Ciprofol exerts anti-tumour effects in hepatocellular carcinoma through the Raf-MEK-ERK signalling pathway

Introduction

Hepatocellular carcinoma (HCC) is one of the most prevalent malignant tumours of the digestive system and the third leading cause of cancer-related deaths worldwide (1). HCC is the most common type of primary liver cancer. Current standard treatment options for HCC include radical resection, liver transplantation, trans-arterial chemoembolization, radioembolization, radiofrequency ablation, and chemotherapy. The emergence and refinement of adjuvant therapies have increased the number of patients eligible for surgical treatment (2).

Although surgical resection is the most important treatment for HCC, postoperative recurrence and metastasis remain critical obstacles that affect patient survival. Intraoperative manipulations may cause cancer cells to be released into the vasculature, lymphatic circulation, or adjacent tissues. The immune function of patients with tumours is often suppressed, preventing immune cells from effectively destroying tumour cells. Additionally, perioperative blood transfusion, hypothermia, hypotension, pain, electrolyte imbalance, and the use of certain anaesthetics further suppress the immune function, thereby increasing the rates of cancer cell metastasis and recurrence in patients undergoing surgery (3).

Numerous reports have indicated that anaesthetics are extensively associated with tumour cell differentiation, proliferation, and apoptosis (4-6). Furthermore, compromised immune function in patients with tumours can be exacerbated by surgery and anaesthesia, thereby affecting the postoperative recurrence and prognosis (7-9). Numerous studies have reported controversial findings regarding the effect of anaesthetics on tumours (10-12). Therefore, the mechanisms by which anaesthetic drugs affect HCC cells should be further explored to enhance the treatment outcomes in patients with HCC.

Propofol, a commonly used intravenous anaesthetic, is employed for sedation, induction, and maintenance of general anaesthesia during surgical procedures, as well as for sedation in the intensive care unit (ICU). Propofol have been reported to regulate the proliferation and migration of bladder cancer cell mainly by suppressing the Hedgehog Pathway (13), inhibit pancreatic cancer by upregulating miR-328 and downregulating ADAM8 (14), and suppress the malignant progression of HCC by inhibiting HOXA11 (15). Most clinical studies have suggested that propofol is associated with better postoperative survival outcomes in patients with cancer (16-18). Ciprofol, a novel compound developed independently in China, is a short-acting intravenous sedative derived from propofol through structural modifications (19). Owing to the advantages mentioned above, ciprofol is widely used in clinical practice. Currently, no study has reported on the impact of ciprofol on HCC; however, we hypothesise that it has the same anti-HCC effects as propofol, potentially guiding anaesthesia induction and maintenance during liver cancer surgical resection.

This research aimed to uncover the impact of ciprofol on the malignant development of HCC and to investigate the pathways by which ciprofol influences HCC. This study showed that ciprofol substantially inhibited the malignant biological behaviour of HCC cells, including proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT). Mechanistically, ciprofol inhibited the phosphorylation of Raf kinase, thereby suppressing the Raf-MEK-ERK signalling pathway, resulting in notable inhibition of the malignant biological behaviour of HCC cells. Importantly, our findings have considerable implications in the selection of perioperative anaesthetics for patients with HCC. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tgh.amegroups.com/article/view/10.21037/tgh-24-115/rc).

Methods

Cell lines and reagents

Human HCC cell lines (Hep3B and HCCLM3) were purchased from the Chinese Academy of Sciences Shanghai Institutes for Biological Sciences (Shanghai, China) and maintained in the Laboratory (Hepatobiliary Center, Jiangsu Provincial Peoples’ Hospital). Genetic background of all cell lines was tested and cross-contamination was confirmed to be free from.

The antibodies against ERK1/2, p-ERK1/2, MEK, p-MEK, Raf and p-Raf were purchased from Cell Signalling Technology (Shanghai, China). The antibodies against Vimentin, N-cadherin, E-cadherin, Snail, Ki-67, β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from Proteintech (Shanghai, China). Sorafenib and epidermal growth factor (EGF) were purchased from MedChemExpress (New Jersey, USA). Propofol was acquired from Sigma-Aldrich (Shanghai, China). Ciprofol was acquired from Haisco (Chengdu, China) (Table S1).

Cell culture

In RPMI-1640 (Thermo Fisher Scientific, Waltham, MA, USA), human HCC Hep3B and HCCLM3 cells were maintained at 37 ℃ and 5% CO₂ and added 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA).

