Rat Prostate Cancer Research Articles

Metastasizing, Luciferase Transduced MAT‑Lu Rat Prostate Cancer Models: Follow up of Bolus and Metronomic Therapy with Doxorubicin as Model Drug

The most fatal outcomes of prostate carcinoma (PCa) result from hormone-refractory variants of the tumor, especially from metastatic spread rather than from primary tumor burden. The goal of the study was to establish and apply rat MAT-Lu prostate cancer tumor models for improved non-invasive live follow up of tumor growth and metastasis by in vivo bioluminescence. We established luciferase transduced MAT-Lu rat PCa cells and studied tumor growth and metastatic processes in an ectopic as well as orthotopic setting. An intravenous bolus treatment with doxorubicin was used to demonstrate the basic applicability of in vivo imaging to follow up therapeutic intervention in these models. In vitro analysis of tissue homogenates confirmed major metastatic spread of subcutaneous tumors into the lung. Our sensitive method, however, for the first time detects metastasis also in lymph node (11/24), spleen (3/24), kidney (4/24), liver (5/24), and bone tissue (femur or spinal cord - 5/20 and 12/20, respectively). Preliminary data of orthotopic implantation (three animals) showed metastatic invasion to investigated organs in all animals but with varying preference (e.g., to lymph nodes). Intravenous bolus treatment of MAT-Lu PCa with doxorubicin reduced subcutaneous tumor growth by about 50% and the number of animals affected by metastatic lesions in lymph nodes (0/4), lung (3/6) or lumbar spine (0/2), as determined by in vivo imaging and in vitro analysis. Additionally, the possible applicability of the luciferase transduced MAT-Lu model(s) to study basic principles of metronomic therapies via jugular vein catheter, using newly established active microport pumping systems, is presented.

Intravenous Injection of Leconotide, an Omega Conotoxin: Synergistic Antihyperalgesic Effects with Morphine in a Rat Model of Bone Cancer Pain

Leconotide (CVID, AM336, CNSB004) is an omega conopeptide similar to ziconotide, which blocks voltage sensitive calcium channels. However, unlike ziconotide, which must be administered intrathecally, leconotide can be given intravenously because it is less toxic. This study investigated the antihyperalgesic potency of leconotide given intravenously alone and in combinations with morphine-administered intraperitoneally, in a rat model of bone cancer pain. Syngeneic rat prostate cancer cells AT3B-1 were injected into one tibia of male Wistar rats. The tumor expanded within the bone causing hyperalgesia to heat applied to the ipsilateral hind paw. Measurements were made of the maximum dose (MD) of morphine and leconotide given alone and in combinations that caused no effect in an open-field activity monitor, rotarod, and blood pressure and heart rate measurements. Paw withdrawal thresholds from noxious heat were measured. Dose response curves for morphine (0.312-5.0 mg/kg intraperitoneal) and leconotide (0.002-200 µg/kg intravenous) given alone were plotted and responses compared with those caused by morphine and leconotide in combinations. Leconotide caused minimal antihyperalgesic effects when administered alone. Morphine given alone intraperitoneally caused dose-related antihyperalgesic effects (ED(50) = 2.40 ± 1.24 mg/kg), which were increased by coadministration of leconotide 20 µg/kg (morphine ED(50) = 0.16 ± 1.30 mg/kg); 0.2 µg/kg (morphine ED(50) = 0.39 ± 1.27 mg/kg); and 0.02 µg/kg (morphine ED(50) = 1.24 ± 1.30 mg/kg). Leconotide caused a significant increase in reversal by morphine of the bone cancer-induced hyperalgesia without increasing the side effect profile of either drug. Translation into clinical practice of the method of analgesia described here will improve the quantity and quality of analgesia in patients with bone metastases. The use of an ordinary parenteral route for administration of the calcium channel blocker (leconotide) at low dose opens up the technique to large numbers of patients who could not have an intrathecal catheter for drug administration. Furthermore, the potentiating synergistic effect with morphine on hyperalgesia without increased side effects will lead to greater analgesia with improved quality of life.

Abstract 5579: Selenomethionine does not protect against testosterone plus 17 beta-estradiol-induced prostate carcinogenesis and prostatic oxidative stress in NBL rats

