Introduction

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths globally1. It has drawn growing attention because of its continuously increasing incidence and high mortality rate. Its invasiveness and poor prognosis have led to the rise in its prevalence2. There is still a need to develop new targeted drugs and treatment regimens. Ciprofol, a derivative of propofol, exhibits a stronger sedative effect in clinical practice3. It is frequently used in gastroscopy and colonoscopy examinations4, as well as in general anesthesia5. However, the impact of Ciprofol on CRC cells has not been reported in previous studies. According to research reports, propofol, a commonly used anesthetic during surgery, can reduce the risk of cancer recurrence, and this process is accompanied by a significant up-regulation of the APC protein6. However, it remains unknown whether Ciprofol, as an iterative drug, can inhibit the progression of CRC by targeting and activating the APC protein.

The adenomatous polyposis coli (APC) gene is the most prevalent tumor suppressor genes in colorectal cancer. Mutations in the APC gene result in the accumulation of β-catenin, thereby activating the Wnt signaling pathway and subsequently triggering TCF/LEF transcription factors and inducing the expression of EMT-associated genes7. Examples include EMT regulatory factors such as Snail, Zeb, and Twist, whose expression levels elevate due to APC inactivation8. Furthermore, APC exerts additional influence on EMT through its regulation of cell adhesion factor functions. Inactivation of APC stimulates glycolysis9. APC acts to inhibit the expression of the glucose transporter GLUT1, consequently constraining the influx of glucose into cells. The absence of functional APC results in heightened GLUT1 expression, fostering augmented glucose absorption and facilitating glycolytic activity. APC also assumes a direct role in glycolysis, governing critical enzymes such as hexokinase and phosphofructokinase. This underscores the intricate interplay of APC with EMT and glycolysis within colorectal cancer cells. The deactivation of APC enhances EMT and glycolysis, thus playing a crucial part in the inception and advancement of CRC. Consequently, APC emerges as a promising target for CRC therapy, presenting innovative avenues through its functional modulation.

Dysregulated activation of the Wnt/β-catenin signaling pathway assumes a role in inducing epithelial cell disruption and stromal cell proliferation, thereby facilitating the process of EMT10,11. Furthermore, the Wnt/β-catenin pathway modulates glycolysis in diverse cancer cells, thus restraining tumor cell invasion and metastasis. The Wnt/β-catenin pathway holds significant importance in the context of colorectal cancer treatment12. Existing literature indicates that propofol, through its modulation of the Wnt/β-catenin signaling pathway, hinders the proliferation and invasion of hepatic cancer cells13. Compared to other anesthetics, propofol is a preferred choice for tumor patients undergoing surgery given the multiple benefits provided by the drug14,15. Ciprofol, a structural derivative of propofol, remains relatively unexplored in terms of its effects on cancer cells, while the anticancer potential of propofol has been extensively documented. Hence, this study primarily investigates the specific mechanisms underlying the effects of ciprofol on the APC protein and the Wnt/β-catenin signaling pathway16,17,18, as well as its impact on CRC cell EMT and glycolysis. The objective is to establish evidence supporting ciprofol as a viable anesthesia option for tumor resection surgery and provide a theoretical foundation for future research.

Methods and materials

Cell culture

Human normal colonic epithelial cells CD841 and human colorectal cancer cells HT29, HCT116, LoVo, and LS174T were procured from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China. Cells were cultured in high-glucose DMEM medium (Gibco, 11,995) containing 10% fetal bovine serum (ExCell Bio, YSN0121) and 1% penicillin–streptomycin solution (Solarbio, P1400). Cultures were maintained at 37 °C in a 5% CO2 incubator (Jiemei Electronics, CI-191C).

CCK8 assay

Following the suspension of cells in a suitable amount of medium, 3000 cells were introduced into each well of a 96-well plate and incubated overnight. Experimental groups received DMEM solutions containing different concentrations of ciprofol (3.125, 6.25, 12.5, 25, 50, 100 μmol/L), while control groups received an equivalent volume of DMSO solution. After 48 h, 10 μL of CCK8 solution (Solarbio, CA1210) was added to each well, and absorbance was measured at 450 nm after 3 h of incubation using a microplate reader (Flash, ReadMax 1200). The calculation process of IC50 can be found in detail in the Supplementary Image Gallery.

