Chitosan encapsulated Naringenin promotes ROS mediated apoptosis in human breast cancer cell MDA-MB-231 through the activation of executioner Caspase-3

Abstract

Abstract We previously reported that chitosan nanoparticles encapsulated Naringenin (CS-NPs/NAR) could scavenge free radicals at lower doses and cytotoxic to cancer cells. As a continuation of that, the current study focuses on the mechanism behind CS-NPs/NAR-induced breast cancer cell (MDA-MB-231) death. MDA-MB-231 cells were treated with higher concentrations (100µg, 200 µg, and 200 µg) of Chitosan nanoparticles (CS-NPs), naringenin (NAR) and chitosan encapsulated naringenin (CS-NPs/NAR). The cell viability, proliferation, oxidative stress parameters such as nitric oxide [NO], xanthine oxidase (XOD), and xanthine dehydrogenase (XDH) levels were analyzed. ROS levels were determined through DCFDA analysis. MTT based cell cytotoxicity and BrdU cell proliferation analysis depicted the cytotoxicity effects (37% and 29% for 24h and 48h) and exhibited a reduction in the proliferation of MDA-MB-231 by CS-NPs/NAR. A significant increase in NO content, XOD, a decrease in XDH, and increase in ROS levels were observed upon treatment with CS-NPs/NAR. Fluorescent images suggested the increase in the ROS level upon treatment with CS-NPs/NAR in cancer cells and the results suggested that it could induce apoptosis. Further, to confirm this, the activity of caspase3 was analyzed through western blotting, and the result suggested that the higher concentration of CS-NPs/NAR has increased the activation of procaspase3 when compared to free NAR. Hence the current investigation concludes that high doses of CS-NPs/NAR induce and increase oxidative stress and so increased activation of procaspase3 may lead to cancer cell apoptosis and reduction in cell proliferation.