Enhanced Chemoprevention of Prostate Cancer by Combining Arctigenin with Green Tea and Quercetin in Prostate-Specific Phosphatase and Tensin Homolog Knockout Mice.

The low bioavailability of most phytochemicals limits their anticancer effects in humans. The present study was designed to test whether combining arctigenin (Arc), a lignan mainly from the seed of Arctium lappa, with green tea (GT) and quercetin (Q) enhances the chemopreventive effect on prostate cancer. We performed in vitro proliferation studies on different cell lines. We observed a strong synergistic anti-proliferative effect of GT+Q+Arc in exposing androgen-sensitive human prostate cancer LNCaP cells. The pre-malignant WPE1-NA22 cell line was more sensitive to this combination. No cytotoxicity was observed in normal prostate epithelial PrEC cells. For an in vivo study, 3-week-old, prostate-specific PTEN (phosphatase and tensin homolog) knockout mice were treated with GT+Q, Arc, GT+Q+Arc, or the control daily until 16 weeks of age. In vivo imaging using prostate-specific membrane antigen (PSMA) probes demonstrated that the prostate tumorigenesis was significantly inhibited by 40% (GT+Q), 60% (Arc at 30 mg/kg bw), and 90% (GT+Q+Arc) compared to the control. A pathological examination showed that all control mice developed invasive prostate adenocarcinoma. In contrast, the primary lesion in the GT+Q and Arc alone groups was high-grade prostatic intraepithelial neoplasia (PIN), with low-grade PIN in the GT+Q+Arc group. The combined effect of GT+Q+Arc was associated with an increased inhibition of the androgen receptor, the PI3K/Akt pathway, Ki67 expression, and angiogenesis. This study demonstrates that combining Arc with GT and Q was highly effective in prostate cancer chemoprevention. These results warrant clinical trials to confirm the efficacy of this combination in humans.

A 17-Gene Genomic Prostate Score Assay Provides Independent Information on Adverse Pathology in the Setting of Combined Multiparametric Magnetic Resonance Imaging Fusion Targeted and Systematic Prostate Biopsy.

PURPOSE: Multiparametric magnetic resonance imaging and biopsy based molecular tests such as the 17-gene Oncotype DX® Genomic Prostate Score™ assay are increasingly performed to improve risk stratification in men with clinically localized prostate cancer. The prostate score assay was previously shown to be a significant independent predictor of adverse pathology findings at radical prostatectomy in men diagnosed by systematic biopsies only. Therefore, we investigated the ability of the prostate score assay to predict adverse pathology findings in the setting of magnetic resonance imaging guided prostate biopsy. MATERIALS AND METHODS: We identified men diagnosed with NCCN® (National Comprehensive Cancer Network®) very low, low or intermediate risk prostate cancer who underwent simultaneous multiparametric magnetic resonance imaging fusion targeted and systematic prostate biopsy with subsequent radical prostatectomy within 6 months. Prostate score assay testing was performed on biopsy tissue with the highest Gleason score. The primary outcome of the study was adverse pathology findings, defined as Gleason score 4 + 3 or greater disease and/or pT3+ at radical prostatectomy. Independent predictors of adverse pathology findings were determined in a multivariable model to adjust for clinical parameters. RESULTS: A total of 134 men were eligible for primary analysis. On univariable analysis the UCLA score, magnetic resonance imaging, prostate score assay results and biopsy Gleason score were significant predictors of adverse pathology findings. After multivariable adjustment prostate score assay values remained a significant predictor of adverse pathology results (prostate score assay per 20 U OR 3.28, 95% CI 1.74-6.62, p <0.001). A wide and overlapping distribution of prostate score assay results was seen across PI-RADS® (Prostate Imaging Reporting and Data System) version 2 scores. CONCLUSIONS: The prostate score assay result is an independent predictor of adverse pathology findings in patients who were diagnosed with very low, low or intermediate risk prostate cancer in the setting of multiparametric magnetic resonance imaging fusion prostate biopsy. This assay can be useful as an independent technology or an adjunct technology to multiparametric magnetic resonance imaging to individualize risk stratification of low and intermediate risk prostate cancer.

