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The abnormal PI3K/AKT/mTOR pathway is one of the most common genomic abnormalities in breast cancers including triple-negative breast cancer (TNBC), and pharmacologic inhibition of these aberrations has shown activity in TNBC patients. Here, we designed and identified a small-molecule Comp34 that suppresses both AKT and mTOR protein expression and exhibits robust cytotoxicity towards TNBC cells but not nontumorigenic normal breast epithelial cells. Mechanically, long noncoding RNA (lncRNA) AL354740.1-204 (also named as NUDT3-AS4) acts as a microRNA sponge to compete with AKT1/mTOR mRNAs for binding to miR-99s, leading to decrease in degradation of AKT1/mTOR mRNAs and subsequent increase in AKT1/mTOR protein expression. Inhibition of lncRNA-NUDT3-AS4 and suppression of the NUDT3-AS4/miR-99s association contribute to Comp34-affected biologic pathways. In addition, Comp34 alone is effective in cells with secondary resistance to rapamycin, the best-known inhibitor of mTOR, and displays a greater in vivo antitumor efficacy and lower toxicity than rapamycin in TNBC xenografted models. In conclusion, NUDT3-AS4 may play a proproliferative role in TNBC and be considered a relevant therapeutic target, and Comp34 presents promising activity as a single agent to inhibit TNBC through regulation of NUDT3-AS4 and miR-99s.

We evaluated if chronic consumption of quercetin (Q) with green tea extract (GTE) enhances the bioavailability of GT polyphenols (GTPs) and reduces methylation activity as previously observed in mouse xenograft tumors. In this prospective, randomized, parallel design, placebo controlled study, thirty-one men with prostate cancer consumed daily 1 gram of GTE (830 mg of GTP) with 800 mg of Q (GT + Q) or placebo (GT + PL) for four weeks before prostatectomy. First morning voided urine was collected at baseline, 3 weeks and the day of surgery, and prostate tissue on the day of surgery. In week 3, plasma concentration of GTPs and Q was measured in blood collected before and 2 hours after the morning dose. Prostate tissue epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) were detected in 67 and 93% of participants in the GT + Q group and 75 and 94% of participants in the GT + PL group. Q was increased 14-fold, 12-fold and 4.5-fold in plasma, urine, and prostate tissue, respectively, in the GT + Q compared to the GT + PL-group. There was a trend for decreased EGC levels in urine collected prior to prostatectomy in the GT + Q compared to GT + PL-group (p = 0.053). Plasma epigallocatechin (EGC) showed a trend to increase (p = 0.066) two hours after capsule intake in the GT + Q vs. the GT + PL-group. There was no significant difference between the groups in GTP content or methylation activity in prostate tissue or RBCs. No liver toxicity was observed. Although our findings are suggestive, further studies are warranted evaluating if Q alters GTP metabolism.

Exosomes are a class of small membrane-bound extracellular vesicles released by almost all cell types and present in all body fluids. Based on the studies of exosome content and their interactions with recipient cells, exosomes are now thought to mediate “targeted” information transfer. Tumor-derived exosomes (TEX) carry a cargo of molecules different from that of normal cell-derived exosomes. TEX functions to mediate distinct biological effects such as receptor discharge and intercellular cross-talk. The immune system defenses, which may initially restrict tumor progression, are progressively blunted by the broad array of TEX molecules that activate suppressive pathways in different immune cells. Herein, we provide a review of the latest research progress on TEX in the context of tumor-mediated immune suppression and discuss the potential as well as challenges of TEX as a target of immunotherapy.

Chronic nicotine exposure significantly increases hypertensive risk in smokers, but the underlying mechanisms are poorly understood. In the kidneys, 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) catalyzes the conversion from active into inactive glucocorticoids and plays a pivotal role in the regulation of blood pressure. We hypothesized that nicotine-induced blood pressure elevation is in part mediated by change in renal 11β-HSD2 leading to higher MR (mineralocorticoid receptor) occupancy. Here, we show that nicotine exposure markedly decreased the expression and activity of renal 11β-HSD2 and increased the mean systolic arterial pressure in C57BL/6J mice. Reduction of renal 11β-HSD2 expression by nicotine was correlated with the suppression of C/EBPβ (CCAAT/enhancer-binding protein-β) and activation of Akt protein kinase phosphorylation (pThr 308 Akt/PKB) within the kidney. Conversely, nicotine-treated mice had elevated renal MR and epithelial sodium channel-α abundance. Treatment with the MR antagonist spironolactone significantly decreased the elevated mean systolic blood pressure and corrected ENaC along with inhibition of pThr 308 Akt/PKB within the kidney in nicotine-treated mice. Suppression of Akt/PKB activation by spironolactone was accompanied by upregulation of renal C/EBPβ and amelioration of nicotine-mediated reduction of 11β-HSD2. Addition of nicotine to mouse renal cortical collecting duct M1 cells downregulated 11β-HSD2 and stimulated MR expression, and these effects are likely mediated by activation of Akt coupled inhibition of C/EBPβ. These findings suggest that nicotine-mediated suppression of 11β-HSD2 in the kidney may contribute to the development of nicotine/smoking-induced hypertension through decreasing the intrarenal deactivation of glucocorticoids. Spironolactone may prove useful in protecting against the hypertensive risks of nicotine/smoking.

Chemokines are a family of small cytokines, which guide a variety of immune/inflammatory cells to the site of tumor in tumorigenesis. A dysregulated expression of chemokines is implicated in different types of cancer including prostate cancer. The progression and metastasis of prostate cancer involve a complex network of chemokines that regulate the recruitment and trafficking of immune cells. The chemokine CCL2 and its main receptor CCR2 have been receiving particular interest on their roles in cancer pathogenesis. The up-regulation of CCL2/CCR2 and varied immune conditions in prostate cancer, are associated with cancer advancement, metastasis, and relapse. Here we reviewed recent findings, which link CCL2/CCR2 to the inflammation and cancer pathogenesis, and discussed the therapeutic potential of CCL2/CCR2 axis in cancer treatment based on results from our group and other investigators, with a major focus on prostate cancer.

