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Resveratrol enhances prostate cancer cell response to ionizing radiation. Modulation of the AMPK, Akt and mTOR pathways

Resveratrol enhances prostate cancer cell response to ionizing radiation. Modulation of the AMPK,... Background: Prostate cancer (PrCa) displays resistance to radiotherapy (RT) and requires radiotherapy dose escalation which is associated with greater toxicity. This highlights a need to develop radiation sensitizers to improve the efficacy of RT in PrCa. Ionizing radiation (IR) stimulates pathways of IR-resistance and survival mediated by the protein kinase Akt but it also activates the metabolic energy sensor and tumor suppressor AMP-Activated Protein Kinase (AMPK). Here, we examined the effects of the polyphenol resveratrol (RSV) on the IR-induced inhibition of cell survival, modulation of cell cycle and molecular responses in PrCa cells. Methods: Androgen-insensitive (PC3), sensitive (22RV1) PrCa and PNT1A normal prostate epithelial cells were treated with RSV alone (2.5-10 μM) or in combination with IR (2-8 Gy). Clonogenic assays, cell cycle analysis, microscopy and immunoblotting were performed to assess survival, cell cycle progression and molecular responses. Results: RSV (2.5-5 μM) inhibited clonogenic survival of PC3 and 22RV1 cells but not of normal prostate PNT1A cells. RSV specifically sensitized PrCa cells to IR, induced cell cycle arrest at G1-S phase and enhanced IR-induced nuclear aberrations and apoptosis. RSV enhanced IR-induced expression of DNA damage (gH2Ax) and apoptosis cip1 kip1 (cleaved-caspase 3) markers as well as of the cell cycle regulators p53, p21 and p27 . RSV enhanced IR- activation of ATM and AMPK but inhibited basal and IR-induced phosphorylation of Akt. Conclusions: Our results suggest that RSV arrests cell cycle, promotes apoptosis and sensitizes PrCa cells to IR cip1 kip1 likely through a desirable dual action to activate the ATM-AMPK-p53-p21 /p27 and inhibit the Akt signalling pathways. cip1 Keywords: radio-sensitizers, clonogenic survival, cell cycle, ATM, p53, p21 Introduction The phosphatidylinositol 3-kinase (PI3k)-Protein Radiotherapy is an effective therapy for localized pros- kinase B/Akt (henceforth: Akt) pathway is known to tate cancer (PrCa) but this disease is highly resistant to promote proliferation, cell cycle progression and resis- ionizing radiation (IR). Conventional radiotherapy doses tance to cytotoxic therapies in PrCa [2]. PI3k is an effec- up to 70 Gy show biochemical failure rates of 30% or tor of the epidermal growth factor receptor (EGFR) [3], that leads to recruitment of Akt and its activators to more in localized disease [1], leading to a need for RT dose escalation, which is associated with rectal and blad- plasma membrane. Akt is activated by phosphorylation der toxicity. Therefore, there is a need for rational on residues T308 and serine S473, both of which are development of effective radiosensitizers for PrCa. required for activation [4]. T308 phosphorylation is mediated by the phosphoinoisitide-dependent kinase 1 (PDK1) [5] but the kinase mediating S473 phosphoryla- * Correspondence: theos.tsakiridis@jcc.hhsc.ca tion (PDK2) is not clearly defined. Candidate kinases Translational Radiation Biology Laboratory, Juravinski Cancer Centre, 699 Concession Street, Hamilton, Ontario, L84 5C2, Canada include the DNA damage sensor Ataxia Telangiectasia Full list of author information is available at the end of the article © 2011 Rashid et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Rashid et al. Radiation Oncology 2011, 6:144 Page 2 of 12 http://www.ro-journal.com/content/6/1/144 Mutated (ATM) [6]. Activated Akt mediates transcrip- Reagents and Antibodies tion of genes involved in survival and inhibition of those Rabbit polyclonal antibodies against total Akt (T-Akt), involved in apoptosis (see [2,7] for review). It promotes phosphorylated-(P)-(S473)-Akt, P-(T308)-Akt, P-mTOR, cell cycle progression through inhibition of the cell cycle total AMPK (T-AMPK), P-(Thr172)-AMPK, T-ATM, P- regulators p53 [8] and the cyclin-dependent kinase inhi- (S1981)-ATM, P-(S139)-gH2Ax, mouse monoclonal cip1 kip1 cip1 kip1 antibodies against p53, p21 ,p27 , actin, an anti-a- bitors (CDKI) p21 and p27 [9,10]. Furthermore, it tubulin antibody conjugated to Alexa Fluor 488 as well regulates metabolic and nuclear processes through acti- as horseradish peroxidase (HRP)-conjugated IgG sec- vation of the mammalian target-of-rapamycin (mTOR). ondary anti-rabbit and anti-mouse antibodies were from Importantly, IR elicits cytoprotective responses mediated in part through activation of the PI3k-Akt pathway [11]. New England Biolabs (Mississauga, ON, Canada). Akt is a mediator of radioresistance and PI3k-Akt path- Hoechst 33258 was from Sigma (Toronto, ON, Canada). way inhibitors are shown to enhance radiosensitivity of RSV and the KuDos Pharma ATM inhibitor KU55933 cancer cells [7,12]. were from Calbiochem (Mississauga, ON, Canada). AMPK is a heterotrimeric enzyme that consists of an Anti-a1and -a2 AMPK siRNA transfection kit was a-catalytic and b-and g-regulatory subunits [13]. It is a obtained from Qiagen (Mississauga, Ontario, Canada). key regulator of carbohydrate and lipid metabolism and of proliferation in normal and cancer cells. AMPK Treatments detects an elevated AMP/ATP ratio in conditions of Cells were treated with 2-8 Gy IR using a Co clinical metabolic stress such as starvation and exercise [13] and unit. For combined RSV or KU55933 and IR treatments, promotes energy conservation by inhibiting protein cells were kept at 37°C with the indicated agent for 1 h synthesis, through mTOR inhibition while it also func- prior to IR treatment. Cells were incubated for 1 h fol- tions as a metabolic checkpoint to induce cell cycle lowing IR exposure, unless otherwise indicated. For cell arrest via p53 [14]. Recently, we showed that IR acti- cycle and clonogenic assays cells were exposed to the vates AMPK in human lung, breast and PrCa cells and treatment agents throughout the experiments (as indi- suggested that AMPK participates in a signaling pathway cated in figure legends). cip1 involving ATM-AMPK-p53-p21 leading to regulation of the cell cycle and survival [15]. siRNA AMPK a Subunit Knockdown Cells were incubated with a mixture of human siRNA RSV (3,4’,5-trihydroxystilbene) is a polyphenolic phy- sequences against the a1and a2 AMPK subunits using toalexin with widely reported anti-aging and anti-cancer HiPerFect vehicle for 72 hours as per the manufacturer’s properties [16,17]. It inhibits cancer cell proliferation protocol. and is suggested to enhance radiation responses [18,19]. RSV has also been reported to increase metabolic rate and reduce fat mass in wild-type mice but not in AMPK Clonogenic Assays a subunitknockoutmice [20]. Further, it wasshown to Clonogenic assays were performed as described earlier suppress tumor growth and metastasis in the mouse [15]. Cells (500-1000) were seeded in triplicates and Lewis lung carcinoma model [21]. RSV is known to reg- allowed to adhere overnight, then were incubated with ulate both Akt and AMPK [22,23] but the effects of this RSV followed by IR treatment (0-2 Gy) followed by compound on the two signaling pathways have not been incubation for 7-10 days. Cells were then fixed and studied in radiated cells. stained with 0.05% methylene blue and colonies (> 50 Here, we investigated the polyphenol RSV due to the cells) were counted. Results are expressed as the surviv- reported ability of this natural compound to modulate ing fraction compared to untreated control. both the radioresistance-mediating Akt and the tumour suppressor AMPK pathways [24,25]. Immunoblotting (IB) IB was performed as described earlier [26]. Approxi- Materials and methods mately 5 × 10 cells were seeded in 6-well plates. After Cell Lines and Cell Culture treatment, cells were washed, lysed and twenty micro- Human PrCa (PC3, 22RV1) and normal prostate epithe- grams of protein were subjected to IB. lial (PNT1A) cell lines were obtained from American Tissue Culture Collection (Manassas, VA, U.S.A.). Cells Immunofluorescence Microscopy Cells (2 × 10 ) were seeded onto glass coverslips and were maintained at 37°C in RPMI media supplemented were incubated without or with RSV (5 μM) 1 h prior with 10% (vol/vol) Fetal Bovine Serum (FBS) and 1% to IR treatment (8 Gy). Ninety six hours after IR expo- (vol/vol) antibiotic-antimycotic (Invitrogen, Burlington, sure, cells were washed lightly with PBS and fixed with ON, Canada). Rashid et al. Radiation Oncology 2011, 6:144 Page 3 of 12 http://www.ro-journal.com/content/6/1/144 3% paraformaldehyde/PBS/0.2% Triton X-100 for 20 RSV enhances the IR-induced inhibition of clonogenic minutes. Cells were stained with Hoechst 33258 (20 survival in PrCa cells μM) nuclear stain or anti-a-tubulin antibody conjugated RSV augmented further the IR-induced inhibition of to Alexa Fluor 488 (1:100 dilution) (followed by Hoechst survival in both PC3 and 22RV1 PrCa cells. RSV (2.5 33258 nuclear staining) and examined by fluorescence and 5 μM) decreased survival of IR-treated PC3 cells by (13.8 ± 0.09% and 20.4 ± 0.9%, Figure 1B). RSV 5 μM