A Mechanical Study Of Synergized Hepatocellular Carcinoma Cell Death Induced By Combined Treatment With Metformin And Glucose Starvation

Objective Type Ⅱ diabetes is a metabolic disease that is prevalent in various countries around the world.It can promote the occurrence of many tumors,especially hepatocellular carcinoma(HCC).Pharmacoepidemiological studies found that the usage of different anti-diabetic medications had distinct effects on HCC: insulin was reported to increase HCC incidence and exacerbate HCC prognosis as well,while metformin decreased HCC incidence.Limited clinical studies found that metformin improved the patients’ prognosis in early-stage HCC patients,but it worsened the prognosis in advanced-stage HCC patients.The effect of metformin on intermediate-stage HCC is yet unknown.The first-line treatment for intermediate-stage HCC is transcatheter artery embolization with or without chemotherapy(TACE or TAE),which can cause hypoxia and metabolic stress.Previous research in our laboratory found that the combination of metformin and glycolysis inhibitor 2-D-glucose promoted HCC cell death.Therefore,this project aims to study the effects of metformin on the survival of HCC cells under metabolic stress(particularly glucose starvation)through in vitro cell experiments and in vivo nude mice HCC xenograft models,and to study the involved molecular mechanisms in the synergized cell death.MethodsFirstly,human HCC cell lines Hep G2,HCC-M,Hep3 B,and Huh7 cells were used and pretreated with two different anti-diabetic drugs(metformin or insulin)for 12 hours,and then cultured in culture medium containing different concentrations of glucose or given inhibitors of glucose transporter,in order to test the effects of metformin or insulin on glucose starvation-induced cell death.Secondly,Hep G2 cells were selected for the following mechanical studies.By detecting mitochondrial oxidative stress,lysosomal functions and changes in the PP2A-GSK3β-Mcl-1 pathway,the mechanisms of action of metformin on glucose starvation-induced HCC cell death were studied.Lastly,a nude mouse HCC xenograft model was used to further investigate the combined effect of metformin and glucose transporter inhibitor on HCC xenograft in vivo by examining the growth of subcutaneous HCC xenograft and cell death-related immunohistochemical indicators.1.Study of the combined effects of anti-diabetic drugs with glucose starvation on cell death:The effects of metformin or insulin combined with glucose starvation on HCC cell survival and proliferation were evaluated using flow cytometry(PI exclusion assay and sub G1 assay),lactate dehydrogenase release experiments,and cloning formation experiments.With detection of apoptotic proteins and the use of anti-apoptotic inhibitors,the type of cell death induced by the combined treatment was then identified.2.Detection of mitochondrial functions:Flow cytometry was used to detect changes in mitochondrial membrane potential and permeability by TMRM assay and JC-1 assay,and to measure intracellular and mitochondrial specific reactive oxygen species(ROS)by DCF assay and Mito Sox assay respectively;ATP biochemical assay kit was utilized to detect the changes of ATP;Transmission electron microscopy was used to monitor mitochondrial damages after the combined treatment of metformin with glucose starvation.3.Detection of lysosomal functions:After staining with Lysotracker Red or Magic Red cathepsin B,flow cytometry was utilized to detect lysosomal function after the combined treatment of metformin and glucose starvation.4.Exploration on the involvement of PP2A-GSK3β-Mcl-1 pathway:Western blotting was used to determine the changes of Mcl-1 and its upstream signaling pathway proteins PP2 A and GSK3β;GSK3β inhibitors were pre-treated to explore the essential role of this signaling pathway on HCC cell death after the combined treatment of metformin and glucose starvation.5.Combined effects of glucose transportation inhibitors and metforminWestern blotting was used to detect the expression levels of glucose transporters SGLT2 and GLUT1 in normal hepatocytes and eight HCC cell lines;Colony formation assay and flow cytometry were used to observe the combined effects of SGLT2 inhibitors canagliflozin(cana)and metformin on the proliferation and cell death of HCC cells6.Animal experimentsHCC cells were inoculated subcutaneously in nude mice to form xenograft tumors.SGLT2 inhibitor canagliflozin(30 mg/kg)were used to simulate the effect of glucose starvation,thereby verifying the synergistic effects of glucose starvation(canagliflozin)and metfomin in vivo.Oral administration of drugs by gacage was performed for 2 weeks,and the