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Purpose

Lignans such as secoisolariciresinol diglucoside (SDG) in flaxseed, are metabolizes to bioactive mammalian lignans of END and ENL. Because mammalian lignans have chemical structural similarity to the natural estrogen, they are thought to behave like selective estrogen receptor modulators (SERM) and therefore have anticancer effect against hormone-related cancers. We isolated a series of lignan compounds, named as Vitexins, from the seed of Chinese herb Vitex Negundo.

Experimental Design

We purified several Vitexin lignan compounds. Cytotoxic and antitumor effects were analyzed in cancer cells and in tumor xenograft models. In vivo metabolism of Vitexins was determined in rat.

Results

Contrasts to the classical lignans, Vitexins were not metabolized to END and ENL. A mixture of Vitexins EVn-50 and purified Vitexin compound VB1 have cytotoxic effect on breast, prostate, and ovarian cancer cells and induces apoptosis with cleavage in PARP protein, up-regulation of Bax, and down-regulation of Bcl-2. This induction of apoptosis seems to be mediated by activation of caspases because inhibition of caspases activity significantly reduced induced apoptosis. We demonstrated a broad antitumor activity of EVn-50 on seven tumor xenograft models including breast, prostate, liver, and cervical cancers. Consistent with in vitro data, EVn-50 treatment induced apoptosis, down-regulated of Bcl-2, and up-regulated Bax in tumor xenografts.

Conclusion

Vitexin is a class of nature lignan compounds, whose action and anticancer effect is mediated by the mechanisms different from the classical lignans. Vitexin induced antitumor effect and cytotoxic activity is exerted through proapoptotic process, which is mediated by a decreased Bcl-2/Bax ratio and activation of caspases.

Lignans and their in vivo metabolites, especially enterolactone (ENL), have attracted substantial interest as potential chemopreventive agents for prostate cancer. Preclinical and clinical interventions performed with lignan-rich flaxseed that use surrogate biomarkers as endpoints suggest that lignans may attenuate prostate carcinogenesis in individuals with increased risk or with diagnosed cancer. No unequivocal prostate cancer risk reduction has been found for lignans in epidemiological studies, suggesting that lignan concentrations found in populations consuming a regular non-supplemented diet are not chemopreventive in prostate cancer. Presumably, the main obstacles in assessing the efficacy of food lignans is limited knowledge of the serum and tissue lignan concentrations required for the putative prevention. Further clinical studies performed with the purified compounds are required to substantiate a health claim.

Although hormone replacement therapy (HRT) is used by post-menopausal women for the relief of menopausal symptoms and the potential reduction of osteoporosis, HRT also increases their risk of Alzheimer’s disease, stroke, breast cancer, and endometrial cancer. Since the majority of these effects are associated primarily with estrogen binding to only one of the estrogen receptors (ER), new assays are needed that can more efficiently evaluate ER-binding and identify ligands selective for ER-α and ER-β. High performance liquid chromatography-tandem mass spectrometry (LC-MS-MS) was combined with ultrafiltration as a new method to investigate the relative binding of compounds to the ERs and to evaluate the structures of these estrogens. Mixtures of estradiol and six equine estrogens including equilin, equilenin, 8,9-dehydroestrone, and their 17β-hydroxyl derivatives were assayed simultaneously to determine their relative binding to human ER-α and ER-β. Estrogens containing a 17β-OH group were found to have higher relative affinities for the estrogen receptors than their ketone analogs. In addition, 17β-EN showed selectivity for binding to ER-β over ER-α. The results were compared to the IC50 values obtained by using a conventional radiolabled estradiol competitive binding assay. Finally, the utility of negative ion electrospray tandem mass spectrometry for the unambiguous identification these estrogen isomers was investigated. Several characteristic recyclization pathways during tandem mass spectrometry were identified, which might be useful for distinguishing related estrogens.

Background

Case–control studies have reported that exogenous estrogen use is associated with increased risk of skin cancer. The effects of menopausal hormone therapy on incidence of nonmelanoma skin cancer and melanoma were evaluated in post hoc analyses of the Women’s Health Initiative randomized placebo-controlled hormone therapy trials of combined estrogen plus progestin (E + P) and estrogen only (E-alone).

Methods

Postmenopausal women aged 50–79 years were randomly assigned to conjugated equine estrogen (0.625 mg/d) plus medroxyprogesterone acetate (2.5 mg/d) or placebo in the E + P trial if they had an intact uterus (N = 16 608) or to conjugated equine estrogen alone or placebo in the E-alone trial if they had a hysterectomy (N =10 739); the mean follow-up was 5.6 and 7.1 years, respectively. Incident nonmelanoma skin cancers (n = 980 [E + P trial]; n = 820 [E-alone trial]) and melanomas (n = 57 [E + P trial]; n =38 [E-alone trial]) were ascertained by self-report. Incident cases of cutaneous malignant melanoma were confirmed by physician review of medical records. Incidences of nonmelanoma skin cancer and melanoma were compared between the two randomization groups within each trial using hazard ratios (HRs), with corresponding 95% confidence intervals (CIs) and Wald statistic P values from Cox proportional hazards models. All statistical tests were two-sided.

