Targeted induction of apoptosis via TRAIL and cryoablation: a ... - Nature

Prostate Cancer and Prostatic Diseases (2007) 10, 175–184 & 2007 Nature Publishing Group All rights reserved 1365-7852/07 $30.00 www.nature.com/pcan

ORIGINAL ARTICLE

Targeted induction of apoptosis via TRAIL and cryoablation: a novel strategy for the treatment of prostate cancer DM Clarke1,2, AT Robilotto1,2, RG VanBuskirk1,2, JG Baust2, AA Gage1 and JM Baust1,2 1

Cell Preservation Services Inc., Owego, NY, USA and 2Institute of Biotechnology, State University of New York, Binghamton, NY, USA

Adjuvant therapies contribute to the successful treatment of cancer. Our previous reports have shown that combining cryoablation with cytotoxic agents enhances cell death. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytotoxic agent that preferentially induces apoptosis in a variety of human cancer cells. Human prostate cancer cells (PC-3) are resistant to many cytodestructive agents, including cryoablation and TRAIL. Here, we evaluated the effects of TRAIL combined with cryoablation on PC-3 and normal prostate (RWPE-1) cell death. Exposure of PC-3 cells to freezing (101C) or TRAIL (500ng/ml) results in minimal cell death, whereas a complete loss of viability is observed with the simultaneous combination. The synergistic effect was found to be due to a marked increase in apoptosis. Western blot analysis revealed a significant level of caspase-8 and -3 cleavage between 12 and 24 h post-exposure. Caspase activation assays provided similar results and also indicated a role for caspase-9. Inhibitors to caspase-8 and -9 along with a pan-caspase inhibitor were incorporated to determine which pathway was necessary for the combined efficacy. Inhibition of caspase-8 significantly blocked the combination-induced cell death compared to cells that did not receive the inhibitor (63% compared to 10% viable). The addition of the caspase-9 inhibitor resulted in only a minimal protection. Importantly, the combination was not effective when applied to normal prostate cells. The results describe a novel therapeutic model for the treatment of prostate cancer and provide support for future in vivo studies. Prostate Cancer and Prostatic Diseases (2007) 10, 175–184; doi:10.1038/sj.pcan.4500920; published online 13 February 2007

Keywords: cryoablation; TRAIL; apoptosis; caspase

Introduction Prostate cancer (CaP) is second in cancer-related deaths in men in the United States, causing approximately 40 000 deaths annually.1,2 One common approach to CaP therapy is management through androgen ablation. Unfortunately, androgen ablation alone is not curative in many patients and the disease recurs in later years. Radical prostatectomy and radiation therapy are considered the ‘gold standard’ treatment options for localized CaP. Both therapies provide less than optimal efficacy in the treatment of the disease.3,4 Indeed, a recent study indicates an average recurrence rate of 25–35% (up to 93% in one group) following radiation therapy.5 Recent scientific and technological advances offer alternatives to these treatment options for CAP with an emphasis on more minimally invasive therapies.6,7 Cryosurgery has emerged as one of the many tools available for the treatment of CaP.5,8 This procedure utilizes the destructive forces of low temperature to ablate

Correspondence: Dr DM Clarke, Cell Preservation Services Inc., 171 Front Street, Owego, NY 13827, USA. E-mail: [email protected] Received 9 August 2006 revised and accepted 25 September 2006; published online 13 February 2007

unwanted tissue.9–12 The center (nearest the cryoprobe) of the cryogenic legion is rendered completely necrotic, but as temperatures elevate further from the probe tip, less is known about the mechanisms of cell death and survival.13–16 In 1998, Hollister et al. demonstrated that DNA from CaP cells frozen to temperatures of 5 to 151C exhibited a non-random, degradation pattern that is typically associated with late-stage apoptosis. Subsequently, other studies have reported on the activation of apoptosis following the application of freezing.17–19 Because of the resistant nature of CaP cells to freezing,18,20 physicians are required to extend the freeze zone significantly beyond the diseased tissue, exposing a greater portion of the non-targeted, healthy tissue to cryoablation.10,12 Inadequate management of the freeze zone periphery (positive freeze margin) can sometimes have a negative effect on the surrounding tissues (i.e. rectum, urethra and nervous tissue), thereby increasing post-freeze morbidity. For this reason and to improve efficacy, there is a focus on understanding the effects of freezing and the development of novel adjunctive therapies that show promise of minimizing the positive freeze margin volume. One strategy under evaluation is the manipulation of freezing-induced apoptosis particularly through adjuvant therapies.17,18,20,21 Numerous cellular factors associated with the apoptotic signaling pathway have been identified. The two