tumor growth,blood glucose fluctuation and weight changes were monitored.Finally,immunohistochemical techniques were used to detect apoptosis and proliferation status in tumor tissues,while Western blotting was used to analyze the expression levels of related proteins.Results1.Metformin combined with glucose starvation significantly promoted HCC cell death in concentration-and time-dependent manners.The release of lactate dehydrogenase(LDH)was correspondingly increased.In contrast,insulin exhibited opposite effects by protecting long-term glucose starvation-induced cell death.Western blotting found that caspase proteins were activated and PARP cleaved after the combined treatment of metformin and glucose starvation.Pretreatment with pan-caspase inhibitor(Z-VAD-FMK),caspase-8 inhibitor(ZIETD-FMK)or caspase-9 inhibitor(Z-LEHD-FMK)reversed the changes of PARP protein and effectively inhibited cell death induced by metformin and glucose starvation,indicating that the combined cell death was mainly apoptosis.2.After the combined treatment of metformin with glucose starvation,the results of flow cytometry and fluorescence microscopy consistently suggested increases of intracellular and mitochondrial ROS accumulation.NAC pretreatment could reverse the accumulation of ROS,thereby suppress cell death caused by the combined treatment of metformin and glucose starvation;flow cytometry showed a significant decrease in mitochondrial membrane potential and an increase in mitochondrial membrane permeability;Next,ATP produced by mitochondria was significantly reduced;Electron microscopy results demonstrated the mitochondria swelling and destruction of mitochondrial ridges.As a result,cells lacked basic energy supplies and consequently died after combined treatment.3.The study of lysosomal function found that the p H of the lysosome increased after the combined treatment of metformin and glucose starvation;furthermore,cathepsin B staining confirmed that the activity of lysosomal enzyme was significantly reduced.Both results suggested that the combination of metformin and glucose starvation suppressed lysosomal function.4.Further mechanical studies showed that the combination of metformin with glucose starvation led to a significant decrease of anti-apoptotic protein Mcl-1,accompanied by the increase of the upstream tumor suppressor PP2 A expression and a decrease in the phosphorylation(Ser9)level of GSK3β.Both GSK3β inhibitors Ⅷ(AR-A014418)and GSK3β inhibitors Ⅻ(TWS119)partially protected cells from cell death induced by the combined treatment,indicating the involvement of PP2A-GSK3β-Mcl-1 signalling in synergized cell death.5.Comparing HCC cell lines with normal L02 hepatocyte,most HCC cells(Huh7,Hep G2,SNU-182 and SNU-387)over-expressed with SGLT2;GLUT1were expressed in all tested HCC cells.Glucose transporter SGLT2 inhibitor canagliflozin or GLUT1 inhibitor phloretin sensitized metformin induced cell death in Huh7 cells.The combination of canagliflozin with metformin could increase ROS production.The above results indicated that canagliflozin may simulate the effect of glucose starvation.6.After successful establishment of HCC xenograft in nude mice,canagliflozin was combined with metformin to intervene tumor growth for 15 days.It was observed that the combined treatment could inhibit the growth of xenograft tumors.The immunohistochemical examination of tissue samples detected a decrease in Ki-67-positive cells but an increase in TUNEL-positive cells,indicating that the cell proliferation in the tumor was suppressed and apoptosis is enhanced.Western blotting analysis of tissue protein showed the decreased expression of Mcl-1,accompanied by increased expression of PP2 A but decreased GSK3β Ser9 phosphorylation,which was consistent with in vitro experiment results.Conclusions1.Metformin could sensitize glucose starvation-induced caspase-dependent cell death in HCC cell lines,while insulin showed protective effects against longterm glucose starvation-induced cell death;2.Metformin and glucose starvation promoted cell death through mitochondria-dependent,ROS-mediated apoptotic pathway;3.The combination of metformin and glucose starvation inhibited lysosome acidification and lysosomal cathepsin B activity,therefore could promote further increase of cellular ROS;4.Metformin sensitized glucose starvation-induced cell death through the PP2A-GSK3β-Mcl-1 pathway;5.The combination of metformin and canagliflozin demonstrated similar synergistic effect as the combination of metformin with glucose starvation to induce HCC cell death;6.The combination of metformin and canagliflozin could inhibit tumor growth in a xenograft nude mice model.