Kinase profiling of liposarcomas using RNAi and drug screening assays identified druggable targets.

BACKGROUND: Liposarcoma, the most common soft tissue tumor, is understudied cancer, and limited progress has been made in the treatment of metastatic disease. The Achilles heel of cancer often is their kinases that are excellent therapeutic targets. However, very limited knowledge exists of therapeutic critical kinase targets in liposarcoma that could be potentially used in disease management. METHODS: Large RNAi and small-molecule tyrosine kinase inhibitor screens were performed against the proliferative capacity of liposarcoma cell lines of different subtypes. Each small molecule inhibitor was either FDA approved or in a clinical trial. RESULTS: Screening assays identified several previously unrecognized targets including PTK2 and KIT in liposarcoma. We also observed that ponatinib, multi-targeted tyrosine kinase inhibitor, was the most effective drug with anti-growth effects against all cell lines. In vitro assays showed that ponatinib inhibited the clonogenic proliferation of liposarcoma, and this anti-growth effect was associated with apoptosis and cell cycle arrest at the G0/G1 phase as well as a decrease in the KIT signaling pathway. In addition, ponatinib inhibited in vivo growth of liposarcoma in a xenograft model. CONCLUSIONS: Two large-scale kinase screenings identified novel liposarcoma targets and a FDA-approved inhibitor, ponatinib with clear anti-liposarcoma activity highlighting its potential therapy for treatment of this deadly tumor.

Prostate Cancer Antigen 3 Score Does Not Predict for Adverse Pathologic Features at Radical Prostatectomy or for Progression-free Survival in Clinically Localized, Intermediate- and High-risk Prostate Cancer.

OBJECTIVE: To evaluate whether preoperative urinary prostate cancer antigen 3 (PCA3) scores predict for adverse pathologic features (APFs) or progression-free survival (PFS) in men with intermediate- or high-risk prostate cancer (PCa) undergoing radical prostatectomy (RP). MATERIALS AND METHODS: One hundred nine men with intermediate- (n = 52) or high-risk (n = 57) PCa who underwent RP were retrospectively identified. Logistic regression analysis was performed to evaluate the association of PCA3 score with various APFs (eg, extracapsular extension, seminal vesicle invasion, etc.). Among 78 men with ≥1 year of follow-up, the association between PCA3 score and PFS was assessed using Cox regression analysis. RESULTS: At RP, 52% of patients had at least 1 APF, and with median follow-up of 2.3 years, overall 3-year PFS was 70%. PCA3 was not a significant predictor of any APF on multivariate analysis (MVA), whereas canonical predictors (eg, biopsy Gleason score and initial prostate-specific antigen) remained predictive of various APFs. No significant predictors for PFS were found on MVA, although certain canonical predictors (eg, National Comprehensive Cancer Network risk group) were significant predictors of PFS on univariate analysis (UVA). PCA3 score was not a significant predictor of PFS on either UVA or MVA. CONCLUSION: Unlike in lower risk cohorts, increasing PCA3 score was not associated with any APF in this higher risk cohort, despite enrichment for APFs, nor was it associated with PFS. Notably, multiple known preoperative predictors for APFs were significant on MVA, and multiple predictors were associated with PFS on UVA. Therefore, PCA3 may not be a useful adjunct predictive marker in men with intermediate- or high-risk PCa.

Sustained-release Formulation of Mitomycin C to the Upper Urinary Tract Using a Thermosensitive Polymer: A Preclinical Study.

