Department of Obstetrics and Gynecology, Peking University People's Hospital, Beijing, China.
Chin Med J (Engl). 2012 Sep;125(18):3273-8.
Histone deacetylase (HDAC) inhibitors are a group of small chemical molecules that inhibit histone deacetylase. At cell level, HDAC inhibitors have multiple biological effects such as cell cycle arrest, apoptosis, cell differentiation and auotophagy. At molecular level, HDAC inhibitors cause histone and nonhistone acetylation and induce gene expression. HDAC inhibitors are widely used in cancer therapy because of its function of inducing apoptosis. However, the mechanisms of apoptosis effect are not fully understood. TSA is a classical HDAC inhibitor and widely used in epigenetic and anti-cancer research. In this study, we selected Trichostatin A (TSA) to investigate the mechanisms of HDAC inhibitors apoptotic effect on cancer cells.
Cervical cancer cell lines such as Hela, Caski and normal human keratinocyte line HaCaT were treated with various concentrations of TSA. Crystal violent assay and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay were performed to determine cell number. PARP cleavage and FITC-AnexinV were performed to determine apoptosis. DNA-methyltransferase (DNMT)1, DNMT3A and DNMT3B were determined by regular PCR, qPCR and Western Blotting. Small interfering RNA (SiRNAi) was used to knock down DNMT3B.
HDAC inhibitors only induce cervical cancer cell apoptosis. At 1 µmol/L of TSA, 86% of Hela cell and 76% of Caski went apoptosis. For normal cells, HDAC inhibitors have no cytotoxic effect at therapeutic dosage, (90.0 ± 8.4)% of normal cell survive after treated with 1 µmol/L of TSA. We compared 1 µmol/L group with untreated control with t-test. There was no significance between 1 µmol/L group and untreated control for normal cell (P > 0.05). HDAC inhibitors decreased DNMT3B in cancer cell but not in normal cell. Manually knock-down of DNMT3B induced Hela and Caski cell apoptosis. More than 99% of Hela and Caski cell went apoptosis after deprived of DNMT3B.
DNMT3B was essential to cervical cancer cell survival. Down-regulated DNMT3B by HDAC inhibitors may play an important role in the toxicity of HDAC inhibitors on cervical cancer cells.
组蛋白去乙酰化酶（HDAC）抑制剂是一组抑制组蛋白去乙酰化酶的小分子化学物质。在细胞水平上，HDAC 抑制剂具有多种生物学效应，如细胞周期停滞、细胞凋亡、细胞分化和自噬。在分子水平上，HDAC 抑制剂引起组蛋白和非组蛋白乙酰化，并诱导基因表达。HDAC 抑制剂由于其诱导细胞凋亡的功能而被广泛应用于癌症治疗。然而，细胞凋亡作用的机制尚未完全阐明。TSA 是一种经典的 HDAC 抑制剂，广泛应用于表观遗传学和抗癌研究。在这项研究中，我们选择 Trichostatin A（TSA）来研究 HDAC 抑制剂对癌细胞凋亡作用的机制。
用不同浓度的 TSA 处理宫颈癌细胞系如 Hela、Caski 和正常人角质形成细胞系 HaCaT。采用结晶紫试验和 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）试验测定细胞数量。PARP 切割和 FITC-AnexinV 用于检测凋亡。用常规 PCR、qPCR 和 Western Blotting 检测 DNA-甲基转移酶（DNMT）1、DNMT3A 和 DNMT3B。用小干扰 RNA（SiRNAi）敲低 DNMT3B。
HDAC 抑制剂仅诱导宫颈癌细胞凋亡。在 1µmol/L 的 TSA 作用下，Hela 细胞的 86%和 Caski 细胞的 76%发生凋亡。对于正常细胞，HDAC 抑制剂在治疗剂量下无细胞毒性作用，用 1µmol/L 的 TSA 处理后，有（90.0±8.4）%的正常细胞存活。我们用 t 检验比较了 1µmol/L 组与未处理对照组。正常细胞中 1µmol/L 组与未处理对照组无显著性差异（P＞0.05）。HDAC 抑制剂降低了癌细胞中的 DNMT3B，但未降低正常细胞中的 DNMT3B。人工敲低 DNMT3B 诱导 Hela 和 Caski 细胞凋亡。DNMT3B 缺失后，Hela 和 Caski 细胞的凋亡率超过 99%。
DNMT3B 是宫颈癌细胞存活所必需的。HDAC 抑制剂下调 DNMT3B 可能在 HDAC 抑制剂对宫颈癌细胞的毒性中起重要作用。
