Transgenic Adenocarcinoma Of Mouse Prostate Mice Research Articles

Alpha-santalol, a derivative of sandalwood oil prevents development of prostate cancer in TRAMP mice

BackgroundAlpha-santalol, a major component of sandalwood oil inhibits growth of cultured prostate cancer cells in vitro by causing apoptosis. However, its in vivo efficacy against prostate cancer development is not established. Hypothesis/purposeProstate cancer is one of the most commonly diagnosed cancers in men. The present study was undertaken to determine the in vivo efficacy of alpha-santalol using TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice as a model. MethodsTRAMP mice were used for the in vivo studies. Immunohistochemical analysis was used to assess prostate/tumor sections and Ki-67 staining. Western blot analysis was used to determine expression of various proteins. TUNEL assay was used to detect apoptotic bodies. ResultsAdministration of alpha-santalol (100 mg/kg body weight) decreased the incidence of prostate tumors, average wet weights of urogenital organs and prostate weight compared to control mice. In addition, the area occupied by normal prostate was significantly higher, along with poorly differentiated carcinoma that was significantly lower in the dorsolateral prostate sections of alpha-santalol-treated mice compared with the controls. Furthermore, the dorsolateral sections of prostate from alpha-santalol-treated mice exhibited decreased cell proliferation (Ki-67 staining) in association with induction of apoptosis (TUNEL-positive cells). In agreement with immunohistochemical analysis, Western blotting analysis of prostate/tumor samples from alpha-santalol-treated group revealed a decrease in survivin, XIAP, PCNA, cyclin D and CDC2 levels compared to control samples. ConclusionThe present study shows for the first time, that alpha-santalol administration inhibits the development of prostate cancer in TRAMP mice by decreasing cell proliferation and inducing apoptosis, and warrants future studies for its clinical development.

NRF2 modulation in TRAMP mice: an in vivo model of prostate cancer.

Prostate cancer (PCa) is one of the most common cancers worldwide and oxidative stress is involved in its occurrence, development and progression. In fact, in transgenic adenocarcinoma of mouse prostate (TRAMP) mice, prostate cancer onset is associated with the methylation of the first five CpG in the nuclear factor erythroid 2-related factor 2 (NRF2) promoter, a key regulator of oxidative stress response, leading to its downregulation and accumulation of reactive oxygen species (ROS). It has been demonstrated that both natural and synthetic compounds can reactivate NRF2 expression inhibiting the methylation status of its promoter by downregulation of DNA methyltransferases (DNMTs) and histone deacetylases (HDACs). Interestingly, NRF2 re-expression significantly reduced prostate cancer onset in TRAMP mice highlighting an important role of NRF2 in prostate tumorigenesis. We analysed the current literature regarding the role of natural and synthetic compounds in modulating NRF2 pathway in TRAMP mice, an in vivo model of prostate cancer, to give an overview on prostate carcinogenesis and its possible prevention. We can conclude that specific natural and synthetic compounds can downregulate DNMTs and/or HDACs inhibiting the methylation status of NRF2 promoter, then reactivating the expression of NRF2 protecting normal prostatic cells from ROS damage and tumorigenesis.

Quercetin-Resveratrol Combination for Prostate Cancer Management in TRAMP Mice.

Prostate Cancer (PCa) is a leading cause of cancer-related morbidity and mortality in men. Therefore, novel mechanistically-driven approaches are needed for PCa management. Here, we determined the effects of grape antioxidants quercetin and/or resveratrol (60 and 600 mg/kg, respectively, in diet) against PCa in Transgenic Adenocarcinoma of Mouse Prostate (TRAMP)-model in prevention and intervention settings. We found resveratrol alone and in combination significantly inhibited prostate tumorigenesis in prevention setting, while the same was seen only in combination after intervention. The observed effects were associated with marked inhibition in proliferation, oxidative stress, and tumor survival markers, and induced apoptosis markers. Utilizing PCa PCR array analysis with prevention tumor tissues, we identified that quercetin–resveratrol modulates genes involved in promoter methylation, cell cycle, apoptosis, fatty acid metabolism, transcription factors, androgen response, PI3K/AKT and PTEN signaling. Ingenuity Pathway Analysis (IPA) identified IGF1 and BCL2 as central players in two gene networks. Functional annotation predicted increased apoptosis and inhibited cell viability/proliferation, hyperplasia, vasculogenesis, and angiogenesis with dual treatment. Furthermore, IPA predicted upstream inhibition of major PCa signaling VEGF, Ca2+, PI3K, CSF2, PTH). Based on PCR array, we identified decreased levels of EGFR, EGR3, and IL6, and increased levels of IGFBP7 and NKX3.1, overall supporting anti-PCa effects of quercetin–resveratrol.

