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 proapoptotic activity of brazilin and its semisynthesized methoxylated (OMe)(3) and acetylated (OAc)(3) derivatives in triple-negative MDA-MB-231, luminal A MCF7 breast cancer cell lines and nontumorigenic MCF10A cells. We assessed cell viability, oxidative stress, and mitochondrial integrity, and analyzed apoptotic features using confocal microscopy, Western blot, 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 a more subtle response. In MDA-MB-231 cells, treatment with brazilin and brazilin-(OAc)(3) induced mild apoptosis-associated features, including chromatin condensation, γH2AX accumulation, and PARP cleavage. These effects were accompanied by a modest increase in oxidative stress and loss of mitochondrial membrane potential. Transcriptomic analysis revealed that in MDA-MB-231 cells brazilin treatment regulates genes associated with endoplasmic reticulum stress and RNA metabolism, among other categories. Only high doses or long exposure to brazilin and brazilin-(OAc)(3) affected the nontumorigenic MCF10A cells. Notably, the methylated compound exhibited minimal biological activity, as it did not significantly affect cell viability across the tested cell lines and induced no substantial transcriptional changes. Collectively, our results suggest that brazilin and its acetylated derivative induce mitochondrial stress and cell death in triple-negative breast cancer cells, potentially involving endoplasmic reticulum stress pathways.