University of Southern California Dissertations and Theses

A randomized, double-blind, placebo-controlled phase II trial to examine the effect of Polyphenon E on endogenous hormone levels

(USC Thesis Other)

A randomized, double-blind, placebo-controlled phase II trial to examine the effect of Polyphenon E on endogenous hormone levels

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content A RANDOMIZED, DOUBLE –BLIND, PLACEBO-CONTROLLED PHASE ΙΙ TRIAL TO EXAMINE THE EFFECT OF POLYPHENON E ON ENDOGENOUS HORMONE LEVELS by Chen Ling ______________________________________________________ A Thesis Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE (APPLIED BIOSTATISTICS AND EPIDEMIOLOGY) May 2009 Copyright 2009 Chen Ling ii ACKNOWLEDGEMENTS I like to acknowledge the chair of my thesis committee Dr. Malcolm C. Pike of USC Department of Preventive Medicine, who served as mentor, role model, shoulder, and driving force throughout my 2-years study life in USC. I would also express my gratitude to Dr. Anna H. Wu and Dr. C. Leigh Pearce for their important data, helpful suggestions and editing throughout the course of my research and in the preparation of this manuscript. My special thanks go to Dr. Frank Z. Stanczyk and Elizabeth Gentzschein at the Reproductive Endocrine Research Laboratory at USC for their precious lessons and help on the hormone assays. iii TABLE OF CONTENTS Acknowledgements ii List of Tables iv List of Figures v Abstract vi A. Introduction 1 B. Background 1 1. Tea and its naturally occurring compounds 1 2. Green tea and human breast cancer 2 3. Green tea and endogenous circulating hormone levels 6 C. Materials and methods 8 1. Study population 8 2. Plasma hormone level measurement 11 3. Plasma hormone level adjustment 12 4. Statistical analysis 14 D. Results 14 1. Baseline characteristics 14 2. Hormone level change 15 E. Discussion 18 F. Conclusion 20 Bibliography 21 iv LIST OF TABLES Table 1: Published results discussing EGCG as a chemoprotective compound and the underlying potential mechanisms at a molecular level 3 Table 2: Black tea consumption and breast cancer risk 5 Table 3: Green tea consumption and breast cancer risk 6 Table 4: Results showing reproducibility of the hormone assays 13 Table 5: Characteristics of study population at baseline by study groups 15 Table 6: Mean percent change of circulating levels of adrostenedione, testosterone, estrone and estradiol 16 Table 7: Mean percent change of circulating levels of adiponectin, IGF1, IGFbp3 and SHBG 17 v LIST OF FIGURES Figure 1: Design of the green tea intervention study 10 Figure 2: Adrostenedione value regression for batch 1 13 vi ABSTRACT In this randomized, double-blind, placebo-controlled phase ΙΙ trial to examine the effect of Polyphenon E [containing the most important polyphenol, epidgallocatechin gallate (EGCG) in green tea] on endogenous hormone profiles, 111 participants were recruited and randomized into three different experimental arms: 400 mg EGCG daily, 800 mg EGCG daily or placebo for a period of 60 days. Four steroid hormones: androstenedione (Andr), testosterone (T), estrone (E1) and estradiol (E2), and two protein hormones: adiponectin (Adip) and insulin-like growth factor 1 (IGF1), plus two hormone binding proteins: IGF binding protein 3 (IGFbp3) and sex hormone binding globulin (SHBG) levels were measured at baseline, at end of month-1 and at end of month-2. This study failed to provide confirmatory evidence of lower serum E1 and E2 values due to green tea consumption. The E1 and E2 values declined in the group treated with EGCG but similar declines were also seen in the placebo group. The only treatment effect suggesting a protective effect against breast cancer was a decline in IGFbp3. 1 A. Introduction There has been accumulating and compelling evidence suggesting that green tea and its naturally occurring compounds exert a preventive effect against breast caner. However, the mechanism underlying this phenomenon remains unknown. Dr. Anna Wu and colleagues reported an inverse association between green tea intake and endogenous hormone levels (estrone and estradiol) in a cross-sectional study [30]. To better understand how green tea and its naturally occurring compound could affect breast cancer risk, a randomized, double-blind, placebo-controlled phase ΙΙ trial to examine the effect of Polyphenon E [containing 200 mg of the most important polyphenol in green tea, epigallocatechin gallate (EGCG) per capsule] on endogenous hormone levels in postmenopausal women was conducted. The objective of the study is to correlate any changes of circulating hormones and their binding proteins with the administration of Polyphenon E. B. Background 1. Tea and its naturally occurring compounds Tea is one of the most popular beverages consumed around the world, second only to water. Tea is derived from the leaf of the plant Camellia sinensis. Polyphenols are the naturally occurring compounds in fresh tea leaves and account for its flavor. The four 2 primary polyphenols in fresh tea leaves are epigallocatechin gallate (EGCG), pigallocatechin, epicatechin gallate and epicatechin, with the most abundant being EGCG. These four catechins account for up to 30% of dry weight of the fresh tea leaves [31]. There are a large number of research results suggesting that EGCG is a promising chemoprotective compound in tea leaves (Table 1). The different methods of processing the fresh tea leaves after harvest produce two major types of tea: green tea and black tea, which are popular in Asian countries (like China and Japan) and western countries (like the United States and Europe) respectively. In the processing of green tea, fresh tea leaves are steamed or heated immediately after harvest, in order to minimize oxidation of the naturally occurring polyphenols in the tea leaves. In the processing of black tea, the tea leaves are dried and crushed upon harvest to encourage oxidation, which converts the indigenous tea polyphenols to other polyphenols (mainly theaflavins and thearubigens). The oxidation of catechins to theaflavins and thearubigens gives it the characteristic red- brown color. 