Icaritin reduces prostate cancer progression via inhibiting high-fat diet-induced serum adipokine in TRAMP mice model.

Objective: Obesity resulting from high-fat diets has a close relationship with the morbidity and mortality associated with Prostate cancer (PCa) in males. The anti-cancer role of Icaritin (ICT, a traditional Chinese herbal medicine) has been reported in several types of cancer including PCa. Adipokines are novel adipocyte-specific secretory protein, which plays a key role in the development of various diseases including obesity, diabetes, atherosclerosis, and cancer. However, the function of ICT and the molecular mechanisms underlying its role in PCa regression through modulation of adipokines have not been studied. Here, we assessed the anti-cancer properties of ICT under the influence of human epidermal growth factor receptor type 2 (HER2) pathway modulating adipokines in obese PCa models.Materials and Methods: In this study, we used transgenic adenocarcinoma of mouse prostate (TRAMP), a well-established animal model for the study of PCa pathogenesis. All the animals were fed on a high-fat diet (HFD with 40% fat) and divided into two groups, one received ICT solution of 30 mg/kg body bwt (i.p) while the other group served as control without any ICT treatment. The mortality rate, tumor formation and fat ratio were assessed by histopathological and magnetic resonance analysis at different time points of 20th, 24th and 28th weeks. The protein expression of HER2 and serum levels of adipokines were measured using western blotting, IHC and multiplex immunoassays. The PCa grade in 12 TRAMP mice were longitudinally evaluated to visualize PCa development and progression upon post-surgery using PET/CT scanning.Results: We observed that ICT treatment significantly reduces the total mortality rate of TRAMP mice (p = 0.045) and the percentage of prostate intraepithelial neoplasia (PIN) or PCa (p = 0.029). Interestingly, significantly decreased levels of leptin (p = 0.006 @20th wk) and the elevated levels of adiponectin (p = 0.030 @20th wk) were observed in different subgroups upon ICT treatment in a time-dependent manner. In addition, a decrease level of HER2 (p = 0.032 @28th wk) and an elevated level of PEA3 (p = 0.014 @28th wk) were also detected in ICT treated group. The PET/CT-based imaging showed that ICT vs non-ICT treated mice had different standard uptake value and metastasis.Discussion and Conclusion: Our results showed potent anti-cancer properties of ICT through the modulation of adipokine secretion may alter the expression and activation of HER2 pathway as an alternative mechanism to prevent PCa progression. Altogether, our findings indicate that ICT could be a promising cancer preventive agent with the potential to target and eradicate tumor cells in obese PCa patients.

Reversal of the Warburg phenomenon in chemoprevention of prostate cancer by sulforaphane.

Inhibition of metabolic re-programming represents an attractive approach for prevention of prostate cancer. Studies have implicated increased synthesis of fatty acids or glycolysis in pathogenesis of human prostate cancers. We have shown previously that prostate cancer prevention by sulforaphane (SFN) in Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model is associated with inhibition of fatty acid metabolism. This study utilized human prostate cancer cell lines (LNCaP, 22Rv1 and PC-3), two different transgenic mouse models (TRAMP and Hi-Myc) and plasma specimens from a clinical study to explore the glycolysis inhibition potential of SFN. We found that SFN treatment: (i) decreased real-time extracellular acidification rate in LNCaP, but not in PC-3 cell line; (ii) significantly downregulated expression of hexokinase II (HKII), pyruvate kinase M2 and/or lactate dehydrogenase A (LDHA) in vitro in cells and in vivo in neoplastic lesions in the prostate of TRAMP and Hi-Myc mice; and (iii) significantly suppressed glycolysis in prostate of Hi-Myc mice as measured by ex vivo1H magnetic resonance spectroscopy. SFN treatment did not decrease glucose uptake or expression of glucose transporters in cells. Overexpression of c-Myc, but not constitutively active Akt, conferred protection against SFN-mediated downregulation of HKII and LDHA protein expression and suppression of lactate levels. Examination of plasma lactate levels in prostate cancer patients following administration of an SFN-rich broccoli sprout extract failed to show declines in its levels. Additional clinical trials are needed to determine whether SFN treatment can decrease lactate production in human prostate tumors.

Nexrutine® preserves muscle mass similar to exercise in prostate cancer mouse model.

