Cell Death Induced by Homoisoflavonoid Brazilin and its Semi-synthetic Derivates on MDA-MB-231 and MCF7 Breast Cancer Cell Lines

ABSTRACT Flavonoids are naturally occurring polyphenolic compounds that have been extensively explored as scaffolds for drug development due to their diverse biological activities. Brazilin, a homoisoflavonoid with reported antitumoral properties, does not fully meet pharmaceutical criteria, and chemical modification of natural compounds is often required to enhance bioactivity and efficacy. Here, we evaluated the pro-apoptotic activity of Brazilin and its semi-synthesized methoxylated (OMe) 3 and acetylated (OAc) 3 derivatives in triple-negative MDA-MB-231 and luminal A MCF7 breast cancer cell lines. We assessed cell viability, proliferation, oxidative stress, and mitochondrial integrity, and analyzed apoptotic features using confocal microscopy, western blotting, and RT-qPCR. In addition, RNA sequencing was performed to characterize transcriptomic changes in MDA-MB-231 cells following treatment with unmodified Brazilin or its derivatives. Brazilin and Brazilin-(OAc) 3 significantly reduced cell viability and proliferation in MDA-MB-231 cells, whereas MCF7 cells exhibited increased viability and growth in response to Brazilin-(OMe) 3 . In MDA-MB-231 cells, treatment with Brazilin and Brazilin-(OAc) 3 induced apoptosis-associated features, including chromatin condensation, γH2AX accumulation, and PARP cleavage. These effects were accompanied by a modest increase in mitochondrial oxidative stress and loss of mitochondrial membrane potential. Notably, no cytotoxic or apoptotic features were detected in non-tumorigenic MCF10A cells. Transcriptomic analysis revealed that Brazilin treatment upregulated genes associated with endoplasmic reticulum stress, including ATF3 , in MDA-MB-231 cells. Collectively, our results indicate that Brazilin and its acetylated derivative selectively induce mitochondrial stress and cell death in triple-negative breast cancer cells, potentially involving ER stress pathways.