Methylanthraquinone from Hedyotis diffusa WILLD induces Ca²⁺-mediated apoptosis in human breast cancer cells

Abstract

Methylanthraquinone from Hedyotis diffusa WILLD exhibited potent anticancer activity in many kinds of cancer cells. However, the exact mechanism and signaling pathway involved in methylanthraquinone-induced apoptosis have not been fully elucidated. Therefore, we explored the mechanisms of methylanthraquinone-mediated apoptosis in MCF-7 human breast cancer cells. When MCF-7 cells were co-incubated with methylanthraquinone, the percentage of apoptotic cell and S phase of cell cycle was markedly increased. In addition, a rise in intracellular calcium levels, phosphorylation of JNK and activation of calpain were found in MCF-7 cells after exposure to methylanthraquinone. With the methylanthraquinone-mediated reduction of mitochondrial membrane potential, cytochrome c was released from mitochondria to cytosol. Moreover, methylanthraquinone strongly induced cleavage of caspase-4, caspase-9 and caspase-7 in MCF-7 cells. These results suggested that methylanthraquinone from Hedyotis diffusa WILLD induced MCF-7 cells apoptosis via Ca²⁺/calpain/caspase-4 pathway.

Introduction

The use of herbal intervention is widespread in all regions of the developing world and is rapidly growing in developed countries (Yan et al., 2006). Medicinal plants are widely used in the treatment of various cancers in many Asian countries and are recognized as an attractive alternative to surgical therapy and radiotherapy (Xie et al., 2009). Hedyotis diffusa WILLD has been known as a traditional Chinese medicine (TCM) for a long time, and widely applied in the treatment of inflammations such as appendicitis, urethritis, and bronchitis, due to its antibacterial activity (Ahmad et al., 2005a, Ahmad et al., 2005b, Lin et al., 2002, Shan et al., 1999). Recently, this herb has gained increasingly attention to its usage as an antitumor herb, such as therapy in liver, lung, colon, brain, pancreas and other cancers (Fang et al., 2004). Up to now, three major classes of this herb compounds, including triterpenes, polysaccharide and anthraquinones, have been reported as bioactive compounds from this herb (Ahmad et al., 2005a, Ahmad et al., 2005b, Li et al., 2008).

In spite of the extensive use of herbal therapies, there is insufficient scientific evidence validating their efficacy and safety. Thus, basic research aimed at elucidating the underlying antitumor mechanisms of Hedyotis diffusa WILLD is very important for the use of this herbal medicine. Recently, scientists have focused on the potential role of extracts of TCM for cancer treatment. Shi et al. reported that two anthraquinones from Hedyotis diffusa WILLD induced HepG2 cell apoptosis via caspase-3 activation (Shi et al., 2008). However, the molecular mechanism is still equivocal up to now. The main aim of this work was to study the possible apoptotic mechanism of methylanthraquinone, which was extracted from Hedyotis diffusa WILLD, in MCF-7 cells. The MCF-7 cells are often used in studies of apoptosis and are well known for its unique characteristic of being deficient in caspase-3 (Kugawa et al., 2004). In addition, breast cancer cells are susceptible to generation of a sustained Ca²⁺ response (Sergeev, 2004a, Sergeev, 2004b).