Visagie Michelle Helen, Birkholtz Lyn-Marie, Joubert Anna Margaretha
Department of Physiology, University of Pretoria, Private Bag X 323, Arcadia, 0007 South Africa.
Department of Biochemistry, Centre for Sustainable Malaria Control, University of Pretoria, Private Bag X20, Hatfield, Pretoria 0028 South Africa.
Cell Biosci. 2015 Apr 22;5:19. doi: 10.1186/s13578-015-0010-5. eCollection 2015.
Research involving antimitotic compounds identified 2-methoxyestradiol (2ME2), as a promising anticancer endogenous metabolite. Owing to its low bioavailability, several in silico-designed 2ME2 analogues were synthesized. Structure-activity relationship studies indicated that an already existing 17-β-estradiol analogue, namely (8R,13S,14S,17S)-2-ethyl-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrane-3,17-diyl bis(sulphamate) (EMBS) to exert potential in vitro anticancer activity.
This study investigated the in vitro apoptotic influence of EMBS in an estrogen receptor-positive breast adenocarcinoma epithelial cell line (MCF-7); an estrogen receptor-negative breast epithelial cell line (MDA-MB-231) and a non-tumorigenic breast cell line (MCF-12A). Cell cycle progression, a phosphatidylserine flip, caspase 6-, 7- and 8 enzyme activity levels, Bcl-2 phosphorylation status at serine 70 and Bcl-2- and p53 protein levels were investigated to identify a possible action mechanism for apoptotic induction.
The xCELLigence real-time label-independent approach revealed that EMBS exerted antiproliferative activity in all three cell lines after 24 h of exposure. A G2M block was observed and apoptosis induction was verified by means of flow cytometry using propidium iodide and Annexin V-FITC respectively. EMBS-treated cells demonstrated a reduced mitochondrial membrane potential. EMBS exposure resulted in a statistically significant increase in p53 protein expression, decreased Bcl-2 protein expression and a decrease in pBcl-2(s70) phosphorylation status in all three cell lines. Results support the notion that EMBS induces apoptosis in all three cell lines.
This study includes investigation into the apoptotic hallmarks exerted by EMBS after exposure of three cell lines namely MCF-7-, MDA-MDA-231- and MCF-12A cells. Increased caspase 6-, caspase 7- and caspase 8 activities, upregulation of p53 protein expression and a decrease in phosphorylation status of Bcl-2 at serine 70 in tumorigenic and non-tumorigenic lines were demonstrated.
涉及抗有丝分裂化合物的研究确定2-甲氧基雌二醇（2ME2）是一种有前景的抗癌内源性代谢物。由于其低生物利用度，合成了几种计算机辅助设计的2ME2类似物。构效关系研究表明，一种已有的17-β-雌二醇类似物，即(8R,13S,14S,17S)-2-乙基-13-甲基-7,8,9,11,12,13,14,15,16,17-十氢-6H-环戊[a]菲-3,17-二基双（氨基磺酸酯）（EMBS）具有潜在的体外抗癌活性。
本研究调查了EMBS对雌激素受体阳性乳腺腺癌上皮细胞系（MCF-7）、雌激素受体阴性乳腺上皮细胞系（MDA-MB-231）和非致瘤性乳腺细胞系（MCF-12A）的体外凋亡影响。研究了细胞周期进程、磷脂酰丝氨酸外翻、半胱天冬酶6、7和8的酶活性水平、丝氨酸70处Bcl-2的磷酸化状态以及Bcl-2和p53蛋白水平，以确定凋亡诱导的可能作用机制。
xCELLigence实时非标记方法显示，暴露24小时后，EMBS在所有三种细胞系中均发挥抗增殖活性。观察到G2M期阻滞，并分别使用碘化丙啶和膜联蛋白V-FITC通过流式细胞术验证了凋亡诱导。经EMBS处理的细胞线粒体膜电位降低。在所有三种细胞系中，EMBS暴露导致p53蛋白表达在统计学上显著增加、Bcl-2蛋白表达降低以及pBcl-2(s70)磷酸化状态降低。结果支持EMBS在所有三种细胞系中诱导凋亡的观点。
本研究包括对三种细胞系（即MCF-7、MDA-MDA-231和MCF-12A细胞）暴露于EMBS后所表现出的凋亡特征的研究。结果表明，在致瘤性和非致瘤性细胞系中，半胱天冬酶6、半胱天冬酶7和半胱天冬酶8的活性增加、p53蛋白表达上调以及丝氨酸70处Bcl-2的磷酸化状态降低。