TRAIL and freezing ablate prostate cancer cells DM Clarke et al

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commonly recognized apoptotic signal-transduction pathways include the receptor-mediated (extrinsic) and the mitochondrial-mediated (intrinsic) pathways. Although each pathway is initially centered on unique initiation events, the final stages generally converge and result in the activation of the caspase cascade.22–24 The mitochondrial pathway can be triggered by multiple stimuli with the signals subsequently affecting the actions of the Bcl-2 family proteins, which are directly linked to the function of the mitochondria.25,26 Following a stress-induced insult, the proapoptotic Bcl-2 members translocate from the cytosol to the mitochondrial membrane leading to the subsequent release of cytochrome c into the cytosol. Cytochrome c interacts with Apaf-1 and pro-caspase-9 (initiator caspase) to activate the caspase cascade. The activation of caspase-9, leads to the further activation of the downstream executioner caspases, including caspase-3 resulting in cell death.25,26 The other apoptotic signaling pathway, the receptormediated pathway, is initiated from the cell surface.24,25 The initiation of this pathway is linked to the activation of key death receptors belonging to the Fas and tumor necrosis factor receptor (TNFR) families. Many of the TNFR family members are located on the cytoplasmic side and binding of TNF or Fas-L to its specific receptor induces formation of a ligand-bound receptor complex. This ‘death domain’ activation results in the interactions of TNFR with the TNFR-associated death domain protein (TRADD) and similarly Fas associates with the Fasassociated death domain protein. These interactions lead to the association and activation of caspase-8, which leads to the activation of downstream caspases and eventual cell death.25,26 Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) is a member of the TNFR superfamily and induces apoptosis through engagement of death receptors.27–29 TRAIL is a cytotoxic agent that preferentially induces apoptosis in a variety of human cancers both in vitro and in vivo, whereas normal cells remain less affected.28 However, some tumor cell types including those of the prostate remain resistant to the apoptogenic effects of TRAIL. Resistance to TRAIL has been shown to be overcome through adjuvant methods, including the combined treatment with conventional chemotherapeutic drugs. For example, in a variety of CaP cells in vitro, the cytotoxic efficacy of TRAIL could be augmented by paclitaxel,30 actinomycin D or gemcitabine,31 doxorubicin,32 amiloride,33 and 5-FU.34 Therefore, agents that sensitize cancer cells to TRAIL-mediated cell death might be of particular importance to the development of novel therapeutic treatment options. In this study, we investigated whether the resistance of human CaP (PC-3) cells to TRAIL could be overcome through the combination of a sublethal exposure to cryoablation.

Materials and methods Cell culture The PC-3 and normal human prostate RWPE-1 cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA). Stock cultures were maintained at 371C in 95% air/5% CO2 in Falcon T-75 cm2 flasks. PC-3 cell cultures were grown in RPMI-1640 Prostate Cancer and Prostatic Diseases

medium (Gibco/BRL Laboratories, Grand Island, NY, USA), and supplemented with 10% fetal calf serum (Atlanta Biological, Norcross, GA, USA) and 1% penicillin/streptomycin (Gibco/BRL). Stock cultures were subcultured every 5–6 days at approximately 95% confluence, and medium was replenished every 3 days. Experiments were performed using cell cultures between passages 3 and 10. Before testing (2 days), cells were seeded into Costar 96-well strip-well plates and cultured to achieve confluence in each well. Soluble TRAIL (Biomol International, Plymouth Meeting, PA, USA) was applied from 0 to 1000 ng/ml for 24 h in cell culture media.

Tissue-engineered CaP models (three-dimensional systems) Rat tail type I collagen solution was obtained from BD Bioscience (Bedford, MA, USA) and gels were formed as per the manufacturer’s recommendations. Briefly, collagen stock solution was diluted to a working concentration of 2 mg/ml in dilution buffer (10 RPMI-1640, 0.1 N NaOH and ddH2O). Cells were directly suspended in collagen solution at 3 106 cells/ml before gel solidification, and 2 ml of the cell/collagen solution was aliquoted into 35 mm Petri dishes yielding a gel thickness of approximately 2 mm. Gels were then solidified by incubation at 371C for 30 min. Cells were cultured 24 h before freezing with media replenishment 10 min before freezing.

Freezing protocol Cell monolayers. Cultures were provided with fresh culture medium 30 min before freezing. Culture flasks/ plates were placed in a refrigerated circulating bath (5 to 201C) (Neslab Instruments, Newington, NH, USA) for 20 min. After 5 min of cooling (2 to 51C sample temperature), ice nucleation was initiated in each sample well by contact with a cold metallic probe. Samples were maintained for the remaining 15 min at their respective temperatures. Following the 20 min freeze interval, samples were removed and thawed at room temperature for 15 min. Samples were then returned to 371C in a cell culture incubator to allow for recovery. Plate freeze configuration was utilized for viability assays and flask configuration was used for both molecular studies and corresponding cell viability studies.

Three-dimensional (3D) systems. An Oncura SeedNet Gold cryosurgical system with 17-gauge argon/helium driven needle cryoprobes (Galil Medical, Plymouth Meeting, PA, USA; Galil Medical, Yokneam, Israel) was utilized to freeze the gels. A single cryoprobe was placed in the center of a 35 mm dish and a 10 min freeze procedure was initiated. The temperature profile of the ice ball was recorded with a series of 5 type T thermocouples at equidistant intervals extending radially from the probe tip using an Omega TempScan 1100 (Omega, Stamford, CT, USA). Following the freeze procedure, samples were thawed at room temperature for 15 min. Once thawed, samples were returned to 371C in a cell culture incubator to allow for recovery.


