Effect And Mechanism Of Curcumin Analogue WZ26 On Cholangiocarcinoma

Objective: The malignant degree of cholangiocarcinoma is very high.Surgery is one of the effective treatments for cholangiocarcinoma,but the 5-year survival rate is not high.Cholangiocarcinoma accounts for about 3% of gastrointestinal malignancies,and the global incidence rate is increasing year by year.The average age of cholangiocarcinoma is about 50 years old,the incidence in men is about 1.5 times than the female.Treatment of cholangiocarcinoma should be based on surgical resection of the comprehensive treatment program,but only less than 1/3 of patients have surgical resection.Chemotherapy effect of cholangiocarcinoma is poor,lacks of relevant research results.It has been reported in the treatment of locally advanced or metastatic unresectable cholangiocarcinoma with gemcitabine and cisplatin.The treatment of cholangiocarcinoma is not yet satisfactory,so the need for more adjuvant therapy combines with the results of clinical trials publication and the new chemotherapy drugs and molecular targeted drug development increased,thereby improving the prognosis of patients with cholangiocarcinoma.Curcumin is a natural component extracts from turmeric,has a variety of biological activities,including antioxidant and anti-tumor effcts,but its chemical instability and low bioavailability has limited its clinical application.In the past few years,our research group has focused on the modification and transformation of curcumin,and synthesized a series of chemically stable monocarbonyl curcuminoids.In vitro screening of the pharmacological activity of cancer cell lines,we found that monocarbonyl curcumin analogue WZ26 showed good anti-cholangiocarcinoma activities.Therefore,this paper focuses on the role of WZ26 in vivo and in vitro anti-cholangiocarcinoma and the mechanism of action.Method:1?Antitumor activity of curcumin analogue WZ26 in vitro and its mechanismIn this study,we have studied the antitumor activity of compound WZ26 on cholangiocarcinoma cells by MTT assay.The effects of compound WZ26 on the cell cycle and apoptosis of cholangiocarcinoma cells are studied by flow cytometry and MTT assay.The changes of pro-caspase3,CDC and cyclin B1 are detected by Western-blot and apoptosis.The effects of WZ26 on cell cycle and apoptosis of cholangiocarcinoma are correlated with ROS by adding ROS inhibitor NAC to Western-blot,flow and electron microscopy.We have also explored the mechanism of WZ26 anti-cholangiocarcinoma cells by Western-blot,scratches and migration.2?Antitumor activity of curcumin analogue WZ26 in vivo and its mechanismSubcutaneous neovascularization of BALB/c nude mice is induced by subcutaneous inoculation of cholangiocarcinoma cells.The pharmacological efficacy of compound WZ26 is analyzed by Western-blot,immunohistochemistry and immuno-fluorescence.Result:Part ? : In vitro antitumor activity of curcumin analogue WZ26 and its mechanism1.WZ26 on the growth inhibition of cholangiocarcinoma cells(RBE,QBC-939,HUCCA)is lower than Curcumin,WZ26 shows better inhibitory activity on cholangiocarcinoma cells' growth.2.WZ26 of different doses(5,10,20?M),promotes cholangiocarcinoma cells'(RBE,QBC-939,HUCCA)apoptosis dose-dependently and induces cholangiocarcinoma G2 / M cycle block.3.WZ26 increases intracellular reactive oxygen species(ROS)to induce apoptosis and cycle changes,which can be reversed by ROS inhibitor NAC.4.JC-1 immunofluorescence can be seen after the compound WZ26 treats by the fluorescence turns from red to green,indicating the mitochondrial membrane's potential failure.Morphological changes of mitochondria are observed by electron microscopy.It is reported that the destruction of mitochondrial mitral plaques caused by WZ26 results in mitochondrial swelling,indicating that the mitochondrial membrane has destroyed5.Use immunofluorescence to detect mitochondrial cytochrome C release to the cytoplasm.The results show that red fluorescence diffuses out of green fluorescence in cells after treating with WZ26(15 ?M)for 24 h,indicating that cytochrome C is released from mitochondria.The activity of caspase9 is also increased after treated with WZ26(15?M)for 24h6.The results of Western blotting show that the activation of STAT3 pathway is inhibited by WZ26 after QBC-939 cells in time and dose-dependent manner.7.WZ26 can inhibit the migration,infiltration and inhibition of TNF-?-induced epithelial-mesenchymal transition of QBC-939 cells which is detected by scratching,Transwell,PCR and Western blotting.8.The results of Western blotting show that WZ26 could inhibit the activation of STAT3 pathway induced by TNF-? and LPS.Part ?:Study on Antitumor Activity and Mechanism of Curcumin Analogue WZ26 in vivoSubcutaneous inoculation of cholangiocarcinoma cells induce BALB/c nude mice subcutaneous tumor model.Immunoprecipitation,immunohistochemistry and immunofluorescence are used to analyze the antitumor pharmacological efficacy.The study found that WZ26 can significantly inhibit the growth of subcutaneous tumor in mice,increase the accumulation of reactive oxygen species in tumor tissue,induce mitochondrial damage and apoptosis.1.Body weight and tumor data indicate that the administration group and control group have no significant difference in body weight,indicating that WZ26 is a basically non-toxic drug in nude mice.However,it can be clearly seen from the tumor volume map that the tumor size decreases after administration of WZ26 in nude mice,indicating that WZ26 can inhibit the proliferation of cholangiocarcinoma cells.2.Western blotting results can be seen in the tumor tissue when pro-apoptotic protein Cleaved-caspase3 expression increases,indicating that WZ26 in vivo can also promote the apoptosis of cholangiocarcinoma cells.3.The results of immunohistochemistry show that proliferation-related protein Ki67 in the administration group has decreased,indicating that WZ26 inhibits nude mice proliferation of cholangiocarcinoma cells in vivo.Conclusion:In vivo and in vitro studies,WZ26 on the one hand increases the level of oxidative stress in cholangiocarcinoma cells and then activates the mitochondrial apoptotic pathway which induces apoptosis,blocks cell cycle,on the other hand inhibits STAT3 phosphorylation,Cholangiocarcinoma cell invasion,migration and interstitial transformation(EMT)to play a role in anti-cholangiocarcinoma.WZ26 has the potential to be a candidate for the treatment of prostate cancer.At the same time,our study also shows that targeting ROS levels and inhibiting the expression of inflammatory factors may be an important strategy for the treatment of cholangiocarcinoma,but currently,the target of this compound and in-depth anticholangiocarcinoma mechanism are not very clear,which need to be further explored.