Ethanol promotes cytotoxic effects of tumor necrosis ...

Prostate Cancer and Prostatic Disease (2013) 16, 16–22 & 2013 Macmillan Publishers Limited All rights reserved 1365-7852/13 www.nature.com/pcan

ORIGINAL ARTICLE

Ethanol promotes cytotoxic effects of tumor necrosis factor-related apoptosis-inducing ligand through induction of reactive oxygen species in prostate cancer cells MK Plante1,2, WT Arscott2, JB Folsom1, SW Tighe2, RJ Dempsey3 and UV Wesley3 BACKGROUND: Effective treatment of prostate cancer (PCa) remains a major challenge due to chemoresistance to drugs including tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Ethanol and ethanol extracts are known apoptosis inducers. However, cytotoxic effects of ethanol on PCa cells are unclear. METHODS: In this study we utilized PC3 and LNCaP cell culture models. We used immunohistochemical analysis, western blot analysis, reactive oxygen species (ROS) measurement, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) Cell Proliferation Assay, Annexin-V staining and flow cytometry for quantification of apoptosis. In vitro soft agar colony formation and Boyden chamber invasion assays were used. Tumorigenicity was measured in a xenotransplantation mouse model. RESULTS: Here, we demonstrate that ethanol enhances the apoptosis-inducing potential of TRAIL in androgen-resistant PC3 cells and sensitizes TRAIL-resistant, androgen sensitive LNCaP cells to apoptosis through caspase activation, and a complete cleavage of poly (ADP)-ribose polymerase, which was in association with increased production of ROS. The cytotoxicity of ethanol was suppressed by an antioxidant N-acetyl cystein pretreatment. Furthermore, ethanol in combination with TRAIL increased the expression of cyclin-dependent kinase inhibitor p21 and decreased the levels of Bcl-2 and phosphorylated-AKT. These molecular changes were accompanied by decreased proliferation, anchorage-independent growth and invasive potential of PC3 and LNCaP cells. In vivo studies using a xenotransplantation mouse model with PC3 cells demonstrated significantly increased apoptosis in tumors treated with ethanol and TRAIL in combination. CONCLUSIONS: Taken together, use of ethanol in combination with TRAIL may be an effective strategy to augment sensitivity to TRAIL-induced apoptosis in PCa cells. Prostate Cancer and Prostatic Disease (2013) 16, 16–22; doi:10.1038/pcan.2012.37; published online 18 September 2012 Keywords: ethanol; TRAIL; apoptosis; reactive oxygen species (ROS)

INTRODUCTION Disrupted apoptotic pathways are associated with decreased sensitivity to current treatments and development of therapy resistant prostate cancer (PCa).1–6 Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a broad range of cancer cells, with little or no effects on normal cells.7–10 Within the PCa-derived cell lines studied, there is extensive variability in their sensitivity to TRAIL-induced apoptosis. Androgenindependent PC3 cells are moderately sensitive to TRAIL. However, androgen-dependent LNCaP cells are refractory to TRAIL-induced apoptosis.7–9 Interestingly, resistance to TRAIL by PCa cells can be overcome when TRAIL is combined with various chemotherapeutic agents.10–19 Ethanol-induced apoptotic cell death has been well recognized in many cell types and animal models.20–24 High concentrations of ethanol can effectively destroy hepatoma tumor as well as normal cells, whereas lower concentrations of ethanol selectively induces apoptosis in hepatoma tumor cells but not in normal hepatocytes.25 Ethanol induces significant apoptosis of purkinje cells in the developing cerebellum.26 Furthermore, ethanol exposure is directly involved in the production of reactive oxygen

species (ROS) and apoptosis. In a dose-dependent manner it promotes lead-induced oxidative stress and induces apoptosis in neuronal cells.27–29 Ethanol also promotes apoptosis through inhibition of mitogenic growth factor signaling, glutathione depletion, DNA damage and endoplasmic reticulum stress.30–34 Clinical studies examining the benefit of prostatic injection with ethanol for symptomatic BPH have demonstrated significant postsurgical improvements.35–37 Furthermore, ethanol promotes tunicamycin- and thapsigargin-induced neuronal cell death, and potentiates TRAIL-induced apoptosis in colon cancer cells and leukemic T-Lymphocytes.38,39 However, little is known about its use as a chemosensitizing agent in PCa cells. In this study, we demonstrate that ethanol enhances the pro-apoptotic effects of TRAIL in PC3 cells, and sensitizes TRAILresistant LNCaP cells to apoptosis through caspase activation and complete poly (ADP) ribose polymerase (PARP) cleavage. Furthermore, ethanol and TRAIL increased the expression of p21 and decreased the levels of pro-survival factors phosphorylated-Akt and Bcl-2. The effects of ethanol were associated with over production of ROS, and blocking of ROS production suppressed the cytotoxic effects of ethanol. These molecular changes were

