Radiosensitivity Of Prostate Cancer Cells Research Articles

Abstract 3339: Silibinin improves radiotherapeutic efficacy in prostate cancer by reducing IR-induced toxicity and EMT

Abstract Radiotherapy of prostate carcinoma is well established and frequently utilized in a significant proportion of cancer patients. However, two major concerns with radiotherapy is the effect on normal tissues and development of more invasive-radioresistant population. We have previously shown that the use of a plant flavonoid, silibinin can radiosensitize prostate cancer cells preferentially in vitro, by inhibiting DNA damage repair. We extended this study further, to validate the effects of silibinin in improving the radiotherapeutic efficacy in vivo. Our results showed that, in vivo, silibinin strongly radiosensitized DU145 tumor xenograft inhibition (84%, P<0.01) with higher apoptotic response (10-fold, P<0.01) and reduced repair of DNA damage as evidenced by reduced Chk2 activation and enhanced pH2A.X foci in tumor tissues. Interestingly, we also observed that silibinin could rescue the mice from IR-caused hematopoietic injury and normal tissue toxicity. Studies have shown that ionizing radiation (IR) increases the vascularity and invasiveness of surviving radioresistant cancer cells. This invasive phenotype of radioresistant cells is an upshot of radiation induced pro-survival and mitogenic signaling in cancer as well as endothelial cells. In the current study, we demonstrate that silibinin can also radiosensitize endothelial cells by inhibiting expression of pro-angiogenic factors. Combining silibinin with IR not only down-regulated endothelial cell proliferation, clonogenicity and tube formation ability rather it significantly reduced migratory and invasive properties of PCa cells which were otherwise marginally affected by IR treatment alone. We have found that most of the pro-angiogenic, migratory and EMT promoting proteins were up regulated in response to IR in PCa cells. All of these invasive and EMT promoting actions of IR were markedly decreased by silibinin. Further, we found that potentiated effect was an end result of attenuation of IR activated mitogenic and pro-survival signaling, including Akt, Erk1/2 and STAT-3 by silibinin. Therefore, the study not only underlines the preferential radiosensitizing ability of silibinin in PCa, but also shows its efficacy in modulating IR-induced toxicity in normal cells and EMT in PCa cells. Citation Format: Dhanya K. Nambiar, Paulraj Rajamani, Anil Jain, Gagan Deep, Rajesh Agarwal, Rana P. Singh. Silibinin improves radiotherapeutic efficacy in prostate cancer by reducing IR-induced toxicity and EMT. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3339. doi:10.1158/1538-7445.AM2015-3339

The TMPRSS2-ERG Gene Fusion Blocks XRCC4-Mediated Nonhomologous End-Joining Repair and Radiosensitizes Prostate Cancer Cells to PARP Inhibition.

Exposure to genotoxic agents, such as ionizing radiation (IR), produces DNA damage, leading to DNA double-strand breaks (DSB); IR toxicity is augmented when the DNA repair is impaired. We reported that radiosensitization by a PARP inhibitor (PARPi) was highly prominent in prostate cancer cells expressing the TMPRSS2-ERG gene fusion protein. Here, we show that TMPRSS2-ERG blocks nonhomologous end-joining (NHEJ) DNA repair by inhibiting DNA-PKcs. VCaP cells, which harbor TMPRSS2-ERG and PC3 cells that stably express it, displayed γH2AX and 53BP1 foci constitutively, indicating persistent DNA damage that was absent if TMPRSS2-ERG was depleted by siRNA in VCaP cells. The extent of DNA damage was enhanced and associated with TMPRSS2-ERG's ability to inhibit DNA-PKcs function, as indicated by its own phosphorylation (Thr2609, Ser2056) and that of its substrate, Ser1778-53BP1. DNA-PKcs deficiency caused by TMPRSS2-ERG destabilized critical NHEJ components on chromatin. Thus, XRCC4 was not recruited to chromatin, with retention of other NHEJ core factors being reduced. DNA-PKcs autophosphorylation was restored to the level of parental cells when TMPRSS2-ERG was depleted by siRNA. Following IR, TMPRSS2-ERG-expressing PC3 cells had elevated Rad51 foci and homologous recombination (HR) activity, indicating that HR compensated for defective NHEJ in these cells, hence addressing why TMPRSS2-ERG alone did not lead to radiosensitization. However, the presence of TMPRSS2-ERG, by inhibiting NHEJ DNA repair, enhanced PARPi-mediated radiosensitization. IR in combination with PARPi resulted in enhanced DNA damage in TMPRSS2-ERG-expressing cells. Therefore, by inhibiting NHEJ, TMPRSS2-ERG provides a synthetic lethal interaction with PARPi in prostate cancer patients expressing TMPRSS2-ERG.