Animal models

Male BALB/c nude mice (Viton Lecer, Shanghai, China) were kept in alternating light/darkness (12 hours), temperature (22–26 ℃), humidity (50–60%) with free access to food and water. The cervical dislocation was performed and euthanised. Animal experiments were performed under a project license (No. 2406081) approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University, in compliance with national guidelines for the care and use of animals. We assign each mouse an ear tag number, then randomize the ear tag numbers and group the mice accordingly. Animals that died before the end of the experiment or developed diseases that significantly affected the experimental outcomes were excluded from the study. A protocol was prepared before the study without registration.

Subcutaneous tumourigenicity model: Hep3B were implanted by subcutaneous injection (1×10⁶ cells/mice) in the armpit of the forelimb. Once palpable tumours reached approximately 50 mm³ in volume, we randomly divided mice into two groups and treated them with ciprofol (20 mg/kg, twice a week) or dimethylsulfoxide (DMSO) via tail vein injection. Tumour growth and body weight were monitored every four days.

Lung metastasis model: HCC cells (1×10⁶ cells/100 µL) was injected into nude mice via tail vein. Mice were randomly assigned to group 7 days later, either ciprofol (20 mg/kg, twice weekly) or DMSO. After 28 days of treatment, the IVIS100 imaging system (Xenogen, Hopkinton, MA, USA) was used for bioluminescence imaging using a 100 mg/kg D-luciferin (Xenogen). Lung tissue was surgically removed and fixed, and the efficacy was assessed with hematoxylin-eosin staining.

Orthotopic transplantation model: Tumours from the subcutaneous tumourigenicity model, which had not received treatment, were cut into 2 mm³ pieces and then transplanted into the livers of mice. Seven days post-transplantation, the mice were randomly allocated to receive either ciprofol (20 mg/kg) or a DMSO solution via tail vein injection twice a week. Tumour growth in liver was obtained and measured after 28 days. All animal experiments were operated according to guidelines specified in the ‘Guide for the Care and Use of Laboratory Animals’ published by the National Institutes of Health.

Cell Counting Kit-8 (CCK-8) assay

For the CCK-8 assay, 1,000/well HCC cells were inoculated into a 96 well plate overnight, 10 µL CCK-8 solution (TargetMol, Boston, MA, USA) was added and incubated at 37 ℃ for 2 hours. Using Multiskan GO (Thermo Fisher) to measure the absorbance at 450 nm. Cells were treated with propofol (0–20 µM) or ciprofol (0–20 µM) for 24, 48, and 72 hours.

Colony formation assay

Hep3B and HCCLM3 cells were inoculated into 6 well plates (600 cells per well), then cultured in 2 mL medium for 14 days until clones were formed. The medium was refreshed every 3 days. Subsequently, the clones were fixed, stained by violate crystal (NCM Biotech, Suzhou, China). Preparation of 0.1–0.5% crystal violet staining solution. Gently cover the plate with the staining solution and incubate for 20 minutes. After staining, aspirate the solution and rinse with deionized phosphate-buffered saline (PBS) to remove excess dye. Finally, the clones were captured and counted. Cells were treated with ciprofol (0, 5, 10 µM) for 15 days. For the group treated with sorafenib, an additional 72-hour pre-treatment with sorafenib (10 nM) is required. For the group treated with EGF, an additional 72-hour pre-treatment with EGF (10 nM) is required.

5-Ethynl-2'-deoxyuridine (EdU) assay

Cells were incubated for 2 days in 96-well plates (5,000 cells per well). Afterward, 50 µM EdU (RiboBio, Guangzhou, China) was added to each well, and the plates were further incubated at 37 ℃ for 2 hours, followed by fixation, permeation and staining with 400 µL Hoechst33342. The cells were counted in five random zones in each well, and the percentage of EdU-positive cells was calculated. Cells were treated with ciprofol (0, 5, 10 µM) for 72 hours.

Flow cytometry analysis of Annexin V-FITC/PI staining

The AnnexinV-FITC/PI Apoptosis Kit (Proteinbio, Beijing, China) was used to measure the apoptosis rates. The cells were harvested and stained as instructed by the manufacturer, and then analyzed by flow cytometry. Apoptosis includes early apoptosis (Annexin V+ PI−) and late apoptosis (Annexin V+ PI+). we calculated the apoptosis rate as the sum of the two. Cells were treated with ciprofol (0, 5, 10 µM) for 72 hours.

