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


CONCLUSIONS
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.
Department of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Li-jing
LJ
Yang
Yan
Y
Chen
Jing
J
He
Sha
S
Yi
Lu
L
Wen
Jie
J
Zhao
Ben-ping
BP
Zhang
Guo-hui
GH
Cui
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy / Q000187:drug effects*; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D006801:Humans; D009101:Multiple Myeloma / Q000188:drug therapy / Q000473:pathology*; D009842:Oligopeptides / Q000494:pharmacology; D053978:Pentacyclic Triterpenes; D014315:Triterpenes / Q000494:pharmacology* / Q000627:therapeutic use; D000094062:Betulinic Acid
D000972:Antineoplastic Agents, Phytogenic; D009842:Oligopeptides; D053978:Pentacyclic Triterpenes; D014315:Triterpenes; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; D053148:Caspase 3; D000094062:Betulinic Acid