Chitosan-encapsulated naringenin promotes ROS mediated through the activation of executioner caspase-3

We previously reported that chitosan nanoparticle-encapsulated Naringenin (CS-NPs/NAR) could scavenge free radicals at lower doses and be cytotoxic to cancer cells. The current study continues to focus on the mechanism behind CS-NPs/NAR-induced breast cancer cell (MDA-MB-231) death. MDA-MB-231 cells were treated with higher concentrations (100, 200, and 200 µg) of Chitosan nanoparticles (CS-NPs), naringenin (NAR), and chitosan-encapsulated naringenin (CS-NPs/NAR). The cell viability, proliferation, and 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 24 and 48 h) 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 an 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 caspase-3 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.