Abstract #3864: Mechanism of 2-Methoxyestradiol-induced G₂/M cell cycle arrest in human breast cancer cells

[Background] 2-Methoxyestradiol (2-MeO-E₂), a nonpolar endogenous metabolite of 7\#946;-estradiol, has strong anti-proliferative, cytotoxic, and pro-apoptotic actions in several human breast cancer cell lines in vitro. Previous studies by us and also others have shown that 2-MeO-E₂ can strongly inhibit cell proliferation by inducing cell cycle arrest at the G₂/M phase as well as apoptosis in several human breast cancer cell lines in culture. However, the biochemical and cellular mechanisms by which 2-MeO-E₂ induces G₂/M cell cycle arrest is still not understood at present. [Methods] Cell viability was determined using MTT assay, and cell cycle was analyzed using flow cytometry. The levels of cell signaling molecules and apoptosis-related proteins were determined by Western blotting using specific antibodies. In addition, RNA interference was used for selective knockdown of the expression of certain target genes of interest. [Results] 2-MeO-E₂ could produce strong growth inhibition and G₂/M phase arrest in two human breast cancer cell lines with different p53 status: the MDA-MB-435s cells (with a mutant p53) and MCF-7 cells (with wild-type p53). Blockadeof the cell cycle at the G₂/M phase in these two cell lines was associated with a similar increase in the levels of p21/WAF1 expression and a decrease in the levels of cyclin B1, Cdc2, and Cdc25C in both cell lines. 2-MeO-E₂ also decreased the functions of Cdc2 by increasing its association with p21/WAF1/Cdc2 and also by increasing thelevels of the inactive phospho-Cdc2 and phospho-Cdc25C proteins. In addition, we found thatc-Jun NH₂-terminal kinase(JNK) also plays an important role in 2-MeO-E₂-induced cell cycle arrest. Chemical inhibition of JNK activity with SP600125 or with JNK small interfering RNA (siRNA) each significantly attenuated the accumulation of inactive phospho-Cdc2 and phospho-Cdc25C proteins and the number of cells arrested at the G₂/M phase. [Conclusion] The results of our present study imply a critical role for the JNK signaling pathway in 2-MeO-E₂-induced G₂/M arrest of humanbreast cancer cells. (Supported, in part, by an NIH grant CA-97109).

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 3864.