TRPM8 channel as a novel molecular target in androgen-regulated prostate cancer cells.

Ethics statement Human high-density

PC tissue microarray was purchased from US Biomax (Rockville, MD). All the prostate tissue samples were obtained from patients (Age: 21 yrs to 83 yrs) undergoing therapeutic surgery at the US Biomax certified hospitals (see Sdata). Cell lines, transfection The cell lines RWPE1, RWPE2, LNCaP, DU145 and PC3 were obtained from the American Type Culture Collection (Manassas, VA) and were maintained under standard conditions as per ATCC guidelines. LNCaP, PC3 and DU145 cells were cultured in RPMI, F-12K and DMEM/F12K (1:1) medium, respectively supplemented with 10% fetal bovine serum (GIBCO BRL, Lewisville, TX) and 1% penicillin/streptomycin. RWPE1 and RWPE2 cells were grown in keratinocyte-serum free media (K-SFM) containing 50 μg/ml bovine pituitary extract, 5 ng/ml epidermal growth factor and 1% penicillin/streptomycin solution. All cells were cultured in a 37°C incubator with 5% CO 2 humidified atmosphere. HEK-293 stable cells overexpressing TRPM8 (HEK-TRPM8) were established and maintained following standard protocol [ 48 ]. All transfection experiments were performed using FuGENE HD transfection reagent (Roche Diagnostics, Indianapolis, IN) (see Sdata). Plasmids, shRNA construct, antibodies and chemical inhibitors TRPM8 rat cDNA cloned in N-terminus myc-tagged pCDNA3 vector for TRPM8 overexpression (TRPM8OE). TRPM8 trafficking investigated by GFP-TRPM8. The siAR and shTRPM8 purchased from Santa Cruz Biotechnology (Santa Cruz, CA). pCDNA3-empty vector used as control. All antibodies purchased from Santa Cruz Biotechnology; anti-TRPM8/CMR-1 (656-680) from Phoenix Pharmaceuticals (Burlingame, CA); anti- DHT/testosterone from Thermo Scientific Pierce (Rockford, IL); Alexa Flour 488 and 594 purchased from Invitrogen (Invitrogen, Carlsbad, CA). Testosterone, DHT (5α-dihydrotestosterone), PYR-41 (UBA1 inhibitor) and hydroxyflutamide (HF; AR inhibitor) was purchased from Sigma (St. Louis, MO).

Chromatin immunoprecipitation

(ChIP) and real time-PCR (RT-PCR) ChIP assay was performed using ChIP-IT TM Express kit from Active Motif (Carlsbad, CA) following supplied protocol. (see SMethods). Sub-cellular protein fractionation, immunoprecipitation, immunoblotting and mass spectrometry Detergent soluble membrane fraction preparation and analysis was done according to standard protocol [ 25 , 49 ].

Ubiquitination and biotinylation assay

For the ubiquitination assay, whole cell lysates (500 μg) were incubated with UbiCapture-Q Matrix (VWR International, Batavia, IL). Biotinylation assays were performed using cell surface protein isolation kit (Thermo Scientific Pierce) (see SMethods). Transmission electron microscopic studies The cells were fixed using fixative solution (2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4). After fixation, samples were buffer rinsed and postfixed with 1% osmium tetroxide, dehydrated (35%, 70%, 95%, 100% ethanol dehydration), and flat embedded in propylene oxide and Epon 812 epoxy resin (Tousimis) in 1:1 ratio at 60 °C for 4 h. A Reichert OMU3 ultramicrotome (Austria) was used to prepare 700A° thin sections that were mounted on 200 copper mesh grids, stained with uranyl acetate and lead citrate. The sections were viewed under a JEOL JEM 100C transmission electron microscope (60 kV). FACS, TUNEL and clonogenic assay The effects of androgen / AR inhibition / TRPM8 overexpression on cancer cell proliferation were examined by FACS, TUNEL and clonogenic assay following standard protocol [ 47 ] (See SMethods).

EMSA and p53 transcription factor assay

The p53 activation in nuclear extracts was monitored using p53 (3) - EMSA (Affymetrix, Santa Clara, CA) and ELISA-based TransAM™ p53 kit (Active Motif) according to the manufacturer's protocol (see SMethods).

Calcium imaging and planar lipid bilayers

Fluorescence measurements of intracellular Ca 2+ concentration of single cells were measured using standard protocol [ 19 , 25 , 48 ]. Planar lipid bilayers measurement and purification of TRPM8 protein from HEK-TRPM8 cells done using standard protocol [ 50 ].