Objective: To investigate the effects of autophagy inhibitor ROC-325 and its combination with bortezomib on the proliferation, apoptosis and autophagy of multiple myeloma cell lines.
Methods: Multiple myeloma cells were treated with ROC-325 at different concentration. The cell proliferation was detected by CCK-8. Apoptosis was determined by Caspase-3/7 and Caspase-9 activity assays. Autophagy was detected by monodansylcadaverine staining. The apoptosis-related proteins (PARP and Caspase-3) and autophagy-related proteins (P62, Beclin-1, and LC3A/B) were analyzed by Western blot. The combined effect with bortezomib on bortezomib-resistant cell line was detected by CCK-8.
Results: ROC-325 inhibited the proliferation of RPMI 8226, RPMI 8226-BTZ100, U266 and IM9 cells in a dose-dependent manner (r=-0.8275, r=-0.9079, r=-0.9422, r=-0.9305), the 72 h IC50 values were 2.795, 4.020, 5.432 and 4.755 μmol/L, respectively. The activity assays of Caspase-3/7 and Caspase-9 showed that their relative activity was increased gradually in proportion to the drug concentration with the statistically significant difference (r=0.9648, r=0.9377, r=0.9318; r=0.9087, r=0.9431, r=0.8914). MDC staining results showed that the number of autophagic vacuoles increased with the rise of ROC-325 concentration (r=0.9565, r=0.9373, r=0.9233). ROC-325 could increase the expression of apoptosis-related proteins (PARP and Caspase-3) and autophagy-related proteins (P62 and LC3-Ⅱ/LC3-Ⅰ), but decrease the expression of Beclin-1 detected by Western blot. The CCK-8 assay showed that ROC-325 combined with bortezomib had synergistic effect on the inhibition of drug resistant cell line RPMI 8226-BTZ100.
Conclusion: ROC-325 can inhibit the proliferation, induce the apoptosis of myeloma cells through the mitochondrial pathway, inhibit the autophagy of myeloma cells by affecting the fusion of autophagosomes and lysosomes, and overcome bortezomib resistance by the combination of ROC-325 with bortezomib.
题目: 自噬抑制剂ROC-325抗多发性骨髓瘤作用的研究.
目的: 探讨自噬抑制剂ROC-325对人多发性骨髓瘤细胞株增殖、凋亡、自噬的影响及其与硼替佐米联合用药的细胞学效应.
方法: 不同浓度的ROC-325作用于骨髓瘤细胞，用CCK-8法检测药物对细胞增殖的作用，用Caspase-3/7、Caspase-9活性测定法检测药物对细胞凋亡的影响，用单丹磺酰戊二胺染色观察自噬小体数量，Western blot检测各组细胞凋亡相关蛋白（PARP和Caspase-3）和自噬相关蛋白（P62、Beclin-1和LC3A/B）的表达情况，用CCK-8法检测ROC-325联合硼替佐米对硼替佐米耐药细胞株的抑制作用.
结果: ROC-325可呈剂量依赖性抑制RPMI 8226、RPMI 8226-BTZ100、U266和IM9细胞增殖（r=-0.8275，r=-0.9079，r=-0.9422，r=-0.9305），72 h IC50 分别为2.795、4.020、5.432和4.755 μmol/L。Caspase-3/7、Caspase-9活性结果显示，随着药物浓度的增加，Caspase-3/7和Caspase-9相对活性逐渐升高（r=0.9648，r=0.9377，r=0.9318；r=0.9087，r=0.9431，r=0.8914）。单丹磺酰戊二胺染色结果显示，细胞内自噬泡数量随着ROC-325浓度的提高逐渐增多（r=0.9565，r=0.9373，r=0.9233）。Western blot结果显示，ROC-325可诱导凋亡相关蛋白PARP、Caspase-3表达上调以及自噬相关蛋白P62、LC3-Ⅱ/LC3-Ⅰ表达上调，Beclin-1表达下调。ROC-325联合硼替佐米的CCK-8法检测结果显示，ROC-325与硼替佐米对耐药细胞株RPMI 8226-BTZ100的抑制具有协同作用.
结论: ROC-325可以抑制骨髓瘤细胞增殖，通过线粒体途径诱导骨髓瘤细胞的凋亡，通过影响自噬体和溶酶体的融合抑制骨髓瘤细胞的自噬，ROC-325联合硼替佐米后可以克服硼替佐米耐药.