Wound healing assay

Hep3B and HCCLM3 were cultured in 6-well plates until the formation of monolayer confluence. Then use a 200 µL pipet to gently scratch straight lines. Subsequently, the medium was replaced by FBS-free DMSO and ciprofol medium. At 0 hours (s1) and 24 hours (s2), measure the scratch width. Calculation of relative mobility (+%) is given by the following formula: (s1 − s2)/s1 × 100%. Cells were treated with ciprofol (0, 5, 10 µM) for 24 hours. For the group treated with sorafenib, an additional 72-hour pre-treatment with sorafenib (10 nM) is required. For the group treated with EGF, an additional 72-hour pre-treatment with EGF (10 nM) is required.

Transwell invasion and migration assays

Transwell chambers (LABSELECT, Hangzhou, China) covered with or without Matrix were tested for invading or migrating capacity of Hep3B and HCCLM3 cells. HCC cells (1×10⁴ cells/well) were placed in the upper chamber with serum-free medium (200 µL), while the lower chambers were filled with serum-containing medium (800 µL). After the cells were incubated for 48 hours, the upper chambers were fixed with paraformaldehyde, then cleaned by PBS, then stained with violate crystal. Cells were treated with ciprofol (0, 5, 10 µM) for 48 hours. For the group treated with sorafenib, an additional 72-hour pre-treatment with sorafenib (10 nM) was required. For the group treated with EGF, an additional 72-hour pre-treatment with EGF (10 nM) was required.

Immunohistochemistry (IHC) and immunofluorescence (IF) assays

Tumour tissue sections, embedded in paraffin, underwent a process of deparaffinization and rehydration before being incubated overnight at 4 ℃ with primary antibodies against Ki-67, E-cadherin, N-cadherin, Vimentin, ERK1/2, p-ERK1/2, MEK, and p-MEK on a shaker. The next day, these sections were incubated at room temperature for 2 hours with secondary antibodies. For the IF assay, Hep3B cells were firstly fixed and then cultured with the relevant primary antibodies. Afterward, they were incubated with the corresponding fluorescent secondary antibodies. Finally, 4’,6-diamidin-2-phenylindole (DAPI) was used to stain the cell nucleus.

RNA sequencing (RNA-seq) analysis

Total RNA from Hep3B cells was extracted using TRIzol reagent (T9424, Sigma-Aldrich, St. Louis, MO, USA), and a corresponding cDNA library was established. Subsequently, sequencing was performed by a BGISEQ 500 instrument (BGI Genomics, Shenzhen, China) to generate a single-end sequencing library.

Western blotting

Total proteins were extracted from HCC cells using a protein extraction kit (KGP2100; Key Gene, Nanjing, China) and separated via 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Based on the above results, the separated proteins were further transferred to polyvinylidene difluoride (PVDF) membranes. After these membranes were blocked, they were incubated at 4 ℃ overnight with specific primary antibodies. Tris Buffered Saline with Tween 20 (TBST) was used to wash the membranes, and the corresponding secondary antibody was used to culture it for 2 hours at room temperature. The proteins were visualized with an ECL kit (Bioprimacy, Shanghai, China), and Chemiluminescent Gel Imaging System (Vilber, Paris, France). For the group treated with sorafenib, an additional 72-hour pre-treatment with sorafenib (10 nM) is required.

Statistical analysis

Statistical analyses were conducted using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA) and SPSS Statistics version 26.0 (IBM, Armonk, NY, USA). Unpaired two-tailed Student’s t-tests were applied for comparisons between two groups, while analysis of variance (ANOVA) with Tukey’s post hoc test was used for comparisons among more than two groups. Kaplan-Meier analysis was used to evaluate overall survival (OS). All experiments were repeated at least three times. Data were expressed as mean ± standard deviation (SD), and a P value of <0.05 was deemed statistically significant.

Results

Ciprofol inhibits the proliferation of HCC

Ciprofol is a novel intravenous anaesthetic in clinical use, similar to propofol in its effects but with advantages such as haemodynamic stability and the absence of injection pain. To determine the effect of propofol and ciprofol on HCC, their effects on the proliferation and viability of Hep3B and HCCLM3 cells were examined using a CCK-8 assay. These results indicate that both drugs inhibited the proliferation of HCC cells in a time- and concentration-dependent manner after 12 hours. However, ciprofol demonstrated a stronger inhibitory effect than propofol, with 5 and 10 µM ciprofol exhibiting significant antitumour activity (Figure 1A). Therefore, we used 5 and 10 µM concentration of ciprofol to further investigate its biological effects on HCC cells.