1 Department of Surgery and Urology, University of Vermont, Burlington, VT, USA; 2Vermont Cancer Center, University of Vermont, Burlington, VT, USA and 3Department of Neurosurgery, School of Medicine and Public Health, University of Wisconsin, Madison, WI, USA. Correspondence: Dr UV Wesley. Current address: Department of Neurosurgery, School of Medicine and Public Health, University of Wisconsin, CSC-K4/670, 600 Highland Avenue, Madison, WI 53792, USA. E-mail: [email protected] Received 25 April 2012; revised 17 August 2012; accepted 20 August 2012; published online 18 September 2012

Ethanol promotes TRAIL-induced cytotoxicity MK Plante et al

17 accompanied by decreased anchorage-independent growth and invasive potential of PCa cells in vitro. In a PC3 mouse tumor xenotransplantation model, ethanol enhanced the apoptotic effects of TRAIL. Taken together, our data suggest the use of ethanol as a promising agent in sensitizing PCa cells to TRAILmediated apoptosis.

Meeting, PA, USA). Ethanol was obtained from the University of Vermont Chem Source. Subconfluent cell cultures were exposed to TRAIL (100 ng ml 1) and ethanol (4%) alone or in combination for 48 h in medium containing 5% FBS. TRAIL and ethanol concentrations were based on work by Shankar et al.12 and Vaculova´ et al.,38 respectively. Cells were treated with 5 mM N-acetyl-L-cystein (NAC) (Sigma Aldrich, St Louis, MO, USA) 1 h before TRAIL and ethanol treatment when necessary. Untreated cells were used as controls.

MATERIALS AND METHODS Cell cultures and exposure to TRAIL and Ethanol

MTT cell proliferation assay

PC3 and LNCaP cells from ATCC, Rockville, MD, USA were grown as previously described.40 TRAIL was purchased from Biomol (Plymouth

Colorimetric assays were performed using MTT assay kit (ATCC) according to manufacturer’s protocol. Briefly, 5 103 cells per well were plated and

Figure 1. Ethanol enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated cytotoxicity and cell growth inhibition in PC3 and LNCaP cells. (a) Bright field photographs showing phenotypic changes in PC3 and LNCaP cells treated with ethanol, TRAIL alone or in combination for 48 h. Morphology of ethanol-treated cells remained mostly similar to untreated cells. TRAIL alone resulted in some changes in cellular morphology. Marked morphological changes were observed in both PC3 and LNCaP cells exposed to TRAIL (100 ng ml 1) and ethanol (4%) in combination. Magnification, 200. (b) The rates of cellular proliferation of PC3 and LNCaP cells were assessed in either untreated or treated with ethanol and TRAIL alone or in combination by MTT assay, as described in Materials and methods. The experiment was repeated twice. Optical density values are expressed as mean±s.d. of triplicates (*Po0.05 compared with control).

Figure 2. Induction of apoptosis by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and ethanol in PC3 and LNCaP cells. (a) Detection of apoptotic cell death after 48 h of either single agent TRAIL at the concentration of 100 ng ml 1 or co-treatment with ethanol at the concentration of 4% by annexin-V-fluorescein isothiocyanate staining using flow cytometry. The percent apoptosis represents mean±s.d. of three independent experiments performed in triplicate (*Po0.05 compared with control). (b) Showing representative annexinV-negative and -positive cells. Original magnification, 200. & 2013 Macmillan Publishers Limited

Prostate Cancer and Prostatic Disease (2013), 16 – 22


18 imaged using a Nikon ECLIPSE TE2000-U (Sterling Heights, MI, USA) inverted microscope connected to a RT Slider Spot digital camera (Diagnostic Instruments, Sterling Heights). Images were acquired using SPOT software version 3.2 (Sterling Heights).

Western blot analysis Antibodies to caspases-3, -8 and -9, PARP, Bcl-2, phospho-Akt and p21 were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-bactin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HRP conjugated secondary antibodies were from Amersham Pharmacia Life Sciences (Rockford, IL, USA). Standard western blot analysis was carried out as previously described.40 Total AKT and actin were used as loading controls.

