natural compounds reverse cancer multi-drug resistance mainly via regulating drug efflux, DNA repair, tumor microenvironment, and related signaling pathways.^597,598,599

Quercetin can inhibit the activation of androgen receptors, PI3K/Akt signaling, and reduce the expression of hnRNPA1 and AR-V7, as well as P‑glycoprotein (P-gp) to enhance the sensitivity for prostate cancer.^600,601 In addition, quercetin could enhance the therapeutic effect of conventional chemotherapy drugs on multi-drug-resistant BEL/5-fluorouracil (5-FU) cells overexpressing ABCB1, ABCC1 and ABCC2.⁶⁰² Apigenin is an ideal adjuvant to improve doxorubicin resistance in breast cancer through regulating JAK2/STAT3/multi-drug resistance 1 (MDR1) axis to inhibit the expression of MDR1, multi-drug resistance-associated proteins, and P-gp.^603,604 Curcumin can be used as a sensitizer to enhances the efficacy of gefitinib by inhibiting Sp1 and blocking its interaction with HADC1, downregulating EGFR activity, and this synergistic effect is confirmed as autophagy-dependent.⁶⁰⁵ In the study of drug resistance in CRC, curcumin modulates TET1-NKD-Wnt signaling to suppress the EMT progress, and also regulates CXC-chemokine/NF-κB signaling to sensitize drug-resistant cells to chemotherapies.⁶⁰⁶ By inhibiting the Akt cascade via activating PTEN, resveratrol attenuates invasive biological features, synergizes with doxorubicin to inhibit gastric tumor growth, and reverses its resistance by inhibiting EMT.⁶⁰⁷ It was reported andrographolide can target to Bax and trigger mitochondrial-mediated apoptosis, thus synergize the cytotoxic effect and reverse resistance of 5-FU on CRC.⁶⁰⁸ Ursolic acid restrains proliferation and reverses drug resistance of ovarian CSCs by downregulating ABCG2 via constricting the expression of hypoxia-inducible factor-1α.⁶⁰⁹ As a key mediator of drug resistance in breast cancer, HuR can be translocated to the nucleus by ursolic acid and decreased MDR1 expression.⁶¹⁰

The sensitization mechanism of the same natural compound to chemotherapy drugs varies for different types of cancer cells, in addition to the heterogeneity of tumors, all of these hindered the positive outcomes of natural compounds. In light of the multi-target characteristic of natural compounds and its complex mechanism of action, future researches should shift the focus from in vitro studies to in vivo studies. Besides, in order to introduce more effective treatment methods for reversing tumor drug resistance into clinical practice, discovery of more effective and low toxicity ingredients is indeed crucial, which can be combined with AI-driven drug discovery, computer-aided drug design, high-throughput screening, etc. technologies to accelerate the development of active substances.

Reducing chemotherapeutic side effects

Due to the limited specificity of chemotherapy drugs, cancer patients often experience adverse reactions or toxicity during treatment, including myelosuppression, gastrointestinal toxicity, cardiotoxicity, hepatotoxicity, neurotoxicity, ototoxicity, etc.^611,612,613 Due to these side effects, patients may reduce the medication dosage or even stop treatment, leading to cancer metastasis or recurrence, seriously affecting their quality of life and survival time. Therefore, it is necessary to explore effective adjuvant strategies to prevent and reduce the side effects caused by chemotherapy. Numerous studies have shown that natural compounds can effectively attenuate the side effects caused by chemotherapy, which undoubtedly provides a potential means for preventing cancer progression.^{614,615,616,617}

Cancer therapy has evolved from traditional therapies to the use of targeted therapy and immunotherapy. However, the cardiotoxicity associated with these therapies has always existed. Currently, dexrazoxane is the only FDA-approved option for preventing cardiotoxicity caused by anthracycline chemotherapy drugs.⁶¹⁸ However, this is insufficient as dexrazoxane is not suitable for addressing all cardiotoxicity and may add additional issues to patient care and treatment.⁶¹⁹ Curcumin, resveratrol, quercetin can significantly reduce the levels of cardiotoxicity markers (creatine kinase and lactate dehydrogenase, etc.) by increasing antioxidant capacity, protecting the myocardium from damage caused by chemotherapy drugs.^{134,620,621,622} He et al. proposed ferroptosis is involved in the occurrence of doxorubicin-induced cardiotoxicity, EGCG treatment can effectively inhibit oxidative stress and abnormal lipid metabolism, and maintain mitochondrial function by upregulating AMPKα2 and activating adaptive autophagy, thereby reducing doxorubicin-induced cardiotoxicity.⁶²³ Moreover, this protective effect is similar to that of the iron chelator dexrazoxane, the only drug approved by the FDA for the clinical treatment of doxorubicin-induced cardiotoxicity.

A chemotherapy drug is usually not limited to producing only one type of side effect, gastrointestinal toxicity, myelosuppression, and nephrotoxicity are also adverse reactions usually caused by chemotherapy drugs. 5-FU is the first-line chemotherapeutic for treating CRC, and the mucosal immune damage increases the possibility of secondary pulmonary infection in the host.⁶²⁴ Curcumin can also protect intestinal mucosa via IL-6/STAT3 signaling pathway.⁶²⁵ Quercetin and its nano-emulsion formulation were also reported to inhibit 5-FU-induced intestinal mucositis by suppressing the formation of ROS, downregulating the expression of NF-κB and hypoxia-induced factor-1α.⁶²⁶

Cisplatin is widely used for the treatment of various solid tumors, and mainly cleared by kidney through glomerular filtration and tubular excretion, thus easy to accumulate in the kidney and lead to nephrotoxicity, which cause negative impacts on clinical outcomes.^627,628 Quercetin attenuates cyclophosphamide-induced nephrotoxicity via modulating the crosstalk of MAPK/ERK and NF-κB signaling.⁶²⁹ EGCG also demonstrates its chemopreventive effect on cisplatin-based ototoxicity as it can restore hearing and outer hair cells in the basal region of the cochlea, mechanismly associated with inhibition of ERK1/2, STAT1, and STAT3.⁶³⁰ Curcumin can also prevent cisplatin-induced nephrotoxicity by improving mitochondrial bioenergetics, ultrastructure, redox balance, and SIRT3 levels.⁶³¹ In addition, curcumin can effectively reduce the concentration of cisplatin in the kidney by improving uric acid, plasma phosphorus, urinary creatinine, and creatinine clearance rate.⁶³² In this study, the researchers also compared the preventive effect of melatonin on cisplatin-induced nephrotoxicity. Although there was no statistical analysis of the therapeutic effects of curcumin and melatonin, it can be found from the comparison of the above data results that melatonin has a weaker renal prevention effect than curcumin. Moreover, the combined use of curcumin and melatonin has a better preventive effect than the use of curcumin or melatonin alone. Besides, curcumin can not only prevent nephrotoxicity caused by cisplatin, but also alleviate ototoxicity by targeting p-STAT3 and Nrf-2 signaling.^633,634