OBJECTIVE: To evaluate the safety and feasibility of single and serial instillations of MitoGel into the upper urinary tract using a preclinical swine animal model. MitoGel is a novel sustained release formulation of mitomycin C (MMC) based on RTGel, a proprietary thermosensitive hydrogel technology. MitoGel is liquid at cold temperatures and solidifies to gel state at body temperature. It is intended as a treatment for upper tract urothelial carcinoma, given its ability to provide sustained release of MMC in the upper urinary tract. MATERIALS AND METHODS: We utilized 23 pigs in a 3-phase design. All animals underwent bilateral nephrostomy tube placement. During phase 1, 3 animals underwent antegrade RTGel instillation, imaging, and euthanasia within 12 hours. In phase 2, 10 pigs underwent single antegrade instillation, unilateral nephrectomy 3 days following instillation, and contralateral nephrectomy and euthanasia 30 days following instillation. During phase 3, 10 animals underwent 6 instillations over 3 weeks, followed by bilateral nephrectomy and necropsy 30 days postinstillation. MitoGel (2 mg/mL and 4 mg/mL), aqueous MMC (2 mg/mL and 4 mg/mL), and RTGel alone were evaluated. RESULTS: MitoGel remained visible within the pelvicalyceal system on fluoroscopic and computed tomography imaging for 4-6 hours. MMC plasma levels were well within acceptable safety thresholds. There was no evidence of urinary obstruction, acute kidney injury, sepsis, or myelosuppression. Histologic changes in the urinary system were mild and transient. CONCLUSION: Antegrade MitoGel delivery to the pelvicalyceal system of Yorkshire swine is feasible and safe. Further evaluation of MitoGel in human clinical trials is warranted.

Effect of Dietary Omega-3 Fatty Acids on Tumor-Associated Macrophages and Prostate Cancer Progression.

BACKGROUND: Preclinical and clinical studies suggest that a fish oil-based diet may play a role in delaying the progression of prostate cancer through a number of different mechanisms involving inflammatory pathways. Given the importance of tumor-associated macrophages (TAMs) in carcinogenesis, we hypothesized that a fish oil-based diet will inhibit TAM infiltration and delay the growth of prostate cancer. METHODS: Androgen sensitive mouse prostate cancer (MycCaP) allograft tumors were grown in fully immunocompetent FVB mice fed a high- fat fish oil (omega-3) or corn oil (omega-6) diet. Gene expression of markers for immune cell populations, cytokines, chemokines, and signaling pathways were determined by real-time PCR and western blot in tumor tissue. Cell proliferation and apoptosis in vitro were measured by MTS assay and flow cytometry. RESULTS: Tumor volumes were significantly smaller in mice in ω-3 versus the ω-6 group (P = 0.048). Gene expression of markers for M1 and M2 macrophages (F4/80, iNOS, ARG1), associated cytokines (IL-6, TNF alpha, IL-10), and the chemokine CCL-2 were also lower in the omega-3 group. Correlative in vitro studies were performed in M1 and M2 polarized macrophages and mirrored the in vivo findings. Dietary fish oil and in vitro omega-3 fatty acid administration reduced protein expression of transcription factors in the nuclear factor kappa B pathway leading to a significant decrease in gene expression of downstream targets (Bcl-2, BCL-XL, XIAP, survivin) in MycCap cells. CONCLUSIONS: These findings underscore the potential of fish oil in modulating the clinical course of human prostate cancer through the immune system. Further preclinical and clinical studies are warranted evaluating fish oil-based therapies for inhibiting the recruitment and function of M1 and M2 tumor infiltrating macrophages. Prostate 76:1293-1302, 2016. © 2016 Wiley Periodicals, Inc.

Randomized clinical trial of brewed green and black tea in men with prostate cancer prior to prostatectomy.