Muscle loss is a debilitating side effect to prostate cancer (PCa) experienced by nearly 60% of men. The purpose of this study was to test the hypothesis that Nexrutine®, a bark extract from the Phellodendrum amurense, can protect against prostate cancer induced muscle loss in a similar manner as exercise, using the transgenic adenocarcinoma of mouse prostate (TRAMP) model. Forty‐five, 8‐ to 10‐week old TRAMP mice were randomized to either control, Nexrutine® (600 mg/kg pelleted in chow) or exercise (voluntary wheel running). Mice were serially sacrificed at weeks 4, 8, 12, and 20, at which time either the left or right gastrocnemius muscle was harvested, weighted, and frozen. Proteolysis inducing factor (PIF), ubiquitin, and NF‐κB concentrations were quantified using ELISA kits. Nexrutine® and exercise were equally able to protect TRAMP mice against PCa‐induced muscle loss (P = 0.04). Both interventions decreased intramuscular PIF concentrations at 20 weeks compared to control (P < 0.05). A treatment effect was also observed when all time points were combined with exercise significantly lowering PIF concentrations (P < 0.01). Exercise significantly lowered intramuscular ubiquitin concentrations in weeks 4, 8, and 20 compared to control mice (P < 0.001). A treatment effect was also observed with exercise significantly lowering ubiquitin compared to control mice (P < 0.001). No significant changes were observed for NF‐κB. The results of this investigation demonstrate that PCa‐induced muscle loss can be attenuated with the herbal supplement Nexrutine®. This investigation provides preliminary evidence to support continued research into Nexrutine® as a potential exercise analog in protecting against muscle loss.

High dietary fat intake lowers serum equol concentration and promotes prostate carcinogenesis in a transgenic mouse prostate model

BackgroundConsumption of diet high in soy products is suggested to contribute to lower prostate cancer incidence in Asian men. But little has been known about the influences of dietary patterns on gut microbiota and microbiota-mediated isoflavone metabolism. Here, we determined the influences of western pattern diet on prostate carcinogenesis, gut microbiota and microbiota-mediated equol metabolism using a transgenic adenocarcinoma of mouse prostate (TRAMP) model.MethodsWe mimicked the western pattern diet using a high fat diet (HFD). TRAMP mice were fed with either control diet (CD) or HFD. At the age of 24 weeks, mice were orally administered daidzein over a 4-day period, and then sacrificed. The serum daidzein and equol were analyzed by ultra high performance liquid chromatography. Fecal microbiome was analyzed with fecal 16S rRNA pyrosequencing, and prostate was dissected and performed with histopathology.ResultsHFD could promote prostate carcinogenesis in TRAMP mice (p = 0.045). The daidzein showed no significant differences between CD and HFD groups, while equol was significantly decreased in HFD group (p = 0.019). Fecal microbiotas differed between the two groups, 21 microbial phylotypes were increased and 11 phylotypes were decreased in abundance in HFD group, including decreased abundance of equol-producing bacterium Adlercreutzia (0.08% vs. 0.27%).ConclusionsHFD may promote prostate carcinogenesis through adversely affecting equol-producing bacterium. Further functional validations are required to ascertain the mechanism of those HFD-responsive bacteria in carcinogenesis.

Association of anti‐inflammatory and antiangiogenic therapies negatively influences prostate cancer progression in TRAMP mice

Chronic inflammation has been implicated in cancer etiology and angiogenesis is stimulated in this disease. In prostate, the crosstalk between malignant epithelial cells and their microenvironment is an essential step of tumorigenesis during which glandular stroma undergo changes designated as reactive stroma. Thus, the aim herewith was to evaluate the effects of associating anti-inflammatory and antiangiogenic therapies on cancer progression, correlating them with steroid hormone receptor (AR and ERα), reactive stroma (vimentin, αSMA, and TGF-β), and cell proliferation (PCNA) markers expression in the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model. TRAMP mice (12-week old) were divided into the groups: Control (TRCON): received the vehicles used for drug dilution; Celecoxib (TRCEL): received oral doses of the anti-inflammatory drug celecoxib (15 mg/kg) twice daily; Nintedanib (TRNTB): received oral doses of the antiangiogenic drug nintedanib (10 mg/kg) daily; Nintedanib+Celecoxib (TRNTCEL): received the combination of drugs. After 6 weeks, mice were euthanized and ventral prostate samples were harvested for morphological, immunohistochemical, and Western blotting analyses. While celecoxib led to fibromuscular hypertrophy attenuation, nintedanib significantly reduced the incidence of well-differentiated adenocarcinoma (WDAC) foci in relation to controls, both when administered per se or in association to celecoxib. Furthermore, drug combination was associated with unique effects, including lower incidence of HGPIN lesions; lower AR stromal distribution; changes in ERα localization from epithelial nuclei to stroma as well as significant decrease of TGF-β levels and associated angiogenesis. In parallel, all treatments applied resulted in reduced inflammatory marker and vimentin (VIM) expression. Celecoxib plus nintedanib is an effective antitumor combination against prostate cancer progression in TRAMP mice, showing remarkable efficacy in relation to isolated therapies. Importantly, this efficacy might be due to drug association effect on driving AR and mainly ERα distribution in the prostatic tissue towards benign patterns. In addition, celecoxib and nintedanib impaired the development of a stromal reaction by reducing the recruitment of reactive stroma cells and maintaining a normal smooth muscle cell-rich prostate stroma in TRAMP mice. Collectively, these findings pointed to the beneficial effects of combining anti-inflammatory and antiangiogenic strategies to prevent or delay prostatic tumorigenesis.

