Oxaloacetate Induced Apoptosis Of Hepatoma Cells By Reversal Of Warburg Effect

BackgroundHepatocellular carcinoma(HCC),a malignant liver tumor,is a malignant disease with short survival,high mortality,difficult treatment,and easy prognosis.At present,the main treatment means for liver cancer are surgical treatment,radiotherapy and chemotherapy.However,it is not easy to diagnose early because the early clinical symptoms of liver cancer are hidden.It is often advanced and metastasize at the time of diagnosis.Even after surgical excision,it is easy to relapse and lack effective treatment drugs and methods.Thus,It is not ideal for the treatment on liver cancer.Nowadays,with the development of basic and clinical research on cancer,biotherapy has become the fourth mode of tumor therapy after surgical treatment,radiotherapy and chemotherapy.Biological therapy mainly includes molecular targeted therapy,gene therapy,immunotherapy and so on.This study was about induced tumor apoptosis through intervention and transformation of tumor metabolism.Abnormal glycometabolism is an important feature of tumor cells differentiated from normal cells.As early as 1920,the German biochemist Warburg proposed a classical theory on tumor cells:tumor cells were characterized by high glucose uptake,active glycolysis and high lactic acid content,even when oxygen was abundant.It was named the Warburg effect.On the one hand,Warburg reveals the characteristics of specific metabolism on tumor cells.On the other hand,Warburg believes that the reason that tumor cells show special aerobic glycolysis is the irreversible damage of mitochondria on tumor cells.In response to the view of Warburg,more and more studies have put forward their own disagreements.People find that not all tumor cells show Warburg effect,some of the tumor cells show a mixture of glycolysis and oxidative phosphorylation(OXPHOS),even some tumor cells are dominated by OXPHOS.In addition,the metabolic pattern of tumor cells is not static.With the changes in the microenvironment,drugs,and genes,the metabolic patterns of tumor cells were also changed.Therefore we changed metabolism of tumor cells though enhanced OXPHOS,so as to reverse the Warburg effect on tumor cells,which induced proliferation inhibition and apoptosis of tumor.Oxaloacetate(OA)is the most critical metabolic substrate in the tricarboxylic acid(TCA)cycle.OA and acetyl-CoA are condensed to form citric acid by citrate synthase catalyzed.This is the starting step as well as the key step on the TCA cycle.The OA concentration influences the rate of TCA cycle.Adding a certain dose of OA can cause TCA accelerate the circulation rate.The whole TCA cycle generates a two molecule FADH₂,and isocitrate,?-ketoglutaric acid and malic acid produce 6molecules of NADH.FADH₂ and NADH are reoxidized by the transmission of the electronic chain,releasing a large amount of ATP.The whole process is called OXPHOS.Therefore,with adding a certain dose of OA to tumor cells,the level of OXPHOS on tumor cells was increased by accelerating the TCA cycle,thereby inhibiting the level of glycolysis and reversing the Warburg effect on tumor cells.Although changes in metabolites can regulate cellular metabolism,genes and related signaling pathways also affect metabolic processes on tumor cells.The Akt/HIF signaling pathway regulates aerobic glycolysis through transcriptional activation of HIF1?,NF?B,c-myc and the subsequent expression of glycolytic enzymes such as hexokinase2(HK2),phosphofructokinase2(PFK2),lactate dehydrogenase(LDH)and pyruvate kinase isozyme type M2(PKM2).In our study,we found that OA at high doses inhibited the phosphorylation of epidermal growth factor receptor(EGFR)and the PI3K/Akt/HIF pathway,which reduced the transcriptional expression of glycolytic enzymes.This inhibited the glycolysis level on tumor cells.MethodsHepatoma cell lines(HepG2),nude mouse xenografts and primary hepatoma cells were used as experimental models.MTT assay was used to test the effect of OA on the proliferation and viability of the hepatoma cells.The effect of OA on the apoptosis of hepatoma cells was detected by Annexin V-PI assay.The effects of OA on glycolysis,TCA cycle and OXPHOS were examined using real-time qPCR,western blot methods and related enzyme activity assay kits.The effect of OA on EGFR phosphorylation and downstream signaling molecules was detected by western blot and immunofluorescence.Finally,bioinformatics was used to screen out cell lines with different levels of glycolysis.ResultsPart I Oxaloacetate promotes the apoptosis on hepatoma cellsHepatoma cell lines(HepG2),nude mouse xenografts,and primary hepatocellular carcinoma cells were used as experimental models.We found that 50mM OA could significantly reduce the activity of HepG2 cells and primary hepatoma cells,and the colony formation rate was also decreased.The nude mice transplanted tumors were significantly reduced in size after OA treatment.Annexin V-PI and TUNEL experiments confirmed that OA could cause apoptosis on hepatoma cells.Subsequently,it was further verified that OA could induce the apoptosis on hepatoma cells by examining the activity and expression of caspase3,cleaved-caspase3,cleaved-PARP,Bax and Bcl-2.Part II The metabolic mechanism of oxaloacetate anticancer effectHepG2 cell,nude mouse xenografts,and primary hepatoma cells were used as experimental models.We found that 50 mM OA could cause a significant decrease in ATP production,the glucose transporter(GLUT)mRNA expression level and glucose uptake on hepatoma cells.We further found that OA could inhibit the mRNA expression and activity of glycolytic key enzymes(HK,PFK and LDH),and increase mRNA expression and activity of TCA cycle-related enzymes(PDH,CS and IDH).At the same time,OA could also increase the protein expression and activity of respiratory chain complex I/II(COXI/II),as well as mitochondrial copy number.These results suggest that OA could increase OXPHOS and decreased the level of glycolysis on hepatoma cells.Part III The signaling mechanism of oxaloacetate anticancer effectHepG2 cell was used as an experimental model.We found that the phosphorylation of EGFR as well as the EGFR mediated downstream PI3K/Akt pathway was inhibited with the increase of OA concentration and action time.The glycolytic enzyme genes(HK,PFK)expression was decreased though inhibition the expression or activity of Akt,HIF1?and c-myc.Therefore,we suggested that OA could reduce the level of glycolysis on HepG2 cell via the signal regulation.Part IV Selective inhibition of oxaloacetateTen tumor cell lines with distinctly different levels of glycolysis were screened outusing bioinformatics.We found that OA has a more obvious anticancer effect on cell lines with high levels of glycolysis and has little effect on cell lines with low levels of glycolysis.ConclusionWe used HepG2 cell,nude mouse xenografts,and primary hepatoma cell as experimental models to confirm that OA could inhibit the proliferation of hepatoma cells and induce the apoptosis.We expounded the mechanism of anticancer effect from two aspects.On the one hand,OA could enhance OXPHOS and break the OXPHOS and glycolysis balance on hepatoma cells,which made glycolysis of hepatoma cell decreased.Hepatoma cells lost the metabolic pattern for survival,which caused supply energy barrier.On the other hand,OA inhibited the expression or activity of Akt,HIF1?,and c-myc by inhibiting EGFR and downstream PI3K/Akt pathway.The expression of glycolysis related target genes was decreased and the glycolysis level was inhibited.Finally,we have initially explored the selectivity of OA on suppressing cancer,that is,OA selectively inhibited tumor cells with high levels of glycolysis.Therefore,OA may become a novel anticancer drug.