The in vitro and in vivo anti-tumor effect of decursin in estrogen receptor-negative human breast cancer cells occurs via downregulation of Akt signaling pathway

Abstract

The need for 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 and leukemia. 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 c-ErbB2, respectively. Cell growth was attenuated by decursin (50-200μM) in a time- and dose-dependent manner. Under these conditions, apoptosis was induced as evidenced by increased sub G1 population, enhanced nuclear fragmentation, up-regulation of Bax and cleaved Bid proteins, down-regulation of Bcl-2 protein. 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 also downregulated as evidenced by reduced phosphorylated proteins. Suppressed Akt activity was seen by reduced phosphorylation of its substates, Bad, p27Kip1 and GSK-3β. Total expression of Bad was also enhanced and such changes in Bad may have contributed to augmented apopotosis by decursin. On the other hand, increased nuclear localization of p27Kip1 was observed, which was consist with reduced phosphorylation at T157 and may have provided for attenuated cell proliferation. Total β-catenin protein as well as Cyclin D1 protein and mRNA were reduced. Moreover, cytoplasmic localization of β-catenin was increased. Therefore, these results strongly suggest the suppression Akt-mediated Wnt/β-catenin signaling pathway by decursin. 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 provide decursin as a potential preventive or therapeutic agent against the more aggressive ER-negative breast cancers through reduced Akt activity and downregulation of EGFR and c-ErbB2.