Scratch assay

A cell density of 5 × 105 cells per well was employed for seeding in a six-well plate, followed by overnight incubation. Using a 10 μL pipette tip, three parallel vertical lines were delicately created at the plate’s base. Then, the previous medium was aspirated, and the cells were rinsed thrice with PBS. Following this, DMEM high-glucose medium supplemented with gradient concentrations of ciprofol (10, 20, 40 μmol/L) was introduced into the wells. After 12 h, images were captured at the intersection of the parallel and vertical lines using a microscope (Shunyu, ICX41). Cell migration rates were analyzed using Image J software.

Transwell assay

The Matrigel matrix (BD, 354,234) was diluted at a ratio of 1:8 using serum-free DMEM, and then applied to the upper chamber of the Transwell inserts. Subsequently, the inserts were incubated at 4 °C for 4 h. Serum-free DMEM containing HT29 cells was introduced into the lower chamber of each well within a six-well plate. Prior to inoculation, cells were subjected to pre-treatment with varying concentrations of ciprofol (10, 20, 40 μmol/L) for 24 h. After 12 h of incubation, cells that invaded the lower chamber were secured, stained, and photographed under a microscope. Invasive cell numbers were quantified using Image J.

Measurement of glucose, lactate, and ATP levels

HT29 cells were subjected to different concentrations of ciprofol treatment for 24 h. Cells were collected and homogenized, and glucose, lactate, and ATP levels were measured using respective assay kits. Absorbance was measured at specific wavelengths using a UV–Vis spectrophotometer. For detailed procedures, please refer to the Supplementary Materials.

Extracellular acidification rate and oxygen consumption rate measurement

Index-phase HT29 cells were digested, and 5 × 103 cells were inoculated per well in a 96-well plate. After transfecting the cells with Si-APC transfection reagent for 24 h, they were treated with ciprofol for 24 h. Extracellular acidification rate and oxygen consumption rate were measured using the glycolysis stress test kit (Agilent, 103346-100) and the mitochondrial stress test kit (Agilent, 103015-100) according to the experimental procedures. The measurements were conducted using a Seahorse XF Pro analyzer from Agilent, USA.

qPCR experiment

HT29 cells in the logarithmic growth phase were subjected to ciprofol treatment for 24 h. Cells were collected in sterile cell scrapers, transferred to 1.5 mL centrifuge tubes, and then mixed with 1 mL of Trizol (Solarbio, 15596026). Extract RNA according to instructions. RNA concentration was determined using a UV–Vis Spectrophotometer Q5000. Reverse transcription was performed using a reverse transcription kit (TIANGEN, KR118), and the resulting cDNA was either immediately used or stored at − 80 °C. cDNA concentration was measured, and qPCR was performed using a real-time fluorescence quantitative instrument (Bio-RAD, CFX Connect) with a qPCR detection kit (TIANGEN, FP313). Raw data were retained for subsequent analysis. QPCR primer sequences and APC interference sequences are detailed in Table S1.

Western blot experiment

Lysis buffer was added to HT29 cells and tumor tissue samples in an appropriate volume, followed by ice lysis for 25 min. Protein quantification was performed using a BCA protein quantification kit (Solarbio, PC0020). Protein samples were heated at 95 °C for 10 min in a water bath, cooled, and then stored at − 80 °C. Gel electrophoresis was performed with increasing voltage (80 V for 35 min, then 120 V for 60 min). The proteins were transferred to PVDF membranes, and these membranes were subsequently treated with a 5% skim milk solution for a duration of 2 h to prevent non-specific binding. Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies. Subsequently, incubate in the corresponding secondary antibody for 2 h. After another round of washing, use a gel imaging system to capture images. Image J 1.5.2a software was used for subsequent data analysis. Antibodies used included HK2 (Affinity, DF6176, 1:1000), LDHA (Huabio, ET1608-57, 1:1000), β-actin (Huabio, ET1701-80, 1:5000), APC (Huabio, ET1610-80, 1:1000), E-Cadherin (ABclonal, A20798, 1:1000), N-Cadherin (ABclonal, A19083, 1:1000), MMP9 (Huabio, ET1704-69, 1:1000), MMP2 (Huabio, ET1606-4, 1:1000), p-GSK3β (Affinity, AF2016, 1:1000), GSK3β (Affinity, AF5016, 1:1000), β-catenin (Cell Signaling, 8480S, 1:1000), goat anti-rabbit IgG (Affinity, S0001, 1:5000), goat anti-mouse IgG (Affinity, S0002, 1:5000). Original Western blot pictures can be seen in detail in the supplementary materials.