Combination protocol. Just before freezing, TRAIL (500 ng/ml) was either removed and fresh culture media added to all samples or TRAIL was added along with fresh culture media. Samples were then frozen following the freezing protocol. Cell viability assessment. Cell viability following the various treatments was assessed both qualitatively and quantitatively. Qualitative assessment was achieved by visualization using light microscopy. Quantitative assessment was accomplished using alamarBlue (Trek Diagnostics, Cleveland, OH, USA). AlamarBlue was diluted 1:20 in Hank’s balanced salt solution (Life Technologies, Gaithersburg, MD, USA) without phenol red. Culture medium from well sample plates was removed and 100 ml per well of the working alamarBlue solution was added. Samples were then incubated in the dark at 371C for 60 min (71 min). Fluorescence was evaluated using a Tecan SPECTRAFluorPlus plate reader (TECAN Systems Inc., San Jose, CA, USA) with a 530 nm excitation/590 nm emission filter set. This assessment was repeated every 2 days for the entirety of the experiment. Cell death analysis YO-PRO-1/PI analysis. PC-3 cells were exposed to either freezing or the combination of freezing and TRAIL as described above. Cell monolayers: After 1, 3, 6, 12 and 24 h recovery periods, cells were assessed for the number of apoptotic, necrotic and live cells present in the samples using the Vybrant Apoptosis assay (Molecular Probes, Eugene, OR, USA). Following protocol, 1 ml of YO-PRO-1 (100 mM stock) and 1 ml of propidium iodide (PI (1 mg/ml stock)) was added to each well containing 100 ml media. In addition, 1 ml of Hoechst 33342 dye (1 mg/ml stock) was added to label live cells. The fluorescent probes were added directly to the wells, and plates were incubated in the dark at room temperature for 20 min. Following incubation, plates were briefly centrifuged (1 min at 100 g). Fluorescent staining was then visualized using a Zeiss Axiovert 200 fluorescent microscope with the AxioVision 4 software (Zeiss, Go¨ttingen, Germany). 3D systems: Apoptotic assessment was similar to that described above described for cell monolayers. Cells staining positively for YO-PRO-1 binding only (green fluorescent membranes) were scored as apoptotic. Cells staining positively with propidium iodide (red fluorescent nuclei) were scored as necrotic, and cells staining positively for Hoechst staining only (blue fluorescent nuclei) were scored as living. Total cell counts were accomplished using a program within the AxioVision 4 software package. Three separate experiments were evaluated. Cell death inhibitor protocol Caspase inhibitors (CalBiochem, La Jolla, CA, USA) were diluted in DMSO to a stock concentration of 10 mM. They were further diluted to a working concentration of 20 mm in cell culture media. For all experiments, the inhibitor was added to the cell culture medium 20 min before freezing and remained in culture for 24 h post-exposure.

Protein analysis Protein isolation. Cell samples were lysed in 80–100 ml of modified RIPA buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 100 mM Tris-HCL pH 7.6, 150 mM NaCl, 5 mM EDTA, 1 mM PMSF, 1 mg/ml leupeptin, 1 mM sodium fluoride and 1 mM sodium vanadate) by manual cell scraping. Lysates were then added to 1.5 ml tubes and stored at 801C. After all samples were collected, samples were thawed and lysed on ice by vortexing the samples 2–3 times followed by centrifugation at 13 000 r.p.m. for 10 min at 41C and the protein containing the supernatant was transferred to new centrifuge tubes and kept on ice.

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Protein quantification Protein extracts were quantified by standard bicinchoninic acid protein assay procedure according to the manufacturer’s protocol (Pierce, Rockford, IL, USA).

Western blot analysis. Protein from each sample (25 mg) was aliquoted into individual tubes and the total volume was adjusted with cell lysis buffer (Pierce). An equal amount of 2 loading buffer (100 mM Tris, pH 6.8, 200 mM DTT, 4% SDS, 20% glycerol and 0.02% bromophenol blue) was added to each sample and samples were heated at 951C for 5 min. Samples were resolved on a discontinuous SDS-polyacrylamide gel (4% acrylamide (29:1) stacking and 12% acrylamide (29:1) resolving gel) at 100 V in 1 running buffer (5 mM Tris, 40 mM glycine and 0.02% SDS). Following electrophoresis, proteins were transferred to PVDF membranes by semidry electrophoretic transfer (Trans-blot SD Semi-Dry Transfer Cell, Bio-Rad, Hercules, CA, USA) at 15 V for 30 min. Once protein transfer was complete, the membranes were blocked in 5% bovine serum albumin (Life Technologies) in 1% TBST (10 mM Tris, pH 7.6, 150 mM NaCl, and 0.05% Tween-20) with gentle agitation for 2 h at room temperature. Membranes were then incubated with primary antibodies overnight at 41C. Following incubation, membranes were washed three times for 10 min each in 1% TBST at room temperature. Membranes were then exposed to secondary antibodies for 1 h at room temperature. Membranes were then washed again. The blots were developed using chemiluminescent detection buffer according to the manufacturer’s protocol (Cell Signaling Technologies, New England Biolabs, Beverly, MA, USA) using a FUJIFILM LAS-3000 Image Reader (FUJIFILM, Stamford, CT, USA).

