The Effect And Regulation Mechanism Of Enhancer Of Zeste Homologue2in Senescence Of Gastric Cancer Cells

Part I The effect of inhibition enhancer of zeste homologue2on senescence induced by doxorubicin in gastric cancer cellsObjective:To assess the effect of doxorubicin (DOX) on the inducement of senescence in gastric cancer cells, explore the outcome of EZH2depletion along with exposure of DOX, and related mechanisms.Methods:Senescence was induced by DOX treatment in gastric cancer cells AGS and MKN28. Senescence associated β galactosidase (SA-β-gal), formation of senescence-associated heterochromatin foci and cell cycle assessed by flow cytometry were used to identify cell senescence. EZH2was down-regulated by transfection with siRNA or treated with (-)-epigallocatechin-3-gallate (EGCG), a targeted inhibitor and using above methods to explore the effect of EZH2depletion along with DOX on cellular senescence. Changes in p53/p21axis activation were detected by Western blotting.Results:We found that DOX could induce gastric cancer cells to senescence effectively and cell proliferative arrest caused by DOX could be promoted by EZH2depletion. Mechanistically, EZH2depletion not only worked in coordination with DNA damage during the progression of cell senescence, but also promoted apoptosis in p53mutant cells. However, it had no cooperative relationship with DOX in p53wild-type cells.Conclusion:These data suggest that DOX could induce gastric cancer cells to senescence effectively and help unravel a crucial role for combination of EZH2inhibition and DOX in senescence and apoptosis in gastric cancer cells and that p53genomic status was associated with different cellular responses to it. Part II Enhancer of zeste homolog2depletion induces cellular senescence by regulating histone methylation along the INK4/ARF locus in gastric cancer cellsObjective:To investigate the effect of EZH2expression on the expression of INK4/ARF and the mechanism involved in regulation of senescence in gastric cancer.Methods:After treatment of EZH2-shRNA, MTT, cell proliferation assay and colony formation assay were used to identify cell proliferation. Senescence-associated-P-galactosidase (SA-β-gal) and cell cycle assessed by flow cytometry were used to identify cell senescence. To investigate effects of EZH2depletion on INK4/ARF in gastric cancer cells, Western blotting and ChIP were employed.Results:We show that in gastric cancer cells, EZH2-shRNA could deplete EZH2and H3K27me3effectively and cause inhibition of cell proliferation. Furthermore, we find that MKN28cells, which don’t express p16INK4a and p21, could be induced to senescence by EZH2depletion via p15INK4b activation. Suppression of p15INK4b reverses senescence progression in MKN28cells induced by EZH2down-regulated. Moreover, INK4/ARF locus is activated to certain extent in consequence of a decrease of H3K27me3along it caused by EZH2silence, which contributes substantially to an increase in the expression of P15INK4b,ARF and p16INK4a and results in cellular senescence ultimately.Conclusions:These data unravel a crucial role of EZH2in the regulation of INK4/ARF expression and senescence procedure of gastric cancer cells, suggesting that therapeutic oncogenic EZH2might serve as a strategy for tumor treatment.