Abstract 4916: Efficiacy of NU7441-encapsulated PLGA nanoparticles in radiation sensitization of prostate cancer cells

Abstract Introduction: Prostate cancer remains the leading cause of cancer-related mortality in men with an estimated 241,740 new cases and 28,170 deaths expected by end of 2012. Conventional cancer treatments such as radiation therapy can be ineffective due to radiation resistance of prostate cancer cells. This resistance arises due to their increased DNA double strand break (DSB) repair ability, especially through Non-Homologous End Joining (NHEJ). In this study, we have developed biodegradable and biocompatible poly lactic-co-glycolic acid (PLGA)-based nanoparticles containing the potent radio-sensitizer NU7441 (8-dibenzothiophen-4-yl-2-morpholin-4-yl-chromen-4-one) for radiation sensitization of prostate cancer cells by inhibiting DNA-dependent protein kinase, which regulates NHEJ. Methods: PLGA nanoparticles encapsulating NU7441 and iron oxide as an imaging and targeting agent were prepared by a standard double emulsion technique and characterized for size and surface charge. R11 peptide was surface conjugated onto the nanoparticles for prostate cancer-specific targeting. Stability studies in de-ionized (DI) water and serum were conducted over 5 days followed by drug release studies in DI water at 37oC. Further, in vitro studies were done to study cytocompatibility with prostate epithelial cells (PZ-HPV7) and cellular uptake by prostate cancer cells (PC3). DSB repair kinetics of prostate cancer cells following nanoparticle uptake was studied using a DSB repair assay. Results: Our nanoparticles had an average size of 274.13+ 79.96 nm and showed good stability in water and serum for 5 days. The nanoparticles also showed bi-phasic NU7441 release within 21 days at 37oC and >80% PZ-HPV7 cell viability up to 2000 µg/ml nanoparticle concentration. Further, the particles were selectively uptake by PC3 cells in a dose and magnetic field-dependent manner and showed effective radiation sensitization of these cells in vitro. Conclusions: Our results thus demonstrate that R11-conjugated PLGA-iron oxide nanoparticles containing NU7441 are biocompatible and can be potentially used to radio-sensitize prostate cancer cells in vivo. Citation Format: Jyothi U. Menon, Vasu Tumati, Jer-Tsong Hsieh, Kytai T. Nguyen, Debabrata Saha. Efficiacy of NU7441-encapsulated PLGA nanoparticles in radiation sensitization of prostate cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4916. doi:10.1158/1538-7445.AM2014-4916

Abstract 845: Silibinin radiosensitizes prostate cancer cells by enhancing radiation-induced cell death and inhibiting nuclear EGFR-mediated DNA repair

