Metformin Facilitates BG45-induced Apoptosis Via The Anti-Warburg Effect In Cholangiocarcinoma

Background:Cholangiocarcinoma(CCA)is a rare but highly lethal malignancy with an often late diagnosis and consequent poor prognosis.Since most of the patients are inoperable,chemotherapy is the exclusive therapeutic strategy for them.But chemoresistance attenuates the efficacy of conventional chemotherapy,thus making the 5-year survival rate of CCA remained at 10%for many years.Compared to normal cells,tumor cells preferentially metabolize glucose to lactate,even under aerobic conditions.Tumor cells also preferentially metabolize glutamine to supply for the aberrant growth and proliferation.Such metabolic alterations not only support the growth and invasion of tumor cells,but also promote their chemoresistance.Objective:The purpose of our study was to explore the role of metformin in regulating the metabolism of CCA,as well as to investigate whether metformin could regulate the abnormal biological functions of CCA and act as a chemosensitizer of the HDAC3 inhibitor BG45,and therefore have potential for the treatment of CCA.Methods:Bioinformatics analysis was performed to detect the potential biological functions and pathways of these genes in CCA.A number of candidate proteins that may be involved in the Warburg Effect and the expression levels of cleaved caspase3 and cleaved PARP were detected by western blotting.Cell Counting Kit(Dojindo;Japan)was used to evaluate the cell viability.The apoptosis was also detected by performing flow cytometry.The oxygen consumption rate(OCR)and extracellular acidification rate(ECAR)were detected in real time with an XF96 Extracellular Flux Analyzer from Seahorse Bioscience,Inc.(North Billerica,MA,USA).GC-MS was used to determine the TCA cycle metabolites and isotopomer enrichment.Finally,we evaluated the antitumor effect in vivo using a CCA cell tumor xenograft model.Results:Through bioinformatics analysis,we found that aberrant metabolism contributed to the proliferation of CCA cells.Seahorse XF96 Extracellular FluxAnalyzer analysis and lactate production analysis showed that metformin could act as a suppressor of the Warburg Effect in CCA cells.Western blotting showed that metformin could decrease the expression of LDHA,which plays a key role in the Warburg Effect.However,suppression of the Warburg Effect was not sufficient to induce CCA cellular apoptosis.According to our previous work,which showed that an HDAC3 inhibitor(MI192)was involved in CCA apoptosis,we observed that metformin combined with BG45(a novel specific HDAC3 inhibitor)could effectively induce the apoptosis of CCA cells in vitro.GC-MS showed that metformin could increase the uptake of glutamine,while BG45 could decrease the utilization of it.Combination of metformin and BG45 inhibit both glucose and glutamine metabolism in CCA cells.Furthermore,in vivo experiments suggested that combined treatment with metformin and BG45 markedly reduced CCA growth in a CCA xenograft model and weight loss of the mice was not found to be significant,which indicated that the combination therapy was safe in vivo.Conclusions:Our data revealed that reversing the Warburg Effect with metformin sensitizes cells to the antitumor effects of HDAC3 inhibitors.This provides a rationale for using the combination of metformin and BG45 as a new therapeutic strategy in the treatment of CCA.