黄腐酚诱导大鼠神经胶质瘤 C6 细胞死亡通过触发线粒体应激。
Xanthohumol-Induced Rat Glioma C6 Cells Death by Triggering Mitochondrial Stress.
机构信息
Center for Mitochondria and Healthy Aging, College of Life Science, Yantai University, Yantai 264005, China.
出版信息
Int J Mol Sci. 2021 Apr 26;22(9):4506. doi: 10.3390/ijms22094506.
AIM
To investigate the underlying mechanisms of xanthohumol (XN) on the proliferation inhibition and death of C6 glioma cells.
METHODS
To determine the effects of XN on C6 cells, cell proliferation and mortality after XN treatment were assessed by SRB assay and trypan blue assay respectively. Apoptotic rates were evaluated by flowcytometry after Annexin V-FITC/PI double staining. The influence of XN on the activity of caspase-3 was determined by Western blot (WB); and nuclear transposition of apoptosis-inducing factor (AIF) was tested by immunocytochemistry and WB. By MitoSOX staining, the mitochondrial ROS were detected. Mitochondrial function was also tested by MTT assay (content of succinic dehydrogenase), flow cytometry (mitochondrial membrane potential (MMP)-JC-1 staining; mitochondrial abundance-mito-Tracker green), immunofluorescence (MMP-JC-1 staining; mitochondrial morphology-mito-Tracker green), WB (mitochondrial fusion-fission protein-OPA1, mfn2, and DRP1; mitophagy-related proteins-Pink1, Parkin, LC3B, and P62), and high-performance liquid chromatography (HPLC) (energy charge). Finally, mitochondrial protein homeostasis of C6 cells after XN treatment with and without LONP1 inhibitor bortezomib was investigated by trypan blue assay (proliferative activity and mortality) and WB (mitochondrial protease LONP1). All cell morphology images were taken by a Leica Microsystems microscope.
RESULTS
XN could lead to proliferation inhibition and death of C6 cells in a time- and dose-dependent manner and induce apoptosis of C6 cells through the AIF pathway. After long incubation of XN, mitochondria of C6 cells were seriously impaired, and mitochondria had a diffuse morphology and mitochondrial ROS were increased. The content of succinic dehydrogenase per cell was significantly decreased after XN insults of 24, 48, and 72 h. The energy charge was weakened after XN insult of 24 h. Furthermore, the MMP and mitochondrial abundance were significantly decreased; the protein expression levels of OPA1, mfn2, and DRP1 were down-regulated; and the protein expression levels of Pink1, Parkin, LC3B-II/LC3B-I, and p62 were up-regulated in long XN incubation times (24, 48, and 72 h). XN incubation with bortezomib for 48 h resulted in lower proliferative activity and higher mortality of C6 cells and caused the cell to have visible vacuoles. Moreover, the protein expression levels of LONP1 was up-regulated gradually as XN treatment time increased.
CONCLUSION
These data supported that XN could induce AIF pathway apoptosis of the rat glioma C6 cells by affecting the mitochondria.
目的
研究黄腐酚（XN）对 C6 神经胶质瘤细胞增殖抑制和死亡的作用机制。
方法
通过 SRB 测定和台盼蓝测定分别评估 XN 对 C6 细胞的影响。用 Annexin V-FITC/PI 双染后通过流式细胞术评估细胞凋亡率。用 Western blot（WB）检测 XN 对 caspase-3 活性的影响；通过免疫细胞化学和 WB 检测凋亡诱导因子（AIF）的核易位。通过 MitoSOX 染色检测线粒体 ROS。通过 MTT 测定（琥珀酸脱氢酶含量）、流式细胞术（线粒体膜电位（MMP）-JC-1 染色；线粒体丰度-线粒体追踪绿色）、免疫荧光（MMP-JC-1 染色；线粒体形态-线粒体追踪绿色）、WB（线粒体融合-分裂蛋白-OPA1、mfn2 和 DRP1；线粒体自噬相关蛋白-Pink1、Parkin、LC3B 和 P62）和高效液相色谱法（HPLC）（能量电荷）检测线粒体功能。最后，用 LONP1 抑制剂硼替佐米处理和不处理 XN 的 C6 细胞的线粒体蛋白稳态，通过台盼蓝测定（增殖活性和死亡率）和 WB（线粒体蛋白酶 LONP1）进行研究。所有细胞形态图像均由徕卡显微镜拍摄。
结果
XN 可呈时间和剂量依赖性抑制 C6 细胞增殖并诱导其死亡，并通过 AIF 途径诱导 C6 细胞凋亡。XN 孵育时间较长时，C6 细胞的线粒体严重受损，线粒体形态弥散，线粒体 ROS 增加。XN 作用 24、48 和 72 h 后，每个细胞的琥珀酸脱氢酶含量明显降低。XN 作用 24 h 后，能量电荷减弱。此外，MMP 和线粒体丰度显著降低；OPA1、mfn2 和 DRP1 的蛋白表达水平下调；Pink1、Parkin、LC3B-II/LC3B-I 和 p62 的蛋白表达水平上调，XN 孵育时间较长（24、48 和 72 h）。XN 与硼替佐米孵育 48 h 后，C6 细胞的增殖活性降低，死亡率升高，细胞出现可见空泡。此外，随着 XN 处理时间的增加，LONP1 的蛋白表达水平逐渐升高。
结论
这些数据支持 XN 通过影响线粒体诱导大鼠神经胶质瘤 C6 细胞 AIF 通路凋亡。
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