Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2021 Jun;29(3):797-804. doi: 10.19746/j.cnki.issn.1009-2137.2021.03.023.
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.