Polymethoxylated flavones induce Ca2+-mediated apoptosis in breast cancer cells

Flavonoids, polyphenolic phytochemicals which include flavones and isoflavones, are present in the common human diet. It has been suggested that these compounds may exert anticancer activity; however, the mechanisms involved remain unknown. We have recently shown (Sergeev, 2004, Biochem Biophys Res Commun 321: 462–467) that isoflavones can activate the novel apoptotic pathway mediated by cellular Ca2+. Here, we report that polymethoxyflavones (PMFs) derived from sweet orange (Citrus sinensis L.) inhibit growth of human breast cancer cells via Ca2+-dependent apoptotic mechanism. The treatment of MCF-7 breast cancer cells with 5-hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone (5-OH-HxMF) and 3′-hydroxy-5,6,7,4′-tetramethoxyflavone (3′-OH-TtMF) induced a sustained increase in concentration of intracellular Ca2+ ([Ca2+]i) resulting from both depletion of the endoplasmic reticulum Ca2+ stores and Ca2+ influx from the extracellular space. This increase in [Ca2+]i was associated with the activation of the Ca2+-dependent apoptotic proteases, μ-calpain and caspase-12, as evaluated with the calpain and caspase-12 peptide substrates and antibodies to active (cleaved) forms of the enzymes. Corresponding non-hydroxylated PMFs, 3,5,6,7,8,3′,4′-heptamethoxyflavone (HpMF) and 5,6,7,3′,4′-pentamethoxyflavone (PtMF), were dramatically less active in inducing Ca2+-mediated apoptosis. Our results strongly suggest that the cellular Ca2+ modulating activity of flavonoids underlies their apoptotic mechanism and that hydroxylation of PMFs is critical for their ability to induce an increase in [Ca2+]i and, thus, activate Ca2+-dependent apoptotic proteases.