Measurement of ROS ROS production was examined using the oxidant-sensitive probe 20 ,70 dichlorofluorescein diacetate (H2DCF-DA) assay, modified for fluorescent microplate reader. The nonfluorescent fluorescin H2DCF-DA (Sigma Aldrich) after being oxidized emits fluorescence in correlation with ROS levels. PC3 cells were plated at a density of 5 103 in 96-well plates, allowed to attach overnight, and exposed to TRAIL and ethanol for 48 h with or without NAC pretreatment. Untreated cells were used as control. The cells were then stained with 10 mmol l 1 H2DCF-DA for 10 min at 37 1C, and ROS levels were analyzed using a multi-mode microplate reader (Biotek, Winooski, VT, USA).

Soft agar colony formation assay To assess the effects of ethanol and TRAIL on anchorage-independent growth ability, PCa cells (PC3 and LNCaP) were seeded on soft agar at a density of 1000 per well in a six-well plate containing one ml of RPMI with 20% fetal bovine serum. Cells were then incubated at 37 1C in a tissue culture incubator. Colony growth was assessed by the number of colonies that exceeded 100 mm after 2 weeks.

3D-matrigel invasion assay Invasion potential in vitro was assessed using matrigel-coated Biocoat cell culture inserts (BD Biosciences, Bedford, MA, USA) with 8 mm pores in 24 wells. A total of 3 104 cells were placed in the upper compartment and the lower compartment was filled with 500 ml RPMI growth medium containing 10% FBS. Cells were exposed to TRAIL (100 ng ml 1), ethanol (4%) alone or in combination. At the end of 24 h, cells on the lower surface were stained with crystal violet and solubilized in extraction buffer (10% acetic acid). Optical density values at 540 nm correlating with cell migration were plotted.

Tumorigenicity in nude mice Figure 3. Western blot analysis of apoptosis-related proteins in the PC3 and LNCaP cells. PC3 and LNCaP cells were either left untreated or treated with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or ethanol either as a single agent or in combination for 48 h and western blot was performed. (a) Western blot analysis indicating the expression and activation of caspase 3, 8, 9 and poly (ADP) ribose polymerase (PARP). Actin was used as a protein loading control. (b) Western blot analysis indicating the levels of pro-survival molecules, BCl2 and, total and Phos-Akt. (c) Western blot analysis showing the expression levels of P21/Waf1 in untreated and TRAIL and ethanol-treated PC3 and LNCaP cells.

We examined the effects of ethanol on enhancing TRAIL-mediated apoptosis of PCa tumor cells in an in vivo environment. Six-week-old male BALB/c (nu/nu) nude mice (Taconic, Hudson, NY, USA) were injected s.c. with 5 106 PC3 cells. Tumors were allowed to grow about 1 cm in diameter. Four groups of tumor bearing animals were randomly chosen for intratumoral injection of either saline solution, 4% ethanol, 100 ng ml 1 TRAIL or combination of ethanol (4%) and TRAIL (100 ng ml 1). Ten animals were used in each group. At day 3 of treatment, mice were killed and the tumors were removed. Tumor sections were analyzed for apoptosis by TUNEL assay. All experiments were approved by the Institutional Animal Care and Use Committee.

cultured in media containing TRAIL (100 ng ml 1) and ethanol (4%) alone or in combination for 48 h in 37 1C incubator with 5% CO2. Untreated cells were used as controls. The absorbance was measured at 570 nm in a microtiter plate reader.

Immunohistochemical analysis

Annexin-V staining and flow cytometry For quantification of apoptosis, cells were exposed to TRAIL (100 ng ml 1) and ethanol (4%) alone or in combination for 48 h. Apoptosis was quantitated by using an annexin-V binding assay kit according to manufacturer’s protocol (BD Biosciences, San Jose, CA, USA). Samples were analyzed at the Vermont Cancer Center Flow Cytometry Core lab using an Epics XL-MCL Flow Cytometry system (Beckman Coulter, Fullerton, CA, USA). Pretreatment of cells with NAC (where indicated) was used to determine if ROS was involved in apoptotic cell death. Stained cells were Prostate Cancer and Prostatic Disease (2013), 16 – 22

Formalin-fixed, paraffin-embedded tumor specimens were processed for standard immunohistochemical staining. Apoptosis was detected by TUNEL assay using an APOPTAG kit (Chemicon International, Temecula, CA, USA) according to manufacturer’s protocol. After staining, slides were mounted and imaged using a Nikon ECLIPSE TE2000-U inverted microscope connected to a RT Slider Spot digital camera (Diagnostic Instruments). Images were acquired using SPOT software version 3.2.