Abstract Previous studies with selenium and/or vitamin E in prostate carcinogenesis animal models have been negative, but these models may not involve oxidative stress mechanisms. Selenium and vitamin E have been the subject of one of the largest trials ever conducted, SELECT, which was discontinued because there was no difference between the study arms and there were side effects, including an increased chance of developing diabetes mellitus with the use of selenium. We previously found that selenomethionine supplementation did not prevent prostate cancer in NBL rats treated with testosterone plus 17 beta-estradiol (T+E2), a model that does involve sex-hormone induced oxidative stress mechanisms and prostatic inflammation; this result was fully predictive of the outcome of the SELECT trial. Based on the outcomes of SELECT and our NBL rat study, we hypothesized that selenium has no protective effect against T+E2-induced prostate cancer development because it does not protect against oxidative stress. In the present study, we examined the potential of selenomethionine to modulate prostatic oxidative stress and induction of dysplasia and inflammation in the T+E2-treated NBL rat. Nbl/Crl rats were treated for 16 weeks with T+E2 by Silastic implants and were fed a natural ingredient control diet (NIH-07) with or with L-selenomethionine at a concentration of 1.5 or 3.0 mg/kg (as selenium). These diets were initiated one week post hormone implantation. T+E2 significantly increased immunohistochemical staining for 8-hydroxy-deoxyguanosine (8-OHdG) in prostatic ducts, and in the lateral and dorsal prostate which are the main sites of T+E2-induced inflammation and preneoplasia after 16 weeks of hormone treatment, and in the seminal vesicles. However, selenomethionine did not reduce these effects and did not, by itself, change 8OHdG staining. There was a lobe-specific differential response and modulation of antioxidant enzymes. Glutathione peroxidase activity and protein expression level of manganese superoxide dismutase were induced by T+E2, which effects were counteracted by selenomethionine in the dorsolateral prostate, which is the main site of T+E2-induced inflammation and preneoplasia occurring after 16 weeks of hormone treatment. However, selenomethionine did not influence the induction by T+E2 of marked inflammation and dysplasia in the lateral prostate. These findings partially explain the negative outcome of our previous NBL rat study and is consistent with the negative results of the SELECT trial. (Supported in part by Grant No. CA104334) 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 5579. doi:10.1158/1538-7445.AM2011-5579

Piceatannol inhibits migration and invasion of prostate cancer cells: possible mediation by decreased interleukin-6 signaling

Piceatannol (trans-3,4,3′,5′-tetrahydroxystilbene) is a polyphenol detected in grapes, red wine and Rheum undulatum; it has also been demonstrated to exert anticarcinogenic effects. In this study, in order to determine whether piceatannol inhibits the lung metastasis of prostate cancer cells, MAT-Ly-Lu (MLL) rat prostate cancer cells expressing luciferase were injected into the tail veins of male nude mice. The oral administration of piceatannol (20 mg/kg) significantly inhibited the accumulation of MLL cells in the lungs of these mice. In the cell culture studies, piceatannol was demonstrated to inhibit the basal and epidermal growth factor (EGF)-induced migration and invasion of DU145 cells, in addition to the migration of MLL, PC3 and TRAMP-C2 prostate cancer cells. In DU145 cells, piceatannol attenuated the secretion and messenger RNA levels of matrix metalloproteinase-9, urokinase-type plasminogen activator (uPA) and vascular endothelial growth factor (VEGF). Piceatannol increased the protein levels of tissue inhibitor of metalloproteinase-2 in a concentration-dependent fashion. Additionally, piceatannol inhibited the phosphorylation of signal transducer and activator of transcription (STAT) 3. Furthermore, piceatannol effected reductions in both basal and EGF-induced interleukin (IL)-6 secretion. An IL-6 neutralizing antibody inhibited EGF-induced STAT3 phosphorylation and EGF-stimulated migration of DU145 cells. Interleukin-6 treatment was also shown to enhance the secretion of uPA and VEGF, STAT3 phosphorylation and the migration of DU145 cells; these increases were suppressed by piceatannol. These results demonstrate that the inhibition of IL-6/STAT3 signaling may constitute a mechanism by which piceatannol regulates the expression of proteins involved in regulating the migration and invasion of DU145 cells.

Abstract 5185: Evaluation of biomarker candidate genes for prostate cancer metastasis

Abstract Prostate cancer is the most frequently diagnosed malignancy in men and although effective treatment is possible for early stage disease, advanced prostate cancer has a very poor prognosis. Thus, identification of biomarkers that can accurately and reliably diagnose prostate cancer in its early stages is urgently needed. MatLyLu and Dunning G are rat prostate cancer cell lines which have different metastatic capabilities. Using microarray analysis we previously identified a set of metastasis-associated genes that are over-expressed in MatLyLu. Genes found to be differentially expressed include several previously associated with the metastatic process, but not previously reported in prostate cancer. We also have found differential expression of genes that code for several proteoglycans involved in epithelial cell-extracellular matrix interaction, cell adhesion molecules, genes related to motility, growth factor receptors, and tumor suppressor genes. We are currently validating the expression of these candidate genes in human prostate cancer cell lines with different metastatic abilities (LNCaP, PC3, PWR-1E) and in clinical samples of prostate cancer at different disease stages using quantitative real time PCR (qRT-PCR) and immunohistochemistry. A better understanding of the molecular events leading to the metastatic prostate cancer phenotype will help to further identify genes or gene-products involved in this process, which may prove useful as biomarkers for diagnosis and/or prognosis of the disease. Project code: 110745921483. Supported by Colciencias Grant # RC No.462-2008 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 5185. doi:10.1158/1538-7445.AM2011-5185

