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Capsaicin Inhibits the Growth of Prostate Cancer Cells

was dissolved in PBS. For all compounds, the diluent was never present at >0.5% in the experiments; and control dishes having the same concentration of diluent had no detectable effect.

MTT and clonogenic assay in soft agar. 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT; Sigma Chemical) and clonogenic assays in soft agar were done as previously described (20, 21).

incubated in 108 mol/L dihydrotestosterone (only LNCaP cells) F 2 104 mol/L capsaicin for 4 and 12 hours. Cells were fixed in 100% methanol and incubated with the primary antibody against AR and p65 (Santa Cruz Biotechnology) at 1 Ag/mL (in PBS with 5% bovine serum albumin and 0.6% Tween 20), at 4jC overnight. A secondary biotinylated antibody and RPE- conjugated streptavidin (DAKO A/S, Denmark) were used for visualization.

Measurement of apoptosis. Terminal deoxynucleotidyl transferase– mediated nick end labeling assay (TUNEL) was done for immunohisto- chemical detection and for the quantification of programmed cell death at the single cell level, based on labeling of DNA strand breaks, the In situ Cell Death Detection kit, POD (Roche, Indianapolis, IN) was used.

Cell cycle analysis. PC-3 cells were exposed to either increasing doses of capsaicin, or vehicle control (10% FCS in RPMI 1640 containing 0.1% or 0.2% ethanol) for 24 hours. Cells were fixed in 75% chilled methanol and stained with propidium iodine. Cell cycle status was analyzed on a Becton Dickinson Flow Cytometer (BD Biosciences, Franklin Lakes, NJ).

Western blotting. Cytoplasmic and nuclear components of the cells were extracted using CelLytic NuCLEAR Extraction kit (Sigma Chemical). Protein concentrations were determined using the Bio-Rad protein assay dye reagent concentrate (Bio-Rad Laboratories, Hercules, CA), according to the manufacturer’s recommendation. Western blotting was done as previously described (20). The following primary antibodies were used: anti- AR (sc-7305), anti-Bax (sc-493), anti-hnRNP A1 (sc10030), anti-InBa (sc847), anti-NF-nB p65 (sc-372-G), anti-p21 (sc-397), anti-p53 (sc126), and anti-PSA (sc-7638) from Santa Cruz Biotechnology (Santa Cruz, CA), and anti– glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Research Diag- nostics (Flanders, NJ).

Real-time reverse transcription-PCR. Total cellular RNAs were isolated from cells using RNeasy mini kit (Qiagen, Valencia, CA) and cDNAs were synthesized using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) according to manufacturer’s recommendation. Expression levels were determined using HotMaster Taq DNA Polymerase (Eppendorf, Westbury, NY) and SYBERGreen I (Molecular Probes, Eugene, OR) with the following primers: AR forward 5V-AGGATGCTCTACTT- CGCCCC-3V, AR reverse 5V-ACTGGCTGTACATCCGGGAC-3V, transient receptor potential vanilloid type 1 (TRPV1) forward 5V-AACTGGACC- ACCTGGAACAC-3V, TRPV1 reverse 5V-GCCTGAAACTCTGCTTGACC-3V. Reactions were done in triplicate using a iCycler iQ system (Bio-Rad Laboratories). Reaction products were visualized on ethidium bromide– stained agarose gels. For each sample, the amount of the target gene and reference gene (18S) was determined from standard curves.

Transfections and luciferase assays. The following promoter reporter constructs were used: PSA P/E-Luc, PSA enhancer E4-LUC, PSA enhancer E4 S-ALL-LUC, and ARE4-E4Lux (21). The NF-nB reporter construct (pGL3- NF-nB) containing four copies of NF-nB binding sequences were cloned into pGL3-basic plasmid (Promega, Madison, WI), a generous gift from Dr. Moshe Arditi (Cedars-Sinai Medical Center, Los Angeles, CA). Cells were transfected with the indicated plasmids using GenePORTER Transfection Reagent (Gene Therapy Systems, San Diego, CA) under serum-free conditions. A PhRLTK vector was included as an internal control for transfection efficacy. Following transfections, cells were incubated in RPMI 1640 with 10% charcoal-stripped FBS either with or without dihydrotes- tosterone and either with or without capsaicin for 24 hours, and were then collected with tissue lysis buffer (Promega). Luciferase activity of the cell lysates was measured by luminometry, and activities were normalized by h-galactosidase activities. Proliferation assays with IKKh- and AR vector– transfected cells were done in either 6- or 12-well plates. Cells were plated on day 0 and grown in RPMI 1640 with 10% FBS at 37jC. Cells were cotransfected on day 1 with an IKKh-expressing vector (kind gift from Prof. R. Gaynor, Eli Lilly, Indianapolis, IN) or an AR vector and pcDNA 3.1 neomycin-resistant vector (also used as control) with LipofectAMINE (Invitrogen) according the manufacturer’s instruction. On day 2, G418 (Invitrogen) was added, and on day 3, capsaicin was added. Between days 10 and 12, colonies were stained with crystal violet (Sigma Chemical) and the growth-inhibitory effect was evaluated by counting the number of colonies.