2. Tea and human breast cancer The incidence rates (IRs) of human breast cancer exhibit significant variation across different ethnic groups in the United States. According to the data from the Surveillance, Epidemiology and End Results (SEER) Program, the age-adjusted incidence rate of human breast cancer between 2001-2005 was 126.1 per 100,000 women per year for all races. While the race-specific incidence rates were 130.6 per 100,000 for Whites and 89.6 3 per 100,000 for Asians respectively [15]. This profound IR difference is likely attributed to variations in dietary and environmental factors. Among these many differences, green Table 1: Published results discussing EGCG as a chemoprotective compound and the underlying potential mechanisms at a molecular level Author Compounds Model Effect Pathway MDA-MB-231 breast cancer cell line (in vitro, ER-) Cells are arrested at G1 cell cycle, proliferation is inhibited Reduction in the expression of Cyclin E, Cyclin D and CDK4 Thangapazham [26] GTP (green tea polyphenol) Epigallocatechin (EGCG) Female athymic nude mice (in vivo) Reduced tumor burden, elevated tumor cell apoptosis Stuart [22] Raloxifene (selective ER modulator) EGCG MDA-MB-231 breast cancer cell line (in vitro, ER-) The apoptotic effect on the cancer cell line induced by EGCG is enhanced when it is combined with raloxifene The combination treatment significantly (modest) reduces the phosphorylation of both EGFR and AKT proteins Farabegoli [4] EGCG MCF-7 , a breast carcinoma cell line with high ER α expression level (in vitro) Inhibited cell proliferation pS2 (ERα activity marker) expression suppressed after 100 ug/ml 24hr treatment MCF-7 , a breast carcinoma cell line with high ER α expression level (in vitro) The combination of GTE and TAM is more effective than either agent given alone on inhibition the proliferation of ER+ human breast cancer cells: MCF-7, ZR75, T47D (in vitro) The GTE blocks the estrogen-induced ERE transactivation. The GTE also blocks the estrogen-induced MAPK activation Sartippour [18] Green tea extract (GTE) Tamoxifen (TAM) Female athymic nude mice (in vivo) The combination of GTE and TAM show more profound anti- tumor effect than either agent given alone The combination of GTE and TAM significantly reduce the expression of ER α Belguise [2] EGCG Her-2/neu- overexpressing mouse mammary tumor cell line EGCG alters the expression of key regulators in the epithelial to mesenchymal (EMT) transition inducing a more epithelial phenotype instead of invasive phenotype Inducing the expression of FOXO3 FOXO3 prevents TGF β-induced EMT. FOXO3 enhances the ER α gene expression. ER α signaling has been shown to promote a more epithelial phenotype 4 tea consumption is higher in Asians. As discussed above, green tea is richer in the more chemoprotective compound EGCG. As described in the review by Sun et al. in 2006 [23], studies on the relationship between black tea and breast cancer risk have given conflicting results. Specifically, black tea intake was positively associated with breast cancer risk in 6 cohort studies [summary odds ratio (OR) =1.19, 95% CI = 1.06-1.32; ORs were calculated from the comparison between the highest exposure level to the lowest exposure level] but was inversely associated with risk in eight case-control studies (summary OR = 0.91, 95% CI = 0.84-0.98) (Table 2). Studies on the relationship between green tea and breast cancer risk have given more consistent results. The meta-analysis of eight green tea studies showed that breast cancer risk was significantly reduced (OR=0.82, 95%CI = 0.75-0.90) (Table 3) in association with green tea intake. 5 Table 2: Black tea consumption and breast cancer risk Author Study period Population Cases/non- cases Exp lvl Lowest exposure level Highest exposure level OR (95%CI) for highest versus lowest exposure level Cohort studies Suzuki [24] 1984-1992 Japan 222/34782 3 never daily 1.44 (0.77-2.69) Michels [13] 1987-1997 Sweden 1271/57765 5 ≤1 cup/day a 4+ cups/day 1.13 (0.91-1.40) Key [7] 1969-1993 Japan 342/34332 3 ≤1 cup/day 5+ cups/day 1.10 (0.82-1.48) Goldboh m [5] 1986-1990 The Netherland 507/1376 6 <1 cup/day 5+ cups/day 1.31 (0.86-1.99) Zheng [33] 1986-1993 USA 1015/10056 4 <monthly 2+ cups/day 1.14 (0.92-1.41) Adebam ow [1] 1991-1999 USA 5 <1/month 2+ cups/day 1.02 (0.81-1.28) Summary OR: cohort studies 1.19 (1.06-1.32) Population-based case-control (PCC) studies Wu [29] 1995-1998 USA 501/593 3 non- drinkers >85 ml/day 0.81 (0.49-1.34) Mannisto [11] 1990-1995 Finland 301/454 5 0 >150 g/day 0.89 (0.50-1.57) McLaug hlin [12] 1982-1984 USA 1617/1617 2 never ever 0.97 (0.81-1.16) Ewertz [3] 1983-1984 Denmark 1474/1322 5 never 5+ cups/day 0.99 (0.69-1.42) Scharier [19] 1973-1980 USA 1510/1882 6 0 5+ cups/day 0.60 (0.20-1.90) Lubin [10] 1975-1979 Israel 804/804 4 0 5+ cups/day 0.80 (0.4-1.80) Summary OR: PCC studies 0.94 (0.81-1.09) Hospital based case-control (HCC) studies Tavani [25] 1983-1994 Italy 5882/5399 2 none ≥1 cup/day 0.94 (0.85-1.03) Rosenber g [17] 1975-1982 USA 2645/1476 4 0 5+ cups/day 0.60 (0.50-0.90) Summary OR: all case-control studies 0.91 (0.84-0.98) Summary OR: all studies 0.99 (0.93-1.06) a Normally, 1cup=120 ml in the U.S. 6 Table 3: Green tea consumption and breast cancer risk Author Study period Population Cases/non- cases Exp lvl Lowest exposure level Highest exposure level OR (95%CI) for highest versus lowest exposure level Cohort studies Suzuki [24] 1984-1992 Japan 103/14306 4 <1 cup/day ≥5 cups/day 1.17 (0.67-2.05) Suzuki [24] 1990-1997 Japan 119/20476 4 <1 cup/day ≥5 cups/day 0.61 (0.36-1.06) Key [7] 1969-1993 Japan 405/34332 3 ≤1 cup/day ≥5 cups/day 0.86 (0.62-1.21) Inoue [6] a 1993-1998 Singapore 172/320 3 < weekly daily 0.45 (0.26-0.79) Inoue [6] 1993-1998 Singapore 208/342 3 < weekly daily 1.04 (0.72-1.50) Summary OR: cohort studies 0.83 (0.68-1.01) Population-based case-control studies Wu [29] 1995-1998 USA 501/593 3 Non- drinkers >85ml/day 0.47 (0.26-0.85) Zhang [32] 2004-2005 China 1009/1009 5 Never/few 2+cups/day 0.48 (0.34-0.68) Zheng [34] 1996-2005 China 3371/3380 2 Never/Ever ever 0.88 (0.79-0.98) Summary OR:case-control studies 0.82 (0.74-0.91) Summary OR: all studies 0.82 (0.75-0.90) a The first OR (0.45, 95% CI:0.26-0.79) was obtained from low folate intake (< 133.4 mg/day) group, the second OR (1.04, 95% CI:0.72-1.50) was obtained from high folate intake ( ≥ 133.4 mg/day) group. 3. Green tea and endogenous circulating hormone levels Many studies have been conducted to explore the underlying protective mechanism of green tea against breast cancer. In addition to the experiments showing the protective effect and possible underlying mechanisms given by the EGCG at molecular level as described in Table 1, researches have been done to explore the cancer prevention effect of tea and its extract at the cellular and organ level. For example: Vergote et al. [27] 7 found that EGCG of green tea can significantly inhibit the growth of breast cancer cell lines (MCF-7 and MDA-MB-231) but not normal epithelial cells by inducing apoptosis. Liang et al. [9] suggested that EGCG can induce cell cycle arrest of human breast carcinoma by inhibition of CDK activities. Leong et al. [8] showed that green tea can directly inhibit mammary tumorigenesis in a mouse model. We are particularly interested in the potential breast cancer prevention effect of green tea via affecting circulating hormone levels. There is extensive evidence that endogenous hormones, such as estrogens and androgens, play a critical role in the etiology of breast cancer [31], and there is some limited evidence of a relationship between green tea intake and blood estrogen levels. Nagata et al. [14] first reported an inverse association between green tea intake and blood estrogen levels in a cross-sectional study conducted in Japan. In this study of premenopausal women, blood estradiol (E2) levels were significantly inversely associated with green tea intake after adjustment for age, body size, and intake of fat and fiber. In another cross-sectional study conducted by Wu et al. [30] in Singapore, serum estrone (E1) levels were found to be 13% lower in regular (at least weekly) green- tea drinkers compared to non or irregular drinkers and no such effect was seen with regular black tea drinkers. To obtain more information on the relationship between green tea intake and circulating hormone levels, we conducted this short-term intervention study to examine the effect of Polyphenon E intake on endogenous hormone levels (6 hormones and 2 hormone binding 8 proteins) in postmenopausal women. Positive results from this double-blind randomized intervention study would lend much credibility to the epidemiological findings on green tea and breast cancer risk and to the results of the cross-sectional studies of hormone levels. C. Material and methods 1. Study population This was a randomized, double-blind, placebo controlled trial using Polyphenon E capsules or placebo capsules. The study population consisted of 111 postmenopausal (at least one year past menses) women recruited over 2 years using advertisements at the University of Southern California campus and local newspapers. Firstly, volunteers were telephone prescreened to preliminarily determine eligibility, then seen for a pre-entry visit to confirm eligibility, obtain informed consent and be randomized. Participants had to meet the following inclusion criteria: 1) Female aged 50 or over and postmenopausal [no vaginal bleeding for 1 year and blood follicle stimulating hormone (FSH)>40 mIU/ml] 2) Able to understand the study, adhere to study schedules and requirements and be willing to provide informed consent and blood samples 3) Willing to refrain from drinking any kind of tea and taking any kind of herbal supplements during the study period 9 Participants with any of the following criteria were not eligible for the study 1) Regular tea drinker (drink tea at least 4 times a month) 2) History of allergic reactions to tea compounds 3) History of cancer (excluding non-melanoma skin cancer) 4) Current or recent (within 3 months) participation in a similar study including any study requiring following a specific dietary regimen 5) Current menopausal hormone therapy (HT) use or HT used within 3 months The study design is shown in Figure 1. Baseline information: Candidates who were eligible and who decided that they would participate were scheduled for the pre-entry clinic visit (v1a). At v1a, a blood specimen was obtained to measure FSH and blood estrogen levels to verify postmenopausal status. Within 2 