Statistical analysis All in vitro studies were performed in triplicate and results are expressed as mean±s.d.. In vivo studies were performed using 10 animals per group and results are expressed as mean±s.d.. Statistical significances were determined by means of Student’s t-test or by ANOVA and multiple & 2013 Macmillan Publishers Limited


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Figure 4. Antioxidant N-acetyl-L-cystein (NAC) decreases reactive oxygen species (ROS) production and suppresses ethanol-tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death in PC3 cells. Quantitative ROS generation was measured by using the oxidant-sensitive probe dichlorofluorescein (H2DCF-DA) assay. PC3 Cells were plated in 96-well plates and exposed to TRAIL and ethanol either alone or in combination for 48 h. PC3 cells were pretreated with 5 mM NAC, 1 h before ethanol-TRAIL treatment. The cells were then stained with H2DCF-DA for 10 min and ROS levels were measured using a microplate reader. The experiment was repeated twice. Dichlorofluorescein (DCF)-fluorescence units are expressed as mean±s.d. of triplicates (*Po0.05 compared with control). (a) DCF fluorescence units of cellular ROS production in untreated or ethanol or TRAIL or ethanol-TRAIL-treated PC3 cells. (b) Fluorescence images of cellular ROS production in untreated or ethanol-TRAIL or ethanol-TRAIL-NAC-treated PC3 cells. (c) Quantitation of ROS production. (d) Detection of apoptotic cell death after 48 h of co-treatment with ethanol (4%)-TRAIL (100 ng ml 1) with or without NAC pretreatment (5 mM) by annexin-Vfluorescein isothiocyanate (FITC) staining using flow cytometry. The percent apoptosis represents mean±s.d. of experiments performed in triplicate (*Po0.05 compared with control). comparisons test. A probability Po0.05 was considered statistically significant in all calculations.

RESULTS Ethanol enhances TRAIL-mediated cell growth inhibition in PC3 cells and sensitizes LNCaP cells for growth inhibition In vitro studies showed that ethanol alone had no significant effect on the morphology of both PC3 and LNCaP cells. TRAIL alone resulted in some changes in cellular morphology. Both PC3 and LNCaP cells exposed to TRAIL (100 ng ml 1) and ethanol (4%) in combination acquired rounded up morphology and started to detach, indicating the strong cytotoxic effects (Figure 1a). In comparison with untreated cells, ethanol alone decreased the cell viability by about 10% in both cells. TRAIL alone resulted in decreased cell viability by about 50% in PC3 cells and 10% in LNCaP cells. TRAIL in combination with ethanol decreased the cell viability of PC3 cells by about 75% and LNCaP cells by about 60% suggesting a possible additive effects between these two agents (Figure 1b). These data show that ethanol augments the growthinhibitory effects of TRAIL on PC3 and LNCaP cells. Ethanol augments TRAIL-induced apoptosis in PC3 cells and sensitizes LNCaP cells to TRAIL-induced apoptosis The growth-inhibitory effects of ethanol and TRAIL correlated with induction of apoptosis. As shown in Figure 2a, untreated and & 2013 Macmillan Publishers Limited

ethanol-treated cells showed 3–5% apoptotic cell death. TRAIL induced moderate levels of apoptosis in PC3 cells (25–28%) and significantly lower level (9–14%) in LNCaP cells. Co-treatment with ethanol and TRAIL increased apoptosis to 55–58% for PC3 and 38– 40% for LNCaP cells. Annexin-positive apoptotic cells are shown in Figure 2b. These data indicate that ethanol significantly enhances TRAIL-mediated apoptosis in PCa cells. Ethanol-TRAIL in combination enhances caspase activation and PARP cleavage Untreated and ethanol-treated PC3 and LNCaP cells did not show caspase activation. TRAIL alone induced moderate levels of caspase-8, -9 and -3 activation in PC3 cells and these effects were significantly enhanced by combined TRAIL and ethanol treatment as indicated by decreased pro-caspase levels and increased levels of their cleavage products (Figure 3a). In LNCaP cells, TRAIL alone did not induce caspase activation. Activation of caspases was evident in LNCaP cells treated with TRAIL and ethanol in combination (Figure 3a). No cleavage of PARP was evident in untreated or ethanol-treated cells. TRAIL alone induced partial cleavage of PARP. This effect was significantly augmented after combined TRAIL and ethanol treatment, as indicated by complete disappearance of full length PARP in PC3 cells. TRAIL alone had no effect on PARP cleavage in LNCaP cells. However, ethanol potentiated TRAIL-induced PARP cleavage in LNCaP cells (Figure 3a). Also, ethanol and TRAIL in combination downregulated the Prostate Cancer and Prostatic Disease (2013), 16 – 22