A number of studies disclosed the benefit of natural compounds for the treatment of tumors, which significantly reduce the side effects caused by chemotherapy drugs. Moreover, these natural compounds have almost no serious side effects and are relatively inexpensive and easy to obtain. However, most current studies did not set up a positive group for effective preventive drugs, making it impossible to evaluate the efficacy of natural compounds compared to existing drugs. Although natural compounds have made great progress in preventing and adverse reactions induced by chemotherapy agents, more clinical studies are needed to further confirm their preventive effects on side effects. In addition, reliable biomarkers are wanted for early identification and monitoring of toxic side effects of chemotherapy agents.

Development of resistance against natural compounds

Despite the important role of natural compounds in cancer prevention and therapy, phenomena of resistance have already occurred. The current research on natural compounds resistance is mainly focused on marketed drugs, including CPT, vinblastine, and paclitaxel. One of the direct mechanisms for developing drug resistance is to increase the expression of ATP-binding cassette transporter family proteins, such as P-gp, breast cancer resistance protein (BCRP) to enhance drug efflux, the overexpression of efflux transporters result in the efflux of paclitaxel, vinblastine, vinblastine, and CPT from cells, thereby reducing therapeutic efficacy.^{635,636,637,638}

Metabolizing enzymes of Cytochrome P450 (CYP) enzymes contributes to drug metabolism, and invasive cancer cells typically exhibit high levels of CYP. CYP3A4 and CYP2C8 are closely related to paclitaxel resistance.^639,640 High levels of CYP enhance the metabolism of paclitaxel, reduce its concentration in cancer cells, thus develop drug resistance, and limit therapeutic efficacy. EMT is associated with cancer metastasis and along with invasion, it also participates in drug resistance.^641,642 It was reported in A549 cells resistant to paclitaxel, Cathepsin L upregulated the expression of EMT-associated transcription factors Snail, Slug, zinc-finger-enhancer-binding protein (ZEB) 1, and ZEB2, downregulated E-cadherin to promote EMT.⁶⁴³ Hypoxia as an environmental stress, can activate tumor cell-related genes, and accelerate angiogenesis and invasion.⁶⁴⁴ Hypoxia induced upregulation of intracellular and extracellular gp96 and increased paclitaxel resistance, and accelerated EMT.⁶⁴⁵ In addition, a series of signaling pathways involved in tumor biological processes are also associated with drug resistance. Paclitaxel, vinca alkaloids have been shown to activate NF-κB pathway through downregulating IκBα and promoting the nuclear translocation of NF-κB thus contributing to chemoresistance.^646,647 Activated PI3K/Akt signaling often contributes to the increased resistance in cancer cells,⁶⁴⁸ and miR-20a and miR-200c were found to upregulate the levels of Akt1 and p-Akt1 to activate PI3K/Akt pathway, thus contributing to paclitaxel resistance.⁶⁴⁹ Downregulation of Notch signaling could also inhibit the EMT process and increase the sensitivity of cervical cancer cells to paclitaxel.⁶⁵⁰ Park et al. demonstrated the prolonged stress associated with paclitaxel treatment stimulates p38 MAPK/p53 network and induces the transcription of EGFR, which activates the EGFR pathway and paclitaxel resistance in NSCLC.⁶⁵¹ Brosseau et al. reported the overexpression of YAP/TEAD signaling was involved in the resistance to paclitaxel chemotherapy in lung cancer.⁶⁵²

Besides, autophagic flux can be driven by paclitaxel to promote resistance in ovarian cancer,⁶⁵³ and non-coding RNA can regulate resistance, such as lncRNA H19,^654,655 MA-linc1,⁶⁵⁶ lincROR,⁶⁵⁷ lncRNA CCAT1,⁶⁵⁸ NONHSAT141924,⁶⁵⁹ miR34a,⁶⁶⁰ miR135b,⁶⁶¹ miR-155,⁶⁶² and miR199a.⁶⁶³ Here, we mainly lists the resistant mechanism of CPT, paclitaxel, and vinca alkaloids, due to the fact that most natural compounds have not been used in clinical for cancer therapy, and current research typically focuses on alleviating resistance with other chemotherapy drugs. With the continuous use of these natural compounds, their resistance might be overcome through the following strategies. The use of inhibitors targeting drug resistance, such as cyclosporine A, can inhibit the function of P-gp, reduce natural compounds being pumped out of cells by P-gp, increase intracellular drug concentration, and enhance the effect on drug-resistant cells. Verapamil and elacridar, the ABCB1 inhibitors, can also reverse resistance through reducing the efflux of drugs.⁶⁶⁴ The combination of different natural compounds or chemotherapeutics may enhance the inhibition of cancer cells by targeting multiple targets, thereby overcoming the resistance of a single natural compound. Natural compounds processed by drug delivery systems is prone to be effectively delivered into cancer cells thus overcoming resistance.⁶⁶⁵ Moreover, drug delivery systems can achieve targeted delivery, increase local drug concentration, and enhance the effect on drug-resistant cells. It is also possible to reduce drug resistance by improving the physicochemical properties of natural compounds processed by drug delivery systems, made it prone to be effectively delivered into cancer cells thus overcoming resistance, such as preparing them as prodrugs or modifying their structures to enhance their absorption and distribution.

