Jafary Hanieh, Ahmadian Shahin, Soleimani Masoud
Department of Biochemistry, Institute of Biochemistry and Biophysics, University of Tehran, P.O. Box 13145-1384, Tehran, Iran.
Mol Biol Rep. 2014 Jun;41(6):3801-12. doi: 10.1007/s11033-014-3246-y. Epub 2014 Mar 5.
Histone deacetylase is strongly associated with epigenetic regulation and carcinogenesis, and its inhibitors can induce cell cycle arrest and apoptosis of the cancer cells. In this study we aimed to examine the antiproliferative effects a combination of the valproate with nicotinamide in MIAPaca2 cell line. We revealed that valproate acted in a synergistic/additive with nicotinamide to inhibit the proliferation and induction of apoptosis in MIAPaca2 cancer cell line. MIAPaca2 was treated with various concentrations of valproate. The MTT assay and colony formation in soft agar indicated that valproate at 0.5 mM, when used alone weakly, suppressed proliferation of cells (37 ± 3.02%) whereas the combination treatment of valproate + nicotinamide significantly suppressed cell proliferation (58 ± 3.5%). The effect of nicotinamide at 25 mM on cell proliferation and cell colonization induced 50% apoptosis of MIAPaca2 cells. To identify the anti-proliferation and apoptotic effects of valproate and nicotinamide we performed flow cytometric and microscopic analyses. The results indicated significant apoptosis induction and nuclear morphological alterations greater than when valproate was used alone. Furthermore, western blot analyses was performed to study the role of acetyl-histone H3 levels, and quantitative RNA expression analyses were performed on expression of thrombospondin (TSP) and maspin genes in MIAPaca2. We found that the combination treatment of valproate + nicotinamide enhanced the expression of maspin and TSP genes and the biological response of the cell line was correlated with the increase of histone H3 acetylation after nicotinamide and valproate application. Together our findings indicate that valproate which act as inhibitor of cell proliferation and inducer of apoptosis in human cancer MIAPaca2 cells when used in combination with nicotinamide makes it a potentially good candidate for new anticancer drug development.
组蛋白去乙酰化酶与表观遗传调控和致癌作用密切相关，其抑制剂可诱导癌细胞的细胞周期停滞和凋亡。在本研究中，我们旨在检测丙戊酸盐与烟酰胺联合使用对MIAPaca2细胞系的抗增殖作用。我们发现丙戊酸盐与烟酰胺协同/相加作用，抑制MIAPaca2癌细胞系的增殖并诱导其凋亡。用不同浓度的丙戊酸盐处理MIAPaca2细胞。MTT法和软琼脂集落形成实验表明，单独使用0.5 mM丙戊酸盐时对细胞增殖的抑制作用较弱（37±3.02%），而丙戊酸盐+烟酰胺联合处理则显著抑制细胞增殖（58±3.5%）。25 mM烟酰胺对细胞增殖和细胞集落形成的作用诱导了MIAPaca2细胞50%的凋亡。为了确定丙戊酸盐和烟酰胺的抗增殖和凋亡作用，我们进行了流式细胞术和显微镜分析。结果表明，与单独使用丙戊酸盐相比，联合处理诱导的凋亡更显著，核形态改变更大。此外，进行了蛋白质印迹分析以研究乙酰化组蛋白H3水平的作用，并对MIAPaca2中血小板反应蛋白（TSP）和丝氨酸蛋白酶抑制剂（maspin）基因的表达进行了定量RNA表达分析。我们发现丙戊酸盐+烟酰胺联合处理增强了maspin和TSP基因的表达，并且在应用烟酰胺和丙戊酸盐后，细胞系的生物学反应与组蛋白H3乙酰化的增加相关。我们共同的研究结果表明，丙戊酸盐在与烟酰胺联合使用时，作为人癌MIAPaca2细胞中细胞增殖的抑制剂和凋亡诱导剂，使其成为新型抗癌药物开发的潜在良好候选物。
