Objective
The purpose of this study was to investigate the effect of epigallocatechin gallate (EGCG) on rat leiomyoma (ELT3) cells in vitro and in a nude mice model.
Study Design
ELT3 cells were treated with various concentrations of EGCG. Cell proliferation, proliferation cell nuclear antigen (PCNA), and cyclin-dependent kinase 4 (Cdk4) protein levels were evaluated. ELT3 cells were inoculated subcutaneously in female athymic nude mice. Animals were fed 1.25 mg EGCG (in drinking water)/mouse/day. Tumors were collected and evaluated at 4 and 8 weeks after the treatment.
Results
Inhibitory effect of EGCG (200 μmol/L) on ELT3 cells was observed after 24 hours of treatment ( P < .05). At ≥50 μmol/L, EGCG significantly decreased PCNA and Cdk4 protein levels ( P < .05). In vivo, EGCG treatment dramatically reduced the volume and weight of tumors at 4 and 8 weeks after the treatment ( P < .05). The PCNA and Cdk4 protein levels were significantly reduced in the EGCG-treated group ( P < .05).
Conclusion
EGCG effectively inhibits proliferation and induces apoptosis in rat ELT3 uterine leiomyoma cells in vitro and in vivo.
Tea is one of the most popular beverages consumed worldwide. Based on the manufacturing process, green, black, and oolong tea are the 3 major commercial types of tea. Green tea, without fermentation, is processed to prevent the oxidation of green leaf polyphenols, although most polyphenols are oxidized in black tea or oolong tea during fermentation production. This fermentation converts catechin to theaflavins and thearubigins, consequently decreasing the catechin content. The polyphenols present in green tea are flavonols, commonly known as catechins, which contain 5 major subtypes: catechin, epicatechin, epicatechin gallate, epigallocatechin, and epigallocatechin gallate (EGCG). These natural compounds show diverse chemical and biologic activities and are nontoxic under daily dose. In recent years, evidences from epidemiologic and animal studies have emerged that have shown chemopreventive and anticancer potential of dietary polyphenols. Several studies have suggested positive correlations between human consumption of green tea and a lower incidence of gastric, esophageal, ovarian, pancreatic, and colorectal cancers. EGCG, the major polyphenol in green tea, was found in animal studies to inhibit carcinogenesis effectively and broadly in various organs, such as the esophagus, stomach, and duodenum.
Uterine leiomyoma (fibroid tumors) are the most common tumors of the reproductive tract in women of reproductive age. It clinically affects 25–30% of women in the United States; however, an incidence of upward of 77% has been reported. Although uterine fibroid tumors are benign tumors and often asymptomatic, they may cause debilitating symptoms, such as abnormal uterine bleeding, abdominal pain, and in some cases infertility. Pregnancy complications that are attributed to uterine fibroid tumors include miscarriage, preterm labor, and postpartum hemorrhage. Approaches that are available for the treatment of uterine fibroid tumors include pharmacologic options, surgical approaches, and uterine artery embolization. We recently reported on the utility of gene therapy approach as a potential alternative treatment for uterine leiomyoma. However, approaches that are minimally invasive and easy to perform and that preserve fertility would be preferable and cost effective. A dietary agent, such as green tea, if proved effective against uterine fibroid tumors, would be a welcome addition, because it is safe, inexpensive, well tolerated, and readily available.
Cancer chemoprevention is defined as the use of natural, synthetic, or biologic chemical agents to reverse, suppress, or prevent either the initial phase of carcinogenesis or the progression of premalignant cells to cancer. The basic difference between cancer chemoprevention and cancer treatment is that the goal of the former approach is to lower the rate of cancer incidence by delaying or suppressing the process of cancer development. Leiomyoma could be an ideal candidate disease for chemoprevention because of its high prevalence and slow progression. The effects of EGCG on leiomyoma cells in vivo (in an animal model), however, remain unclear. This study was designed to investigate the effects of EGCG in vivo on leiomyoma lesions that developed in a nude mice-based model.
Materials and Methods
Materials
Smooth muscle cell basal media were purchased from Lonza (Walkersville, MD). EGCG was purchased from Sigma Chemical Company (St. Louis, MO). EGCG was dissolved in distilled water and filtered through a 0.22-μm filter to have 10 mmol/L stock solution. All other chemicals and biochemicals were of the highest quality available from commercial resources. The 60-day released 17β-estrodials were purchased from Innovative Research of America (Sarasota, FL).
Cell culture of uterine leiomyoma cells
The Eker rat tumor-derived ELT3 uterine leiomyoma cell line was provided by Dr Cheryl Walker (MD Anderson Cancer Center, Houston, TX). The cells were cultured in smooth muscle cell basal medium that was supplemented with 5% fetal bovine serum, 0.1% insulin, 0.2% human fibroblast growth factor–basic, 0.1% gentamicin, amphotericin B–1000, and 0.1% human epidermal growth factor (Lonza). The cells were maintained at 37 ° C and 5% CO 2 -95% humidified air.
Morphologic observation
For assessment of morphologic changes, the cells that were treated with desired concentrations of EGCG for various durations were observed with the use of a phase-contrast microscope and photographs were taken with the Nikon Eclipse TE2000-S microscope (Nikon Instruments Inc, Melville, NY).
Leiomyoma cell proliferation assay
The proliferative response of ELT3 cells to EGCG was determined by the methylthiazolyldiphenyl-tetrazolium bromide ([MTT] assay; Sigma Chemical Company), as we have described previously. Briefly, ELT3 cells were plated at density of 2 × 10 3 cells/well in 96-well plates. After 24 hours, the cells were treated with various concentrations of EGCG (0, 1.0, 50, 100, and 200 μm) for up to 7 days. Media were changed every other day. At designed time points, the cells that were treated with various concentrations of EGCG were incubated with 50 μL/well of 0.5% MTT solution for 4 hours at 37°C. The MTT solution was removed, and the dye was solubilized with 150 μL/well of dimethyl sulfoxide for 5 minutes. The optical density of each well was measured with a spectrophotometer at 570 nm. For each concentration of EGCG, 3 wells were assayed. Mean values of optical density for each concentration were calculated. The MTT data for EGCG treatment were collected at the following time points: days 1, 3, 5, and 7 after treatment.
Western blotting for PCNA and cyclin-dependent kinase 4 (Cdk4) in uterine leiomyoma cells and tumor tissue
For Western blotting, ELT3 rat leiomyoma cells that were treated with the indicated concentrations of EGCG for 48 hours were lysed, and the proteins were harvested. Proteins from tumor tissues that had developed in nude mice were isolated with a standard protocol at 4 and 8 weeks after the inoculation of ELT3 cells. Equivalent amounts of protein extracts, from ELT3 cells or tumor tissue, were separated by NuPAGE Novex 10% Bis-Tris Gel (Invitrogen Life Technologies, Carlsbad, CA) under a reducing condition with 200 V for 50 minutes, as we have described earlier. The proteins were then electrophoretically transferred onto polyvinylidene fluoride membranes (Millipore Corp, Billerica, MA) with the use of the XCell II Blot Module (Invitrogen Life Technologies). After nonspecific binding sites were blocked by incubation for 1 hour with phosphate-buffered saline solution that contained 5% fat-free milk and 0.1% Tween 20, the membranes were incubated with the corresponding primary antibodies overnight at 4°C. Immunologic detection was performed with primary antibodies against human proliferation cell nuclear antigen (PCNA; 1:500 dilution) (Santa Cruz Biotechnology, Inc, Santa Cruz, CA) and Cdk4 (1:1000 dilution; Sigma Chemical Company). The membranes were then incubated for 1 hour with horseradish peroxidase conjugated secondary antibodies diluted 1:5000 with blocking buffer. The antigen-antibody complexes were detected with the enhanced chemiluminescence detection system (Amersham Bioscience, Piscataway, NJ). The membranes were reprobed with a monoclonal antibody raised against β-actin (diluted1:5000; Sigma Chemical Company) as an internal control for protein loading and normalization between samples. Films that had been exposed to blots were scanned, and optical densities of the positive signals were quantified.
TUNEL staining for EGCG-treated leiomyoma cells
ELT3 leiomyoma cells were seeded onto BioCoat CultureSlide (BD Bioscience, San Jose, CA) and treated with various concentrations of EGCG (0, 1.0, 50, 100, and 200 μm) for 48 hours. Apoptosis in ELT3 cells was determined by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) technique with the DeadEnd Fluorometric TUNEL System (Promega, Madison, WI), as we have described earlier. Briefly, the slides were fixed in 4% paraformaldehyde solution and then placed in equilibration buffer. The slides were incubated with terminal deoxynucleotidyl transferase, recombinant, enzyme (rTdT) incubation buffer, which consisted of equilibration buffer, nucleotide mix, and rTdT enzyme at 37°C for 1 hour. Positive control was performed with 10 units/mL DNase I–treated slides (Sigma Chemical Company) and negative control-omitted rTdT enzyme. The reaction was terminated with 2× saline-sodium citrate buffer. The nuclei were stained by 1 μg/mL of propidium iodide solution (Sigma Chemical Company). Slides were examined under an Eclipse TE2000-S, fluorescence microscope (Nikon Instruments Inc). Cells with green fluorescence were scored as apoptotic.
Animal experiments
Female athymic nude mice (Hsd:Athymic Nude- Foxn1 nu / Foxn1 + ) that were 5–6 weeks old were purchased from Harlan Sprague Dawley (Indianapolis, IN). The mice were maintained in a specific pathogen germ-free environment. The animal experiments were conducted according to the guidelines of experimental animals by the Institutional Animal Care and Use Committee at Meharry Medical College. After quarantine, one 60-day estrogen pellet (17β-estradiol 1.7 mg; Innovative Research of America, Sarasota, FL) was implanted surgically under the neck skin of each mouse under aseptic conditions. Three days later, all mice were anesthetized and inoculated subcutaneously with 1 × 10 7 ELT3 uterine leiomyoma cells in 300-μL serum-free medium, in the right flank region. All surgical procedures were performed under aseptic conditions. The animals were then randomized into 2 groups: the control group (n = 10) was fed with autoclaved distilled water; the experimental group (n = 10) was fed with 1.25-mg EGCG/mouse/day dissolved in autoclaved distilled water and dispensed in the drinking water bottles, starting from the day of the inoculation. The animals were fed with standard laboratory diet during the experiment. Animals were examined weekly for bodyweight, and the size of tumors was measured with calipers in 3 dimensions. The tumor volume was calculated from the following formula: length × width × height × 0.5326. Five animals from each group were sacrificed at week 4 after injection. The tumors were excised, weighed, and stored in 10% formalin solution or at –80°C until further biochemical analysis. The remaining 5 animals in each group were allowed to remain in the study until 8 weeks. The tumors and organs were collected at this point for histologic and biochemical analysis. The experiment was terminated at 8 weeks when control mice showed severe morbidity with tumor volume exceeding 30% of bodyweight, as per approved animal protocol. Animal experiments were repeated twice.
Histologic and immunohistochemical analysis
Tumor samples for histologic evaluation and immunohistochemistry were fixed in 10% neutral formalin, dehydrated, and embedded in paraffin. The blocks were sectioned at a thickness of 5 μm for hematoxylin and eosin staining. For PCNA immunohistochemical staining, the primary PCNA antibody (Santa Cruz Biotechnology, Inc) was diluted 1:500 in Dulbecco’s phosphate-buffered saline solution and incubated for 1 hour at room temperature. After being washed with Dulbecco’s phosphate-buffered saline solution, a biotinylated secondary antibody (diluted 1:200) was used for 10 minutes, after streptavidin-conjugated peroxidase for 15 minutes. The sections were stained with diaminobenzidine substrate and counterstained with hematoxylin. The PCNA-positive cells were counted in 4 randomly selected high-power fields as a percentage of total cells, as we have described earlier.
Statistical analysis
The data were expressed as the mean ± SD of the values. Statistical significance was determined by analysis of variance. A difference with a probability value of < .05 was considered statistically significant.
Results
Effects of EGCG on cell morphologic findings in uterine leiomyoma cells
ELT3 rat uterine leiomyoma cells displayed oval or short-spindle morphologic condition and grew fast in complete medium ( Figure 1 , A). The cells that were treated with 1μmol/L of EGCG displayed similar morphologic condition, compared with that of untreated controls. Change in cell morphologic condition was observed after 72 hours in cells that were treated with ≥50 μmol/L EGCG. These cells grew slowly and less crowded than the cells that were treated with lower concentrations of EGCG ( Figure 1 , B and C). The cell morphologic condition changed markedly as blebbing formation, and apoptotic vesicles were observed in cells that were treated with 100 μmol/L EGCG for 72 hour ( Figure 1 , D). After treatment with 200 μmol/L EGCG for 48 hours, the ELT3 cells were no longer robust and had no cellular crowding. The cells were shrunken and fewer in number and had irregular-shaped nuclei.