This study investigated the inhibitory effect of arctigenin, a novel anti-inflammatory lignan, on prostate cancer in obese conditions both in vitro and in vivo. In vitro obese models were established by co-culture of mouse adipocytes 3T3-L1 with androgen-sensitive LNCaP human prostate cancer cells, or by culturing LNCaP cells in adipocytes-conditioned medium. Arctigenin significantly inhibited LNCaP proliferation, along with decreased androgen receptor (AR) and increased Nkx3.1 cellular expression. Male severe combined immunodeficiency mice were subcutaneously implanted with human prostate cancer LAPC-4 xenograft tumors for in vivo study. Mice were fed high-fat (HF) diet and orally given arctigenin at 50 mg/kg body weight daily or vehicle control for 6 weeks. Tumor bearing HF control mice showed a significant increase in serum free fatty acids (FFAs) and decrease in subcutaneous/peritoneal fat depots compared to non-tumor bearing control mice. Arctigenin intervention significantly reduced tumor growth by 45%, associated with decreased circulating FFAs and adipokines/cytokines including IGF-1, VEGF, and MCP-1, along with decreased AR, Ki67, and microvessel density and increased Nkx3.1 expression in tumors. These results indicate the strong ability of arctigenin to co-target obesity and tumor itself in inhibition of prostate tumor growth at a lower concentration compared to most phytochemicals.

Colorectal cancer is one of the leading causes for mortalities worldwide. The most common cause of colorectal cancer mortality is hepatic metastasis. There has been a limited advancement in the targeted-therapies for metastatic colorectal cancer. Conventional chemotherapeutic agent 5-fluorouracil has been used for various cancer treatments including colorectal cancer. Development of drug resistance and severe toxicity are major hurdles for its use in clinical setting. Resveratrol is a natural polyphenolic compound which has protective effects against aging-related diseases. In this study, we have tested whether combined treatments of resveratrol and 5-FU enhanced inhibitory effects against colorectal cancer cell growth. We herein showed that resveratrol and 5-FU combination treatments caused the anti-cancer activities by simultaneously inhibiting STAT3 and Akt signaling pathways. Resveratrol treatment induced S-phase specific cell cycle arrest, and when combined with 5-FU, it showed further increase in colorectal cancer cell apoptosis. Combined treatments of resveratrol and 5-FU inhibited epithelial-mesenchymal transition. Notably, resveratrol showed anti-inflammatory effects by downregulating inflammatory biomarkers, pSTAT3 and pNFκB. Resveratrol and 5-FU treatments inhibited STAT3 phosphorylation and its binding to the promoter region of human telomerase reverse transcriptase (hTERT). Our data provide the first evidence that resveratrol can enhance anti-telomeric and pro-apoptotic potentials of 5-FU in colorectal cancer, hence lead to re-sensitization to chemotherapy.

Background: Colorectal cancer is the third leading cause of cancer-related mortality in most developed countries. This mortality is mainly due to the metastatic progression to the liver with frequent recurrence. Colorectal cancer remains a therapeutic challenge and this has intensified the search for new drug targets. In an effort to establish a novel targeted-therapy, we studied the molecular mechanisms of cancer stem cell inhibitor salinomycin. Materials and Methods: Co-immunoprecipitation was performed to examine STAT3-STAT1 protein interactions. Telomerase activity was measured by polymerase chain reaction (PCR) and ELISA assays. Apoptosis and cell stress arrays were analyzed to identify key proteins responding to salinomycin treatments. Results: IL-6 and TNF-α induced STAT3 and STAT1 interactions, however the interactions were abolished by salinomycin challenge. Salinomycin reduced cancer stem cell phenotype and decreased telomerase activity of colorectal cancer cells. Conclusion: Our work uncovers a new mechanism through which salinomycin inhibits cancer stemness suggesting a novel targeted-therapy for metastatic colorectal cancer.

Chemotherapy with docetaxel (Doc) is a standard treatment for metastatic and castration-resistant prostate cancer. However, chemoresistance and side effects of Doc limit its clinical success. We investigated whether natural products green tea (GT) and quercetin (Q), a flavonoid from apples and onions, will enhance the efficacy of Doc in androgen-independent (AI) prostate cancer cells. Two cell lines including LAPC-4-AI and PC-3 were treated in vitro with 40 μM of (-)-epigallocatechin gallate (EGCG), 5 μM of Q, 2 or 5 nM of Doc alone or in combination. The mixture of EGCG+Q+Doc increased the antiproliferative effect by threefold in LAPC-4-AI cells and eightfold in PC-3 cells compared to Doc alone. EGCG, Q and Doc in combination significantly enhanced cell cycle arrest at G2/M phase and increased apoptosis in both LAPC-4-AI and PC-3 cells compared to Doc alone. The mixture increased the inhibition of PI3K/Akt and the signal transducer and activator of transcription (Stat) 3 signaling pathways compared to Doc alone, and decreased the protein expression of multidrug resistance-related protein. In addition, the combination with EGCG and Q increased the inhibition of tumor cell invasion and colony formation in both LAPC-4-AI and PC-3 cells compared to Doc alone, and decreased the percentage of CD44(+)/CD24(-) stem-like LAPC-4-AI cells. In summary, GT and Q enhanced the therapeutic effect of Doc in castration-resistant prostate cancer cells through multiple mechanisms including the down-regulation of chemoresistance-related proteins. This study provides a novel therapeutic modality to enhance the efficacy of Doc in a nontoxic manner.

The low bioavailability of most phytochemicals limits their translation to humans. We investigated whether arctigenin, a novel anti-inflammatory lignan from the seeds of Arctium lappa, has favorable bioavailability/potency against prostate cancer. The anticarcinogenic activity of arctigenin was investigated both in vitro using the androgen-sensitive LNCaP and LAPC-4 human prostate cancer cells and pre-malignant WPE1-NA22 cells, and in vivo using xenograft mouse models. Arctigenin at lower doses (

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 [NFkB]) 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 NFkB was significantly decreased in RP tissue of men consuming GT (P ¼0.013) but not BT (P ¼0.931) 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 ¼0.03). GT, but not BT or water, also led to a small but statistically significant decrease in serum prostate-specific antigen (PSA) levels (P ¼0.04). Conclusion. Given the GT-induced changes in NFkB 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. Prostate # 2014 Wiley Periodicals, Inc.

Scope We investigated whether a combination of two promising chemopreventive agents arctigenin (Arc) and quercetin (Q) increases the anticarcinogenic potency at lower concentrations than necessary when used individually in prostate cancer. Methods and results Androgen-dependent LAPC-4 and LNCaP prostate cancer cells were treated with low doses of Arc and Q alone or in combination for 48 h. The antiproliferative activity of Arc was 10- to 20-fold stronger than Q in both cell lines. Their combination synergistically enhanced the antiproliferative effect, with a stronger effect in androgen receptor (AR) wild-type LAPC-4 cells than in AR mutated LNCaP cells. Arc demonstrated a strong ability to inhibit AR protein expression in LAPC-4 cells. The combination treatment significantly inhibited both AR and PI3K/Akt pathways compared to control. A protein array analysis revealed that the mixture targets multiple pathways particularly in LAPC-4 cells including Stat3 pathway. The mixture significantly inhibited the expression of several oncogenic microRNAs including miR-21, miR-19b, and miR-148a compared to control. The mixture also enhanced the inhibition of cell migration in both cell lines compared to individual compounds tested. Conclusion The combination of Arc and Q that target similar pathways, at low physiological doses, provides a novel regimen with enhanced chemoprevention in prostate cancer.