Inhibition of Glycolysis in Prostate Cancer Chemoprevention by Phenethyl Isothiocyanate.

We have shown previously that dietary administration of phenethyl isothiocyanate (PEITC), a small molecule from edible cruciferous vegetables, significantly decreases the incidence of poorly differentiated prostate cancer in Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice without any side effects. In this study, we investigated the role of c-Myc-regulated glycolysis in prostate cancer chemoprevention by PEITC. Exposure of LNCaP (androgen-responsive) and 22Rv1 (castration-resistant) human prostate cancer cells to PEITC resulted in suppression of expression as well as transcriptional activity of c-Myc. Prostate cancer cell growth inhibition by PEITC was significantly attenuated by stable overexpression of c-Myc. Analysis of the RNA-Seq data from The Cancer Genome Atlas indicated a significant positive association between Myc expression and gene expression of many glycolysis-related genes, including hexokinase II and lactate dehydrogenase A Expression of these enzyme proteins and lactate levels were decreased upon PEITC treatment in prostate cancer cells, and these effects were significantly attenuated by ectopic expression of c-Myc. A normal prostate stromal cell line (PrSC) was resistant to lactic acid suppression by PEITC treatment. Prostate cancer chemoprevention by PEITC in TRAMP mice was associated with a significant decrease in plasma lactate and pyruvate levels. However, a 1-week intervention with 10 mg PEITC (orally, 4 times/day) was not sufficient to decrease lactate levels in the serum of human subjects. These results indicated that although prostate cancer prevention by PEITC in TRAMP mice was associated with suppression of glycolysis, longer than 1-week intervention might be necessary to observe such an effect in human subjects. Cancer Prev Res; 11(6); 337-46. ©2018 AACR.

Comparing The Effects Of Nexrutine And Exercise In Modulating The Pathophysiology Of Cachexia In Treatment Naïve Prostate Cancer Mouse Model

Oncologists encourage nutritional and physical interventions to improve outcomes in cancer patients with cachexia; however, it is inconclusive how these interventions affects the pathophysiology of cachexia. PURPOSE: To compare the effects of Nexrutine® (Nex; a natural bark extract of the Amur cork tree) and exercise in modulating the pathophysiology associated with cachexia in treatment naive transgenic adenocarcinoma of mouse prostate (TRAMP) model. METHODS: Forty-five, 10-week old male TRAMP mice were randomized to control (Con), Nex (600 mg/kg pelleted into chow) or exercise (Ex; voluntary wheel running). At 4, 8, 12 and 20 weeks, gastrocnemius muscle was collected to quantify intramuscular IGF-1, myostatin, TNF-α, proteolysis-inducing factor (PIF) and ubiquitin (Ub). An ANOVA with Tukey’s post hoc test was done with significance set at p<0.05. RESULTS: Analysis of gastrocnemius mass revealed significant group differences (F=4.159, p=0.02) with both Nex and Ex groups having greater mass compared to Con (p<0.05). A treatment response was observed for myostatin (F=4.762; p=0.01), PIF (F= 8.633, p=0.001) and Ub (F=19.55, p<0.001). Specifically, Ex mice had significantly lower concentrations of myostatin, PIF and Ub compared to Con (p<0.01). Group comparisons at 20 weeks showed significantly lower concentrations of PIF (F= 22.85, p<0.001) with Ex (p<0.001) and Nex (p=0.03) significantly lowering PIF concentrations compared to Con. Time point comparisons for Ub revealed significant differences at weeks 4 (F=32.35, p<0.001) and week 8 (F=16.24,p=0.002), respectively, with Ex mice having significantly lower concentrations of Ub compared to Con mice (p=0.004) at both time points. CONCLUSION: The results of this study suggest that Nex and Ex similarly maintain muscle mass in treatment naïve TRAMP mice by reducing tumor specific cachectic protein PIF. Exercise was capable of reducing downstream Ub; however, the mechanisms by which Nex elicits a protective effect require further study.