microscopy. Four hundred cells were evaluated for caused only a minor reduction of SF2 in 22RV1 cells nuclear aberrations (fragmentation, micro-nuclei and (non-significant) but at 10 μM the drug induced a sig- multi-nucleation) in four representative areas of each nificant 2.5-fold inhibition in SF2. RSV (5 μM) pre-treat- slide in 3 independent experiments. ment did not decrease further SF2 of PNT1A prostate Cell Cycle Analysis epithelial cells but this was achieved when RSV was (1 × 10 cells/mL) were seeded in 10-cm dishes. After increased to 10 μM (Figure 1B). treatment, cells were trypsinized, washed, fixed with To better demonstrate the potential clinical benefit 70% ethanol and stored overnight at -20°C followed by of RSV in combination with radiotherapy, we used SF2 washing and staining with a solution containing 100 μL values to estimate tumour cell survival after 40 frac- Triton X-100 and 50 μg/mL propidium iodide. Cells tions of radiotherapy (2 Gy each), which consists a were subjected to flow cytometric cell cycle analysis standard approach in clinical PrCa management [1]. using a Beckman Coulter Epics XL flow cytometer. We determined (SF2) as the tumour cell fraction sur- viving after “n” fractions of radiotherapy. Assuming Statistical Analysis iso-effectiveness of all fractions, we plotted the pro- Data are expressed as the mean ± standard error (SE). jected tumour survival over 40 consecutive fractions Statistical analysis was performed using unpaired t-test (0-80 Gy) (Figure 1C). As typical human prostates with SPSS v16.0 software (SPSS, Chicago, IL). Statistical range between 35-100 gr (5 - 10 × 10 cells) we significance was considered at p < 0.05. plotted projected tumour size reductions (tumour kill- -11 ing) on a scale of 1 - 1 × 10 . Figure 1C estimates Results that RSV (5 μM) maybe abletoreduce the dose of RSV inhibits PrCa cell survival radiotherapy needed to eliminate 22RV1 type (hor- mone-sensitive) PrCa tumours (from about 47 to 40 As a single agent, RSV effectively inhibited survival of Gy) and may be able to eliminate PC3 type (hormone- both PC3 and 22RV1 PrCa cells with significant inhibi- insensitive) PrCa tumours (with about 54 Gy) that tion achieved at even the lower doses of 2.5 and 5 μM would otherwise be incurable with doses even higher (p < 0.05) (Figure 1A). The IC values were approxi- mately 10 μMand 2.5 μM for PC3 and 22RV1 cells, than 80 Gy.Further,thisapproachalso demonstrated respectively. The PC3 response to RSV was dose-depen- the complete lack of radio-sensitization of normal dent. RSV 2.5 μM significantly decreased survival in epithelial cells (PNT1A) by 5 μM RSV. Based on these 22RV-1 cells to 40 ± 3.06% of control without any results, we pursued all subsequent studies with PrCa further decrease at higher doses. In contrast, PNT1A cells alone and with doses of 2.5 - 5 μMRSV. normal epithelial cells were less responsive to RSV showing only 5 and 10% inhibition of survival at 2.5 and RSV inhibits IR-mediated cell cycle arrest and induces cell 5 μM, respectively (Figure 1A). Only the higher dose of accumulation at G1-S and sub-G1 phases consistent with 10 μM caused significant decrease in normal epithelial apoptosis PNT1A cell survival (to 60 ± 12.26% of control) (p < We utilized the 22RV1 cell line in our cell cycle analy- 0.05). PTN1A cells were used here as a non-malignant sis experiments because it is a cloned cell line [28] control. consisting of a more isogenic population of cells than PC3 cells, and is hence appropriate for cell cycle analy- IR-induced inhibition of PrCa cell survival sis. As expected, IR induced an arrest of cells at the PC3 cells showed greater resistance to IR alone com- G2-M interphase (Control: 13.4% vs. IR: 31.7%, Figure pared to 22RV1 cells (surviving fractions after 2 Gy 2) but pre-treatment with RSV (5 μM) prevented this (SF2) of 60 ± 5.30% vs 40 ± 3.53%, respectively, Figure event (RSV+IR: 8.3% vs. 31.7% for IR, Figure 2). RSV 1B). Their IR sensitivity was similar to that of PNT1A caused accumulation of radiated cells into the G1-S phases of the cycle (IR: 58% vs. RSV + IR: 70.7%) and cells (SF2 of 62.9 ± 2.26%, Figure 1B). In clonogenic increased the population of cells in the sub-G1 region assays we opted to use the standard therapeutic IR dose by 2-fold compared to those treated with IR alone (IR: of 2 Gy and not high doses of IR, for clinical relevance 10.3% vs. RSV + IR: 21.0%), indicating induction of and because higher doses (i.e. 6-8 Gy) were highly toxic apoptosis. to PrCa cells in agreement with previous reports [27]. Rashid et al. Radiation Oncology 2011, 6:144 Page 4 of 12 http://www.ro-journal.com/content/6/1/144 Figure 1 Effects of resveratrol (RSV) and ionizing radiation (IR) on survival of prostate cancer (PrCa) cells. A. Cells were incubated with increasing doses of RSV (0-10 μM) and allowed to form colonies. Colonies (> 50 cells) were counted. Results are expressed as the surviving fraction compared to untreated control (see Methods). ‘*’ p < 0.05 compared to untreated PC3 cells, ‘#’ p < 0.05 compared to untreated 22RV1 cells, ‘+’ p< 0.05 compared to untreated PNT1A cells. B. Cells were pre-treated with RSV (0-10 μM) followed by treatment with a single fraction of IR (2 Gy) and allowed to grow and form colonies. Treatment values were normalized to untreated controls and surviving fractions are presented as percent of control. Shown is the mean of 4-5 independent experiments ± standard error (SE). ‘*’ p < 0.05 compared to 2 Gy treated PC3 cells, ‘#’ p < 0.05 compared to 2 Gy treated 22RV1 cells, ‘+’ p < 0.05 compared to 2 Gy treated PNT1A cells. C. Projected proportion of tumour survival after treatment with 0 - 80 Gy was estimated using the Surviving Fraction at 2 Gy (SF2) values for control and RSV (5 μM) treated PNT1A, PC3 and 22RV1 cells. SF2 was determined as the tumour proportion surviving after “n” 2 Gy fractions, assuming iso-effectiveness of each fraction. Values were -11 10 plotted up to a scale of 1 × 10 to reflect elimination of tumour of 50 - 100 gr expected to contain 5 - 10 × 10 cells. RSV enhances the IR induction of cell cycle inhibitors levels of this established apoptosis marker. IR increased To examine whether the effects of RSV on IR regulation caspase-3 cleavage within 1 hour of IR exposure in both of the cell cycle were associated with enhancement of PC3 and 22RV1 cells (Figure 3A). Pre-treatment with molecular pathways of cell cycle regulation, we exam- RSV (2.5 and 5 μM) enhanced the IR-induced cleaved ined the expression of p53 and the cyclin-dependent caspase-3 levels in a dose-dependent fashion. cip1 kip1 kinase inhibitors (CDKI) p21 and p27 .Figure2B To evaluate morphological markers of apoptosis, cells shows that IR induced expression of both p53 and the were stained with Hoechst 33258 and examined with CDKIs in 22RV1 cells and that RSV enhanced this microscopy. Cells with nuclear aberrations such as frag- further. Similar results were obtained for the two CDKIs mentation, micro-nuclei and multi-nucleation (polys- in p53-null PC3 cells. omy), which are markers predictive of apoptosis and/or mitotic cell death (mitotic catastrophe), were counted in RSV induces expression of apoptotic markers and nuclear each treatment condition. Figure 3B shows representa- aberrations tive images of treated PC3 cells that were either stained Using immunoblotting with a cleaved caspase-3-specific with Hoechst 33258 alone (low magnification, Figure antibody, we examined the effects of RSV and IR on the 3B) or additional immunostaining of tubulin (high Rashid et al. Radiation Oncology 2011, 6:144 Page 5 of 12 http://www.ro-journal.com/content/6/1/144 Figure 2 Effects of resveratrol (RSV) and ionizing radiation (IR) on prostate cancer cell cycle progression and expression of cycle regulators. A. 22RV1 cells were pre-treated or not with RSV (5 μM) for 1 hour followed by treatment with 8 Gy IR. Cells were allowed to cycle for 48 hours after which they were fixed, treated with a triton-X and stained with propidium iodide (PI) solution as described in Methods. Shown are representative results of 3 independent experiments. Pie charts illustrate the proportion of cells in each phase of cell cycle. B. 22RV1 and PC3 cells were pre-treated with RSV (5 μM) for 1 hour followed by 8 Gy IR treatment. Cells were lysed 1 hour after IR exposure and subjected to immunoblot analysis using antibodies against the indicated cell cycle markers. Shown are representative immunoblots of 3 independent experiments. magnification), which was utilized to better demonstrate total cell counts and reduced overall cellular viability the effects of the treatments on nuclear and cellular (Figure 3B). morphology (Figure 3C). The results of four indepen- dent experiments (as in Figure 3B) were quantitated (as Effects of RSV on molecular pathways of radio-resistance described in Methods)and areshown in Figure 3D). IR and tumour suppression alone induced significant nuclear aberrations (43 ± RSV inhibits basal and IR-induced levels of phosphorylated 2.18%, Figure 3B), while RSV alone (5 μM) increased Akt nuclear abnormalities by 6% (Figure 3B). However, RSV We examined the effects of RSV on the well-described significantly