Epigenetics describes heritable alterations of gene expression and chromatin organization without changes in DNA sequence. Both hypermethylation and hypomethylation of DNA can affect gene expression and the multistep process of carcinogenesis. Epigenetic changes are reversible and may be targeted by dietary interventions. Bioactive compounds from green tea (GT) such as (–)-epigallocatechin gallate have been shown to alter DNA methyltransferase activity in studies of esophageal, oral, skin, Tregs, lung, breast and prostate cancer cells, which may contribute to the chemopreventive effect of GT. Three out of four mouse model studies have confirmed the inhibitory effect of (–)-epigallocatechin gallate on DNA methylation. A human study demonstrated that decreased methylation of CDX2 and BMP-2 in gastric carcinoma was associated with higher GT consumption. It is the goal of this review to summarize our current knowledge of the potential of GT to alter epigenetic processes, which may be useful in chemoprevention.

Background Chemotherapy with docetaxel (Doc) remains the standard treatment for metastatic and castration-resistance prostate cancer (CRPC). However, the clinical success of Doc is limited by its chemoresistance and side effects. This study investigated whether natural products green tea (GT) and quercetin (Q) enhance the therapeutic efficacy of Doc in CRPC in mouse models. Methods Male severe combined immunodeficiency (SCID) mice (n = 10 per group) were inoculated with androgen-independent prostate cancer PC-3 cells subcutaneously. When tumors were established the intervention started. Mice were administered with GT + Q, Doc 5 mg/kg (LD), GT + Q + LD Doc, Doc 10 mg/kg (HD) or control. The concentration of GT polyphenols in brewed tea administered as drinking water was 0.07 % and Q was supplemented in diet at 0.4 %. Doc was intravenously injected weekly for 4 weeks, GT and Q given throughout the study. Results GT + Q or LD Doc slightly inhibited tumor growth compared to control. However, the combination of GT and Q with LD Doc significantly enhanced the potency of Doc 2-fold and reduced tumor growth by 62 % compared to LD Doc in 7-weeks intervention. A decrease of Ki67 and increase of cleaved caspase 7 were observed in tumors by the mixture, along with lowered blood concentrations of growth factors like VEGF and EGF. The mixture significantly elevated the levels of tumor suppressor mir15a and mir330 in tumor tissues. An increased risk of liver toxicity was only observed with HD Doc treatment. Conclusions These results provide a promising regimen to enhance the therapeutic effect of Doc in a less toxic manner.

Acute and chronic inflammation and dyslipidemia play a critical role in the development of various diseases, including cardiovascular disease. Green tea polyphenols possess potent antioxidative and anti-inflammatory properties that contribute to the beneficial effects on heart health. The present study was carried out to determine if administration of a green tea extract (Polyphenon(®) E [PPE]; Mitsui Norin Co., Ltd., Tokyo, Japan) at 0.2% in the diet reduces cardiovascular risk factors, including dyslipidemia, inflammation, adiposity, and oxidative stress, in rats fed an atherogenic (high fat, cholesterol, and sugar) diet with dextran sodium sulfate (DSS) in drinking water. DSS treatment increased serum total cholesterol, low-density lipoprotein (LDL)-cholesterol, C-reactive proteins (CRP), and markers of liver toxicity and decreased high-density lipoprotein (HDL)-cholesterol significantly. Adding PPE to the atherogenic diet (PPE-diet) was associated with lower total cholesterol and LDL-cholesterol (P

Bioactive constituents of pecan nuts such as γ-tocopherol and flavan-3-ol monomers show antioxidant properties in vitro, but bioavailability in humans is not known. We examined postprandial changes in plasma oxygen radical absorbance capacity (ORAC) and in concentrations of tocopherols, catechins, oxidized LDL, and malondialdehyde (MDA) in response to pecan test meals. Sixteen healthy men and women (23-44 y, BMI 22.7 ± 3.4) were randomly assigned to 3 sequences of test meals composed of whole pecans, blended pecans, or an isocaloric meal of equivalent macronutrient composition but formulated of refined ingredients in a crossover design with a 1-wk washout period between treatments. Blood was sampled at baseline and at intervals up to 24 h postingestion. Following the whole and blended pecan test meals, plasma concentrations of γ-tocopherols doubled at 8 h (P < 0.001) and hydrophilic- and lipophilic-ORAC increased 12 and 10% at 2 h, respectively. Post whole pecan consumption, oxidized LDL decreased 30, 33, and 26% at 2, 3, and 8 h, respectively (P < 0.05), and epigallocatechin-3-gallate concentrations at 1 h (mean ± SEM; 95.1 ± 30.6 nmol/L) and 2 h (116.3 ± 80.5 nmol/L) were higher than at baseline (0 h) and after the control test meal at 1 h (P < 0.05). The postprandial molar ratio of MDA:triglycerides decreased by 37, 36, and 40% at 3, 5, and 8 h, respectively (P < 0.05), only when whole and blended pecan data were pooled. These results show that bioactive constituent of pecans are absorbable and contribute to postprandial antioxidant defenses.

The efficacy of NovaSil clay (NS) to reduce aflatoxin (AF) biomarkers of exposure was evaluated in 656 blood samples and 624 urine samples collected from study participants during a 3-month phase IIa clinical intervention trial in Ghana. NS was delivered before meals via capsules. Serum AFB (1)-albumin adduct was measured by radioimmunoassay and urinary AFM (1) metabolites were quantified by immunoaffinity-high-performance liquid chromatography (HPLC)-fluorescence methods. Levels of AFB (1) -albumin adduct in serum samples collected at baseline and at 1 month were similar (p = 0.2354 and p = 0.3645, respectively) among the placebo (PL), low dose (LD, 1.5 g NS day (-1)), and high dose (HD, 3.0 g NS day (-1)) groups. However, the levels of AFB (1)-albumin adduct at 3 months were significantly decreased in both the LD group (p < 0.0001) and the HD group (p < 0.0001) compared with levels in the PL group. Levels of AFM(1) in urine samples collected at baseline and at 1 month were not statistically different among the three study groups. However, a significant decrease (up to 58%) in the median level of AFM (1) in samples collected at 3 months was found in the HD group when compared with the median level in the PL group (p < 0.0391). In addition, significant effects were found for dose, time, and dose-time interaction with serum AFB(1)-albumin adduct and dose-time interaction with urinary AFM (1) metabolites. The results suggest that capsules containing NS clay can be used to reduce effectively the bioavailability of dietary AF based on a reduction of AF-specific biomarkers.

