Induction of apoptosis in MCF-7 human breast cancer cells by phytochemicals from Anoectochilus formosanus.

The antitumor activity of Anoectochilus formosanus Hayata (Orchidaceae), a popularly used folk medicine in the treatment of cancers in Asia, was investigated in MCF-7 human mammary carcinoma cells. Plant extracts of A. formosanus were observed to induce apoptosis of MCF-7 cells as evidenced by cell-morphological changes, an early redistribution of plasma membrane phosphatidylserine, and DNA content distribution studies. Bioactivity-guided fractionation of A. formosanus extracts produced a specific ethyl acetate (EA)-partitioned fraction in which apoptotic activity was enriched. The chemical profile and candidate index compounds of the active EA fraction were obtained using HPLC and various spectral analyses. Western blot analysis showed that upon treatment of MCF-7 cells with the EA fraction, cleavage of pro-caspases-8, -9, and -7, and poly(ADP-ribose) polymerase as well as significant release of mitochondrial cytochrome c into the cytosol were readily observed. Flow cytometry showed that the Fas ligand protein was overexpressed in EA-treated MCF-7 cells. Functional genomic studies indicated that specific genes related to cytoskeleton rearrangement, apoptotic signal transduction, and various transcription factors were differentially regulated in EA-treated MCF-7 cells. Putative apoptotic signaling pathways of MCF-7 cells in response to the EA extract of A. formosanus are proposed.