Results

Rates of incident nonmelanoma skin cancer and melanoma were similar between the active hormone (combined analysis of E + P and E-alone) and placebo groups (nonmelanoma skin cancer: HR = 0.98, 95% CI = 0.89 to 1.07; melanoma: HR = 0.92, 95% CI = 0.61 to 1.37). Results were similar for the E + P and E-alone trials when analyzed individually.

Conclusions

Menopausal hormone therapy did not affect overall incidence of nonmelanoma skin cancer or melanoma. These findings do not support a role of menopausal estrogen, with or without progestin, in the development of skin cancer in postmenopausal women.

The Women’s Health Initiative found that combination estrogen and progesterone hormone replacement therapy increases breast cancer and cardiovascular disease risk, which compelled many women to seek herbal alternatives such as black cohosh extract (BCE) to relieve their menopausal symptoms. While several clinical trials document the efficacy of BCE in alleviating menopausal symptoms, preclinical studies to determine how BCE works have yielded conflicting results. Part of this is because there is not a universally accepted method to standardize the dose of black cohosh triterpenes, the presumed active ingredients in the extract. Although the mechanism by which BCE relieves symptoms is unknown, several hypotheses have been proposed: it acts 1) as a selective estrogen receptor modulator, 2) through serotonergic pathways, 3) as an antioxidant, or 4) on inflammatory pathways. We found that while the most prominent triterpene in BCE, 23-epi-26-deoxyactein, suppresses cytokine-induced nitric oxide production in brain microglial cells, the whole BCE extract actually enhanced this pathway. A variety of activities have been reported for black cohosh and its compounds, but the absorption and tissue distribution of these compounds is unknown.

Background

Mother's diet during pregnancy is important, since plant lignans and their metabolites, converted by the intestinal microflora to enterolignans, are proposed to possess multiple health benefits. Aim of our study was to investigate whether a dietary intervention affects lignan concentrations in the serum of pregnant women.

Methods

A controlled dietary intervention trial including 105 first-time pregnant women was conducted in three intervention and three control maternity health clinics. The intervention included individual counseling on diet and on physical activity, while the controls received conventional care. Blood samples were collected on gestation weeks 8-9 (baseline) and 36-37 (end of intervention). The serum levels of the plant lignans 7-hydroxymatairesinol, secoisolariciresinol, matairesinol, lariciresinol, cyclolariciresinol, and pinoresinol, and of the enterolignans 7-hydroxyenterolactone, enterodiol, and enterolactone, were measured using a validated method.

Results

The baseline levels of enterolactone, enterodiol and the sum of lignans were higher in the control group, whereas at the end of the trial their levels were higher in the intervention group. The adjusted mean differences between the baseline and end of the intervention for enterolactone and the total lignan intake were 1.6 ng/ml (p = 0.018, 95% CI 1.1-2.3) and 1.4 ng/mg (p = 0.08, 95% CI 1.0-1.9) higher in the intervention group than in the controls. Further adjustment for dietary components did not change these associations.

Conclusion

The dietary intervention was successful in increasing the intake of lignan-rich food products, the fiber consumption and consequently the plasma levels of lignans in pregnant women.

Trial registration

ISRCTN21512277, http://www.isrctn.org

Cardiovascular disease (CVD) is more common in postmenopausal than premenopausal women, suggesting vascular protective effects of estrogen. Vascular estrogen receptors ERα, ERβ and a transmembrane estrogen-binding protein GPR30 have been described. Also, experimental studies have demonstrated vasodilator effects of estrogen on the endothelium, vascular smooth muscle and extracellular matrix. However, randomized clinical trials have not supported vascular benefits of menopausal hormone therapy (MHT), possibly due to the subjects' advanced age and age-related changes in estrogen synthesis and metabolic pathways, the vascular ERs number, distribution and integrity, and the post-ER vascular signaling pathways. Current MHT includes natural estrogens such as conjugated equine estrogen, as well as synthetic and semi-synthetic estrogens. New estrogenic formulations and hormone combinations have been developed. Phytoestrogens is being promoted as an alternative MHT. Specific ER modulators (SERMs), and selective agonists for ERα such as PPT, ERβ such as DPN, and GPR30 such as G1 are being evaluated. In order to enhance the vascular effectiveness of MHT, its type, dose, route of administration and timing may need to be customized depending on the subject's age and pre-existing CVD. Also, the potential interaction of estrogen with progesterone and testosterone on vascular function may need to be considered in order to maximize the vascular benefits of MHT on senescent blood vessels and postmenopausal CVD.

