Yang Bohan, Yu Dandan, Liu Jingwen, Yang Kunyu, Wu Gang, Liu Hongli
Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. 1277 Jiefang Avenue, Wuhan, 430022, Hubei, China.
Tumour Biol. 2015 Jul;36(7):5051-61. doi: 10.1007/s13277-015-3156-1. Epub 2015 Feb 4.
Suberoylanilide hydroxamic acid (SAHA; vorinostat), the second generation of histone deacetylase (HDAC) inhibitor, has been approved for the treatment of cutaneous manifestations of cutaneous T cell lymphoma (CTCL). It has also shown its anticancer activity over a large range of other hematological and solid malignancies, but few studies have been reported in B cell lymphoma. In this study, we aimed to investigate the antitumor activity of SAHA on murine B cell lymphoma cell line A20 cells. We treated A20 cells with different concentrations of SAHA. The effect of SAHA on the proliferation of A20 cells was studied by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay in vitro; the anti-proliferation activity in vivo was evaluated by proliferating cell nuclear antigen (PCNA) of xenograft tumor tissues through immunocytochemical staining. Apoptosis were detected by Hoechst 33258 staining and Annexin V/propidium iodide (PI) double-labeled cytometry in vitro. The effect of SAHA on cell cycle of A20 cells was studied by a propidium iodide method. Autophagic cell death induced by SAHA was confirmed by transmission electron microscopy (TEM). Angiogenesis marker (CD31) was measured by immunocytochemical staining to investigate the anti-angiogenic effect of SAHA. Western blot was used to detect the expression of signaling pathway factors (phospho-AKT, phospho-ERK, AKT, ERK, Nur77, HIF-1α, and VEGF). Our results showed that SAHA inhibited the proliferation of A20 cells in a time- and dose-dependent manner, induced cell apoptosis and G0/G1 phase arrest of cell cycle, promoted autophagic cell death, and suppressed tumor progress in NCI-A20 cells nude mice xenograft model in vivo. SAHA decreased the activation of AKT (phospho-AKT: p-AKT) and ERK1/2 (phospho-ERK: p-ERK) proteins and inhibited the expression of pro-angiogenic factors (VEGF and HIF-1α), downregulated its downstream signaling factor (Nur77), which might be contributed to the antitumor mechanisms of SAHA.
伏立诺他（SAHA；异羟肟酸苯丁酯）是第二代组蛋白去乙酰化酶（HDAC）抑制剂，已被批准用于治疗皮肤T细胞淋巴瘤（CTCL）的皮肤表现。它在其他多种血液系统恶性肿瘤和实体瘤中也显示出抗癌活性，但关于B细胞淋巴瘤的研究报道较少。在本研究中，我们旨在探讨SAHA对小鼠B细胞淋巴瘤细胞系A20细胞的抗肿瘤活性。我们用不同浓度的SAHA处理A20细胞。通过体外3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2H-四氮唑溴盐（MTT）法研究SAHA对A20细胞增殖的影响；通过免疫细胞化学染色检测异种移植瘤组织中增殖细胞核抗原（PCNA）来评估体内抗增殖活性。体外通过Hoechst 33258染色和膜联蛋白V/碘化丙啶（PI）双标记流式细胞术检测细胞凋亡。通过碘化丙啶法研究SAHA对A20细胞细胞周期的影响。通过透射电子显微镜（TEM）证实SAHA诱导的自噬性细胞死亡。通过免疫细胞化学染色检测血管生成标志物（CD31）以研究SAHA的抗血管生成作用。采用蛋白质免疫印迹法检测信号通路因子（磷酸化AKT、磷酸化ERK、AKT、ERK、Nur77、HIF-1α和VEGF）的表达。我们的结果表明，SAHA以时间和剂量依赖性方式抑制A20细胞的增殖，诱导细胞凋亡和细胞周期G0/G1期阻滞，促进自噬性细胞死亡，并在体内NCI-A20细胞裸鼠异种移植模型中抑制肿瘤进展。SAHA降低了AKT（磷酸化AKT：p-AKT）和ERK1/2（磷酸化ERK：p-ERK）蛋白的活化，并抑制促血管生成因子（VEGF和HIF-1α）的表达，下调其下游信号因子（Nur77），这可能是SAHA抗肿瘤机制的原因。