BACKGROUND: Preclinical and epidemiologic studies suggest chemopreventive effects of green tea (GT) and black tea (BT) in prostate cancer. In the current study we determined the effect of GT and BT consumption on biomarkers related to prostate cancer development and progression. METHODS: In this exploratory, open label, phase II trial 113 men diagnosed with prostate cancer were randomized to consume six cups daily of brewed GT, BT or water (control) prior to radical prostatectomy (RP). The primary endpoint was prostate tumor markers of cancer development and progression determined by tissue immunostaining of proliferation (Ki67), apoptosis (Bcl-2, Bax, Tunel), inflammation (nuclear and cytoplasmic nuclear factor kappa B [NFκB]) and oxidation (8-hydroxydeoxy-guanosine [8OHdG]). Secondary endpoints of urinary oxidation, tea polyphenol uptake in prostate tissue, and serum prostate specific antigen (PSA) were evaluated by high performance liquid chromatography and ELISA analysis. RESULTS: Ninety three patients completed the intervention. There was no significant difference in markers of proliferation, apoptosis and oxidation in RP tissue comparing GT and BT to water control. Nuclear staining of NFκB was significantly decreased in RP tissue of men consuming GT (P = 0.013) but not BT (P = 0.931) compared to water control. Tea polyphenols were detected in prostate tissue from 32 of 34 men consuming GT but not in the other groups. Evidence of a systemic antioxidant effect was observed (reduced urinary 8OHdG) only with GT consumption (P = 0.03). GT, but not BT or water, also led to a small but statistically significant decrease in serum prostate-specific antigen (PSA) levels (P = 0.04). CONCLUSION: Given the GT-induced changes in NFκB and systemic oxidation, and uptake of GT polyphenols in prostate tissue, future longer-term studies are warranted to further examine the role of GT for prostate cancer prevention and treatment, and possibly for other prostate conditions such as prostatitis.

Effect of a low-fat fish oil diet on proinflammatory eicosanoids and cell-cycle progression score in men undergoing radical prostatectomy.

We previously reported that a 4- to 6-week low-fat fish oil (LFFO) diet did not affect serum insulin-like growth factor (IGF)-1 levels (primary outcome) but resulted in lower omega-6 to omega-3 fatty acid ratios in prostate tissue and lower prostate cancer proliferation (Ki67) as compared with a Western diet. In this post hoc analysis, the effect of the LFFO intervention on serum pro-inflammatory eicosanoids, leukotriene B4 (LTB4) and 15-S-hydroxyeicosatetraenoic acid [15(S)-HETE], and the cell-cycle progression (CCP) score were investigated. Serum fatty acids and eicosanoids were measured by gas chromatography and ELISA. CCP score was determined by quantitative real-time reverse transcriptase PCR (RT-PCR). Associations between serum eicosanoids, Ki67, and CCP score were evaluated using partial correlation analyses. BLT1 (LTB4 receptor) expression was determined in prostate cancer cell lines and prostatectomy specimens. Serum omega-6 fatty acids and 15(S)-HETE levels were significantly reduced, and serum omega-3 levels were increased in the LFFO group relative to the Western diet group, whereas there was no change in LTB4 levels. The CCP score was significantly lower in the LFFO compared with the Western diet group. The 15(S)-HETE change correlated with tissue Ki67 (R = 0.48; P < 0.01) but not with CCP score. The LTB4 change correlated with the CCP score (r = 0.4; P = 0.02) but not with Ki67. The LTB4 receptor BLT1 was detected in prostate cancer cell lines and human prostate cancer specimens. In conclusion, an LFFO diet resulted in decreased 15(S)-HETE levels and lower CCP score relative to a Western diet. Further studies are warranted to determine whether the LFFO diet antiproliferative effects are mediated through the LTB4/BLT1 and 15(S)-HETE pathways.

Enhanced inhibition of prostate cancer xenograft tumor growth by combining quercetin and green tea.

The chemopreventive activity of green tea (GT) is limited by the low bioavailability and extensive methylation of GT polyphenols (GTPs) in vivo. We determined whether a methylation inhibitor quercetin (Q) will enhance the chemoprevention of prostate cancer in vivo. Androgen-sensitive LAPC-4 prostate cancer cells were injected subcutaneously into severe combined immunodeficiency (SCID) mice one week before the intervention. The concentration of GTPs in brewed tea administered as drinking water was 0.07% and Q was supplemented in diet at 0.2% or 0.4%. After 6-weeks of intervention tumor growth was inhibited by 3% (0.2% Q), 15% (0.4% Q), 21% (GT), 28% (GT+0.2% Q) and 45% (GT+0.4% Q) compared to control. The concentration of non-methylated GTPs was significantly increased in tumor tissue with GT+0.4% Q treatment compared to GT alone, and was associated with a decreased protein expression of catechol-O-methyltransferase and multidrug resistance-associated protein (MRP)-1. The combination treatment was also associated with a significant increase in the inhibition of proliferation, androgen receptor and phosphatidylinositol 3-kinase/Akt signaling, and stimulation of apoptosis. The combined effect of GT+0.4% Q on tumor inhibition was further confirmed in another experiment where the intervention started prior to tumor inoculation. These results provide a novel regimen by combining GT and Q to improve chemoprevention in a non-toxic manner and warrant future studies in humans.

