南方医科大学学报

南方医科大学学报 ›› 2020, Vol. 40 ›› Issue (09): 1288-1294.doi: 10.12122/j.issn.1673-4254.2020.09.10

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紫草素通过抑制人睾丸癌I-10和精原细胞瘤TCAM-2糖酵解诱导细胞死亡

姚 越，张 冲，韩 兵，汤玉锐，熊言骏，汪 盛   

  • 出版日期:2020-09-20 发布日期:2020-09-20

Shikonin induces cell death by inhibiting glycolysis in human testicular cancer I-10 and seminoma TCAM-2 cells

  • Online:2020-09-20 Published:2020-09-20

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摘要： 目的 探究紫草素对睾丸癌细胞I-10和精原细胞瘤TCAM-2死亡形式的影响，并从线粒体功能和糖酵解方面探讨其可能机制。方法 I-10细胞组加入浓度分别为0 μmol/L（对照组）、1.2、1.4、1.6 μmol/L的紫草素，TCAM-2细胞组加入浓度分别为0 μmol/L（对照组）、0.5、1、1.5 μmol/L的紫草素，JC-1试剂盒和ROS试剂盒分别用来检测线粒体膜电位和活性氧的变化；乳酸试剂盒检测胞内乳酸变化；MTT法检测细胞增殖抑制；透射电镜观察细胞死亡形式；Annexin V-FITC/PI双染法检测细胞凋亡；Western blot法检测线粒体通路相关凋亡蛋白Bax、Bcl-2、cleaved caspase 3和自噬相关蛋白LC3B以及糖酵解相关蛋白PKM2、GLUT1、HK2的相对表达水平。结果 MTT结果表明，紫草素能够随时间和剂量依赖性的抑制I-10和TCAM-2细胞的增殖（P<0.05），I-10组24、48、72 h的IC50值分别为1.8、1.36和1.16 μmol/L，TCAM-2组24、48、72 h的IC50值分别为2.37、0.8和0.41 μmol/ L；紫草素能通过下调I-10和TCAM-2细胞线粒体膜电位和增加细胞活性氧水平而影响其线粒体功能（P<0.05），抑制乳酸水平影响细胞糖酵解能力（P<0.05）；透射电镜和Annexin V-FITC/PI双染法检测出紫草素能够诱导I-10和TCAM-2细胞凋亡和过度自噬现象（P<0.05）；Western blot证明，紫草素可以通过下调线粒体通路相关凋亡蛋白Bax、Bcl-2、cleaved caspase 3的表达而影响I-10和TCAM-2细胞线粒体功能，下调PKM2、GLUT1、HK2影响其糖酵功能，通过上调LC3B增加细胞自噬（P<0.05）。结论 紫草素对睾丸癌细胞I-10和TCAM-2具 有增殖抑制及诱导凋亡和增加自噬作用，其机制可能为紫草素影响I-10和TCAM-2细胞能量代谢功能。

关键词: 睾丸癌, 精原细胞瘤, 紫草素, 糖酵解, 死亡形式

Abstract: Objective To investigate the pattern of shikonin-induced cell death in testicular cancer cell I-10 and seminoma TCAM-2 cells and explore the possible mechanism in light of mitochondrial function and glycolysis. Methods I-10 cells treated with 0, 1.2, 1.4 and 1.6 μmol/L shikonin and TCAM-2 cells treated with 0, 0.5, 1 and 1.5 μmol/L shikonin were examined for mitochondrial membrane potential and production of reactive oxygen species (ROS) using JC-1 kit and ROS kit, respectively. The levels of intracellular lactic acid in the cells were detected using a lactic acid kit. The inhibitory effect of shikonin on the proliferation of the cells was assessed with MTT assay. The death patterns of the cells were observed by transmission electron microscopy, and annexin V-FITC/PI double staining was used to detect cell apoptosis. Western blotting was used to detect the relative expression levels of the apoptotic proteins Bax, Bcl-2, and cleaved caspase-3, the autophagy- related protein LC3B and glycolysis- related proteins PKM2, GLUT1 and HK2. Results MTT assay showed that shikonin significantly inhibited the proliferation of I-10 and TCAM-2 cells in a time- and dose-dependent manner (P<0.05). The IC50 values of shikonin in I-10 cells at 24, 48, and 72 h were 1.8, 1.36 and 1.16 μmol/L, as compared with 2.37, 0.8 and 0.41 μmol/L in TCAM-2 cells, respectively. Shikonin treatment significantly reduced mitochondrial membrane potential, increased ROS levels and lower the level of lactic acid in both I-10 and TCAM-2 cells (P<0.05). Transmission electron microscopy and annexin V-FITC/PI double staining demonstrated that shikonin induced apoptosis and excessive autophagy in I-10 and TCAM-2 cells (P<0.05). In both I-10 and TCAM cells, shikonin treatment significantly down- regulated the expressions of Bax, Bcl-2, cleaved caspase-3, PKM2, GLUT1 and HK2, and up-regulated the expression of autophagy-related protein LC3B (P<0.05). Conclusion Shikonin can inhibit the proliferation, induce apoptosis and increase autophagy in both I-10 and TCAM-2 cells probably by affecting energy metabolism of the cells.

Key words: testicular cancer, seminoma, shikonin, glycolysis, cells death pattern