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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

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.

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

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

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.