weeks, a baseline visit was made (v1b) to obtain a second blood specimen [30 ml of blood (morning fasting sample)] for blood hormone levels and to perform other baseline studies. A structured interview and brief physical examination were conducted to obtain the participant’s baseline information including: weight, height, age, race, menstrual history, reproductive history and use of HT. Randomization: Participants were randomized to 1 of 3 study drug administration arms. The randomization information was kept by the Investigational Drug Service to maintain the blinding of investigators, staff, nurses, and subjects. Study drug and placebo capsules were identical in appearance. Drug administration and blood sample collection: The three drug administration arms were: 400 mg EGCG daily for 60 days, 800 mg EGCG daily for 60 days, and placebo. During the two months intervention, all participants were asked to keep a daily log to record the time of day when they took the Polyphenon E/placebo capsule. On study evaluation: Month-1: Assess study drug compliance, concomitant medication and adverse events, and tea consumption. Complete a 3-day diet history questionnaire. Perform body size measurements, make a blood draw and collect an overnight urine sample. Month-2: Assess study drug compliance, concomitant medication and adverse events, and tea consumption. Complete a 3-day diet history questionnaire. Perform body size measurement, make a blood draw and collect an overnight urine sample. Figure.1 Design of the green tea intervention study Screening of postmenopausal (at least one year past menses) women age ≥50 years, not on a specific diet, no current HT use, who do not consume four or more cups of tea/month Baseline assessment Randomized 400 mg EGCG daily for 60 days (Target =50) 800 mg EGCG daily for 60 days (Target = 50) placebo daily for 60 days (Target = 50) Assessment at the end of Month-1 Assessment at the end of Month-2 10 11 2. Plasma hormone level measurement A 10 ml sample of fasting venous blood was collected in heparinized collection tubes or EDTA sterile vacutainers before 10 a.m from each participant upon each visit (baseline, month-1, month-2). Blood samples were immediately centrifuged (2500 g, 15mins, 4 o C), separated and stored in 1.5ml tubes at -80 o C for hormone measurements. These specimens were then delivered to the Reproductive Endocrine Research Laboratory at the University of Southern California, directed by Dr Frank Stanczyk. Radioimmunoassays, previously validated in his laboratory [21], were used to measure plasma levels of androstenedione (Andr), testosterone (T), E1 and E2. Prior to quantification, the hormones were first extracted with hexane:ethyl acetate (1:1) and then separated from interfering metabolites by the use of celite column partition chromatography. Separated hormones were then dried and suspended in PBS buffer. The immunoradiometric assay was applied to measure each hormone. The adiponectin was measured by using the Human Adiponectin RIA Kit (Cat. #HADP-61 HK) from Linco Research (St. Charles, Missouri). IGF-1, IGFbp3, SHBG were quantified by solid-phase, enzyme-labeled chemiluminescent immunometric assays, using the Immulite 2000 (Siemens Medical Solutions Diagnostics, LA, CA). Bioavailable (non-SHBG bound) T and E2 concentrations were calculated using a validated algorithm on the basis of total testosterone, total E2, and SHBG measurements [16,20,28]. I conducted all the 4 steroid hormone assays (Andr, T, E1 and E2) and the results of the other 4 were obtained from another technician in Dr Frank Stanczyk’s lab. A total of 333 (111 ×3) plasma samples 12 were measured in the hormones assays. These samples were measured in 10 different batches. The 3 samples (baseline, month-1, month-2) obtained from one person were measured in the same batch. In a test experiment to check the reproducibility of hormones assays, replicate-blinded quality control samples were included to check reproducibility of the hormone assays; the intra-assay correlation coefficients between duplicates for androstenedione, testosterone, E1 and E2 were all above 0.95. In addition, quality control samples with low, medium and high concentrations (two samples per level) were included in each batch. The inter-assay coefficients of variation for androstenedione, testosterone, E1 and E2 were 6.58%, 1.37%, 6.42% and 7.65% respectively (Table 4) 3. Plasma hormone level adjustment Three quality controls (low medium high) were included in each batch. The measured values of these 3 quality controls (the quality controls were drawn from a large pool which has been quantified and aliquoted previously) were compared to their standard values. The regression formula was then applied to rest of the samples values in the batch to adjust for batch-to-batch variation for the four steroid hormones. For example, figure 2 shows the adjustment of androstenedione for batch 1. The theoretical value of low, medium and high quality controls were 5.22, 6.32 and 7.36 pg/ml (log scale), and the mean of measured value of these quality controls were 4.87, 5.90 and 6.94 respectively. The regression formula: (adjusted value) = 1.037 × (measured value) + 0.179 was applied to the rest of the samples as shown in Figure 2. Table 4: Results showing reproducibility of the hormone assays Batch 1 a Batch 2 Inter-assay coefficient of variation Dup.1 b Dup.2 Stand. value Dup.3 Dup.4 Stand. value Androstendione Low 5.24 5.60 5.22 4.70 5.19 5.22 11.41% Medium 6.15 6.46 6.32 5.78 6.27 6.32 3.97% High 7.55 7.28 7.36 7.04 7.20 7.36 4.37% R 