Background: Prostate cancer is the most common non-cutaneous malignancy and second leading cause of male cancer deaths nationally. Tumor Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL) or antibodies to TRAIL receptors are potential agents in treating human cancers. TRAIL induces apoptosis in cancer cells while sparing most benign tissues. Ethanol (EtOH) has been demonstrated to enhance TRAIL induced apoptosis in a colon cancer cell line. We investigated the effects of EtOH and TRAIL in the androgen-independent human prostate cancer cell line PC-3.
Methods:
Cell Culture
PC-3 cells(American Type Culture Collection) were cultured with 5% CO₂ at 37°C
Experimental Design
Cells were plated at 1x10 ⁶ cells/plate, grown to 70% confluence, treated with: media alone, TRAIL 300ng/ml, 4% EtOH, or both TRAIL 300ng/ml and 4% EtOH for 24 hrs, harvested, washed in PBS, divided among each assay, and experiments repeated x 4.
Cell Viability
Cells were incubated with 50 ug/ml PI for 15 min at RT and analyzed using flow cytometry(FC).
Active Caspase-3
Cells were fixed/permeabilized, washed, resuspended in PE-conjugated anti-active caspase-3 antibody for 30 min, washed, and analyzed by FC.
TUNEL
Cells were fixed, washed, resuspended in 70% EtOH at -20°C for 24 hrs, washed, incubated with bromolated dUTP/TdT enzyme for 1hr at 37°C, washed, and resuspended in FITC-labeled anti-BrdU for 30 min at RT. PI/RNase buffer was added, incubated for 30 min, and analyzed by FC.
Statistical Analysis
Four independent experiments were expressed as means +/- SD and statistical significance (p<0.05) was determined by ANOVA. A non-parametric ANOVA was used for the caspase-3 assay due to differences in variances.
Results:
Cell Viability
The untreated, TRAIL, and 4% EtOH groups all retained > 90% viability (TRAIL p=0.03; 4% EtOH p=0.33). Cells exposed to TRAIL and 4% EtOH had decreased viability (51%; p=<0.0001).
Caspase-3 Activity
Cells exposed to TRAIL had elevated caspase 3 activity (55% ; p<0.0001) while cells exposed to 4% EtOH had low active caspase 3 (3%; p=0.02). Cells exposed to both TRAIL and 4% EtOH had elevated active caspase 3 (66%; p=.0004).
TUNEL
Cells exposed to TRAIL had little DNA fragmentation (4%; p=0.17) as did cells exposed to 4% EtOH (3%; p=0.11). However, TRAIL and 4% EtOH resulted in increased TUNEL positive cells(57%; p<0.0001).
Conclusions: Exposure to 4% EtOH and TRAIL for 24hrs enhances cell death (decreases cell viability), caspase 3 activity, and DNA fragmentation in PC-3 cells. We postulate that EtOH accelerates TRAIL-mediated apoptosis in PC-3 cells.