Apoptosis-inducing activity of hydroxylated polymethoxyf lavones and polymethoxyflavones from orange peel in human breast cancer cells

Sweet orange (Citrus sinensis L.) peel is a rich resource of flavonoids, especially polymethoxyflavones (PMFs). Citrus flavonoids exert a broad spectrum of biological activity, including antiproliferative and proapoptotic effects in cancer cells. We have recently shown that individual PMFs from orange peel induce Ca²⁺-mediated apoptosis in human breast cancer cells and that hydroxylation of PMFs is critical for enhancing their proapoptotic activaty. Here, we report that the fraction of orange peel extract containing a mixture of non-hydroxylated PMFs (75.1%) and hydroxylated PMFs (5.44%) and the fraction containing only hydroxylated PMFs (97.2%) induce apoptosis in those cells as well. Treatment of MCF-7 breast cancer cells with these fractions inhibited growth and induced apoptosis associated with an increase in the basal level of intracellular Ca²⁺. Effective concentrations of the hydroxylated PMFs fraction in inhibiting growth, inducing apoptosis, and increasing intracellular Ca²⁺ were lower than those of the non-hydroxylated PMFs fraction. Our results strongly imply that bioactive PMFs from orange peel exert proapoptotic activity in human breast cancer cells, which depends on their ability to induce an increase in intracellular Ca²⁺ and thus, activate Ca²⁺-dependent apoptotic proteases.