Gyovai András, Minorics Renáta, Kiss Anita, Mernyák Erzsébet, Schneider Gyula, Szekeres András, Kerekes Erika, Ocsovszki Imre, Zupkó István
Department of Pharmacodynamics and Biopharmacy, University of Szeged, Szeged, Hungary.
Department of Organic Chemistry, University of Szeged, Szeged, Hungary.
Front Pharmacol. 2018 Jul 27;9:825. doi: 10.3389/fphar.2018.00825. eCollection 2018.
19-Nortestosterone C-17 epimers with prominent antiproliferative properties have been previously described. In our present study, five novel 17α-19-nortestosterones () were synthesized to increase their beneficial biological activities with no associated undesired hormonal effects. The compounds were screened by a viability assay against a panel of human adherent gynecological cancer cell lines. Three of the tested derivatives (-) exhibited a remarkable inhibitory effect on the proliferation of HeLa cells with IC values lower than that of our reference agent cisplatin (CIS). These three active agents also displayed considerable cancer selectivity as evidenced by their weaker growth inhibitory effect on non-cancerous fibroblast cells compared to CIS. The most potent newly synthesized 17α-chloro derivative () was selected for additional experiments in order to characterize its mechanism of action. Since nandrolone (19-nortestosterone, ) is a structural analog with selective antiproliferative action on cervical carcinoma cells, it was utilized as a positive control in these studies. A lactate dehydrogenase (LDH) assay demonstrated a moderate cytotoxic effect of the test compounds. Cell cycle disturbance and the elevation of the hypodiploid population elicited by the test agents were detected by flow cytometry following propidium staining. The proapoptotic effects of the tested steroids were confirmed by fluorescent microscopy and a caspase-3 activity assay. Treatment-related caspase-9 activation without a substantial change in caspase-8 activity indicates the induction of the intrinsic apoptotic pathway. The selected agents directly influence the rate of tubulin assembly as evidenced by a polymerization assay. Yeast-based reporter gene assay revealed that the androgenic activity of the novel 19-nortestosterone derivative is by multiple orders of magnitude weaker than that of the reference agent . Based on the behavior of the examined compounds it can be concluded that a halogen substitution of the 19-nortestosterone scaffold at the 17α position may produce compounds with unique biological activities. The results of the present study support that structurally modified steroids with negligible hormonal activity are a promising basis for the research and development of novel anticancer agents.
先前已描述过具有显著抗增殖特性的19-去甲睾酮C-17差向异构体。在我们目前的研究中，合成了五种新型17α-19-去甲睾酮，以增强其有益的生物学活性，且无相关不良激素作用。通过针对一组人贴壁妇科癌细胞系的活力测定对这些化合物进行筛选。三种测试衍生物对HeLa细胞的增殖表现出显著的抑制作用，其IC值低于我们的参考药物顺铂（CIS）。与CIS相比，这三种活性剂对非癌性成纤维细胞的生长抑制作用较弱，也显示出相当的癌症选择性。选择最有效的新合成的17α-氯衍生物进行额外实验，以表征其作用机制。由于诺龙（19-去甲睾酮）是对宫颈癌细胞具有选择性抗增殖作用的结构类似物，因此在这些研究中用作阳性对照。乳酸脱氢酶（LDH）测定显示测试化合物具有中等细胞毒性作用。经碘化丙啶染色后，通过流式细胞术检测测试剂引起的细胞周期紊乱和亚二倍体群体增加。通过荧光显微镜和半胱天冬酶-3活性测定证实了测试类固醇的促凋亡作用。与治疗相关的半胱天冬酶-9激活而半胱天冬酶-8活性无实质性变化表明诱导了内源性凋亡途径。聚合测定表明，所选药物直接影响微管蛋白组装速率。基于酵母的报告基因测定显示，新型19-去甲睾酮衍生物的雄激素活性比参考药物弱多个数量级。根据所研究化合物的行为可以得出结论，19-去甲睾酮支架在17α位的卤素取代可能产生具有独特生物学活性的化合物。本研究结果支持，激素活性可忽略不计的结构修饰类固醇是新型抗癌药物研发的有前途的基础。