Polyphenols in brewed green tea inhibit prostate tumor xenograft growth by localizing to the tumor and decreasing oxidative stress and angiogenesis.

It has been demonstrated in various animal models that the oral administration of green tea (GT) extracts in drinking water can inhibit tumor growth, but the effects of brewed GT on factors promoting tumor growth, including oxidant damage of DNA and protein, angiogenesis and DNA methylation, have not been tested in an animal model. To explore these potential mechanisms, brewed GT was administered instead of drinking water to male severe combined immunodeficiency (SCID) mice with androgen-dependent human LAPC4 prostate cancer cell subcutaneous xenografts. Tumor volume was decreased significantly in mice consuming GT, and tumor size was significantly correlated with GT polyphenol (GTP) content in tumor tissue. There was a significant reduction in hypoxia-inducible factor 1-alpha and vascular endothelial growth factor protein expression. GT consumption significantly reduced oxidative DNA and protein damage in tumor tissue as determined by 8-hydroxydeoxyguanosine/deoxyguanosine ratio and protein carbonyl assay, respectively. Methylation is known to inhibit antioxidative enzymes such as glutathione S-transferase pi to permit reactive oxygen species promotion of tumor growth. GT inhibited tumor 5-cytosine DNA methyltransferase 1 mRNA and protein expression significantly, which may contribute to the inhibition of tumor growth by reactivation of antioxidative enzymes. This study advances our understanding of tumor growth inhibition by brewed GT in an animal model by demonstrating tissue localization of GTPs in correlation with inhibition of tumor growth. Our results suggest that the inhibition of tumor growth is due to GTP-mediated inhibition of oxidative stress and angiogenesis in the LAPC4 xenograft prostate tumor in SCID mice.

Phase II prospective randomized trial of a low-fat diet with fish oil supplementation in men undergoing radical prostatectomy.

Preclinical studies suggest lowering dietary fat and decreasing the ratio of omega-6 to omega-3 polyunsaturated fatty acids decreases the risk of prostate cancer development and progression. We conducted a phase II randomized trial to test the effect of decreasing dietary fat combined with decreasing the dietary omega-6:omega-3 ratio on biomarkers related to prostate cancer development and progression. Patients undergoing radical prostatectomy were randomly assigned to receive a low-fat diet with 5 grams of fish oil daily (dietary omega-6:omega-3 ratio of 2:1) or a control Western diet (omega-6:omega-3 ratio of 15:1) for four to six weeks prior to surgery. The primary endpoint was change in serum insulin-like growth factor I (IGF-1) between arms. Secondary endpoints were serum IGFBP-1, prostate prostaglandin E2 levels, omega-6:omega-3 fatty acid ratios, COX-2, and markers of proliferation and apoptosis. Fifty-five patients were randomized and 48 completed the trial. There was no treatment difference in the primary outcome. Positive secondary outcomes in the low-fat fish oil versus Western group were reduced benign and malignant prostate tissue omega-6:omega-3 ratios, reduced proliferation (Ki-67 index), and reduced proliferation in an ex vivo bioassay when patient sera was applied to prostate cancer cells in vitro. In summary, four to six weeks of a low-fat diet and fish oil capsules to achieve an omega-6:omega-3 fatty acid ratio of 2:1 had no effect on serum IGF-1 levels, though in secondary analyses, the intervention resulted in decreased prostate cancer proliferation and decreased prostate tissue omega-6:omega-3 ratios. These results support further studies evaluating reduction of dietary fat with fish oil supplementation on modulating prostate cancer biology.