Figure 1 Effects of ciprofol on HCC cell proliferation and apoptosis in vitro. (A) The effects of propofol and ciprofol on Hep3B cells were verified using the CCK-8 assay. Hep3B cells were treated with propofol (0–20 μM) or ciprofol (0–20 μM) for 24, 48, and 72 hours. (B,C) Colony formation assays assessed the proliferation capacity of HCC cells treated with ciprofol for 14 days. The clones were fixed, stained by violate crystal. (D,E) The EdU assay evaluated the proliferation of HCC cells exposed to varying concentrations of ciprofol for 72 hours (scale bars, 50 μm). (F,G) Flow cytometry analyzed the apoptosis of HCC cells treated with ciprofol for 72 hours. *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant. CCK-8, Cell Counting Kit-8; EdU, 5-ethynl-2'-deoxyuridine; HCC, hepatocellular carcinoma; Cip, ciprofol; Pro, propofol.

Colony formation assays revealed a notable reduction in the number of HCC cell colonies with increasing concentrations of ciprofol (Figure 1B,1C). EdU assays demonstrated that ciprofol substantially reduced the proliferation of Hep3B and HCCLM3 cells in a concentration-dependent manner (Figure 1D,1E). Overall, these results provide compelling evidence that ciprofol effectively inhibits HCC cell proliferation in vitro.

Flow cytometry analysis revealed that the ciprofol-treated groups had a higher number of apoptotic cells (Figure 1F,1G). Collectively, these results strongly indicate that ciprofol inhibits HCC cell proliferation in vitro.

Ciprofol Inhibits the migration and invasion of HCC

The wound healing assay, which measures the closure of the wound area, was employed to determine the migratory ability of HCC cells and further confirm the effect of ciprofol on the migration of Hep3B and HCCLM3 cells. The results of the wound healing assay indicated that at 0 hours, the wound areas in the control and experimental groups (5 and 10 µM) were comparable. However, after 24 hours of culture, wound healing in the ciprofol-treated Hep3B and HCCLM3 cells was substantially inhibited compared with that in the control group (Figure 2A,2B).

Figure 2 Ciprofol inhibited the EMT process as well as the migration and invasion abilities of HCC cells in vitro. (A,B) HCC cell migration was assessed using wound healing assays (scale bars, 200 μm). Cells were treated with ciprofol (0, 5, 10 μM) for 24 hours. (C-E) Transwell experiments were conducted to evaluate the migration and invasion abilities of HCC cells (scale bars, 200 μm). Cells were treated with ciprofol (0, 5, 10 μM) for 48 hours. The upper chambers were fixed and then stained with violate crystal. (F,G) The expression of EMT markers in response to different concentrations of ciprofol were evaluated by western blotting. Cells were treated with ciprofol (0, 5, 10 μM) for 72 hours. (H,I) The expression of EMT markers in response to different concentrations of ciprofol was evaluated by multicolor fluorescence assays (scale bars, 10 μm). Cells were treated with ciprofol (0, 5, 10 μM) for 72 hours. *, P<0.05; **, P<0.01; ***, P<0.001. Cip, ciprofol; DAPI, 4',6-diamidino-2-phenylindole; EMT, epithelial-mesenchymal transition; HCC, hepatocellular carcinoma.

Additionally, using non-coated extracellular matrix (ECM) migration chambers, a clear trend was observed that correlated with the ciprofol concentration. With increasing concentrations of ciprofol, a decrease in the number of cells that migrated through the chamber membrane was observed. At a concentration of 10 µM, the number of migrating cells was approximately one-third of that in the control group. In invasion assays, the number of Hep3B and HCCLM3 cells penetrating the ECM membrane was markedly higher in the control group compared to those treated with ciprofol (Figure 2C-2E). Transwell assays for invasion and migration further demonstrated a significant decrease in the migratory potential of Hep3B and HCCLM3 cells after ciprofol treatment, reinforcing the role of ciprofol in HCC inhibition.

EMT is often associated with uncontrolled cell migration and invasion in various cancers, thereby enhancing the metastatic potential of malignant tumours (20). In this study, we investigated the influence of ciprofol on the protein expression levels of EMT markers (including E-cadherin, N-cadherin, Vimentin, and SNAI-1) in HCC cells by western blot analysis. Data revealed that ciprofol exposure upregulated E-cadherin expression and downregulated N-cadherin, Vimentin, and SNAI-1 expression in a concentration-dependent manner (Figure 2F,2G). Moreover, IF staining indicated that ciprofol increased the expression of E-cadherin and decreased the expression of Vimentin in Hep3B cells compared to that in the control group (Figure 2H,2I). These observations suggest that ciprofol inhibits the EMT and migration of HCC cells.

