Pterostilbene induces mitochondrially derived apoptosis in breast cancer cells in vitro
Pterostilbene induces mitochondrially derived apoptosis in breast cancer cells in vitro
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DOI：
10.1016/j.jss.2012.04.027
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发表时间：
2013-04-01
影响因子：
2.2
通讯作者：
McFadden, David
中科院分区：
文献类型：
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作者：
Moon, Dora;McCormack, Denise;McFadden, David
背景：乳腺癌细胞逃避凋亡的能力在肿瘤进展和治疗敏感性中起关键作用。已经发现肿瘤细胞中高水平的Bcl-2相关X蛋白（Bax）促进细胞凋亡并使细胞对抗癌疗法敏感。Bcl-2相关的X蛋白重新分布到线粒体膜导致促凋亡因子的释放，包括细胞色素C、第二线粒体衍生的半胱天冬酶激活剂/具有低PI的凋亡结合蛋白的直接抑制剂（Smac/DIABLO）和Ca 2+。我们的目的是在用天然抗氧化剂3，5-二甲氧基-4-羟基二苯乙烯处理的乳腺癌细胞系中探索这一途径方法：我们在酶联免疫吸附测定中使用来自细胞系MCF-7和MDA-MB-231的全细胞裂解物+/- Bax SiRNA来定量Bax、细胞色素C、Smac/DIABLO表达，和锰超氧化物歧化酶（MnSOD）活性。我们使用来自胞质和线粒体组分的组蛋白相关DNA复合物定量细胞死亡，并使用甲基噻唑四氮唑测定来分析细胞增殖，在沉默或乱序RNA的存在下。我们测量了细胞内钙的变化，使用比率钙敏感染料Fura-2-AM使用倒置的比率monochromaticmicroscope.Results：治疗MCF-7和MDA-MB-231（MDA）细胞与紫檀芪引起浓度依赖性增加细胞内Bax在所有剂量的测试。Bax的RNA沉默导致两种细胞类型的细胞凋亡率降低，并增加细胞存活率时，用紫檀芪处理。我们观察到细胞色素C在MDA细胞处理后，紫檀芪的增加。MCF-7细胞表现出细胞溶质细胞色素C的净增加，与线粒体细胞色素C与50和75 μ mol/L的紫檀芪处理后相应的减少。我们在两种细胞类型中的Smac/DIABLO表达中再次观察到这一点。在MCF-7细胞中，紫檀芪处理引起细胞质中的增加，但线粒体Smac/DIABLO蛋白浓度的降低。紫檀芪能显著提高MDA-MB-231细胞MnSOD活性。最后，pterostilbene导致在胞浆Ca 2 + concentration.Conclusions的显着增加：天然膳食化合物pterostilbene具有抗增殖作用，并诱导凋亡的乳腺癌细胞在体外通过Bax的激活和过度表达，导致MnSOD，Smac/DIABLO，细胞色素C的活性和胞浆Ca 2+超载。(c)2013 Elsevier Inc. All rights reserved.
Background: The ability of a breast cancer cell to evade apoptosis has a key role in tumor progression and sensitivity to treatment. High levels of Bcl-2-associated X protein (Bax) in tumor cells have been found to promote apoptosis and sensitize cells to anti-cancer therapies. Bcl-2-associated X protein redistribution to the mitochondrial membrane results in the release of proapoptotic factors including cytochrome C, second-mitochondrial-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low PI (Smac/DIABLO), and Ca2+. We aimed to explore this pathway in cancerous breast cell lines treated with the naturally occurring antioxidant 3,5-dimethoxy-4-hydroxystilbene (pterostilbene).Methods: We used whole cell lysates +/- Bax SiRNA from the cell lines MCF-7 and MDA-MB-231 in an enzyme-linked immunosorbent assay to quantify Bax, cytochrome C, Smac/DIABLO expression, and manganese superoxide dismutase (MnSOD) activity after treatment with pterostilbene. We quantified cell death using histone-related DNA complexes from cytosolic and mitochondrial fractions and used methylthiazol tetrazolium assay to analyze cell proliferation, in the presence of Bax-silencing or scrambled RNA. We measured changes in cytosolic calcium using the ratiometric calcium-sensitive dye fura-2-AM using an inverted ratiometric monochromator microscope.Results: Treatment of MCF-7 and MDA-MB-231 (MDA) cells with pterostilbene caused concentration-dependent increases in intracellular Bax at all doses tested. RNA silencing of Bax resulted in reduced rates of apoptosis in both cells types and increased cell survival when treated with pterostilbene. We observed an increase in cytochrome C in MDA cells after treatment with pterostilbene. The MCF-7 cells showed a net increase in cytosolic cytochrome C, with a corresponding reduction in mitochondrial cytochrome C after treatment with 50 and 75 mu mol/L pterostilbene. We observed this again in Smac/DIABLO expression in both cell types. In MCF-7 cells, pterostilbene treatment caused an increase in cytosolic but a decrease in mitochondrial Smac/DIABLO protein concentrations. Pterostilbene significantly increase MnSOD activity in MDA-MB-231 cells. Finally, pterostilbene resulted in significant increases in cytosolic calcium concentrations.Conclusions: The natural dietary compound pterostilbene has an anti-proliferative effect and induces apoptosis in breast cancer cells in vitro via Bax activation and overexpression, resulting in increased MnSOD, Smac/DIABLO, and cytochrome C activity and cytosolic Ca2+ overload. (c) 2013 Elsevier Inc. All rights reserved.