Epicatechins Purified from Green Tea (Camellia sinensis) Differentially Suppress Growth of Gender-Dependent Human Cancer Cell Lines

The anticancer potential of catechins derived from green tea is not well understood, in part because catechin-related growth suppression and/or apoptosis appears to vary with the type and stage of malignancy as well as with the type of catechin. This in vitro study examined the biological effects of epicatechin (EC), epigallocatechin (EGC), EC 3-gallate (ECG) and EGC 3-gallate (EGCG) in cell lines from human gender-specific cancers. Cell lines developed from organ-confined (HH870) and metastatic (DU145) prostate cancer, and from moderately (HH450) and poorly differentiated (HH639) epithelial ovarian cancer were grown with or without EC, EGC, ECG or EGCG. When untreated cells reached confluency, viability and doubling time were measured for treated and untreated cells. Whereas EC treatment reduced proliferation of HH639 cells by 50%, EGCG suppressed proliferation of all cell lines by 50%. ECG was even more potent: it inhibited DU145, HH870, HH450 and HH639 cells at concentrations of 24, 27, 29 and 30 µM, whereas EGCG inhibited DU145, HH870, HH450 and HH639 cells at concentrations 89, 45, 62 and 42 µM. When compared with EGCG, ECG more effectively suppresses the growth of prostate cancer and epithelial ovarian cancer cell lines derived from tumors of patients with different stages of disease.


Introduction
There is accruing evidence that green tea may have anticancer activity (1), but the mechanisms for this action are poorly understood. Green tea is produced from the shrub Camellia sinensis (Fig. 1); leaves are dried but not fermented so that the green coloration attributed to polyphenols is retained. Commercially prepared green tea extracts contain $60% polyphenols (1). These polyphenols are the source of bioflavonoids, which have strong antioxidant activity.
Epicatechins have apparent activity against human cancer: they reportedly may promote apoptosis (2)(3)(4)(5)(6), arrest metastasis by inhibiting metalloproteinases (7,8), impair angiogenesis (9,10) and reverse multidrug resistance (11,12). Although all epicatechins except EC can potentially suppress cell proliferation (13)(14)(15)(16)(17)(18), EGCG appears the most promising and is therefore under clinical investigation in chemoprevention trials (19). However, given the wide range in physiologic potency of the different catechins, an exclusive focus on EGCG is probably short-sighted. EGCG is reportedly more effective than EGC in decreasing the intestinal absorption of cholesterol (20) and it is the most potent catechin inhibitor of HIV-1 reverse transcriptase (21), but ECG has the strongest collagenase inhibitory effect (22) and the highest antioxidant potential (23). By contrast, only EGC is a potent mediator of oxidative modification and an inhibitor of xanthine oxidase during hepatic catabolism of purines (24).
We hypothesized that the in vitro anticancer action of the various catechins varies with the type and stage of malignancy. We tested this hypothesis by examining proliferation of catechin-treated cell lines derived from organ-confined or metastatic prostate cancer (CaP) and from moderately or poorly differentiated epithelial ovarian cancer (EOC). The goal was to obtain data that would be useful for developing chemopreventive and therapeutic clinical trials in patients with gender-specific and non-specific solid tumors.

Human Cancer Cell Lines
Four gender-specific human cancer cell lines were used. The HH870 androgen-receptor-negative CaP cell line was developed at Hoag Cancer Center, Newport Beach, CA, from an organ-confined primary tumor that had been resected from a 56-year-old, previously untreated Caucasian (25). This tumor was Gleason Grade 3/4, with no evidence of vascular or perineural invasion or extracapsular extension (stage T2b). The DU145 metastatic CaP cell line (American Type Culture Collection line HTB-81) was derived from a brain lesion of 69-year-old male Caucasian. It is androgen insensitive and does not express prostate-specific antigen. Two EOC cell lines developed at Hoag Cancer Center were also used: HH639 was from a poorly differentiated clear cell, Grade 3 carcinoma in the omentum and left ovary of a 56year-old Caucasian female; HH450 was from moderately differentiated metastatic cells recovered from the abdominal fluid of a 52-year-old Asian female.
All four cell lines were cryopreserved in liquid nitrogen freezer at À70 o C. For recovery of cryopreserved cells, the vials were transferred to a 37 o C water bath for 15-30 s, further thawed at room temperature and then transferred to a 15 ml polypropylene tube with a Pasteur pipette. An aliquot of 9 ml of RPMI-9% fetal bovine serum (FBS) was added in drops. The cells were allowed to settle for 5 min and then centrifuged at 4 o C for 10 min at 300 g. Supernatant was removed, and cells were suspended in fresh RPMI, gently tapped and vortexed. Cell viability was monitored by 0.2% trypan blue dye exclusion, and cell count was determined using a hemocytometer.  Cells recovered from cryovials were grown in RPMI-1640 with glutamine (Invitrogen, Carlsbad, CA) supplemented with 9% FBS, HEPES buffer, gentamycin (5 mg%) and fungizone (0.5 mg%), at 37 C in a humidified atmosphere of 5% CO 2 . Upon confluency, cells were detached with sterile EDTA-dextrose (137 mM sodium chloride, 5.4 mM potassium chloride, 5.6 mM dextrose, 0.54 mM ethylene diamine tetra acetate (EDTA), 7.1 mM sodium bicarbonate) at 37 C for 5-15 min (or $45 min for HH639), recovered with cold RPMI-1640-9% FBS and resuspended in the same medium. Use of trypsin was avoided for harvesting the cells. Cell viability and cell count were reassessed before cells were seeded in culture flasks.