Prostate cancer chemoprevention by sulforaphane in a preclinical mouse model is associated with inhibition of fatty acid metabolism.

Increased de novo synthesis of fatty acids is a rather unique and targetable mechanism of human prostate cancer. We have shown previously that oral administration of sulforaphane (SFN) significantly inhibits the incidence and/or burden of prostatic intraepithelial neoplasia and well-differentiated adenocarcinoma in TRansgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice. The present study used cellular models of prostate cancer and archived plasma/adenocarcinoma tissues and sections from the TRAMP study to demonstrate inhibition of fatty acid synthesis by SFN treatment in vitro and in vivo. Treatment of androgen-responsive (LNCaP) and castration-resistant (22Rv1) human prostate cancer cells with SFN (5 and 10 μM) resulted in downregulation of protein and mRNA levels of acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FASN), but not ATP citrate lyase. Protein and mRNA levels of carnitine palmitoyltransferase 1A (CPT1A), which facilitates fatty acid uptake by mitochondria for β-oxidation, were also decreased following SFN treatment in both cell lines. Immunohistochemistry revealed a significant decrease in expression of FASN and ACC1 proteins in prostate adenocarcinoma sections of SFN-treated TRAMP mice when compared with controls. SFN administration to TRAMP mice resulted in a significant decrease in plasma and/or prostate adenocarcinoma levels of total free fatty acids, total phospholipids, acetyl-CoA and ATP. Consistent with these results, number of neutral lipid droplets was lower in the prostate adenocarcinoma sections of SFN-treated TRAMP mice than in control tumors. Collectively, these observations indicate that prostate cancer chemoprevention by SFN in TRAMP mice is associated with inhibition of fatty acid metabolism.

High-fat diet-induced adipokine and cytokine alterations promote the progression of prostate cancer in vivo and in vitro.

High-fat diet (HFD) -induced obesity is associated with more aggressive and lethal prostate cancer (PCa) in males, although the exact underlying mechanisms remain unclear. In the present study, transgenic adenocarcinoma of mouse prostate (TRAMP) models fed on an HFD (40% fat) or a control diet (CD; 16% fat) were generated, and cancer differentiation, local invasion and metastasis were compared at 20, 24 and 28 weeks. Mouse sera from each group were collected, and adipokines and cytokines were measured using multiplex immunoassays. HFD-sera and CD-sera were additionally processed into conditioned media (2.5% mixed sera), and in vitro studies were conducted to determine the proliferation, migration and invasion of cancer cells when conditioned media were used for culture. In TRAMP mice, HFD feeding increased body weight and adipose tissue deposition, and promoted the progression of PCa, specifically with regard to poorer differentiation, increased local invasion and metastasis rate. Sera from HFD-fed TRAMP mice contained increased levels of leptin, and a time-dependent increasing trend in the levels of CC chemokine ligand (CCL)3, CCL4, CCL5 and CXC chemokine ligand (CXCL)10 was observed. However, no alterations were detected in the levels of adiponectin, interleukin (IL)-4, IL-5, IL-6, IL-12p70, interferon-γ, tumor necrosis factor-α, CCL2, CCL7, CCL11, CXCL1 and CXCL2. In vitro studies determined that HFD-sera-conditioned medium promoted proliferation, migration and invasion of DU145 cells, as compared with CD-sera-conditioned medium and serum-free medium. In conclusion, the results of the present study suggested that the circulating adipokine and cytokine alterations in response to excess adipose tissue deposition induced by HFD feeding contributed to PCa progression.

