The Chemotherapeautic Sensitization Effect Of PPIs And Metformin On Cholangiocarcinoma Cells

Background and Objects:Cholangiocarcinoma is a rare but fatal malignant tumors with pore early diagnostic rates and more than 70%of cases are on inoperable stage when the diagnosis is made.Chemotherapy is an optional treatment for cholangiocarcinoma.However,a poor efficiency response to chemotherapy was found according to several studies.Combination chemotherapeutic agents with high efficient and low toxic chemosensitizers may be one of the best chances to improve the effects and the sensitivity of chemotherapy.Recent studies indicate the phenomenon of acidity microenvironment of tumors.The pH gradient of tumors may effect the distribution of chemotherapeutic agents and contribute to the drug resistance.Vacuolar type ATPase(V-ATPase)plays a critical role in maintaining the pH gradient with high expression in tumors.Proton pump inhibitors(PPIs)was found to effectively inhibit the expression and activation of V-ATPase and exchange the pH gradient,leading to reversing the drug resistance.In addition,PPIs may exert anti-tumor effects and increase the chemosensitivity via the phosphoinositide-3 kinase(PI3K)/protein kinase A(Akt)/mammalian target of rapamycin(mTOR)pathway.One of the purposes of this study is to ditermine the influence of PPIs on proliferation and apoptosis of cholangiocarcinoma cells and its possible mechanism.Recently,a case-control report in the U.S.confirmed diabetes as independent risk factor for intrahepatic cholangiocarcinoma(ICC)and a 60%reduction in ICC risk in diabetic patients.Metformin is an oral hypoglycemic agent of the biguanide family which is one of the first choices of type 2 diabetes.Several studies showed that metformin may be associated with a decreased risk of multiple tumors.More and more experiments in vitro showed the anti-proliferation and apoptosis induction effects of metformin and metformin may be a chemosensitizer in future tumor treatment.Metformin treatment resulted in enhanced adenosine monophosphate activated protein kinase(AMPK)phosphorylation and reduced mTOR phosphorylation.The part two of this study is to explore the influence of metformin on proliferation and apoptosis of cholangiocarcinoma cells via the AMPK/mTOR pathway.The mammalian target of rapamycin(mTOR)is a key signal molecule associated with cell cycle,proliferation,survival and metabolism.The mTOR is a common downstream molecule of PI3K/Akt and AMPK pathways.Hypoxia inducible factor la(HIF-la)which is located in the downstream of mTOR palys an important role in the response to hypoxia,angiogenesis and drug resistance of tumor cells.Based on the first two parts of this study,our third part is focused on whether PPIs and metformin act synergistically in cholangiocarcinoma cells via the dural fuction of PI3K/Akt/mTOR/HIF-1? and AMPK/mTOR/HIF-1? pathways.PART1 Anti-proliferation and apoptosis-induction effect of pantoprazole(PPZ)on HCCC9810 cells and its possible mechanismObjects:To explore the effect of proliferation and apoptosis of PPZ on HCCC9810 cells and its possible mechanismMethods:Various concentrations of PPZ were given to HCCC9810 cells.The cell counting kit-8(CCK8)was used to evaluate proliferation inhibition rate.Annexin V-FITC-PI apoptosis detection kit was used to analyze the apoptosis rate.The expression of V-ATP,PI3K,Akt,mTOR,HIF-1?,MRP1 proteins after the treatment of PPZ were determined by western blot.The V-ATP siRNA was used to inhibit the expression of V-ATP in the HCCC9810 cells.The above proteins were measured after the interference.Results:1.PPZ had the proliferation inhibition effect on HCCC9810 cells in a dose and time-dependent manner.2.PPZ had the apoptosis-induction effect on HCCC9810 cells in a dose-dependent manner.3.PPZ could inhibit the expression of V-ATP and influence a reduce in proteins of PI3K,Akt,mTOR,HIF-1? and MRP1.4.V-ATP siRNA could inhibit the expression of V-ATP and lead to the same changes of the proteins above.Conclution:PPZ inhibits the expression of V-ATP and have anti-proliferation and apoptosis-inducing effects which are related to the inhibition of the PI3K/Akt/mTOR/HIF-1? pathway in HCCC9810 cells.PART2 Anti-proliferation and apoptosis-induction effect of metformin on HCCC9810 cells and its possible mechanismObjects:To explore the effect of proliferation and apoptosis of metformin on HCCC9810 cells and its possible mechanismMethods:Various concentrations of metformin were given to HCCC9810 cells.The cell counting kit-8(CCK8)was used to evaluate proliferation inhibition rate.Annexin V-FITC-PI apoptosis detection kit was used to analyze the apoptosis rate.The expression of AMPK,mTOR,HIF-1?,MRP1 proteins after the treatment of metformin were determined by western blot.Results:5.Metformin had the proliferation inhibition effect on HCCC9810 cells in a dose and time-dependent manner.6.Metformin had the apoptosis-induction effect on HCCC9810 cells in a dose-dependent manner.7.Metformin could influence a boost in proteins of AMPK and a reduce in proteins of mTOR,HIF-1?and MRP1.Conclution:Metformin have anti-proliferation and apoptosis-inducing effects which are related to the inhibition of the AMPK/mTOR/HIF-1? pathway in HCCC9810 cells.Part3 The chemotherapeautic sensitization effect of PPZ and metformin on HCCC9810 cells and its possible mechanismObjects:To explore the PPZ and metformin's sensitization effect to cisplatin(DDP)on HCCC9810 cells and its possible mechanismMethods:The experiment is divided into five groups:1)DDP;2)PPZ+DDP;3)metformin+DDP;4)PPZ+metformin;5)PPZ+metformin+DDP(the triad for short)The PPZ concentration is lOug/ml;the metformin concentration is 5mM;the DDP concentration is 0,1,5,10,20,50,100ug/ml respectively.The cell counting kit-8(CCK8)was used to evaluate proliferation inhibition rate.Annexin V-FITC-PI apoptosis detection kit was used to analyze the apoptosis rate.The expression of mTOR and HIF-1? proteins after the treatment of metformin and PPZ were determined by western blot.The expression of mTOR,HIF-1?,MRP1 proteins after the treatment of rapamycin were determined by western blot.Results:9.Whether DDP combined with PPZ or metformin all showed higher proliferation inhibition rates than a drug alow and the same to the PPZ combined with metformin;the triad showed the highest proliferation inhibition rates;DDP,PPZ and metformin act synergistically.10.A reduce in proteins of mTOR,HIF-1? and MRPlwere observed after the drug intervetion.Conclution:PPZ and metformin act synergistically and have a sensitization effect to cisplatinin in cholangiocarcinoma cells probably via the dural fuction of PI3K/Akt/mTOR/HIF-la and AMPK/mTOR/HIF-1? pathways.