Inhibition of glioma growth by flavokawain B is mediated through endoplasmic reticulum stress induced autophagy.
- NORLUX Neuro-Oncology Laboratory
Flavokawain B (FKB), a natural kava chalcone, displays potent antitumor activity in various types of cancer. The mechanism of action, however, remains unclear. Here, we evaluated the efficacy of FKB in the treatment of human glioblastoma multiforme (GBM) as well as the molecular basis for its inhibitory effects in cancer. Approximately 60% of GBM cells, U251, U87, and T98, became senescent after treatment with FKB (3 mug/mL for 48 h) as assessed in the senescence-associated (SA)-GLB1/SA-beta-galactosidase assay. The cellular process of macroautophagy/autophagy potentially contributed to the establishment of senescence. Transmission electron microscopy revealed the formation of autophagic vesicles under FKB treatment, and MAP1LC3B (microtubule associated protein 1 light chain 3 beta)-II, a protein associated with autophagy, was increased. Transfection of ATG5 or ATG7 small interfering RNAs (siRNAs) inhibited FKB-induced autophagy in U251 cells. Western blot analysis revealed that molecular components of the endoplasmic reticulum stress pathway were activated, including ATF4 (activating transcription factor 4) and DDIT3 (DNA damage inducible transcript 3), while levels of TRIB3 (tribbles pseudokinase 3) increased. In addition, based on the phosphorylation status, the AKT-MTOR-RPS6KB1 pathway was inhibited, which induced autophagy in GBM cells. Inhibition of autophagy by pharmacological autophagy inhibitors 3-methyladenine and chloroquine or knockdown of ATG5 or ATG7 caused FKB-treated U251 cells to switch from senescence to apoptosis. Finally, in orthotopic implantations of luciferase-transduced U251 cells in mice, knockdown of ATG5 or treatment with chloroquine in combination with FKB, significantly inhibited tumor growth. Our results demonstrated that FKB induced protective autophagy through the ATF4-DDIT3-TRIB3-AKT-MTOR-RPS6KB1 signaling pathway in GBM cells, indicating that the combination treatment of FKB with autophagy inhibitors may potentially be an effective therapeutic strategy for GBM.