Abstract Nearly 60% of all cancer patients undergo radiotherapy at some juncture of their treatment schedule. However, the problem of therapeutic resistance and side effects to normal dividing cells wanes the positive facets of radiotherapy. Radiosensitizing agents have met with limited success in clinical settings. Hence, development of novel radiosensitizers with a selective response is greatly desired. Herein, we analyzed the radiosensitizing effects of silibinin, a natural plant flavonoid on prostate cancer (PCa) cells in vitro. Clonogenic assay and soft agar colony formation assay revealed that silibinin (25-100µM) could significantly enhance radiation (2.5-10 Gy) induced inhibition in colony formation selectively in PCa cells. G2/M phase is the most sensitive phase for radiation-induced damage. Silibinin enhanced and prolonged radiation-induced G2/M arrest in both DU145 and PC-3 cells. Even though low doses of silibinin (25µM) alone did not induce significant cell death, it could substantially enhance radiation-induced apoptosis. This could also be due to increased and prolonged ROS production observed in combination treatment. Foremost among the pro-survival pathways activated by radiation which contributes to therapeutic resistance is the EGFR pathway. Radiation-induced activation of EGFR subdues the response, both by induction of pro-survival pathway and by activating DNA repair after its nuclear translocation. We observed that silibinin could reduce radiation-induced EGFR signaling and consequently reverse the resistance mediating mechanisms within the cell. Silibinin down regulated levels of anti-apoptotic BCl-2 and survivin, enhanced by radiation. Inhibiting DNA repair would essentially enhance therapeutic index of radiation. Hence, we examined whether silibinin can down-regulate the repair pathways activated in response to radiation. Silibinin reversed the radiation-induced activation of CHK1 and CHK2 at both mRNA and protein levels. We further analyzed its effect on radiation-induced nuclear translocation of EGFR. Our study revealed that silibinin inhibited the nuclear translocation of EGFR and subsequently also affected the nuclear localization of DNA-PK, which is known to be one of the most important mediators of DSB repair in human cells. This was further evident by the increase in the number of pH2AX (ser139) foci suggesting lesser DNA repair in these cells. Thus, we found that silibinin not only enhanced the cell death by reversing the pro-survival signaling activated by radiation; but also suppressed the repair of DSBs by inhibiting nuclear-EGFR mediated DNA-PK activation. Overall, since silibinin is already in phase II clinical trial for prostate cancer patients, the present findings indicate that a combinatorial approach involving silibinin with radiation could prove to be more effective against radioresistant PCa. Citation Format: Dhanya K. Nambiar, Paulraj Rajamani, Rana P. Singh. Silibinin radiosensitizes prostate cancer cells by enhancing radiation-induced cell death and inhibiting nuclear EGFR-mediated DNA repair. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 845. doi:10.1158/1538-7445.AM2014-845

Inhibition of soluble adenylyl cyclase increases the radiosensitivity of prostate cancer cells

Pharmacological modulation of tumor radiosensitivity is a promising strategy for enhancing the outcome of radiotherapy. cAMP signaling plays an essential role in modulating the proliferation and apoptosis of different cell types, including cancer cells. Until now, the regulation of this pathway was restricted to the transmembrane class of adenylyl cyclases. In the present study, the role of an alternative source of cAMP, the intracellular localized soluble adenylyl cyclase (sAC), in the radiosensitivity of prostate cancer cells was investigated. Pharmacological inhibition of sAC activity led to marked suppression of proliferation, lactate dehydrogenase release, and induction of apoptosis. The combination of ionizing radiation with partial suppression of sAC activity (~50%) immediately after irradiation synergistically inhibited proliferation and induced apoptosis. Overexpression of sAC in normal prostate epithelial PNT2 cells increased the cAMP content and accelerated cell proliferation under control conditions. The effects of radiation were significantly reduced in transformed PNT2 cells compared with control cells. Analysis of the underlying cellular mechanisms of sAC-induced radioresistance revealed the sAC-dependent activation of B-Raf/ERK1/2 signaling. In agreement with this finding, inhibition of ERK1/2 in prostate cancer cells enhanced the cytotoxic effect of irradiation. In conclusion, the present study suggests that sAC-dependent signaling plays an important role in the radioresistance of prostate cancer cells. This article is part of a Special Issue entitled: The role of soluble adenylyl cyclase in health and disease.

Suppression of Radiation-Induced c-Met Activation Leads to Radiosensitization of Prostate Cancer Cells

Suppression of Radiation-Induced c-Met Activation Leads to Radiosensitization of Prostate Cancer Cells

Polymeric nanoparticles for targeted radiosensitization of prostate cancer cells.

One of the many issues of using radiosensitizers in a clinical setting is timing daily radiation treatments to coincide with peak drug concentration in target tissue. To overcome this deficit, we have synthesized a novel nanoparticle (NP) system consisting of poly (lactic-co-glycolic acid) (PLGA) NPs conjugated with prostate cancer cell penetrating peptide-R11 and encapsulated with a potent radio-sensitizer 8-dibenzothiophen-4-yl-2-morpholin-4-yl-chromen-4-one (NU7441) to allow prostate cancer-specific targeting and sustained delivery over 3 weeks. Preliminary characterization studies showed that the R11-conjugated NPs (R11-NU7441 NPs) had an average size of about 274 ± 80 nm and were stable for up to 5 days in deionized water and serum. The NPs were cytocompatible with immortalized prostate cells (PZ-HPV-7). Further, the particles showed a bi-phasic release of encapsulated NU7441 and were taken up by PC3 prostate cancer cells in a dose- and magnetic field-dependent manner while not being taken up in nonprostate cancer cell lines. In addition, R11-NU7441 NPs were effective radiation sensitizers of prostate cancer cell lines in vitro. These results thus demonstrate the potential of R11-conjugated PLGA NPs as novel platforms for targeted radiosensitization of prostate cancer cells.

