Ethanol and Tumor Necrosis Factor Related Apoptosis Inducing

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					Ethanol and Tumor Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL) synergistically induce apoptosis in an androgen-independent prostate cancer cell line
Holoch P.A. Folsom J.B., Naud S.(Biostatistics) & Plante M.K.

Introduction Prostate cancer is the most common non-cutaneous malignancy and the second leading cause of cancer-related deaths in men in the United States [1]. Despite available screening with DRE and serum PSA, 10 to 20% of patients present with clinically advanced disease not amenable to curative radical prostatectomy [2]. Approximately 2030% of such cancers will not respond to androgen deprivation. The median survival after the diagnosis of hormone-refractory prostate cancer is 40-60 weeks and current chemotherapeutic agents offer a survival advantage of only 2 months with significant systemic toxicities [2, 3]. Among novel targeted therapies, Tumor Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL) or antibodies directed at TRAIL receptors have emerged as potential agents in treating many human cancers. TRAIL appears to induce apoptosis in cancer cells while most normal cells are resistant to this effect [4]. TRAIL initiates apoptosis by binding to death receptors in the TNF receptor family [5]. The death receptors DR4 and DR5 are upregulated in prostate cancer cells treated with chemotherapeutic agents [6]. Shankar et al. showed that sequential treatment of prostate cancer cell lines with chemotherapeutic agents followed by TRAIL enhanced the induction of apoptosis by TRAIL [5]. Ethanol has been shown to have a similar effect in sensitizing a human colon cancer cell line to TRAIL-induced apoptosis [7]. We investigated the effects of ethanol on TRAIL sensitivity in the androgen-independent human prostate cancer cell line PC-3. Materials and Methods Reagents PE-conjugated monoclonal active Caspase-3 antibody apoptosis kit and terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) kit were purchased from BD Biosciences (San Jose, CA). Soluble TRAIL was purchased from Biomol International (Plymouth Meeting, PA). Cell Culture Conditions Human prostate cancer PC-3 cells were obtained from American Type Culture Collection (Manassas, VA). PC-3 cells are androgen independent cells derived from a bone metastasis of a grade IV prostate adenocarcinoma. Cells were grown in Ham’s F12K medium with 2 mM L-glutamine, 10% fetal bovine serum, 10 IU penicillin and 10mg/ml streptomycin. Cells were grown in tissue culture dishes at 37°C with 5% CO2. Experimental Design Once the PC-3 cells had reached approximately 70% confluence, they were treated with either TRAIL 300ng/ml with media, 4% ethanol in media or both TRAIL 300ng/ml and 4% EtOH in media for 24 hours. Control cells were treated with media alone for 24 hours. The cells were then harvested and washed twice in cold PBS. Cells from each dish 1

were divided among the cell viability, caspase-3 and TUNEL assays. Experiments were repeated 4 times. Cell Viability Assay Cells were incubated with 50 ug/ml propidium iodide for 15 minutes at room temperature and then analyzed using a Coulter EPICS XL flow cytometer. Active Caspase-3 Activity Cells were fixed and permeabilized in 4% (w/v) paraformaldehyde and saponin for 20 minutes on ice. They were washed twice in buffer and resuspended in PE-conjugated monoclonal anti-active caspase-3 antibody for 30 minutes, then washed again in buffer and analyzed by flow cytometry. TUNEL Assay Cells were fixed and permeabilized in 1% (w/v) paraformaldehyde and saponin for 3060 minutes on ice. They were washed twice in buffer and resuspended in 70% ethanol at -20°C for a minimum of 24 hours. Cells were then washed twice in buffer and incubated with bromolated deoxyuridine triphosphate and TdT enzyme for 60 minutes at 37°C. They were then rinsed twice and resuspended in FITC-labeled anti-BrdU for 30 min at room temperature. Buffer containing propidium iodide and RNase was then added and the cells were incubated for another 30 minutes at room temperature before analysis by flow cytometry. Statistical Analysis The results of four independent experiments were expressed as means +/- standard deviation. For each experiment, statistical significance (p<0.05) was determined by ANOVA. For the TRAIL + 4% ethanol groups, statistical significance was measured for a synergistic effect (greater than additive). A non-parametric ANOVA was also run for the caspase-3 assay due to differences in variances. Results Cell Death After 24 hours of treatment, cells in the untreated, TRAIL and 4% ethanol groups all retained greater than 90% viability (TRAIL p=0.03; 4% EtOH p=0.33). Cells exposed to a combination of TRAIL and 4% ethanol had an average viability of 51% indicating a synergistic effect (p=<0.0001; fig. 1). Caspase-3 Activity Cells exposed to TRAIL alone had high levels of caspase-3 activity (average of 55% positive; p<0.0001) while cells exposed to 4% ethanol alone had very little active caspase-3 (3% positive; p=0.02). A synergistic effect was observed in cells exposed to both TRAIL and 4% EtOH (average of 66% positive; p=.0004).


