A Study On Reducing The Level Of Autophagy Potentiated Diisopropylamine Dichloroacetate Anti-cholangiocarcinoma Effect

Background and objectiveCholangiocarcinoma(CCA)is one of the most highly malignant tumors in alimentary tract.CCA has triggered researchers' attention because of its increase in morbidity,difficulty in early diagnosis,low resection rate and poor prognosis.To explore new therapeutic methods is of great significance for the further improvement of the curative effect of bile duct cancer.Warburg effect was observed in most cancers,which may be the basis of the tumor resistance to chemotherapy and radiotherapy.Domestic and foreign researches show that inhibition of some key enzymes in glycolysis,like hexokinase,6-phosphofructokinase 1 and pyruvate kinase,could significantly destroy the energy metabolism of tumor cells.Pyruvate dehydrogenase kinase(PDK)is a negative regulator of pyruvate dehydrogenase(PDH),one member of pyruvate dehydrogenase complex(PDC)in mitochondria.There are four kinds of isozymes in PDK:PDK1 is mainly expressed in the heart,while PDK2 in mostly tissues,PDK3 in the testis and PDK4 in the heart and skeletal muscle.It has been shown that PDKs are overexpreesed in many cancers containing head and neck cancer,gastric cancer,nonsmall-cell lung cancer,glioma,colon cancer and PDKs have been proposed to contribute to cancer development and progression.However,the role of PDKs in cholangiocarcinoma has not yet been understood.Dichloroaceticacid(DCA)represents a PDK inhibitor,currently approved for the treatment of congenital lactic acidosis.In 2007,the Michelakis team at the University of Alberta in Canada found that DCA had anti-cancer activity,however,one of the factors limiting its clinical use in a continuous oral regimen is a dose-related,reversible neurotoxicity,including peripheral neuropathy and encephalopathy.Diisopropylamine Dichloroacetate(DPDA),a novel PDK inhibitor,structurally similar to DCA,mainly is used for the treatment of fatty liver,intrahepatic cholestasis and liver injury.Compared with DCA,DPDA is more safe,and almost no peripheral neurotoxicity and other adverse reactions were reported.The purpose of this study was to investigate the effects of DPDA on the proliferation and autophagy in cholangiocarcinoma cells,and to investigate whether autophagy inhibitors and siRNA ATG5 small interfering could enhance the anti-tumor effect of DPDA.MethodsCell Counting Kit-8(CCK-8)was used to assess cell viability after the treatment with PDK inhibitor DPDA in different cell lines(RBE and QBC-939).After the treatment with DPDA,Western Blotting was used to detect autophagy maker LC3;and transmission electron microscopy was used to monitor the formation of autophagosomes and autophagolysosomes.CCK-8 was used to determine the combination effect of DPDA and autophagy inhibitors chloroquine in CCA cells.Using small interfering RNA inhibits ATG 5 expression in CCA cells followed by DPDA;CCK-8 was used to determine cell proliferation,apoptosis and cell cycle detection kit for detection of apoptosis and cell cycle arrest.ResultsCCK-8 assay showed that inhibition of PDK by DPDA time-dependently and dose-dependently inhibited the proliferation of three human cholangiocarcinoma cell lines with an IC50 from 18.2 mM to 33 mM at 48 h.DPDA also significantly induced protective-autophagy in CCA cell lines.SiRNA ATG5 combined with DPDA could significantly inhibit CCA cell proliferation through promoting cell apoptosis and cycle arrest.ConclusionsThe PDK inhibitor DPDA inhibits CCA cell lines proliferation both in time-dependent and dose-dependent way.Furthermore,DPDA induces a protective autophagy in CCA cells.Autophagy inhibitors bafilomycin A1 and chloroquine enhance the sensitivity of CCA cells to DPDA.Moreover,inhibiting ATG5 expression by siRNA in CCA cells followed by DPDA could significantly potentiate cell proliferation.