Betulinic acid inhibits autophagic flux and induces apoptosis in human multiple myeloma cells in vitro

Abstract

Aim: 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 KM3 cells was measured with MTT assay. FITC/PI double-labeled flow cytometry (FCM) and Hoechst 33258 staining were used to analyze the cell apoptosis. Caspase 3, PARP, Beclin1, LC3-II, and P62 were detected using Western blotting. Results: Treatment of KM3 cells with BA (5−25 μg/mL) suppressed the cell proliferation in time-and dose-dependent manners. The IC 50 values at 12, 24, and 36 h were 22.29, 17.36, and 13.06 μg/mL, respectively. BA treatment dose-dependently induced apoptosis of KM3 cells, which was associated with the activation of caspase 3. However, Z-DEVD-FMK, a specific inhibitor of caspase 3, 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 P62 in KM3 cells, representing the inhibition of autophagic flux. Furthermore, BA treatment dose-dependently downregulated the expression of Beclin 1, an important inducer of autophagy, in KM3 cells. In the presence of BA, Z-DEVD-FMK induced autophagy and increased the amount of LC3-II in KM3 cells, which may occur via attenuating BA-induced decrease in the level of Beclin 1. Similarly, rapamycin, an autophagy inducer, increased the amount of LC3-II in KM3 cells. In the presence of BA, rapamycin caused further increase in the amount of LC3-II. Furthermore, rapamycin sensitized BA-treated KM3 cells to apoptosis. Conclusion:The results demonstrate that BA induces apoptosis and blocks autophagic flux in KM3 cells. Furthermore, in addition to activation of caspase 3, the inhibition of autophagic flux also contributes to the BA-mediated apoptosis of KM3 cells.

Betulinic Acid and its Derivatives as Potential Antitumor Agents

“…Furthermore, BA combined with rapamycin, an autophagic inducer, causes the synergic accumulation of p62 and LC3‐II and induction of apoptosis. Collectively, BA may have a different effect in the process of autophagy in different cancer cell lines …”

“…Collectively, BA may have a different effect in the process of autophagy in different cancer cell lines. 136 B10, a glycosylated derivative of BA, not only induces apoptosis, but also induces autophagy. B10 destabilizes lysosomes and releases lysosomal hydrolases, cathepsin B and Z, to the cytoplasm and induces cell death.…”

Betulinic acid-induced mitochondria-dependent cell death is counterbalanced by an autophagic salvage response

“… 17 , 22 Previously, BetA and a derivative of BetA, B10, have been shown to induce autophagosome formation in multiple myeloma cells and glioblastoma cells. 23 , 24 In these studies it was suggested that the autophagic flux was prevented, leading to an accumulation of undigested autophagosomes. Although both studies observed autophagy, the role of autophagy as a cell death mechanism was not addressed for BetA.…”

“…Although both studies observed autophagy, the role of autophagy as a cell death mechanism was not addressed for BetA. 23 , 24 …”

Anti‑cancer effects of a novel Pan‑RAF inhibitor in a hepatocellular carcinoma cell line

“…Early apoptotic cells are Annexin V positive and PI negative (Annexin V-FITC + /PI -), whereas late apoptotic cells are Annexin V/PI double-positive (Annexin V-FITC + /PI + ) (14,15). The total number of apoptotic cells is the sum of early and late apoptotic cells (16)(17)(18). Cells were treated with tested compounds for 48 h, harvested, washed twice with PBS and then resuspended in 500 µl of binding buffer.…”