Health benefits of green tea polyphenols (GTPs) have been reported in many animal models, but human studies are inconclusive. This is partly due to a lack of biomarkers representing green tea consumption. In this study, GTP components and metabolites were analyzed in plasma and urine samples collected from a phase II intervention trial carried out in 124 healthy adults who received 500- or 1,000-mg GTPs or placebo for 3 months. A significant dose-dependent elevation was found for (-)-epicatechin-3-gallate (ECG) (p

Arctigenin is a novel anti-inflammatory lignan derived mainly from the seeds of Arctium lappa which is an herb widely used in traditional Chinese medicine to treat inflammation related diseases. We previously demonstrated that arctigenin strongly inhibited prostate tumor cell growth in cell culture and mouse models in non-obese state. The present study investigated the tumor inhibitory effect of arctigenin in obese state. An in vitro obese setting was created by co-culture of mouse adipocytes 3T3-L1 with androgen-sensitive LAPC-4 and LNCaP prostate cancer cells. An ELISA analysis of 3T3-L1 conditioned medium revealed that three cytokines/growth factors were prominently secreted by 3T3-L1 cells, including IGF-1, VEGF, and MCP-1, and the sensitivity of prostate cancer cells to arctigenin was decreased in the obese state. However, arctigenin at a moderate concentration (10 µM) significantly inhibited the proliferation of both LAPC-4 and LNCaP cell by 40-50% at 96h in the co-culture system. Male severe combined immunodeficiency (SCID) mice was implanted subcutaneously with LAPC-4 xenograft tumors to confirm the tumor-inhibitory effect of arctigenin in vivo. Mice were fed high-fat diet containing 45% energy from fat, and treated with arctigenin at 50mg/kg b.w. orally or vehicle control for 6 weeks (n=10 per group). The tumor growth in arctigenin group was significantly inhibited by 40% compared to control, along with decreased blood concentrations of several cytokines including IGF-1, VEGF, and MCP-1. Immunohistochemistry analysis is ongoing to determine the molecular changes involved in tumor cell proliferation and apoptosis in response to arctigenin treatment. This study provides a promising natural compound to enhance chemoprevention of prostate cancer especially in obese patients. These results warrant future clinical trial studies to confirm the anti-carcinogenic effect of arctigenin in humans. Citation Format: Piwen Wang, Tanya Diaz, Susanne Henning, Jaydutt Vadgama. Arctigenin inhibits prostate tumor growth in vitro and in vivo in obese state [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5253. doi:10.1158/1538-7445.AM2017-5253

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.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.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).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.

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 (AR) and phosphatidylinositol 3-kinases (PI3K)/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.

The polyphenols in green tea (GT) and black tea (BT) are very different due to the different post-harvest processing of the tea leaves, which result in polymerization of GT polyphenols as a result of auto-oxidation. Eighty percent of tea is consumed as BT, while the majority of the literature on the health benefits of tea concerns GT. GT contains higher concentrations of monomeric polyphenols such as epigallocatechin gallate (EGCG), while BT contains polymeric polyphenols such as theaflavins and thearubigins. We have studied the distribution of GT and BT polyphenols in urine and prostate tissue in humans after consumption of brewed GT or BT prior to prostatectomy. Overall, higher concentrations of polyphenols are found in the circulation after GT consumption. While EGCG, 4″-O-MeEGCG and ECG are present in prostate tissue following GT consumption, BT polymers are not found in either blood or prostate tissue. In contrast to human prostate tissue, mouse and rat tissues have detectable levels of both gallated and non-gallated GTPs, suggesting differences in their tissue metabolism of GTPs compared to humans. In both humans and mice, the majority of gallated GTPs are found in the free form, whereas 100% of non-gallated GTPs are conjugated and excreted in the urine. In humans and mice, about 50% of GTPs occur in the methylated form. Future research is needed to investigate how to increase the bioavailability of GTPs.

Arctigenin is a novel anti-inflammatory lignan derived mainly from the seeds of Arctium lappa, an herb widely used in traditional Chinese medicine to treat inflammation related diseases such as cough, cold and swelling of throat. The present study was designed to investigate whether arctigenin is a potent inhibitor of prostate cancer using both in vitro and xenograft mouse models. The treatments with arctigenin at lower doses (< 2μM) for 48h dose-dependently inhibited the proliferation of two androgen-sensitive prostate cancer cell lines, LNCaP and LAPC-4, by 40-50% compared to control. There was no cytotoxicity observed in normal prostate epithelial PrEC cells with arctigenin doses up to 10μM. An intervention study was then conducted in severe combined immunodeficiency (SCID) mice to confirm the tumor-inhibitory effect of arctigenin in vivo. Male SCID mice (n = 10 per group) were inoculated with 5×10⁁5 LAPC-4 cells subcutaneously. One week later when tumors were palpable the intervention started. Mice were administered with arctigenin at 50mg/kg (LD) or 100mg/kg (HD) or vehicle control daily for 6 weeks by oral gavage. After 4 weeks of intervention the tumor growth was significantly inhibited by both of the arctigenin doses. After 6 weeks of intervention, the tumor volumes were reduced by 48% (LD arctigenin) and 67% (HD arctigenin) compared to control. There was no significant difference in food or water consumption or body weight among groups. An ELISA assay of growth factors demonstrates that arctigenin treatments significantly decreased the concentrations of VEGF, EGF, NGF-β, TNF-α, and FGF-β in tumor tissues compared to control. Western blot analysis revealed a significantly reduced expression of galectin-4 in tumor tissues by arctigenin treatment. Previous studies have shown that strong expression of galectin-4 can be induced in different types of cancer, associated with tumor growth and progression. A microRNA PCR array assay was performed to measure the tumor concentrations of 84 microRNAs involved in prostate cancer development and progression. A panel of microRNAs including miR-126 and miR-135 were identified to be responsive to arctigenin treatment. Further mechanistic investigation using tissue microarray assay is ongoing to measure the protein markers of proliferation and apoptosis in tumor tissues. This study provides a promising non-toxic agent to enhance chemoprevention of prostate cancer. These results warrant future clinical trial studies to confirm the anti-carcinogenic effect of arctigenin in humans. Citation Format: Piwen Wang, Susanne M. Henning, Jaydutt V. Vadgama. A lignan compound arctigenin inhibits prostate tumor growth in vitro and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2626A.