921: Importance of Mcl-1 and the Mcl-1-modulating enzyme USP9x for radiosensitivity in prostate cancer cells

921: Importance of Mcl-1 and the Mcl-1-modulating enzyme USP9x for radiosensitivity in prostate cancer cells

Berberine inhibits the expression of hypoxia induction factor-1alpha and increases the radiosensitivity of prostate cancer.

BackgroundThe radiation resistance of prostate cancer remains the primary obstacle to improve patient survival. This study aimed to investigate the effects of berberine, a commonly used natural product, on the radiosensitivity of prostate cancer.MethodsProstate cancer cell line LNCaP and DU-145 were subjected to hypoxia and/or ionizing radiation (IR), in the presence or absence of berberine treatment. Cell growth and colony formation, and apoptosis were evaluated. Moreover, LNCaP cells were xenografted into nude mice and subjected to IR and/or berberine treatment. The expression of HIF-1α and VEGF in prostate cancer cells and xenografts was detected by Western blot analysis.ResultsBerberine increased radiosensitivity of prostate cancer cells and xenografts in a dose dependent manner, and this was correlated with the inhibition of HIF-1α and VEGF expression.ConclusionsBerberine may inhibit the expression of HIF-1α and VEGF and thus confer radiosensitivity on prostatic cancer cells. Berberine has potential application as an adjuvant in radiotherapy of prostatic cancer.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1519827543125021.

Metformin sensitizes prostate cancer cells to radiation through EGFR/p-DNA-PKCS in vitro and in vivo.

Although neo-adjuvant radiotherapy is generally successful in treatment of advanced prostate cancer, radioresistance is still a major therapeutic problem in many patients. In the current study, we investigated the effects of metformin (1,1-dimethylbiguanide hydrochloride), a widely used antidiabetic drug, on tumor cell radiosensitivity in prostate cancer. Through clonogenic survival assays, we found that metformin treatment enhanced radiosensitivity of prostate cancer cells with a dose enhancement factor. Moreover, irradiation of subcutaneous C4-2 tumors in mice treated with metformin resulted in an increase in radiation-induced tumor growth delay (17.3 days to 29.5 days, P < 0.01), which indicates that the tumor radiosensitivity increased by metformin in vivo. We also measured the sublethal damage repair and analyzed double-strand breaks (DSBs) in X-irradiated cells. γ-H2AX, as an indicator of DSBs, had significantly more foci per cell in the group treated with metformin and radiation compared to groups treated with metformin or irradiation, respectively. Moreover, mice with subcutaneous tumor implants lived longer after a combined treatment of metformin and radiation. In addition, the reduced phosphorylation of DNA-PKcs caused by EGFR/PI3K/Akt down-regulation is essential for metformin to induce radiosensitivity in prostate cancer cells. Our results indicate that metformin enhances prostate cancer cell radiosensitivity in vitro and in vivo. Exposure to metformin before radiation therapy could be a beneficial option for the treatment of prostate cancer.

Inhibition of Both EGFR and IGF1R Sensitized Prostate Cancer Cells to Radiation by Synergistic Suppression of DNA Homologous Recombination Repair

