dactolisib and Fibrosis

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.

Topics: Actins; Animals; Cell Proliferation; Cells, Cultured; Collagen Type I; Collagen Type I, alpha 1 Chain; Dose-Response Relationship, Drug; Fibrosis; Imidazoles; Interleukin-6; Male; Mitogen-Activated Protein Kinase Kinases; Pancreatic Stellate Cells; Pancreatitis, Chronic; Phenylurea Compounds; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Pyridines; Pyridones; Pyrimidinones; Quinolines; Rats, Inbred Lew; Signal Transduction; Time Factors; TOR Serine-Threonine Kinases; Transforming Growth Factor beta1

The mammalian target of rapamycin (mTOR) regulates cellular activity in many diseases, but the complex interplay with PI3K/Akt pathway may hampers its function.. This study was undertaken to determine the activity of PI3K/Akt/mTOR signaling in the fibroblasts from systemic sclerosis (SSc) patients, and compare the effects of vertical inhibiting PI3K/Akt/mTOR by BEZ235 and inhibiting mTOR alone by rapamycin in fibroblast activation and in two complementary established mouse model of SSc.. Pharmaceutical specific inhibitors BEZ235 and rapamycin were used to vertical inhibit PI3K/Akt/mTOR signaling and mTOR signaling alone in cultured fibroblasts and in mice. SSc mouse model was established by daily injecting bleomycin subcutaneously or by overexpression of constitutively active type I TGF-β receptor (TβRI(ca)). To delineate the mechanisms underlying the antifibrotic effects of BEZ235 and rapamycin, activity of PI3K/Akt/mTOR signaling was analyzed by determining the expressions of phosphorylated Akt, GSK-3β, mTOR and S6 ribosomal protein (S6).. Primary dermal fibroblasts demonstrated hyperactivity of PI3K/Akt and mTOR signaling. mTOR inhibitor rapamycin failed to inhibit dermal fibrosis in an established SSc mouse model. However, administration of a dual inhibitor for PI3K/Akt and mTOR signaling BEZ235 attenuated dermal fibrosis by reversing increased dermal thickness and collagen deposition in two SSc mouse models. Furthermore, BEZ235 showed superior inhibitory effect on fibroblast activation relative to rapamycin in vitro. Also both BEZ235 and rapamycin could prevent the phosphorylation of mTOR and S6 completely. BEZ235 also blocked the activation of Akt and GSK-3β dramatically, whereas rapamycin has been shown to increase further upregulation of phosphorylated Akt on Ser473 both in vitro and in vivo.. These data show that blocking PI3K/Akt/mTOR with BEZ235 leads to superior inhibitory effect for dermal fibrosis, suggesting that vertical inhibition of PI3K/Akt/mTOR signaling may have therapeutic potential for SSc.

Topics: Animals; Cells, Cultured; Fibrosis; Humans; Imidazoles; Mice; Mice, Inbred C57BL; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinolines; Scleroderma, Systemic; Signal Transduction; Skin; TOR Serine-Threonine Kinases; Transforming Growth Factor beta