Ciprofol inhibits malignancy and metastatic potential of HCC in vivo

To assess the effects of ciprofol on tumour progression and metastasis in vivo, we established a subcutaneous HCC mouse model. Hep3B cells were subcutaneously injected into BALB/c nude mice. Once all mice developed subcutaneous tumours of approximately 50 mm³, they were treated with multiple doses of ciprofol (25 mg/kg, twice a week) (Figure 3A). Tumours developed in all the mice in both the control and treated groups (Figure 3B). However, compared to the control group, the ciprofol-treated group exhibited smaller tumour volumes at the end of weeks 1, 2, 3, and 4 (Figure 3C). Additionally, the average weight of subcutaneous tumours in the control group was 874.7 mg, whereas it was 380.9 mg in the treated group, which was significantly lower than in the control group (Figure 3D). After 28 days of treatment, subcutaneous tumours were collected, subjected to HE staining, and analysed for Ki-67 and Vimentin expression by IHC. These results indicate that ciprofol reduced the proliferation rate of Hep3B cells in vivo and attenuated EMT (Figure 3E). Furthermore, there were no significant difference was observed in the body weights of mice between the DMSO and ciprofol groups throughout the experiment (Figure S1).

Figure 3 Ciprofol suppressed HCC growth in vivo. (A) The schematic diagram illustrates the method of subcutaneous tumour inoculation and the orthotopic transplantation in nude mice with the drug administration protocol. (B) Macroscopic images of subcutaneous tumours (n=6). (C,D) Tumour volume and weight in the subcutaneous tumour models (n=6). (E) H&E staining, Ki-67, and vimentin immunohistochemistry of xenograft tumours (scale bars, 50 μm). (F) Macroscopic appearance of liver tumours (scale bars, 5 mm) (n=6). The red-dotted circle highlights the liver cancer tissue in the image. (G,H) The volume and weight of the orthotopic HCC model in nude mice (n=6). (I) Overall survival of orthotopic transplantation mice injected with ciprofol and DMSO (n=10). *, P<0.05; **, P<0.01. DMSO, dimethylsulfoxide; HCC, hepatocellular carcinoma; H&E, hematoxylin and eosin; Cip, ciprofol.

To further evaluate Hep3B cell proliferation in the liver, we also established an orthotopic liver tumour transplantation model (Figure 3A). After 28 days, the mice were euthanised and their livers were examined to assess the impact of ciprofol on liver tumour growth. The tumour volume and weight in ciprofol-treated mice were lower than those in the control group (Figure 3F-3H). Survival analysis also indicated that ciprofol improved the OS of orthotopic liver tumour-bearing mice (Figure 3I).

Additionally, to evaluate the capacity of ciprofol in HCC metastasis, a lung metastasis mouse model was constructed, and the effects of ciprofol on HCC metastatic potential were assessed using bioluminescence imaging (Figure 4A). Bioluminescence imaging revealed that ciprofol significantly inhibited the lung metastatic capacity of HCC cells (Figure 4B,4C). Finally, hematoxylin and eosin (H&E) staining of lung tissues from metastatic mice confirmed that ciprofol reduced the number of HCC metastases to the lungs compared with that in the control group (Figure 4D,4E). Survival analysis of mice with lung metastasis showed that the OS rate in the ciprofol group was significantly higher than that in the control group (Figure 4F). In summary, our results demonstrate that ciprofol effectively inhibits the malignant progression of HCC cells in vivo.

Figure 4 Ciprofol inhibited the HCC metastasis in vivo. (A) Flow chart of the mice lung metastasis model. (B,C) Bioluminescence images of lung of mice treated with ciprofol or DMSO and the relative luciferase activity (n=6). (D) H&E staining of lung tissue in the nude mice lung metastasis model. Red arrows indicate metastatic lesions of HCC in lung tissue. (E) Histogram of metastases in the lung (n=6). (F) Percent survival of the mice lung metastasis model injected with ciprofol and DMSO (n=10). *, P<0.05; ***, P<0.001. Cip, ciprofol; DMSO, dimethylsulfoxide; HCC, hepatocellular carcinoma; H&E, hematoxylin and eosin.

Ciprofol inhibits the Raf-MEK-ERK signalling pathway in HCC

To elucidate how ciprofol suppresses the malignant biological behaviour of HCC, RNA-seq was performed on Hep3B cells treated with or without ciprofol. Principal component analysis (PCA) of the sequencing data revealed a clear separation between the treated and untreated groups, indicating that ciprofol significantly altered the transcriptomic profiles of Hep3B cells (Figure 5A). Volcano plots and heat maps demonstrate the changes in gene expression caused by ciprofol treatment (Figure 5B,5C). Further Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the differentially expressed genes (DEGs) revealed that the GO terms were primarily associated with GTPase regulation, Ras protein signal transduction, and other pathways (Figure 5D). KEGG analysis highlighted the most significant changes in the MAPK pathway (Figure 5E). Additionally, based on the structural information of ciprofol predicted by the PharmMapper website, molecular docking and scoring algorithms were used to predict the potential targets of small molecules. Pathway enrichment analysis indicated that the MAPK pathway had a higher score and gene count than most other signalling pathways (Figure S2).