2c 0.99 0.99 Average 6.58% Testosterone Low 4.79 4.82 4.94 4.70 4.70 4.94 1.40% Medium 5.71 5.92 5.99 5.80 5.86 5.99 0.10% High 7.20 6.90 7.00 6.82 6.76 7.00 2.60% R 2 0.99 0.99 Average 1.37% Estrone Low 3.46 3.87 3.22 3.20 3.09 3.22 13.6% Medium 4.08 4.41 4.32 4.04 4.09 4.32 1.69% High 5.44 5.59 5.35 5.32 5.15 5.35 3.98% R 2 0.95 0.99 Average 6.42% Estradiol Low 2.37 2.46 2.56 2.38 2.34 2.56 0.15% Medium 3.36 3.86 3.64 2.95 3.36 3.64 10.20% High 4.51 4.74 4.62 3.90 4.37 4.62 12.60% R 2 1.00 0.99 Average 7.65% a Results shown are for log transformed values, where untransformed values are measured in pg/ml. b Duplicate 1-4 are measurement results of 4 identical samples blindly tested in two different assay batches. The standard values are theoretical values of these testing samples. c Correlation coefficient between the measured and standard values. Figure.2 Adrostenedione value regression for batch 1 y = 1.0372x + 0.1787 R 2 = 0.9998 0 2 4 6 8 02 46 Measurement value Theoretical value 8 13 14 4. Statistical analysis Since we are still blinded to the full group information, the only comparison that can be done at present is that between treatment group and placebo group. The mean values of each hormone at each visit were calculated within each group. One sample (treatment month minus baseline value) t-tests were applied to compare the hormone levels of either month -1 or month -2 versus baseline. All p-values quoted are 2-tailed. Calculations were performed using Stata 10 (Stata Corp, College Station, TX) D. Results 1. Baseline characteristics We succeeded in recruiting 111 subjects in the period available for recruitment. Five of these 111 subjects were excluded from the analysis since they had abnormally high estrone levels (> 60 pg/ml) at baseline. Descriptive characteristics by study groups are presented in Table 5. There were 73 and 33 subjects in the treatment group and placebo group respectively. The treatment group was slightly older than the placebo group (60.2 vs 56.9, p=0.03). The placebo group also had a greater proportion of Asians (15.2% vs 4.1%) and a lower proportion of Whites. The groups were approximately the same in terms of age at menarche, age at menopause, height, weight and BMI. The FSH level of the treatment group was slightly lower than the placebo group (62.0 vs 69.3 mIU/ml), but 15 this difference was not statistically significant (p = 0.18). The placebo group had a greater proportion of participants who had simple hysterectomy history (18.2% vs 4.1%). Table 5: Characteristics of study population at baseline by study group Treatment group Placebo group No. women 73 33 Mean age at baseline (SD),y 60.2 (7.89) 56.9 (6.39) Age at menarche (SD),y 12.7 (1.32) 12.6 (1.40) Age at menopause (SD),y 49.8 (4.97) 47.5 (6.00) Mean height (SD),cm 159.0 (7.04) 160.0 (8.10) Mean weight (SD),kg 75.0 (15.1) 74.5 (19.0) Mean BMI (SD),kg/m 2 29.4 (5.38) 29.2 (6.36) FSH (SD), mIU/ml 62.0 (25.0) 69.3 (29.3) Ethnicity (%) Asian 3 (4.1) 5 (15.2) Africa American 11 (15.1) 6 (18.2) Hispanic 41(56.2) 18 (54.6) White 18 (24.7) 5 (15.2) Parity (%) Ever pregnant 65 (89.0) 31 (93.9) Never pregnant 6 (8.2) 2 (6.1) Missing 2 (2.8) 0 Uterine surgery history (%) None 67 (91.8) 25 (75.8) Simple hysterectomy 3 (4.1) 6 (18.2) Hysterectomy & unilateral oophorectomy 1 (1.4) 0 (0) Hysterectomy & bilateral oophorectomy 1 (1.4) 1 (3.0) Missing 1(1.4) 1 (3.0) 2. Hormone level change Percent change of circulating levels of the four steroid hormone levels: Andr, T, E1 and E2 are presented in Table 6. Adip, IGF1, IGFbp3, SHBG are presented in Table 7. 16 Table 6: Mean percent change of circulating levels of androstenedione, testosterone, estrone and estradiol Treatment group Placebo group Month-1Month-2Month-1 Month-2 Andr N737032 32 % change2.93% -0.1%-10.1% -0.65% p a 0.40 0.98 0.01 0.90 p b 0.02 0.94 T (total) N737032 33 % change-3.40%-3.90% 2.1% 2.42% p0.310.200.57 0.57 p 0.33 0.23 T (free) c N726932 33 % change-0.91%-1.61%1.66% 4.03% p0.800.620.70 0.34 p 0.68 0.31 E1 N737032 33 % change-2.18%-7.13%-1.16% -6.30% p0.42 0.05 0.77 0.10 p 0.83 0.88 E2 (total) N736932 33 % change1.24%-5.62%-4.39% -12.2% p0.660.220.37 0.03 p 0.55 0.35 E2 (free) c N726832 33 % change 0.94% -3.69% -4.72% -11.3% p0.740.440.35 0.04 p 0.29 0.30 a p-value is obtained via one sample t-test: Ha: hormone level of month-1 or month-2 ≠ baseline. b p-value is obtained via two sample t-test: Ha: change of hormone level of treatment group in month-1 or month-2 ≠ change of hormone level of placebo group. c % change in free T equals the % change in non-SHBG bound T; similar for free E2 and non-SHBG bound E2. 17 Table 7: Mean percent change of circulating levels of adiponectin, IGF1, IGFbp3 and SHBG Treatment group Placebo group Month-1Month-2Month-1 Month-2 Adip N727031 33 % change-4.07%-1.10%6.44% 7.62% p0.180.800.37 0.25 p 0.11 0.27 IGF1 N727032 33 % change-0.48%-0.98%-1.25% -1.30% p0.810.570.62 0.63 p 0.82 0.91 IGFbp3 N727032 33 % change-1.38%-3.46%-1.02% 0.80% p0.15 0.01 0.43 0.65 p 0.83 0.03 SHBG N727032 33 % change-2.83%-3.93%0.52% -2.31% p0.09 0.05 0.81 0.23 p 0.24 0.61 Two different circulating forms (total and free) of T and E2 are reported separately. The percent change was derived as follows: let y = log hormone level value; change from baseline to Month-1 is z1 = (y1-yb) and from baseline to Month-2 is z2 = (y2-yb) where yb, y1 and y2 are values found at Baseline, Month-1 and Month-2 respectively with mean values z1m and z2m respectively. The mean percent change are then exp(z1m) × 100 and exp(z2m) × 100 respectively. One sample two-sided