Natural compounds serving as drugs against cancers in clinical

The current clinical research status of cancer chemoprevention with natural compounds

The potential applications of various natural compounds in preventing cancer have been extensively studied. Increasingly in vitro and in vivo data indicate the excellent efficacies on cancer prevention, prompting scientists to conduct clinical trials. However, currently, few clinical studies using natural compounds alone as a preventive agent, mostly in combination with natural compounds or in combination with chemotherapy drugs. Moreover, these studies mainly focused on the pharmacokinetics, efficiency, and safety of natural compounds. At present, there are a large number of natural compounds-related studies registered on ClinicalTrials.gov, but most of the completed projects have not published their results, and most of the studies are in phase I/II clinical trials, with only a few studies in phase III. Therefore, there is still a long way to go in verifying the chemopreventive effect of natural compounds, as not all results indicate that natural compounds is beneficial for cancer chemoprevention. The existing clinical studies on natural compounds have many common issues: small sample size, not the multicenter clinical research, single-arm or phase II trial without a comparator, lack of follow-up studies or short follow-up time, or the dosage may be ineffective. In addition, the patient is unwilling to undergo invasive or radiation-damaging examinations such as colonoscopy, gastroscopy, computed tomography, etc. at the end of treatment. These factors constrain the true efficacy of natural compounds, and with people’s understanding and attention to health, these issues will be overcome in future. In the next section, we listed the clinical research with research results in the past decade (Table 2).

Table 2 Clinical trials of natural compounds in cancer prevention

Clinical trials with a single natural compound on cancer

Curcumin

In a randomized double-blind placebo-controlled trial, patients with liver cirrhosis were randomly divided into two groups to receive 1000 mg/day curcumin or placebo for 3 months, beneficial effects of curcumin supplementation were observed in downregulating disease activity scores and severity of cirrhosis in patients.⁶⁶⁶ Although it provided evidence that curcumin has a beneficial effect and has the potential to prevent the progression of liver cirrhosis, and reducing the development of liver cancer, this study only used liver enzyme activity to evaluate liver status, without conducting computed tomography, magnetic resonance imaging, or liver biopsy. Besides, it is reported oral administration of curcumin for 6 months (1440 mg/day) did not significantly affect the overall cessation time of intermittent androgen deprivation in prostate cancer patients, but the intake of curcumin significantly inhibited the elevation of prostate-specific antigen (PSA) during this period, indicated curcumin has potential beneficial effects on prostate cancer patients.⁶⁶⁷ Although this study failed to demonstrate the difference of the off-treatment duration in the curcumin group, it is the first clinical study to use curcumin alone on prostate cancer. If the dosage and duration of curcumin administration were increased in this study, better results might be achieved. Cruz-Correa et al. evaluated the efficacy and safety of curcumin in treatment of intestinal adenomas in 44 patients with FAP, patients were randomly assigned to curcumin group (3000 mg/day) and placebo group, and continued to receive treatment for 12 months.⁶⁶⁸ At the end of treatment, there was no significant difference in the mean number of polyps between curcumin group (22.6; 95% CI, 12.1–33.1) and placebo group (18.6; 95% CI, 9.3–27.8); so did the polyp size between the curcumin group (2.3 mm; 95% CI, 1.8–2.8) and the placebo group (2.1 mm; 95% CI, 1.5–2.7). The insufficient dosage of curcumin might be the main factor leading to insufficient regression of adenomas. While it is gratifying that after 12 months of treatment with curcumin, the tolerance was good and there was no significant difference in the incidence of adverse events compared to the placebo group, indicating the safety of long-term use of curcumin. UC and Crohn’s disease is a risk factor for CRC.^669,670 Sadeghi et al. conducted a randomized double-blind clinical trial to assess the preventive effect of curcumin, 70 patients with mild-to-moderate UC were randomly assigned to curcumin (1500 mg/day) or placebo intake for 8 weeks, and it was found taking curcumin supplements combined with medication treatment can significantly improve clinical outcomes and quality of life in patients with mild-to-moderate UC.⁶⁷¹

Berberine

It is widely accepted that adenoma-carcinoma sequence represent the roadmap of most CRC arise.⁶⁷² A multicenter, double-blinded, randomized controlled study was identified orally administrated with 0.3 g berberine twice a day was safe and can be effectively reduce the risk of recurrence of colorectal adenomas, and can be used as an option for postoperative chemoprevention after polypectomy.⁶⁷³ During the 2 years of follow-up, 155 (36%) participants in the berberine group and 216 (47%) in the placebo group were found to have recurrent adenoma (relative RR, 0.77; P = 0.001), revealed berberine reduced the risk of recurrence.

Application of natural compounds in clinical combination/adjuvant therapy for cancer prevention

Genistein

Given the anti-CRC activity of genistein, a phase I/II pilot study was executed to estimate the safety and tolerability of genistein in combination with FOLFOX or FOLFOX-bevacizumab.⁶⁷⁴ In the 13 evaluable patients, RR in cycle 6 was 46%, with best overall response of 61.5%, median PFS of 11.5 months, and 15.4% patients were found in progression of disease. Seven of these patients continued to undergo surgical resection of the primary tumor (if not previously removed) and metastatic disease. In a previous study, the PFS was reported to be 9.4 months in chemotherapy combined with bevacizumab, and 8.0 months in the chemotherapy group alone,⁶⁷⁵ it suggested genistein in combination with chemotherapy may have potential to improve the therapeutic efficacy and chemoprevention of CRC.

EGCG

Acute radiation-induced esophagitis (ARIE) is a major dose-dependent side effect in unresectable stage III NSCLC and esophageal cancer patients receiving radiotherapy and chemotherapy, which may lead to unplanned treatment interruptions, and severe reduction in tumor control and survival rates. A phase I study of EGCG therapy protection of the esophagus from damage induced by chemoradiotherapy displayed 22 NSCLC patients had a rapid regression of esophagitis to level 0/1 in 24 patients within 2 weeks, the pain score also decreased from an initial average of 4.58 to 1.13 per week.⁶⁷⁶ In further phase II study, EGCG administration was found to be superior to traditional therapeutic applications in preventing ARIE, and the average adjusted esophagitis index of prophylactic EGCG (3.56 ± 2.90) was significantly lower than that of therapeutic EGCG (5.19 ± 2.73), indicated the prophylactic application of EGCG had an advantage over therapeutic use in adjuvant therapy for lung cancer.⁶⁷⁷ In another single-arm study related to esophageal cancer, the ARIE exhibited significant declines after EGCG treatment.⁶⁷⁸ Radiation-induced oral mucositis has a dismal outcome with limited treatment options, and EGCG mouthwash can effectively alleviate the integrity of oral mucosa in patients receiving head and neck radiotherapy.⁶⁷⁹ The 20 enrolled patients did not develop grade 3 or higher mucositis, and the score of mucositis-related pain was significantly reduced after administration EGCG mouthwash, and the recommended dose is determined to be 1760 μmol/L. Radiation dermatitis (RID) is the most common adverse event of radiotherapy for breast cancer, which may lead to the interruption of treatment, and then affect the progress of cancer. A phase II clinical trial enrolled 180 breast cancer patients of whom 165 [EGCG (660 μmol/L), n = 111; placebo, n = 54] were evaluable for efficacy.⁶⁸⁰ The occurrence of grade 2 or higher RID was significantly lower in the EGCG group (50.5%; 95% CI, 41.2–59.8%) than that in the placebo group (72.2%; 95% CI, 60.3–84.1%). This study once again confirms that prophylactic use of EGCG is an effective adjuvant therapy for cancer.

