The Research About The Effect Of KA By Inhibiting GAPDH On The Survival And Apoptosis Of Non-Small Cell Lung Cancer

Background:The metabolic mode of tumor cells is quite different from normal cells,especially in glycolysis metabolism.The differential expression of various glycolysis metabolizing enzymes in tumor cells makes it have higher metabolic capacity.Glyceraldehyde-3-phosphate dehydrogenase(GAPDH)is a differential protein that we screened by 2D-DIGE binding mass spectrometry in clinical samples of tumor tissues and paracanceroust tissues.Previous studies have found that the expression of GAPDH in tumor tissues is significantly higher than paracancerous tissues.With sh RNA-induced GAPDH expression silenced in stable transfected cell line NCIH520 cells and nude mice,The proliferation of tumor cells significantly reduced and promoted Platinum-induced apoptosis in NCI-H520 cells.Some related studies in the literature indicate that overexpression of GAPDH can increase the phosphorylation of Akt,which can increase the content of Bcl-xl in cells to avoid apoptosis of tumor cells.Koningic acid(KA)is a small molecule chemical derived from fungi that specifically inhibits GAPDH in cells.Objective: This study preliminarily uses KA to inhibit GAPDH and investigated whether KA and chemotherapeutic drug carboplatin(CBP)have synergistic effect to non-small cell lung cancer cells,then explores the effect of KA on apoptosis and mechanism of KA-induced apoptosis.Methods: 1.Cell viability of four kinds of cells including lung cancer cells NCIH520,lung adenocarcinoma A549,lung large cell carcinoma NCI-H460 and normal bronchial cells HBE cells at different KA concentrations by CCK-8 assay.,plot the cell survival curve.2.Using CCK-8 assay to determine the survival rate of the above four kinds of cells under different carboplatin(CBP)concentrations,and plot the cell survival curve.3.Using hoechst33342/pi fluorescent staining method to explore the way Ka induces the death of NCI-H520 and A549 cells.4.Using NCI-H520 and A549 as the target,explore the synergistic effect of KA on the chemotherapeutic drug CBP under different CBP concentrations;and use HBE as a control to explore the effect of KA+CBP on normal cells to estimate the adverse reactions of the combination of the two.5.Western blotting was used to detect A549 and NCI-H520,and the changes of related apoptotic proteins in A549 and NCI-H520 under the action of IC50 KA concentration of KA,such as Akt,Bcl-2,Bcl-xl,Caspase-3,etc..Results: 1.CCK-8 results showed that the sensitivity of four kinds of cells to KA was:NCI-H460>A549>NCI-H520>HBE.The results of CCK-8 showed that the sensitivity of four kinds of cells to CBP was: HBE>NCI-H520>A549>NCI-H460.3.The way in which KA induces NCI-H520 and A549 cell death is apoptosis.4.Combination therapy showed that KA has synergistic effect on CBP inhibiting the survival of NCIH520 cells and A549 cells.It has less effect on HBE in normal cells.5.Western blotting showed that compared with HBE cells,the Akt content in KA-induced lung cancer cells increased(p<0.05),the p-Akt content decreased(p<0.05),and the Fox O1 content increased(p< 0.05);Bcl-2 and Bcl-xl levels decreased(p<0.05);Caspase3content decreased(p<0.05),and Phospho-Caspase3 content in-creased(p<0.05).Conclusion: 1.In vitro results show that KA has a better inhibitory effect on tumor cell survival than CBP,a common clinical chemotherapy drug.2.KA has a synergistic effect on CBP inhibition of tumor survival and reduces the impact on nor-mal cells.In A549 and NCI-H520,KA specifically inhibits the expression of GAPDH,which down-regulates the phosphorylation of Akt,which leads to the down-regulation of Bcl-2 and Bcl-xl,thereby inducing apoptosis and also promoting Caspase3.Expression thereby induces apoptosis of the cells.