Antibodies Monoclonal anti-caspase-3, anti-PARP, anti-TRADD, anti-b-tubulin and polyclonal anti-caspase-9 were purchased from Transduction Laboratories (BD Biosciences, San Jose, CA, USA). Monoclonal anti-caspase-8 was purchased from Cell Signaling Technologies (Danvers, MA, USA). Goat anti-mouse and goat anti-rabbit HRP secondary antibodies were purchased from Pierce (Rockford, IL, USA). Prostate Cancer and Prostatic Diseases


Data analysis Fluorescence units were converted to percent survival based upon experimental controls (371C). Calculations of standard error (s.e.m.) were performed and statistical significance was determined using single-factor analysis of variance (ANOVA). s.e.m. ANOVA-based P-values were calculated to determine the statistical significance of the data. Cell viability experiments were repeated a minimum of three times with an intra-experimental repeat of 8 (N ¼ 24). Western blot analysis experiments were repeated on a minimum of three separate experimental isolations (N ¼ 3).

Results CaP cells are resistant to TRAIL To investigate the effects of TRAIL-induced cytotoxicity on human CaP cells, PC-3 cells were treated with TRAIL (100–1000 ng/ml) for 24 h. Before treatment, cells were grown to confluent monolayers to reflect the growth characteristics of prostate tumors in vivo. After 24 h, cell viability was assessed and no significant cytotoxicity was observed with any of the concentrations tested (data not shown). In each case, cell viability returned to control levels by 3 days post-exposure. Additionally, subconfluent cells were tested in a similar manner to confirm that the resistance to TRAIL cytotoxicity was not due to cell division. Again, PC-3 cells were found to be relatively resistant to TRAIL cytotoxicity (data not shown). The data obtained were comparable to a previous report demonstrating the resistance of CaP cells to TRAIL.29 Cryoablation enhances TRAIL-induced cytotoxicity PC-3 cells were exposed to TRAIL (500 ng/ml), freezing (5, 10, 15, 201C), TRAIL for 24 h before freezing, or TRAIL and freezing combined at the same time. Cell viability was then assessed 24 h post-exposure to evaluate the efficacy of each of the conditions (Figure 1). Again, TRAIL exposure alone resulted in a minimal loss of viability. Freezing alone to temperatures of 5, 10, Prostate Cancer and Prostatic Diseases

Effects of TRAIL (1 day) and Freezing on Human Prostate Cancer Cell (PC-3) Viability

% Viability (Metabolic Activity)

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Caspase activation assay protocol Caspase-8 Activity Assay (EMD Biosciences, San Diego, CA, USA), and caspase-9 and caspase-3 BD ApoAlert caspase fluorescent assay kits (BD Biosciences, San Jose, CA, USA) were used according to the manufacturer’s protocol to assess activity. Briefly, 50 mg of protein was combined with an equal volume of 2 reaction buffer/ DTT mix followed by addition of 5 ml of caspase substrate to each tube. The reagents were mixed and incubated in a 371C water bath for 1 h. Enzyme activity was then read using the Tecan SPECTRAFluorPlus plate reader to assess caspase-8 and -3, and caspase-9 activity. Two separate controls were used in each experiment (blank, containing no cells; negative control, containing untreated cells). Fold increase in activity was calculated by subtracting fluorescent blank fluorescent values from each sample followed by comparison of negative control to treated samples. Each assay was performed three separate times.

C

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Condition

Figure 1 Effects of freezing on TRAIL-induced cytotoxicity on human prostate cancer PC-3 cell viability. Cells were treated with TRAIL (500 ng/ml), freezing (5 to 201C), the combination of TRAIL before freezing (TRAIL then Freeze), or the simultaneous combination of TRAIL and freezing (Freeze and TRAIL). Cell survival was determined by the AlamarBlue metabolic activity assay every other day for 7 days with the first assessment 24 h postfreezing. Error bars represent s.e.m.

15 and 201C resulted in a 5, 15, 35 and 70% decrease in viability respectively. When TRAIL exposure was followed by freezing, a significant increase in cell death was evident at all temperatures tested. Following this combination, a 25, 80, 90 and 100% decrease in viability was observed when TRAIL was followed by each of the freezing temperatures (Figure 1; TRAIL then Freeze). In each case, except for those exposed to 201C, cells began to repopulate over the recovery period. When TRAIL and freezing were applied simultaneously, a greater loss in viability was noted (Figure 1; TRAIL and Freeze). Applying this combination to PC-3 cultures led to a 50% initial decrease with the TRAIL and 51C exposure, whereas combinations with each of the other temperatures yielded 100% ablation. After 7 days of analysis, the TRAIL/51C combination was the only condition that resulted in cell survival (Figure 1). Similar trends were also found using lower concentrations of TRAIL (data not shown).