Curcumin

In a study of 50 mesalamine-treated patients with active mild-to-moderate UC, addition of curcumin to the treatment group resulted in better induction of clinical and endoscopic relief compared to the placebo group with no adverse reactions, indicated curcumin can be a safe and promising UC treatment drug and cancer chemopreventive agent.⁶⁸¹ In addition, multiple oncology clinical studies have explored curcumin can be safely administered with standard chemotherapy agents such as gemcitabine,^682,683 5-FU,⁶⁸⁴ docetaxel,⁶⁸⁵ etc. Gbolahan et al. investigated the combination of curcumin with irinotecan which share the same metabolic enzymes of UDP-glucuronosyltransferases.⁶⁸⁶ It was found the combination of up to 4 g of Meriva^® (lectinized curcumin) and intravenous injection irinotecan was well-tolerated, and there was no increase in irinotecan-related adverse reaction. Another parallel-group comparative clinical study was designed to estimate the efficacy and safety of curcumin (CUC-01, 300 mg) combined with paclitaxel as compared to paclitaxel plus placebo in patients with metastatic and advanced breast cancer.⁶⁸⁷ After 12 weeks of treatment, a significant difference in the auxiliary analysis of the change in Response Evaluation Criteria in Solid Tumors (RECIST) score and ORR (51% vs. 33%) compared to that in the placebo group, indicated the greater benefit than the placebo. The median PFS in the curcumin group (27.0 weeks) was 2.4 weeks longer than that in the placebo group (24.6 weeks). In addition, a slight beneficial effect on reducing fatigue was also be discovered. Pastorelli et al. conducted a phase II study to investigate Meriva^® as complementary therapy of advanced pancreatic cancer of gemcitabine.⁶⁸⁸ It was found the disease control rate was 61.3% with a median PFS of 8.4 months, and a median OS of 10.2 months (95%CI, 8.8–11.7). The OS observed in this study was higher than that observed with gemcitabine alone (OS, 5.9 months),⁶⁸⁹ suggesting the complementary adminstration of phytosome complex of curcumin could increase the therapy of gemcitabine in advanced pancreatic cancer.

Resveratrol

Paller et al. conducted a phase I study of MPX (containing ellagic acid, quercetin, and resveratrol) on patients with biochemically recurrent prostate cancer (BRPC) to estimate its safety, tolerability, and dose determination.⁶⁹⁰ The cohort (n = 14) had a median follow-up of 19.2 months, and suggested 4000 mg of MPX was safe for future research. In the phase II trial of MPX in BRPC, 125 patients form multi-cancer were treated for at least 6 months.⁶⁹¹ Although MPX did not significantly prolong prostate-specific antigen doubling time (PSADT) in BRPC patients, it is worth noting that PSADT pre-to-post increase was significant in the 27 genotyped patients with SOD2 Alanine/Alanine genotype (rs4880 T > C polymorphism) of MPX group (6.4 months), with the control group of 1.8 months. In another 12 month randomized pilot study of combination phytotherapy (containing turmeric, resveratrol, green tea, and broccoli) in BRPC patients,⁶⁹² it was found the phytotherapetic intervention was well-tolerated, and the treatment group experienced a non-significant increase in the log-slope of PSADT (pre treatment and post-treatment was 10.2 and 5.5 months, respectively), while the placebo group experienced no change in the log-slope of PSADT (pre treatment and post-treatment was 10.8 and 10.9 months, respectively). It suggested this phytotherapeutic intervention is feasible in BRPC patients.

CPT

The toxicity of CPT greatly limits its application, current clinical research mostly focuses on evaluating the effectiveness of NLG207 (formerly CRLX101), novel cyclodextrin-containing polymer conjugate of CPT. A phase I/II trial of CRLX101 with capecitabine and radiotherapy as neoadjuvant treatment for locally advanced rectal cancer was conducted by Sanoff el al. The maximum tolerated dose and recommended phase 2 dose of weekly administration of CRLX101 was determined to be 15 mg/m², and 6/31 (19%) of patients achieved overall pathologic CR, with an additional 18/31 (58%) patients experienced a moderate response. Therefore, the addition of CRLX101 to capecitabine-based CRT might be a feasible combination strategy with good tolerance for locally advanced rectal cancer patients.⁶⁹³ The team of Haas conducted a phase II trial of CRLX101 in combination with bevacizumab versus standard of care (approved targeted agents in the third-/fourth-line setting) in 111 patients with metastatic renal cell carcinoma.⁶⁹⁴ However, there was no significant difference in PFS between the two groups with CRLX101 plus bevacizumab group (3.7 months; 95% CI, 2.0–4.3) and standard of care group (3.9 months; 95% CI, 2.2–5.4). Schmidt et al. conducted another clinical trial of NLG207 with enzalutamide in treating advanced metastatic castration-resistant prostate cancer post-enzalutamide. In this study, a total of 4 patients were included, but only 2 patients were able to evaluate for toxicity, and 2 patients had a decrease in PSA levels compared to baseline. This study suggests that the combination of 12 mg/m² NLG207 and 160 mg enzalutamide is an intolerable regimen in metastatic castration-resistant prostate cancer patients who have undergone multiple treatments, with the probable bladder toxicity caused by long-tern exposure to CPT in the bladder, even at a low concentration.⁶⁹⁵

Vinblastine

Methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC) was considered as one of the most effective chemotherapy regimens for eurothelial cancer. Choueirii et al. explored the efficacy and safety of neoadjuvant dose-dense MVAC (dd-MVAC) with pegfilgrastim support in muscle-invasive urothelial cancer (MIUC).⁶⁹⁶ It was concluded this chemotherapy regimen was effective as 49% patients achieved pathologic response on cystectomy, and 1-year disease-free survival was 89% vs. 67% for patients who achieved pathologic response compared with those who did not. Moreover, dd-MVAC chemotherapy is also effective in nonmetastatic muscle-invasive bladder cancer (MIBC) patients. A randomized phase III controlled study comparing the efficacy of dd-MVAC or gemcitabine and cisplatin (GC) in MIBC patients was designed.^697,698 The dd-MVAC arm was associated with a significantly longer time to tumor progression (TTP, 3-year rate: 69% vs. 58%,), and dd-MVAC also improved 3-years PFS over GC arm (66% vs. 56%), suggested dd-MVAC regimen is superior and it should be considered as a treatment option for MIBC patients in the future.

