Effect of dasatinib on TGF&beta-induced fibroblast-to-myofibroblast differentiation

Purpose: The purpose of this study is to determine the effect of dasatinib, a broad spectrum non-receptor tyrosine kinase inhibitor, on myofibroblast differentiation as a means to identify therapeutic targets for fibrosis-related health issues and drug development.

Methods: After incubation to 70% confluence, mouse embryonic fibroblasts were treated with 4 ng/ml of transforming growth factor-β (TGFβ) in Dulbecco’s modified eagle medium (DMEM) for 48 hours. At 48 hours, the fibroblasts were further treated with 4 ng/ml TGFβ and dasatinib at doses of 0, 10, 5, 2.5, and 1 nM. At 72 hours, cells were lysed and subjected for Western-blot analysis of alpha smooth muscle actin (α-SMA), phospho-Src, Src, phospho-ERK, and ERK. Current investigation in the lab is focused on identifying the role of Src and/or other tyrosine kinases in myofibroblast differentiation using genetically modified cells (Src, Yes and Fynn knockout [SYF^-/-]fibroblasts), plasmids encoding different variants of Src as well as specific inhibitors.

Results: Our data indicate that dasatinib at doses suggested for cancer cells (100 nM) results in substantial toxicity to mouse embryonic fibroblasts to the extent that molecular targets cannot be identified through Western Blot analysis. From a dose-response study, we identified that dasatinib at a dose of 1 nM was optimal to decrease α-SMA without significant toxicity.

Conclusion: Our study so far demonstrates that dasatinib inhibits TGFβ-induced fibroblast-to-myofibroblast differentiation at a dose several fold lower than that is used for cancer cells. While our study suggests that low-dose dasatinib can be re-purposed for the treatment of fibrotic diseases such as pulmonary fibrosis, future research in our lab will identify additional targets for therapeutic interventions.