The Expression Of Pyruvate Dehydrogenase Kinase In Cholangiocarcinoma And The Potential Therapeutic Effects Of Its Inhibitor Diisopropylamine Dichloroacetate On Cholangiocarcinoma

Backgroud and ObjectCholangiocellular carcinoma(CCA)is the most frequent malignant neoplasm originating from the epithelium of the biliary duct system.In the past decades,the incidence of cholangiocarcinoma has been shown to increase.The overall prognosis has remained poor despite surgery combined with chemotherapy and radiotherapy.Thus,there is an urgent need to develop novel effective therapeutics against this neoplasm,which will provide new insights into improving these patientsPyruvate dehydrogenase kinase(PDHK)acts as a negative regulator of mitochondrial pyruvate dehydrogenase complex(PDC)and play an crucial role in the regulation of oxidative glycolysis.There are four isoforms of PDHK(1,2,3,4).A lot of evidence have showed that PDHK isoforms' expression is related with malignant phenotype of cancers and drug resistance induced by hypoxia.Therefore,PDHK may be a new and useful target for improving chemotherapy and drug resistanceDichloroacetate(DCA)is a PDHK inhibitor,which activates pyruvate dehydrogenase complex(PDC)by inhibiting PDHK to restore the resistance to anticancer drug and enhance the sensitivity of tumor cells to chemotherapeutics.However,DCA also has symptomatic adverse reaction of peripheral neuropathy and liver injury.Therefore,it is urgent to explore a safer and more potent drug that could sensitize cancer cells to treat cancers.Diisopropylamine dichloroacetate(DADA)was originally obtained from apricot nuclei and called pangamic acid or“vitamin B15",which is an analogue to DCA.Previous studies have demonstrated that DADA is a more effective anti-cancer drug than DCA in inhibiting cell proliferation and inducing apoptosis in malignant cells.A recent study has showed that DADA cansuppress proliferation of esophageal squamous cell carcinoma(ESCC)and enhance radiosensitization in ESCC by increasing mitochondria-derived reactive oxygen species levels.To date,the role of PDHK1-4 isoforms and the antitumor activity of DADA are not known in CCA.In the present study,PDK 1-4 isoforms' expression in CCA was assessed and its association with the prognosis of CCA patients was evaluated.Also,in vitro studies were performed to assess the effect of DADA treatment in metabolism,proliferation and apoptosis of CCA cell lines.Furthermore,whether low-dose DADA can enhance the anticancer effect of cisplatin will be investigated in vivo and in vitro studies.The current study will provide the evidence for the further clinical trials.Methods1.Immunohistochemical assays were performed to analyze PDHK1-4 isoforms in CCA and adjacent tissues.We next examined the relationship between the PDHK1-4 isoforms expression in tumor tissues and various clinicopathological characteristics.Survival curves were calculated using the Kaplan-Meier method and compared using the log-rank test to investigate the level of PDHK1-4 isoforms expression with the prognosis of CCA patients.2.Human CCA cell lines RBE and QBC 939 were chosen as the study objects.We used CCK-8 assay to test the influence of DADA on cell proliferation at different concentrations.Annexin V-FITC-PI staining was used to assess apoptosis.Western Blotting was used to detect the apoptosis markers PARP,Caspase9,bcl-2 and bax.Flow cytometry was applied to analyze cell cycle progression and western blotting was used to detect the cell cycle markers CyclinDl and Cdc2.Glucose consumption,lactate production,ATP level,PDH activity,and reactive oxygen species(ROS)level were also detected after the treatment of DADA.Western Blotting was performed to detect glycolytic markers PDHK1-4 isoforms,PDH,and Phosphorylated PDH 293(pPDH Ser293).ROS scavenger was used to investigate whether the effect of DADA on the apoptosis was induced by ROS.3.CCK-8 assays were used for analysis of synergy between DADA and cisplatin,cells were exposed