Introduction

Cancer is caused by the uncontrolled proliferation of any cell in the body. There are over a hundred distinct forms of cancer, and their behavior and treatment response can vary substantially1. In the US, 1,958,310 new cases and 609,820 deaths due to different types of cancer have been reported in 2023 which become a point of serious concern in the world of cancer2. Cervical cancer is ranked fourteenth among all the cancer types and fourth most occurring gynecologic malignancy in the globe3. More than half a million women have been diagnosed with cervical cancer which causes more than 300,000 deaths worldwide4. In most cases, the activation of oncogenes and/or the inactivation of tumor suppressor genes leads to an uncontrolled progression of the cell cycle and an inactivation of apoptosis mechanisms5. Primary prevention and screening is the best method to decrease the burden of cervical cancer and rate of mortality6. Surgical excision (complete/radical hysterectomy), radiotherapy, and chemotherapy are presently available therapeutic options for cervical cancer management7. All of these therapeutic strategies have overall low survival rate and numerous side effects in the cervical cancer patients. Thus, the focus of researchers has been shifted to develop alternative strategies which become safe and effective against cervical cancer. Natural products have been studied for their therapeutic potential since ancient times and more extensively during the last two decades due to their vast diversity and underlying physiologically active components8.

Apoptosis, also known as programmed cell death, is a natural process that occurs in multicellular organisms to maintain tissue homeostasis by eliminating damaged, unnecessary or potentially harmful cells. It plays a vital role in normal development, tissue remodeling and removal of damaged cells. However, cancer cells often develop mechanisms to evade apoptosis, which leads to their uncontrolled growth and resistance to treatment (Diepstraten et al., 2022).

Cancer cells may have genetic alterations or dysregulation of signaling pathways that interfere with the apoptotic process. Some common mechanisms by which cancer cells evade apoptosis include overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-xL or Mcl-1, which prevent the activation of caspases and block the apoptotic pathway and loss of pro-apoptotic proteins such as p53, Bax or Bak, which are critical for initiating apoptosis (Choudhary et al., 2015).

Medicinal plants include a wide range of biologically active chemicals from a variety of chemical families terpenes, saponinic glycosides, steroids, alkaloids, and flavonoids9. Some of these chemicals, especially monoterpenes, have been identified in essential oils derived from plants and have been linked to pharmacological activities10. Phytochemicals, such as monoterpenes, are bioactive substances generated naturally in plants that provide numerous health advantages in various medical conditions, like different types of cancer, viral and bacterial infections and neurodegenerative diseases, and have little hazardous effects11. For many years, medical professionals have used phytochemicals and medicinal plants to cure and control cancer. Several plants and phytochemicals have previously been claimed to have anticancer properties. Nonetheless, additional research is needed to determine their efficacy12 Monoterpenes constitute about 90% of essential oils and have several biological and pharmacological effects, such as antibacterial, antihypertensive, antioxidant, antiarrhythmic and antispasmodic qualities13.

Pulegone is a naturally ocervical cancerurring monoterpene obtained from the essential oils of a variety of plants, including Nepeta cataria (catnip), Mentha piperita, and pennyroyal14. It is found predominantly in peppermint and pennyroyal (Mentha spp). Other plants that contain pulegone in high quantity are Ziziphora taurica M. Bieb. (81.86%), Mentha spicata L. (72.1%), Cyclotrichium niveum (Boiss.) Manden and Scheng. (76.84%) and Micromeria cilicica Hausskn. ex P.H. Davis (66.55%)15. Pulegone is a clear, colorless oily liquid with a pleasant odor and reminiscent of pennyroyal, peppermint and camphor. It is used in flavoring, perfumery and aromatherapy16 Pulegone has been shown to have a variety of pharmacological effects, including cardiovascular, antibacterial, antioxidant, antinociceptive, analgesic, anti-inflammatory, and antispasmodic properties15. The present study was designed to evaluate the anticancer potential of Pulegone against cervical cancer cells via induction of apoptosis, natural compounds have apoptotic potential, and Pulegone is a natural compound it may have apoptotic and anti-proliferative activities against cervical cancer cell.

Materials and methods

Culturing of HeLa and VERO cell lines CaSKi and SiHa

HeLa and VERO CaSKi and SiHa cells were obtained from cell culture lab The University of Lahore, Lahore-Pakistan. Cells were cultured according to Maqbool T protocol17, briefly cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (high glucose) (Caisson’s Lab, USA. Cat # NC1121414), along with 10% fetal bovine serum (Sigma Aldrich, USA. Cat # F1051), 100 µg/ml streptomycin and 100 units/ml penicillin (Caisson’s Lab, USA. PSL01 100 ml) and incubated in CO2 incubator. When cells reached at 70–80% confluency, they were sub-cultured and plated.