Nexrutine, a Viable Exercise Mimetic for Prostate Cancer Prevention

Epidemiological data suggests that physical activity (PA) has the potential to decrease the risk of high-grade prostate cancer (PCa). However, for various reasons, cancer patients fail to meet minimum standards for physical activity. For this reason, efforts need to be made to discover biologics that confer the physiological benefits of exercise, serving as an “exercise mimetic.” Previous work by our group has discovered that the natural product Nexrutine® (Nx), a bark extract of the phellodendrun amurense, can inhibit tumor development in prostate, pancreatic and skin cancers. PURPOSE: To compare the effectiveness of Nx and exercise in modulating carcinogenesis of the prostate using the transgenic adenocarcinoma of mouse prostate (TRAMP) model. METHODS: 10-week old, male TRAMP mice were randomized to exercise, Nx or control groups (n=15 each). Mice randomized to the exercise group were given access to a running wheel and Nx treated mice were fed 600 mg/kg pelleted into their chow. Mice were scarified at weeks 4, 8, 12 and 20 weeks. Mice were monitored weekly for tumor development, activity and food consumption. Efficacy of exercise and Nexrutine was determined by histopathological evaluation of the prostate and tissue expression of pAkt and p65, key signaling proteins for carcinogenesis. One-way analysis of variance was performed with significance set at p<0.05. RESULTS: No significant pathological changes were observed as a function of time, therefore, data were pooled for analysis. Animals on exercise intervention group ran an average of 4.4 km/day. Both exercise and Nx groups presented with palpable tumors 4 weeks later than the control group. While 100% of animals developed tumors (varying stages), Nx treated and exercising TRAMP mice had fewer poorly differentiated tumors compared to controls (p<0.05). Only Nexrutine expressed lower pAkt in tumors. No differences were seen in p65 expression. CONCLUSIONS: Our data provides preliminary evidence that Nx can act as an exercise mimetic in protecting against tumor development in prostate cancer. Though both Nx and exercise decreased advanced stage tumors, only Nx has lower pAkt expression. Therefore, continued efforts need to be made to decipher the mechanisms by which exercise reduces tumors development.

Fatty Acid Synthesis Intermediates Represent Novel Noninvasive Biomarkers of Prostate Cancer Chemoprevention by Phenethyl Isothiocyanate.

Increased de novo synthesis of fatty acids is a distinctive feature of prostate cancer, which continues to be a leading cause of cancer-related deaths among American men. Therefore, inhibition of de novo fatty acid synthesis represents an attractive strategy for chemoprevention of prostate cancer. We have shown previously that dietary feeding of phenethyl isothiocyanate (PEITC), a phytochemical derived from edible cruciferous vegetables such as watercress, inhibits incidence and burden of poorly differentiated prostate cancer in transgenic adenocarcinoma of mouse prostate (TRAMP) model. The current study was designed to test the hypothesis of whether fatty acid intermediate(s) can serve as noninvasive biomarker(s) of prostate cancer chemoprevention by PEITC using archived plasma and tumor specimens from the TRAMP study as well as cellular models of prostate cancer. Exposure of prostate cancer cells (LNCaP and 22Rv1) to pharmacologic concentrations of PEITC resulted in downregulation of key fatty acid metabolism proteins, including acetyl-CoA carboxylase 1 (ACC1), fatty acid synthase (FASN), and carnitine palmitoyltransferase 1A (CPT1A). The mRNA expression of FASN and CPT1A as well as acetyl-CoA levels were decreased by PEITC treatment in both cell lines. PEITC administration to TRAMP mice also resulted in a significant decrease in tumor expression of FASN protein. Consistent with these findings, the levels of total free fatty acids, total phospholipids, triglyceride, and ATP were significantly lower in the plasma and/or prostate tumors of PEITC-treated TRAMP mice compared with controls. The current study is the first to implicate inhibition of fatty acid synthesis in prostate cancer chemoprevention by PEITC. Cancer Prev Res; 10(5); 279-89. ©2017 AACR.

Prostate Cancer Xenograft Inhibitory Activity and Pharmacokinetics of Decursinol, a Metabolite of Angelica gigas Pyranocoumarins, in Mouse Models.

We have previously shown that the ethanol extract of dried Angelica gigas Nakai (AGN) root exerts anticancer activity against androgen receptor (AR)-negative human DU145 and PC-3 prostate cancer xenografts and primary carcinogenesis in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. The major pyranocoumarin isomers decursin (D) and decursinol angelate (DA), when provided at equi-molar intake to that provided by AGN extract, accounted for the inhibitory efficacy against precancerous epithelial lesions in TRAMP mice. Since we and others have shown in rodents and humans that D and DA rapidly and extensively convert to decursinol, here we tested whether decursinol might be an in vivo active compound for suppressing xenograft growth of human prostate cancer cells expressing AR. In SCID-NSG mice carrying subcutaneously inoculated human LNCaP/AR-Luc cells overexpressing the wild type AR, we compared the efficacy of 4.5[Formula: see text]mg decursinol per mouse with equi-molar dose of 6[Formula: see text]mg D/DA per mouse. The result showed that decursinol decreased xenograft tumor growth by 75% and the lung metastasis, whereas D/DA exerted a much less effect. Measurement of plasma decursinol concentration, at 3[Formula: see text]h after the last dose of respective dosing regimen, showed higher circulating level in the decursinol-treated NSG mice than in the D/DA-treated mice. In a subsequent single-dose pharmacokinetic experiment, decursinol dosing led to 3.7-fold area under curve (AUC) of plasma decursinol over that achieved by equi-molar D/DA dosing. PK advantage notwithstanding, decursinol represents an active compound to exert in vivo prostate cancer growth and metastasis inhibitory activity in the preclinical model.