20 expression of the anti-apoptotic protein Bcl-2 and Phos-AKT in both PC3 and LNCaP cells (Figure 3b). In addition, these effects were associated with increased levels of cyclin-dependent kinase (cdk) inhibitor p21/waf1, an important apoptosis inducer (Figure 3c). Ethanol-TRAIL cytotoxicity is mediated through ROS generation The ROS production was significantly increased in PC3 cells treated with ethanol and TRAIL in combination, as compared with untreated control and single-agent treatment (Figure 4a). PC3 cells treated with ethanol-TRAIL in combination showed increased DCF fluorescence, an indicator of ROS production and pretreatment of cells with the antioxidant NAC decreased the ROS levels as shown by fluorescence imaging (Figure 4b). Further quantification showed that pretreatment with NAC decreased ROS production by 50–60% (Figure 4c). Suppression of ethanol-TRAIL-mediated ROS production by NAC correlated with decreased number of apoptotic cells by 55–60% as compared with ethanol-TRAIL combined treatment (Figure 4d). Similar results were obtained in LNCaP cells (data not shown). These data suggest that ethanolTRAIL induce PCa cell cytotoxicity, at least in part through ROS production. Ethanol and TRAIL in combination inhibit anchorage-independent growth and invasive potential of PC3 and LNCaP cells Ethanol alone had no significant effect on number of soft agar colonies formed as compared with control PC3 or LNCaP cells. TRAIL alone suppressed clonal growth by 45–50% in PC3 cells with no significant effect on LNCaP cells. However, inhibition of colony formation with combined ethanol and TRAIL treatment was more than 75% in PC3 and more than 50% in LNCaP cells (Figure 5a). Also the ability of PC3 and LNCaP cells to migrate and invade through matrigel-coated filters was significantly reduced by ethanol and TRAIL combination treatment (50% in PC3 and 40% in LNCaP cells) (Figure 5b). Ethanol enhances apoptotic effects of TRAIL on PC3 cells in a xenotransplantation mouse model In accordance with our in vitro data, the additive effects of ethanol and TRAIL on increased apoptosis of tumor cells was evident after 3 days of treatment. Apototic cells are shown in Figure 6a. Apoptotic cell death was significantly increased by ethanol and TRAIL combination treatment (65%) as compared with TRAIL alone (48%), ethanol alone (21%) and untreated control (15%) (Figure 6b). These data indicate that combination of ethanol and TRAIL more effectively induces apoptosis of PC3 xenografts in vivo than TRAIL alone. DISCUSSION Although most PCa initially respond to therapy, tumor cells eventually acquire resistance to therapy-induced apoptosis.1–6 Within the PCa-derived cell lines studied, androgen-independent PC3 cells are previously shown to be moderately sensitive, and LNCaP cells are reported to be resistant to TRAIL-induced apoptosis.11–20 Given the established role of ethanol in inducing cell death, it is a promising agent for promoting tumor cell death in combination with known therapeutic drugs such as TRAIL. Indeed, a critical role for ethanol in promoting apoptotic events in various cell lines and animal models has been well documented.20–29 Also, ethanol stimulates the TRAIL-induced apoptosis in leukemic T-lymphocytes and colon cancer cells in vitro.38,39 Despite these data, an interactive effect of ethanol and TRAIL in promoting PCa cell death has not been studied. Our current studies have demonstrated an important role for ethanol in overcoming TRAIL resistance and enhancing sensitization of PCa cells to the TRAIL-induced cytotoxicity. Ethanol Prostate Cancer and Prostatic Disease (2013), 16 – 22