enhanced the IR-induced nuclear aberra- radio-resistance pathway of Akt-mTOR. We observed tions to 70 ± 3.11% (an increase of 27%, Figure 3D) of that IR alone induced a significant time-dependent Rashid et al. Radiation Oncology 2011, 6:144 Page 6 of 12 http://www.ro-journal.com/content/6/1/144 Figure 3 Resveratrol (RSV) enhances ionizing radiation (IR)-induced cell death. Induction of cleaved caspase-3 and nuclear aberrations. A. PC3 cells were pre-treated or not with RSV (5 μM) for 1 hour after which they were treated with IR (8 Gy). Cells were lysed 1 hour after IR exposure and subjected to immunoblot analysis using the indicated antibodies. Shown are representative immunoblots of 3-4 independent experiments. B. and C. Cells were treated or not with RSV (5 μM) prior to IR treatment (8 Gy) followed by staining with nuclear stain Hoeschst 33258 (low magnification, in B) or Hoeschst 33258 and immunostaining of tubulin (high magnification, in C) used to illustrate better cellular morphology. Morphology was assessed using fluorescence microscopy. Representative images from 4 independent experiments are shown. D. Average ± SE values (% Nuclear Aberrations: as indicated by the proportion of cells with features of nuclear fragmentation, micro-nuclei and multi-nucleation) were extracted through quantitation of 4 independent experiments (in B), as described in Methods. increase in the levels of phosphorylated Akt (P-Akt) exposure to allow demonstration of the effects of RSV (both S473 and T308 sites) in PC3 cells, with no effects on basal Akt phosphorylation which are significantly on total levels of the protein (Figure 4A). Highest levels lower than in radiated cells (as shown in Figure 4A and of P-Akt were reached 30 minutes to 1 hour after IR 4C). Pre-treatment with RSV led to significant inhibition exposure. Akt phosphorylation was associated with of IR-induced P-Akt with no effects on total levels of increased activity of this enzyme indicated by the IR- the protein (Figure 4C). Although IR exposure led to an induced phosphorylation of mTOR, a key effector of increase in P-Akt, RSV pre-treatment (2.5 and 5 μM) Akt (Figure 4A). Similar results were obtained in 22RV1 significantly reduced and/or prevented IR-induced phos- phorylation of Akt. The effects of RSV on IR-induced PrCa cells (results not shown). Treatment of PC3 and 22RV1 cells with RSV inhibited Akt phosphorylation on S473 were quantitated in four basal levels of P-Akt (Figure 4B). Of note, the images independent experiments (Figure 4D). Similar results shown were obtained after significantly longer film were obtained for Akt T308phosphorylation.A Rashid et al. Radiation Oncology 2011, 6:144 Page 7 of 12 http://www.ro-journal.com/content/6/1/144 Figure 4 Resveratrol (RSV) inhibits basal and ionizing radiation (IR)-induced Akt phosphorylation. A. Effects of IR alone: PC3 cells were treated with 0 or 8 Gy IR and lysed according to the indicated time points following IR exposure. Then cells were subjected to immunoblot analysis. B. RSV inhibits basal Akt phosphorylation: PC3 and 22RV-1 cells were treated with RSV (0, 2.5 or 5 μM) for 1 hour and subjected to lysis and immunoblot analysis using the indicated antibodies. C. RSV inhibits IR-induced Akt phosphorylation. PC3 and 22RV-1 cells were pre-treated with the indicated concentrations of RSV 1 hour prior to IR treatment. Cells were lysed 1 hour after IR exposure and subjected to immunoblot analysis. Shown are representative immunoblots of 4 independent experiments. D. Densitometric quantitation of Akt (S473) phosphorylation and total Akt was performed. P-Akt was normalized to T-Akt levels and presented as fold change compared to untreated control cells (mean ± SE) from 3-4 independent immunoblotting experiments as in C). * p < 0.05 compared to cells treated with IR alone. representative immunoblot from PC3 cells is shown (ACC). RSV induced a robust activation of AMPK in (Figure 4C). both PC3 and 22RV1 cells at both 2.5 and 5 μM (Figure RSV and IR effects on AMPK 5B). Furthermore, RSV pre-treatment enhanced signifi- We reported earlier that IR activates AMPK in lung, cantly the IR-induced AMPK phosphorylation in both breast and prostate cancer cells [15]. Similarly, we 22RV1 and PC3 cells (Figure 5C). Total AMPK levels observed here that IR induced a robust phosphorylation remained unchanged after both IR and RSV treatments. of AMPK on Thr172 of the catalytic a subunitthatwas The results of 3-4 independent experiments were quan- detectable within 15 min. Highest levels of phosphory- titated and are shown in Figure 5D. lated AMPK (P-AMPK) were detected 1 hour after IR exposure and returned to almost basal levels 4 hours The pathway of radiation activation of Akt and AMPK after radiation (Figure 5A). This was associated with Akt regulation activation of AMPK indicated by the detected phosphor- Recent reports suggest that Akt activation by IR may be ylation of the AMPK substrate Acetyl CoA Carboxylase regulated by ATM [29]. We examined this notion in Rashid et al. Radiation Oncology 2011, 6:144 Page 8 of 12 http://www.ro-journal.com/content/6/1/144 Figure 5 Resveratrol (RSV) enhances basal and ionizing radiation (IR)-induced AMPK phosphorylation. A.EffectsofIRalone:PC3 cells were treated with 0 or 8 Gy IR and lysed at the indicated time points after IR exposure. The adjacent panel shows PC3 cells treated with 8 Gy IR, which were lysed 1 hour after IR treatment. Samples were subjected to immunoblot analysis using the indicated antibodies. B. RSV enhances basal AMPK phosphorylation: PC3 and 22RV-1 cells were treated with the indicated concentrations of RSV for 1 hour followed by lysis and immunoblotting analysis. C. RSV enhances IR-induced AMPK phosphorylation. PC3 and 22RV-1 cells were pre-treated with the indicated concentrations of RSV 1 hour prior to 8 Gy IR treatment. Cells were lysed 1 hour after IR exposure and subjected to immunoblotting using indicated antibodies. Shown are representative immunoblots of at least 3-4 independent experiments. D. Densitometry-based quantitation. Density values of P-AMPK were normalized to T-AMPK levels and presented as fold change compared to untreated control cells (mean ± SE) from 2-3 independent experiments in C. is shown. * p < 0.05 compared to cells treated with IR alone. cip1 PrCa cells using the ATM-specific inhibitor KU55933 subunits of AMPK blocked p53 and p21 induction by [30]. Pre-incubation with KU55933 prevented IR- IR (Figure 6C). Interestingly, RSV pre-treatment induced ATM phosphorylation but also inhibited IR enhanced IR-induced phosphorylation of ATM and of phosphorylation of Akt at S473 and activation of its its substrate histone H2Ax (gH2Ax), as well as phos- cip1 kinase activity as indicated by reduced phosphorylation phorylation of AMPK and induction of p21 . of mTOR (Figure 6A). AMPK Regulation Discussion We attempted to verify, in PrCa cells, our earlier obser- IR and RSV effects on PrCa cell clonogenic survival vations in lung cancer cells [15] of AMPK participation RSV inhibits survival and proliferation of cancer cells as cip1 in an ATM-AMPK-p53/p21 pathway activated by IR. a single agent and induces radiosensitization in human We observed a robust phosphorylation of ATM and cervical cancer cells [19]. Similarly, we observed that at cip1 AMPK as well as induction of p21 in PC3 PrCa cells low doses (2.5-10 μM) RSV inhibited clonogenic survival in response to IR. IR induced ATM and AMPK phos- of PrCa cells. Several studies have reported IC values cip1 phorylation and p21 induction were all inhibited by for cell growth inhibition by RSV in the range of 5 to treatment with KU55933 (Figure 6B). To verify that 10 μM [31,32]. Free RSV has a low bioavailability in- cip1 AMPK truly acts as a mediator of p53/p21 induction vivo as it is rapidly metabolized to glucoronide and sul- fate conjugates [32]. A human study reported plasma in response to IR in PrCa cells, we used wild-type p53 expressing 22RV1 cells to perform AMPK knockdown concentrations of free RSV of 21 nM after oral dose of experiments. siRNA knockdown of both a1and a2 25 mg RSV [32]. However, all combined RSV Rashid et al. Radiation Oncology 2011, 6:144 Page 9 of 12 http://www.ro-journal.com/content/6/1/144 Figure 6 Mechanism of Akt and AMPK regulation by resveratrol (RSV) and ionizing radiation (IR). A.and B. Inhibition of ATM blocks IR- induced activation of the Akt and AMPK signaling pathways: PC3 cells were treated or not with the ATM inhibitor KU55933 (10 μM) for 1 hour followed by 0 or 8 Gy IR treatment. Cells were lysed 1 hour after IR exposure and subjected to immunoblot analysis using the indicated cip1 antibodies. C. AMPK mediates the IR induction of p53 and p21 : 22RV-1 cells were incubated with siRNA complexes against the a1 and a2 subunits of AMPK for 72 hours as described in Methods. Cells were then treated with 0 or 8 Gy IR and lysed 1 hour after IR exposure and cip1 subjected to immunoblot analysis. D. RSV enhances signalling along the ATM-AMPK-p21 pathway: Cells were pre-treated with RSV (5 μM) for 1 hour followed by IR of 8 Gy. Cells were lysed 1 hour after IR exposure and subjected to immunoblot analysis using the indicated antibodies. Shown