In vivo experiment

Twelve male BALB/c Nude Mice, aged 4–6 weeks and weighing 17–20 g, were purchased from Henan Skysource Biotechnology Co., Ltd. License No.: SCXK (Yu) 2020-0005. These mice were housed in the Animal Experiment Center of the Fourth Hospital of Hebei Medical University (License No.: 2023205). After 7 days of acclimatization, subcutaneous injections of HT29 colorectal cancer cells were performed. The nude mice were randomly divided into two groups: the control group and the treatment group, with 6 mice in each group. On the 7th day after tumor inoculation, the treatment group was intraperitoneally injected with ciprofol solution (40 mg/kg), and subsequent injections were given every 3 days. The control group was administered an equal volume of normal saline19. Fourteen days after treatment, the mice were euthanized via intraperitoneal injection of 150 mg/kg pentobarbital sodium (Suzhou Yakoo Technology Co., Ltd., W0091, China). Approximately 5 min later, death was confirmed by the dilation of pupils and the cessation of heartbeat and breathing. Subsequently, the subcutaneous tumor tissues were collected, weighed, photographed, and stored in liquid nitrogen. The formula for calculating the tumor volume is: V = (L × W2) / 2. The subcutaneous tumor tissues were collected, weighed, photographed and stored in liquid nitrogen. The formula for calculating the tumor volume is: V = (L × W2)/2.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 9.5.0. Measured data were expressed as mean ± standard deviation (SD). For multi—factor Western blot assays, two-way analysis of variance (ANOVA) was used, with Tukey’s test for post—hoc analysis. For other in vitro experiments, one-way analysis of variance (ANOVA) was adopted, and Bonferroni’s test was chosen for post-hoc analysis. In in-vivo experiments, the data between two groups were analyzed by t-test. All experimental data were obtained from parallel experiments conducted three times or more. A P- value < 0.05 was considered to indicate statistical significance.

Results

Ciprofol can inhibit the proliferative ability of CRC cells

First, we evaluated the effect of Ciprofol on the proliferative ability of CRC cells through the CCK8 assay. Figure 1A shows the chemical structure of Ciprofol. The results of the CCK8 assay indicated that Ciprofol had no significant impact on the proliferative ability of CCD841 (human normal colonic epithelial) cells, but could inhibit the proliferative ability of CRC cells. We calculated the IC50 of Ciprofol for each type of CRC cell. The results were as follows: the IC50 for HT29 cells was 37.62 ± 3.85 μmol/L, for LS174T cells was 74.6133 ± 12.91 μmol/L, for LOVO cells was 97.25 ± 17.47 μmol/L, and for HCT116 cells was 48.33 ± 5.99 μmol/L. Among them, Ciprofol had the lowest IC50 for HT29 cells, confirming that Ciprofol had the strongest inhibitory effect on the proliferation of HT29 cells (Fig. 1B). As is well known, Ciprofol is a short-acting intravenous sedative based on the structural modification of Propofol3. Therefore, we also focused on the inhibitory effect of Propofol on HT29 cells. The results showed that the IC50 of Propofol for HT29 cells was 105.84 ± 16.15 μmol/L. The results suggested that Ciprofol had a stronger inhibitory effect on the proliferation of HT29 cells compared with Propofol (Fig. 1C). The calculated IC50 curves for each cell line can be found in Figure S1 in detail.

