Piwen Wang

Limitations of MTT and MTS-based assays for measurement of antiproliferative activity of green tea polyphenols.

Background The chemopreventive effect of green tea polyphenols, such as (-)-epigallocatechin-3-gallate (EGCG), has been well demonstrated in cell culture studies. However, a wide range of IC50 concentrations has been observed in published studies of the anti-proliferative activity of EGCG from different laboratories. Although the susceptibility to EGCG treatment is largely dependent on cancer cell type, the particular cell viability and proliferation assays utilized may significantly influence quantitative results reported in the literature. Methodology/Principal Findings We compared five widely used methods to measure cell proliferation and viability after EGCG treatment using LNCaP prostate cancer cells and MCF-7 breast cancer cells. Both methods using dyes to quantify adenosine triphosphate (ATP) and deoxynucleic acid (DNA) showed accuracy in the measurement of viable cells when compared to trypan blue assay and results showed good linear correlation (r = 0.95). However, the use of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) as indicators of metabolically active mitochondria overestimated the number of viable cells by comparison with the ATP, DNA, or trypan blue determinations. As a result, the observed IC50 concentration of EGCG was 2-fold higher using MTT and MTS compared to dyes quantifying ATP and DNA. In contrast, when cells were treated with apigenin MTT and MTS assays showed consistent results with ATP, DNA, or trypan blue assays. Conclusions/Significance These results demonstrate that MTT and MTS -based assays will provide an underestimation of the anti-proliferative effect of EGCG, and suggest the importance of careful evaluation of the method for in vitro assessment of cell viability and proliferation depending on the chemical nature of botanical supplements.

NovaSil clay intervention in Ghanaians at high risk for aflatoxicosis: II. Reduction in biomarkers of aflatoxin exposure in blood and urine

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 AFB1–albumin adduct was measured by radioimmunoassay and urinary AFM1 metabolites were quantified by immunoaffinity-high-performance liquid chromatography (HPLC)-fluorescence methods. Levels of AFB1–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 AFB1–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 AFM1 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 AFM1 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 AFB1–albumin adduct and dose–time interaction with urinary AFM1 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.

Phenolic acid concentrations in plasma and urine from men consuming green or black tea and potential chemopreventive properties for colon cancer

SCOPE 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. METHODS AND RESULTS 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. CONCLUSION The consumption of both, GT and BT, was associated with a significant increase in urinary and serum phenolic acids.

Green Tea Polyphenols and Metabolites in Prostatectomy Tissue: Implications for Cancer Prevention

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.

Chemopreventive effects of tea in prostate cancer: green tea versus black tea.

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.

Quercetin increased bioavailability and decreased methylation of green tea polyphenols in vitro and in vivo

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.

Pecans Acutely Increase Plasma Postprandial Antioxidant Capacity and Catechins and Decrease LDL Oxidation in Humans

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.

Epigenetic effects of green tea polyphenols in 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.

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.

Quercetin increased the antiproliferative activity of green tea polyphenol (-)-epigallocatechin gallate in prostate cancer cells.

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.