Tea polyphenols are metabolized by the colonic microflora yielding phenolic metabolites, which may contribute to the health benefits of tea. We determined the serum and urine concentrations of phenolic acids, hippuric acid, and polyhydroxyphenyl-γ-valerolactones during green tea (GT) and black tea (BT) administration. The effects of (-)-epigallocatechin gallate (EGCG) and 3,4-dihydroxyphenylacetic acid (3,4-DHPAA) alone and in combination on bioavailability, intracellular metabolism, and antiproliferative activity were determined in HCT-116 colon cancer cells.The concentration of phenolic metabolites was quantified by HPLC with electrochemical detection and MS. Urine concentrations of 4-hydroxyphenylacetic acid (4-HPAA), 3-hydroxyphenylacetic acid (3-HPAA), and polyhydroxy-γ-valerolactones were increased significantly in men drinking GT compared to control. Urine concentration of 3-O-methylgallic acid (3OMGA) was significantly increased in men drinking BT compared to control. Serum 3,4-DHPAA was significantly increased after consumption of GT and BT and 4-HPAA after GT consumption. In vitro treatment of HCT-116 colon cancer cells with 3,4-DHPAA and EGCG exhibited an additive antiproliferative effect, while methylation of 3,4-DHPAA was significantly decreased. 3OMGA exhibited the strongest antiproliferative activity among the phenolic acids.The consumption of both, GT and BT, was associated with a significant increase in urinary and serum phenolic acids.

We previously demonstrated that 50% of (-)-epigallocatechin gallate (EGCG) was present in methylated form (4″-MeEGCG) in human prostate tissue, which is less bioactive. We therefore investigated whether quercetin, a natural inhibitor of catechol-O-methyl transferase (COMT), will inhibit EGCG methylation leading to enhanced antiproliferative activity of EGCG in prostate cancer cells. Incubation with both quercetin and EGCG for 2 h increased the cellular concentrations of EGCG by 4- to 8-fold and 6- to 10-fold in androgen-independent PC-3 cells and androgen-dependent LNCaP cells, respectively. Concurrently, the percent of 4″-MeEGCG in the total EGCG was decreased from 39% to 15% in PC-3 cells and from 61% to 38% in LNCaP cells. Quercetin and EGCG in combination synergistically inhibited cell proliferation, caused cell cycle arrest, and induced apoptosis in PC-3 cells. In LNCaP cells, EGCG and quercetin exhibited a stronger antiproliferative activity leading to an additive effect. The synergistic effect of these 2 agents in PC-3 cells could be based on the fact that EGCG primarily inhibited COMT activity, whereas quercetin reduced the amount of COMT protein. In summary, quercetin combined with EGCG in vitro demonstrated enhanced inhibition of cell proliferation by increasing the intracellular concentration of EGCG and decreasing EGCG methylation.

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 (GSTp1) to permit reactive oxygen species promotion of tumor growth. GT inhibited tumor 5-cytosine DNA methyltransferase 1 (DNMT1) 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.

The polyphenol compositions of green tea (GT) and black tea (BT) are very different due to post-harvest processing. GT contains higher concentrations of monomeric polyphenols, which affect numerous intracellular signaling pathways involved in prostate cancer (CaP) development. BT polymers, on the other hand, are poorly absorbed and are converted to phenolic acids by the colonic microflora. Therefore, after consumption of GT, higher concentrations of polyphenols are found in the circulation, whereas after BT consumption the phenolic acid levels in the circulation are higher. The majority of in vitro cell culture, in vivo animal, and clinical intervention studies examine the effects of extracts of GT or purified (-)-epigallocatechin-3-gallate (EGCG) on prostate carcinogenesis. These studies provide strong evidence supporting a chemopreventive effect of GT, but results from epidemiological studies of GT consumption are mixed. While the evidence for a chemopreventive effect of BT is much weaker than the body of evidence with regard to GT, there are several animal BT intervention studies demonstrating inhibition of CaP growth. This article will review in detail the available epidemiological and human clinical studies, as well as animal and basic mechanistic studies on GT and BT supporting a chemopreventive role in CaP.

We determined whether nine common herbs (basil, chili, cilantro, dill, garlic, ginger, lemongrass, oregano, and parsley) and one herb mixture (Italian Herbs) retain the antioxidant capacity (AC) and content of phenolics and characteristic marker compounds during processing to dry and paste forms. Oregano exhibited the highest AC among the herbs tested in dry and fresh forms. Compared with fresh herbs, the AC in dry form was decreased in garlic, chili, dill, oregano and parsley and paste form of oregano and basil. With the exception of dried garlic and lemongrass in fresh and paste form, all herbs in dry, paste, and fresh form contained significant AC. The AC was correlated significantly to the total phenolic content in both dry and fresh form. However, there was no significant correlation between the AC and the concentration of chemical marker compounds. In summary, processed herbs contribute significant amounts of AC to the diet.

Epidemiologic, preclinical, and clinical trials suggest that green tea consumption may prevent prostate cancer through the action of green tea polyphenols including (−)-epigallocatechin-3-gallate (EGCG). To study the metabolism and bioactivity of green tea polyphenols in human prostate tissue, men with clinically localized prostate cancer consumed six cups of green tea (n = 8) daily or water (n = 9) for 3 to 6 weeks before undergoing radical prostatectomy. Using high-performance liquid chromatography, 4″-O-methyl EGCG (4″-MeEGCG) and EGCG were identified in comparable amounts, and (−)-epicatechin-3-gallate was identified in lower amounts in prostatectomy tissue from men consuming green tea (38.9 ± 19.5, 42.1 ± 32.4, and 17.8 ± 10.1 pmol/g tissue, respectively). The majority of EGCG and other green tea polyphenols were not conjugated. Green tea polyphenols were not detected in prostate tissue or urine from men consuming water preoperatively. In the urine of men consuming green tea, 50% to 60% of both (−)-epigallocatechin and (−)-epicatechin were present in methylated form with 4′-O-MeEGC being the major methylated form of (−)-epigallocatechin. When incubated with EGCG, LNCaP prostate cancer cells were able to methylate EGCG to 4″-MeEGCG. The capacity of 4″-MeEGCG to inhibit proliferation and NF-κB activation and induce apoptosis in LNCaP cells was decreased significantly compared with EGCG. In summary, methylated and nonmethylated forms of EGCG are detectable in prostate tissue following a short-term green tea intervention, and the methylation status of EGCG may potentially modulate its preventive effect on prostate cancer, possibly based on genetic polymorphisms of catechol O-methyltransferase. Cancer Prev Res; 3(8); 985–93. ©2010 AACR.

