Research On The Targets Of Anti-multiple Myeloma Cells Proliferation Using Two New Natural Medicinal Monomers

Part I. Effects of gambogic acid on the activation of caspase-3and downregulation of SIRT1in RPMI-8226multiple myeloma cells via the accumulation of ROSAim:To investigate the effect of gambolgic acid (GA) on the proliferation and apoptosis of multiple myeloma (MM) RPMI-8226cells and explore the underlying mechanisms.Methods:The effect of GA on the proliferation of RPMI-8226cells was assayed by3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. Annexin V-FITC/propidium iodide (PI) double-labeled flow cytometry (FCM) and Hoechst33258staining were applied to detect the apoptosis rate and nuclear fragmentation of RPMI-8226cells treated with GA. The intracellular reactive oxygen species (ROS) generation of cell was estimated by FCM using the2’,7’-dichlorfluorescein-diacetate (DCFH-DA), while the protein levels of activated caspase-3, PARP, and SIRT1were detected by western blot.Results:GA presents a potent activity to inhibit the proliferation and induce the apoptosis of RPMI-8226cells in a dose-dependent manner. Besides, GA results in the accumulation of intracellular ROS, activation of caspase-3, cleavage of PARP, and downregulation of SIRT1. Moreover, ROS scavenger N-acetylcysteine (NAC) released the accumulation of ROS and blocked the apoptosis in RPMI-8226cells induced by GA. At the same time, NAC antagonised the capacity of GA to activate caspase-3and downregulate SIR1expression.Conclusion:GA exerted potent potential to induce RPMI-8226cells apoptosis mainly by the mechanisms of contributing to the intracellular ROS accumulation, followed by the downregulation of SIRT1and activation of caspase-3. These suggested that GA could be a promising new experimental anticancer agent in the treatment of MM. Part II. Betulinic acid inhibits autophagic flux and induces apoptosis in human multiple myeloma cells in vitroAim:To investigate the effects of betulinic acid (BA) on apoptosis and autophagic flux in multiple myeloma cells and the relationship between the two processes.Methods:The proliferation of human multiple myeloma KM3cells was measured with MTT assay. FITC/PI double-labeled flow cytometry (FCM) and Hoechst33258staining were used to analyze the cell apoptosis. Caspase-3, PARP, Beclinl, LC3-II, and P62were detected using Western blotting.Results:Treatment of KM3cells with BA (5-25μg/mL) suppressed the cell proliferation in time-and dose-dependent manners. The IC50values at12,24, and36h were22.29,17.36, and13.06μg/mL, respectively. BA treatment dose-dependently induced apoptosis of KM3cells, which was associated with the activation of caspase3. However, Z-DEVD-FMK, a specific inhibitor of caspase3, did not decrease, but rather sensitized the cells to BA-induced apoptosis, suggesting an alternative mechanism involved. On other hand, BA treatment dose-dependently increased the accumulation of LC3-Ⅱ and P62in KM3cells, representing the inhibition of autophagic flux. Furthermore, BA treatment dose-dependently downregulated the expression of Beclin1, an important inducer of autophagy, in KM3cells. In the presence of BA, Z-DEVD-FMK induced autophagy and increased the amount of LC3-Ⅱ in KM3cells, which may occur via attenuating BA-induced decrease in the level of Beclin1. Similarly, rapamycin, an autophagy inducer, increased the amount of LC3-Ⅱ in KM3cells. In the presence of BA, rapamycin caused further increase in the amount of LC3-Ⅱ. Furthermore, rapamycin sensitized BA-treated KM3cells to apoptosis.Conclusion:The results demonstrate that BA induces apoptosis and blocks autophagic flux in KM3cells. Furthermore, in addition to activation of caspase3, the inhibition of autophagic flux also contributes to the BA-mediated apoptosis of KM3cells.