BackgroundThe 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 FindingsWe 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/SignificanceThese 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.

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.

Epidemiologic, preclinical, and clinical trials suggest that green tea (GT) consumption may prevent prostate cancer via the action of green tea polyphenols including (-)-epigallocatechin-3-gallate (EGCG). In order to study the metabolism and bioactivity of green tea polyphenols in human prostate tissue, men with clinically localized prostate cancer consumed 6 cups of GT (n=8) daily or water (n=9) for 3-6 weeks prior to 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 (ECG) in lower amounts in prostatectomy tissue from men consuming GT (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 GT, 50-60% of both (-)-epigallocatechin (EGC) and (-)-epicatechin were present in methylated form with 4′-O-MeEGC being the major methylated form of EGC. 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 to EGCG. In summary, methylated and non-methylated forms of EGCG are detectable in prostate tissue following a short-term GT intervention and the methylation status of EGCG may potentially modulate its preventive impact on prostate cancer, possibly based on genetic polymorphisms of catechol O-methyltransferase.