Solamargine triggers cellular necrosis selectively in different types of human melanoma cancer cells through extrinsic lysosomal mitochondrial death pathway.
Solamargine triggers cellular necrosis selectively in different types of human melanoma cancer cells through extrinsic lysosomal mitochondrial death pathway.
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DOI：
10.1186/s12935-016-0287-4
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发表时间：
2016
影响因子：
5.8
通讯作者：
Adham SA
中科院分区：
文献类型：
--
作者：
Al Sinani SS;Eltayeb EA;Coomber BL;Adham SA
先前的报告表明，类固醇糖生物碱 Solamargine 可抑制非黑色素瘤皮肤癌细胞的增殖。然而，Solamargine 并未针对不同类型的黑色素瘤细胞进行系统测试，也未同时针对正常细胞进行测试。在本研究中，我们旨在研究 Solamargine 的作用以及抑制不同类型黑色素瘤细胞生长的机制。在正常细胞和另外三种黑色素瘤细胞系上测试了 Solamargine 的效果。分别使用垂直生长期转移性和原发性黑色素瘤细胞系WM239和WM115以及径向生长期良性黑色素瘤细胞WM35。使用 Alamarblue 测定法测定 Solamargine 的半数抑制浓度 IC50。分别使用光显微镜和透射电子显微镜评估细胞和亚细胞的变化。使用流式细胞术测量经历凋亡和坏死的细胞的百分比。使用蛋白质印迹法检测和测量不同的蛋白质表达。通过进行克隆形成测定来确定 Solamargine 的功效。对收集的数据进行统计分析，对至少三次独立重复实验的三次重复的平均值进行统计分析，对参数数据使用单向方差分析检验，对非参数数据使用 Kruskal-Wallis 检验。当 P 值小于 0.05 时，认为差异显着。因此，我们证明 Solamargine 能够快速、选择性和有效地分别抑制转移性和原发性黑色素瘤细胞 WM239 和 WM115 的生长，而对正常和良性 WM35 细胞的影响最小。 Solamargine 通过快速诱导溶酶体膜通透作用，引起两种恶性黑色素瘤细胞系（WM115、WM239）的细胞坏死，组织蛋白酶 B 上调证实了这一点，触发了以细胞色素 c 释放和 TNFR1 上调为代表的外源性线粒体死亡途径。仅在 WM115 和 WM239 细胞中，Solamargine 破坏了 hILP/XIAP 下调所揭示的内在凋亡途径，导致 caspase-3 裂解、Bcl-xL 和 Bcl2 上调以及 Apaf-1 和 Bax 下调。 Solamargine 在体外表现出高效，特别是针对垂直生长期黑色素瘤细胞。我们的研究结果表明 Solamargine 是一种有前途的抗恶性黑色素瘤药物，值得进一步关注。本文的在线版本 (doi:10.1186/s12935-016-0287-4) 包含补充材料，可供授权用户使用。
Previous reports showed that the Steroidal Glycoalkaloid Solamargine inhibited proliferation of non-melanoma skin cancer cells. However, Solamargine was not tested systematically on different types of melanoma cells and was not simultaneously tested on normal cells either. In this study we aimed to investigate the effect of Solamargine and the mechanism involved in inhibiting the growth of different types of melanoma cells. Solamargine effect was tested on normal cells and on another three melanoma cell lines. Vertical growth phase metastatic and primary melanoma cell lines WM239 and WM115, respectively and the radial growth phase benign melanoma cells WM35 were used. The half inhibitory concentration IC50 of Solamargine was determined using Alamarblue assay. The cellular and subcellular changes were assessed using light and Transmission Electron Microscope, respectively. The percentage of cells undergoing apoptosis and necrosis were measured using Flow cytometry. The different protein expression was detected and measured using western blotting. The efficacy of Solamargine was determined by performing the clonogenic assay. The data collected was analyzed statistically on the means of the triplicate of at least three independent repeated experiments using one-way ANOVA test for parametric data and Kruskal–Wallis for non-parametric data. Differences were considered significant when the P values were less than 0.05. Hereby, we demonstrate that Solamargine rapidly, selectively and effectively inhibited the growth of metastatic and primary melanoma cells WM239 and WM115 respectively, with minimum effect on normal and benign WM35 cells. Solamargine caused cellular necrosis to the two malignant melanoma cell lines (WM115, WM239), by rapid induction of lysosomal membrane permeabilization as confirmed by cathepsin B upregulation which triggered the extrinsic mitochondrial death pathway represented by the release of cytochrome c and upregulation of TNFR1. Solamargine disrupted the intrinsic apoptosis pathway as revealed by the down regulation of hILP/XIAP, resulting in caspase-3 cleavage, upregulation of Bcl-xL, and Bcl2, and down regulation of Apaf-1 and Bax in WM115 and WM239 cells only. Solamargine showed high efficacy in vitro particularly against the vertical growth phase melanoma cells. Our findings suggest that Solamargine is a promising anti-malignant melanoma drug which warrants further attention. The online version of this article (doi:10.1186/s12935-016-0287-4) contains supplementary material, which is available to authorized users.