β1 integrin- and JNK-dependent tumor growth upon hypofractionated radiation.

Radiation therapy is an effective cancer treatment modality although tumors invariably become resistant. Using the transgenic adenocarcinoma of mouse prostate (TRAMP) model system, we report that a hypofractionated radiation schedule (10 Gy/day for 5 consecutive days) effectively blocks prostate tumor growth in wild type (β1wt /TRAMP) mice as well as in mice carrying a conditional ablation of β1 integrins in the prostatic epithelium (β1pc-/- /TRAMP). Since JNK is known to be suppressed by β1 integrins and mediates radiation-induced apoptosis, we tested the effect of SP600125, an inhibitor of c-Jun amino-terminal kinase (JNK) in the TRAMP model system. Our results show that SP600125 negates the effect of radiation on tumor growth in β1pc-/- /TRAMP mice and leads to invasive adenocarcinoma. These effects are associated with increased focal adhesion kinase (FAK) expression and phosphorylation in prostate tumors in β1pc-/- /TRAMP mice. In marked contrast, radiation-induced tumor growth suppression, FAK expression and phosphorylation are not altered by SP600125 treatment of β1wt /TRAMP mice. Furthermore, we have reported earlier that abrogation of insulin-like growth factor receptor (IGF-IR) in prostate cancer cells enhances the sensitivity to radiation. Here we further explore the β1/IGF-IR crosstalk and report that β1 integrins promote cell proliferation partly by enhancing the expression of IGF-IR. In conclusion, we demonstrate that β1 integrin-mediated inhibition of JNK signaling modulates tumor growth rate upon hypofractionated radiation.

Copper as a target for prostate cancer therapeutics: copper-ionophore pharmacology and altering systemic copper distribution.

Copper-ionophores that elevate intracellular bioavailable copper display significant therapeutic utility against prostate cancer cells in vitro and in TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice. However, the pharmacological basis for their anticancer activity remains unclear, despite impending clinical trails. Herein we show that intracellular copper levels in prostate cancer, evaluated in vitro and across disease progression in TRAMP mice, were not correlative with copper-ionophore activity and mirrored the normal levels observed in patient prostatectomy tissues (Gleason Score 7 & 9). TRAMP adenocarcinoma cells harbored markedly elevated oxidative stress and diminished glutathione (GSH)-mediated antioxidant capacity, which together conferred selective sensitivity to prooxidant ionophoric copper. Copper-ionophore treatments [CuII(gtsm), disulfiram & clioquinol] generated toxic levels of reactive oxygen species (ROS) in TRAMP adenocarcinoma cells, but not in normal mouse prostate epithelial cells (PrECs). Our results provide a basis for the pharmacological activity of copper-ionophores and suggest they are amendable for treatment of patients with prostate cancer. Additionally, recent in vitro and mouse xenograft studies have suggested an increased copper requirement by prostate cancer cells. We demonstrated that prostate adenocarcinoma development in TRAMP mice requires a functional supply of copper and is significantly impeded by altered systemic copper distribution. The presence of a mutant copper-transporting Atp7b protein (tx mutation: A4066G/Met1356Val) in TRAMP mice changed copper-integration into serum and caused a remarkable reduction in prostate cancer burden (64% reduction) and disease severity (grade), abrogating adenocarcinoma development. Implications for current clinical trials are discussed.

Gene expression signatures associated with suppression of TRAMP prostate carcinogenesis by a kavalactone-rich Kava fraction.