Treatment of hela, VERO, CaSKi and SiHa cells

For treatment, 1 molar aqueous stock solution of pulegone (Sigma Aldrich CAS # 89-82-7) was prepared by dissolving 0.15233 g of pulegone (Molecular weight 152.33 g/mol) in 1 ml sterile water, further dilutions were prepared in DMEM. On the basis of treatment, cell cultures were divided into five groups in the case of MTT of HeLa and VERO, 1st untreated group (negative control, cells with DMEM) 2nd, 3rd, 4th treated group (0.1, 1 and 10 µM doses respectively) and 5th positive control (with cisplatin10 µM dose). While count & viability, apoptosis and western blotting treatment cell cultures were divided into three groups for HeLa cells i.e. untreated group (negative control, cells with DMEM), treatment group with maximum cytotoxicity 10 µM and positive control group (cisplatin with 10 µM dose).

MTT assay

MTT was performed according to Maqbool T protocol17, briefly HeLa cells, VERO cells, CaSKi cells and SiHa cells were seeded into 96-well plates at a density of 5,000 cells per well overnight then were treated with cisplatin and different concentrations of pulegone. After 48 h of treatment, 0.5% MTT (Amresco. Cat # 0793-1G) solution was added and incubated for 3 h. Then, 100 µL of DMSO was added to dissolve the formazan crystals. Finally, absorbance was obtained at 570 nm.

$$\% {\text{ Cell viability}}\,=\,{\text{Experimental }}\left( {{\text{OD57}}0} \right)/{\text{Control }}\left( {{\text{OD57}}0} \right) \times {\text{1}}00$$

Apoptosis via Annexin V

HeLa cells were cultured in 6 well plates and treated. Apoptosis was performed according to kit manual briefly after 48 h of treatment, cells were centrifuged at 2000 rpm for 5 min supernatant discarded, pellet dissolved in annexin V & Dead Cell kit for 30 min (Cat Number: MCH100105) and apoptosis evaluated with the “Muse”™ (Merck-Millipore) automated cell counter/ analyzer.

Hoechst 33,342 staining

Cells were exposed to pulegone (10 µM) for 48 h and cells without treatment served as control. After treatment cells were fixed with 4% paraformaldehyde for 15 min and 0.1% Triton X-100 used to permibilize for 5 min, then stained with 10 µl Hoechst33342 for 15 min18. Finally, cells were viewed under Floid Cell Imaging Station.

Immunostaining

Immunocytochemistry was performed according to technique followed by Maqbool T17. After treatment with drug medium was removed from all wells for staining. The cells were washed three times with TBS-T, 4% Para formaldehyde (PFA. Cat# 30525-89-4) was added in each well for 30 min at room temperature. Cells were again washed with TBS-T and treated with 5% BSA (blocker, Cat# A9418) to block nonspecific binding for 25 min. BSA was removed and again washed with TBS-T incubated with p53 primary antibody for 1.5 h at 37 °C. After that, again washed three times with PBS-T and incubated at 37 °C with FITC conjugated secondary antibody for 1.5 h. Cells were stained with DAPI (nuclear stain) for 15 min followed by three times washing. After that cells were again washed and observed under Floid Cell Imaging Station.

Western blotting

Western blot was performed according to Akhtar S19. Briefly, Hela cells were seeded in a 6-well plate at a concentration of 106 cells / well, incubated overnight and treated with pulegone for 48 h. The cells were then lysed and total protein was determined using the Bio-Rad Bradford Assay Kit (Bio-Rad, UK). The cell lysate was separated by protein electrophoresis and blotted onto a nitrocellulose membrane. The membrane was blocked in 5% bovine serum albumin (BSA) overnight and the solution was diluted in Tris-buffered saline supplemented with 0.1% Tween 20 at 4 °C. Then, the membrane was incubated with the primary antibodies of BAX, BCL2 and B actin 1:500 (1 mg/ml main stock) and then secondary antibody 1:1000 (1 mg/ml main stock) dilutions overnight at low temperature and for 1 h at room temperature, respectively.

Statistics

All the experiments were performed in triplicate. GraphPad Prism, Version 5.01 (GraphPad Software, San Diego, California, USA) URL: https://www.graphpad.com/scientific-software/prism/ was used to analyze the data. Image analyses were performed using ImageJ software, Version 1.53t, developed by the National Institutes of Health (NIH), USA.