to different fixed-ratio combinations of DADA(dose range,10-50?M)and cisplatin(dose range,1.5-7.5 ?M)and cell proliferation.The combination index(CI)of DADA and cisplatin was determined using CompuSyn software,based on the median-effect model of Chou-Talalay.Nude mice implanted with QBC939 were divided into four groups as follows:untreated controls,mice treated with DADA alone,mice treated with cisplatin alone or mice treated with a combination of DADA and cisplatin.We evaluated the inhibition of different drugs on Xenograft tumor growth.Results1.The expression levels of PDHK1,PDHK2,PDHK3,and PDHK4 were higher in CCA than in adjacent tissues(P<0.05).We next examined the relationship between the PDHK1-4 expression in tumor tissues and various clinicopathological characteristics.The expression of PDHK1 was not correlated with age,sex,the tumor size,location,vascular invasion,nerve invasion,lymph node metastasis,and TNM stage(P>0.05).Higher PDK2 expression levels were associated with location,vascular invasion,and TNM stage(P<0.05),however,no association was found between PDHK2 and age,sex,the tumor size,CA199 level,AFP level,CEA,vascular invasion,nerve invasion,and lymph node metastasis(P>0.05).Higher PDK3 expression levels were associated with tumor differentiation and TNM stage(P<0.05).Higher PDK4 expression levels were associated with CEA level,location,and nerve invasion(P<0.05).The median survival time was 13 months in high PDHK1 expression group and 19 months in low PDHK1 expression group,respectively(P=0.037).The median survival time was 12 months in high PDHK3 expression group and 19 months in low PDHK3 expression,respectively(P=0.024).The median survival time for high PDHK2 expression group was similar to that for low PDHk2 expression group(P=0.220).Also,there was no significant difference for survival time between high PDHK4 expression and low PDHK4 expression groups.2.CCK8 assay showed that DADA dose-dependently inhibited the proliferation of two human cholangiocarcinoma cell lines.IC50 was 33mM for RBE and 18.2mM for QBC939,respectively.DADA treatment caused a significant increase in CCA intracellular ROS levels,and dose-dependently induced cell apoptosis,which could be caused by regulating Bcl-2 family proteins.In addition,simultaneous treatment with the thiol antioxidant N-acetylcysteine(NAC)inhibited the elevated ROS levels and restored the effect of DADA on apoptosis of RBE and QBC939.3.DADA induced G2/M cell cycle arrest of RBE via regulation of Cdc2 expression,and induced G0/1 cell cycle arrest of QBC939 via down-regulation of CyclinDl.4.DADA enhanced PDH activity,and dose-dependently inhibited the consumption of glucose,the productions of lactate,and ATP in both CCA cell lines.Furthermore,western blotting showed that PDHK1,PDHK2,PDHK3,PDHK4,and PDH expressions were not altered by DADA,however,the expression level of the phosphorylated forms of PDH(PDHE1(S293))was dose-dependently decreased by DADA.5.DADA in combination with cisplatin synergistically inhibited cell proliferation of CCA cells(CI<1).DADA significantly inhibited Xenograft tumor growth.DADA/cisplatin combination was more effective in tumor growth inhibition compared with individual drugs(47.9%vs 69.1%,P<0.05).ConclusionsPDHK 1-4 isoforms are overexpressed in CCA compared to adjacent tissues.PDHK1 and PDHK3 expression correlate negatively with CCA patients' outcome,which suggests that PDHK1 and PDHK3 be regarded as a marker of poor prognosis for CCA.DADA inhibits CCA cell proliferation,induces apoptosis,and cell cycle arrest,which may be caused by the alteration of glucose metabolism by DADA.DADA/cisplatin combination synergistically inhibits cell proliferation of CCA cells,and also may enhance cytotoxity in nude mice xenograft models of human CCA.Therefore,our results suggest that PDHK expression may play a role in CCA development and DADA in combination with cisplatin may provide novel and promising strategies to improve the treatment of cholangiocarcinoma.