A study of aflatoxin (AF) exposure and the levels of vitamins A and E was carried out with a group of 507 Ghanaian participants. AFB(1)-albumin adducts (AFB-AA) were measured by radioimmunoassay and vitamins A and E were measured by high-performance liquid chromatography (HPLC). The average level of serum AFB-AA was 0.94 +/- 0.64 (range = 0.1-4.44) pmol mg(-1) albumin. Mean levels of vitamins A and E were 1.32 +/- 0.48 (range = 0.41-4.85) micromol l(-1) and 15.68 +/- 4.12 (range = 6.35-30.40) micromol l(-1), respectively. A significantly negative correlation was found between serum AFB-AA and vitamin A levels (r = -0.110, p = 0.013). An even stronger, significant negative, correlation was found between serum AFB-AA and vitamin E levels (r = -0.149, p < 0.001). Serum AFB-AA levels were statistically higher (median = 0.985 pmol mg(-1) albumin) in subjects who had low levels of both vitamins A and E as compared with the levels (median = 0.741 pmol mg(-1) albumin) subjects who had high vitamins A and E levels (p(trend) = 0.001). To verify these findings, blood samples were again collected from 165 of the 507 people 3 months after the initial collection. Significantly negative correlations were confirmed between levels of serum AFB-AA and both vitamins A (r = -0.232, p = 0.003) and E (r = -0.178, p = 0.023). Again, high serum AFB-AA concentrations (median = 1.578 pmol mg(-1) albumin) were found in subjects with low levels of vitamins A and E compared with the concentrations (median = 1.381 pmol mg(-1) albumin) in subjects with high levels of vitamins A and E (p(trend) = 0.002). These data show that AF exposure was associated with decreased levels of serum vitamins A and E in high-risk human populations, which may significantly influence the incidence of AF-related adverse health effects.

Our recent study demonstrated a bone-protective role of green tea polyphenols (GTPs), extracted from green tea, in estrogen-deficiency-induced bone loss of middle-aged female rats through an increa...

Chemotherapy with docetaxel (Doc) is a standard treatment for metastatic and castration-resistant prostate cancer. However, the development of chemoresistance and its side effects limit the clinical success of Doc. Previously we demonstrated that the combined use of natural products green tea (GT) and quercertin (Q) with Doc synergistically enhanced the anti-proliferative effect in androgen-independent prostate cancer PC-3 cells. The objective of the present study was to confirm the combined effect of GT, Q and Doc in xenograft prostate cancer mouse models and determine underlying mechanisms. Male severe combined immunodeficiency (SCID) mice (n = 10 per group) were inoculated with 5×105 PC-3 cells subcutaneously. When tumors reached a size of about 100mm3 the intervention started. Mice were administered with GT+Q, Doc 5mg/kg (LD), GT+Q+LD Doc, Doc 10mg/kg (HD) or control. The concentration of GT polyphenols in brewed tea administered as drinking water was 0.07% and Q was supplemented in diet at 0.4%. Doc was given through tail vein once a week for 4 weeks. There was a nonsignificant inhibition of tumor growth by GT+Q or LD Doc alone compared to control. However the combination treatment with GT+Q+LD Doc significantly inhibited the tumor growth by 62% compared to LD Doc alone after 7 weeks intervention. The combined effect was comparable to that by HD Doc treatment. Western blot analysis demonstrated a significantly increased ratio of Bax to Bcl-2 protein expression by GT+Q+LD Doc treatment compared to GT+Q or LD Doc. ELISA analysis of blood samples demonstrated that the combination treatment significantly decreased the blood concentrations of several growth factors, including VEGF, EGF, TGF-β, FGF-β, and TNF-α compared to GT+Q or LD Doc alone. A liver toxicity evaluation revealed that the blood alanine aminotransferase (ALT) level was significantly elevated with HD Doc treatment compared to control, while not changed by other groups. Further mechanistic investigations using protein array and tissue microarray assays are ongoing. This study provides a promising regimen by combining GT and Q with Doc to enhance chemotherapeutic effect in advanced prostate cancer in a less toxic manner. The results warrant future clinical trials to confirm the combined effect of the mixture in humans. Citation Format: Piwen Wang, Susanne Henning, David Heber, Jaydutt Vadgama. Enhanced inhibition of PC-3 xenograft prostate tumor growth by combination of green tea and quercetin with docetaxel. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5345. doi:10.1158/1538-7445.AM2015-5345

Seasonal variations in the concentration of microcystin-LR (MCLR) in Buffalo Springs Lake (BSL) and Lake Ransom Canyon (LRC; both locations in Lubbock, TX, USA) were monitored from 2003 to 2004. In BSL, the average concentrations of MCLR were 1.78 +/- 1.43 microg/L (mean +/- SD; range, 0.177-4.914 microg/L) in spring, 0.41 +/- 0.096 microg/L (range, 0.191-0.502 microg/L) in summer, 0.46 +/- 0.41 microg/L (range, 0.205-1.598 microg/L) in fall, and 1.04 +/- 0.71 microg/L (range, 0.096-2.428 microg/L) in winter. In LRC, the corresponding concentrations were 1.08 +/- 1.29 microg/L (range, 0.2-5.83 microg/L) in spring, 0.83 +/- 0.46 microg/L (range, 0.315-1.671 microg/L) in summer, 0.44 +/- 0.03 microg/L (range, 0.368-0.555 microg/L) in fall, and 0.78 +/- 0.52 microg/L (range, 0.225-2.130 microg/L) in winter. In both lakes, the seasonal fluctuation of MCLR concentrations correlated positively with dissolved oxygen and negatively with temperature and pH.

Background: Green tea polyphenols (GTPs) have been shown to play a role in the regulation of cell proliferation, apoptosis and inflammation in prostate cancer cells in vitro and in animal models using human prostate cancer cell xenografts. While there is extensive epidemiological and basic scientific evidence for these effects of GTPs, data from human intervention studies is much more limited. Objective: We conducted a phase II intervention study to evaluate the anticarcinogenic effects of green tea in prostate cancer by administering green tea in men prior to prostatectomy. Design: 79 men, diagnosed with prostate cancer and scheduled for prostatectomy, were randomized to either 6 cups of brewed green tea or water daily for 3-8 weeks prior to surgery. 67 men (GT group, N=34; control group, N=33) completed the intervention. Blood and urine samples were collected before and after the intervention and sections of prostate tissue were frozen as OCT blocks following the pathology evaluation. Serum prostate specific antigen (PSA) concentration was determined by ELISA assay and prostate tissue PSA protein concentration was measured by Western blot. The prostate concentration of GTPs and their metabolites was measured using high-performance liquid chromatography (HPLC) with CoulArray electrochemical detection. The protein expression of Ki67 (proliferation), Bcl2, Bax (apoptosis) and nuclear factor kappa B (NFκB) (inflammation) were determined by immunostaining in prostate tissue of confirmed tumor areas. Results: Statistical analysis within each treatment arm showed that the serum PSA concentration was significantly decreased in the GT group (final vs. baseline blood) (P Conclusion: Green tea consumption may contribute to the inhibition of prostate tumor growth through reducing inflammation and stimulating apoptosis. Analyses of additional markers are needed to confirm these findings. Citation Format: Susanne M. Henning, Piwen Wang, William J. Aronson, Catherine L. Carpenter, David Heber. Inhibition of NFκB and proapoptotic effect of green tea in prostate tumor tissue: A phase II clinical trial in men with prostate cancer. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B67.