Freeze and TRAIL combination induces apoptosis The viability data in the previous section revealed that TRAIL cytotoxicity could be significantly enhanced when combined with cryoablation. Additional studies were then designed to examine whether the combination led to an increase in PC-3 cell apoptosis. PC-3 cells were grown in a collagen gel (2 mm thick) to form a 3D environment. The cells were then exposed to freezing or the simultaneous combination as described in the Materials and methods section. The freezing process, similar to in vivo situations, generates a freeze zone extending from the center of the gel. Furthermore, a temperature gradient is produced with the lowest temperature (851C) at the center and elevated temperatures (01C) at the periphery. After 24 h, 3D cultures were stained with YO-PRO-1 to assess the modes of cell death involved. Using this assay, it was apparent that whereas both conditions induced apoptosis (green), the combination treatment resulted in a far greater induction


(Figure 2a; Freeze vs Combo). The increase in apoptotic cell number also correlated with a diminished number of live (blue) cells after exposure to the combination and is comparable with the data in Figure 1. To evaluate this closer, specific temperatures were observed over a 24 h time course. PC-3 cells were exposed to the various conditions as discussed in Figure 1 along with freezing to 10 or 151C and evaluated for apoptotic induction at 0, 3, 6, 12 and 24 h post-exposure. In this experimental series, TRAIL exposure resulted in little cell death over the entire time course and was comparable to control cultures. Freezing (101C) resulted in an initial increase in necrotic (red) cells followed by a small rise in apoptotic cells between 3 and 12 h (Figure 2b). Although both freezing and the combination reveal similar

a

patterns of cell death directly after exposure, a significant burst of apoptosis was induced at 6 h post-treatment with the combination treatment. The apoptotic event peaked at 12 h and appeared to lessen by 24 h, although this may be due to the lack of viable (blue) cells present at this time (Figure 2b; 24 h). In each study, few viable cells remained at 24 h following exposure to the combination.

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Effects of freezing and the freezing/TRAIL combination on caspase activation Considering the results depicted in the previous figures, studies were performed to determine which apoptotic

Effects of Freezing and TRAIL/Freeze Combination on PC-3 Cells Grown in 3D –30 °C

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Figure 2 Involvement of apoptosis following treatment with TRAIL, freezing, or the combination. (a) PC-3 cells were grown in 3D in a collagen gel and exposed to freezing or the freeze/TRAIL combination. (b) Cells were exposed to TRAIL (500 ng/ml), freezing (101C), and the combinations of TRAIL before or during freezing. In each case, cells were stained with YO-PRO-1, PI, and Hoechst post-treatment, and cell death was visualized with a fluorescent microscope. Apoptotic cells stained green, necrotic/freeze rupture cells stained red, and live cells stained blue. Micrographs are representative of three separate experiments. Prostate Cancer and Prostatic Diseases


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pathway was activated when TRAIL and freezing were combined. First, PC-3 cells were exposed to either freezing or the combination treatment, and then cell lysates were collected over a 24 h time course. Western blot analysis revealed that the combination of freezing with TRAIL induced TRADD activation (Figure 3). TRADD (34 kDa) levels decreased by 6 h after exposure to the combination and continued to decrease over the remainder of the 24 h time course. Freezing also appears to induce a change in TRADD levels around 6 h, but this is a minimal decrease as levels return to controls by 24 h. Protein analysis also shows that procaspase-8 (57 kDa) was cleaved to the intermediate/active (41/43 kDa) by 6 h following exposure to the combination. The cleaved caspase-8 persisted at both the 12 and 24 h time points. Caspase-8 was also cleaved following freezing alone, but only a small amount of the cleaved form was noted (Figure 3; 6 h). The results thus far indicated that the combination treatment may induce a caspase-8-mediated death cascade, and also suggested a membrane-mediated involvement in freezing-induced cell death. Interestingly, the combined treatment also resulted in an apparent activation of caspase-9. Procaspase-9 (47 kDa) decreased at 6 and 12 h post-exposure before returning to control levels by 24 h (Figure 3). Freezing alone did not induce a similar change in procaspase-9 levels. Protein analysis also demonstrated downstream apoptotic events including caspase-3 (32 kDa) and PARP (113 kDa) cleavage following exposure to either condition (Figure 3). It should be noted that a greater enhancement of cleavage was promoted by the cytotoxicity induced by the freeze/ TRAIL combination. TRAIL alone did not result in caspase activation (data not shown). To evaluate the results further, additional experiments were performed to quantitatively assess the caspase involvement induced by the freeze/TRAIL combination. Caspase cleavage assays were incorporated to provide data to support the information illustrated in Figure 3. Levels of caspase-8 activation increased 8–10-fold by 3 h in cells exposed to the combination treatment compared Molecular Changes in PC-3 cells following Exposure to Freezing or the Combination of Freezing with TRAIL (500 ng) PARP

