Abstract 1709: Molecular inhibition of autophagy enhances dihydroceramides-induced cell death in glioblastoma multiforme cell lines.

Abstract Introduction: Study aim was to characterize dihydroceramide-related cell death in the glioblastoma multiforme cell lines, A-172 and T98G. Methods: Cytotoxicity was evaluated using fluorescence digital image microscopy (DIMSCAN). Apoptosis was analyzed by Annexin V-FITC and TUNEL assays. Mitochondrial depolarization and Reactive Oxygen Species (ROS) were assayed by JC1 staining and DCFDA staining and flow cytometry. Autophagy marker, LC3BII, and caspase-3 cleavage were assayed by immunoblotting. Autophagy inhibition was by bafilomycin A1 and siRNA against ATG7 and BECN1. Sphingolipid levels were assayed by radiolabeling and mass spectrometry. Results: Fenretinide (4-HPR) increased dihydroceramides coincident with cytotoxicity. Artificial L-threo-sphinganine analogs, safingol (S) and C20-base safingol (C20-S), were incorporated into L-threo variant dihydroceramides coincident with synergistic enhancement of 4-HPR cytotoxicity. 4-HPR, alone or in combination with S or C20-S, resulted in a significant (p<0.05) increase (∼5-fold) in ROS formation in T98G and ∼2-fold induction in A-172 cells; L-threo sphinganines alone did not increase ROS formation. At +36h, 4-HPR induced greater mitochondrial membrane depolarization (62.3 +/- 4.1%) in T98G cells in comparison to A-172 (27.4 +/- 2.3%) cells. Mitochondrial depolarization with 4-HPR-alone trended greater than combination treatments in both cell lines at all time points (+12 - 36h). At +12h and +24h, 4-HPR +/- S/C20-S did not increase Annexin V-FITC+/PI- staining in either cell line, suggesting lack of early apoptosis, but Annexin V-FITC+/PI- staining increased in A-172 cells at +36h. Although cytotoxicity was present at +24h, TUNEL assay and caspase-3 cleavage were negative at +12h and +24 h. TUNEL-positivity increased with a concomitant increase of cleaved caspase-3 at +36 h in remaining cells. Autophagy was strongly activated (+6 - 36h) in response to 4-HPR +/- S/C20-S exposure as evidenced by increased conversion of LC3-I to the autophagic vesicle-associated form, LC3-II. Pharmacological inhibition of autophagy using bafilomycin A1 demonstrated an active autophagic flux in response to treatments. Suppression of autophagy by siRNA (BECN1, ATG7) significantly (p<0.05) decreased cell viability in 4-HPR + S/C20-S -exposed cells at +24 h. Conclusions: Results suggest that multiple, distinct, cell death pathways are induced by 4-HPR + S/C20-S treatment. Early cell death was non-apoptotic and caspase-independent; late cell death was by apoptosis with caspase-3 cleavage. Inhibition of autophagy synergized/accelerated 4-HPR + S/C20-S -induced cell death, indicating a pro-survival role for autophagy in these cell lines. The results suggest that strategies that exploit concurrent autophagy inhibition may be useful therapeutic adjuncts for 4-HPR and dihydroceramide-based strategies. Citation Format: Nikhil Vad, Dong Wang, Barry J. Maurer. Molecular inhibition of autophagy enhances dihydroceramides-induced cell death in glioblastoma multiforme cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1709. doi:10.1158/1538-7445.AM2013-1709