are representative immunoblots of at least 3 independent experiments. Rashid et al. Radiation Oncology 2011, 6:144 Page 10 of 12 http://www.ro-journal.com/content/6/1/144 metabolites were reported to reach about 2 μM [16]. For activation of caspase-, p53-, or Fas ligand-dependent this, we pursued our studies with low RSV concentra- pathways [39,40]. In our studies, IR-mediated nuclear tions (2.5 - 10 μM). damage showed early signs of mitotic catastrophe and As a singleagent,RSV inhibitedmorepotentlysurvi- RSV potentiated such nuclear aberrations (Figure 3C-D). val of 22RV1 compared to PC3 cells but the response of Mitotic cell death predominates in cells with defects in 22RV1 cells did not display dose-dependence. Overall, cycle checkpoints that prevent cycle arrest and DNA PC3 cells displayed greater resistance to both IR and repair when cells are exposed to genotoxic stress such RSV alone, consistent with other studies [33]. PC3 cells as IR [41]. The features of mitotic catastrophe seen in our studies in PC3 cells (p53-null)after RSVandIR are deficient in key tumour suppressors including PTEN and p53 [34]. Lack of PTEN allows aberrant Akt activa- treatments are consistent with those observed earlier in tion [35], which in combination with the lack of p53, other p53-null cancer models [41]. may confer such cells a survival advantage and IR- and RSV-resistance. PNT1A cells were less responsive to Modulation of the Akt and AMPK pathways RSV, indicating a potential for this drug to achieve a IR and RSV exert opposite effects on Akt. IR mediated positive therapeutic ratio in-vivo. RSV inhibited signifi- Akt phosphorylation on T308 and S473 and enzymatic cantly PNT1A cells at 10 μM and for that we focused activation indicated by mTOR phosphorylation (Figure our work on 2.5 and 5 μM RSV. 4A). This suggests activation of both PDK1 and PDK2 We demonstrated that RSV can sensitize PrCa cells to activity by IR. However, we observed that RSV is an IR (Figure 1). Concentrations of RSV (2.5 and 5 μM), effective inhibitor of this radioresistance-associated path- similar to those that can be achieved in human serum, way (Figure 4). Inhibition of IR-induced T308 and S473 enhanced the cytotoxicity of a conventional RT fraction phosphorylation by RSV suggests an ability to regulate (2 Gy) in PrCa cells without additional toxicity to nor- both PDK1 and PDK2. The enhancement of IR-induced mal epithelial cells. The potential clinical utility of our cell death by RSV may indeed be mediated through finding is illustrated in Figure 1C, which suggests that modulation of Akt and its downstream targets as sug- low RSV doses have the potential to reduce the dose of gested earlier for uterine cancer [25] and PrCa cells [42]. radiotherapy required to treat human hormone- and Using the ATM-specific inhibitor KU55933, [30] we radiation-sensitivePrCaand maybeabletomakecur- observed that ATM mediates IR-induced Akt S473 phos- able hormone- and radiation-insensitive tumours that phorylation and activation, suggesting that ATM may may otherwise be incurable with even modern dose indeed be the IR responsive PDK2, as suggested earlier [6]. escalated radiotherapy. This notion needs to be verified However, despite inhibition of Akt, RSV enhanced ATM in in-vivo models of human PrCa. phosphorylation and activation indicated by histone H2Ax phosphorylation (gH2Ax) (Figure 6D). This points to a Regulation of cell cycle and apoptosis complex regulatory action of RSV to enhance ATM activa- RSV is reported to arrest PrCa and other cells at G0-G1 tion but inhibit its PDK2 action on Akt downstream. This and/or S-phases of the cycle leading to senescence concept needs to be explored further in future studies. cip1 [36,37] and cause p21 -mediated G1-phase arrest and We aimed to evaluate, in PrCa cells, the DNA damage cip1 apoptosis in A431 cells [38]. Consistently, we observed a responsive ATM-AMPK-p21 pathwayweproposed significant arrest of radiated PrCa cells at G1-S (Figure earlier in lung cancer cells [15] and found that IR 4). RSV’s effects on survival were additive to those of IR indeed mediates AMPK activation downstream of ATM but the two agents mediate different regulation of cell (Figure 6B). We observed that AMPK mediates p53 and cip1 cycle. Whereas IR induces G1-S and more so G2-M p21 induction in response to IR in PrCa cells (Figure cycle arrest, RSV prevented the latter (Figure 2), likely 6C). RSV and IR stimulated phosphorylation/activation due to induction of an earlier checkpoint (G1-S). The of AMPK, in both PC3 and 22RV1 PrCa cells, which cip1 kip1 potentiation of IR-induced expression of p53 and CDK was associated with induction of p21 and p27 cip1 kip1 inhibitors p21 and p27 by RSV (Figure 2B), (Figure 6D). Although, we did not examine whether the known to regulate the G1 and S phase checkpoints, may latter is regulated by AMPK activity, this notion is well provideamolecular pathwayofactionfor RSV’sinduc- described by other studies [43]. The enhancement of IR- tion of the G1-S phase arrest seen in our studies. stimulated ATM activity by RSV provides a framework cip1 RSV also caused re-distribution of cells into the sub- for upregulation of the ATM-AMPK-p21 pathway in G1 or apoptotic range that was associated with cleavage RSV-treated cancer cells and a rationale for the of caspase-3 and induction of extensive nuclear aberra- observed inhibition of cell cycle and survival. This tions (Figure 3). Apoptosis appears to be the primary notion is supported by observations that the AMPK mode of cell death induced by RSV. Studies demon- inhibitor compound C inhibits IR-induced cytotoxicity strated such cell death effects in PrCa cells through in PrCa (results not shown). Rashid et al. Radiation Oncology 2011, 6:144 Page 11 of 12 http://www.ro-journal.com/content/6/1/144 Figure 7 Model of the mechanism of action of resveratrol (RSV) and ionizing radiation (IR) to regulate cell cycle and survival in PrCa cells. A. IR induces DNA strand breaks which are detected by ATM, leading to AMPK activation and induction of the cell cycle inhibitors p53, cip1 kip1 p21 and p27 . IR also activates Akt to increase protein synthesis through mTOR and stimulate cell cycle progression, proliferation and survival. B. The work presented here suggests that RSV i) enhances the effects of IR on AMPK activation, leading to an early cell cycle arrest, and ii) inhibits Akt to reduce gene expression and proliferation and enhance radiation cytotoxicity. L8S 4L8, Canada. Department of Pathology and Molecular Medicine, Conclusions McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S 4L8, Figure 7 illustrates a model of the effects of RSV on the 4 Canada. Department of Community Health Science, Brock University, 500 IR responses of PrCa cells. Low (μM) concentrations of Glenridge Avenue, St. Catharines, Ontario, L2S 3A1, Canada. Department of Radiation Oncology, Princess Margaret Hospital, University of Toronto, 610 RSV, that can be achieved in human serum, inhibit University Avenue, Toronto, Ontario, M5G 2M9, Canada. PrCa cell survival and enhance IR-induced cytotoxicity. RSV enhances IR-induced DNA-damage response sig- Authors’ contributions AR carried out the cellular/molecular experiments (clonogenic assays), cell nals (ATM), apoptosis markers (cleaved caspase-3) and cycle studies and helped draft the manuscript. CL aided in the cell cycle cip1 kip1 cell cycle inhibitors (p53, p21 and p27 ). Further, it studies. TS helped carry out the immunofluorescence experiments. TT arrests cycling at the G1-S phase and enhances IR- conceived the study, directed the study design, supervised all experimental work and prepared the manuscript. ET, GS, RB participated in the design of induced nuclear aberrations. RSV exerts a desirable dual the study and advised TT on experimental approaches. All authors have read action to inhibit the Akt radioresistance pathway while and approved the final manuscript. enhancing the tumour suppressor AMPK, leading to cip1 kip1 Competing interests potentiation of an ATM-AMPK-p53-p21 /p27 sig- The authors declare that they have no competing interests. naling axis. We conclude that RSV is a promising agent that deserves further investigation as an adjunct to IR in Received: 4 August 2011 Accepted: 26 October 2011 Published: 26 October 2011 in-vivo models of PrCa to help elucidate its potential for clinical use in combination with RT. References 1. 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Verbeuren TJ, Cohen RA: Polyphenols stimulate AMP-activated protein doi:10.1186/1748-717X-6-144 kinase, lower lipids, and inhibit accelerated atherosclerosis in diabetic Cite this article as: Rashid et al.: Resveratrol enhances prostate cancer LDL receptor-deficient mice. Diabetes 2006, 55(8):2180-2191. cell response to ionizing radiation. Modulation of the AMPK, Akt and mTOR pathways. Radiation Oncology 2011 6:144. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Radiation Oncology Springer Journals

Resveratrol enhances prostate cancer cell response to ionizing radiation. Modulation of the AMPK, Akt and mTOR pathways

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Copyright © 2011 by Rashid et al; licensee BioMed Central Ltd.
