Xie Zonggang, Xie Ye, Xu Youjia, Zhou Haibin, Xu Wei, Dong Qirong
Department of Orthopedics, The Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215004, P.R. China.
Mol Med Rep. 2014 Aug;10(2):1103-7. doi: 10.3892/mmr.2014.2281. Epub 2014 May 29.
The purpose of the present study was to investigate the effects of bafilomycin A1 (BafA1) on proliferation, apoptosis and autophagy in MG63 osteosarcoma cells. The growth rate of MG63 cells was determined using a Cell Counting Kit‑8 assay. The mitochondrial membrane potential (Δψ) was measured using a fluorescent probe, JC‑1, and the inhibition of autophagy and apoptosis was monitored by transmission electron microscopy. In addition, the inhibition of autophagy was monitored by western blot analysis of microtubule‑associated protein 1 light chain 3 (LC3), and the ratio of LC3‑II/LC3‑I protein levels was calculated as an indicator of the extent of autophagy. Furthermore, the expression levels of specific proteins associated with autophagy, including p53, Beclin1 and p62, were detected in cultured MG63 cells by western blotting. It was shown that the viability of MG63 cells was inhibited following the use of BafA1, and an induction in the expression levels of the apoptosis‑related protein p53 and the autophagic protein Beclin1 was detected. Furthermore, a collapse in Δψ was observed, together with an induction of apoptotic cell death, following treatment with BafA1. Therefore, following BafA1‑mediated inhibition of autophagy, the inhibition of MG63 cell proliferation and induction of apoptosis were observed.
本研究的目的是探讨巴弗洛霉素A1（BafA1）对MG63骨肉瘤细胞增殖、凋亡和自噬的影响。使用细胞计数试剂盒-8检测法测定MG63细胞的生长速率。使用荧光探针JC-1测量线粒体膜电位（Δψ），并通过透射电子显微镜监测自噬和凋亡的抑制情况。此外，通过对微管相关蛋白1轻链3（LC3）进行蛋白质印迹分析监测自噬的抑制情况，并计算LC3-II/LC3-I蛋白水平的比值作为自噬程度的指标。此外，通过蛋白质印迹法检测培养的MG63细胞中与自噬相关的特定蛋白的表达水平，包括p53、Beclin1和p62。结果显示，使用BafA1后MG63细胞的活力受到抑制，并且检测到凋亡相关蛋白p53和自噬蛋白Beclin1的表达水平升高。此外，用BafA1处理后观察到Δψ崩溃，同时诱导凋亡细胞死亡。因此，在BafA1介导的自噬抑制后，观察到MG63细胞增殖受到抑制并诱导凋亡。
