Gene expression status at the protein and mRNA levels in both xenograft and spontaneous breast tumors were detected by western blot assays and real time PCR. As indicated in Figure 5A left panel, first row kinase inhibitor Ruxolitinib and Figure 5B left panel, GE treatment alone and combin ation treatment of GE and TAM induced Inhibitors,Modulators,Libraries significant ER protein re expression in mice breast xenografts. Consistently, ER mRNA level, was sistent with its expression at the mRNA level. In terms of the expression status of DNMT1 and HDAC1, dietary Inhibitors,Modulators,Libraries GE caused a gradual reduction of the expression of these enzymes at the protein and mRNA levels in both tested mouse mod els, especially when GE and TAM were acting together. These results indicate that epigenetic mechan isms may contribute to GE induced ER re activation leading to increased sensitivity of TAM therapy toward intractable ER negative breast cancer.

Inhibitors,Modulators,Libraries Epigenetic enzymatic activities changes in response to GE and TAM treatment in vivo Our observations on expression changes of DNMT1 and HDAC1 indicated that GE alone or combined with TAM treatment led to a significant decrease in expression of these two important epigenetic enzymes. We next sought to investigate whether this reduced expression can result in direct enzymatic activ ities changes in vivo that may contribute to epigenetic mechanisms modulated gene expression alteration such as ER re activation. We assessed the epigenetic enzym atic activities of HDACs and DNMTs in both xenograft and spontaneous breast tumors.

As shown in Figure 7A, both GE and TAM treatment alone and Inhibitors,Modulators,Libraries in combination r than spon taneous breast tumors, suggesting that GE exposure time could be a key factor influencing TAM induced epigenetic regulation. However, as to DNMTs activity shown in Figure 7B, only GE treatment caused a slight inhibition suggesting that dietary GE treatment is pri marily mediated through histone remodeling rather than DNA methylation, which is consistent with our previous in vitro studies. We found that TAM, acting as an anti hormone Inhibitors,Modulators,Libraries drug, may exert its anti cancer properties by interacting with epigenetic modulators such as DNMTs or HDACs. This may explain our previous results indicating that TAM enhanced GE induced anti cancer properties through, at least in part, ER reactivation. TAM may influence epigenetic pathways that facilitate the epigenetic effects of GE leading to ER activation.

These results suggest an important synergistic inter action between GE and TAM against ER negative breast cancer. In summary, our results indicate that dietary GE may affect ER expression via modulating epigenetic pathways, especially, histone modification. In addition, dietary GE reinforced TAM caused anti cancer effects thenthereby through increased therapeutic target via up regulated ER and po tential interaction between these two compounds resulting in epigenetic modulations of more relevant genes.