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Medicine & Public Health; Oncology; Radiotherapy
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Abstract

Background: Prostate cancer (PrCa) displays resistance to radiotherapy (RT) and requires radiotherapy dose escalation which is associated with greater toxicity. This highlights a need to develop radiation sensitizers to improve the efficacy of RT in PrCa. Ionizing radiation (IR) stimulates pathways of IR-resistance and survival mediated by the protein kinase Akt but it also activates the metabolic energy sensor and tumor suppressor AMP-Activated Protein Kinase (AMPK). Here, we examined the effects of the polyphenol resveratrol (RSV) on the IR-induced inhibition of cell survival, modulation of cell cycle and molecular responses in PrCa cells. Methods: Androgen-insensitive (PC3), sensitive (22RV1) PrCa and PNT1A normal prostate epithelial cells were treated with RSV alone (2.5-10 μM) or in combination with IR (2-8 Gy). Clonogenic assays, cell cycle analysis, microscopy and immunoblotting were performed to assess survival, cell cycle progression and molecular responses. Results: RSV (2.5-5 μM) inhibited clonogenic survival of PC3 and 22RV1 cells but not of normal prostate PNT1A cells. RSV specifically sensitized PrCa cells to IR, induced cell cycle arrest at G1-S phase and enhanced IR-induced nuclear aberrations and apoptosis. RSV enhanced IR-induced expression of DNA damage (gH2Ax) and apoptosis cip1 kip1 (cleaved-caspase 3) markers as well as of the cell cycle regulators p53, p21 and p27 . RSV enhanced IR- activation of ATM and AMPK but inhibited basal and IR-induced phosphorylation of Akt. Conclusions: Our results suggest that RSV arrests cell cycle, promotes apoptosis and sensitizes PrCa cells to IR cip1 kip1 likely through a desirable dual action to activate the ATM-AMPK-p53-p21 /p27 and inhibit the Akt signalling pathways. cip1 Keywords: radio-sensitizers, clonogenic survival, cell cycle, ATM, p53, p21 Introduction The phosphatidylinositol 3-kinase (PI3k)-Protein Radiotherapy is an effective therapy for localized pros- kinase B/Akt (henceforth: Akt) pathway is known to tate cancer (PrCa) but this disease is highly resistant to promote proliferation, cell cycle progression and resis- ionizing radiation (IR). Conventional radiotherapy doses tance to cytotoxic therapies in PrCa [2]. PI3k is an effec- up to 70 Gy show biochemical failure rates of 30% or tor of the epidermal growth factor receptor (EGFR) [3], that leads to recruitment of Akt and its activators to more in localized disease [1], leading to a need for RT dose escalation, which is associated with rectal and blad- plasma membrane. Akt is activated by phosphorylation der toxicity. Therefore, there is a need for rational on residues T308 and serine S473, both of which are development of effective radiosensitizers for PrCa. required for activation [4]. T308 phosphorylation is mediated by the phosphoinoisitide-dependent kinase 1 (PDK1) [5] but the kinase mediating S473 phosphoryla- * Correspondence: theos.tsakiridis@jcc.hhsc.ca tion (PDK2) is not clearly defined. Candidate kinases Translational Radiation Biology Laboratory, Juravinski Cancer Centre, 699 Concession Street, Hamilton, Ontario, L84 5C2, Canada include the DNA damage sensor Ataxia Telangiectasia Full list of author information is available at the end of the article © 2011 Rashid et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Rashid et al. Radiation Oncology 2011, 6:144 Page 2 of 12 http://www.ro-journal.com/content/6/1/144 Mutated (ATM) [6]. Activated Akt mediates transcrip- Reagents and Antibodies tion of genes involved in survival and inhibition of those Rabbit polyclonal antibodies against total Akt (T-Akt), involved in apoptosis (see [2,7] for review). It promotes phosphorylated-(P)-(S473)-Akt, P-(T308)-Akt, P-mTOR, cell cycle progression through inhibition of the cell cycle total AMPK (T-AMPK), P-(Thr172)-AMPK, T-ATM, P- regulators p53 [8] and the cyclin-dependent kinase inhi- (S1981)-ATM, P-(S139)-gH2Ax, mouse monoclonal cip1 kip1 cip1 kip1 antibodies against p53, p21 ,p27 , actin, an anti-a- bitors (CDKI) p21 and p27 [9,10]. Furthermore, it tubulin antibody conjugated to Alexa Fluor 488 as well regulates metabolic and nuclear processes through acti- as horseradish peroxidase (HRP)-conjugated IgG sec- vation of the mammalian target-of-rapamycin (mTOR). ondary anti-rabbit and anti-mouse antibodies were from Importantly, IR elicits cytoprotective responses mediated in part through activation of the PI3k-Akt pathway [11]. New England Biolabs (Mississauga, ON, Canada). Akt is a mediator of radioresistance and PI3k-Akt path- Hoechst 33258 was from Sigma (Toronto, ON, Canada). way inhibitors are shown to enhance radiosensitivity of RSV and the KuDos Pharma ATM inhibitor KU55933 cancer cells [7,12]. were from Calbiochem (Mississauga, ON, Canada). AMPK is a heterotrimeric enzyme that consists of an Anti-a1and -a2 AMPK siRNA transfection kit was a-catalytic and b-and g-regulatory subunits [13]. It is a obtained from Qiagen (Mississauga, Ontario, Canada). key regulator of carbohydrate and lipid metabolism and of proliferation in normal and cancer cells. AMPK Treatments detects an elevated AMP/ATP ratio in conditions of Cells were treated with 2-8 Gy IR using a Co clinical metabolic stress such as starvation and exercise [13] and unit. For combined RSV or KU55933 and IR treatments, promotes energy conservation by inhibiting protein cells were kept at 37°C with the indicated agent for 1 h synthesis, through mTOR inhibition while it also func- prior to IR treatment. Cells were incubated for 1 h fol- tions as a metabolic checkpoint to induce cell cycle lowing IR exposure, unless otherwise indicated. For cell arrest via p53 [14]. Recently, we showed that IR acti- cycle and clonogenic assays cells were exposed to the vates AMPK in human lung, breast and PrCa cells and treatment agents throughout the experiments (as indi- suggested that AMPK participates in a signaling pathway cated in figure legends). cip1 involving ATM-AMPK-p53-p21 leading to regulation of the cell cycle and survival [15]. siRNA AMPK a Subunit Knockdown Cells were incubated with a mixture of human siRNA RSV (3,4’,5-trihydroxystilbene) is a polyphenolic phy- sequences against the a1and a2 AMPK subunits using toalexin with widely reported anti-aging and anti-cancer HiPerFect vehicle for 72 hours as per the manufacturer’s properties [16,17]. It inhibits cancer cell proliferation protocol. and is suggested to enhance radiation responses [18,19]. RSV has also been reported to increase metabolic rate and reduce fat mass in wild-type mice but not in AMPK Clonogenic Assays a subunitknockoutmice [20]. Further, it wasshown to Clonogenic assays were performed as described earlier suppress tumor growth and metastasis in the mouse [15]. Cells (500-1000) were seeded in triplicates and Lewis lung carcinoma model [21]. RSV is known to reg- allowed to adhere overnight, then were incubated with ulate both Akt and AMPK [22,23] but the effects of this RSV followed by IR treatment (0-2 Gy) followed by compound on the two signaling pathways have not been incubation for 7-10 days. Cells were then fixed and studied in radiated cells. stained with 0.05% methylene blue and colonies (> 50 Here, we investigated the polyphenol RSV due to the cells) were counted. Results are expressed as the surviv- reported ability of this natural compound to modulate ing fraction compared to untreated control. both the radioresistance-mediating Akt and the tumour suppressor AMPK pathways [24,25]. Immunoblotting (IB) IB was performed as described earlier [26]. Approxi- Materials and methods mately 5 × 10 cells were seeded in 6-well plates. After Cell Lines and Cell Culture treatment, cells were washed, lysed and twenty micro- Human PrCa (PC3, 22RV1) and normal prostate epithe- grams of protein were subjected to IB. lial (PNT1A) cell lines were obtained from American Tissue Culture Collection (Manassas, VA, U.S.A.). Cells Immunofluorescence Microscopy Cells (2 × 10 ) were seeded onto glass coverslips and were maintained at 37°C in RPMI media supplemented were incubated without or with RSV (5 μM) 1 h prior with 10% (vol/vol) Fetal Bovine Serum (FBS) and 1% to IR treatment (8 Gy). Ninety six hours after IR expo- (vol/vol) antibiotic-antimycotic (Invitrogen, Burlington, sure, cells were washed lightly with PBS and fixed with ON, Canada). Rashid et al. Radiation Oncology 2011, 6:144 Page 3 of 12 http://www.ro-journal.com/content/6/1/144 3% paraformaldehyde/PBS/0.2% Triton X-100 for 20 RSV enhances the IR-induced inhibition of clonogenic minutes. Cells were stained with Hoechst 33258 (20 survival in PrCa cells μM) nuclear stain or anti-a-tubulin antibody conjugated RSV augmented further the IR-induced inhibition of to Alexa Fluor 488 (1:100 dilution) (followed by Hoechst survival in both PC3 and 22RV1 PrCa cells. RSV (2.5 33258 nuclear staining) and examined by fluorescence and 5 μM) decreased survival of IR-treated PC3 cells by (13.8 ± 0.09% and 20.4 ± 0.9%, Figure 1B). RSV 5 μM microscopy. Four hundred cells were evaluated