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Figure 3 Membrane-mediated death cascade activated with the freeze/TRAIL combination. Cells were exposed to freezing (10) or the freeze/TRAIL (500 ng/ml) combination and harvested at 0, 6, 12 and 24 h post-exposure. Untreated (Con) cells were also harvested to serve as a control. Cell lysates were then subjected to immunoblotting for PARP, TRADD, caspase-8, caspase-9 and caspase-3. b-Tubulin was used to confirm the amount of protein loaded in each lane. Prostate Cancer and Prostatic Diseases

to controls (Figure 4a). By 6 h, caspase-8 levels had peaked at nearly 13–15-fold, whereas cells exposed to freezing alone demonstrated only a 4–5-fold increase. Levels returned to controls by 24 h both in freezing alone and in the combination. Caspase-9 levels followed a very similar trend, with levels peaking in both around 6 h (Figure 4b). Again, a greater activation of caspase-9 (23vs 6-fold) was associated with the cultures exposed to the combination. Furthermore, caspase-9 activation persisted at 24 h following exposure to freezing and TRAIL. Changes to caspase-3 activation closely resembled the changes observed with caspase-9 (Figure 4c). Although the data demonstrate that caspases are activated in both treatments, the activation is much more profound when cells are exposed to the combination treatment.

Inhibition of caspase-8 blocks combination-induced cell death The previous data demonstrated an increase in caspase-8 and -9 activation following exposure to the freeze/ TRAIL combination treatment. These results imply that both the membrane- and mitochondrial-mediated pathways actually may be necessary for maximum effectiveness of the combination. To examine which of the pathways is critical to the synergistic effect, specific apoptotic inhibitors were employed. PC-3 cells were exposed to each of the previous conditions alone or in the presence of caspase inhibitors specific to caspase-8 or -9, or with a pan-caspase inhibitor (zVAD–FMK). Caspase-8 and -9 inhibitors did not provide any benefit to cells exposed to freezing alone (Figure 5). However, the caspase-8 inhibitor did provide significant protection to cultures exposed to the combination treatment. The combination without the inhibitors induced a 93% reduction in viability whereas the addition of the inhibitor led to only a 40% reduction (Figure 5). Although the caspase-9 inhibitor provided some protection (19%), the amount was much less than with the caspase-8 inhibitor present. As expected, experiments incorporating the pan-caspase inhibitor resulted in the greatest protection (Figure 5). The addition of the inhibitor also protected a significant population of cells from freezing-induced apoptotic injury as demonstrated in a previous study.35 The viability of cells exposed to TRAIL alone was not affected (data not shown). The effect of the inhibitors on cell viability was further confirmed by evaluating their ability to block apoptosis induction. PC-3 cells were exposed to similar conditions as noted in Figure 5 and modes of cell death were assessed over a 24 h time course using the YO-PRO-1 apoptosis assay. As was demonstrated previously, the freeze/TRAIL combination promoted a significant activation of apoptosis (green) between 6 and 12 h (Figure 6). Some of the early apoptosis appears to be prevented when the caspase-9 inhibitor is added, but by 24 h many of the cells have died. The amount of apoptosis is largely diminished with the addition of the caspase-8 inhibitor as depicted in the micrographs (Figure 6). The pancaspase inhibitor appeared to block essentially all of the apoptotic events. Cell counts generated from the micrographs are summarized in Table 1. The simultaneous combination caused a substantial increase in apoptotic events compared to freezing and TRAIL alone (25, 12 and


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Caspase 8 Activity Following Freezing or the Combination of Freezing and TRAIL (500 ng/ml) in PC-3 Cells 15

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Caspase 9 Activity Following Freezing or the Combination of Freezing and TRAIL (500 ng/ml) in PC-3 Cells

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Figure 4 Effects of freezing and the combination on caspase-8 (a), caspase-9 (b) and caspase-3 (c) activation. PC-3 cells were exposed to freezing or the freezing/TRAIL combination and harvested over a 24 h post-thaw time course. Using equal amounts of protein, cell lysates were then assessed for changes in caspase activity as described in Materials and methods. Untreated cells served as an internal control for baseline caspase activity.

Freeze/TRAIL combination is ineffective when applied to normal prostate cells Previous reports provided data demonstrating that TRAIL cytotoxicity is specific to cancer cells.27 We have demonstrated here that although CaP cells are resistant to TRAIL, the cytotoxicity can be significantly enhanced when combined with freezing. The combination was then applied to normal human prostate cells to evaluate potential therapeutic effects. RWPE-1 prostate cells were cultured and treated with TRAIL, freezing, or the combination treatment and effects on cell viability were observed. RWPE-1 cell viability was unaffected by TRAIL when applied as a single agent (Figure 7). RWPE-1 cells are less resistant to freezing compared to the PC-3 cells (compare Figure 7 with Figure 1). Although freezing to 101C and 151C resulted in a loss of 60 and 80% respectively at 24 h post-exposure, the combination of freezing and TRAIL resulted in losses of

Caspase Inhibition on Freeze/TRAIL Combination Efficacy 125 % Viability (Metabolic Activity)

3% respectively) after 6 h. Furthermore, capsase-8 inhibition prevented a large number of early-induced apoptotic events and decreased the efficacy of the combination treatment.

