The mode of action of sorafenib in MDA-MB-231 breast carcinoma cells involves components of apoptotic, necroptotic and autophagy-dependent cell death pathways

Abstract

We report the identification of an interesting mode of action by sorafenib (SF) (Nexavar) in triple-negative breast adenocarcinoma MDA-MB-231 cells. The dying cells presented features of apoptosis, such as externalization of phosphatidylserine and cleaved caspase-3, and autophagy-mediated cell death, such as formation of autophagosomes and autolysosomes, the overexpression of LC3-II, and the presence of LAMP1-positive vacuoles, while displaying insufficient autophagic flux. Components of endoplasmic reticulum stress (ER stress; PERK and CHOP) and of necroptosis (p-MLKL) were also elevated considerably. Investigating potential target proteins that could modulate this form of cell death, we next investigated the role of tubulin disruption, which is known to induce necroptosis, apoptosis, and autophagy-dependent cell death. Interactions of SF with purified tubulin were investigated in detail using a combination of cellular and biophysical assays, transmission electron microscopy, and computer simulations. A marked reduction in the intrinsic tryptophan fluorescence of tubulin, a concentration-dependent elevation of anilinonaphthalene sulfonate–tubulin complex fluorescence, electron micrographs of deformed in vitro–assembled microtubules, and disrupted and hyper-stabilized cellular microtubules evinced the ability of SF to target tubulin and disrupt cellular microtubules. Molecular docking and molecular dynamic simulations positioned the drug between the α and β subunits of tubulin with considerable stability (ΔG_bind, −31.43 kcal/mol), suggesting that drug-induced perturbation of tubulin could contribute to this mode of cell death.