Abstract 1658: Fenretinide induces lethal autophagy in pancreatic cancer cells

Abstract

Abstract Background: Recent studies show that the synthetic vitamin A analog fenretinide (4-HPR) is cytotoxic in a number of human tumor cell lines and is a chemopreventative agent that is well-tolerated clinically. 4-HPR has been shown to augment intracellular levels of reactive oxygen species (ROS) and ceramides, and to activate the MAPK family of kinases. Pancreatic cancer cells are sensitive to increased levels of ROS, ceramides, and MAPK phosphorylation, suggesting 4-HPR will be an effective agent against this disease. Results: 4-HPR exposure markedly decreased viability of MIA PaCa-2 and PANC-1 cells, as shown by a dose-dependent decrease in absorbance from an MTS assay and a 3-fold increase in propidium iodide staining. A 50% reduction in [3H]thymidine incorporation showed that cell proliferation was halted as well. 4-HPR treatment caused a 3-fold increase in de novo ceramide production at 24 h and a time- and dose-dependent generation of ROS. 4-HPR led to a minor increase in caspase 3/7 activity and in FITC-Annexin V-positive cells. However, other measures of apoptosis were lacking, suggesting apoptosis was not the major death pathway elicited by 4-HPR. Further investigation of the mechanism of cell death suggests that the dominant pathway triggered by 4-HPR is lethal autophagy. After 4-HPR treatment, microscopic analysis of cells exposed to acridine orange showed bright red autophagic vesicles, and Western blot showed a robust increase in LC3 II protein expression. Pretreatment with the autophagy inhibitor 3-methyladenine (3-MA) or the ROS scavenger vitamin E (VE) abrogated the increase in LC3 II expression caused by 4-HPR exposure, and both 3-MA and VE partially preserved viability. A sustained, dose-dependent increase in phosphorylation of the stress kinases JNK and p38 was observed following incubation with 4-HPR, whereas phosphorylation of the mitogenic kinase ERK 1/2 decreased. Pretreatment with myriocin (an inhibitor of de novo ceramide production), 3-MA, or VE attenuated the increase in JNK and p38 phosphorylation caused by 4-HPR. Additionally, VE pretreatment blocked the decrease in ERK 1/2 phosphorylation elicited by 4-HPR. Conclusions: These data suggest that 4-HPR induces lethal autophagy in human pancreatic cancer cells by a mechanism that involves generation of ROS and engagement of the MAPK and sphingolipid signaling pathways. 4-HPR used alone or in combination with current therapies or signaling pathway modulators may be a promising avenue for future pancreatic cancer therapies. [Support: NIH/NIGMS grant no. GM077391] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1658.