Shikonin kills glioma cells through necroptosis mediated by RIP-1.
Shikonin kills glioma cells through necroptosis mediated by RIP-1.
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DOI：
10.1371/journal.pone.0066326
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Ge P
中科院分区：
文献类型：
--
作者：
Huang C;Luo Y;Zhao J;Yang F;Zhao H;Fan W;Ge P
紫草素可诱导白血病细胞坏死性凋亡，但可诱导胶质瘤细胞凋亡。因此，紫草素是否能引起胶质瘤细胞的坏死性凋亡及其机制有待进一步研究。本实验采用紫草素与大鼠C6胶质瘤细胞系和人U87胶质瘤细胞系进行实验。MTT法检测细胞活力。流式细胞术结合Annexin V-FITC和PI双染色分析细胞死亡模式。应用Hoechst 33342和PI双染荧光显微镜和透射电镜观察紫草素对C6胶质瘤细胞形态学的影响。采用氧化还原敏感染料DCFH-DA检测细胞内活性氧水平，Western blotting检测细胞凋亡相关蛋白RIP-1的表达水平。紫草素诱导C6和U87胶质瘤细胞死亡，呈剂量和时间依赖性。坏死凋亡抑制剂necrotatin-1可抑制C6和U87胶质瘤细胞的死亡，而pan-caspase抑制剂z-VAD-favor则不能抑制C6和U87胶质瘤细胞死亡。紫草素处理的C6胶质瘤细胞在形态学上表现为胞浆电子透明，质膜完整性丧失，核膜完整。Necrostatin-1可抑制紫草素引起的ROS水平升高，而抗氧化剂NAC阻断ROS可挽救C6和U87胶质瘤细胞中紫草素诱导的细胞死亡。紫草素还能上调RIP-1的表达，且呈剂量和时间依赖性，NAC则抑制RIP-1的表达。我们证明紫草素在C6和U87胶质瘤细胞中引起的细胞死亡主要是通过坏死性凋亡。此外，RIP-1通路和氧化应激均参与了紫草素诱导的坏死性凋亡的激活过程。
Shikonin was reported to induce necroptosis in leukemia cells, but apoptosis in glioma cell lines. Thus, it is needed to clarify whether shikonin could cause necroptosis in glioma cells and investigate its underlying mechanisms. Shikonin and rat C6 glioma cell line and Human U87 glioma cell line were used in this study. The cellular viability was assayed by MTT. Flow cytometry with annexin V-FITC and PI double staining was used to analyze cellular death modes. Morphological alterations in C6 glioma cells treated with shikoinin were evaluated by electronic transmission microscopy and fluorescence microscopy with Hoechst 33342 and PI double staining. The level of reactive oxygen species was assessed by using redox-sensitive dye DCFH-DA. The expressional level of necroptosis associated protein RIP-1 was analyzed by western blotting. Shikonin induced cell death in C6 and U87 glioma cells in a dose and time dependent manner. The cell death in C6 and U87 glioma cells could be inhibited by necroptosis inhibitor necrotatin-1, not by pan-caspase inhibitor z-VAD-fmk. Shikonin treated C6 glioma cells presented electron-lucent cytoplasm, loss of plasma membrane integrity and intact nuclear membrane in morphology. The increased ROS level caused by shikonin was attenuated by necrostatin-1 and blocking ROS by anti-oxidant NAC rescued shikonin-induced cell death in both C6 and U87 glioma cells. Moreover, the expressional level of RIP-1 was up-regulated by shikonin in a dose and time dependent manner as well, but NAC suppressed RIP-1 expression. We demonstrated that the cell death caused by shikonin in C6 and U87 glioma cells was mainly via necroptosis. Moreover, not only RIP-1 pathway, but also oxidative stress participated in the activation of shikonin induced necroptosis.
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