Breast cancer is the most common and the leading cause of cancer death in women worldwide; treating invasive breast carcinomas is challenging due to the side effects of chemotherapeutics. Compounds isolated from natural sources have been proposed as potential molecules for cancer therapy; for instance, the homoisoflavonoid brazilin has shown pharmacological properties, including anti-tumoral and anti-inflammatory activities. In this study, we isolated brazilin from the heartwood of Haematoxylum brasiletto; then, we performed a semi-synthesis by adding three methyl or acetyl groups to the core structure of brazilin. We confirmed the identity of brazilin and its derivatives by spectroscopic data (¹H NMR and ¹³C NMR) and measured their purity by optical rotation. Then, we analyzed the effects of brazilin and its derivatives in three mammary gland-derived cell lines: the TNBC MDA-MB-231, the ERα(+) MCF7, and the non-tumorigenic MCF10A. We evaluated the cell viability by MTT assays, cell migration by wound-healing assays, and focal adhesion kinase (FAK) activation by Western blot. Regarding biological assays, the MTT assay showed that these compounds showed cytotoxic effects on the MCF7 and MDA-MB-231 breast cancer cells at 20 µM but was not toxic in non-tumorigenic MCF10A mammary epithelial cells. Specifically, the greatest effects found from treatment with the compounds were in the MDA-MB-231 cell line, where the IC50 of brazilin was 49.92 μM, and for MCF7, the brazilin-(OAc)₃ was 49.97 μM. These effects were dose- and time-dependent, as well as being associated with a decrease in the levels of cell migration and FAK activation.

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.

Breast cancer is the most common and the leading cause of cancer death in women worldwide; treating invasive breast carcinomas is challenging due to the side effects of chemotherapeutics. Compounds isolated from natural sources have been proposed as potential molecules for cancer therapy; for instance, the homoisoflavonoid brazilin has shown pharmacological properties, including anti-tumoral and anti-inflammatory activities. In this study, we isolated brazilin from the heartwood of Haematoxylum brasiletto (Pacific coast of Mexico and Central America); then, we performed a semi-synthesis by adding three methyl or acetyl groups to the core structure of brazilin. We confirmed brazilin and its derivatives identity by spectroscopic data (1H NMR and 13C NMR) and measured their purity by optical rotation. Then, we analyzed the effects of brazilin and derivatives in three mammary gland-derived cell lines: the TNBC MDA-MB-231, the ER(+) MCF7, and the non-tumorigenic MCF10A. We evaluated the cell viability by MTT assays, cell migration by wound-healing assays and focal adhesion kinase (FAK) activation by Western blot. Regarding biological assays, the MTT assay showed that brazilin and its derivatives showed cytotoxic effects on MCF7 and MDA-MB-231 breast cancer cells at 20 µM but was not toxic in non-tumorigenic MCF10A mammary epithelial cells. These effects were dose and time-dependent, as well as a decrease in the levels of cell migration and FAK activation.