Figure 5. Ethanol and tumor necrosis factor-related apoptosisinducing ligand (TRAIL) inhibit anchorage-independent growth and invasive potential of PC3 and LNCaP Cells in vitro. (a) PC3 and LNCaP cells plated in triplicate in agar were treated with either TRAIL or ethanol alone or in combination. After incubation for 14 days, the numbers of colonies formed were determined using an inverted microscope. Results shown are mean number of colonies±s.d. (*Po0.05 compared with control). (b) Effect of TRAIL and ethanol either as a single agent or in combination on PC3 and LNCaP cell migration/invasion in a 3D-matrigel was determined using Boyden chamber cell invasion assay. Optical densities correlating with the number of cells invaded were read at 540 nm. Results shown are mean values±s.d. of triplicates (*Po0.05 compared with control).

significantly increased the apoptosis-inducing effects of TRAIL in PCa cells by activation of both intrinsic and extrinsic caspase pathways and complete cleavage of PARP. It is likely that the crosstalk between extrinsic and intrinsic apoptosis pathways augment a death signal mediated by TRAIL, leading to a more effective implementation of apoptosis. Furthermore, we also observed increased levels of p21/waf1 and decreased levels of Bcl-2 and phospho-AKT in ethanol-TRAIL-treated cells. Pro-survival factors, particularly Akt is a constitutively active in PCa cells, promoting their survival and resistance to chemotherapy.40–44,47,48 Thus, targeting Akt activity with a combination of agents is a promising approach to promote responsiveness to TRAIL as supported by our current results. Additionally, ethanol enhanced TRAIL’s effects including caspase activation and PARP cleavage in LNCaP cells, which are inherently resistant to TRAIL. The differential sensitivity of PC3 and LNCaP cells to TRAIL-induced apoptosis is associated with the varying levels of expression of TRAIL receptors. Indeed PC3 cells express high levels of death receptors DR4 and DR5 and thus are more responsive to TRAIL.9 It is very likely that ethanol overcomes resistance to TRAIL-induced apoptosis particularly in LNCaP through upregulation of death receptors via activation of ROS-mediated signaling pathways. Indeed ROS is shown to upregulate the expression of proapoptotic TRAIL receptors DR4 and DR5.45 Overall, our results & 2013 Macmillan Publishers Limited


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Figure 6. Measurement of intratumoral apoptotic activity using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Six-week-old nude mice were injected s.c. with 5 106 PC3 cells. PC3 Tumors were allowed to grow about 1 cm in diameter. Four groups of tumor bearing animals were randomly chosen for intratumoral injection of either saline solution, 4% ethanol, 100 ng ml 1 tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), or combination of ethanol and TRAIL. Ten animals were used in each group. (a) TUNEL assay indicating the additive effects of TRAIL and ethanol on induction of apoptosis in PC3 tumor cells. (b) TUNEL-positive apoptotic cells were counted in five different high power fields. The data are expressed as percent TUNEL-positive cells. Results shown are mean percent apoptotic cells±s.d. *P-value o0.05, combination treatment compared with ethanol or TRAIL alone.

suggest that ethanol may be useful not only to enhance TRAIL effects, but also potentially to overcome TRAIL resistance. Emerging studies show that ethanol induces ROS production,27–29 and increased ROS promote TRAIL-induced tumor cell death.7–10,46,49,50 In agreement with these reports, in both PC3 and LNCaP cells, ethanol-TRAIL combined treatment enhanced apoptosis through increased ROS. The increase in ROS is likely linked to mitochondrial dysfunction resulting in activation of intrinsic pathway as well as extrinsic pathways of apoptosis in PCa cells. As the main goal of chemotherapy is to provoke apoptosis, ROS induction may represent an attractive approach for the development of new cancer therapeutics. Overall, this study found that ethanol had additive effects and potentiated TRAIL-induced apoptosis both in vitro and in vivo mouse model of PC3. Future studies using various dose and time regimens, especially in an orthotopic model of PCa can further determine the anti-tumor effects of combined ethanol-TRAIL treatments. Also, identifying upstream components involved in decisions about PCa cell survival verses cell death, and how ethanol and TRAIL participate in these decisions may shed light on specific molecular mechanisms involved in ethanol-TRAILmediated PCa cell death. Taken together, use of ethanol in combination with TRAIL may be an effective strategy to augment sensitivity to TRAIL-induced apoptosis in PCa cells. CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS This work was supported in part by Lake Champlain Cancer Research Organization (LCCRO) at the University of Vermont, Burlington, VT, Department of Neurosurgery, University of Wisconsin, Madison, WI, and NEAUA post residency research scholarship award. We thank Core Facilities of COBRE-Neuroscience (2P20RR16435-6) and Vermont Cancer Center (VCC) DNA analysis and Flow Cytometry Facility for their technical help.

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