| Background:Cholangiocarcinoma(CCA),comprising the malignant transformation of epithelial cells in bile ducts,is the second most common primary hepatic cancer worldwide.It is characterized by early invasion,poor prognosis,and is a highly lethal cancer,and in recent years,the incidence and mortality of cholangiocarcinoma have increased year by year.The current treatment is mainly surgery,but there is still a high recurrence rate,and it is resistant to chemotherapy drugs such as cisplatin.Therefore,there is an urgent need to find effective treatments and drugs to improve the survival time and prognosis of patients with cholangiocarcinoma.Pterostilbene(Pte),as a natural compound found in plant extracts,has anti-inflammatory,anti-tumor,antioxidation and other pharmacological effects.Previous experimental studies have shown that pterostilbene has anti-tumor activity,including lung cancer,breast cancer,prostate cancer,and colon cancer.However,its effect on cholangiocarcinoma and its specific mechanism are not clear.In this study,we found that pterostilbene has the effect of inhibiting the proliferation of cholangiocarcinoma cells by inducing S-phase arrest,triggering non-caspase-dependent cell vacuolar death and enhancing Beclin-1/ATG5-mediated autophagy to play the anti-cholangiocarcinoma role.In addition,by constructing a tumor-bearing mouse model of cholangiocarcinoma,the safety and effectiveness of pterostilbene in the treatment of cholangiocarcinoma in vivo were verified,and provided new experimental basis and theoretical basis for the treatment of cholangiocarcinoma.Objective:To explore whether pterostilbene has the effect of inhibiting the proliferation of cholangiocarcinoma cells and its possible mechanism,as well as the effectiveness and biosecurity of pterostilbene in the treatment of cholangiocarcinoma in vivo,providing new directions for the development and treatment of cholangiocarcinoma drugs.Methods:1.The study of anti-cholangiocarcinoma activity and mechanism of pterostilbene in vitro.1)The effect of pterostilbene on cholangiocarcinoma.After treatment of cholangiocarcinoma cells with different concentrations of pterostilbene for 24,48,72 h,CCK was used to detect the cell viability and calculate the IC50 of the drug.Cell counting experiments and cloning experiments were used to test the effect of pterostilbene on the proliferation of cholangiocarcinoma cells.2)Flow cytometry and Western Blot were used to detect the effects of pterostilbene on the cell cycle and cell cycle-related proteins expression,then quantify cycle-related proteins expression.3)The morphological changes of the cells were observed by light microscope and electron microscope after the treatment of pterostilbene.Flow cytometry was used to detect the effects of drugs on the apoptosis rate of cholangiocarcinoma.Cell viability and cell morphology were observed after the combination of apoptosis inhibitor,ZVAD and pterostilbene treatment on cholangiocarcinoma cells.4)The study of autophagy induced by pterostilbene on the cholangiocarcinoma.The expression of Beclin-1,ATG5,LC3 I / II,and p62,which are autophagy-related proteins,was detected by Western blot;the effects of drugs on cell morphology and the formation of autophagosomes were observed by electron microscopy;Confocal microscopy was used to observe the amount of LC3 puncta accumulation in the cells.Cell viability,cell morphology,and the amount of LC3 puncta accumulation were observed after intervening the cells with pterostilbene and autophagy inhibitor 3-MA.2.The bioavailability and safety of pterostilbene in the treatment of cholangiocarcinoma-bearing mice in vivo.To construct HCCC-9810 cholangiocarcinoma subcutaneous tumor-bearing mouse model,intraperitoneal injection of pterostilbene,to measure tumor growth rate,final volume and weight of control group,low-dose pterostilbene group,high-dose pterostilbene group to study inhibition of pterostilbene on the growth of cancer,to explore the effectiveness of drugs;to measure the weight change of mice and the pathological sections of organs to detect the toxicity of pterostilbene,confirming its safety.Results:1.The study of anti-cholangiocarcinoma activity and mechanism of pterostilbene in vitro.1)Pterostilbene has the effect of inhibiting the activity,proliferation and cloning capacity of cholangiocarcinoma cells.This anti-cholangiocarcinoma capacity is inversely proportional to the concentration and time of drug action.2)Pterostilbene induces the expression of S-phase related proteins Cyclin A2,CyclinE1,and P53 to cause cholangiocarcinoma cells S-phase arrest.3)Cytoplasmic vacuoles were observed in pterostilbene-treated cholangiocarcinoma cells under light microscope and electron microscope and this phenomenon shown in a dose-dependent manner.However,it did not show typical apoptosis characteristics such as cytoplasmic contraction?nuclear fragmentation and apoptotic body.The results of apoptosis flow cytometry further proved pterostilbene does not inhibit cholangiocarcinoma through the apoptotic-dependent pathway.Combining pterostilbene with the inhibitor of apoptosis Z-VAD to deal with cells does not reverse pterostilbene effect on the morphology and viability of cholangiocarcinoma cells.4)It can be observed by electron microscopy that after pterostilbene treatment,the cytoplasmic autophagosomes increase in cholangiocarcinoma cells.Pterostilbene could increase the expression of Beclin-1,ATG5,and LC3 II in cholangiocarcinoma cells,while the expression of LC3 I and p62 decreased in a dose-dependent manner.Confocal results show that pterostilbene can induce cholangiocarcinoma cells to produce a large number internal LC3 particles with increased fluorescence intensity.The autophagy inhibitor 3-MA can reverse the effect of pterostilbene on the viability of cholangiocarcinoma cells,cellular vacuolation,and LC3 particle formation.These data indicate that pterostilbene can stimulate the autophagy signaling in human cholangiocarcinoma cells,and the cytoplasmic vacuole formation is associated with autophagy.2.The bioavailability and safety of pterostilbene in the treatment of cholangiocarcinoma-bearing mice in vivo.Pterostilbene significantly inhibited the tumor growth rate of cholangiocarcinoma tumor-bearing mice.The size and weight of the xenografts tumors were smaller than that of the control group.There was no difference in the body weight and the pathological sections of heart,liver,spleen,lung and kidney between the control group and the tumor-bearing mice.It was confirmed that at an effective concentration,pterostilbene can effectively inhibit the growth of bile duct cancer without any toxic reaction,providing effective evidence for the biosafety and effectiveness of pterostilbene.Conclusion:1.Pterostilbene can significantly inhibit the viability and proliferation ability of cholangiocarcinoma cells in a time-and dose-dependent manner.2.Pterostilbene can enhance the expression of CyclinA2,CyclinE1,and p53 proteins,thereby inducing S-phase arrest in cholangiocarcinoma cells.3.Pterostilbene mainly exerts its inhibitory effect by activating the autophagy flow of cholangiocarcinoma cells rather than by inducing apoptosis,and the enhanced autophagy flow is positively related to the phenomenon of cytoplasmic vacuole induced by pterostilbene in cholangiocarcinoma.4.Pterostilbene can effectively inhibit the tumor growth of cholangiocarcinoma in vivo without any adverse reactions,so it has the potential to be a candidate adjuvant drug for the treatment of cholangiocarcinoma. |
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