The chemopreventive activity of green tea (GT) and green tea polyphenols (GTPs) in prostate cancer has been demonstrated in preclinical cell culture and animal models. However, extensive methylation of GTPs in vivo to less active methyl-metabolites may be limiting the anti-cancer activity of GT. The objective of the present study was to investigate whether quercetin (Q), a natural inhibitor of catechol-O-methyltransferase (COMT), will increase tissue concentrations of non-methylated GTPs and enhance the inhibition of xenograft tumor growth in severe combined immune deficient (SCID) mice. Androgen-sensitive LAPC-4 prostate cancer cells were injected subcutaneously into SCID mice (n=12 per group). SCID mice were randomly assigned to four groups: control, GT, Q, and GT + Q treatment. The interventions were initiated when tumors reached volume of 10mm3. The final concentration of GTPs in brewed tea administered as drinking water was 0.07% and Q was supplemented in AIN-93G diet at a concentration of 4g/kg diet. There were no differences in food intake, body weight or water consumption during the intervention. Tissue bioavailability of GTPs was increased significantly by 2 to 3-fold in lung, kidney and xenograft tumors and showed a trend to increase by 1.3-fold in liver tissue by GT and Q treatment compared to GT alone. Methylation of epigallocatechin gallate (EGCG), the main polyphenol in green tea, was decreased in tissues from mice in the GT+Q group. After 6 weeks, tumor growth was inhibited by 16% (Q alone), 21% (GT alone), and 45% (GT+Q) compared to the control group. Western blot analysis demonstrated a significant inhibition of methyl transferases such as COMT (31%) and DNA methyltransferase (DNMT) (52%) in tumors from the GT+Q group compared to the control. It has been demonstrated previously that EGCG can act as androgen receptor (AR) antagonist as well as inhibit angiogenesis via inhibition of the vascular endothelial growth factor (VEGF). Together Q+GT inhibited protein expression of AR (35%), prostate-specific antigen (PSA) (39%), and VEGF (25%) more than either GT or Q alone. This study demonstrates an enhanced inhibition of prostate tumor growth when GT and Q are co-administered and this is likely due to inhibition of methylation resulting in enhanced anti-tumor activity of EGCG via effects on angiogenesis, and androgen-mediated tumor growth. This study sets the stage for future human interventions to confirm the increase in bioavailability and chemoprevention using GT in combination with Q. This project was supported by DOD grant W81XWH-10-1-0298 and NIH grant RO3 CA150047-01. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 607. doi:1538-7445.AM2012-607

The extensive methylation of green tea polyphenols (GTPs) in vivo may limit their chemopreventive potential. We investigated whether quercetin, a natural inhibitor of catechol-O-methyltransferase (COMT) and multidrug resistance proteins (MRPs), will differentially increase the intracellular concentration and decrease the methylation of GTPs in different cancer cell lines. Intrinsic COMT activity was lowest in lung cancer A549 cells, intermediate in kidney 786-O cells and highest in liver HepG2 cells. Quercetin increased the cellular absorption of epigallocatechin gallate (EGCG) four-fold in A549 cells with a decreased methylation rate from 63 to 19%, 2-fold in 786-O cells with a decreased methylation from 97% to 56%, while no significant effect was observed in HepG2 cells. The combination significantly decreased the activity and protein expression of COMT and decreased the protein expression of MRP1 compared to individual treatments. The combination exhibited the strongest increase in antiproliferation in A549 cells, an intermediate effect in 786-O cells and lowest effect in HepG2 cells. The effect of quercetin on bioavailability and metabolism of GTPs was confirmed in vivo. Severe combined immunodeficiency (SCID) mice were administered brewed green tea (GT) and a diet supplemented with 0.4% quercetin alone or in combination for 2 weeks. We observed a 2- to 3-fold increase of total and non-methylated EGCG in lung and kidney and an increasing trend in liver. In summary, combining quercetin with GT provides a promising approach to enhance the chemoprevention of GT. Responses of different cancers to the combination may vary by tissue depending on the intrinsic COMT and MRP activity.

Green tea polyphenls (GTPs) are metabolized through extensive methylation which reduces their chemopreventive potential. Previously we found that more than 50 percent of (−)-epigallocatechin-3-gallate (EGCG) was methylated to 4″-O-methyl EGCG in mouse tissues and in human prostate after green tea consumption. Methylation significantly decreased the antiproliferative effects of EGCG in prostate cancer cells. Quercetin (Q) is a natural inhibitor of multidrug resistance proteins and catechol-O-methyltransferase (COMT), which determines the absorption and the methylation of GTPs. In order to determine whether co-treatment with Q will increase the bioavailability and decrease the methylation of GTPs in cell culture and mouse tissues, three different human cancer cell lines with different COMT activity were studied in vitro. COMT activity was lowest in lung cancer A549 cells, intermediate in kidney cancer 786-O cells, and highest in liver cancer Hep G2 cells. Corresponding to the COMT activity, A549 cells showed the highest cellular content of EGCG and lowest methylation rate (66.1 pmol/mg cytosolic protein and 63% methylation rate); followed by 786-O cells (39.6, 97% methylated) and Hep G2 cells (25.1, 98% methylated) when incubated with 80 μmol/L EGCG for 2h. Treatment with Q and EGCG increased cellular absorption of EGCG most in A549 cells (4-fold) with decreased methylation rate (19%), less in 786-O cells (2- fold) and decreased methylation rate (56%), while there was little effect on methylation in HepG2 cells. The strongest additive effect was observed on proliferation of A549 cells, followed by renal 786-O cells, and least in Hep G2 cells. In order to examine the impact of Q treatment on GTP bioavailability in vivo, two groups of SCID mice (n=5) fed the AIN-93G diet were provided with brewed tea (one Celestial Seasonings authentic green tea bag brewed in 240ml boiling water for 5min) in drinking water and either diet alone or diet supplemented with 0.4% Q for 2 weeks. Demonstrating the impact of Q on bioavailability and methylation in vivo there was a 2-3 fold increase of non-methylated EGCG and other GTPs observed in lung and kidney tissues. In vivo but not in vitro, the levels of methylated Q increased markedly suggesting that Q acted as a substrate for methylation sparing the GTPs and increasing unmethylated GTPs. These observations demonstrate that Q inhibits methylation of GTPs and when used in combination with GTPs may increase bioavailability. Studies are underway in SCID mice with prostate cancer xenografts to determine whether this increased bioavailability will translate to an enhanced chemopreventive activity. 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 4615. doi:10.1158/1538-7445.AM2011-4615