Reduced sensitivity of prostate cancer (PC) cells to radiation therapy poses a significant challenge in the clinic. Activation of epidermal growth factor receptor (EGFR), type 1 insulin-like growth factor receptor (IGF1R), and crosstalk between these two signaling pathways have been implicated in the development of radiation resistance in PC. This study assessed the effects of targeting both receptors on the regulation of radio-sensitivity in PC cells. Specific inhibitors of EGFR and IGF1R, Erlotinib and AG1024, as well as siRNA targeting EGFR and IGF1R, were used to radio-sensitize PC cells. Our results showed that co-inhibiting both receptors significantly dampened cellular growth and DNA damage repair, and increased radio-sensitivity in PC cells. These effects were carried out through synergistic inhibition of homologous recombination-directed DNA repair (HRR), but not via inhibition of non-homologous end joining (NHEJ). Furthermore, the compromised HRR capacity was caused by reduced phosphorylation of insulin receptor substrate 1 (IRS1) and its subsequent interaction with Rad51. The synergistic effect of the EGFR and IGF1R inhibitors was also confirmed in nude mouse xenograft assay. This is the first study testing co-inhibiting EGFR and IGF1R signaling in the context of radio-sensitivity in PC and it may provide a promising adjuvant therapeutic approach to improve the outcome of PC patients to radiation treatment.

NVP-BEZ235, Dual Phosphatidylinositol 3-Kinase/Mammalian Target of Rapamycin Inhibitor, Prominently Enhances Radiosensitivity of Prostate Cancer Cell Line PC-3

Aberrant activation of phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway may account for development of radioadaptation and is not rare in prostate cancer. Neither PI3K nor mTOR blockade could completely inhibit the pathway owing to paradoxical feedback, so we anticipate dual PI3K/mTOR blockade by NVP-BEZ235 to radiosensitize prostate cancer cells. We investigated into the radiosensitizing effect of NVP-BEZ235 on PC-3 cells, which are devoid of androgen receptors. Clonogenic survival and MTT assays were performed, and to pursue underlying cellular changes flow cytometric analysis of cell cycle and apoptosis as well as western blot were carried out. Exposure to NVP-BEZ235 and irradiation caused a greater degree of survival inhibition than ionizing radiation (IR) or BEZ235 alone. Dual PI3K/mTOR blockade along with IR induced a G2/M arrest and enhanced proapoptotic effect. NVP-BEZ235 radiosensitized PC-3 cells through counteracting constitutive as well as IR-triggered activation of Akt/mTOR signaling. Our study demonstrated that the dual PI3K/mTOR inhibitor NVP-BEZ235 prominently improved the radiosensitivity of PC-3 cells. It sensitized tumor cells to irradiation via interruption of cell cycle progression and augmentation of cell apoptosis, which was due to its constraint on constitutive and IR-elicited PI3K/Akt/mTOR signaling activation.

Novel Hsp90 inhibitor NVP-AUY922 radiosensitizes prostate cancer cells

Outcomes for poor-risk localized prostate cancers treated with radiation are still insufficient. Targeting the “non-oncogene” addiction or stress response machinery is an appealing strategy for cancer therapeutics. Heat-shock-protein-90 (Hsp90), an integral member of this machinery, is a molecular chaperone required for energy-driven stabilization and selective degradation of misfolded “client” proteins, that is commonly overexpressed in tumor cells. Hsp90 client proteins include critical components of pathways implicated in prostate cancer cell survival and radioresistance, such as androgen receptor signaling and the PI3K-Akt-mTOR pathway. We examined the effects of a novel non-geldanamycin Hsp90 inhibitor, AUY922, combined with radiation (RT) on two prostate cancer cell lines, Myc-CaP and PC3, using in vitro assays for clonogenic survival, apoptosis, cell cycle distribution, γ-H2AX foci kinetics and client protein expression in pathways important for prostate cancer survival and radioresistance. We then evaluated tumor growth delay and effects of the combined treatment (RT-AUY922) on the PI3K-Akt-mTOR and AR pathways in a hind-flank tumor graft model. We observed that AUY922 caused supra-additive radiosensitization in both cell lines at low nanomolar doses with enhancement ratios between 1.4–1.7 (p < 0.01). RT-AUY922 increased apoptotic cell death compared with either therapy alone, induced G2-M arrest and produced marked changes in client protein expression. These results were confirmed in vivo, where RT-AUY922 combination therapy produced supra-additive tumor growth delay compared with either therapy by itself in Myc-CaP and PC3 tumor grafts (both p < 0.0001). Our data suggest that combined RT-AUY922 therapy exhibits promising activity against prostate cancer cells, which should be investigated in clinical studies.