Figure 5 The Raf-MEK-ERK signalling pathway is the downstream target of ciprofol. (A) PCA analysis plot of the ciprofol-treated group versus the control group (n=3). (B) The volcano plot shows the differentially expressed genes between the ciprofol-treated group and the control group (n=3). (C) The heatmap shows the differentially expressed genes and their approximate quantities between the ciprofol-treated group and the control group (n=3). (D) The bar plot shows the results of the GO pathway enrichment analysis. (E) The bubble chart shows the results of the KEGG pathway enrichment analysis. The red box highlights the most significantly differentially expressed pathway. (F) The multicolor fluorescence assay showed the expression levels of Raf, MEK, ERK, and their phosphorylated forms under different concentrations of ciprofol treatment for 72 hours (scale bars, 20 μm). (G,H) Western blotting showed the protein levels of Raf, MEK, ERK, and their phosphorylated forms under different concentrations of ciprofol treatment for 72 hours. *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant. AGE, advanced glycation endproduct; BP, biological process; CC, cellular component; cip, ciprofol; con, control; DAPI, 4',6-diamidino-2-phenylindole; FC, fold change; FoxO, forkhead box O; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; MF, molecular function; MAPK, mitogen-activated protein kinase; PCA, principal component analysis; RAGE, receptor of advanced glycation endproduct; TNF, tumour necrosis factor.

The Raf-MEK-ERK signalling pathway is a classic component of the MAPK pathway and promotes tumour cell proliferation, invasion, and metastasis in HCC. We further investigated the impact of ciprofol treatment on the Raf-MEK-ERK signalling axis in HCC cells. IF experiments showed that in Hep3B cells, while the total levels of Raf, MEK, and ERK remained unchanged with increasing concentrations of ciprofol, the levels of phosphorylated and activated forms of Raf, MEK, and ERK gradually decreased (Figure 5F). This was corroborated by the western blot analysis (Figure 5G,5H). In vivo validation was also performed. Multiplexed immunohistochemical (mIHC) assays of subcutaneous tumours in nude mice showed a significant decrease in the expression of key Raf-MEK-ERK signalling molecules, including p-MEK and p-ERK following ciprofol treatment (Figure S3). Collectively, these results demonstrate that ciprofol inhibits the malignant progression of liver cancer by suppressing the activation of the Raf-MEK-ERK signalling pathway.

Ciprofol’s suppression of HCC is blocked by inhibitors of the MAPK/ERK pathway

To further determine whether inhibition of the MAPK/ERK signalling pathway is essential for the antitumour effects of ciprofol, we utilised the MAPK/ERK pathway inhibitor sorafenib. Sorafenib has been reported to effectively blocked the MAPK/ERK signalling pathway and inhibited HCC progression. To assess the role of MAPK/ERK pathway inhibition, HCC cells were pretreated with 10 nM sorafenib prior to ciprofol treatment. Notably, Western blot analysis showed that sorafenib pretreatment significantly prevented ciprofol-induced reduction in ERK and MEK phosphorylation without affecting the total levels of ERK and MEK in Hep3B cells (Figure 6A,6B).

Figure 6 The inhibition of Raf-MEK-ERK signalling pathway is the core of the ciprofol anti-tumour effect. (A,B) The total and phosphorylated protein levels of Raf, MEK and ERK in HCC cells by western blotting. Cells were treated with ciprofol (10 μM), sorafenib (10 nM) or their combination for 72 hours. (C,D) Colony formation assay was used to assess the proliferation capacity of HCC cells after with different treatments. Cells were treated with ciprofol (10 μM), sorafenib (10 nM) or their combination for 14 days. The clones were fixed, stained by violate crystal. (E,F) The migration and invasion abilities of HCC cells in each treatment group were assessed using the transwell assay (scale bars, 200 μm). Cells were treated with ciprofol (10 μM), sorafenib (10 nM) or their combination for 72 hours. The upper chambers were fixed and then stained with violate crystal. (G,H) Wound healing assay showed the migration ability of HCC cells (scale bars, 200 μm). Cells were treated with ciprofol (10 μM), sorafenib (10 nM) or their combination for 72 hours. (I) Macroscopic appearance of liver tumours (scale bars, 5 mm) (n=6). The red-dotted circle highlights the liver cancer tissue in the image. (J,K) The volume and weight of the orthotopic HCC model in nude mice (n=6). *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant. Cip, ciprofol; DMSO, dimethylsulfoxide; HCC, hepatocellular carcinoma.

