Mo1954 Rottlerin Promotes Apoptosis and Autophagy in Pancreatic Stellate Cells via AMPK Activation

Abstract

Background and Significance: Phytochemicals may play roles to reduce carcinogenesis by modulating central metabolic pathways in cancer and tumor stromal cells. AMP-activated kinases (AMPK) are key regulators of cellular metabolism, cell growth and proliferation. Activation of AMPK kinases by low energy status inhibits anabolic processes such as protein and fatty acid synthesis, activates autophagy and can lead to apoptosis. AMPK actions are mediated at least in part by inhibition of mTOR complex-1 (TORC1). Previous studies from our group showed that rottlerin, a phytochemical from the kamala tree, reduced pancreatic tumor volume in an orthotopic model of pancreatic cancer. Here we investigated the effects of rottlerin on AMPK/mTORC1 pathways in the pancreatic stellate cell (PaSC), a key cell type in the tumor microenvironment that promotes pancreatic carcinogenesis. Methods: Culture-activated primary mouse PaSC were incubated for up to 72 h in 10% FBS in the absence (control) or presence of rottlerin (0.5-10 μM). We then measured 1) activation of AMPK and Akt/mTORC1 pathways by Western blot analysis to assess phosphorylated states of intermediate proteins, 2) autophagy by Western blot and immunofluorescence to detect levels of and localize autophagic markers, 3) apoptosis by caspase-3 activity and ELISA to detect internucleosomal DNA fragmentation, and 4) cell viability/proliferation by MTT assay/ cell counting. Results: Rottlerin at concentrations as low as 0.5 μM induced rapid, sustained activation of AMPK, as determined by a significant increase in PaSC levels of phosphoAMPKα (Thr172). A marked inhibition of the Akt/mTOR pathway was indicated by reduced phosphorylation of Akt (Ser473) and the mTORC1 substrates p70 S6 kinase and 4EBP1. Further, mTORC1 inhibition blocked protein translation, and induced endoplasmic reticulum (ER) stress as indicated by phosphorylation of the translation initiator protein eiF2α (Ser51) and upregulation of the proapoptotic transcription factor CHOP. In addition, rottlerin treatment rapidly (within 15 min) induced LC3I-LC3II conversion that was sustained at least 48 h, and significantly increased p62/SQSTM1 protein expression. Together, these data suggest accumulation of autophagosomes as well as reduced autophagic flux in rottlerintreated PaSC. Interestingly, preincubation with the AMPK inhibitor compound C (20 μM) greatly reduced rottlerin-induced ER stress and autophagy. Furthermore, rottlerin dosedependently induced PaSC apoptosis, to a 6-fold increase over control at 1 μM and 36-fold at 10 μM after 72 h, and reduced cell viability by 35% at 0.5 μM, and by 70% at 10 μM. Conclusion: Our data elucidate the AMPK/mTOR pathways as a key target of rottlerin in PaSC and reinforce the notion that phytochemicals are therapeutically useful to reduce pancreatic cancer progression by eliminating pancreatic stellate cells.