Bioassay-guided isolation of cantharidin from blister beetles and its anticancer activity through inhibition of epidermal growth factor receptor-mediated STAT3 and Akt pathways.

Cantharidin is an active constituent of blister beetles (cantharides) which have traditionally been used for cancer treatment. Several studies have shown that cantharidin has a cytotoxic effect on various cancer cells. However, few studies have examined the effect of cantharidin on signal transducer and activator of transcription 3 (STAT3) signaling in cancer. In this study, we isolated cantharidin from cantharides by bioassay-guided fractionation and examined its inhibitory effect on STAT3 activation in human breast cancer MDA-MB-231 cells, expressing high level of phosphorylated STAT3. Cantharides were extracted with acetonitrile and separated into hexane, methylene chloride/acetonitrile, and water fractions. The methylene chloride/acetonitrile fraction was further separated into four fractions by preparative high-throughput high-performance liquid chromatography. Cantharidin was then isolated from the third fraction by countercurrent chromatography and structurally determined by comparing nuclear magnetic resonance and high-resolution mass spectrometry data. Cantharidin inhibited STAT3 tyrosine phosphorylation in MDA-MB-231 cells. Cantharidin suppressed epidermal growth factor (EGF)-induced STAT3 and PI3K/Akt signaling pathways through inhibition of EGF receptor phosphorylation. Moreover, cantharidin reduced cell proliferation and induced apoptosis with downregulation of STAT3 target genes, such as Bcl-2, COX-2, and cyclin D1. Taken together, this study provides evidence that cantharidin may be a potential therapeutic agent for triple-negative breast cancer by reducing EGFR-mediated STAT3 and Akt signaling pathways.