Cell Viability
1.2 1
% Viability

0.8 0.6 0.4 0.2 0
Co ntr ol TR AIL EtO H 4% TR AIL +4 % EtO H

Figure 1. Effects of 24 hour exposure to TRAIL and 4% EtOH alone or in combination on cell viability. Values are means +/- SD from four independent experiments.

Active Caspase 3
% Caspase 3 Positive

1 0.8 0.6 0.4 0.2
Co ntr ol TR AIL EtO H EtO H TR AIL +4 %


Figure 2. Effects of 24 hour exposure to TRAIL and 4% EtOH alone or in combination on caspase-3 activity. Values are means +/- SD from four independent experiments.


TUNEL Assay
0.8 0.7
% TUNEL Positive

0.6 0.5 0.4 0.3 0.2 0.1
EtO H TR AIL +4 %

TR AIL Co ntr ol 4% EtO H

Figure 3. Effects of 24 hour exposure to TRAIL and 4% EtOH alone or in combination on TUNEL activity. Values are means +/- SD from four independent experiments.

TUNEL Assay The TUNEL assay measures DNA fragmentation as a marker of apoptosis. DNA fragmentation is a late stage in the process, occurring after the activation of caspase 3. Cells exposed to TRAIL alone showed very little DNA fragmentation (4%; p=0.17) as did cells exposed to 4% ethanol alone (3%; p=0.11). However, the two in combination caused very high levels of TUNEL positive cells indicating high levels of DNA fragmentation (mean of 57%; p<0.0001) again demonstrating a synergistic effect (fig. 3). Discussion TRAIL is a novel anticancer agent with a high degree of specificity for inducing apoptosis in cancer cells [4]. TRAIL triggers apoptosis via the extrinsic death receptor pathway, leading to activation of caspases 8 and 10 which may then be sufficient to activate the “executioner” caspases 3, 6 and 7 in certain cells [8]. The intrinsic p53mitochondrial apoptotic pathway is initiated by mitochondrial damage leading to increased cytoplasmic concentrations of cytochrome c and subsequent activation of caspase-9, which in turn activated the executioner caspases [8]. This intrinsic pathway is dependent on cellular expression of p53. Mutations in p53 are commonly observed in