t-tests were applied to analyze the hormone values changes after 1-month or 2-months treatment. Two-sample two-sided 18 t-test were applied to analyze if the change in treatment group is different from the change in placebo group. Significant differences are highlighted in the table. Andr decreased 10.10% in the placebo group at Month-1 (p = 0.01) and the difference from the treatment group of 13.0% was also statistically significant (p = 0.02). E1 decreased 7.13% in the treatment group at Month-2 (p = 0.05) but the difference from the placebo group of 0.83% was not statistically significant (p = 0.88). E2 decreased 12.2% in the placebo group at Month-2 (p = 0.03) but the difference from the treatment group of 6.6% was not statistically significant (p = 0.35). E2 (free) decreased 11.3% in the placebo group at Month-2 (p = 0.04) but the difference from the treatment group of 7.6% was not statistically significant (p = 0.30). ). The IGFbp3 level decreased at Month- 2 by 3.46% in the treatment group (p = 0.01) while increasing by 0.80% in placebo group; this difference was statistically significant (p = 0.03). SHBG decreased 3.93% in the treatment group at Month-2 (p = 0.05) but the difference from the placebo group of 1.62% was not statistically significant (p = 0.61) E. Discussion This is only a preliminary evaluation of the experimental data, since we are still blind to the distinction between the active treatment group assignments, both the 400 mg EGCG daily and 800 mg EGCG daily groups have had to be combined together as the “treatment” group in the analysis. The data will be re-evaluated after the group assignments are unblinded. 19 Before discussing the findings, some methodological features of the study should be considered. The study population was relatively small (106 participants); the groups were not well balanced in terms of race and uterine surgery history (Table 5). Also the treatment group was on average 3.3 years older than the placebo group. However, if we are looking for an effect on the change of hormone levels within each person, the imbalance between the two groups will not jeopardize our analysis. The validation hormone assays gave fairly good reproducibility: the inter-assay coefficients of variation for Andr, T, E1 and E2 hormones were 6.58%, 1.37%, 6.42% and 7.65% respectively (Table 4). The percent changes in the values of each hormone are reported in Tables 6 and 7. The changes shown are calculated using unadjusted values. Calculations using adjusted values give very similar results. To our knowledge, this is the first intervention study of the effect of EGCG on circulating hormone levels in postmenopausal women. Nagata et al. [14] first reported an inverse association between green tea intake and blood estrogen levels in a cross-sectional study in premenopausal women. It was reported in their paper that green tea intake was inversely correlated with E2 on Day 11 of the menstrual cycle (r = -0.32, p = 0.04), but no actual hormone levels were given. There is only a single study of the effect of green tea consumption in postmenopausal women. This study was a cross-sectional study conducted by Dr Wu et al. in Singapore [30], which provided the basis of the study reported here. They found that serum E1and E2 levels were 12.5% and 8.0% lower in regular (weekly/daily) green-tea drinkers compared to none or irregular tea drinkers. The 20 dose of EGCG in our intervention study was chosen to be the equivalent of 5 and 10 cups of green tea per day, so we expected to observe declines in E1 and E2 in the EGCG treatment group in this study of similar magnitudes. We did observe declines in E1 and E2 of 7.1% and 5.6% in the treatment group (with the decline in E1 being just statistically significant), but there were also declines in the control group of 6.3% and 12.2% and the differences between the treated and control group were not statistically significant. The declines in the treated group were not much less than those reported in the study of Wu et al. [30], but the declines in the placebo group were unexpected and we have been unable to identify any factor (such as a change in weight) which would explain these latter declines. We are also unable to explain the decline in Andr at Month-1 in the placebo group. The only statistically significant result to suggest a protective effect of green tea against breast cancer was that for IGFbp3. IGFbp3 level was decreased by 3.46% (p = 0.01) in the treatment group at Month-2 and this change was statistically significant compared to the increase of 0.80% in the placebo group (p=0.03). F. Conclusion In summary, this study failed to provide confirmatory evidence of lower serum E1 and E2 values due to green tea consumption. The E1 and E2 values declined in the group treated with EGCG but similar declines were also seen in the placebo group. The only treatment effect suggesting a protective effect against breast cancer was a decline in IGFbp3. 21 Bibliography: 1. Adebamowo CA, Cho E, Sampson L, Katan MB, Spiegelman D, Willett WC, Holmes MD. Dietary flavonols and flavonol-rich foods intake and the risk of breast cancer. Int J Cancer. 2005; 114:628-33. 2. Belguise K, Guo S, Sonenshein GE. Activation of FOXO3a by the green tea polyphenol epigallocatechin-3-gallate induces estrogen receptor alpha expression reversing invasive phenotype of breast cancer cells. Cancer Res. 2007; 67:5763- 70. 3. Ewertz M, Gill C. Dietary factors and breast-cancer risk in Denmark. Int J Cancer. 1990; 46:779-84. 