Castration Therapy Results in Decreased Ku70 Levels in Prostate Cancer

Neoadjuvant castration improves response to radiotherapy of prostate cancer. Here, we determine whether castration therapy impairs nonhomologous end-joining (NHEJ) repair of DNA double-strand breaks (DSB) by downregulating Ku70 protein expression. Twenty patients with locally advanced prostate cancer were enrolled, and 6 to 12 needle core biopsy specimens were taken from the prostate of each patient before treatment. Bilateral orchidectomy was conducted in eight patients and 12 patients were treated with a GnRH agonist. After castration, two to four similar biopsies were obtained, and the levels of Ku70 and γ-H2AX foci were determined by immunofluorescence in verified cancer tissues. We observed that the androgen receptor binds directly to Ku70 in prostate tissue. We also found a reduction of the Ku70 protein levels in the cell nuclei in 12 of 14 patients (P < 0.001) after castration. The reduction in Ku70 expression correlated significantly with decreased serum prostate-specific antigen (PSA) levels after castration, suggesting that androgen receptor activity regulates Ku70 protein levels in prostate cancer tissue. Furthermore, a significant correlation between the reductions of Ku70 after castration versus changes induced of castration of γ-H2AX foci could be seen implicating a functional linkage of decreased Ku70 levels and impaired DNA repair. Castration therapy results in decreased levels of the Ku70 protein in prostate cancer cells. Because the Ku70 protein is essential for the NHEJ repair of DSBs and its downregulation impairs DNA repair, this offers a possible explanation for the increased radiosensitivity of prostate cancer cells following castration.

Radiosensitization of prostate cancer cells by the dual PI3K/mTOR inhibitor BEZ235 under normoxic and hypoxic conditions

Background and purposeDespite appropriate radiotherapy, high-risk prostate cancer patients often experience local relapse and progression to metastatic disease. Radioresistance may be due to tumor-hypoxia but also due to the PTEN mutation/deletion present in 70% prostate cancers. We investigated whether the novel PI3K/mTOR inhibitor BEZ235 might sensitize prostate cancer cells to radiation and reduce hypoxia-induced radioresistance. Materials and methodsThe potential radiosensitizing properties of BEZ235 were investigated in vitro and in vivo using two prostate cancer cell lines, PC3 (PTEN−/−) and DU145 (PTEN+/+), under normoxic (21% O2) and hypoxic (0.5% O2) conditions. ResultsBEZ235 rapidly inhibited PI3K and mTOR signaling in a dose dependent manner and limited tumor cell proliferation and clonogenic survival in both cell lines independently of PTEN status. In vivo, BEZ235 pretreatment enhanced the efficacy of radiation therapy on PC3 xenograft tumors in mice without inducing intestinal radiotoxicity. In culture, BEZ235 radiosensitized both cell lines in a comparable manner. Moreover, BEZ235 inhibited PI3K/mTOR activation and radiosensitized both cell lines under normoxia and hypoxia. BEZ235 radiosensitizing effects correlated with a decrease in γH2AX foci repair and increased G2/M cell cycle arrest. ConclusionsBEZ235 is a potent radiosensitizer of normoxic and hypoxic prostate cancer cells.

A Possible Role for Perforin and Granzyme B in Resveratrol‐Enhanced Radiosensitivity of Prostate Cancer

Perforin and granzyme B are expressed primarily by activated lymphocytes (cytotoxic T cells, natural killer cells, and natural killer T cells) and function together to induce apoptosis of target cells. Typically, these proteins are not expressed in tumor cells. In the present study, we established the constitutive expression of perforin and granzyme B by the PC-3 and DU145 prostate cancer (PCA) cell lines with reverse transcription polymerase chain reaction, immunohistochemistry, Western blot, or a combination of techniques. The combination of radiation and resveratrol (XRT/RSV) additively/synergistically decreased survival of PCA because, at least in part, of increased apoptosis. We further demonstrated that treatment with RSV up-regulated the expression of both perforin and granzyme B, whereas treatment with XRT up-regulated the expression of granzyme B, but not that of perforin. Combined XRT/RSV treatment of PCA cells further increased the expression of both perforin and granzyme B compared with RSV or XRT alone. Thus, increased radiosensitivity of prostate cancer cells induced by RSV correlated with up-regulation of perforin and granzyme B, demonstrating a possible mechanism for tumor apoptosis. These findings might be helpful in devising new strategies for treating PCA.