Moreover, recovery experiments demonstrated that ciprofol reduced HCC cell proliferation, but this effect was nullified by sorafenib pretreatment (Figure 6C,6D). Additionally, Transwell and scratch assays indicated that sorafenib reversed the reduction in migration and invasion capabilities induced by ciprofol treatment (Figure 6E-6H). In vivo experiments using a mouse orthotopic cancer model showed that ciprofol treatment reduced tumour volume and weight; however, this effect was blocked by sorafenib (30 mg/kg) (Figure 6I-6K). These findings were further validated using HCCLM3 cells (Figure S4).

Activation of the Raf-MEK-ERK pathway reverses ciprofol’s antitumour effects

To further elucidate the role of ciprofol in the Raf-MEK-ERK signalling pathway, we used EGF to activate the MAPK/ERK pathway. The inhibitory effects of ciprofol on HCC cell proliferation, migration, and invasion were significantly reversed by EGF exposure (10 nM) (Figure S5). In summary, these results suggest that the activation of the Raf-MEK-ERK pathway mitigates ciprofol-induced suppression of the malignant progression of HCC.

In conclusion, these data demonstrate that sorafenib counteracts ciprofol-induced suppression of HCC cell malignancy, suggesting that ciprofol primarily exerts its antitumour effects by inhibiting the MAPK/ERK signalling pathway (Figure 7).

Figure 7 Schematic diagram of ciprofol inhibiting the HCC progression. Ciprofol targets Raf protein and inhibits its kinase activity, thereby blocking the Raf-MEK-ERK signalling pathway and consequently suppressing the malignant biological progression of HCC cells. GABA, γ-aminobutyric acid; HCC, hepatocellular carcinoma; RTK, receptor tyrosine kinase.

Discussion

Due to immune evasion by cancer cells and immune suppression induced by the tumour microenvironment, patients with liver cancer often experience compromised immune function. Although surgical resection remains the most common treatment for HCC, surgical procedures, anaesthesia, and perioperative analgesia can further disrupt a patient’s immune function, potentially contributing to tumour progression and metastasis. Extensive research has demonstrated that various anaesthetic and analgesic agents are broadly associated with tumour cell differentiation, proliferation, and apoptosis. For example, inhalational anaesthetics can enhance the proliferation, migration, invasion, and angiogenesis of various types of cancer cells. Isoflurane can induce elevated levels of hypoxia-inducible factor-1α (HIF-1α), increasing proliferation and migration abilities in prostate cancer, but this effect can be blocked by propofol (21). Isoflurane’s carcinogenic effects are also linked to the suppression of natural killer (NK) cell cytotoxicity and the induction of T lymphocyte apoptosis, both of which play crucial roles in immune surveillance and anti-metastatic immunity (22). In contrast, intravenous anaesthetics such as propofol (23), etomidate (24), cisatracurium (25), and lidocaine (5,6,26) have been shown to inhibit malignant progression in various cancer cells. The proliferative and apoptotic effects of opioid analgesics may depend on their concentration or duration of exposure, with low or single doses potentially promoting immune suppression and tumour growth (27). Therefore, the selection of anaesthetic agents with antitumour effects is critical for improving the prognosis of cancer patients.

Ciprofol, a novel intravenous anaesthetic independently developed in China, is a structural analogue of propofol and a classic 2,6-disubstituted phenolic derivative. Its mechanism of action involves acting as a gamma-aminobutyric acid (GABA) receptor agonist, enhancing GABA-mediated chloride influx for sedation or anaesthesia. In contrast, propofol has a range of adverse effects (e.g., hypotension, metabolic acidosis, cardiac arrest, and injection pain) (19). Ciprofol is rapidly metabolised with lower accumulation, with fewer adverse reactions, more stable haemodynamics, less injection pain, and reduced incidence of nausea and vomiting, while its sedative effect is comparable to that of propofol (28). An animal study has reported that ciprofol can inhibit increases in myocardial injury enzymes, reduce pro-inflammatory cytokine expression, and diminish myocardial apoptosis (29). Additionally, ciprofol has been shown to improve intraoperative mean arterial pressure and cerebral oxygen saturation, and reduce the incidence of postoperative delirium (30). Animal experiments also confirmed that Nrf2 mediates the protective effect of ciprofol against ischaemia-reperfusion injury by reducing oxidative stress (31).

