| Background and ObjectiveColon cancer has been the third leading cause of death in the world with high morbidity and mortality. The main event of tumorigenesis is escaping from cellular senescence and cellular death into permanent growth. Therefore, re-inducing tumor cells senescence and apoptosis has become an important way for anticancer treatment.Enhancer of Zeste Homologe-2 (EZH2), one of the histone methyltransferases, is the catalytic subunit of Polycom Repressive Complex 2 (PRC2) that methylates H3K27me3 and results in epigenetic control of cancer genes expression. EZH2 has been found abnormal expressed in many kinds of malignant cancers, including colorectal carcinomas, and inhibition of EZH2 can induce cancer cell senescence. Thus, targeting of EZH2 could be an effect method of anticancer therapy in colon cancer. However, the epigenetic mechanism and role of EZH2 in the cell senescence of colon cancer induced by enedyine antitumor antibiotic Lidamycin (LDM) is still unclear. To sum up, we aim to explore the role of EZH2 in cell senescence induced by LDM and EZH2 inhibitor DZNep in human colon cancer cells.MethodsCellular viability and proliferation was measured by MTT and colony assay. Cell senescence-like phenotypes were measured by SA-β-Gal staining. Cell cycle and apoptosis were analyzed by flow cytometry. The protein and mRNA levels of EZH2 were measured by Western Blot and Real-time PCR, respectively. The level of y-H2AX was assayed with flow cytometry. Luciferase reporter assay was used to detect p21 promoter activity. Immunohistochemistry was performed with colon cancer tissue array to compare the expression of EZH2 and p21. EZH2 was knocked down with siRNA transfection or overexpressed with lentivirus infection, to explore the role of EZH2 in cell senescence. p21 was knocked down with siRNA to explore the role of p21 in cell senescence. Chromatin Immunoprecipitation was used to detect the binding of protein and gene primer.ResultsLDM significantly inhibited the cellular viability and proliferation in HCT116 and SW620 cells. Cellular senescence in both HCT116 and SW620 was observed by SA-β-Gal staining after LDM treatment for 48 h-120 h. Moreover, G2/M cell cycle arrest and apoptosis were observed after treatment with LDM, simultaneously. The level of DNA damage marker y-H2AX was increased by LDM as well. In addition, LDM inhibited EZH2 expression and increased p21 expression at both protein level. Immunohistochemical result showed the expression of EZH2 in colon carcinoma tissues was significantly higher than that in adjacent tissues. Using RNA interference, knockdown of EZH2 expression resulted in cellular senescence and G2/M cell cycle arrest as well. Expectedly, over-expression of EZH2 reduced cellular senescence, and blocked p21 and the level of y-H2AX up-regulation upon LDM exposure in HCT116 cells. ChIP assay showed direct binding of EZH2 and H3K27me3 to the p21waf1/ciP1 promoter region in SW620 cells.DZNep significantly inhibited the cellular viability and proliferation in HCT116 cells. Cellular senescence was observed by SA-β-Gal staining after treatment with DZNep in HCT116 cells, similier with treatment with siEZH2. Moreover, DZNep caused G1 cell cycle arrest and apoptosis in a time-dependent manner. DZNep inhibited EZH2 expression with increased p21 and p27 expression and decreased p-RB and p-cdc2 expression. PARP cleavage was increased after treated with DZNep in HCT116 cells.ConclusionIn this study, we first find that LDM can inhibit EZH2 expression in colon cancer cells, and demonstrate the mechanism of LDM-induced cell senescence by inhibition of EZH2 and increased p21 expression through an epigenetic way. Meanwhile, we first demonstrate that DZNep can induce cell senescence in HCT116 cells and inhibite the growth and survival by inducing G1 cell arrest and apoptosis. The study provides a novel relation between EZH2 and cell senescence in human colon cancer cells, and also provides a strong evidence for EZH2-target anticancer therapeutic strategies. |
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