Hormones Department, National Research Centre, Dokki, Giza, Egypt.
Chemistry Department, Faculty of Science, Cairo University, Cairo, Egypt.
J Steroid Biochem Mol Biol. 2018 Oct;183:125-136. doi: 10.1016/j.jsbmb.2018.06.006. Epub 2018 Jun 10.
A series of pregnenolone derivatives were synthesized and assessed for anti-cancer activity against hepatocellular carcinoma cell line (HepG2). The synthesized hetero-steroids (compounds 3, 4, 5, 6, 7, 8a and 8b) were evaluated for their cytotoxic activities using MTT (3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) assay. Apoptotic activity was assessed using dual acridine orange/ethidium bromide staining method and DNA fragmentation assay. Pro-apoptotic genes (Bax and Bak) and anti-apoptotic genes (Bcl-2 and Bcl-xL) were analyzed using quantitative real time PCR. The results revealed that compounds 4 and 6 displayed cytotoxic activity (IC, 36.97 ± 2.18 and 18.46 ± 0.64 μM, respectively), while compounds 5 and 7 exhibited weak cytotoxic activity (IC, 93.87 ± 8.30 μM and 93.48 ± 4.14 μM, respectively). All synthesized heterocyclic pregnenolone derivatives induced apoptosis through DNA fragmentation. Compounds 4 and 6 increased early and late apoptotic cell percentages while compounds 3, 5, 7 and 8b increased either early or late apoptotic cell percentage. Moreover, compounds 3, 6 and 8b up-regulated the expression level of Bak gene. On the other hand, compounds 4, 5, 7 and 8a down-regulated the Bcl-2 expression level, besides, compounds 5, 7 and 8a down-regulated the Bcl-xL expression level. Compounds 5, 7, 8a and 8b increased the Bak/Bcl-xL ratio, besides, compound 8a raised the Bax/Bcl-xL ratio whereas compound 5 elevated Bax/Bcl-2 and Bak/Bcl-2 ratios. The present work introduced novel pro-apoptotic pregnenolone derivatives that acted against HepG2 cells through DNA fragmentation, apoptotic morphological changes and were able to increase the pro-apoptotic/anti-apoptotic ratios of Bcl-2 family genes. This study particularly revealed that the cytotoxic compound 4 is the most promising pro-apoptotic compound among other synthesized derivatives where it induced apoptosis (late and early) through the down-regulation of Bcl-2 gene expression level.
合成了一系列孕烯醇酮衍生物，并评估了它们对肝癌细胞系（HepG2）的抗癌活性。使用 MTT（3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴盐）测定法评估合成的杂甾体（化合物 3、4、5、6、7、8a 和 8b）的细胞毒性活性。通过双重吖啶橙/溴化乙锭染色法和 DNA 片段化测定法评估凋亡活性。使用实时定量 PCR 分析促凋亡基因（Bax 和 Bak）和抗凋亡基因（Bcl-2 和 Bcl-xL）。结果表明，化合物 4 和 6 表现出细胞毒性活性（IC，分别为 36.97 ± 2.18 和 18.46 ± 0.64 μM），而化合物 5 和 7 表现出较弱的细胞毒性活性（IC，分别为 93.87 ± 8.30 μM 和 93.48 ± 4.14 μM）。所有合成的杂环孕烯醇酮衍生物均通过 DNA 片段化诱导细胞凋亡。化合物 4 和 6 增加了早期和晚期凋亡细胞的百分比，而化合物 3、5、7 和 8b 增加了早期或晚期凋亡细胞的百分比。此外，化合物 3、6 和 8b 上调了 Bak 基因的表达水平。另一方面，化合物 4、5、7 和 8a 下调了 Bcl-2 的表达水平，此外，化合物 5、7 和 8a 下调了 Bcl-xL 的表达水平。化合物 5、7、8a 和 8b 增加了 Bak/Bcl-xL 比值，此外，化合物 8a 增加了 Bax/Bcl-xL 比值，而化合物 5 增加了 Bax/Bcl-2 和 Bak/Bcl-2 比值。本工作介绍了新型的促凋亡孕烯醇酮衍生物，它们通过 DNA 片段化、凋亡形态变化作用于 HepG2 细胞，并能够增加 Bcl-2 家族基因的促凋亡/抗凋亡比值。本研究特别表明，细胞毒性化合物 4 是其他合成衍生物中最有前途的促凋亡化合物，它通过下调 Bcl-2 基因表达水平诱导细胞凋亡（晚期和早期）。
