Experimental evidence indicates that prostate apoptosis response-4 (Par-4, also known as

Experimental evidence indicates that prostate apoptosis response-4 (Par-4, also known as PAWR) is a key regulator of cancer cell survival and may be a target for cancer-selective targeted therapeutics. BID expression. Par-4 overexpression did not affect the cell cycle profile. However, MCF-7 cells with increased Par-4 expression showed reduced ERK phosphorylation, suggesting that the inhibition of cell proliferation promoted by Par-4 may be mediated by the MAPK/ERK1/2 pathway. MCF-7 cells with increased Par-4 expression showed a marginal increase in early apoptotic cells. Importantly, we found that Par-4 expression modulates apoptosis in response to docetaxel in MCF7 breast cancer cells. Par-4 exerts growth inhibitory effects on breast cancer cells and chemosensitizes them to docetaxel. forward, 5-ACTTTCCAGAGCTACAAC ATG-3; reverse 5-GTCCATGGGGTTAAGAATCAA-3. Relative expression was calculated by 2?CT (CT = fluorescence threshold value; CT = CT of the target gene ? CT of the reference gene (GADPH); CT = CT of the target sample ? CT of the reference sample). The average value of the control cells served as the reference sample. The results were expressed as n-fold differences in mRNA expression relative to expression of GAPDH and the reference sample. Western blot analysis Cells were harvested and total 1314890-29-3 IC50 cell lysates were prepared in lysis buffer (50 mM Na pyrophosphate, 50 mM NaF, 5 mM NaCl, 5 mM PMSF, 100 mM Na3VO4), followed by centrifugation at 13,000 rpm for 15 min CSF3R at 4C. Thirty micrograms of protein lysate were separated on a 10% SDS-PAGE gel and blotted onto nitrocellulose membranes (Pierce Biotechnology, Rockford, IL, USA). Blots were incubated with anti-Par-4 monoclonal antibody (Abcam, Cambridge, MA, USA), anti-p-ERK, anti-ERK (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti–actin mouse monoclonal antibody (Millipore, Temecula, CA, USA) for 2 h at room temperature. Membranes were washed in TBS-T (25 mM Tris, 125 mM NaCl and 0.1% Tween-20) and incubated with appropriate peroxidase-conjugated secondary IgG antibody for 2 h at room temperature. Incubations were performed in 5% skim milk diluted in TBS-T. Specific proteins were detected using an enhanced chemiluminescence system (ECL? Western Blotting Detection Reagents, GE Healthcare, Buckinghamshire, UK) and exposed to Hyperfilm ECL film (GE Healthcare). Cell proliferation assay Cell proliferation and viability were measured using a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT, Molecular Probes, Invitrogen) assay following the manufacturers instructions. Cells were seeded in 96-well plates (1104 1314890-29-3 IC50 cells/well) and maintained in RPMI-1640 medium without phenol red supplemented with 5 or 0.5% FBS. Cell growth was assessed at 0, 48, 72, 96 and 144 h. At the end of 1314890-29-3 IC50 incubation, the absorbance was measured at 570 nm using the Biotrak 1314890-29-3 IC50 II Plate reader (Amersham Biosciences, Cambridge, UK). Imunocytochemistry MCF-7 cells were cultured in 8-well chamber slides in RPMI-1640 medium without phenol red and supplemented with 5% FBS or 5% dextran-coated charcoal-treated FBS (stripped serum, ST) for 48 h before treatment. For immunocytochemistry, cells were fixed using 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. Next, cells were incubated with primary mouse monoclonal anti-Par-4 antibody 1:50 (Santa Cruz, Biotechnology; catalog sc-1666) and rabbit polyclonal anti-tubulin antibody 1:300 (OriGene), followed by the appropriate secondary antibodies conjugated with Alexa Fluor 546 and Alexa Fluor 488 1:300 (Invitrogen, OR, USA). Nuclei were counterstained with Hoechst 33342 1:3000 (Invitrogen). Cells were visualized with Carl Zeiss LSM 510 Meta (Oberkochen, Germany) laser scanning confocal microscopy. All immunofluorescence images were recorded at magnification 20 and 63. Cell cycle analysis Floating and adherent cells were collected, washed once with PBS, fixed with 70% ice-cold ethanol and stored at ?20C until analysis by flow cytometry. The fixed cells were washed twice with PBS, resuspended in PBS containing 200 (27) reported that Par-4 mRNA and protein levels rapidly and progressively increase after trophic factor withdrawal in cultured rat hippocampal neurons and that Par-4 acts early in apoptosis (i.e., before mitochondrial dysfunction, caspase activation and nuclear changes). Previous results from our group also showed that withdrawal of estrogens and growth factors from the serum led to significantly increased Par-4 expression compared with the expression observed in MCF-7 cells maintained in media supplemented with 5% FBS (22). Furthermore, our group has demonstrated that Par-4 expression is negatively regulated by IGF-1 and the hormone 17-estradiol (22). These findings are in agreement with results from another study, in which cholangiocarcinoma cells cultured in the absence of serum exhibited Par-4 protein increased expression associated with a significant increase in BAX protein (21). In the present study, we used RNAi to reduce Par-4 expression and the expression vector pCMV6-XL6-PAR4 to increase Par-4 expression in MCF-7 cells. MCF-7 cells with reduced Par-4 expression.