Homoharringtonine

The clinical trials of homoharringtonine is mainly focused on the treatment of leukemia. The HAG regimen with a low-dose homoharringtonine, cytarabine, and granulocyte colony-stimulating factor was evaluated by Liu et al. used as an induction therapy for untreated AML patients. The CR was 65.5% (19/29), with ORR of 86.1%.⁶⁹⁹ There were 9 cases of recurrence in CR patients, and the median disease-free survival of 19 CR patients were 12 months, with the median OS of 20 months.⁶⁹⁹ Homoharringtonine-based regimen is also beneficial to childhood AML. A multicenter, randomized clinical trial was performed with 1258 patients, the overall CR of the homoharringtonine-based (H arm) was remarkable higher than that in the etoposide-based (E arm) (79.9% vs. 73.9%), and according to the intention-to-treat analysis, the 3-year OS was 69.2%, 62.8% in H arm and E arm, respectively; the 3-year event-free survival was 61.1%, 53.4% in H arm and E arm, respectively.⁷⁰⁰ It is worth noting that in the subgroup analysis, patients with AML₁-ETO positivity in the H + ATRA-based arm showed a better 3-year event-free survival compared with those in the E + cytarabine-based arm (73.6% vs. 52.8%).⁷⁰⁰ The homoharringtonine-based regimen may be a better choice for treating newly diagnosed children with AML, especially those with AML₁-ETO. Refractory/relapsed acute myeloid leukemia (R/R AML) is a very challenging complication in AML treatment, with the prognosis dismal. The addition of homoharringtonine might bring new hope to R/R AML patients. A BHA regimen consisted with bortezomib, homoharringtonine, and cytarabine was evaluated for patients with R/R AML. After one course of BHA treatment, the ORR of 21 patients was 52.4%, with composite complete remission rate [CRc, CR plus CR with incomplete hematologic recover (CRi)] of 38.1%, and 9 cases harbored FLT3 mutations exhibited an astonishing higher ORR than that without FLT3 mutations (66.7% vs. 16.7%), indicated BHA regimen as a well-tolerated and effective chemotherapy for patients with R/R AML, especially for FLT3 mutations.⁷⁰¹ Another drug combination of venetoclax, azacytidine, and homoharringtonine (VAH) was executed by Jin et al. to investigate the activity and tolerability. The CRc of 96 enrolled patients was 70.8%, with ORR of 78.1%, in the patients with CRc, measurable residual disease (MRD)-negative was achieved in 58.8%. The median follow-up time was 14.7 months, with the median OS was 22.1 months, as well as event-free survival was 14.3 months.⁷⁰² In another study, sorafenib was added to VAH combination to be used as a salvage therapy for R/R AML patients, and the CRc was 76.5% (39/51), with ORR of 82.4% (42/51), the median follow-up of 17.7 months and the OS was 18.1 months.⁷⁰³ The therapy of VAH and combination therapy of sorafenib and VAH both achieved a high CRc and well-tolerance, suggest this regimen might be a suitable treatment option for R/R AML. Li et al. reported a phase II clinical trial for azacitidine with homoharringtonine, idarubicin, and cytarabine (HIA) in treating newly diagnosed AML patients.⁷⁰⁴ The results indicated that the combination therapy has high efficacy for young or elderly patients fit for intensive chemotherapy as first-line induction therapy with 95% (19/20) of patients gained ORR, 89.5% (17/19) had undetectable minimal residual disease. The median OS and RFS were not achieved during the follow-up period (median follow-up was 733 days), and the estimated 2-year OS and RFS were 87.5% and 87.1%, respectively, demonstrated this regimen was an effective and well-tolerance for previously untreated young or fit older patients with AML.⁷⁰⁴

Currently, compared to synthetic drugs/marketed drugs, there are relatively few studies on natural compounds-based cancer treatment/prevention with available data, and the administration and follow-up time is shorter. Due to the long time required to determine the benefits of cancer chemoprevention, longer administration and follow-up periods are necessary. Long-term intervention also increases the likelihood of research subjects being lost to follow-up, so it is recommended to have a large sample size as much as possible. The current effectiveness evaluation of cancer chemoprevention lacks clear biomarkers, although it can be assessed by detecting tumor markers or gene expression levels, these indicators may lack specificity and sensitivity for cancer prevention, making it difficult to assess the safety of long-term effects and determining the optimal dosage and duration of use of preventive agents. In addition, necessary imaging examinations will make the results more objective, and the advantages of the main results are significantly higher than the form of survey questionnaire. Moreover, as the long-term use of preventive agents may lead to the development of drug resistance and reduce the preventive effect, it is necessary to conduct in-depth research and evaluation on long-term safety.