The software is freely available at: https://imagej.nih.gov/ij/. All the data was expressed as mean ± standard deviation (SD), one-way analysis of variance (ANOVA) used for statistical comparison along with Bonferroni’s; compare all pairs of the column. Where * showing p ≤ 0.05, ** p ≤ 0.01 and *** showing p ≤ 0.001.

Results

Cytotoxicity assay

MTT assay was used to assess cytotoxicity of pulegone on the growth of HeLa (cancer) and VERO (normal) cells, which is a reliable method for measuring cell cytotoxicity dependent on substrate conversion to the chromogenic product by live cells. All the cells were treated with increasing concentration of pulegone (0.1, 1 and 10 µM) for 12, 24 and 48 h, respectively. Pulegone caused inhibition of cell viability in case of HeLa cells while there was not much difference in case of VERO cells at a time in a dose-dependent manner as shown in Fig. 1. While, at the dose of 10 µM for 48 h showed more cytotoxicity in case of HeLa cells compared to positive control and untreated group.

Fig. 1
figure 1figure 1

Effects of Pulegone on (A) HeLa (cervical cancer), (B) VERO (normal cells) cells (C) CaSki cells and (D) SiHa cells. HeLa, VERO, CaSki and SiHa cells treated with increasing concentration of Pulegone for 12, 24 and 48 h, respectively. Data are the mean ± SD of at least three independent experiments. Pulegone 10 µM dose showed significantly more cytotoxicity compared with all other groups including Cisplatin (positive control). Where ***, **, * showing P ≤ 0.001, P ≤ 0.01, P ≤ 0.05 respectively.

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Apoptosis via Annexin V

For the investigation of the apoptotic effect of pulegone, HeLa cells were treated with 10 µM pulegone for 48 h in contrast with positive and negative control. The proportion of apoptotic cells was quantified by Annexin V staining. As shown in Fig. 2, untreated HeLa cells exhibited less Annexin V 24.55 ± 3.11%, apoptosis with positive control 44.65 ± 2.55%. On the other hand, pulegone treated HeLa cells showed an increased (49.4 ± 2.71%) degree of Annexin V staining in comparison to untreated and positive control groups (Fig. 2).

Fig. 2
figure 2

Effect of Pulegone on HeLa cells (cervical cancer) via cell apoptosis. Early apoptotic cells are shown in the lower-right quadrant of the scatter plot, and live cells are in the lower-left quadrant. Pulegone and cisplatin induced apoptosis measured by the Muse Annexin V and Dead Cell assay. Where (A) showing untreated group (B) Pulegone treated group (C) Cisplatin group. Where Pulegone showed more apoptotic cells compared with positive control and untreated group.

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Hoechst staining

It was also assessed the effect of pulegone on HeLa cell, apoptosis by fluorescent Hoechst 33,342 staining. The nuclei of control cells were regular in morphology, but some nuclei were fragmented and condensed after pulegone treatment, which was typical apoptotic morphology (Fig. 3).

Fig. 3
figure 3

is showing hoechest staining where (A) is untreated (control) (B) is Pulegone treated HeLa cells and (C) is cisplatin treated HeLa cells, pulegone causes morphological changes in the nucleus of HeLa cells. Cells were exposed to 10 µM dose of pulegone, cisplatin (Positive Control) and untreated (negative control) for 48 h then stained with 1ug/ml Hoechst 33,342. As the blue arrows indicate, condensed cells morphology in pulegone, while more condensed nucleus can be seen in pulegone compared with cisplatin, and untreated cells in shape (x200) (Scale bar 100 μm).

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Immunocytochemistry

Immunocytochemistry was performed to assess the level of apoptosis by using p53 antibody, a principal factor in apoptosis. Figure 4 showed that the HeLa cells treated with pulegone showed higher levels of apoptosis compared to controls and cisplatin.

Fig. 4
figure 4

Expression of P53 in Normal, Pulegone and cisplatin treated Hela cells. Where A1-A3 showing Normal cancer cells with reduced P53 level. B1-B3 Hela cells treated with Pulegone higher expression of P53. C1-C3 HeLa cells treated with cisplatin also showed higher expression of Annexin-V. Where more level of P53 in Pulegone treatment group compared with positive control and untreated group. (Antibody stained in green, nuclei stained with DAPI in blue color while A, B, C 3 were the merge of green and blue) (X200) (Scale bar 100 μm).

