Figure 5

Activated SXR is necessary and sufficient to inhibit the growth of breast cancer cells. A) Constitutively active SXR is sufficient to decrease the growth of MCF-7 cells. MCF-7 cells were transfected with VP16 or VP16-SXR in the presence of pDSRed, and red fluorescent cells were sorted, plated and grown for an additional 4 days. Cell proliferation was measured by fluorescence assay as above and the experiment was repeated at least twice. ** represents P ≤ 0.01 in comparison to VP-16 (by Student's t test). B) SXR expression is reduced by siRNA directed against SXR. MCF-7 cells were transfected with siRNA against SXR (siRNA) or scrambled siRNA (SCR) (100 nM each) for 72 hrs was tested for SXR protein expression using anti-412 SXR antibody. The chemiluminescent bands were analyzed by spot densitometry analysis using FluorChem AlphaEase FC software (Alpha Innotech) C) Ligand induced up-regulation of iNOS is directly regulated by SXR. SXR-siRNA but not SCR transfection blocked the ability of SXR ligands (RIF and ANA) to induce up-regulation of CYP3A4 and iNOS. Un-transfected MCF-7 cells, or cells transfected with siRNA or SCR sequence (36 nM each) were treated with 10 μM RIF or ANA for 48 hr. Target gene expression was tested by QRT-PCR. The data are shown as average fold induction relative to solvent control in triplicates ± S.E.M. * represents P ≤ .05 in comparison to SCR (by student's t test). D) SXR activation induced apoptosis is blocked by SXR siRNA. Apoptosis was measured in equal number of MCF-7 cells induced with SXR activator rifampicin or solvent control DMSO for 72 hours after knocking down SXR expression using SXR specific siRNA. The cells transfected with a non-specific scrambled siRNA or un-transfected cells were used as a control. The experiment was repeated twice in triplicates. The absorbance at 405 nM represents apoptotic cells and data are shown as mean absorbance in triplicates ± S.E.M. * represents P ≤ .05 in comparison to DMSO solvent control.