Abstract 2073: Parthenolide-mediated redox modification of Keap1 leads to differential effects on radiosensitivities of prostate cancer and normal cells

Abstract Elevated oxidative stress is more frequently observed in cancer cells than in normal cells. Thus, further exposure to exogenous reactive oxygen species (ROS) is expected to push cancer to cell death, whereas normal cells may maintain redox homeostasis through ROS mediated adaptive responses. We previously demonstrated that parthenolide selectively enhances radiosensitivity of prostate cancer cells through activation of a NADPH oxidase-dependent redox signaling pathway. Our present study identifies the status of the redox sensitive Kelch-like ECH-associated protein-1 (Keap1) as a central regulator responsible for the parthenolide-mediated differential redox modification. This results in both a radiosensitization effect in prostate cancer cells and a radioresistance effect in normal prostate epithelial cells. Mechanistically, parthenolide increases the reduced state of Keap1 in prostate cancer cells in a thioredoxin-dependent manner but causes oxidation of Keap1 in normal prostate epithelial cells. Selective knock-down of thioredoxin leads to Keap1 oxidation and subsequent activation of the NF-E2-related factor 2 (Nrf2). These results suggest that redox modification of Keap1 is a major mechanism for the parthenolide effect on radiation therapy in cancer and normal cells. Consistent with the differential effect of parthenolide in cancer and normal cells, the levels of superoxide, hydrogen peroxide and peroxynitrite were selectively increased in parthenolide treated prostate cancer cells but not in normal prostate epithelial cells, indicating the selective increase of ROS in cancer cells. Parthenolide reduces the levels of Nrf2 targets, antioxidant proteins including manganese superoxide dismutase and heme oxygenase in prostate cancer cells, but increases the levels of these proteins in normal cells. This suggests that an antioxidant response is critical for the protection of normal cells against ROS generated from parthenolide. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2073. doi:1538-7445.AM2012-2073

Abstract B27: The PARP inhibitor rucaparib radiosensitizes prostate cancer cells, most effectively those that are PTEN-deficient and are expressing ETS gene fusion proteins, which inhibit NHEJ DNA repair

Abstract Exposure to genotoxic agents, such as irradiation produces DNA damage and its toxicity is augmented when the DNA repair is impaired. Poly ADP ribose polymerase (PARP) inhibitors, such as rucaparib (CO-338; formerly known as AG014699 and PF-01367338) have been also found to be most effective in cells deficient in DNA repair. Radiation dose and scheduling experiments were performed in the prostate cancer cell line C4-2 in conditions mimicking brachytherapy with low dose radiation (Ir-192) as compared to external beam radiotherapy (EBRT). These studies have established the effectiveness of rucaparib to radiosensitize prostate cancer cells when the two clinical modalities are used: EBRT or brachytherapy, with a clear benefit of lower doses administered over a longer period of time, that caused DNA damage due to DNA replication fork collapse when rucaparib was present. Clonogenic survival assay were then performed on a panel of prostate cancer cell lines (LNCaP, C4-2, PC3, DU145, MDaPc2B, CWR22rv1, and VCaP) following radiation and rucaparib, used either alone or together. The combination index (CI) calculated for these various combinations revealed synergistic interactions, which was strongest for LNCaP cells. In addition to apoptosis, senescence was an important response, particularly in PTEN-negative tumors, as it was not observed in Du145 cells that have a wild-type PTEN allele. Absence of PTEN therefore allows activation of senescence, which contributes to decreased clonogenic survival. Increased radiosensitivity in presence of rucaparib was associated with persistent DNA breaks as determined by gamma-H2AX and p53BP1 foci. Interestingly, VCaP cells showed such foci constitutively, indicative of persistent DNA damage that was not present if the TMPRSS2/ERG gene fusion was depleted by siRNA. This response correlated with the ability of the gene fusion protein to bind DNA-PKcs on the chromatin and, as a result, inhibited its kinase activity. DNA-PKcs functional deficiency caused by TMPRSS2/ERG destabilized critical nonhomologous end-joining (NHEJ) components, such as Ku70 and XRCC4. When the TMPRSS2/ERG gene fusion protein was depleted by siRNA the DNA-PKcs kinase activity was restored and the DNA damage response (constitutive or induced) diminished, as measured by gamma-H2AX and p53BP1 foci. Therefore, the presence of the TMPRSS2/ERG fusion gene protein, by inhibiting NHEJ DNA repair, sensitized prostate cancer cells to TREATMENT WITH radiation and/or rucaparib. These results support the clinical testing of rucaparib in combination with radiotherapy. Late stage metastatic tumors, which harbor a gene fusion and are defective in PTEN could benefit most from a combination therapy using brachytherapy and rucaparib in a neoadjuvant setting. Citation Format: Payel Chatterjee, Gaurav Choudhary, Warren D. Heston, Eric A. Klein, Alex Almasan. The PARP inhibitor rucaparib radiosensitizes prostate cancer cells, most effectively those that are PTEN-deficient and are expressing ETS gene fusion proteins, which inhibit NHEJ DNA repair [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B27.

