Klisovic Dino D, Katz Steven E, Effron David, Klisovic Marko I, Wickham Joseph, Parthun Mark R, Guimond Martin, Marcucci Guido
William H Havener Eye Center, The Ohio State University, Columbus, Ohio 43210, USA.
Invest Ophthalmol Vis Sci. 2003 Jun;44(6):2390-8. doi: 10.1167/iovs.02-1052.
Uveal melanoma (UM) is the most common primary malignant ocular tumor in adults. No effective chemotherapy regimens are available for either intraocular or metastatic uveal melanoma. Therefore, the ability of the histone deacetylase inhibitors (HDACIs), depsipeptide, sodium butyrate (NaB) and trichostatin A (TSA), to induce apoptosis and inhibit cell growth of UM cell lines in vitro was examined.
Three primary and two metastatic UM cell lines were treated in vitro with different concentrations of histone deacetylase inhibitors (HDACIs). Cell proliferation was studied in 24-well plates. Induction of apoptosis was studied by flow cytometry. Changes in gene expression of Fas/FasL, p21(Waf/Cip1), and p27(Kip1) were studied by RT-PCR. Western blot analysis was used to study histone acetylation, Fas/FasL, p21(Waf/Cip1), p27(Kip1) and caspase-3 protein levels. Real-time PCR was used to study changes in bcl-2/bax gene expression.
A dose-dependent increase in histone acetylation was observed in all cell lines. This corresponded to significant inhibition of cell growth and induction of apoptosis in all melanoma cell lines in a concentration-dependent manner. Western blot analysis revealed dose-dependent increases in the amount of caspase-3, Fas/FasL, p21(Waf/Cip1), and p27(Kip1) proteins. However, no changes in bcl-2/bax gene expression were detected by real-time PCR.
HDACIs are potent inhibitors of primary and metastatic UM cell growth in vitro. The apoptosis is probably mediated through the Fas/FasL signaling pathway, whereas bcl-2 appears not to be involved. These data support further clinical evaluation of depsipeptide and other HDACIs in patients with primary and metastatic UM.
葡萄膜黑色素瘤（UM）是成人中最常见的原发性恶性眼肿瘤。目前尚无针对眼内或转移性葡萄膜黑色素瘤的有效化疗方案。因此，研究了组蛋白去乙酰化酶抑制剂（HDACIs）、缩肽、丁酸钠（NaB）和曲古抑菌素A（TSA）在体外诱导UM细胞系凋亡和抑制其细胞生长的能力。
用不同浓度的组蛋白去乙酰化酶抑制剂（HDACIs）体外处理三种原发性和两种转移性UM细胞系。在24孔板中研究细胞增殖。通过流式细胞术研究凋亡诱导情况。通过逆转录聚合酶链反应（RT-PCR）研究Fas/FasL、p21（Waf/Cip1）和p27（Kip1）基因表达的变化。采用蛋白质印迹分析研究组蛋白乙酰化、Fas/FasL、p21（Waf/Cip1）、p27（Kip1）和半胱天冬酶-3蛋白水平。采用实时聚合酶链反应研究bcl-2/bax基因表达的变化。
在所有细胞系中均观察到组蛋白乙酰化呈剂量依赖性增加。这与所有黑色素瘤细胞系中细胞生长的显著抑制和凋亡的诱导呈浓度依赖性相关。蛋白质印迹分析显示半胱天冬酶-3、Fas/FasL、p21（Waf/Cip1）和p27（Kip1）蛋白量呈剂量依赖性增加。然而，实时聚合酶链反应未检测到bcl-2/bax基因表达的变化。
HDACIs是体外原发性和转移性UM细胞生长的有效抑制剂。凋亡可能通过Fas/FasL信号通路介导，而bcl-2似乎未参与其中。这些数据支持对缩肽和其他HDACIs在原发性和转移性UM患者中进行进一步的临床评估。