Day 1

100 75 50 25 0

Control TRAIL Freeze Freeze Freeze Freeze Combo Combo Combo Combo (500ng) (–10) (Inh 8) (Inh 9) (Inh VI) (Inh 8) (Inh 9) (Inh VI)

Condition

Figure 5 Effects of caspase inhibitors on the freeze/TRAIL combination-induced cytotoxicity. (a) PC-3 cells were exposed to TRAIL (500 ng/ml), freezing (101C), and the combination alone or in the presence of a caspase-8, caspase-9 or a pan-caspase (Inh VI) inhibitor. Each inhibitor (20 mm) was applied at the same time for each of the conditions evaluated and cell viability was determined by AlamarBlue metabolic activity assay 24 h post-exposure.

77% (101C) and 88% (151C). More significantly, cell recovery approaching controls (untreated) was observed with all conditions by 7 days post-exposure (Figure 7). Prostate Cancer and Prostatic Diseases


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TRAIL/Inh 8 TRAIL/Inh 9 TRAIL/Inh VI

Freeze (–10) and

TRAIL (500ng)

Effects of TRAIL and Freezing on PC-3Cell Death

0 hour-post

3 hour-post

6 hour-post

12 hour-post

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Figure 6 Effects of caspase inhibitors on the freeze/TRAIL combination-induced apoptosis. Cells were exposed to the combination of freezing (101C) and TRAIL (500 ng/ml) alone or in the presence of caspase inhibitors. Cells were stained as described in Figure 2 and visualized with a fluorescent microscope. Micrographs are representative of three separate experiments.

Table 1 Effects of TRAIL and freezing on PC-3 cell death (YO-PRO counts)

Control TRAIL Freeze (10) Combo Combo+Inh 8 Combo+Inh 9 Combo+Inh VI

6 h post

Day 1 Day 3

% live

% apoptotic

% live

% apoptotic

95 93 59 28 49 36 53

2 3 12 25 11 21 2

94 89 72 7 64 27 70

3 5 3 17 5 13 1

Percent of apoptotic cell death involved in TRAIL, freezing and combination treatment-induced cytotoxicity. PC-3 cells were exposed to TRAIL (500 ng/ ml) or freezing (101C) or the freeze/TRAIL combination alone or in the presence of caspase inhibitors. Cells were stained at 6 and 24 h post-treatment as described in Figure 2, visualized with a fluorescent microscope, and cell counts were made using a program within the AxioVision 4 software. Data were collected from three separate fields.

The combination of TRAIL before freezing had no effect on RWPE-1 cell viability, further demonstrating the resistance of normal cells to the combination effects of freezing and TRAIL treatment.

Discussion Despite continued progress in the diagnosis and management of early-stage disease, the management of advanced CaP remains challenging. Numerous studies have reported on the resistant nature of CaP cells to Prostate Cancer and Prostatic Diseases

Effects of TRAIL and Freezing on Human Prostate Cells (RW PE-1) Viability

24 h post % Viability (Metabolic Activity)

Condition

125

Day 5

100

75

50

25

0

Control TR (500ng)

F-10

TR then F TR then F

F-15

TR then F TR then F

Condition

Figure 7 Effects of the freeze/TRAIL combination on RWPE-1 normal prostate cell viability. RWPE-1 cells were treated with TRAIL (500 ng/ml), freezing (10 or 151C), the combination of TRAIL before freezing (TRAIL then Freeze), or the simultaneous combination of TRAIL and freezing (Freeze and TRAIL). Cell survival was determined by AlamarBlue metabolic activity assay every other day for 7 days with the first assessment 24 h postfreezing. Error bars represent s.e.m. from at least three separate experiments.

various therapies due in large part to their inability to activate apoptosis.36–38 Studies directed at understanding the reasons for this resistance may assist in the development of novel and more effective methods to treat the disease. In the current study, we show that applying sublethal freezing temperatures with TRAIL