Immunofluorescence. PC3 cells (p65 staining) and LNCaP cells (AR staining) were grown overnight on coverslips in six-well plates and then

Proteasome function assay. PC3 cells were treated with capsaicin (2 104, 24 or 48 hours), harvested, and a proteasome function assay was done as previously described (22). The chymotrypsin-like, trypsin-like, and PGPH activities of the proteasome were determined by the ability to degrade the appropriate fluorogenic substrates: Suc-LLVY-AMC (Sigma Chemical), Z-ARR-AMC (Calbiochem, Darmstadt, Germany), and Z-LLE- AMC (Sigma Chemical).

In vivo animal treatment protocol. Three-week-old BNX nu/nu male mice (Harlan Sprague-Dawley, Inc., Indianapolis, IN) were maintained in pathogen-free conditions and fed irradiated chow. PC-3 cells (5 106) in 0.1 mL of Matrigel (Collaborative Biological Products, Bedford, MA) were injected s.c. into the bilateral flanks of each mouse and treatment (5 mg/kg/d in 100 AL of PBS containing 0.3% ethanol per day) was started the next day and continued for 4 weeks. Five control mice received vehicle (100 AL PBS containing 0.3% ethanol per day). Both groups received the agent by gavage 3 days per week (Monday, Wednesday, and Friday). Tumor sizes were measured every week using the formula A B C 0.5236 (A, length; B, width; C, height; all measured in millimeters). The mice were sacrificed by carbon dioxide asphyxiation after 4 weeks and tumor weights were measured. Differences of tumor sizes and weights between mice in control and experimental groups at the end of the study were analyzed by Student’s t test (statistical tests were two-sided).

Results

Effect of capsaicin on clonal proliferation and cell cycle of LNCaP, PC-3, and DU-145. We tested the effect of increasing doses of capsaicin on the clonogenic growth in soft agar of the prostate cancer cell lines LNCaP, PC-3, and DU-145. Capsaicin inhibited the clonal proliferation of each in a dose-dependent manner (Fig. 1A). At 5 104 mol/L, the growth of each cell type was completely inhibited. For cell cycle analysis, PC-3 cells were treated with either increasing doses of capsaicin or vehicle for 24 hours and examined by flow cytometry. The population of cells in the G0/G1 phase increased and those in S phase decreased in a capsaicin dose- dependent manner (Fig. 1B).

Capsaicin induces apoptosis in prostate cancer cells. Cells were exposed to increasing concentrations of capsaicin for 24 hours and apoptosis was measured by TUNEL assay. Capsaicin induced apoptosis in a dose-dependent manner, with the percentage of apoptotic cells ranging from 3% at 1 104 mol/L capsaicin to 75% at 5 104 mol/L capsaicin for PC-3 cells, and from 9% at 1 104 mol/L capsaicin to 93% at 5 104 mol/L in LNCaP cells (Fig. 3C).

Capsaicin modulates the levels of proteins associated with apoptosis and cell cycle in a time-dependent manner. To characterize the molecular mechanism of capsaicin-induced cell cycle arrest followed by apoptosis, we examined the levels of several apoptotic- and cell cycle–related proteins in capsaicin- treated (2 104 mol/L) LNCaP cells (Fig. 1D). Expression of p21 Waf1 protein increased after 6 hours, and levels of Bax and p53 increased after 3 and 12 hours exposure to capsaicin, respectively.

Effect of capsaicin on the transcriptional activity of androgen-responsive reporter constructs. PSA is an androgen- responsive gene produced by prostate epithelial cells. To investigate the effect of capsaicin on PSA transcription, androgen-sensitive LNCaP cells were transfected with a PSA promoter/enhancer- luciferase reporter vector (PSA P/E-Luc). These cells were incubated

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Cancer Res 2006; 66: (6). March 15, 2006

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