Departamento de Biotecnologia y Bioingenieria, Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional, Av. IPN 2508, San Pedro Zacatenco, CDMX, 07360, Mexico.
Laboratorio de Diseno y Desarrollo de Nuevos Farmacos e Innovacion Biotecnologica (Laboratory for the Design and Development of New Drugs and Biotechnological Innovation), Escuela Superior de Medicina, Instituto Politecnico Nacional, Plan de San Luis y Diaz Miron s/n, Casco de Santo Tomas, CDMX, 11340, Mexico.
Curr Mol Pharmacol. 2021 Oct 25;14(4):570-578. doi: 10.2174/1874467213666200730113828.
Valproic acid (VPA) is an HDAC inhibitor (HDACI) with an anticancer activity, but is hepatotoxic. N-(2-hydroxyphenyl)-2-propylpentanamide (o-OH-VPA) is a VPA aryl derivative designed in silico as a selective inhibitor of HDAC8 with biological properties against HeLa, rhabdomyosarcoma and breast cancer cell cultures.
We studied the epigenetic mechanism of o-OH-VPA as an HDACI and evaluated whether it was toxic to normal cells.
HeLa cells and primary human fibroblasts were used for this study as carcinogenic and normal cells, respectively. Cell survival was evaluated by MTT assay, whereas viability and doubling time were determined by the Trypan-blue method. HDAC activity was tested using the colorimetric HDAC activity assay. The expression of p21 was analyzed by PCR and HDAC8 expression was also evaluated by real-time PCR. Cell cycle and caspase-3 activity were analyzed by flow cytometry and caspase-3 colorimetric assay, respectively.
o-OH-VPA (IC = 0.1 mM) was fifty-eight times more effective than VPA (IC = 5.8 mM) to reduce HeLa cell survival. Furthermore, o-OH-VPA increased the doubling time of HeLa cells by 33% with respect to the control. o-OH-VPA acted as HDACI in HeLa cells without affecting the HDAC8 expression, arresting the cell cycle of HeLa cells in the G0/G1 phase due to the increase in p21 expression with the inhibition of caspase-3 activity without exhibiting toxicity toward normal cells.
Our results revealed that o-OH-VPA is an HDACI with a selective effect against HeLa cells but without the known toxicity exerted by most pan-HDACIs on normal cells.
丙戊酸（VPA）是一种具有抗癌活性的组蛋白去乙酰化酶抑制剂（HDACI），但具有肝毒性。N-(2-羟基苯基）-2-丙基戊酰胺（o-OH-VPA）是一种 VPA 芳基衍生物，通过计算机设计为 HDAC8 的选择性抑制剂，对 HeLa、横纹肌肉瘤和乳腺癌细胞培养物具有生物学特性。
我们研究了 o-OH-VPA 作为 HDACI 的表观遗传机制，并评估了它是否对正常细胞有毒。
本研究分别使用 HeLa 细胞和原代人成纤维细胞作为致癌细胞和正常细胞。通过 MTT 测定评估细胞存活率，通过台盼蓝法测定细胞活力和倍增时间。使用比色 HDAC 活性测定法测试 HDAC 活性。通过 PCR 分析 p21 的表达，通过实时 PCR 评估 HDAC8 的表达。通过流式细胞术分析细胞周期和 caspase-3 活性，通过 caspase-3 比色法分析 caspase-3 活性。
o-OH-VPA（IC = 0.1 mM）比 VPA（IC = 5.8 mM）更有效地降低 HeLa 细胞存活率，效力高出 58 倍。此外，o-OH-VPA 使 HeLa 细胞的倍增时间相对于对照增加了 33%。o-OH-VPA 在不影响 HDAC8 表达的情况下在 HeLa 细胞中作为 HDACI 发挥作用，通过抑制 caspase-3 活性和增加 p21 表达使 HeLa 细胞周期停滞在 G0/G1 期，而对正常细胞没有毒性。
我们的结果表明，o-OH-VPA 是一种对 HeLa 细胞具有选择性作用的 HDACI，但对大多数 pan-HDACIs 对正常细胞的已知毒性没有作用。