Oral administration of piceatannol inhibits the lung metastasis of prostate cancer cells

Piceatannol (trans‐3,4,3′,5′‐tetrahydroxystilbene) is a polyphenol detected in grapes, red wine, and Rheum undulatum; it has been demonstrated to exert anticarcinogenic effects. In this study, in order to determine whether piceatannol inhibits the lung metastasis of prostate cancer cells, MAT‐Ly‐Lu (MLL) rat prostate cancer cells expressing luciferase were injected into the tail veins of male nude mice. The oral administration of piceatannol (20 mg/kg) significantly inhibited the accumulation of MLL cells in the lungs of these mice. In the cell culture studies, piceatannol was demonstrated to inhibit the basal and EGF‐induced migration and invasion of DU145 human prostate cancer cells. Piceatannol attenuated the mRNA levels and secretion of MMP‐9, uPA and VEGF and increased the protein levels of TIMP‐2 in a concentration‐dependent fashion. Additionally, piceatannol inhibited the phosphorylation of signal transducer and activator of transcription (STAT)3. Furthermore, piceatannol effected reductions in both basal and EGF‐induced IL‐6 secretion. An IL‐6 neutralizing antibody inhibited EGF‐induced STAT3 phosphorylation and EGF‐stimulated migration of DU145 cells. These results demonstrate that the inhibition of IL‐6/STAT3 signaling may constitute a mechanism by which piceatannol regulates the expression of proteins involved in regulating the migration and invasion of DU145 cells.

401 ORAL APOCYNIN SENSITIZES PROSTATE CANCER XENOGRAFTS TO RADIATION

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 2011401 ORAL APOCYNIN SENSITIZES PROSTATE CANCER XENOGRAFTS TO RADIATION Alex Wolf, Doug Boreham, and Jehonathan Pinthus Alex WolfAlex Wolf Hamilton, Canada More articles by this author , Doug BorehamDoug Boreham Hamilton, Canada More articles by this author , and Jehonathan PinthusJehonathan Pinthus Hamilton, Canada More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.490AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Radiation therapy (RT) is a well established therapy for prostate cancer (PC), but treatment failure occurs in 45% of patients. We have previously shown that the basal levels reactive oxygen species (bROS) in PC cells negatively correlate with their radiosensitivity, and that Apocynin, an NADPH oxidase (NOX) inhibitor can sensitize in-vitro PC cells to radiation by lowering their bROS levels. The primary objective of this study was to take these observations a step forwards and verify it in-vivo. The second objective was to determine the optimal timing of anti-oxidative therapy which will not interfere with the therapeutic effect of RT that is based itself on the induction of acute oxidative attack. The third objective was to test if apocynin can sensitize non-cancerous neighboring tissue (skin) to radiation and thus increase toxicity of RT. METHODS MATLyLu (rat PC cell line) served as a model because it is a very aggressive PC model for which RT (10Gy given at 2Gy/day) can delay progression but is not curative. Thus, related experimental manipulation can result in a readable response. N=98 male Copenhagen rats were implanted s.c with MATLyLu cells. When tumours reached 5mm diameter rats were divided into 5 groups: N=19 received no treatment (control), n=21 received 5mM Apocynin in drinking water x 14 days with no RT, n=20 received RT only, n=18 received 5mM Apocynin in drinking water followed by RT and n=20 received 5mM Apocynin in drinking water 5 days before RT, 5 days during RT and 4 days after RT. Rats were followed for survival and were euthanized if tumours reached a diameter>35mm, had skin ulceration over the tumor or were ill. Kaplan Meyer curves were constructed and the differences in survival between the different groups were calculated by the log-rank test. RESULTS Control untreated rats and rats receiving apocynin only had the same minimal survival (median survival of 19 and 20.9 days respectively). RT significantly extended survival (median survival time of 34.3 days p<0.0001). Survival was significantly increased in rats which received apocynin orally throughout a course of radiotherapy (p=0.0418), but not if it was discontinued before radiotherapy (p=0.208). Apocynin did not sensitize normal surrounding skin to radiation (p=0.185). CONCLUSIONS 1. Apocynin promotes the in-vivo response of PC to RT without toxic effects on neighbouring normal tissues 2. Manipulating the oxidative capacity of PC sensitized it to RT only when applied concomitantly with the radiation. This may suggest that patients who take commonly used anti-oxidants vitamins may not have to cease it during RT. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e162 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Alex Wolf Hamilton, Canada More articles by this author Doug Boreham Hamilton, Canada More articles by this author Jehonathan Pinthus Hamilton, Canada More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

