Trametinib and dactolisib but not regorafenib exert antiproliferative effects on rat pancreatic stellate cells

Abstract

Modulation of the stroma response is considered a promising approach for the treatment of chronic pancreatitis and pancreatic cancer. The aim of this study was to evaluate the effects of three clinically available small molecule kinase inhibitors, regorafenib, trametinib and dactolisib, on effector functions of activated pancreatic stellate cells (PSCs), which play a key role in pancreatic fibrosis. Cultured rat PSCs were exposed to small molecule kinase inhibitors. Proliferation and cell death were assessed by measuring the incorporation of 5-bromo-2'-deoxyuridine and cytotoxicity, respectively. Levels of mRNA were determined by real-time PCR, while protein expression and phosphorylation were analyzed by immunoblotting. Interleukin-6 levels in culture supernatants were quantified by ELISA. Zymography assays were performed to monitor collagenase activity in culture supernatants. The MEK inhibitor trametinib and the dual phosphatidylinositol 3-kinase/mTOR inhibitor dactolisib, but not the multi-kinase inhibitor regorafenib, efficiently inhibited PSC proliferation. Trametinib as well as regorafenib suppressed the expression of two autocrine mediators of PSC activation, interleukin-6 and transforming growth factor-beta1. Dactolisib-treated cells expressed less alpha1 type I collagen and lower levels of alpha-smooth muscle actin, a marker of the myofibroblastic PSC phenotype. Simultaneous application of dactolisib and trametinib displayed additive inhibitory effects on cell growth without statistically significant cytotoxicity. Activity of matrix metalloproteinase-2 was not affected by any of the drugs. We suggest the combination of two drugs, that specifically target two key signaling pathways in PSC, Ras-Raf-MEK-ERK (trametinib) and phosphatidylinositol 3-kinase-AKT-mTOR (dactolisib), as a concept to modulate the activation state of the cells in the context of fibrosis.