Effect of altering dietary omega-6/omega-3 fatty acid ratios on prostate cancer membrane composition, cyclooxygenase-2, and prostaglandin E2.

PURPOSE: To determine whether altering the dietary content of omega-6 (n-6) and omega-3 (n-3) polyunsaturated fatty acids affects the growth of androgen-sensitive prostate cancer xenografts, tumor membrane fatty acid composition, and tumor cyclooxygenase-2 and prostaglandin E(2) (PGE(2)) levels. EXPERIMENTAL DESIGN: Individually caged male severe combined immunodeficiency mice were fed isocaloric 20% kcal fat diets with the fat derived either primarily from n-6 fatty acids (n-6 group) or with the fat consisting of n-6 and n-3 fatty acids in a ratio of 1:1 (n-3 group), and injected s.c. with Los Angeles Prostate Cancer 4 (LAPC-4) cells. Tumor volumes and mouse weights were measured weekly, caloric intake was measured 3 days per week, and tumors and serum were harvested at 8 weeks postinjection. RESULTS: Tumor growth rates, final tumor volumes, and serum prostate-specific antigen levels were reduced in the n-3 group relative to the n-6 group. The n-3 group tumors had decreased proliferation (Ki67 staining) and increased apoptosis (terminal nucleotidyl transferase-mediated nick end labeling staining). In vitro proliferation of LAPC-4 cells in medium containing n-3 group serum was reduced by 22% relative to LAPC-4 cells cultured in medium containing serum from the n-6 group. The n-6/n-3 fatty acid ratios in serum and tumor membranes were lower in the n-3 group relative to the n-6 group. In addition, n-3 group tumors had decreased cyclooxygenase-2 protein and mRNA levels, an 83% reduction in PGE(2) levels, and decreased vascular endothelial growth factor expression. CONCLUSION: These results provide a sound basis for clinical trials evaluating the effect of dietary n-3 fatty acids from fish oil on tumor PGE(2) and membrane fatty acid composition, and serum and tumor biomarkers of progression in men with prostate cancer.

Risk group assessment and clinical outcome algorithm to predict the natural history of patients with surgically resected renal cell carcinoma.

PURPOSE: To create a comprehensive algorithm that can predict postoperative renal cell carcinoma (RCC) patient outcomes and response to therapy. PATIENTS AND METHODS: A prospective cohort study was performed with outcome assessment on the basis of chart review of 814 patients who underwent nephrectomy between 1989 and 2000. At diagnosis, M1 or N1/N2M0 metastatic disease (M) was present in 346 patients (43%), whereas 468 patients had no metastatic disease (NM) (N0M0). On the basis of UCLA Integrated Staging System category and the presence of metastases, patients were divided into low-risk (LR), intermediate-risk (IR), and high-risk (HR) groups. Decision boxes integrating tumor-node-metastasis staging, tumor grade, and performance status were compiled for determining a patient's risk group. RESULTS: NM-LR patients had 91% disease-specific survival at 5 years, lower recurrence rate, and better disease-free survival compared with NM-IR and HR patients. Disease progressed in 50% of NM-HR patients. Disease-specific survival of NM-HR patients who received immunotherapy (IMT) for recurrent disease was similar to that of M-LR patients treated with cytoreductive nephrectomy and adjuvant IMT. Time from recurrence to death for NM-HR patients was inferior to that for M-LR patients. After IMT, approximately 25% of M-LR and 12% of M-IR patients had long-term progression-free survival. M-HR patients did poorly despite IMT. CONCLUSION: Stratifying RCC patients into high-, intermediate-, and low-risk subgroups provides a clinically useful system for predicting outcome and provides a unique tool for risk assignment and outcome analysis. Subclassifying RCC into well-defined risk groups should allow better patient counseling and identification of both NM-HR subgroups that need adjuvant treatment and nonresponders who need alternative therapies.