효과적인 항암효능측정을 위한 발광 전립선 세포의 개발 및 평가

기존의 동물모델에서 암의 성장은 caliper를 이용하여 고형암 부피를 측정으로써 조사하였으나, 암 조직 속의괴사와 부종으로 인하여 부피측정에 신뢰성이 결여 되어 있다. 이러한 문제점을 해결하기 위해 발광 암세포를 이용하여 광학생체영상적으로 분석하는 방법이 개발 되었다. 본 연구에서는 전립선 발광 암세포를 제조하여 고형암 동물모델에서 B16 발광 암세포와 암 성장을 비교 측정하여 신규발광 암세포를 평가하였다. In vitro에서 세포 수와 발광강도는 높은 상관관계를 보였고(<TEX>$R^2$</TEX>=0.99), 고형암 동물모델에서 암 성장 측정은 괴사에 의한 오차를 줄였다. 이러한 발광신호를 기반으로 한 측정방법은 caliper의 부피 측정에 비하여 높은 항암효과를 보임으로써 기존의 발광 암세포보다 신규 발광전립선 암세포의 유용성을 증명하였다. Caliper measurements of tumor volume have been widely used in the assessment of tumors in animal models. However, experiments based on caliper data have resulted in unreliable estimates of tumor growth, due to necrotic areas of tumor mass. To overcome this systematic bias, we engineered a new luciferase-expressing rat prostate cancer cell line (MLL-Luc) that produces bioluminescence from viable cancer cells. MLL-Luc cells showed a strong correlation between bioluminescence intensity and cell number (<TEX>$R^2$</TEX>=0.99) and also accurately quantified tumor growth, with reduced bioluminescence signals caused by necrotic cells in a subcutaneous MLL-Luc xenograft model. The accurate quantification of tumor growth with bioluminescence imaging (BLI) was confirmed by a better antitumor effect of combination chemotherapy, compared to that based on caliper measurements with a correlation between the bioluminescence signal and tumor volume (<TEX>$R^2$</TEX>=0.84). These data suggest that bioluminescent MLL xenografts are a powerful and quantitative tool for monitoring tumor growth and are useful in evaluating the efficacy of anticancer drugs, with less systematic bias.

Selectively Hydrogenated Soybean Oil Exerts Strong Anti‐Prostate Cancer Activities

Prostate cancer is the second leading cause of male deaths due to cancer in the United States. Hydrogenated vegetable oils have been suspected of inducing adverse health effects, including atherosclerosis and cancer. Here we report that a selectively hydrogenated soybean oil (SHSO) containing a high quantity of conjugated linoleic acids showed remarkably strong anticarcinogenic activity against prostate cancer in the rat model (Copenhagen rats with MAT-LyLu syngeneic rat prostate cancer cells) study in vivo and human prostate carcinoma cell lines studies in vitro, as compared with native soybean oil. A 5% dietary supplementation with SHSO inhibited the growth of prostate cancer by 80% in vivo. The TUNEL method and immunohistochemical staining assays of bax, bcl-2, and survivin clearly showed that SHSO induced prostate cancer cell apoptosis in the tested rats. DNA fragmentation analysis in vitro further confirmed the apoptotic activity of SHSO on the MAT-LyLu prostate cancer cells. The SHSO also showed strong cytotoxicity on human prostate cancer cells (DU145 and PC3). This represents the first report demonstrating the significant anticancer activities of hydrogenated vegetable oils at low levels of dietary supplementation.

Vascular Endothelial Growth Factor Receptor 2-Specific Microbubbles for Molecular Ultrasound Detection of Prostate Cancer in a Rat Model

To investigate whether rat prostate cancer can be detected by means of molecular ultrasound (US) using target-specific microbubbles versus a nonspecific contrast agent. A total of 20 Copenhagen rats were randomly examined 75 to 104 days after orthotopic implantation of G-Dunning rat prostatic tumor cells was using a high-end US system and either 1.2 mL/kg of the nonspecific agent A or 0.1 mL/kg of the target-specific agent B, containing vascular endothelial growth factor receptor 2 binding peptide. Contrast inflow (areas under the curve analysis) was determined during the first 30s, and all microbubbles were destroyed in the scan plane by means of the flash technique 20 minutes after intravenous administration to investigate specific accumulation of individual bubbles in tumors. Differences between normal prostate tissue and tumor were analyzed using luminance images. Sonographically determined tumor localization and extent were compared with magnetic resonance imaging and histology. The median tumor size in the 20 rats determined on US (2.3 mm) and magnetic resonance imaging (2.4 mm) showed a very high correlation (r = 0.92, P = 0.01). Both agent A and agent B demonstrated higher vascularization of tumor periphery compared with normal prostate (P < 0.05) based on contrast inflow and areas under the curve analysis. Twenty minutes after administration, luminance for agent B in the tumor was significantly higher (P = 0.003) compared with nonspecific agent A (11.8-0.1). In consensus reading, the increase in signal intensity of the tumor compared with normal prostate tissue was significantly higher for agent B (P = 0.005), whereas no significant difference was found for agent A (P = 0.180). The target-specific contrast agent was superior to the unspecific US contrast agent both with regard to early inflow analysis and specific accumulation in prostate cancer after 20 minutes.

