Autophagy Induced By Dca, Pi3k Inhibition Or Starvation Results In Reduced Lactate Production Measured In Real-Time By Dnp 13c Mrs

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL INTRODUCTION: Autophagy is a cellular degradation response to starvation or stress where cellular proteins, organelles and cytoplasm are engulfed, digested and recycled to sustain cellular metabolism [Mathew et al. Nat Rev Cancer (2007)]. Increased TCA cycle activity is predicted during autophagy, as the amino acids and fatty acids generated by the autophagic process are utilised in the TCA cycle. AIM: To investigate the effects of autophagy on TCA cycle activation in cells by dynamic nuclear polarisation (DNP) and 13C magnetic resonance spectroscopy (MRS) of pyruvate metabolism, in order to develop a non-invasive marker for autophagy. METHODS: Bax deficient colon carcinoma HCT116 cells (Baxko) were used to induce autophagy with 24 hrs of starvation or 10µM PI-103 (a PI3K inhibitor). These cells were also treated with 75mM dichloroacetate (DCA), a PDK inhibitor, to study the effect of TCA cycle activation [Bonnet et al. Cancer Cell (2007)] on lactate production. Electron microscopy, flow cytometry and western blots provided markers of autophagy and apoptosis. Real-time lactate production rate was monitored by [1-13C] pyruvate DNP assay and 13C MRS. Culture media and extracts from the treated and control cells were analysed by 1H MRS. Lactate dehydrogenase (LDH) expression and activity were examined. Surprisingly, autophagy was induced in HCT116 Baxko cells by DCA. This effect was further examined in HT29 (colon) and PC3 (prostate) cancer cells. RESULTS: Markers of autophagy by western blots (increased LC3II expression) and electron microscopy (presence of autophagosomes) confirmed the induction of autophagy in HCT116 Baxko cells by starvation, DCA or PI103 treatment and in DCA treated PC3 and HT29 cells. All treated groups exhibited minimal apoptosis or necrosis. Significant reductions in the rate of real-time lactate production by DNP 13C MRS were found in all treated groups. Decreases in steady-state lactate production were found in media from all treated groups. No change in LDH expression/activity or its co-factor NAD+ concentration was found in any treated group. DISCUSSION: Autophagy was induced in 3 cancer cell lines by 3 different methods. To our knowledge, this is the first study to show DCA induced autophagy in cancer cells. The observed reduction in real-time (and steady-state) lactate production was associated with autophagy. This finding together with unchanged cellular NAD+ and LDH could be due to more pyruvate being diverted to the TCA cycle; and/or LDH being sequestrated in autophagic vacuoles [Houri et al. Biochem J (1995)]. CONCLUSIONS: A reduction in lactate production measured by DNP 13C MRS, and unchanged NAD+, may provide a non-invasive means of assessing autophagy. We acknowledge CRUK, EPSRC, MRC and Department of Health for the Cancer Imaging Centre, NHS funding to the NIHR Biomedical Research Centre and Dr Paul Clarke for HCT116 Baxko cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3788. doi:10.1158/1538-7445.AM2011-3788