Research strategies for the discovery of low abundance and high anticancer activity natural compounds

The investigation on anticancer drugs has never stopped searching for better and less side effect agents. In addition to chemically synthesized anticancer compounds, natural compounds have been proven to be a highly promising alternative source for the discovery of anticancer drugs, and facilitate drug discovery by providing novel and unique compounds.^705,706,707 Compared to typical synthetic small molecules, natural compounds are rich in biologically active compounds covering a wider chemical space.⁷⁰⁵ They serve as both a direct source of drugs and leading compounds.⁷⁰⁸ Currently, multitude of high abundance components have been repeatedly discovered, however, natural compounds with low abundance but high activity abound in nature, and they were considered to be important treasure trove for drug development. Due to the low abundance of these ingredients, they are difficult to be analyzed and detected or far below the detection limit of instruments, and are even insufficient for biological evaluation in multiple consecutive phenotypes or target-based assays.⁷⁰⁹ Nevertheless, they may exert a significant role in cancer prevention and treatment, and act as leading compounds for novel drug discovery. With the continuous innovation and cross fusion of modern analytical technology, bioinformatics, AI, multi-omics and other technologies, the discovery of low-abundance and high-activity anticancer ingredients has been greatly promoted (Fig. 6).^705,710

Fig. 6

Research strategies for the discovery of low-abundance and high anticancer activity natural compound. Here we simply show through constructing the database via literature search and experience inheritance, omics technologies integration method based on molecular network construction at different levels of each omics, and AI (machine learning and deep learning)-driven assist the discovery of low-abundance and high anticancer activity natural compound. The ultimate goal of data integration is to discover low-abundance and high-activity anticancer components from complex natural product system and elucidate their biological significance. This figure was created with BioRender.com

Natural compounds databases inspired leading compounds

The digital revolution is driving significant changes in a novel mean for storing, distributing, and using information. Massive amounts of data are organized, standardized, and stored as database, providing valuable resources for researchers. The establishment of database can better administrate, analyze, collect, and investigate information on chemical composition, structure, properties, biological activity, etc., and assist in various stages of drug discovery and development.⁷¹¹ There currently exists specialized natural compound databases, such as LOTUS,⁷¹² NPASS,⁷¹³ Super Natural II,⁷¹⁴ etc., which involved structural information, physical and chemical properties, and cross-references with other resources. Besides, herbal resource databases that include natural compounds and targets of action are helpful, such as TCMSP,⁷¹⁵ TCMID,⁷¹⁶ SymMap,⁷¹⁷ BATMAN-TCM 2.0,⁷¹⁸ etc. Universal compound databases of DrugBank, ChemSpider,⁷¹⁹ PubChem,⁷²⁰ also presented abundant resources for the discovery of natural compounds. However, the current pattern of natural compounds-related databases is highly fragmented, repetitive, and difficult to integrate. It was reported over 120 different natural product databases were available including commercial and non-commercial resource.⁷²¹ Therefore, in order to fully utilize the information in the database, it is necessary to coordinate and integrate the data in database, which is crucial for both users and developers. The cost of using commercial databases is also a barrier, although some companies can offer discounts to some researchers or institutions, it is expected to significantly reduce this barrier by creating high-quality FAIR-compliant (with the principles of findable, accessible, interoperable and reusable) resources.^722,723,724 Increasingly scientists are using more precise analysis and extensive calculation methods to mine data from massive databases, accelerating the discovery of low-abundance and high anticancer activity natural compounds.

Omics technologies assisted acceleration

Historically, bioactivity-guided separation was the main method to isolate and identify individual ingredients from complex natural compound mixtures.⁷²⁵ Due to the time-consuming, low-abundance components are prone to disappear during the separation process, currently, scientists have shifted away from bioactivity-guided separation to omics technologies to guide natural compounds discovery.^725,726 Various high-throughput screening techniques, represented by genomics, transcriptomics, proteomics, metabolomics, chinmedomics, spatial multi-omics, etc., can efficiently and quickly reveal detailed biological information of diseases and drug efficacy, playing significant role in promoting human disease research.⁷²⁷ With the reduction of detection costs and the rapid development of high-throughput analytical instruments, the advantages of omics technologies in guiding natural compound discovery are gradually emerging.⁷²⁶ Each type of omics data provides differentially expressed disease-related molecules that can serve as biomarkers during disease progression and as indicators of changes that occur in the body under the influence of internal and external environments.^728,729,730 Omics science focuses on the interactions between systematicity, integrity, and interactions within internal environment of living organisms, exploring the relationships between genes, environment, and phenotype, and thus assisting in the discovery of bioactive natural compounds.⁷³¹ Omics technologies can assist in the discovery of natural compounds from the following aspects: 1) explore known structures with novel functions, 2) known functions with novel structures, 3) unknown structures with unknown functions.⁷³² Li et al. employed network pharmacology to predict tricin is a bioactive component of Weijing formula, and discovered key differential metabolites sphingosine-1-phosphate and its upstream metabolic enzymes sphingosinase 1 and sphingosinase 2 by metabolomics, it was verified through further experiments that tricin mainly inhibits NSCLC tumor growth by suppressing PRKCA/SPHK/S1P signaling.⁷³³ Through screening the natural compound database, Hao et al. identified a diterpenoid compound named nodosin, might have potential anti-bladder cancer activity. Through the joint analysis of transcriptomics and proteomics, nodosin was found can prevent cell proliferation by inducing apoptosis, blocking the S phase cell cycle, destroying the cytoskeleton to inhibit migration, and significantly inhibit the tumor size of xenograft tumor mice.⁷³⁴ In addition, chinmedomics was considered as an effective strategy to discover bioactive natural compounds by integrating serum pharmacochemistry of traditional Chinese medicine, metabolomics, big data analysis, and bioinformatics.^{735,736,737,738,739,740,741}

AI-enabled exploration

In the era of big data, the volume and diversity of data are dramatically increasing. The global data volume has increased from ~1.5 zettabytes in 2009 to an estimated 175 zettabytes by 2025.^722,742 With the emergence of new technologies and open data initiatives, computational methodologies have brought a paradigm shift in the field of drug discovery, providing indispensable tools for the entire process of drug development. These technologies greatly reduce costs and enhance the effectiveness of the process of identifying and producing new medications.⁷⁴³ The rapid development of AI with the wider accessibility of supercomputing resources, and graphics processing unit-accelerated computing made large-scale computer simulation become feasible.⁷⁴⁴ AI provides powerful technologies for extensive data analysis and identifying compounds with high therapeutic potential to accelerate the drug discovery process. Machine learning and deep learning are the mainly subsets of AI, and they are increasingly being applied to drug discovery pipelines, mainly because they can facilitate data-driven modeling processes to learn from large datasets.^745,746 Through the use of algorithms, statistical models, and deep neural networks to study and execute virtual drug screening, predict chemical structures, drug-related parameters, activities, and targets of action, the accuracy of predicting anticancer potential of natural compounds has been greatly enhanced.^709,747,748