Targeting castration‐induced tumour hypoxia enhances the acute effects of castration therapy in a rat prostate cancer model

What's known on the subject? and What does the study add? Castration therapy has rather modest effects on cell death in tumours but can be enhanced by other treatments targeting tumour stroma and vasculature. This study shows that the prostate becomes hypoxic following castration and that targeting hypoxic cells during castration therapy potently enhances the effects of castration. To explore the effects of castration therapy, the standard treatment for advanced prostate cancer, in relation to tumour hypoxia and to elicit its importance for the short- and long-term therapeutic response. We used the androgen-sensitive rat Dunning H prostate tumour model that transiently responds to castration treatment followed by a subsequent relapse, much like the scenario in human patients. Tumour tissues were analysed using stereological methods in intact, 1 and 7 days after castration therapy. Hypoxia was transiently up-regulated after castration therapy and correlated with the induction of tumour cell apoptosis. When castration therapy was combined with tirapazamine (TPZ), a drug that targets hypoxic cells and the vasculature, the effects on tumour cell apoptosis and tumour volume were enhanced in comparison to either castration or TPZ alone. The present study suggests that castration-induced tumour hypoxia is a novel target for therapy.

Momordica charantia leaf extract suppresses rat prostate cancer progression in vitro and in vivo

Cancer metastasis is a major cause of death in cancer patients, with invasion as a first step greatly contributing to the failure of clinical treatments. Any compounds with an inhibitory influence on this process are therefore of prime interest. Momordica charantia (bitter melon) is widely consumed as a vegetable and especially as a folk medicine in Asia. Here, we investigated the anti-invasive effects of bitter melon leaf extract (BMLE) on a rat prostate cancer cell line (PLS10) in vitro and in vivo. The results indicated that non-toxic concentrations of BMLE significantly inhibited the migration and invasion of cells in vitro. The results of zymography showed that BMLE inhibited the secretion of MMP-2, MMP-9 and urokinase plasminogen activator (uPA) from PLS10. Real-time RT-PCR revealed that BMLE not only significantly decreased gene expression of MMP-2 and MMP-9, but also markedly increased the mRNA level of TIMP-2, known to have inhibitory effects on the activity of MMP-2. An EnzChek gelatinase/collagenase assay showed that collagenase type IV activity was partially inhibited by BMLE. In the in vivo study, intravenous inoculation of PLS10 to nude mice resulted in a 100% survival rate in the mice given a BMLE-diet as compared with 80% in the controls. The incidence of lung metastasis did not show any difference, but the percentage lung area occupied by metastatic lesions was slightly decreased in the 0.1% BMLE treatment group and significantly decreased with 1% BMLE treatment as compared with the control. Thus, the results indicate for the first time an anti-metastatic effect of BMLE both in vitro and in vivo.

Abstract 967: Serine proteases as targets for prostate cancer prevention: Identification by analysis of differential gene expression in rodent and human prostate cancers

Abstract Patterns of gene expression in normal and neoplastic prostate tissues from rats and humans were compared to identify novel molecular targets for prostate cancer chemoprevention. Differentially expressed genes in human prostate cancers were identified by reviewing published genome-wide screening studies and hypothesis-driven studies of gene expression in prostate cancer patients. Differentially expressed genes in rat prostate cancers were identified by microarray analysis (using Affymetrix Rat 230 2.0 GeneChips) of paired malignant and phenotypically normal prostatic tissues harvested from Wistar-Unilever rats treated with N-methyl-N-nitrosourea [MNU] + testosterone. Patterns of differential gene expression in human prostate cancers demonstrated only limited concordance between studies in clinical populations performed by different groups. Most differentially expressed genes were identified in only a single human study; only two genes (AMACR [α-methylacyl-CoA racemase] and HPN [hepsin, a protease inhibitor]) were found to be differentially expressed in &amp;gt;25% of the human prostate cancer microarray studies evaluated. Genes found to be differentially expressed in multiple hypothesis-driven studies of human prostate cancers included E2F1 (transcription factor), MUC1 (mucin 1), PLAB (placental bone morphogenetic protein), and ST14 (serine protease); these genes were not identified in genome-wide screening studies of human prostate cancers. In tissues harvested from rats treated with MNU + testosterone, 890 genes were identified as differentially regulated (fold change &amp;gt; 1.5 and p &amp;lt; 0.001) in prostate cancers in comparison to paired phenotypically normal prostate tissues. These 890 genes included four serine protease genes (Prss 11, Prss22, Prss 25, and Prss35) that were significantly up-regulated (by 1.7 to 11.4-fold). In addition, five serine protease inhibitor genes (Serpins e1, e2, f1, g1, h1) were over-expressed in rat prostate cancers (by 2.2 to 23.5-fold), suggesting possible up-regulation in response to increased protease activity in prostatic neoplasms. When human and rat gene expression data are considered together, serine proteases were the only class of molecules that demonstrated a reproducible pattern of differential gene expression in prostate cancers in both species. These data suggest that modulation of protease activity may provide a useful target for prostate cancer prevention. This conclusion is supported by our finding that Bowman-Birk Inhibitor, a mixture of soy-based protease inhibitors, inhibits carcinogenesis in the rat prostate (Nutr. Cancer, 57, 184, 2007). Development of small molecule protease inhibitors may be a useful strategy for prostate cancer chemoprevention. (Supported by IR&amp;D funds from IIT Research Institute, NCI-N01-CN-25017, and NCI-N01-CN-85177) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 967.