Fig. 1
figure 1

Inhibitory Effects of Ciprofol on HT29 Cell Migration and Invasion. (A) Molecular structure and relative molecular mass of ciprofol. (B) The CCK8 assay was used to detect the changes in the viability of CCD841 (human normal colonic epithelial cells), HT29, LOVO, LS174T and HCT116 cells after treatment with different concentration gradients of Ciprofol for 48 h. (C) The CCK8 assay was used to detect the changes in the viability of HT29 cells after treatment with different concentration gradients of Propofol for 48 h. (D) Western blot experiment measuring the relative protein expression levels of HK2 and LDHA. (* indicates statistical significance compared between the two groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

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Our study found that APC protein was under-expressed in four strains of colorectal cancer cells compared to normal colonic epithelial cells. Notably, APC protein expression was lowest in HT29 cells, while HCT116 cells exhibited the highest APC protein expression among the tested colorectal cancer cell lines(Fig. 1D). Regarding the selection of Ciprofol concentration for subsequent experiments, we set the maximum concentration to be around the IC50 of HT29 cells, which is 40 μmol/L. A concentration around the IC50 can not only largely retain the cytotoxic effect of Ciprofol on CRC cells, but also avoid drug toxicity caused by an overly high concentration, ensuring that it is the targeted killing rather than non-specific toxicity20. Moreover, the addition of 100 μmol/L Ciprofol has no effect on the proliferative ability of CCD841 cells, and a concentration of 40 μmol/L is also non—toxic to colonic epithelial cells. In conclusion, we set the maximum concentration of Ciprofol as 40 μmol/L in the subsequent experiments.

Ciprofol suppresses EMT and glycolysis in colorectal cancer cells

To highlight the role of Ciprofol in targeting and activating APC, in the phenotypic experiments verifying the effects of different concentration gradients of Ciprofol (10, 20, 40 μmol/L) on EMT and glycolysis in CRC cells, we selected HT29 cells, which have the lowest APC expression among the four CRC cell lines, as the research subject. The EMT process is a crucial factor in the invasion and metastasis of CRC, and glycolysis provides energy for this process21. Therefore, we investigated the effects of Ciprofol on the EMT process and glycolysis in CRC cells. Moreover, ciprofol effectively suppressed the invasion and migration of colorectal cancer cells, with enhanced inhibition observed at higher concentrations (Fig. 2A,B).

Fig. 2
figure 2

Inhibitory Effects of Ciprofol on HT29 Cell Migration and Invasion. (A) Scratch assay measuring the influence of ciprofol on HT29 cell migration. (B) Transwell assay evaluating the effect of ciprofol on HT29 cell migration. (C) Western blot experiment analyzing the expression levels of E-cadherin, N-cadherin, MMP2, and MMP9 proteins. (* indicates statistical significance compared between the two groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

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Subsequently, we detected the relative expression levels of EMT-related marker proteins N-cadherin and E-cadherin22, as well as cell metastasis-related marker proteins MMP2 and MMP923. The results showed that Ciprofol led to a decrease in the relative expression levels of MMP2, MMP9, and N-cadherin proteins, while the relative expression level of E-cadherin protein was upregulated. This further confirmed the potential effect of Ciprofol on the EMT process in colorectal cancer cells (Fig. 2C). Having established ciprofol’s capacity to restrain colorectal cancer cell proliferation, invasion, and migration, our subsequent focus shifted toward exploring its potential capacity to hinder glycolysis. Our findings demonstrated that ciprofol effectively impeded ATP levels, lactate production, and glucose generation in colorectal cancer cells. This inhibition was further substantiated by downregulation of HK2, LDHA mRNA and protein expression levels, suggesting ciprofol’s capability to suppress glycolytic metabolism in colorectal cancer cells (Fig. 3A,B).

Fig. 3
figure 3

Inhibitory Effects of Ciprofol on HT29 Cell Glycolysis. QPCR experiment assessing the expression levels of HK-2 and LDHA mRNA. Changes in ATP levels, lactate production, and glucose metabolism in HT29 cells after ciprofol treatment. (B) Western blot experiment measuring the relative protein expression levels of HK2 and LDHA. (* indicates statistical significance compared between the two groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

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Ciprofol suppresses colorectal cancer Cell EMT process and glycolysis through apc protein activation

To elucidate whether ciprofol suppresses colorectal cancer cell glycolysis and EMT processes through APC activation, we focused on HCT116 cells, which exhibited the highest APC expression among the four colorectal cancer cell strains. Experimental groups were divided into four categories (Control, Ciprofol, NC, and Ciprofol + Si-APC), with the latter involving the construction of APC-knockdown HCT116 cell lines in vivo. QPCR results demonstrated significant downregulation of APC mRNA expression by all three Si-APC sequences, with Si-APC2 sequence displaying the most pronounced decrease (Fig. 4A). Accordingly, Si-APC2 was selected as the subsequent interference sequence.