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Western blotting

Western blotting was performed by using controlled HeLa cells and cells treated with pulegone. BAX and BCL2 were detected in blot as shown in Fig. 5. Beta-actin was used as house-keeping gene to ensure the equal distribution of sample. Bands were analyzed via image J software. BAX is a pro-apoptotic protein which was up-regulated in treated HeLa cells as compared to untreated cells. BAX was 1.5-folds increased in pulegone treated cells as compared to untreated. BCL2 is anti-apoptotic protein which was down-regulated in treated HeLa cells in contrast to untreated cells. BCL2 was four folds decreased in pulegone treated cells in comparison to untreated cells.

Fig. 5
figure 5

Figure is showing immunoblots (A1-3) and protein fold increase (B) graphs (A = untreated B = Pulegone treated) of HeLa cells containing negative control group and Pulegone treated group where A1 showing Beta-Actin which act as house-keeping protein at 45KDA, A2 showing BAX (increased level) apoptotic protein at 26KDA while A3 showing BCL2 (decreased level) anti-apoptotic protein at 20KDA. B is showing Protein Fold increase.

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Discussion

Pulegone is a naturally cervical cancerurring monoterpene ketone found primarily in the essential oils of various plants, including peppermint and spearmint. Studies have shown that pulegone has multiple biological activities, including antibacterial, anti-inflammatory and anticancer effects20. Regarding anticancer activity, different reports have shown that pulegone can induce apoptosis in cancer cell lines (Nikolic et al., 2014 and Ghavam et al., 2023). Specifically, it has demonstrated good efficacy against prostate and breast cancer cells21. Pulegone has also imparted its anticancer effects by inhibiting angiogenesis, the process by which new blood vessels are formed in tumors for promoting their growth and metastasis22. In addition to numerous health benefits of pulegone, it has hepatotoxic activity thus further research is required to determine its safety and efficacy23.

The present study showed that pulegone was proved to be a potent anticancer agent when tested against cervical carcinoma cells. HeLa, VERO, CaSKi and SiHa cells were treated with pulegone for cytotoxicity analysis via MTT assay. It is a cytotoxicity assay in which mitochondrial enzymes convert MTT reagent into formazan in live cells, if cells are dead in wells, then reduction of formazan cervical cancerous and hence absorbance is less noted24,25. MTT assay was performed via application of different doses of drug i.e. 0.1, 1, 10 µM for 12, 24 and 48 h. Pulegone with 10 µM dose has shown increased cytotoxicity at 48 h in case of HeLa cells while, no difference was observed in VERO cells. The current research work was aimed to determine the anticancer potential of pulegone against HeLa cells. However, VERO cells were also checked whether drug was cytotoxic for normal cells. Based on MTT results of the current study, it was demonstrated that pulegone has shown cytotoxic effects in cervical cancer cell line, HeLa without causing any damage to normal cells. As 10 µM dose had more cytotoxicity so the rest of the assays were applied on HeLa cells at the dose of 10 µM for 48 h.

Several apoptotic markers were used to examine the apoptotic index, however; P53 was widely used marker to estimate the cellular apoptosis in different cancer cell lines25,26. The findings of the current study revealed that there was more apoptosis in HeLa cells after treating these cells with pulegone resulting in enhanced p53 release (Fig. 04), as reported previously (Ishfaq et al., 2023). Furthermore, Hoechst staining was done to assess apoptosis of cells treated with pulegone exhibited typical apoptotic morphological changes as reported previously27, with nuclei showing chromatin condensation or splitting into smaller structures by Hoechst 33,342. A cervical cancer according to the results, pulegone showed more chromatin condensation or splitting into smaller structures compared with control. These findings suggested that pulegone is a potential therapeutic compound in case of cervical cancer. Protein expression was analyzed by western blotting which showed that administration of pulegone in HeLa cells shifted balance of BCL2/BAX in favor of BAX and limits proliferation of cervical cancer cells. Therefore, pulegone can play a promising role as an anticancer agent against cervical cancer.

Conclusion

The present study showed that pulegone has dramatic tumor suppressive and antiapoptotic effects, and induced anti-proliferative morphological changes in cervical cancer cell lines. Thus, pulegone can be used alone or in combination with the traditional therapeutic strategies like radiotherapy and chemotherapy in the treatment of cervical cancer. This curative approach cannot only minimize the adverse effects of conventional anticancer therapy by decreasing its dosage but also reduces the economic burden on the cancer patients. As pulegone has very little harmful effects as compared to routine anticancer medications so, it can be routinely prescribed by oncologists to cervical cancer patients after proper recommendation by drug regulatory authorities.