Numerous studies have reported on the effects of propofol on tumour cells. Notably, propofol inhibits bladder cancer cell proliferation and migration by suppressing the Hedgehog Pathway (13). Additionally, it suppresses pancreatic cancer through the upregulation of miR-328 and downregulation of ADAM8 (14), and inhibits colon cancer metastasis by activating WIF-1 and suppressing the Wnt signalling pathway (32). Extensive research has been conducted on propofol’s effects on HCC, revealing that it can inhibit HCC proliferation, invasion, and migration by downregulating miR-374a and suppressing the Wnt/β-catenin and PI3K/AKT pathways, as well as by inhibiting HOXA11 to suppress malignant progression (15). However, no studies have addressed the antitumour effects of ciprofol in HCC. Therefore, we determined the inhibitory effects of ciprofol on HCC cells in vitro and established three kinds of HCC animal tumour models to confirm the significant suppressive effects of ciprofol on the malignant biological behaviour of HCC cells in vivo.

We performed RNA-seq analysis to explore the underlying mechanism of ciprofol inhibition of HCC progression. These results indicate that the MAPK signalling pathway is crucial for the antitumour effects of ciprofol on HCC. The classical MAPK cascade includes four pathways: ERK, JNK, P38, and ERK5. Research suggests that the JNK and P38 MAPK pathways are primarily associated with cellular stress and apoptosis, whereas the ERK MAPK pathway, the most widely studied, is closely linked to cell proliferation and differentiation (33,34).

ERK is a serine/threonine kinase that is crucial for mitogen signal transmission. Normally located in the cytoplasm, ERK translocate to the nucleus upon activation, where it regulates transcription factors and gene expression (35). Elevated ERK expression has been observed in various cancers including ovarian, colon, breast, lung, and liver cancers. The MAPK cascade is a central signalling pathway that regulates essential cellular processes including proliferation, differentiation, and stress responses. Among these, the ERK cascade is highly regulated and crucial for cellular processes, such as proliferation and differentiation. Overactivation of upstream proteins and kinases within the ERK pathway has been linked to various diseases, including cancer, inflammation (36,37), developmental disorders, and neurological conditions (38).

The ERK/MAPK signalling pathway is closely associated with tumourigenesis and tumour progression. This pathway regulates essential cellular functions, including cell proliferation, differentiation, cell cycle control, apoptosis, and tissue formation-I, and is also implicated in cancer development (34,39). Activation of the ERK/MAPK pathway promotes cell proliferation and exerts anti-apoptotic effects. Inhibition of this signalling pathway can suppress tumour cell proliferation and resistance to apoptosis, thereby promoting differentiation (40,41).

Consequently, we focused on the MAPK/ERK pathway and validated our findings by IF and western blotting. We found that ciprofol inhibited ERK phosphorylation without altering the total protein levels of ERK. To further investigate the mechanism underlying the suppression of ERK phosphorylation, we examined its upstream components and found that phosphorylation of MEK and Raf was similarly inhibited. Ciprofol inhibits the phosphorylation activation of Raf, thereby suppressing the phosphorylation of MEK and ERK, leading to a reduction in the expression of the downstream target genes of ERK and, consequently, the suppression of the malignant phenotype of HCC.

Conclusions

In summary, our study demonstrates that ciprofol inhibits HCC progression by suppressing the Raf-MEK-ERK signalling pathway. These findings elucidate the pharmacological effects of ciprofol on HCC cells and provide new clinical guidance for perioperative intravenous anaesthetic drug use in patients with HCC.

Acknowledgments

We want to thank the technical support from Hepatobiliary Center, The First Affiliated Hospital of Nanjing Medical University, Key Laboratory of Liver Transplantation, Chinese Academy of Medical Sciences, NHC Key Laboratory of Living Donor Liver Transplantation, for helping with technical assistance, providing experimental conditions, and offering valuable guidance throughout our research process in this study.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://tgh.amegroups.com/article/view/10.21037/tgh-24-115/rc

Data Sharing Statement: Available at https://tgh.amegroups.com/article/view/10.21037/tgh-24-115/dss

Peer Review File: Available at https://tgh.amegroups.com/article/view/10.21037/tgh-24-115/prf

Funding: This study was supported in part by grants from the Major Program of the National Natural Science Foundation of China (Nos. 31930020, 82103440), and Jiangsu Provincial Health Commission Medical Research Project (No. M2021059).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tgh.amegroups.com/article/view/10.21037/tgh-24-115/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Animal experiments were performed under a project license (No. 2406081) approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University, in compliance with national guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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