Li et al. exploited a web server named CDRUG with a relative frequency-weighted fingerprint to describe the molecules, and a hybrid score was introduced to test molecular similarity.⁷⁴⁹ Subsequently, they screened the 21334 compounds from TCM Database@Taiwan, and a total of 5278 compounds were predicted exhibiting anticancer activity, with 75% of them were highly similar to approved anticancer medications, and 346 of them exerted high activity in 60 cell line tests.⁷⁵⁰ Rayan et al. employed ISE algorithm to construct a prediction model to detect natural compounds with anticancer activity with 2892 natural compounds as the test subjects, and 12 natural compounds (neoechinulin, colchicine, piperolactam, etc.) were disclosed as potential anticancer candidates.⁷⁵¹ Furthermore, there are few researches reported on these screened natural compounds, and further research is needed to test their activity.⁷⁵¹ Nguyen et al. used machine learning approach incorporated with evolutionary computer to construct a computing framework, termed iANP-EC, to identify natural compounds with anticancer activity. By screening of collected 997 compounds, it was proved that iANP-EC was a stable, robust, and effective framework with an AUC value of 0.9193.⁷⁵² Ouyang et al. developed an AI-powered omics strategy for innovative drug discovery to take pyroptosis therapy for TNBC as a paradigm.⁷⁵³ By using large-scale TNBC queues and drug databases for data mining, a neural network for regulating biological factors was established, which can quickly screen and optimize compound with pyroptosis drugs, and the combination of glycyrrhetinic acid and mitoxantrone has been ultimately identified as candidate drug.⁷⁵³ Furthermore, a nanococrystal of glycyrrhetinic acid and mitoxantrone was formed to improve its solubility and exhibit stronger antitumor activity than the combination of abraxane and anti-PD-1 drugs. Despite the powerful capabilities of AI, its use for potential natural compounds drug discovery is still in the infant stage, and many issues still need to be addressed. For example, the challenges from data, such as the scale, growth, diversity, and uncertainty of data. With the increasing availability of high-quality and ideal labeled training data, more appropriate models will be created for the discovery of anticancer bioactive natural compounds, which will also lead to the discovery of more natural compounds, thereby improving the diversity of training data, further promote the development of the field. In addition, a well-maintained and accessible database is crucial for developing, testing, and establishing AI frameworks. It is also necessary to strengthen the close cooperation between computer and biology/pharmacy experts, so that the developed AI models can truly meet the needs of reality, and can be effectively validated and continuously adjusted to demonstrate strong performance in the real world and be widely used by researchers.

Based on natural compounds-related databases, omics techniques, AI, and bioinformatics technologies, they effectively associate natural compounds with targets and diseases. Especially the datasets generated by databases and multi-omics technologies are massive and complex, making them suitable for joint application with AI. The combination use can greatly improve data analysis, biological interpretation, and predictive statistical modeling.⁷⁵⁴ However, each stage of joint application brings unique challenges that require specialized methods and close collaboration among various researchers to overcome existing bottlenecks. Firstly, coordinating data scale is challenging as multidimensional data units vary depending on omics types and instruments.^755,756 In addition, the significant differences in metabolite concentrations and the presence of isomers complicates the process of finding meaningful correlation with other omics data, and the influence of dietary and environmental factors increases this complexity.⁷⁵⁷ Therefore, choosing appropriate data integration method is crucial, meanwhile, the interpretation of multi-omics data results requires the employment of appropriate interpretation models to analyze important variables under disease background. Although AI can quickly adapt to large amounts of new data, it still needs to be monitored and updated to ensure the accuracy of analysis. Moreover, AI requires substantial computational resources and professional knowledge, which are not easily accessible in the existing medical environment. Although the rapid development of sequencing and spectroscopic technologies, as well as the availability of computational tools have stimulated research on natural compounds worldwide, there is currently limited research on the discovery of active ingredients from multi-omics aspect.

Moving toward precision cancer chemoprevention

Although there are diversified strategies for cancer prevention currently, the preventive effect is not ideal. Due to individual differences in behavior, lifestyle, and environmental exposure, as well as the presence of tumor heterogeneity, gene mutations of different cancer cells in the same patient may vary. Moreover, in the process of cancer prevention and treatment, new mutations or fusion genes might occur, which may disrupt the targets of drugs and render chemopreventive agents ineffective.⁷⁵⁸ Patients also have different reactions to medications, resulting in poor preventive effects.⁷⁵⁹ Precision medicine is defined as tailoring clinical strategies based on genomic, genetic, behavioral, and environmental backgrounds of individual patients.^{758,760,761,762} With the development of molecular biology, high-throughput multi-omics, bioinformatics and AI, precision therapy is exerting an increasingly prominence role in oncology.^763,764,765 Personalized cancer chemoprevention has the potential to improve prevention effectiveness, prolong patient survival time, and enhance their quality of life. Cancer chemoprevention is also undergoing revolutionary changes, shifting towards precision prevention, gradually improving effectiveness, and patient-centeredness. The United States Preventive Services Task Force (USPSTF) has incorporated precision prevention into evidence-based recommendations at the population level.^766,767,768 The individuals age between 50-59 who have a life expectancy and risk of cardiovascular disease of at least 10 years, and are willing to take low-dose aspirin daily and are not at risk of bleeding, the USPSTF recommended to use low-dose aspirin to prevent cardiovascular disease and CRC.⁷⁶⁹ In addition, tamoxifen and raloxifene are proved to have significant efficacy in preventing ER-positive breast cancer.^770,771 However, studies have shown that SERMs have potential hazards in prevention, which will increase the risk of uterine cancer among patients taking tamoxifen. In order to eliminate this hazard, a breast cancer prevention trial is investigating the efficacy of tamoxifen delivered through breast skin to achieve local prevention (NCT02993159).