Fig. 4
figure 4

Relief of Ciprofol’s Inhibitory Effects on HCT116 Cell Glycolysis and Invasion upon APC Protein Downregulation. (A) The relative expression level change of APC mRNA in HCT116 cells was detected by qPCR assay 48 h after transfection with siAPC—infected RNA sequence. (B) The relative expression level of APC protein in HCT116 cells was detected by Western blot assay. (C) Transwell experiment assessing changes in cell invasion ability. (D) QPCR analysis of HK2 and LDHA mRNA expression levels. (E) Measurement of ATP levels, lactate production, and glucose metabolism in HT29 cells treated with ciprofol. (F)Measurement of HT29 cell ECAR (extracellular acidification rate) and OCR (oxygen consumption rate) at different time intervals. (G) Western blot analysis of E-cadherin, N-cadherin, MMP2, and MMP9 protein expression levels. (H) Western blot analysis of HK2, LDHA and PKM2 protein expression levels. (*indicates statistical significance compared between the two groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

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Our study demonstrated that the down-regulation of APC attenuated the inhibitory effects of Ciprofol on the invasion and migration of HCT116 cells (Fig. 4B,C). After treating HCT116 cells with Ciprofol, the down—regulation of APC enhanced the relative expression levels of HK2 and LDHA mRNA (Fig. 4D), and up-regulated the accumulation of ATP, glucose, and lactate (Fig. 4E). This further confirmed that Ciprofol inhibits the EMT process and glycolysis by targeting and activating APC. ECAR and OCR are also important indicators for evaluating glycolysis24. We found that Ciprofol could inhibit the levels of ECAR and OCR in HCT116 cells, while siAPC could counteract the inhibitory effect of Ciprofol and enhance the expression levels of ECAR and OCR (Fig. 4F). Subsequently, we detected the expression levels of EMT and glycolysis-related marker proteins through Western blot assay. The results showed that Ciprofol could up—regulate the relative expression level of E-cadherin, and down-regulate the relative expression levels of N—cadherin, MMP9, MMP2, HK2, LDHA, and PMK2 proteins. In contrast, the addition of siAPC had the opposite regulatory effects (Fig. 4G,H). This further strengthened our conclusion.

Ciprofol suppresses HT29 Cell EMT and glycolysis via inhibition of Wnt/β-Catenin signaling pathway

Derivative83 is an activator of the Wnt/β-catenin signaling pathway. To investigate whether ciprofol exerted its pharmacological effects through the Wnt/β-catenin signaling pathway, experiments were divided into four groups (Control, Ciprofol, Pathway Activator, and Ciprofol + Pathway Activator) to explore ciprofol’s impact on HT29 cells. Transwell experiments and glycolysis-related indicator assessments confirmed that the introduction of Wnt/β-catenin signaling pathway activators mitigated ciprofol’s inhibition of HT29 cell invasion and glycolysis (Fig. 5A,B). Western blot analysis revealed that ciprofol treatment reduced β-catenin protein expression while increasing p-GSK-3β protein expression versus the control group. GSK-3β protein expression remained unaltered. The addition of Wnt/β-catenin signaling pathway activators counteracted ciprofol’s effects (Fig. 5C). These results underscored that ciprofol’s pharmacological effects on HT29 cells were likely achieved through the suppression of the Wnt/β-catenin signaling pathway.