for caused only a minor reduction of SF2 in 22RV1 cells nuclear aberrations (fragmentation, micro-nuclei and (non-significant) but at 10 μM the drug induced a sig- multi-nucleation) in four representative areas of each nificant 2.5-fold inhibition in SF2. RSV (5 μM) pre-treat- slide in 3 independent experiments. ment did not decrease further SF2 of PNT1A prostate Cell Cycle Analysis epithelial cells but this was achieved when RSV was (1 × 10 cells/mL) were seeded in 10-cm dishes. After increased to 10 μM (Figure 1B). treatment, cells were trypsinized, washed, fixed with To better demonstrate the potential clinical benefit 70% ethanol and stored overnight at -20°C followed by of RSV in combination with radiotherapy, we used SF2 washing and staining with a solution containing 100 μL values to estimate tumour cell survival after 40 frac- Triton X-100 and 50 μg/mL propidium iodide. Cells tions of radiotherapy (2 Gy each), which consists a were subjected to flow cytometric cell cycle analysis standard approach in clinical PrCa management [1]. using a Beckman Coulter Epics XL flow cytometer. We determined (SF2) as the tumour cell fraction sur- viving after “n” fractions of radiotherapy. Assuming Statistical Analysis iso-effectiveness of all fractions, we plotted the pro- Data are expressed as the mean ± standard error (SE). jected tumour survival over 40 consecutive fractions Statistical analysis was performed using unpaired t-test (0-80 Gy) (Figure 1C). As typical human prostates with SPSS v16.0 software (SPSS, Chicago, IL). Statistical range between 35-100 gr (5 - 10 × 10 cells) we significance was considered at p < 0.05. plotted projected tumour size reductions (tumour kill- -11 ing) on a scale of 1 - 1 × 10 . Figure 1C estimates Results that RSV (5 μM) maybe abletoreduce the dose of RSV inhibits PrCa cell survival radiotherapy needed to eliminate 22RV1 type (hor- mone-sensitive) PrCa tumours (from about 47 to 40 As a single agent, RSV effectively inhibited survival of Gy) and may be able to eliminate PC3 type (hormone- both PC3 and 22RV1 PrCa cells with significant inhibi- insensitive) PrCa tumours (with about 54 Gy) that tion achieved at even the lower doses of 2.5 and 5 μM would otherwise be incurable with doses even higher (p < 0.05) (Figure 1A). The IC values were approxi- mately 10 μMand 2.5 μM for PC3 and 22RV1 cells, than 80 Gy.Further,thisapproachalso demonstrated respectively. The PC3 response to RSV was dose-depen- the complete lack of radio-sensitization of normal dent. RSV 2.5 μM significantly decreased survival in epithelial cells (PNT1A) by 5 μM RSV. Based on these 22RV-1 cells to 40 ± 3.06% of control without any results, we pursued all subsequent studies with PrCa further decrease at higher doses. In contrast, PNT1A cells alone and with doses of 2.5 - 5 μMRSV. normal epithelial cells were less responsive to RSV showing only 5 and 10% inhibition of survival at 2.5 and RSV inhibits IR-mediated cell cycle arrest and induces cell 5 μM, respectively (Figure 1A). Only the higher dose of accumulation at G1-S and sub-G1 phases consistent with 10 μM caused significant decrease in normal epithelial apoptosis PNT1A cell survival (to 60 ± 12.26% of control) (p < We utilized the 22RV1 cell line in our cell cycle analy- 0.05). PTN1A cells were used here as a non-malignant sis experiments because it is a cloned cell line [28] control. consisting of a more isogenic population of cells than PC3 cells, and is hence appropriate for cell cycle analy- IR-induced inhibition of PrCa cell survival sis. As expected, IR induced an arrest of cells at the PC3 cells showed greater resistance to IR alone com- G2-M interphase (Control: 13.4% vs. IR: 31.7%, Figure pared to 22RV1 cells (surviving fractions after 2 Gy 2) but pre-treatment with RSV (5 μM) prevented this (SF2) of 60 ± 5.30% vs 40 ± 3.53%, respectively, Figure event (RSV+IR: 8.3% vs. 31.7% for IR, Figure 2). RSV 1B). Their IR sensitivity was similar to that of PNT1A caused accumulation of radiated cells into the G1-S phases of the cycle (IR: 58% vs. RSV + IR: 70.7%) and cells (SF2 of 62.9 ± 2.26%, Figure 1B). In clonogenic increased the population of cells in the sub-G1 region assays we opted to use the standard therapeutic IR dose by 2-fold compared to those treated with IR alone (IR: of 2 Gy and not high doses of IR, for clinical relevance 10.3% vs. RSV + IR: 21.0%), indicating induction of and because higher doses (i.e. 6-8 Gy) were highly toxic apoptosis. to PrCa cells in agreement with previous reports [27]. Rashid et al. Radiation Oncology 2011, 6:144 Page 4 of 12 http://www.ro-journal.com/content/6/1/144 Figure 1 Effects of resveratrol (RSV) and ionizing radiation (IR) on survival of prostate cancer (PrCa) cells. A. Cells were incubated with increasing doses of RSV (0-10 μM) and allowed to form colonies. Colonies (> 50 cells) were counted. Results are expressed as the surviving fraction compared to untreated control (see Methods). ‘*’ p < 0.05 compared to untreated PC3 cells, ‘#’ p < 0.05 compared to untreated 22RV1 cells, ‘+’ p< 0.05 compared to untreated PNT1A cells. B. Cells were pre-treated with RSV (0-10 μM) followed by treatment with a single fraction of IR (2 Gy) and allowed to grow and form colonies. Treatment values were normalized to untreated controls and surviving fractions are presented as percent of control. Shown is the mean of 4-5 independent experiments ± standard error (SE). ‘*’ p < 0.05 compared to 2 Gy treated PC3 cells, ‘#’ p < 0.05 compared to 2 Gy treated 22RV1 cells, ‘+’ p < 0.05 compared to 2 Gy treated PNT1A cells. C. Projected proportion of tumour survival after treatment with 0 - 80 Gy was estimated using the Surviving Fraction at 2 Gy (SF2) values for control and RSV (5 μM) treated PNT1A, PC3 and 22RV1 cells. SF2 was determined as the tumour proportion surviving after “n” 2 Gy fractions, assuming iso-effectiveness of each fraction. Values were -11 10 plotted up to a scale of 1 × 10 to reflect elimination of tumour of 50 - 100 gr expected to contain 5 - 10 × 10 cells. RSV enhances the IR induction of cell cycle inhibitors levels of this established apoptosis marker. IR increased To examine whether the effects of RSV on IR regulation caspase-3 cleavage within 1 hour of IR exposure in both of the cell cycle were associated with enhancement of PC3 and 22RV1 cells (Figure 3A). Pre-treatment with molecular pathways of cell cycle regulation, we exam- RSV (2.5 and 5 μM) enhanced the IR-induced cleaved ined the expression of p53 and the cyclin-dependent caspase-3 levels in a dose-dependent fashion. cip1 kip1 kinase inhibitors (CDKI) p21 and p27 .Figure2B To evaluate morphological markers of apoptosis, cells shows that IR induced expression of both p53 and the were stained with Hoechst 33258 and examined with CDKIs in 22RV1 cells and that RSV enhanced this microscopy. Cells with nuclear aberrations such as frag- further. Similar results were obtained for the two CDKIs mentation, micro-nuclei and multi-nucleation (polys- in p53-null PC3 cells. omy), which are markers predictive of apoptosis and/or mitotic cell death (mitotic catastrophe), were counted in RSV induces expression of apoptotic markers and nuclear each treatment condition. Figure 3B shows representa- aberrations tive images of treated PC3 cells that were either stained Using immunoblotting with a cleaved caspase-3-specific with Hoechst 33258 alone (low magnification, Figure antibody, we examined the effects of RSV and IR on the 3B) or additional immunostaining of tubulin (high Rashid et al. Radiation Oncology 2011, 6:144 Page 5 of 12 http://www.ro-journal.com/content/6/1/144 Figure 2 Effects of resveratrol (RSV) and ionizing radiation (IR) on prostate cancer cell cycle progression and expression of cycle regulators. A. 22RV1 cells were pre-treated or not with RSV (5 μM) for 1 hour followed by treatment with 8 Gy IR. Cells were allowed to cycle for 48 hours after which they were fixed, treated with a triton-X and stained with propidium iodide (PI) solution as described in Methods. Shown are representative results of 3 independent experiments. Pie charts illustrate the proportion of cells in each phase of cell cycle. B. 22RV1 and PC3 cells were pre-treated with RSV (5 μM) for 1 hour followed by 8 Gy IR treatment. Cells were lysed 1 hour after IR exposure and subjected to immunoblot analysis using antibodies against the indicated cell cycle markers. Shown are representative immunoblots of 3 independent experiments. magnification), which was utilized to better demonstrate total cell counts and reduced overall cellular viability the effects of the treatments on nuclear and cellular (Figure 3B). morphology (Figure 3C). The results of four indepen- dent experiments (as in Figure 3B) were quantitated (as Effects of RSV on molecular pathways of radio-resistance described in Methods)and areshown in Figure 3D). IR and tumour suppression alone induced significant nuclear aberrations (43 ± RSV inhibits basal and IR-induced levels of phosphorylated 2.18%, Figure 3B), while RSV alone (5 μM) increased Akt nuclear abnormalities by 6% (Figure 3B). However, RSV We examined the effects of RSV on the