4. Farabegoli F, Barbi C, Lambertini E, Piva R. (-)-Epigallocatechin-3-gallate downregulates estrogen receptor alpha function in MCF-7 breast carcinoma cells. Cancer Detect Prev. 2007; 31:499-504. 5. Goldbohm RA, Hertog MG, Brants HA, van Poppel G, van den Brandt PA. Consumption of black tea and cancer risk: a prospective cohort study. J Natl Cancer Inst. 1996; 88:93-100. 6. Inoue M, Robien K, Wang R, Van Den Berg DJ, Koh WP, Yu MC. Green tea intake, MTHFR/TYMS genotype and breast cancer risk: the Singapore Chinese Health Study. Carcinogenesis. 2008; 29:1967-72. 7. Key TJ, Sharp GB, Appleby PN, Beral V, Goodman MT, Soda M, Mabuchi K. Soya foods and breast cancer risk: a prospective study in Hiroshima and Nagasaki, Japan. Br J Cancer. 1999; 81:1248-56. 8. Leong H, Mathur PS, Greene GL. Inhibition of mammary tumorigenesis in the C3(1)/SV40 mouse model by green tea. Breast Cancer Res Treat. 2008; 107:359- 69. 9. Liang YC, Lin-Shiau SY, Chen CF, Lin JK. Inhibition of cyclin-dependent kinases 2 and 4 activities as well as induction of Cdk inhibitors p21 and p27 during growth arrest of human breast carcinoma cells by (-)-epigallocatechin-3- gallate. J Cell Biochem. 1999; 75:1-12. 10. Lubin F, Ron E, Wax Y, Modan B. Coffee and methylxanthines and breast cancer: a case-control study. J Natl Cancer Inst. 1985; 74:569-73. 22 11. Männistö S, Pietinen P, Virtanen M, Kataja V, Uusitupa M. Diet and the risk of breast cancer in a case-control study: does the threat of disease have an influence on recall bias? J Clin Epidemiol. 1999; 52:429-39. 12. McLaughlin CC, Mahoney MC, Nasca PC, Metzger BB, Baptiste MS, Field NA. Breast cancer and methylxanthine consumption. Cancer Causes Control. 1992; 3:175-8. 13. Michels KB, Holmberg L, Bergkvist L, Wolk A. Coffee, tea, and caffeine consumption and breast cancer incidence in a cohort of Swedish women. Ann Epidemiol. 2002; 12:21-6. 14. Nagata C, Kabuto M, Shimizu H. Association of coffee, green tea, and caffeine intakes with serum concentrations of estradiol and sex hormone-binding globulin in premenopausal Japanese women. Nutr Cancer. 1998; 30:21-4. 15. National Cancer Institute: http://seer.cancer.gov/statfacts/html/breast.html 16. Rinaldi S, Déchaud H, Toniolo P, Kaaks R. Reliability and validity of direct radioimmunoassays for measurement of postmenopausal serum androgens and estrogens. IARC Sci Publ. 2002; 156:323-5. 17. Rosenberg L, Miller DR, Helmrich SP, Kaufman DW, Schottenfeld D, Stolley PD, Shapiro S. Breast cancer and the consumption of coffee. Am J Epidemiol. 1985; 122:391-9. 18. Sartippour MR, Pietras R, Marquez-Garban DC, Chen HW, Heber D, Henning SM, Sartippour G, Zhang L, Lu M, Weinberg O, Rao JY, Brooks MN. The combination of green tea and tamoxifen is effective against breast cancer. Carcinogenesis. 2006; 27:2424-33. 19. Schairer C, Brinton LA, Hoover RN. Methylxanthines and breast cancer. Int J Cancer. 1987; 40:469-73. 20. Södergård R, Bäckström T, Shanbhag V, Carstensen H. Calculation of free and bound fractions of testosterone and estradiol-17 beta to human plasma proteins at body temperature. J Steroid Biochem. 1982; 16:801-10. 21. Stanczyk FZ, Shoupe D, Nunez V, Macias-Gonzales P, Vijod MA, Lobo RA. A randomized comparison of nonoral estradiol delivery in postmenopausal women. Am J Obstet Gynecol. 1988; 159:1540-6. 22. Stuart EC, Scandlyn MJ, Rosengren RJ. Role of epigallocatechin gallate (EGCG) in the treatment of breast and prostate cancer. Life Sci. 2006; 79:2329-36. 23 23. Sun C-L, Yuan J-M, Koh1 WP, Yu MC. Green tea, black tea and breast cancer risk: a meta-analysis of epidemiological studies. Carcinogenesis 2006; 27:1310– 1315. 24. Suzuki Y, Tsubono Y, Nakaya N, Suzuki Y, Koizumi Y, Tsuji I. Green tea and the risk of breast cancer: pooled analysis of two prospective studies in Japan. Br J Cancer. 2004; 90:1361-3. 25. Tavani A, Pregnolato A, La Vecchia C, Favero A, Franceschi S. Coffee consumption and the risk of breast cancer. Eur J Cancer Prev. 1998; 7:77-82. 26. Thangapazham RL, Singh AK, Sharma A, Warren J, Gaddipati JP, Maheshwari RK. Green tea polyphenols and its constituent epigallocatechin gallate inhibits proliferation of human breast cancer cells in vitro and in vivo. Cancer Lett. 2007; 245:232-41. 27. Vergote D, Cren-Olivé C, Chopin V, Toillon RA, Rolando C, Hondermarck H, Le Bourhis X. (-)-Epigallocatechin (EGC) of green tea induces apoptosis of human breast cancer cells but not of their normal counterparts. Breast Cancer Res Treat. 2002; 76:195-201. 28. Vermeulen A, Verdonck L, Kaufman JM. A critical evaluation of simple methods for the estimation of free testosterone in serum. J Clin Endocrinol Metab. 1999; 84:3666-72. 29. Wu AH, Yu MC, Tseng C-C, Hankin J, Pike MC. Green tea and risk of breast cancer in Asian Americans. Int J Cancer 2003; 106: 574–579. 30. Wu AH, Arakawa K, Stanczyk FZ, Van Den Berg D, Koh W-P, Yu MC. Tea and circulating estrogen levels in postmenopausal Chinese women in Singapore. Carcinogenesis 2005; 26:976-980. 31. Wu AH, Yu MC. Tea, hormone-related cancers and endogenous hormone levels. Review Mol Nutr Food Res 2006; 50:160-169. 32. Zhang M, Holman CD, Huang JP, Xie X. Green tea and the prevention of breast cancer: a case-control study in Southeast China. Carcinogenesis. 2007; 28:1074-8. 33. Zheng W, Doyle TJ, Kushi LH, Sellers TA, Hong CP, Folsom AR. Tea consumption and cancer incidence in a prospective cohort study of postmenopausal women. Am J Epidemiol. 1996; 144:175-82. 34. Zheng W, Shrubsole MJ, Lu W, Chen Z, Shu XO, Dai Q, Cai Q, Gu K, Ruan ZX, Gao YT. Drinking green tea modestly reduces breast cancer risk. The Journal of Nutrition. 2009; 139: 310-316.

