RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Cancer is an inflammatory disease because carcinogenesis and tumor progression depend on intrinsic and extrinsic inflammatory pathways. Although species of the genus Aspidosperma are widely used to treat tumors, and there is ethnopharmacological evidence for traditional use of the species A. subincanum as an anti-inflammatory agent, its antineoplastic potential is unknown. AIM OF THE STUDY: To evaluate toxic effects of the indole alkaloid-rich fraction (IAF) of A. subincanum on the MCF7 cell line and identify some of the anti-inflammatory mechanisms involved. MATERIALS AND METHODS: Chromatographic analyses were performed by ultra-high-performance liquid chromatography with electrospray ionization mass spectrometry, and cytotoxic and antiproliferative effects of IAF were verified by MTT and clonogenic assays. Cell cycle alterations were analyzed by measuring DNA content, while propidium iodide and acridine orange staining was performed to determine the type of induced cell death. The expression of apoptosis markers and proteins involved in cell proliferation and survival pathways was analyzed by immunoblotting, RT-qPCR, and ELISAs. Interference with redox status was investigated using a DCFH-DA probe and by measuring catalase activity. RESULTS: Chromatographic analyses showed that IAF is a complex mixture containing indole alkaloids. IAF selectively exerted toxic and antiproliferative effects, elevating the Bax/Bcl-xL ratio and inducing apoptosis in MCF7 cells. IAF decreased intracellular reactive oxygen species levels and increased catalase activity, while reducing the IL-8 level and suppressing COX-2 expression. CONCLUSIONS: IAF induces apoptosis in MCF7 cells by suppressing COX-2 expression while reducing IL-8 levels and intracellular content of reactive oxygen species.
Assuntos
Anti-Inflamatórios/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Aspidosperma , Alcaloides Indólicos/farmacologia , Extratos Vegetais/farmacologia , Linhagem Celular Tumoral , Fenômenos Fisiológicos Celulares/efeitos dos fármacos , Ciclo-Oxigenase 2/genética , Humanos , Interleucina-8/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
COVID-19 is a pandemic with no end in sight. There is only one approved antiviral agent but global stocks are deemed insufficient. Despite in vitro antiviral activity, clinical trials of chloroquine and hydroxychloroquine were disappointing, and they may even impair outcomes. Chloroquine causes zebroid deposits reminiscent of Fabry disease (α-galactosidase A deficiency) and endothelial cells are key targets of COVID-19. We have explored the effect of chloroquine on cultured endothelial cells and its modulation by recombinant α-galactosidase A (agalsidase). Following dose-response studies, 0.5 µg/mL chloroquine was added to cultured human endothelial cells. Neutral red and Lysotracker were used to assess lysosomes. Cytotoxicity was evaluated by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) - MTT assay and cell stress by assessing reactive oxygen species (ROS) and nitric oxide (NO). In endothelial cells, chloroquine induced dose-dependent cytotoxicity at in vitro test concentrations for COVID-19 therapy. At a sublethal concentration, chloroquine significantly induced the accumulation of acid organelles (P < 0.05), increased ROS levels, and decreased NO production (P < 0.05). These adverse effects of chloroquine on endothelial cell biology were decreased by agalsidase-ß (P < 0.05). Chloroquine-induced endothelial cell cytotoxicity and stress is attenuated by agalsidase-ß treatment. This suggests that endothelial cell injury may contribute to the failure of chloroquine as therapy for COVID-19 and may be at least in part related to causing dysfunction of the lysosomal enzyme α-galactosidase A.