well-described significantly enhanced the IR-induced nuclear aberra- radio-resistance pathway of Akt-mTOR. We observed tions to 70 ± 3.11% (an increase of 27%, Figure 3D) of that IR alone induced a significant time-dependent Rashid et al. Radiation Oncology 2011, 6:144 Page 6 of 12 http://www.ro-journal.com/content/6/1/144 Figure 3 Resveratrol (RSV) enhances ionizing radiation (IR)-induced cell death. Induction of cleaved caspase-3 and nuclear aberrations. A. PC3 cells were pre-treated or not with RSV (5 μM) for 1 hour after which they were treated with IR (8 Gy). Cells were lysed 1 hour after IR exposure and subjected to immunoblot analysis using the indicated antibodies. Shown are representative immunoblots of 3-4 independent experiments. B. and C. Cells were treated or not with RSV (5 μM) prior to IR treatment (8 Gy) followed by staining with nuclear stain Hoeschst 33258 (low magnification, in B) or Hoeschst 33258 and immunostaining of tubulin (high magnification, in C) used to illustrate better cellular morphology. Morphology was assessed using fluorescence microscopy. Representative images from 4 independent experiments are shown. D. Average ± SE values (% Nuclear Aberrations: as indicated by the proportion of cells with features of nuclear fragmentation, micro-nuclei and multi-nucleation) were extracted through quantitation of 4 independent experiments (in B), as described in Methods. increase in the levels of phosphorylated Akt (P-Akt) exposure to allow demonstration of the effects of RSV (both S473 and T308 sites) in PC3 cells, with no effects on basal Akt phosphorylation which are significantly on total levels of the protein (Figure 4A). Highest levels lower than in radiated cells (as shown in Figure 4A and of P-Akt were reached 30 minutes to 1 hour after IR 4C). Pre-treatment with RSV led to significant inhibition exposure. Akt phosphorylation was associated with of IR-induced P-Akt with no effects on total levels of increased activity of this enzyme indicated by the IR- the protein (Figure 4C). Although IR exposure led to an induced phosphorylation of mTOR, a key effector of increase in P-Akt, RSV pre-treatment (2.5 and 5 μM) Akt (Figure 4A). Similar results were obtained in 22RV1 significantly reduced and/or prevented IR-induced phos- phorylation of Akt. The effects of RSV on IR-induced PrCa cells (results not shown). Treatment of PC3 and 22RV1 cells with RSV inhibited Akt phosphorylation on S473 were quantitated in four basal levels of P-Akt (Figure 4B). Of note, the images independent experiments (Figure 4D). Similar results shown were obtained after significantly longer film were obtained for Akt T308phosphorylation.A Rashid et al. Radiation Oncology 2011, 6:144 Page 7 of 12 http://www.ro-journal.com/content/6/1/144 Figure 4 Resveratrol (RSV) inhibits basal and ionizing radiation (IR)-induced Akt phosphorylation. A. Effects of IR alone: PC3 cells were treated with 0 or 8 Gy IR and lysed according to the indicated time points following IR exposure. Then cells were subjected to immunoblot analysis. B. RSV inhibits basal Akt phosphorylation: PC3 and 22RV-1 cells were treated with RSV (0, 2.5 or 5 μM) for 1 hour and subjected to lysis and immunoblot analysis using the indicated antibodies. C. RSV inhibits IR-induced Akt phosphorylation. PC3 and 22RV-1 cells were pre-treated with the indicated concentrations of RSV 1 hour prior to IR treatment. Cells were lysed 1 hour after IR exposure and subjected to immunoblot analysis. Shown are representative immunoblots of 4 independent experiments. D. Densitometric quantitation of Akt (S473) phosphorylation and total Akt was performed. P-Akt was normalized to T-Akt levels and presented as fold change compared to untreated control cells (mean ± SE) from 3-4 independent immunoblotting experiments as in C). * p < 0.05 compared to cells treated with IR alone. representative immunoblot from PC3 cells is shown (ACC). RSV induced a robust activation of AMPK in (Figure 4C). both PC3 and 22RV1 cells at both 2.5 and 5 μM (Figure RSV and IR effects on AMPK 5B). Furthermore, RSV pre-treatment enhanced signifi- We reported earlier that IR activates AMPK in lung, cantly the IR-induced AMPK phosphorylation in both breast and prostate cancer cells [15]. Similarly, we 22RV1 and PC3 cells (Figure 5C). Total AMPK levels observed here that IR induced a robust phosphorylation remained unchanged after both IR and RSV treatments. of AMPK on Thr172 of the catalytic a subunitthatwas The results of 3-4 independent experiments were quan- detectable within 15 min. Highest levels of phosphory- titated and are shown in Figure 5D. lated AMPK (P-AMPK) were detected 1 hour after IR exposure and returned to almost basal levels 4 hours The pathway of radiation activation of Akt and AMPK after radiation (Figure 5A). This was associated with Akt regulation activation of AMPK indicated by the detected phosphor- Recent reports suggest that Akt activation by IR may be ylation of the AMPK substrate Acetyl CoA Carboxylase regulated by ATM [29]. We examined this notion in Rashid et al. Radiation Oncology 2011, 6:144 Page 8 of 12 http://www.ro-journal.com/content/6/1/144 Figure 5 Resveratrol (RSV) enhances basal and ionizing radiation (IR)-induced AMPK phosphorylation. A.EffectsofIRalone:PC3 cells were treated with 0 or 8 Gy IR and lysed at the indicated time points after IR exposure. The adjacent panel shows PC3 cells treated with 8 Gy IR, which were lysed 1 hour after IR treatment. Samples were subjected to immunoblot analysis using the indicated antibodies. B. RSV enhances basal AMPK phosphorylation: PC3 and 22RV-1 cells were treated with the indicated concentrations of RSV for 1 hour followed by lysis and immunoblotting analysis. C. RSV enhances IR-induced AMPK phosphorylation. PC3 and 22RV-1 cells were pre-treated with the indicated concentrations of RSV 1 hour prior to 8 Gy IR treatment. Cells were lysed 1 hour after IR exposure and subjected to immunoblotting using indicated antibodies. Shown are representative immunoblots of at least 3-4 independent experiments. D. Densitometry-based quantitation. Density values of P-AMPK were normalized to T-AMPK levels and presented as fold change compared to untreated control cells (mean ± SE) from 2-3 independent experiments in C. is shown. * p < 0.05 compared to cells treated with IR alone. cip1 PrCa cells using the ATM-specific inhibitor KU55933 subunits of AMPK blocked p53 and p21 induction by [30]. Pre-incubation with KU55933 prevented IR- IR (Figure 6C). Interestingly, RSV pre-treatment induced ATM phosphorylation but also inhibited IR enhanced IR-induced phosphorylation of ATM and of phosphorylation of Akt at S473 and activation of its its substrate histone H2Ax (gH2Ax), as well as phos- cip1 kinase activity as indicated by reduced phosphorylation phorylation of AMPK and induction of p21 . of mTOR (Figure 6A). AMPK Regulation Discussion We attempted to verify, in PrCa cells, our earlier obser- IR and RSV effects on PrCa cell clonogenic survival vations in lung cancer cells [15] of AMPK participation RSV inhibits survival and proliferation of cancer cells as cip1 in an ATM-AMPK-p53/p21 pathway activated by IR. a single agent and induces radiosensitization in human We observed a robust phosphorylation of ATM and cervical cancer cells [19]. Similarly, we observed that at cip1 AMPK as well as induction of p21 in PC3 PrCa cells low doses (2.5-10 μM) RSV inhibited clonogenic survival in response to IR. IR induced ATM and AMPK phos- of PrCa cells. Several studies have reported IC values cip1 phorylation and p21 induction were all inhibited by for cell growth inhibition by RSV in the range of 5 to treatment with KU55933 (Figure 6B). To verify that 10 μM [31,32]. Free RSV has a low bioavailability in- cip1 AMPK truly acts as a mediator of p53/p21 induction vivo as it is rapidly metabolized to glucoronide and sul- fate conjugates [32]. A human study reported plasma in response to IR in PrCa cells, we used wild-type p53 expressing 22RV1 cells to perform AMPK knockdown concentrations of free RSV of 21 nM after oral dose of experiments. siRNA knockdown of both a1and a2 25 mg RSV [32]. However, all combined RSV Rashid et al. Radiation Oncology 2011, 6:144 Page 9 of 12 http://www.ro-journal.com/content/6/1/144 Figure 6 Mechanism of Akt and AMPK regulation by resveratrol (RSV) and ionizing radiation (IR). A.and B. Inhibition of ATM blocks IR- induced activation of the Akt and AMPK signaling pathways: PC3 cells were treated or not with the ATM inhibitor KU55933 (10 μM) for 1 hour followed by 0 or 8 Gy IR treatment. Cells were lysed 1 hour after IR exposure and subjected to immunoblot analysis using the indicated cip1 antibodies. C. AMPK mediates the IR induction of p53 and p21 : 22RV-1 cells were incubated with siRNA complexes against the a1 and a2 subunits of AMPK for 72 hours as described in Methods. Cells were then treated with 0 or 8 Gy IR and lysed 1 hour after IR exposure and cip1 subjected to immunoblot analysis. D. RSV enhances signalling along the ATM-AMPK-p21 pathway: Cells were pre-treated with RSV (5 μM) for 1 hour followed by IR of 8 Gy. Cells were lysed 1 hour