Abstract (if available)

Abstract In this randomized, double-blind, placebo-controlled phase II trial to examine the effect of Polyphenon E [containing the most important polyphenol, epidgallocatechin gallate (EGCG) in green tea] on endogenous hormone profiles, 111 participants were recruited and randomized into three different experimental arms: 400 mg EGCG daily, 800 mg EGCG daily or placebo for a period of 60 days. Four steroid hormones: androstenedione (Andr), testosterone (T), estrone (E1) and estradiol (E2), and two protein hormones: adiponectin (Adip) and insulin-like growth factor 1 (IGF1), plus two hormone binding proteins: IGF binding protein 3 (IGFbp3) and sex hormone binding globulin (SHBG) levels were measured at baseline, at end of month-1 and at end of month-2. This study failed to provide confirmatory evidence of lower serum E1 and E2 values due to green tea consumption. The E1 and E2 values declined in the group treated with EGCG but similar declines were also seen in the placebo group. The only treatment effect suggesting a protective effect against breast cancer was a decline in IGFbp3.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Association between endogenous sex hormone levels and cognition: the Women’s Isoflavone Soy Health (WISH) trial

PDF

Effects of post-menopausal hormone therapy on arterial stiffness in the ELITE trial

PDF

The effects of hormone therapy on carotid artery intima-media thickness and serum lipids by ApoE4 genotype

PDF

The effect of vitamin D supplementation on the progression of carotid intima-media thickness and arterial stiffness in elderly African American women: Results of a randomized placebo-controlled trial

Asset Metadata

Creator Ling, Chen (author)

Core Title A randomized, double-blind, placebo-controlled phase II trial to examine the effect of Polyphenon E on endogenous hormone levels

School Keck School of Medicine

Degree Master of Science

Degree Program Applied Biostatistics

Publication Date 04/22/2009

Defense Date 03/22/2009

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag hormone,OAI-PMH Harvest,phase II trial,Polyphenon E

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Pike, Malcolm C. (committee chair), Pearce, Celeste Leigh (committee member), Wu, Anna (committee member)

Creator Email chenling@usc.edu,ling-c02@hotmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m2101

Unique identifier UC179639

Identifier etd-Ling-2739 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-221552 (legacy record id),usctheses-m2101 (legacy record id)

Legacy Identifier etd-Ling-2739.pdf

Dmrecord 221552

Document Type Thesis

Rights Ling, Chen

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu