The in vitro and in vivo Anti-Tumor Effect of Decursin in Estrogen Receptor-Negative Human Breast Cancer Cells via Downregulation of Akt Signaling Pathway

Abstract

The need to development of an effective therapeutic agent to Estrogen Receptor(ER)-negative breast cancer cells has been recognized because they are more aggressive and resistant to conventional hormonal therapy. Decursin ((S)-8,8-Dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano(3,2-g)chromen-7-yl 3-methyl-2-butenoic acid), a naturally existing coumarin compound originated from the root of Korean angelica, has been shown to possess therapeutic potential in various cancers including prostate cancer cells and leukemia cells. Thus we tested the anti-tumor effect of decursin in ER-negative breast cancer cell lines, MDA-MB-231 and SK-BR-3, which overexpress epidermal growth factor receptor (EGFR) and/or c-ErbB2 , respectively as well as in vivo xenograft model. Reduced cell numbers under in vitro culture were observed upon decursin treatment at 50 to 200 M for 72 to 120 h as assessed by MTT assay. Under these conditions, apoptosis was induced as evidenced by increased sub G1 population and enhanced nuclear fragmentation shown by flow cytometry and DAPI staining, respectively. The expression of pro-apoptotic proteins, Bax was enhanced and Bid was cleaved, while anti-apoptotic protein Bcl-2 was reduced. Decursin also affected signaling pathways involved in cell growth and proliferation. The expression of membrane receptors, EGFR and c-ErbB2, were reduced and their downstream effectors, Akt and Erk, were downregulated as evidenced by reduced phosphorylated proteins. The expression of Cyclin D1 and the phosphorylated form of GSK-3 at S9 was reduced, suggesting the inhibitory role of decursin in canonical Wnt signaling pathway. In effect of Decursin on cell cycle regulators, it also enhanced the expression of Cdk inhibitor, p27Kip1 both at the transcript and protein levels. Moreover, phosphorylated form of p27 at T157 was reduced, which may have resulted from reduced Akt activity. Finally, the in vivo anti-tumor effect of decursin was seen in the mouse xenograft model. When MDA-MB-231 or SK-BR-3 cells were engrafted into nude mice by subcutaneous injection, tumor growth was significantly reduced in decursin-administered mice orally for 28 days. By showing the anti-cancer effect of decursin both in vitro and in vivo and suggest cell cycle arrest and apoptosis as a molecular mechanism, our novel findings indicates that decursin is an effective therapeutic agent against the more aggressive ER-negative breast cancers through reduced Akt activity and downregulation of EGFR and c-ErbB2.