The Relationship Between Pentose Phosphate Pathway And Mitochondrial Function In Tumorigenesis

Tumor cells exists abnormal metabolism,converted oxidative phosphorylation and warburg effect in glycometabolism disorder.As a significant branch of glycolysis,PPP showns to be up-regulated in a variety of tumor cells.Studies shows that the rate-limiting enzyme G6PD in PPP,which activity is also significantly increased in various tumor tissues or cell lines.It indicates whether we can achieve the purpose of tumor treatment by inhibiting PPP.Mitochondria is the energy centers in cells,which have important effects on the occurrence,growth,metastasis and apoptosis of tumors.Studies have shown that mitochondrial fragmentation is closely related to tumorigenesis,but there are few reports on the relationship between pentose phosphate pathway in tumorigenesis,mitochondrial fragmentation and apoptosis.This study used RNAi and DHEA(G6PD inhibitors)to inhibit G6PD of PPP,tested cell vitality of HepG2,and detected cell migration ability,the glucose consumption and lactic acid production quantity,changes in the nucleus,mitochondria fusion and fission protein,ROS content,cell apoptosis,Discusses the change in NADPH,fragmented mitochondria and apoptosis.We hope to obtain the relationship between G6PD and mitochondrial dysfunction and apoptosis.The following research results were obtained:1.The recombinant plasmid against G6PD gene was successfully constructed.After transfecting the recombinant plasmid,the inhibition efficiency of G6PD in mRNA and protein levels in HepG2 cells was detected.The results showed that the inhibition rate at the mRNA level and the protein level was about 70%and 50%respectively.2.After RNAi with G6PD,the activity of HepG2 cells decreased,but there was no significant difference.The effect of 50?M DHEA concentration on cell viability was the most significant.Both treatments significantly reduced the cell migration ability.The results showed that G6PD inhibition had a effect on tumor cells.3.After RNAi with G6PD,HepG2 cell glucose consumption was significantly down-regulated and the amount of lactic acid production was extremely significant down-regulated.Glucose consumption was significantly down-regulated,and lactic acid production was extremely significant decreased in the 50 ?M,200 ?M,and 300 ?M DHEA treatment groups while there was no significant change in 100 ?M DHEA.4.After RNAi with G6PD,the expression of apoptosis-related genes was detected.At mRNA levels,Bax and caspase3 were significantly down-regulated,but at protein levels,Bax,cleaved casase9,cleaved caspase3 and AMPK were significantly up-regulated;ROS content was significantly up-regulated;nuclear chromatin marginalized,nuclear volume decreased and cleavaged as well.These results suggest that G6PD gene silencing further activates the endogenous apoptotic pathway.Bax and caspase3 were significantly down-regulated at the level of mRNA,but Bax and cleaved caspase3 were up-regulated at the level of protein,suggesting that G6PD gene silencing activated the existing Bax and caspase3 proteins in cells,thus triggering apoptosis.After low concentration DHEA treatment,ROS increased earlier than the expression of Bax,cleaved caspase3 and AMPK,but synchronized with cleaved caspase9.These results suggest that DHEA treatment may induce apoptosis through ROS-Tom20-Bax-caspase3 pathway.Caspase9 expression was significantly up-regulated at low concentration(25 M DHEA),suggesting similar sensitivity to ROS for metabolic changes in G6PD or PPP pathway.Taken together,results above indicate that apoptosis was activated by RNAi and DHEA treatment in HepG2 cells.5.After RNAi with G6PD,the protein expression of mitochondrial fission protein drpl and mitochondrial fusion protein mfn2 was detected.The results shown that the protein expressions of drpl and mfn2 were both significantly down-regulated in HepG2 cells.After treatment with 50 ?M,100 ?M,200 ?M,and 300 ?M DHEA of HepG2 cells,the expression levels of drpl and mfn2 were significantly down-regulated.These results indicate that the mitochondrial division and fusion motility were inhibit by RNAi and DHEA,thus stabilized mitochondrial function and inhibited cancer cell growth.Conclusion:RNA interference and inhibitor DHEA treatment can both inhibit the activity of G6PD in HepG2 cells.Down-regulated the G6PD activity,PPP is inhibited to a certain extent,NADPH content is decreased,ROS level is increased,mitochondrial endogenous apoptosis pathway is activated,mean while mitochondria tend to be stable,so that the growth,proliferation and migration of tumor cells decreased.However,after HepG2 cells interfered,there was no significant change in cell viability;after DHEA treatment,the effect on the level of apoptosis-related gene protein was higher than mRNA.The above experimental results showed that the effect is better treatment with inhibitors than RNAi,DHEA may be a potential drug for the treatment of cancer.