In addition to individual differences in metabolic enzymes, individual characteristics that affect drug transporter action are important factors affecting natural compounds and other agent response. For example, BCRP is a key transport protein, which is encoded by ABCG2 gene and participates in the special function of patients in drug transport.⁷⁷² There is a common genetic polymorphism in the ABCG2 gene worldwide, the common ABCG2 mutations can affect the efficacy of cancer drug therapy and the occurrence of adverse drug reactions, leading to changes in drug reactions and disease risk. Apigenin has good binding affinity with BCRP,⁷⁷³ compared with normal rats, the expression of BCRP/ABCG2 protein in the liver and intestine was significantly reduced after apigenin pretreatment, which can improve the systemic bioavailability and absorption rate of dasatinib.⁷⁷⁴ When compared to using doxorubicin alone, exposure to 2 μmol/L apigenin can increase the accumulation of doxorubicin by 29%.⁷⁷⁵ This indicates that the differences in ABCG2 genes may have impact on the chemoprevention of natural compounds. Cytokine gene polymorphism plays key role in immune regulation,^776,777 for example, IL-10 is an important anti-inflammatory cytokine, and plays crucial role in maintaining intestinal homeostasis and balancing gut microbiota with the digestive system.⁷⁷⁸ The single nucleotide polymorphisms in IL genes may dramatically affect protein expression levels or alter their function, leading to gastritis or ulcer, and eventually promote cancer occurrence.⁷⁷⁹ McCann et al. indicated rs1800896 single nucleotide polymorphism in the promoter region of the IL-10 gene linked with the reduction of IL-10 and severity of IBD severity.⁷⁸⁰ Curcumin can attenuate the reduction of IL-10 mutations in a dose-dependent manner and alleviate the severity of IBD, thus, IBD patients with tested gene variants may benefit from increased intake of curcumin. Cancer genome sequencing revealed widespread genetic heterogeneity and profound genomic instability in advanced tumors,⁷⁸¹ therefore, gene sequencing is necessary for precision medicine, as it can provide more information about gene mutations and is crucial for selecting more effective drugs and dosages.

The current progress in cancer chemoprevention is still limited, but generous work is still being carried out, researchers are actively exploring biomarkers for various types of cancer and translating them into clinical applications. The rapid development of spatial omics approaches further enable comprehensively understand the interactions between cells from a multidimensional perspective. In addition, the continuous development of patient-derived organoids and organoids co-culture model systems, the structure, unique cancer characteristics, and genetic features of tumors are faithfully reproduced, which can better guide personalized prevention. With the continuous in-depth research on preventive agents that can be used for cancer chemoprevention, such as natural compounds, and the publication of multiple related clinical trial results in the future, more cancer chemoprevention agents will assist achieve personalized prevention for individuals, providing unprecedented opportunities for people, and improving the current dilemma in oncology.

Conclusion and future perspectives

Cancer chemoprevention represents a promising strategy for mitigating the incidence and mortality rates associated with tumor development. Discovering novel, safe, and effective chemopreventive agents is crucial for gaining more acceptance of cancer chemoprevention. The development and application of preventive agents have gradually shifted from drugs with a single target and stronger side effects to natural compounds with less toxicity regulated by multiple pathways and targets. The multi-target and multi-pathway properties of natural compounds enable them to effectively counteract the biological complexity of cancer, indicating natural compounds provide extensive and promising resources for cancer chemoprevention (Fig. 7). However, cancer prevention agents based on natural compounds still have a long way to go before clinical translation. Most chemopreventive agents are currently in the preclinical research stage, and the preventive effects of natural compounds on cancer can be easily discovered in cell lines or animal models, however, it is difficult to apply the results as translational evidence for direct entry into clinical trials. Considering the duration of clinical trials for discovering cancer chemopreventive drugs, selecting appropriate preclinical research models that fully simulate the process of human tumor occurrence and development may result in greater success of candidate compounds in clinical trials. Burgeoning technologies such as patient-derived organoids and organoid co-culture model systems can be combined. Moreover, most studies evaluate only the short-term effects of natural compounds, and the mid-term/long-term effects should also be examined. In addition, the appropriate dosage is also crucial for the success of clinical trials, because depending solely on dietary cancer chemoprevention in high-risk populations appears unreasonable.⁷⁸² Therefore, conducting phase 0 and phase I clinical trials on potential natural compounds is necessary. Appropriate clinical endpoints (including the potential to utilize surrogate, early, or quality-of-life endpoints) appear to be equally important for the development of novel preventive agents, as evidenced by past experiences, such as the approval of drugs for AK treatment and the accelerated approval of celecoxib, it should be tailored to specific disease environments to make endpoints clinically meaningful.¹⁵

Fig. 7

The roles of natural compounds in chemoprevention. Natural compounds have shown great promise in cancer chemoprevention by targeting on signaling pathways, immune checkpoints and gut microbiota, reducing adverse reactions, as well as improving chemotherapy drug sensitivity. In addition, drug delivery systems also enhance the bioavailability of natural compounds, making them more robust for cancer prevention. Natural compounds are not only treasure reservoir for drug discovery, but will also achieve precise cancer chemoprevention in the future to better serve human health. This figure was created with BioRender.com

Natural compounds generally possess characteristics of low bioavailability, and the development of synthetic analogs with better pharmacokinetic properties using natural compounds as scaffold is an alternative method. For organs with unique characteristics, local administration can be attempted for cancer prevention during research, and the efficacy obtained may be more significant than that obtained by systemic administration, for example, inhalation administration can be attempted for lung cancer. In addition, the development of novel drug delivery systems and building new drug loading platforms to accurately control drug-targeted delivery to targets, along with limiting systemic toxicity, is also a development direction.

The definition of high-risk populations without obvious characteristics, the uncertainty of the time from precancerous lesions to invasive cancer, metastasis, and recurrence have hindered chemoprevention research. Screening reliable biomarkers is of great significance for promoting the field of cancer chemoprevention, which assisting in screening high-risk populations, evaluating the effectiveness of cancer prevention agents, and assessing prognosis. The risk and prediction models established with the help of AI and big data also provide assistance for preclinical and clinical research on cancer chemoprevention. The development of cancer chemoprevention agents involves several disciplines, and it was advocated for data sharing between institutions. Nonetheless, determining how to better protect patient privacy is also an important task.

To summarize, the development of novel cancer chemopreventive agents is an alternative method to alleviate the burden caused by cancer, and natural compounds have excellent broad prospect as cancer chemopreventive agents, it provides a window of opportunity for the discovery of cancer chemopreventive agents, regardless of numerous obstacles. Although a few approved cancer chemopreventive agents are available today, the discovery and validation results of cancer chemopreventive agents are not exciting, while, without taking a single step, one can travel thousands of miles. With the continuous emergence of various technologies and the collaborative efforts of personnel from various disciplines, we believe that cancer chemoprevention can inspire people greatly.