Fig. 5
figure 5

Derivative83 Counteracts Ciprofol’s Inhibition of Wnt/β-Catenin Signaling in HT29 Cells. Transwell experiment assessing changes in cell invasion ability. (B) Western blot analysis of p-GSK3β, GSK3β, and β-catenin protein expression levels. (C) QPCR analysis of HK2 and LDHA mRNA expression levels, and measurement of ATP levels, lactate production, and glucose metabolism in colorectal cancer cells. (* indicates statistical significance compared between the two groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

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To further confirm the regulatory relationship between APC and the Wnt/β-catenin signaling pathway, we established a cell line with overexpressed APC in HT29 cells, which have the lowest APC expression. The results of the RT-qPCR assay showed that, compared with the Vector group, the relative expression level of APC mRNA in HT29 cells in the oe-APC group increased significantly, demonstrating the successful establishment of our APC-overexpressing cell model (Figure S2A). Subsequently, we found that, compared with the control group, both overexpression of APC and treatment with Ciprofol alone could significantly inhibit the activity of the Wnt/β-catenin signaling pathway (Figure S2B).

Ciprofol suppresses tumor tissue growth in subcutaneous murine xenografts

In previous studies, we successfully demonstrated that Ciprofol can inhibit the EMT process and glycolysis in CRC cells. Moreover, this process may be achieved by targeting and activating APC, which in turn activates the Wnt/β-catenin signaling pathway. To explore the effects of ciprofol on HT29 cells in vivo, a xenograft model was established by subcutaneously injecting HT29 cells into mice. The procedures for establishing tumor—bearing mouse models and the treatment process with Ciprofol are shown in Fig. 6A.

Fig. 6
figure 6

Ciprofol Inhibits Xenograft Tumor Growth in Mice. (A) Schematic representation of the process of establishing HT29 xenograft tumor models in mice. (B) Overhead view of tumor-bearing mice and axillary view of tumor tissue. (C) Tumor volume and weight analysis. (D) Measure the length and width of the tumor tissue every 3 days. Calculate the change in the tumor volume of the mice over time. Meanwhile, measure the change in the body weight of the mice. (E) Western blot analysis of E-cadherin, N-cadherin, MMP9, MMP2, HK2, LDHA, PMK2, GSK3β, p-GSK3β, and β-cateninprotein expression levels in tumor tissue. (* indicates statistical significance compared between the two groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

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The top-view anatomical images of tumor tissues from the Ciprofol-treated group and the control group are shown in Fig. 6B. Compared with the control group, the experimental group treated with ciprofol exhibited a significant decrease in both tumor volume and weight (Fig. 6C). On this basis, we focused on the dynamic changes in tumor volume over time during ciprofol treatment. The results clearly showed that, compared with the control group, the ciprofol-treated group could effectively inhibit the growth of tumor tissues. In addition, during the experiment, it was observed that the body weight of the mice in the ciprofol-treated group increased steadily compared with that of the mice in the control group (Fig. 6D). These results fully demonstrate that ciprofol has a good inhibitory effect on CRC cells in the in-vivo environment. Subsequent Western blot analysis of invasion and glycolysis-related proteins aligned with our in vitro findings, providing preliminary evidence of ciprofol’s ability to suppress HT29 cell invasion and glycolytic metabolism within an in vivo context(Fig. 6E).

Discussion

The novelty of this study lies in its groundbreaking revelation of ciprofol’s inhibitory effects on colorectal cancer, thereby demonstrating its anticancer potential and offering preliminary insights into its mechanism of action. LL Y et al.25 treated colorectal cancer cells with propofol at a concentration of 20 μg/mL (113 μmol/L), resulting in approximately 50% inhibition of cell viability compared to the control group. The research performed by YJ L et al. indicated that propofol achieved a 50% inhibition of colorectal cell viability at a concentration of 12 μg/mL (67 μmol/L). In contrast, ciprofol exhibited an IC50 of 37.62 μmol/L for inhibiting colorectal cancer cell viability. These findings suggest that ciprofol exerts a more potent inhibitory effect on colorectal cancer cells when compared to propofol at equivalent drug concentrations.

