Abstract
Abstract The goal of this study is to evaluate SapC-DOPS, a novel cancer nanotherapeutic, for glioblastoma multiforme (GBM). SapC-DOPS delivered intravenously (i.v) was found to specifically target intracranial tumors in mice bearing spontaneous brain tumor, as well in nude mice intracranially implanted with human GBM cells. Treatment of tumor bearing mice with SapC-DOPS (i.v.) significantly increased survival: 25% and 75% long-term survivors in U87ΔEGFR-Luc and X12v2 implanted mice, respectively (P<.0001). Western blot analysis of primary GBM neurospheres treated with SapC-DOPS did not show activation of apoptosis as measured by cleaved caspase 9 and cleaved PARP, and lacked activation of DNA damage markers phosphor-ATM and γ-H2AX. Consistent with this, treatment with a pan-caspase inhibitor Z-VAD-FMK did not rescue SapC-DOPS-induced killing (P>0.05). In contrast, SapC-DOPS treatment increased levels of an autophagic marker LC3-II via western blot. Autophagosome formation was also confirmed through transmission electron microscopy. Utilizing a stable GBM cell line expressing a GFP-LC3 fusion protein, we observed punctuated GFP expression following treatment, indicative of autophagosome formation. Quantification of GFP punctated cells showed a significant increase in SapC-DOPS treated cells compared to control (P<.001). Analysis of red/green fluorescence following acridine orange staining showed an induction of acidic vesicular organelles indicative of autophagolysosomes. In addition, inhibition of autophagosome formation using 3-methyladeneine or inhibition of auotphagic vacuole maturation with bafilomycin A1 resulted in a significant rescue of SapC-DOPS-induced killing (P<.001). Knockdown of ATG5 using siRNA also resulted in a rescue of SapC-DOPS-induced cell death and autophagy induction (P<.001). Interestingly, we did not observe a decrease in the activation of mTOR as determined by the phosphorylation of 4EBP1 and p70S6K which are typically induced during autophagy. This led us to test the use of rapamycin, a known inhibitor of mTOR and inducer of autophagy, in combination with SapC-DOPS. By using the Chou Talalay analysis, we observed strong synergy for multiple drug combinations in primary GBM neurospheres (combination index < .4). To investigate whether SapC-DOPS induced autophagy could be preferentially targeting mitochondria (mitophagy), we utilized MitoTracker Green (mitochondria) and LysoTracker Red (autophagolysosomes). By using confocal microscopy analysis, we were able to observe a decrease in mitochondrial mass in cells treated with SapC-DOPS as well as co-localization of mitochondria with autophagolysosomes. In addition to this, we observed a significant decrease in ATP levels following SapC-DOPS treatment (P<.01). These findings suggest therapeutic implications for treating GBM by using SapC-DOPS alone and in combination with an AKT/mTOR inhibitor. Citation Format: Jeffrey Wojton, Naduparambil K. Jacob, Nicholas Denton, Nina Dmitrieva, Hiroshi Nakashima, Chang-Hyuk Kwon, Lionel Chow, Ennio A. Chiocca, Arnab Chakravarti, Balveen Kaur, Xiaoyang Qi. SapC-DOPS induces lethal mitophagy in glioblastoma. [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 2169. doi:10.1158/1538-7445.AM2013-2169
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