Abstract 2515: Inverse relationship between PSA and IL-8 in prostate cancer: An insight into the mechanisms of NF-κB-mediated radioresistance

Abstract Nuclear factor κB (NF-κB), an inflammatory responsive transcription factor, is involved in tumorigenesis and resistance of cancer cells to therapy-induced cytotoxicity. Our previous studies demonstrated that activation of RelB-mediated NF-κB alternative pathway contributes to tumorigenicity of prostate cancer, decreased the circulating levels of prostate specific antigen (PSA), and enhanced the level of interleukin-8 (IL-8). The present study extends to directly test the role of RelB on radioresistance of prostate cancer in vivo, investigate the relationship between IL8 and PSA expression, and determine their role in the response of prostate cancer to radiation. We found that expression of RelB in prostate cancer cells with a low basal level of RelB (LNCaP) reduced their radiosensitivity and suppression of RelB in prostate cancer cells with a high basal level of RelB (PC3) enhanced their radiosensitivity in vitro and in xenograft tumors. The RelB-mediated inverse relationship between PSA and IL-8 expression was verified through direct modulation of RelB levels by transfection of RelB cDNA and RNAi in multiple prostate cancer cell lines. Interestingly, when the cellular levels of PSA and IL-8 were manipulated, by treating cultural cells with pure compounds or genetic manipulations, we found that up-regulation of IL-8 with concurrent down-regulation of PSA reduced radiosensitivity of prostate cancer cells. In contrast, up-regulation of PSA with concurrence down-regulation of IL-8 resulted in an increase in radiosensitivity. Together, the results reveal a previously unrecognized role of IL-8 on PSA expression, identify an inverse relationship between PSA and IL-8 as an indicator of radiosensitivity and implicate inhibition of the NF-κB alterative pathway as a promising approach for prostate cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2515. doi:10.1158/1538-7445.AM2011-2515

The alkylphospholipid, perifosine, radiosensitizes prostate cancer cells both in vitro and in vivo

BackgroundPerifosine is a membrane-targeted alkylphospholipid developed to inhibit the PI3K/Akt pathway and has been suggested as a favorable candidate for combined use with radiotherapy. In this study, we investigated the effect of the combined treatment of perifosine and radiation (CTPR) on prostate cancer cells in vitro and on prostate cancer xenografts in vivo.MethodsHuman prostate cancer cell line, CWR22RV1, was treated with perifosine, radiation, or CTPR. Clonogenic survival assays, sulforhodamine B cytotoxity assays and cell density assays were used to assess the effectiveness of each therapy in vitro. Measurements of apoptosis, cell cycle analysis by flow cytometry and Western blots were used to evaluate mechanisms of action in vitro. Tumor growth delay assays were used to evaluate radiation induced tumor responses in vivo.ResultsIn vitro, CTPR had greater inhibitory effects on prostate cancer cell viability and clonogenic survival than either perifosine or radiation treatment alone. A marked increase in prostate cancer cell apoptosis was noted in CTPR. Phosphorylation of AKT-T308 AKT and S473 were decreased when using perifosine treatment or CTPR. Cleaved caspase 3 was significantly increased in the CTPR group. In vivo, CTPR had greater inhibitory effects on the growth of xenografts when compared with perifosine or radiation treatment alone groups.ConclusionsPerifosine enhances prostate cancer radiosensitivity in vitro and in vivo. These data provide strong support for further development of this combination therapy in clinical studies.