Both in vitro and in vivo experiments demonstrated ciprofol’s inhibitory impact on colorectal cancer cells, particularly HT29 cells, characterized by the suppression of invasion, migration, and glycolysis. These results align with the reported effects of propofol26,27. Recent studies have highlighted the clinical relevance of E-cadherin and N-cadherin in various cancers, where the downregulation of E-cadherin and the overexpression of N-cadherin are pivotal factors in the process of EMT28,29. Our observations revealed that ciprofol downregulated MMP2, MMP9, and N-cadherin while upregulating E-cadherin protein expression, providing initial evidence of ciprofol’s potential to hinder colorectal cancer cell EMT. As EMT necessitates significant energy consumption, tumor tissues rely on aerobic glycolysis for energy generation, resulting in increased energy production rates. Furthermore, the incomplete oxidation of glucose leads to the accumulation of metabolites, benefiting tumor cells30. Our experimental results indicated that ciprofol diminishes ATP levels, lactate production, and glucose metabolism in colorectal cancer cells (HT29). This is corroborated by the decreased mRNA and protein expression of HK2 and LDHA. These findings suggest that ciprofol may disrupt energy supply through glycolysis in colorectal cancer cells, thereby affecting the EMT process. This resembles propofol’s actions in inhibiting EMT in bladder and breast cancers31,32,33.

The APC gene plays a crucial role in glycolytic metabolism regulation. It is involved in activating and stabilizing ATP hydrolytic enzymes, thus regulating the rate and direction of glycolysis. Mutations in the APC gene can lead to aberrant glycolytic metabolism34. Our study demonstrated that ciprofol activated APC, resulting in a significant reduction in the glycolytic rate of colorectal cancer cells. Furthermore, cell invasion and migration were inhibited, and these regulatory effects were restored upon APC downregulation. This establishes a potential connection between ciprofol and the tumor suppressor gene APC.

Subsequently, we delved into the closely related Wnt/β-catenin pathway, which is intricately connected to the APC gene35. Drawing from substantial preclinical evidence, the Wnt/β-catenin pathway assumes a crucial function in transcriptional regulation, cytokine generation, and rearrangement of cellular cytoskeleton. Previous research has also demonstrated propofol’s ability to inhibit the Wnt/β-catenin signaling pathway36,37. Thus, we initially investigated the Wnt/β-catenin pathway as a downstream signaling pathway of ciprofol. Consistent with propofol’s results, ciprofol was found to suppress β-catenin protein expression and activate GSK-3β phosphorylation, indicating its inhibition of the Wnt/β-catenin pathway. This inhibitory effect was counteracted by the addition of Derivative83, affirming the role of Wnt/β-catenin as a downstream signaling pathway of ciprofol. Furthermore, the addition of Derivative83 rescued the inhibitory effects of ciprofol on glycolysis, invasion, and migration in HT29 cells, further supporting the involvement of Wnt/β-catenin in EMT and metabolic processes.

This study presents the first evidence of ciprofol’s inhibitory effects on colorectal cancer cell glycolysis and EMT, shedding light on a novel aspect of ciprofol’s interaction with the Wnt/β-catenin pathway. We also took into account the significance of using ciprofol as a clinical surgical anesthetic for CRC patients. During surgery, tumor cells may detach and disseminate due to surgical manipulation, entering the bloodstream or surrounding tissues, which can lead to postoperative recurrence and metastasis. If ciprofol is used as an anesthetic for cancer surgery, its inhibitory effect on tumor cells may reduce the risk of tumor cell dissemination caused by surgical procedures, thereby decreasing the likelihood of postoperative recurrence. Secondly, the physical function of cancer patients is already compromised due to tumor consumption, and the surgical trauma further exacerbates the body’s burden. In animal experiments, ciprofol did not cause severe toxic reactions, and the body weight of mice in the treatment group increased steadily. This implies that it may have good safety and tolerability. For cancer patients with relatively weak physical conditions, it may be more tolerable, helping to reduce the incidence of anesthesia—related complications. However, there are still numerous challenges to overcome in translating basic research findings into clinical applications. Although positive effects have been observed in cell and animal models, the human physiological environment is far more complex. There may be differences in aspects such as pharmacokinetics and the interactions between the drug and various human tissues and organs. Additionally, for clinical applications, precise determination of the drug dosage and its interactions with other commonly used drugs during surgery need to be considered. Therefore, rigorous clinical trials are required in the future to verify the safety and effectiveness of ciprofol as an anesthetic for cancer surgery. The treatment of CRC with the combination of ciprofol and nanomaterials is also a novel therapeutic strategy. Charles Chidi Okechukwu et al. enhanced the therapeutic effect of 5-fluorouracil through this technology38. Currently, it remains unclear how ciprofol enters cells, which will be one of the key focuses of our future research.