探討西達苯胺(愛普沙)加速非小細胞肺癌細胞衰老之機制

近年來許多文獻指出細胞衰老是不可逆的細胞週期停滯，在腫瘤形成的時候扮演極為重要的生理角色，因此促進細胞衰老反應被認為是具有潛力的治療方針。 而造成細胞衰老的主要原因為增加氧化壓力以及抑制端粒酶活性兩類。 Chidamide(西達苯胺)屬於新苯胺類(Benzamide-type)的組蛋白去乙醯酶抑制劑(Histone deactylase inhibitor, HDACi)。 目前的研究發現Chidamide不僅能夠誘發細胞凋亡亦能抑制細胞生長，而本實驗室先前研究也發現Suberoylanilide hydroxamic acid (SAHA)能夠透過去甲基化抑制端粒酶活性。 因此，本實驗欲探討Chidamide是否透過表觀基因調控抑制端粒酶活性進而造成肺癌細胞衰老(Cellular senescence)。 本實驗先以MTT assay及Clonogenic assay證實Chidamide能抑制肺癌細胞增殖及對肺癌細胞造成細胞毒性。利用Flow cytomery分析細胞週期發現Chidamide會造成A549肺癌細胞G1期停滯(G1 arrest)，我們以Senescence- Associated β-galactosidase activity staining (SA-β-gal)和Acridine orange stain(AO)及C12FDG染色證明細胞衰老現象，並利用RT-PCR與Western blotting證實Chidamid會增加細胞衰老相關基因的p21、p27 mRNA和蛋白質表現。 從TRAP和Telomere Length Assay發現Chidamide能夠有效抑制端粒酶活性但卻不會縮短端粒長度，並發現Chidamide能夠有效減少hTERT的表現量。 Chidamide能抑制DNA methyltransferase 1 (DNMT1)以及DNA methyltransferase 3b (DNMT3b) 的表現量，並造成hTERT promoter去甲基化而減少hTERT的表現量。此外Chidamide還能誘發miR-129-3p的產生而且也發現利用Transfection方式發現，將miR-129-3p過度表現的時候就會降低hTERT蛋白的表現量。利用Flow cytomery分析Chidamide會增加Reactive oxygen species (ROS)的產生，此外利用抗氧化劑NAC(ROS Scavenger)發現抑制ROS的產生，能夠部分抑制Chidamide所誘發的細胞衰老。綜合以上結論：Chidamide經由hTERT啟動子去甲基化與誘發miR-129-3p產生導致降低hTERT表現而抑制端粒酶活性，並透過G1期停滯與ROS促使肺癌細胞走向細胞衰老

Abundant evidence points to a crucial physiological role for cellular senescence, a stable cell cycle arrest, in combating tumorigenesisis. For the reasons, senescence is considered as a potential strategy for cancer therapy. ROS production and telomerase inhibition mainly contribute to senescencec. Chidamide belongs to a novel benzamide-type histone deacetylase inhibitor (HDACi) and the literatures show that HDACi inhibits cancer cells growth and induces apoptosis. Our previous research reveals a HDACi, SAHA, inhibits telomerase activity via epigenetic regulation in lung cancer. Therefore, the present study is to investigate the mechanisms of Chidamide inhibited telomerase activity and induced cellular senescence in non-small lung cancer cells. Chidamide can inhibit the proliferation of A549 lung cancer cells via MTT assay and clonogenic assay. By flow cytometry, we demonstrated that Chidadmide arrested cell growth at G1 phase. Using Senescence-Associated β-galactosidase activity staining (SA-β-gal), C12FDG staining and Acridine orange staining (AO) exhibited the phenomenon of cell senescence by chidamide. Chidamide increases p21and p27 but decreases pRb expression in A549 lung cancer cells by RT-PCR and western blotting. Chidamide also resulted in inhibition of telomerase activity but not shortened of telomere length on TRAP assay and Telomere Length Assay. Chidamide reduced expression of hTERT expression by RT-PCR, Real-time PCR (q-PCR) and Western blotting. In addition, chidamide caused hTERT promoter demethylation to reduce the expression of hTERT mRNA through downregulation DNA methyltransferase 1(DNMT1) and DNA methyltransferase 3b (DNMT3b) expression. Chidamide indeed induced miR-129-3p production when overexpression miR-129-3p downregulated hTERT protein expression. Chidamide also induced reactive oxygen species (ROS) generation. We demonstrate that the effects of chidamide on telomerase activity and senescence can be blunted by ROS inhibitor N-acetyl cysteine (NAC). In conclusions, this is the first study to demonstrate that chidamide induces ROS generation, epigenetic regulation and miR-129-3p expression that inhibit telomerase activity and induce cellular senescence.