Study On The Mechanism Of The Effect Of Shikonin Combined With CM-272 On Non-small Cell Lung Cancer

Objective:1.To investigate the effect of G9a/DNMT inhibitor CM-272 combined with shikonin on the proliferation and apoptosis of non-small cell lung cancer cells and the possible mechanism.2.To investigate the effect of G9a/DNMT inhibitor CM-272 combined with shikonin on the glycolytic capacity of non-small cell lung cancer cells.3.To investigate the effect of G9a/DNMT inhibitor CM-272 combined with shikonin on autophagy in non-small cell lung cancer cells and the possible mechanism.Methods:1.Shikonin(0.03125,0.0625,0.125,0.25,0.5,1,2,4 μmol-L-1)and CM-272(0.0625,0.125,0.25,0.5,1,2,4,8 μmol-L-1)alone and 0.25 μmol-L-1 CM-272 with complain(0.5,1,2 μmol-L-1)in combination with shikonin(0.5,1,2,4,8 μmol-L-1)on non-small cell lung cancer cells A549;cell viability was detected using the CCK-8 assay.The CI of the drug combination was calculated using Compu Syn with reference to the results of CCK-8 assay;a CI value greater than 1 indicated the antagonistic effect of the drug combination,while a CI value less than 1 indicated the synergistic effect of the drug combination,and the concentration with the smallest CI value was selected as the concentration for subsequent experiments.3.Clone formation assay was performed to detect the inhibition of proliferation of A549 cells by CM-272 and shikonin alone and in combination,and to detect whether the effect of the combination of the two drugs was stronger than that of the use of the drugs alone.4.A concentration of 0.25 μmol-L-1 CM-272 and 2 μmol-L-1 shikonin were applied to A549 cells alone or in combination,and apoptosis was detected using Annexin V/PI doublestaining assay;changes in cellular glycolysis and maximum glycolytic capacity were detected using glycolytic stress test.5.The expression of m TOR,p-m TOR,BAX,BCL-2,LC3 B,Cyto-C proteins in A549 cells was detected using Western Blot.Results:1.CM-272 and shikonin alone or in combination reduced the activity of A549 non-small cell lung cancer cells,and the combined effect was stronger than that alone;0.25 μmol-L-1CM-272 and shikonin(0.5,1,2 μmol-L-1)were used to inhibit the viability of A549 cells alone or in combination,and the cell viability was measured by CCK-8.The CI values were calculated by Compu Syn software,and it was found that the combination of several concentrations had a synergistic effect(i.e.,CI value less than 1)on the inhibition of cell viability of non-small cell lung cancer cells,and the combination of 0.25 μmol-L-1 CM-272 and 2 μmol-L-1 shikonin had the best effect(i.e.,the smallest CI value).2.Clonogenesis assay showed that CM-272 and shikonin alone or in combination could inhibit the proliferation of non-small cell lung cancer cells,and the combined effect was better than that alone.3.Annexin V/PI double-staining assay showed that CM-272 and shikonin alone or in combination could promote apoptosis of non-small cell lung cancer cells,and the proapoptotic effect of the combination was better than that of the combination alone.4.The results of glycolytic stress test showed that CM-272 and shikonin alone or in combination could reduce the glycolytic and maximal glycolytic capacity of non-small cell lung cancer cells,and the pro-apoptotic effect of the combination was better than that of the combination alone.5.Western Blot results showed that the ratio of apoptosis-related protein Bax to Bcl-2was more significantly up-regulated and the expression of cleaved-caspase 8 was significantly increased in the combination group compared with CM-272 or shikonin alone;the semiquantitative results of autophagy-related protein LC-3 Ⅱ were significantly increased;and the phosphorylation level of pathway-related protein m TOR was significantly decreased.Conclusion:1.The combined action of CM-272 and shikonin inhibited the proliferation of non-small cell lung cancer cells in vitro.2.Combination of CM-272 and shikonin may induce apoptosis in non-small cell lung cancer cells in vitro by inhibiting PI3K/AKT/mTOR signaling pathway.3.The combination of CM-272 and shikonin may diminish the glycolytic capacity and enhance the autophagy of NSCLC cells to exert a synergistic anti-cancer effect.