malignant cells, especially those with metastatic potential. PC-3 cells do not express p53 [9]. TRAIL is thus an ideal candidate for bypassing the intrinsic pathway and inducing apoptosis in malignant cells that have lost p53. Our results indicate a synergistic effect of 4% ethanol and TRAIL on programmed cell death in PC-3 cells after 24 hours of treatment. Since DNA fragmentation was greatly increased and viability was greatly decreased only in cells treated with both TRAIL and ethanol, we suspect that the process of TRAIL-mediated apoptosis is accelerated by ethanol. This may occur via ethanol mediated mitochondrial damage and activation of the intrinsic apoptotic pathway. Alternatively, ethanol may increase the expression of the death receptors DR4 and DR5 which are believed to mediate TRAIL-induced apoptosis. Another possibility is that ethanol interferes with the expression or activity of an antiapoptotic factor downstream of the death receptors. Levels of active caspase-3 were elevated among cells exposed to TRAIL alone or in combination with ethanol because both populations were undergoing activation of the caspase cascade. However, it is unknown whether the intrinsic pathway was activated in either group since caspase-3 is activated by both pathways. Current clinical trials have focused on human monoclonal antibodies to TRAIL receptors rather than recombinant TRAIL. It is felt that this may reduce potential systemic toxicity by targeting a single type of receptor and circumvent the problem of the short half-life of TRAIL in plasma [8]. A phase 2 trial of HGS-ETR1, a human monoclonal agonistic antibody to TRAIL R1 is currently underway in patients with advanced malignancies [8]. The effects of activating the TRAIL receptors in vivo may be enhaced by chemotherapeutic drugs or ethanol. Obviously, systemic administration of 4% ethanol is not possible. However, Plante et al. have shown that direct transurethral intraprostatic injection with absolute ethanol in dogs resulted in coagulative necrosis with minimal systemic absorption and no damage to surrounding structures, although the extent of the necrosis is variable [10, 11]. Future studies are needed to follow the pattern of caspase-3 activation and DNA fragmentation over time and to look for caspase-8 and 10 activity (indicating activation of the extrinsic pathway) versus caspase-9 activity (indicating activation of the intrinsic pathway) in malignant prostate cells treated with ethanol and TRAIL. We would expect to see more caspase-8 and 10 activity in the TRAIL group and all three initiator caspases activated in the TRAIL + ethanol group. In vivo studies, such as nude mouse xenografts, would be very useful for evaluating the response of solid human prostate cancer tumors to direct injections with ethanol in the setting of systemic administration of TRAIL or agonistic TRAIL receptor antibodies. In summary, we have found that 24 hour exposure to 4% ethanol and TRAIL synergistically enhances cell death, DNA fragmentation and activation of caspase-3 in human prostate cancer (PC-3) cells. We postulate that ethanol accelerates TRAILmediated apoptosis in PC-3 cells. References [1] American Cancer Society. Cancer facts and figures 2005. Atlanta: American Cancer Society; 2005. [2] Tannock et al. Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer. New England Journal of Medicine. 2004; 351:1502-1512.


[3] Tanagho E.A. and McAninch J. W. (2000) Smith’s General Urology, fifteenth edition. [4] Bouralexis S., Findlay D.M. and Evdokiou A. Death to the bad guys: Targeting cancer via Apo2L/TRAIL. Apoptosis. 2005; 10: 35-51. [5] Shankar S., Xufen C. and Srivastava R.K. Effects of sequential treatments with chemotherapeutic drugs followed by TRAIL on prostate cancer in vitro and in vivo. The Prostate. 2004; 9999:1-22. [6] Munshi A., McDonnell T.J. and Meyn R.E. Chemotherapeutic agents enhance TRAIL-induced apoptosis in prostate cancer cells. Cancer Chemotherapy Pharmacology. 2002; 50: 46-52. [7] Vaculova A. et al. Ethanol acts as a potent agent sensitizing colon cancer cells to the TRAIL-induced apoptosis. Federation of European Biochemical Societies. 2004; 577:309-313. [8] Rowinsky E.K. Targeted induction of apoptosis in cancer management: the emerging role of tumor necrosis factor-related apoptosis-inducing ligand receptor activating agents. Journal of Clinical Oncology. 2005; 23:9394-9407. [9] Mikata K. et al. Inhibition of growth of human prostate cancer xenograft by transfection of p53 gene: gene transfer by electroporation. Molecular Cancer Therapeutics. 2002; 1:247-252. [10] Plante M.K., Folsom J.B. and Zvara P. Prostatic tissue ablation by injection: a literature review. Journal of Urology. 2004; 172:20-26. [11] Plante M.K. et al. Intraprostatic ethanol chemoablation via transurethral and transperineal injection. British Journal of Urology. 2003; 91:94-98.