High consumption of fruit-, vegetable-, and plant-based beverages has been associated with a reduced incidence and mortality of various chronic diseases including cancer. A wide variety of phytochemicals contribute to these chemopreventive effects. The largest group of phytochemicals belongs to the family of flavonoids, which all share the common polyphenol structure. Considerable effort has been made to quantitate the dietary intake and main food sources of flavonoids. In the U.S., based on the USDA flavonoid database and NHANES 1999-2002 24-h dietary recall data, the average daily intake of flavonoids is 189.7 mg/d. The main daily mean food sources of flavonoids were tea (157 mg), citrus fruit juices (8 mg), wine (4 mg), and citrus fruit (3 mg) (1). In Spain, the mean flavonoid intake was 313 mg/d with the main sources being apples (23%), red wine (21%), unspecified fruit (12.8%), and oranges (9.3%) (2). Tea polyphenols are consumed in such high quantity due to the popularity of tea worldwide and the high concentration of polyphenols per cup of tea (150 mg). Tea leaves from the plant Camellia sinensis are manufactured in nonfermented form as green tea or fermented form as black tea. Green tea contains the gallated polyphenols (-)-epigallocatechin-3-gallate (EGCG) and (-)-epicatechin-3-gallate (ECG) as well as non-gallated (-)-epigallocatechin (EGC) and (-)-epicatechin (EC). During fermentation to black tea these green tea polyphenols (GTP) undergo polymerization to theaflavin and thearubigin. Other examples of large polymeric polyphenols are ellagitannins from pomegranate and procyanidins from grape seed extract and cocoa. Daily pomegranate juice consumption has been shown to decrease the rate of rise of PSA in men treated for prostate cancer increasing the PSA doubling time from 15 to 54 weeks. We have conducted extensive studies of the bioavailability, metabolism by colonic microflora, and conjugation after absorption of polyphenols. For example, pomegranate ellagitannins are converted to gallagic acid (GA), ellagic acid (EA), urolithin A (UA) and B (UB), and tea polyphenols to phenolic acids, such as 3,4-dihydroxyphenyl acetic acid (3,4DHPAA) (3). These products can be found in the blood and urine and have biological activity. During the absorption process and in the tissues polyphenols and their metabolites are glucuronidated, sulfated and methylated. Interestingly, there are strong interindividual differences observed in the plasma and urine content of metabolites most likely due to differences in microbial composition, polymorphisms in metabolizing enzymes and transport proteins. Epidemiological studies show conflicting evidence of the chemopreventive effect of green tea. However, for prostate cancer, one randomized controlled trial (RCT) from Italy suggested that the consumption of GTP supplement inhibited the progression of premalignant lesions to prostate cancer and furthermore, that green tea catechins might positively influence quality-of-life issues in patients with high-grade prostate intraepithelial neoplasia. Another intervention study in the U.S. demonstrated a decrease in serum prostate-specific antigen, hepatocyte growth factor, and vascular endothelial growth factor after the consumption of 800 mg of EGCG for an average of 6 weeks. In an ongoing randomized pre-prostatectomy trial at UCLA we determined the concentration of GTPs and metabolites in prostate and urine of participants consuming 6 cups of green tea for 3-6 weeks. In the prostate only gallated tea polyphenols such as EGCG, 4″-O-MeEGCG and ECG were found with more than 50% in the nonconjugated free form. In urine the nongallated forms such as EGC, 4′-O-MeEGC and EC were found (3). The ratio of methylated EGCG to nonmethylated EGCG varies considerably among participants. Catechol-O-methyl transferase (COMT), the enzyme responsible for methylation of EGCG and other catechols, has three polymorphic sites. On codon 158 (exon 4) the G->A transition leading to Val->Met amino acid change is associated with a four time lower enzyme activity compare to wild type. An epidemiological cohort study by Wu et al. demonstrated that breast cancer risk reduction was only associated with green tea intake in women with at least one low-activity allele in COMT (4). However, in addition to the influence of the single nucleotide polymorphism on COMT activity, EGCG itself inhibits COMT activity. We demonstrated in PC3 prostate cancer cells that EGCG inhibited COMT enzyme activity significantly. The same effect was observed in vivo in severe combined immunodeficiency mice (SCID) implanted subcutaneously with LAPC4 prostate cancer cells. The administration of brewed green tea, instead of drinking water, lead to a decrease in tumor growth and COMT m-RNA expression. Methylation and conjugation may also alter the biologic activities of the parent compound. For example, our cell culture experiments demonstrated differences in the antiproliferative activity, anti-inflammatory activity (NFkB stimulation), and stimulation of apoptosis of methylated EGCG compared to the parent compound in androgen dependent (LNCaP) and androgen independent (PC-3) prostate cancer cells. In the case of EGCG, methylation leads to a significant decrease of biological activity. However, in case of quercetin, there was no significant difference in the antiproliferative activity between methylated quercetin and the parent compound. Since quercetin is known as a strong natural methylation inhibitor we tested the effect of co-treatment of EGCG and quercetin on EGCG methylation and bioavailability. In cell culture experiments combining EGCG with quercetin decreased EGCG methylation and increased intracellular EGCG 10-fold. These results demonstrate that combination of polyphenols with differential effects on metabolism may be useful to enhance bioavailability and bioactivity. In summary, in vitro mechanistic investigations with translational potential, will need to be performed with the appropriate metabolites and concentrations found in vivo. References: 1. Chun OK, Chung SJ, Song WO. Estimated dietary flavonoid intake and major food sources of U.S. adults. J Nutr. 2007, 137(5):1244-52. 2. Zamora-Ros R, Andres-Lacueva C, Lamuela-Raventós RM, Berenguer T, Jakszyn P, Barricarte A, Ardanaz E, Amiano P, Dorronsoro M, Larrañaga N, Martínez C, Sánchez MJ, Navarro C, Chirlaque MD, Tormo MJ, Quirós JR, González CA. Estimation of dietary sources and flavonoid intake in a Spanish adult population (EPIC-Spain). J Am Diet Assoc. 2010, 110(3):390-8. 3. Wang P,Aronson WJ, Huang M, Heber D, Henning SM. Methyl-Metabolites of Green Tea Polyphenols and Their Bioactivity in Prostate — Results of a Phase II Clinical Trial in Men with Prostate Cancer. Cancer Prev. Res. 2010, 3(8):985-93. 4. Wu AH, Tseng CC, Van Den Berg D, Yu MC. Tea intake, COMT genotype, and breast cancer in Asian-American women. Cancer Res. 2003, 63(21):7526-9. Citation Information: Cancer Prev Res 2010;3(12 Suppl):CN06-03.