Abstract 319: Combination of miR-30e overexpression and proanthocyanidin treatment inhibits autophagy and induces apoptosis in human glioblastoma stem cells and SNB19 cells

Abstract

Abstract Glioblastoma is the most common and malignant brain tumor that harbors heterogeneic abnormal astrocytic cells. Hypoxia in solid tumors including glioblastoma creates a metabolic stress for induction of a basal level of autophagy to recycle and supply cellular ingredients to surviving tumor cells that become significantly resistant to apoptosis. Even a small number of human glioblastoma stem cells (GSC), which are highly resistant to radiotherapy and chemotherapy, may eventually produce huge number of heterogeneic glioblastoma cells to regrow the brain tumor. Inhibition of autophagy and induction of apoptosis in GSC as well as in other glioblastoma cells could greatly cause inhibition of overall growth of glioblastoma. We used sodium sulfite to imitate hypoxia for induction of basal levels of autophagy in GSC and glioblastoma SNB19 cells and then examined efficacy of combination of miR-30e overexpression and proanthocyanidin (PAC) treatment in promoting apoptosis in both GCS and SNB19 cells. Our flow cytometry and acridine orange staining indicated that use of 2 mM sodium sulfite could induce 16% and 25% autophagy in GSC and SNB19 cells, respectively. Our reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting showed significant increases in mRNA and protein expression of Beclin 1, an important inducer of autophagy, in the cells due to hypoxia. A search in microRNA database (miRDB) suggested that miR-30e could inhibit expression of not only the autophagy inducer Beclin 1 but also inhibit the apoptosis inhibitors such as Aven (caspase activation inhibitor) and BIRC-6 (Bruce). Combination of miR-30e overexpression and PAC treatment significantly reduced viability in cells under hypoxia. Apoptotic mode of cell death due to this combination therapy was manifested in morphological features (as evidenced from Wright staining) and also in an early biochemical marker (as confirmed from Annexin V-FITC/PI staining). GSC conferred more resistance to apoptosis than SNB19 following this combination therapy. RT-PCR results showed significant decreases in mRNA expression of Aven and BIRC-6, indicating induction of apoptosis in the cells. Western blotting subsequently confirmed that the combination therapy was indeed highly potent to induce extrinsic pathway of apoptosis with activation of caspase-8 and also intrinsic pathway of apoptosis with a raise in Bax:Bcl-2 ratio to trigger mitochondrial release of pro-apoptotic factors into the cytosol for activation of caspase-9 and caspase-3. Increases in calpain and caspase-3 activities cleaved α-spectrin to generate calpain-specific 145 kD SBDP and caspase-3-specific 120 kD SBDP, respectively, in cells for apoptosis. Collectively, combination of miR-30e overexpression and PAC treatment could serve as a promising therapeutic strategy to inhibit autophagy and induce apoptosis in human GSC and SNB19 cells. Citation Format: Mrinmay Chakrabarti, Swapan K. Ray. Combination of miR-30e overexpression and proanthocyanidin treatment inhibits autophagy and induces apoptosis in human glioblastoma stem cells and SNB19 cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 319. doi:10.1158/1538-7445.AM2014-319