We offer the possi bility that extranuclear ER may be detected more fre quently in PKC expressing tumors that are regressing Bioactive compound possibly indicating a response to treatment. It remains to be seen whether other techniques will be developed that may improve the detection of extranuclear ER in clinical specimens. We have previously suggested that PKC may be used as predictive biomarker for the use of E2 or an E2 like compound to effect tumor regression, and in fact the utility of using E2 was demonstrated. We report here that not only E2, but RAL is capable of eliciting T47D,A18 PKC tumor regression, despite the fact that these tumors are TAM resistant. Further we have shown that Inhibitors,Modulators,Libraries following 5 years of TAM treatment, these tumors are still sensitive to RAL induced tumor regression.

Although RAL may Inhibitors,Modulators,Libraries be considered as a potential treatment for patients with PKC expressing breast cancers, RAL is not as durable as E2 to elicit complete tumor regression. Since RAL has poor bioavailability, we are cur rently Inhibitors,Modulators,Libraries testing a series of benzothiophene analogues in our T47D,A18 PKC preclinical model for improved tumor in hibitory activity. Conclusions In summary, we report for the first time the involvement of extranuclear ER in an endocrine resistant tumor model to be associated with tumor regression and not growth stimulation. Key to this phenomenon may be ex pression of PKC, frequently associated with endocrine resistance Inhibitors,Modulators,Libraries and a potential biomarker for the use of E2 or RAL like compounds for the treatment of endocrine resistant breast cancer.

Methods Reagents For in vitro experiments dimethylsulfoxide, ethanol, E2, 4 OHT and RAL were obtained from Sigma Aldrich. For in vivo experi ments E2 and TAM were obtained from Sigma. RAL was Inhibitors,Modulators,Libraries purchased from the University of Illinois at Chicago Hospital Pharmacy. Cell culture reagents were obtained from Life Technologies. Tis sue cultureware was purchased from Becton Dickinson. The following antibodies were used, rabbit monoclonal ER, mouse monoclonal ER, rabbit polyclonal ER, and mouse monoclonal caveolin 1. Sec ondary antibodies included, anti rabbit Alexa Fluor 488, anti mouse third Cy3 and HRP cojungated anti rabbit and anti mouse. Cell culture conditions T47D,A18 neo and T47D,A18 PKC cells were maintained in RPMI 1640 with phenol red supplemented with 10% fetal bovine serum and G418 at 37 C, 5% CO2. Prior to experiments cell lines were placed in phenol red free RPMI 1640 supplemented with 10% stripped FBS for 3 days and maintained in the same manner for the duration of experi ments. Cell lines were tested for Mycoplasm contamination on a regular basis. Cell lines were not au thenticated by the authors.