Growth Conditions
All experiments used 25 ml sterile polystyrene tissue culture flasks with a vented blue plug seal cap (Beckton Dickinson, Franklin Lakes, NJ, Cat. No. 353107). Each flask contained stock solution with or without epicatechin in concentrations of 50 mM (five flasks for each epicatechin and five flasks for control) and 25, 75 and 100 mM (three flasks for each epicatechin and three flasks for control). Cells (0.25 · 10 6 ) suspended in 10 ml of the RPMI-1640-FBS solution described above were transferred to each flask and allowed to grow until control cells reached confluency. The cells were detached with sterile EDTA-dextrose at 37 C for 5 min, recovered with cold RPMI-1640-FBS medium and resuspended in the same medium.
Cells were counted using a hemocytometer; trypan blue dye exclusion was used to determine the number of viable versus dead cells. The interval between seeding and confluent growth of control cells was used to calculate the doubling time and the number of cell cycles. The 50% inhibitory concentration (IC50) of each catechin in each cell line was calculated using a software program (Microcal Origin Corp, OriginLab Corporation, Northampton, MA). The cells were photographed directly from the flask using light microscopy (Olympus IX-70, Japan).

Statistics
Analyses of variance and Fisher's least significant difference (LSD) method were used for pairwise comparisons of values significant at the 0.05 level.

ECG as a Better Growth Suppressor Than EGCG: Microscopic Observations
Organ-confined prostate cancer cell line HH870 and primary and metastatic epithelial ovarian cancer cell lines (HH450 and HH639) seeded (2.5 · 10 5 cells) in flasks with or without various concentrations (25,50,75 or 100 mM) of ECG or EGCG were photographed under a light microscope after the untreated control cells reached confluency (Fig. 3). Both ECG and EGCG significantly affected the density of each cell line at or above 75 mM. The decrease in cell density at higher concentrations is much pronounced for ECG than for EGCG, a finding significant considering recommendations of clinical trials with EGCG (19).

ECG Suppresses Viable Cell Density Better Than EGCG
The mean density or viable cell number (in millions) (n ¼5 per treatment) of different cell lines was examined with or without   catechins (50 mM) (Fig. 4). The cell density was measured when growth of untreated cells reached confluency. Statistical analysis by ANOVA as well as by pairwise comparison showed that both ECG and EGCG significantly affected the cell density. ECG decreased the cell density of prostate cancer cells DU145, HH870 and ovarian cancer cell line HH639 more potently than EGCG. But EGCG inhibited the growth of ovarian cancer cell line HH450 better than ECG, suggesting the need to determine relative efficacy of ECG and EGCG in clinical trials for different cancers.
Tumor Cell Doubling Time: ECG versus EGCG Figure 5 shows the influence of the four epicatechins on cell doubling time. ECG and/or EGCG prolonged the doubling        (14) EGC and ECG inhibited the growth of a human lung cancer cell line, PC-9 cells as potently as did EGCG, but EC did not show significant growth inhibition. The mechanism of growth inhibition by EGCG was studied in relation to cell-cycle regulation. EGCG (50 and 100 mM) increased the percentages of cells in the G The relative cytotoxicity (CTX) of ECG to carcinoma HSC-2 cells and normal HGF-2 fibroblasts cells from the human oral cavity, as compared with other polyphenols in tea, was evaluated. For the HSC-2 carcinoma cells, ECG, CG and EGCG grouped as highly toxic, EGC as moderately toxic, and C and EC as least toxic. For the HGF-2 fibroblasts, ECG and CG grouped as highly toxic, EGCG as moderately toxic, and EGC, C and EC as least toxic. The CTX effects of the polyphenols were more pronounced to the carcinoma, than to the normal, cells.   Table 1 summarizes the effects of EC, ECG, EGC and EGCG on viability, doubling time and cycling of the four cell lines. Untreated cells from each line reached confluency in about 2.5 cell cycles. EC did not affect the proliferation of DU145, HH870 or HH450 cells but it reduced the proliferation of HH639 cells by half (P < 0.05) and prevented their confluent growth (Table 1). EGC did not affect the proliferation of any cell line (Table 1), whereas EGCG arrested proliferation of all four lines. ECG, followed by EGCG, was the most potent inhibitor of cell growth and cycling.