significantly reduces PC-3 cell viability when compared to each applied as a single treatment. Although TRAIL alone is unable to activate apoptosis, the addition of freezing promotes a significant level of apoptosis. The combination treatment led to caspase-8, -9 and -3 activation indicating involvement of both the membraneand mitochondrial-mediated pathways. Subsequent caspase inhibitor analysis revealed that although both pathways were activated, caspase-8 cleavage was necessary for the combined efficacy. Finally, we show that although the freeze/TRAIL combination effectively leads to CaP cell death, the combination is noticeably less effective when applied to normal prostate cells. Owing to the resistance of CaP cells to most therapies, it has been recognized that combined therapies are necessary for the effective ablation of CaP. We have demonstrated in our previous studies that the combination of freezing with chemotherapeutic agents was more effective than either treatment alone.18 In these studies, it was found that pretreatment of CaP cells with a subtoxic concentration of these agents led to a significant increase in cell death when followed by a sublethal exposure to freezing. Similarly, our data here indicate that the combination of freezing and TRAIL results in a significant synergistic decrease in cell viability (Figure 1). Interestingly, we found that the simultaneous combination was more effective than the addition of TRAIL before freezing, which is in contrast to our previous data showing that a greater combined efficacy is attained when the cytotoxic agent is applied before freezing.18,39 Many studies have documented the resistance of CaP cells to TRAIL cytotoxicity.29,33,40 In each case, the resistance to TRAIL was decreased when it was combined with other cytotoxic agents, resulting in apoptosis. It has previously been shown that the application of freezing can induce apoptosis.19,35 Before these reports, freeze rupture and necrosis were the only forms of cell death thought to be associated with cryoablation.12 The advent of apoptosis activation provided a novel opportunity to manipulate freezing-induced cell death in an attempt to improve procedural efficacy. In our earlier study, we suggested that some cells died through the mitochondrial-mediated apoptotic pathway and a shift in the Bax/Bcl-2 ratio. Furthermore, the cells that survived the freezing episode revealed increased levels of Bcl-2, thereby shifting the ratio toward that of protection.18 When chemotherapeutic drugs were combined with freezing, Bcl-2 protein levels were prevented from increasing whereas Bax levels increased significantly. Similarly, other groups have reported that the manipulation of the Bcl-2 family of proteins enhances TRAIL cytotoxicity.34,41 We found in the current study that the combination of freezing and TRAIL results in a dramatic induction of apoptosis (Figure 2a and b). The molecular studies indicate that the combination results in the activation of membrane-mediated (TRADD, caspase8) events as well as mitochondrial-mediated (caspase-9) events (Figures 3 and 4a, b). Interestingly, whereas caspase-9 activity increased, Bcl-2 levels remained unchanged (data not shown). These data support, for the first time, a role for the membrane-mediated pathway in cell death associated with freezing. Despite the fact that both pathways were activated with the freeze/TRAIL combination, we observed with

the use of specific caspase inhibitors that caspase-8 activation and the membrane-mediated pathway were necessary for the combination to be successful (Figures 5 and 6). Although an increase in caspase-9 activity was found, the addition of the caspase-9 inhibitor did not provide a significant amount of protection (Figure 5). While an interesting result, this finding is not surprising as many reports have documented the interconnection between the two pathways.26,42 It is entirely plausible that the cleavage of caspase-8 led to downstream cleavage of Bid to tBid, stimulating the release of cytochrome c and the resultant activation of caspase9.42 Furthermore, the pan-caspase inhibitor was the only inhibitor to provide any protection to the cells exposed to freezing alone (Figure 5). The data support the idea that the freeze/TRAIL combination treatment initiates a specific pathway. As TRAIL alone cannot stimulate the cascade in PC-3 cells, the freezing must be initiating a physical or molecular change in the cells that facilitates the cytotoxic effects of TRAIL. A potential explanation is that a sublethal application of freezing leads to changes to the cell membrane leading to expression of the death receptors (DR4, DR5). Although we did not investigate a role for death receptors in this study, future work will be directed at understanding the effects that freezing may have on the cell membrane. With the increase in cryosurgical application, the development of new strategies to improve efficacy will become increasingly valuable.5,43,44 Targeted induction of programmed cell death via the extrinsic apoptotic pathway represents an additional unexploited therapeutic strategy that can be combined with cryoablation to destroy cancer cells. The adjuvant strategy is an effective method to reduce the critical freezing temperature, thereby reducing the need to extend the freeze zone beyond the targeted tissue. The impact of reducing the critical temperature can be visualized clearly in Figure 2a (compare freeze only to TRAIL combo). This would then lessen the chance of affecting the surrounding organs. The freeze/TRAIL combination represents a particularly exciting therapeutic model because molecules that directly activate the TRAIL receptors, such as agonistic monoclonal antibodies and recombinant TRAIL, are currently being developed.45 It may be possible to place TRAIL ‘seeds’ in a similar manner to that which is done with brachytherapy, and target them toward the periphery of the diseased tissue along with the cryosurgical application. The adjuvant application of TRAIL may also reduce the harmful cytotoxic side effects typically associated with current chemotherapeutic agents especially as the freeze/TRAIL combo appears to be less toxic to normal prostate cells (Figure 7). Furthermore, knowledge of the molecular mechanisms involved in both the resistance of the cancer and the efficacy of the therapy will provide significant benefits. For instance, small molecules designed to inhibit caspase-8 could be used to protect specific areas to protect them against the destructive measures of the freeze/TRAIL combination. Our findings demonstrate that freezing enhances TRAILinduced cell death by increasing apoptosis through a caspase-8-mediated pathway. Overall, the study described herein provides data illustrating a potentially effective therapeutic model for the cryodestructive treatment of CaP and provides the framework for future studies.

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Acknowledgements This project was supported in part by Galil Medical, USA.

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