Wang Ji-Zhen, Chen Jun-Min, Zeng Zhi-Yong, Qiu Dong-Biao
Department of Hematology and Rheumatology, The First Affiliated Hospital of Fujian Medical University, First Clinical Medical College of Fujian Medical University, Fuzhou 350035, Fujian Province, China.
Department of Hematology and Rheumatology, The First Affiliated Hospital of Fujian Medical University, First Clinical Medical College of Fujian Medical University, Fuzhou 350035, Fujian Province, China. E-mail:
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2018 Jun;26(3):817-823. doi: 10.7534/j.issn.1009-2137.2018.03.031.
To investigate the effects of autophagy activator (rapamycin, RAPA) and autophagy inhibitor (hydroxychloroquine, HCQ and 3-methyl adenine, 3-MA) on the proliferation, apoptosis and autophagy of multiple myeloma cell line of RPMI8226.
RPMI8226 cells were treated with autophagy regulating drugs of different concentrations. The proliferation and apoptosis of cells were determined by CCK-8 and flow cytometry, respectively. The expressions of apoptosis-related proteins BCL-2, caspase-3 and PARP protein were assessed by Western blot. Autophagy was detected by monodansylcadaverine staining. Autophagic protein (LC-3b) and apoptosis-related proteins (caspase-3, PARP and BCL-2) were analyzed by Western blot.
RAPA and HCQ inhibited the proliferation of RPMI8226 in a concentration- and time-dependent manner, and increased the apoptosis. However, 3-MA did not show significantly inhibitory effect on the proliferation and apoptosis of RPMI8226. MDC staining showed that the more autophagic vacuoles could be detected in the higher concentration of RAPA, but the less autophagic vacuoles in the higher concentration of HCQ and 3-MA. Western blot showed that RAPA increased the expression of LC3-II/LC3-I, caspase-3 and PARP, but inhibited the expression of BCL-2. HCQ inhibited the expression of LC3-II/LC3-I and BCL-2, but increased the expression of caspase-3 and PARP. 3-MA inhibited the expression of LC3-II/LC3-I, but had no effect on the expression of caspase-3, PARP or BCL-2.
Rapamycin can inhibit the proliferation, induce apoptosis and autophagy of RPMI 8226, the hydroxychloroquine can inhibit autophagy and proliferation of RPMI 8226, and induce apoptosis, the 3-MA can inhibit autophagy of RPMI 8226, but hardly has any effects on proliferation and apoptosis of RPMI 8226 cells.
探讨自噬激活剂（雷帕霉素，RAPA）和自噬抑制剂（羟氯喹，HCQ和3-甲基腺嘌呤，3-MA）对多发性骨髓瘤细胞系RPMI8226增殖、凋亡及自噬的影响。
用不同浓度的自噬调节药物处理RPMI8226细胞。分别采用CCK-8法和流式细胞术检测细胞的增殖和凋亡情况。通过蛋白质免疫印迹法评估凋亡相关蛋白BCL-2、半胱天冬酶-3和聚（ADP-核糖）聚合酶（PARP）蛋白的表达。采用单丹磺酰尸胺染色检测自噬。通过蛋白质免疫印迹法分析自噬蛋白（LC-3b）和凋亡相关蛋白（半胱天冬酶-3、PARP和BCL-2）。
RAPA和HCQ以浓度和时间依赖性方式抑制RPMI8226的增殖，并增加凋亡。然而，3-MA对RPMI8226的增殖和凋亡未显示出明显的抑制作用。单丹磺酰尸胺染色显示，在较高浓度的RAPA中可检测到更多的自噬泡，但在较高浓度的HCQ和3-MA中自噬泡较少。蛋白质免疫印迹法显示，RAPA增加了LC3-II/LC3-I、半胱天冬酶-3和PARP的表达，但抑制了BCL-2的表达。HCQ抑制了LC3-II/LC3-I和BCL-2的表达，但增加了半胱天冬酶-3和PARP的表达。3-MA抑制了LC3-II/LC3-I的表达，但对半胱天冬酶-3、PARP或BCL-2的表达无影响。
雷帕霉素可抑制RPMI 8226的增殖，诱导其凋亡和自噬；羟氯喹可抑制RPMI 8226的自噬和增殖，并诱导凋亡；3-MA可抑制RPMI 8226的自噬，但对RPMI 8226细胞的增殖和凋亡几乎没有影响。