Dosimetric Analysis of Growth to Tumor cells: IC50 of ECG is Superior to EGCG
Proliferation of each cell line (n ¼ 3 per treatment) was monitored with or without ECG or EGCG at concentrations of 0, 25, 50, 75 and 100 mM. The dosimetric results plotted in Fig. 6 shows concentration-dependent suppression of cell growth by ECG and EGCG. The suppressive effect on cell density was striking at higher concentrations of ECG and EGCG. ECG was a more potent inhibitor of cell growth than EGCG. At 25 mM of EGCG, cell numbers for HH870 and DU145 were significantly higher than control values. Based on the results plotted in Fig. 6, IC50 values were calculated. The IC50 values are 24-30 mM for ECG, versus 42-89 mM for EGCG (Table 2). ECG suppressed growth at all higher concentrations tested (Fig. 6), whereas EGCG significantly (P < 0.05) enhanced proliferation of CaP cells at 25 mM, a finding relevant to chemoprevention trials with EGCG only.

Discussion
Green tea is widely consumed in Japan and China and its polyphenolic components have a chemopreventive effect against cancer in vitro and in vivo (39). A cup of green tea contains 100-150 mg catechins, of which 8% are EC, 15% are EGC, 15% are ECG and 50% are EGCG (40). Although numerous investigations have shown the role of EGCG in cancer chemoprevention, only a few studies have attempted to compare the relative antitumor efficacy of all four catechins (Table 3). When we used a systematic approach to assess the effect of various catechins on cell lines derived from gender-based cancers, we found that each catechin's antitumor activity depended on the type of tumor. EGCG was not always the most potent chemopreventive agent.
Most of the earlier literature (Table 3) indicates that EGCG is the most potent growth inhibitor of cell lines from glioblastoma, melanoma and cancers of the breast, colon, lung, prostate (androgen-receptor-positive), pancreas, liver and mouth. EGCG prevents proliferation of DU145 cells by arresting the cell cycle at G 0 /G 1 -phase (19). Gupta and others (26) have documented that G 0 /G 1 -phase arrest is independent of p53 mutation, and EGCG treatment of DU145 induces the cyclin kinase inhibitor WAF1/p21. These observations suggest that EGCG imposes a cell-cycle checkpoint (19). However, our results showed that ECG may be more potent than EGCG for inhibition of primary and metastatic CaP and EOC cells (Fig. 4, Tables 1 and 2). ECG significantly reduced cell proliferation (Table 1, Figs 2 and 3) and increased mean doubling time (Table 1, Fig. 4).
The in vitro effect of chemopreventive agents can be studied when tumor cells are in a matrix (1,4,27) or in a suspension (28,29). We used the suspension method because it exposes the entire cell surface to the chemotherapeutic agent. Our findings confirm an earlier report that used the matrix method to show that ECG is more potent than EGCG in suppressing the proliferation of DU145 CaP cells (4). Thus reported differences in the relative efficacy of different catechins may not be due to differences in methodology.
Not all tumor cells are killed by catechins. In our study, ECG (50 mM) induced death of most but not all HH639 cells. Doubling ECG's IC50 concentration might increase the tumor kill rate if ECG does not epimerize to CG. Our in vitro dose of 100 mM is equivalent to 29 mg (EC/EGC) to 45 mg (EGCG/ ECG), far less than the 100-150 mg (50% of which is EGCG) in one cup of green tea. However, Lee et al. (41) reported that plasma levels of EGCG and EGC in healthy volunteers increased to 78 and 223 ng ml À1 , respectively, 20 min after drinking brewed green tea (1.2 g of tea solids in 200 ml hot water). This suggests that drinking more than 10 cups of green tea may be necessary to maintain a plasma concentration of EGCG equivalent to that used in vitro by a dose of 50 mM or 22.5 mg. Kaegi (42) suggested a daily intake of 13 cups of green tea as a chemopreventive measure. Because this level of tea consumption is impractically high, chemoprevention of cancer with catechins may require administration of the appropriate catechin in a purified form.
In conclusion it may be stated that both green and black tea polyphenols are important components of antitumor aspect of complementary and alternative medicine (CAM), which play a significant role in the American health care system and in patients who suffer from chronic problems (43). While green tea catechin gallates such as EGCG and ECG possess potent antitumor activities, their epimers, commonly found in black tea, act as potent inhibitor of proteases involved in replication of viruses, including coronoviruses (44). There is a need to understand preventive and therapeutic potential of catechin gallates from both green and black teas. We are currently designing a phase I chemopreventive study to examine the effects of purified EGCG and ECG in patients who have been chosen observational management of organ-confined prostate cancer.