after IR exposure and subjected to immunoblot analysis using the indicated antibodies. Shown are representative immunoblots of at least 3 independent experiments. Rashid et al. Radiation Oncology 2011, 6:144 Page 10 of 12 http://www.ro-journal.com/content/6/1/144 metabolites were reported to reach about 2 μM [16]. For activation of caspase-, p53-, or Fas ligand-dependent this, we pursued our studies with low RSV concentra- pathways [39,40]. In our studies, IR-mediated nuclear tions (2.5 - 10 μM). damage showed early signs of mitotic catastrophe and As a singleagent,RSV inhibitedmorepotentlysurvi- RSV potentiated such nuclear aberrations (Figure 3C-D). val of 22RV1 compared to PC3 cells but the response of Mitotic cell death predominates in cells with defects in 22RV1 cells did not display dose-dependence. Overall, cycle checkpoints that prevent cycle arrest and DNA PC3 cells displayed greater resistance to both IR and repair when cells are exposed to genotoxic stress such RSV alone, consistent with other studies [33]. PC3 cells as IR [41]. The features of mitotic catastrophe seen in our studies in PC3 cells (p53-null)after RSVandIR are deficient in key tumour suppressors including PTEN and p53 [34]. Lack of PTEN allows aberrant Akt activa- treatments are consistent with those observed earlier in tion [35], which in combination with the lack of p53, other p53-null cancer models [41]. may confer such cells a survival advantage and IR- and RSV-resistance. PNT1A cells were less responsive to Modulation of the Akt and AMPK pathways RSV, indicating a potential for this drug to achieve a IR and RSV exert opposite effects on Akt. IR mediated positive therapeutic ratio in-vivo. RSV inhibited signifi- Akt phosphorylation on T308 and S473 and enzymatic cantly PNT1A cells at 10 μM and for that we focused activation indicated by mTOR phosphorylation (Figure our work on 2.5 and 5 μM RSV. 4A). This suggests activation of both PDK1 and PDK2 We demonstrated that RSV can sensitize PrCa cells to activity by IR. However, we observed that RSV is an IR (Figure 1). Concentrations of RSV (2.5 and 5 μM), effective inhibitor of this radioresistance-associated path- similar to those that can be achieved in human serum, way (Figure 4). Inhibition of IR-induced T308 and S473 enhanced the cytotoxicity of a conventional RT fraction phosphorylation by RSV suggests an ability to regulate (2 Gy) in PrCa cells without additional toxicity to nor- both PDK1 and PDK2. The enhancement of IR-induced mal epithelial cells. The potential clinical utility of our cell death by RSV may indeed be mediated through finding is illustrated in Figure 1C, which suggests that modulation of Akt and its downstream targets as sug- low RSV doses have the potential to reduce the dose of gested earlier for uterine cancer [25] and PrCa cells [42]. radiotherapy required to treat human hormone- and Using the ATM-specific inhibitor KU55933, [30] we radiation-sensitivePrCaand maybeabletomakecur- observed that ATM mediates IR-induced Akt S473 phos- able hormone- and radiation-insensitive tumours that phorylation and activation, suggesting that ATM may may otherwise be incurable with even modern dose indeed be the IR responsive PDK2, as suggested earlier [6]. escalated radiotherapy. This notion needs to be verified However, despite inhibition of Akt, RSV enhanced ATM in in-vivo models of human PrCa. phosphorylation and activation indicated by histone H2Ax phosphorylation (gH2Ax) (Figure 6D). This points to a Regulation of cell cycle and apoptosis complex regulatory action of RSV to enhance ATM activa- RSV is reported to arrest PrCa and other cells at G0-G1 tion but inhibit its PDK2 action on Akt downstream. This and/or S-phases of the cycle leading to senescence concept needs to be explored further in future studies. cip1 [36,37] and cause p21 -mediated G1-phase arrest and We aimed to evaluate, in PrCa cells, the DNA damage cip1 apoptosis in A431 cells [38]. Consistently, we observed a responsive ATM-AMPK-p21 pathwayweproposed significant arrest of radiated PrCa cells at G1-S (Figure earlier in lung cancer cells [15] and found that IR 4). RSV’s effects on survival were additive to those of IR indeed mediates AMPK activation downstream of ATM but the two agents mediate different regulation of cell (Figure 6B). We observed that AMPK mediates p53 and cip1 cycle. Whereas IR induces G1-S and more so G2-M p21 induction in response to IR in PrCa cells (Figure cycle arrest, RSV prevented the latter (Figure 2), likely 6C). RSV and IR stimulated phosphorylation/activation due to induction of an earlier checkpoint (G1-S). The of AMPK, in both PC3 and 22RV1 PrCa cells, which cip1 kip1 potentiation of IR-induced expression of p53 and CDK was associated with induction of p21 and p27 cip1 kip1 inhibitors p21 and p27 by RSV (Figure 2B), (Figure 6D). Although, we did not examine whether the known to regulate the G1 and S phase checkpoints, may latter is regulated by AMPK activity, this notion is well provideamolecular pathwayofactionfor RSV’sinduc- described by other studies [43]. The enhancement of IR- tion of the G1-S phase arrest seen in our studies. stimulated ATM activity by RSV provides a framework cip1 RSV also caused re-distribution of cells into the sub- for upregulation of the ATM-AMPK-p21 pathway in G1 or apoptotic range that was associated with cleavage RSV-treated cancer cells and a rationale for the of caspase-3 and induction of extensive nuclear aberra- observed inhibition of cell cycle and survival. This tions (Figure 3). Apoptosis appears to be the primary notion is supported by observations that the AMPK mode of cell death induced by RSV. Studies demon- inhibitor compound C inhibits IR-induced cytotoxicity strated such cell death effects in PrCa cells through in PrCa (results not shown). Rashid et al. Radiation Oncology 2011, 6:144 Page 11 of 12 http://www.ro-journal.com/content/6/1/144 Figure 7 Model of the mechanism of action of resveratrol (RSV) and ionizing radiation (IR) to regulate cell cycle and survival in PrCa cells. A. IR induces DNA strand breaks which are detected by ATM, leading to AMPK activation and induction of the cell cycle inhibitors p53, cip1 kip1 p21 and p27 . IR also activates Akt to increase protein synthesis through mTOR and stimulate cell cycle progression, proliferation and survival. B. The work presented here suggests that RSV i) enhances the effects of IR on AMPK activation, leading to an early cell cycle arrest, and ii) inhibits Akt to reduce gene expression and proliferation and enhance radiation cytotoxicity. L8S 4L8, Canada. Department of Pathology and Molecular Medicine, Conclusions McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S 4L8, Figure 7 illustrates a model of the effects of RSV on the 4 Canada. Department of Community Health Science, Brock University, 500 IR responses of PrCa cells. Low (μM) concentrations of Glenridge Avenue, St. Catharines, Ontario, L2S 3A1, Canada. Department of Radiation Oncology, Princess Margaret Hospital, University of Toronto, 610 RSV, that can be achieved in human serum, inhibit University Avenue, Toronto, Ontario, M5G 2M9, Canada. PrCa cell survival and enhance IR-induced cytotoxicity. RSV enhances IR-induced DNA-damage response sig- Authors’ contributions AR carried out the cellular/molecular experiments (clonogenic assays), cell nals (ATM), apoptosis markers (cleaved caspase-3) and cycle studies and helped draft the manuscript. CL aided in the cell cycle cip1 kip1 cell cycle inhibitors (p53, p21 and p27 ). Further, it studies. TS helped carry out the immunofluorescence experiments. TT arrests cycling at the G1-S phase and enhances IR- conceived the study, directed the study design, supervised all experimental work and prepared the manuscript. ET, GS, RB participated in the design of induced nuclear aberrations. RSV exerts a desirable dual the study and advised TT on experimental approaches. All authors have read action to inhibit the Akt radioresistance pathway while and approved the final manuscript. enhancing the tumour suppressor AMPK, leading to cip1 kip1 Competing interests potentiation of an ATM-AMPK-p53-p21 /p27 sig- The authors declare that they have no competing interests. naling axis. We conclude that RSV is a promising agent that deserves further investigation as an adjunct to IR in Received: 4 August 2011 Accepted: 26 October 2011 Published: 26 October 2011 in-vivo models of PrCa to help elucidate its potential for clinical use in combination with RT. References 1. 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Verbeuren TJ, Cohen RA: Polyphenols stimulate AMP-activated protein doi:10.1186/1748-717X-6-144 kinase, lower lipids, and inhibit accelerated atherosclerosis in diabetic Cite this article as: Rashid et al.: Resveratrol enhances prostate cancer LDL receptor-deficient mice. Diabetes 2006, 55(8):2180-2191. cell response to ionizing radiation. Modulation of the AMPK, Akt and mTOR pathways. Radiation Oncology 2011 6:144.

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Radiation OncologySpringer Journals

Published: Oct 26, 2011

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