Abstract LB-422: A novel mechanism for the anti-cancer effects of licofelone against testosterone and estradiol-17ß-induced prostate carcinogenesis: Selective modulation of the cross-talk between estrogen receptor and NF-κB

Abstract The chemopreventive and therapeutic potential of licofelone, an anti-inflammatory drug, has been reported in preclinical models against the cancers of the colon, breast, pancreas, and bladder, including prostate cancer (PCa). In contrast to nonsteroidal anti-inflammatory drugs (NSAIDs) and selective cyclooxygenase-2 (COX-2) inhibitors, licofelone exerts significantly less adverse effects under chronic use, and is currently undergoing phase III trials for osteoarthritis. However, the anticancer mechanism of licofelone is unclear. To elucidate the molecular mechanisms of licofelone, we used a short-term Noble (NBL) rat PCa model. Male NBL rats at 10 weeks of age were randomly assigned to control and three experimental groups (n=18 per group), and they were fed a semi-purified AIN-93M diet containing 0, 100, 200, and 300ppm of licofelone. After 2 weeks on control and licofelone diets, hormonal implants, the silastic tubing packed with testosterone (T) and estradiol-17 (E2), were placed subcutaneously in rats under anesthesia. Empty silastic implants (n=5 rats) served as vehicle control, and were fed semi-purified AIN-93M diet only. Dietary regimen was resumed immediately after the hormonal implantation, and all rats were sacrificed at the end of 16 weeks. To examine the anti-cancer activities of licofelone, histological, cell proliferation (ki67 staining), and apoptosis (TUNEL assay) analyses were performed using rat dorsolateral prostate (DLP) tissues. Serum T and E2 levels were measured using radioimmunoassay (RIA) kits. The levels of TNF-α, PGE2, and LTB4 in DLP tissues were analyzed using enzyme immunoassay EIA) kits. Real-time PCR, Western blot and immunohistochemical methods were used to determine the molecular targets at the mRNA and protein levels. Dietary administration of licofelone in NBL rats did not induce any toxic effect or weight loss. A 16-week hormonal treatment contributed chronic inflammation, and developed prostate intraepithelial neoplasia (PIN) in 88% of the rats (n=18). Histological analysis revealed that licofelone treatment suppressed PIN lesions significantly, in a dose-dependent manner; suppressed 21% at 100ppm, 47% at 200ppm, and 68% at 300ppm compared to the untreated control (p&amp;lt;0.001). Further, licofelone at 300ppm significantly decreased the rate of tumor cell proliferation by 68%, and enhanced apoptosis by 8-fold in contrast to the untreated controls. T + E2 treatment-induced production of critical inflammatory mediators were significantly decreased by licofelone treatment at 300ppm (p&amp;lt;0.001); prostaglandin E2 (PGE2) was reduced from 674 to 128 pg/mg protein, leukotriene B4 (LTB4) from 169 to 17 pg/mg protein, and tumor necrosis factor alpha (TNF-α) from 540 to 84 pg/mg protein. However, licofelone treatment had a minimal effect on COX-2 expression at the mRNA and protein level, and COX-1 level was not altered. These findings provide evidence that licofelone effectively suppressed the hormone-induced prostate carcinogenesis in NBL rats, independent of COX-2. Since estrogen receptors (ERα and ERß) are the main mediators of estrogen action in the prostate, it is plausible that a positive interaction between ERα and NF-κB (NF-κB) may trigger the transcriptional activation of pro-inflammatory NF-κB signal transduction pathway and may lead to cancer progression. To test this hypothesis, we further examined the expression of ERα, ERß, and NF- Bp65 expression in PIN lesions of the DLP. Our findings revealed that licofelone treatment at 300ppm significantly downregulated ERα and NF-κBp65 expression, whereas ERß expression was upregulated compared to untreated control (p&amp;lt;0.001). In summary, our findings suggest that licofelone modulated several pro-inflammatory targets, and effectively suppressed the hormone-induced PIN development in Noble rats, without significantly affecting COX-2. Most interestingly, our findings are the first to report evidence that licofelone may selectively bind to ERα, and inhibit ERα-mediated activation of NF-κB signaling in prostate carcinogenesis. Further studies are in progress to confirm this unique molecular mechanism of licofelone binding to ER in PCa chemoprevention (Supported by NIH/NCI R03 CA133929 grant to N. Narayanan). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-422.

Selenomethionine and α-Tocopherol Do Not Inhibit Prostate Carcinogenesis in the Testosterone plus Estradiol–Treated NBL Rat Model

Previous studies with selenium and/or vitamin E in prostate carcinogenesis animal models have been negative, but these models may not involve oxidative stress mechanisms. In this study, we examined the potential of selenomethionine and alpha-tocopherol to modulate prostate cancer development in the testosterone plus estradiol-treated NBL rat, a model that does involve sex hormone-induced oxidative stress mechanisms and prostatic inflammation. One week following the implantation with hormone-filled Silastic implants, rats were fed diets containing l-selenomethionine (1.5 or 3.0 mg/kg), DL-alpha-tocopherol acetate (2,000 or 4,000 mg/kg), or a natural ingredient control diet (NIH-07). The development of prostate carcinomas was not affected by dietary treatment with either agent. Food intake, body weight, and mortality were also not affected. The high dose of selenomethionine reduced the severity of epithelial dysplasia in the lateral prostate that was not associated with inflammation, and alpha-tocopherol reduced in a dose-related fashion the incidence of marked inflammation and marked epithelial dysplasia in the lateral prostate, regardless of whether these lesions were associated with inflammation. alpha-Tocopherol significantly increased the incidence of adenocarcinomas of the mammary glands at both dietary concentrations. Collectively, our findings suggest that selenomethionine and alpha-tocopherol supplementation does not prevent prostate cancer in rats fed diets with nutritionally adequate levels of selenium and vitamin E. Importantly, the results of the current animal studies and those reported previously were fully predictive of the outcome of the Selenium and Vitamin E Cancer Prevention Trial.

Abstract PL06-01: Targeted inhibition of chromatin-modifying enzymes as cancer therapeutics: The HDAC inhibitor vorinostat and beyond

Abstract PL06-01: Targeted inhibition of chromatin-modifying enzymes as cancer therapeutics: The HDAC inhibitor vorinostat and beyond

Protein kinase A and regulation of neonatal Nav1.5 expression in human breast cancer cells: Activity-dependent positive feedback and cellular migration

Voltage-gated Na+ channels (VGSCs) are expressed in excitable cells (e.g. neurons and muscles), as well as in some classically ‘non-excitable’ cells (e.g. fibroblasts), and in carcinomas. In general, functional expression of VGSCs in plasma membrane (PM) is hierarchical and dynamic. Previously, we have shown that an activity-dependent positive feedback mechanism involving cAMP-dependent protein kinase A (PKA) plays a significant role in upregulation of VGSCs in strongly metastatic rat prostate cancer Mat-LyLu cells expressing Nav1.7. Here, we investigated the possible role of PKA in VGSC regulation and its functional consequences in strongly metastatic human breast cancer (BCa) MDA-MB-231 cells, where the neonatal splice form of Nav1.5 (nNav1.5) is the predominant VGSC present. Treatment with the PKA activator forskolin for 24h increased mRNA and PM protein levels of nNav1.5, without changing the total VGSC protein level. Opposite effects were obtained by application of the PKA inhibitor KT5720 or the highly specific VGSC blocker tetrodotoxin (TTX), the latter implying activity-dependent upregulation. We tested the possibility, therefore, that the activity dependence of VGSC (nNav1.5) expression involved PKA. Indeed, TTX pretreatment reduced the level of phosphorylated PKA and eliminated basal and PKA-stimulated cellular migration. These data suggested that activity-dependent positive feedback mediated by PKA plays an important role in the functional expression of nNav1.5 in BCa, and in turn, this enhances the cells’ metastatic potential.

Adenosine A3 Receptor Suppresses Prostate Cancer Metastasis by Inhibiting NADPH Oxidase Activity

Prostate cancer is the most commonly diagnosed and second most lethal malignancy in men, due mainly to a lack of effective treatment for the metastatic disease. A number of recent studies have shown that activation of the purine nucleoside receptor, adenosine A(3) receptor (A(3)AR), attenuates proliferation of melanoma, colon, and prostate cancer cells. In the present study, we determined whether activation of the A(3)AR reduces the ability of prostate cancer cells to migrate in vitro and metastasize in vivo. Using severe combined immunodeficient mice, we show that proliferation and metastasis of AT6.1 rat prostate cancer cells were decreased by the administration of A(3)AR agonist N(6)-(3-iodobenzyl) adenosine-5'-N-methyluronamide. In vitro studies show that activation of A(3)AR decreased high basal nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity present in these cells, along with the expression of Rac1 and p47(phox) subunits of this enzyme. Inhibition of NADPH oxidase activity by the dominant-negative RacN17 or short interfering (si)RNA against p47(phox) reduced both the generation of reactive oxygen species and the invasion of these cells on Matrigel. In addition, we show that membrane association of p47(phox) and activation of NADPH oxidase is dependent on the activity of the extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase pathway. We also provide evidence that A(3)AR inhibits ERK1/2 activity in prostate cancer cells through inhibition of adenylyl cyclase and protein kinase A. We conclude that activation of the A(3)AR in prostate cancer cells reduces protein kinase A-mediated stimulation of ERK1/2, leading to reduced NADPH oxidase activity and cancer cell invasiveness.

Tranilast suppresses prostate cancer growth and osteoclast differentiation in vivo and in vitro

In bone metastatic sites, prostate cancer cells proliferate on interacting with osteoclasts. Tranilast, which is used for an antiallergic drug, has been shown to inhibit growth of several cancers and stromal cells. The present study was conducted to assess suppressive effects of Tranilast on prostate cancer growth and osteoclast differentiation in vivo and in vitro. In vivo, rat prostate cancer tissue was transplanted onto cranial bones of F344 rats and Tranilast was given for 9 days at doses of 0, 200, or 400 mg/kg/day. In vitro, human prostate cancer cell lines, LNCaP, PC3, and DU145, the rat prostate cancer cell line, PLS-10, and rat bone marrow cells were similarly treated with the agent. In vivo, tumor volumes were significantly decreased in the high dose group. While cell proliferation did not appear to be affected, apoptosis was induced and tumor necrosis was apparent. Cranial bone defects were decreased in the high dose group. In vitro, cell proliferation rates of all four cell lines were reduced by Tranilast and increased apoptosis was observed in LNCaP and PLS-10. In addition, Tranilast significantly reduced osteoclast differentiation of rat bone marrow cells. Western blot analysis of PLS-10 and LNCaP revealed that phospho-GSK3beta was up-regulated and phospho-Akt was down-regulated. Tranilast here suppressed rat prostate cancer growth and osteoclast differentiation. Growth of human prostate cancer cells was also inhibited. Thus, this agent deserves consideration as a candidate for conventional therapy of bone metastatic prostate cancer.

In vivo characterization of a reporter gene system for imaging hypoxia-induced gene expression

To characterize a tumor model containing a hypoxia-inducible reporter gene and to demonstrate utility by comparison of reporter gene expression to the uptake and distribution of the hypoxia tracer (18)F-fluoromisonidazole ((18)F-FMISO). Three tumors derived from the rat prostate cancer cell line R3327-AT were grown in each of two rats as follows: (1) parental R3327-AT, (2) positive control R3327-AT/PC in which the HSV1-tkeGFP fusion reporter gene was expressed constitutively, (3) R3327-AT/HRE in which the reporter gene was placed under the control of a hypoxia-inducible factor-responsive promoter sequence (HRE). Animals were coadministered a hypoxia-specific marker (pimonidazole) and the reporter gene probe (124)I-2'-fluoro-2'-deoxy-1-beta-d-arabinofuranosyl-5-iodouracil ((124)I-FIAU) 3 h prior to sacrifice. Statistical analysis of the spatial association between (124)I-FIAU uptake and pimonidazole fluorescent staining intensity was then performed on a pixel-by-pixel basis. Utility of this system was demonstrated by assessment of reporter gene expression versus the exogenous hypoxia probe (18)F-FMISO. Two rats, each bearing a single R3327-AT/HRE tumor, were injected with (124)I-FIAU (3 h before sacrifice) and (18)F-FMISO (2 h before sacrifice). Statistical analysis of the spatial association between (18)F-FMISO and (124)I-FIAU on a pixel-by-pixel basis was performed. Correlation coefficients between (124)I-FIAU uptake and pimonidazole staining intensity were: 0.11 in R3327-AT tumors, -0.66 in R3327-AT/PC and 0.76 in R3327-AT/HRE, confirming that only in the R3327-AT/HRE tumor was HSV1-tkeGFP gene expression associated with hypoxia. Correlation coefficients between (18)F-FMISO and (124)I-FIAU uptakes in R3327-AT/HRE tumors were r=0.56, demonstrating good spatial correspondence between the two tracers. We have confirmed hypoxia-specific expression of the HSV1-tkeGFP fusion gene in the R3327-AT/HRE tumor model and demonstrated the utility of this model for the evaluation of radiolabeled hypoxia tracers.