Kava (Piper methysticum Forster) extract and its major kavalactones have been shown to block chemically induced lung tumor initiation in mouse models. Here we evaluated the chemopreventive effect of a kavalactone-rich Kava fraction B (KFB), free of flavokavains, on carcinogenesis in a transgenic adenocarcinoma of mouse prostate (TRAMP) model and characterized the prostate gene expression signatures. Male C57BL/6 TRAMP mice were fed AIN93M diet with or without 0.4% KFB from 8 wk of age. Mice were euthanized at 16 or 28 wk. The growth of the dorsolateral prostate (DLP) lobes in KFB-treated TRAMP mice was inhibited by 66% and 58% at the respective endpoint. Anterior and ventral prostate lobes in KFB-treated TRAMP mice were suppressed by 40% and 49% at 28 wk, respectively. KFB consumption decreased cell proliferation biomarker Ki-67 and epithelial lesion severity in TRAMP DLP, without detectable apoptosis enhancement. Real time qRT-PCR detection of mRNA from DLP at 28 wk showed decreased expression of cell cycle regulatory genes congruent with Ki-67 suppression. Microarray profiling of DLP mRNA indicated that "oncogene-like" genes related to angiogenesis and cell proliferation were suppressed by KFB but tumor suppressor, immunity, muscle/neuro, and metabolism-related genes were upregulated by KFB in both TRAMP and WT DLP. TRAMP mice fed KFB diet developed lower incidence of neuroendocrine carcinomas (NECa) (2 out of 14 mice) than those fed the basal diet (8 out of 14 mice, χ2 = 5.6, P < 0.025). KFB may, therefore, inhibit not only TRAMP DLP epithelial lesions involving multiple molecular pathways, but also NECa. © 2016 Wiley Periodicals, Inc.

Prenatal exposure to soy and selenium reduces prostate cancer risk factors in TRAMP mice more than exposure beginning at six weeks.

Diets high in soy and selenium (Se) decrease prostate cancer risk factors in healthy rats. The purpose of this study was to determine whether treatment with high levels of soy and/or supplemental Se would decrease prostate cancer risk factors in the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mouse, and whether timing of the introduction of these nutrients would affect risk reduction. Male hemizygous [C57BL/6 × FVB]F1 TRAMP mice were exposed to stock diets high or devoid of soy, with or without a supplement of Se-methylselenocysteine (MSC) starting at conception (10 mg Se/L in drinking water of pregnant/nursing dams; daily bolus of 4 mg Se/kg body weight to pups after weaning) or at 6 weeks of age in a 2 × 2 factorial design. Mice were killed at 12 weeks (n per dietary treatment = 20-30). Liver and serum Se concentrations were increased by MSC supplementation (P < 0.001), high-soy diet (P < 0.05), and initiation of dietary treatments at conception (P < 0.05). MSC supplementation had greater effects in mice fed the zero-soy basal diet, compared to the high-soy formulation (Pinteraction < 0.01). These same three interventions, individually and interactively, decreased body weight and epididymal fat pad weights, and steady state levels of mRNA for Cyp19a1 (aromatase) and Srd5a1 (5α-reductase). In contrast, MSC was the only treatment that decreased urogenital tract weights (P < 0.001), serum IGF-1 levels (P < 0.002), and Gleason scores (P < 0.05). Supplemental MSC reduces risk of prostate cancer in TRAMP mice. Basal diet composition (zero- vs. high-soy) can modify MSC's chemopreventive effects. Initiation of dietary treatments from conception maximizes chemopreventive effects of MSC. Prenatal Se status may have long-lasting effects on development and progression of prostate cancer.

Nimbolide-Induced Oxidative Stress Abrogates STAT3 Signaling Cascade and Inhibits Tumor Growth in Transgenic Adenocarcinoma of Mouse Prostate Model.

Prostate cancer (PCa) is one of the most commonly diagnosed cancers worldwide. Currently available therapies for metastatic PCa are only marginally effective, hence novel treatment modalities are urgently required. Considerable evidence(s) suggest that deregulated activation of oncogenic transcription factor, signal transducer and activator of transcription 3 (STAT3) plays a pivotal role in the development and progression of PCa. Thus, agents that can abrogate STAT3 activation could form the basis of novel therapy for PCa patients. In the present study, we analyzed whether the potential anticancer effects of nimbolide (NL), a limonoid triterpene derived from Azadirachta indica, against PCa cell lines and transgenic adenocarcinoma of mouse prostate (TRAMP) model are mediated through the negative regulation of STAT3 pathway. Data from the in vitro studies indicated that NL could significantly inhibit cell viability, induce apoptosis, and suppress cellular invasion and migration. Interestingly, NL also abrogated STAT3 activation and this effect was found to be mediated via an increased production of reactive oxygen species (ROS) due to GSH/GSSG imbalance. Oral administration of NL significantly suppressed the tumor growth and metastasis in TRAMP mouse model without exhibiting any significant adverse effects. The present study demonstrates the critical role of GSH/GSSG imbalance-mediated ROS production contributing to the STAT3 inhibitory and tumor-suppressive effect of NL in PCa. Overall, our findings indicate that NL exhibits significant anticancer effects in PCa that may be primarily mediated through the ROS-regulated inhibition of STAT3 signaling cascade.