Cancer stem–like cells (CSCs) are a cell subpopulation that can reinitiate tumors, resist chemotherapy, give rise to metastases and lead to disease relapse because of an acquired resistance to apoptosis. Especially, epigenetic alterations play a crucial role in the regulation of stemness and also have been implicated in the development of drug resistance. Hence, in the present study, we examined the cytotoxic and apoptotic activity of valproic acid (VPA) as an inhibitor of histone deacetylases (HDACs) against breast CSCs (BCSCs). Increased expression of stemness markers were determined by western blotting in mammospheres (MCF-7s, a cancer stem cell-enriched population) propagated from parental MCF-7 cells. Anti-growth activity of VPA was determined via ATP viability assay. The sphere formation assay (SFA) was performed to assess the inhibitory effect of VPA on the self-renewal capacity of MCF-7s cells. Acetylation of histon H3 was detected with ELISA assay. Cell death mode was performed by Hoechst dye 33342 and propidium iodide-based flouresent stainings (for pyknosis and membrane integrity), by M30 and M65 ELISA assays (for apoptosis and primary or secondary necrosis) as well as cytofluorimetric analysis (caspase 3/7 activity and annexin-V-FITC staining for early and late stage apoptosis). VPA exhibited anti-growth effect against both MCF-7 and MCF-7s cells in a dose (0.6–20 mM) and time (24, 48, 72 h) dependent manner. As expected, MCF-7s cells were found more resistant to VPA than MCF-7 cells. It was observed that VPA prevented mammosphere formation at relatively lower doses (2.5 and 5 mM) while the acetylation of histon H3 was increased. At the same doses, VPA increased the M30 levels, annexin-V-FITC positivity and caspase 3/7 activation, implying the induction of apoptosis. The secondary necrosis (late stage of apoptosis) was also evidenced by nuclear pyknosis with propidium iodide staining positivity. Taken together, inhibition of HDACs is cytotoxic to BCSCs by apoptosis. Our results suggested that targeting the epigenetic regulation of histones may be a novel approach and hold significant promise for successful treatment of breast cancer.

癌干样细胞（CSC）是一种细胞亚群，由于获得的对细胞凋亡的抗性，它可以重新引发肿瘤，抵抗化学疗法，引起转移并导致疾病复发。特别地，表观遗传改变在茎的调节中起关键作用，并且还与耐药性的发展有关。因此，在本研究中，我们研究了丙戊酸（VPA）作为组蛋白脱乙酰酶（HDACs）抑制剂对乳腺癌CSC（BCSC）的细胞毒性和凋亡活性。通过Western印迹在从亲本MCF-7细胞繁殖的乳球（MCF-7s，富含癌症干细胞的人群）中确定了干性标记的表达增加。VPA的抗生长活性是通过ATP活力测定法确定的。进行球形成测定（SFA）以评估VPA对MCF-7s细胞自我更新能力的抑制作用。用ELISA法检测组蛋白H3的乙酰化。细胞死亡模式由Hoechst染料33342和基于碘化丙啶的荧光染色（针对融合度和膜完整性），M30和M65 ELISA分析（针对凋亡和原发性或继发性坏死）以及细胞荧光分析（胱天蛋白酶3/7活性）进行和膜联蛋白-V-FITC染色用于早期和晚期凋亡。VPA表现出对MCF-7和MCF-7s细胞的抗生长作用，呈剂量（0.6–20 mM）和时间（24、48、72 h）依赖性。不出所料，发现MCF-7s细胞对VPA的抵抗力要比MCF-7细胞高。观察到VPA在相对较低的剂量下可防止形成乳球（2。5和5 mM），而组蛋白H3的乙酰化增加。在相同剂量下，VPA可提高M30水平，膜联蛋白-V-FITC阳性和caspase 3/7激活，这暗示了细胞凋亡的诱导。继发性坏死（细胞凋亡后期）也由碘化丙锭染色阳性的核固缩证明。两者合计，抑制HDAC通过凋亡对BCSC具有细胞毒性。我们的结果表明，靶向组蛋白的表观遗传调控可能是一种新颖的方法，对于成功治疗乳腺癌具有重要的前景。继发性坏死（细胞凋亡后期）也由碘化丙锭染色阳性的核固缩证明。两者合计，抑制HDAC通过凋亡对BCSC具有细胞毒性。我们的结果表明，靶向组蛋白的表观遗传调控可能是一种新颖的方法，对于成功治疗乳腺癌具有重要的前景。继发性坏死（凋亡后期）也由碘化丙锭染色阳性的核固缩证明。两者合计，抑制HDAC通过凋亡对BCSC具有细胞毒性。我们的结果表明，靶向组蛋白的表观遗传调控可能是一种新颖的方法，对于成功治疗乳腺癌具有重要的前景。