Epidemiological studies have shown that cardiovascular disease (CVD) is less common in pre-menopausal women (Pre-MW) compared to men of the same age or post-menopausal women (Post-MW), suggesting cardiovascular benefits of estrogen. Estrogen receptors (ERs) have been identified in the vasculature, and experimental studies have demonstrated vasodilator effects of estrogen/ER on the endothelium, vascular smooth muscle (VSM) and extracellular matrix. Several natural and synthetic estrogenic preparations have been developed for relief of menopausal vasomotor symptoms. However, whether menopausal hormone therapy (MHT) is beneficial in postmenopausal CVD remains controversial. Despite reports of vascular benefits of MHT from observational and experimental studies, randomized clinical trials (RCTs), such as the Heart and Estrogen/progestin Replacement Study (HERS) and the Women’s Health Initiative (WHI), have suggested that, contrary to expectations, MHT may increase the risk of CVD. These discrepancies could be due to age-related changes in sex hormone synthesis and metabolism, which would influence the effective dose of MHT and the sex hormone environment in Post-MW. Age-related changes in the vascular ER subtype, structure, expression, distribution, and post-ER signaling pathways in the endothelium and VSM, along with factors related to the design of RCTs, preexisting CVD condition, and structural changes in the blood vessels architecture have also been suggested as possible causes of MHT failure in CVD. Careful examination of these factors should help in identifying the causes of the changes in the vascular effects of estrogen with age. The sex hormone metabolic pathways, the active versus inactive estrogen metabolites, and their effects on vascular function, the mitochondria, the inflammatory process and angiogenesis should be further examined. Also, the genomic and non-genomic effects of estrogenic compounds should be viewed as integrated rather than discrete responses. The complex interactions between these factors highlight the importance of careful design of MHT RCTs, and the need of a more customized approach for each individual patient in order to enhance the vascular benefits of MHT in postmenopausal CVD.

Objective

To provide information and recommendations to women with a previous diagnosis of breast cancer and their physicians regarding hormone replacement therapy (HRT).

Outcomes

Control of menopausal symptoms, quality of life, prevention of osteoporosis, prevention of cardiovascular disease, risk of recurrence of breast cancer, risk of death from breast cancer.

Evidence

Systematic review of English-language literature published from January 1990 to July 2001 retrieved from MEDLINE and CANCERLIT.

Recommendations

· Routine use of HRT (either estrogen alone or estrogen plus progesterone) is not recommended for women who have had breast cancer. Randomized controlled trials are required to guide recommendations for this group of women. Women who have had breast cancer are at risk of recurrence and contralateral breast cancer. The potential effect of HRT on these outcomes in women with breast cancer has not been determined in methodologically sound studies. However, in animal and in vitro studies, the development and growth of breast cancer is known to be estrogen dependent. Given the demonstrated increased risk of breast cancer associated with HRT in women without a diagnosis of breast cancer, it is possible that the risk of recurrence and contralateral breast cancer associated with HRT in women with breast cancer could be of a similar magnitude. · Postmenopausal women with a previous diagnosis of breast cancer who request HRT should be encouraged to consider alternatives to HRT. If menopausal symptoms are particularly troublesome and do not respond to alternative approaches, a well-informed woman may choose to use HRT to control these symptoms after discussing the risks with her physician. In these circumstances, both the dose and the duration of treatment should be minimized.

Validation

Internal validation within the Steering Committee on Clinical Practice Guidelines for the Care and Treatment of Breast Cancer; no external validation.

Sponsor

The steering committee was convened by Health Canada.

Completion date

October 2001.

Background

Studies conducted in Asian populations have suggested that high consumption of soy-based foods that are rich in isoflavone phytoestrogens is associated with a reduced risk of breast cancer. However, the potential associations of other dietary phytoestrogens — i.e., the lignans or their bioactive metabolites, the enterolignans — with the risk of breast cancer are unclear.

Methods

We prospectively examined associations between the risk of postmenopausal invasive breast cancer and dietary intakes of four plant lignans (pinoresinol, lariciresinol, secoisolariciresinol, and matairesinol) and estimated exposure to two enterolignans (enterodiol and enterolactone), as measured with a selfadministered diet history questionnaire, among 58 049 postmenopausal French women who were not taking soy isoflavone supplements. Relative risks (RRs) and 95% confidence intervals (CIs) were estimated using multivariable Cox proportional hazards regression models. Analyses were further stratified by the combined estrogen and progesterone receptor (ER/PR) status of the tumors. Statistical tests were two-sided.

Results

During 383 425 person-years of follow-up (median follow-up, 7.7 years), 1469 cases of breast cancer were diagnosed. Compared with women in the lowest intake quartiles, those in the highest quartile of total lignan intake (>1395 μg/day) had a reduced risk of breast cancer (RR = 0.83, 95% CI = 0.71 to 0.95, Ptrend = .02, 376 versus 411 cases per 100 000 person-years), as did those in the highest quartile of lariciresinol intake (RR = 0.82, 95% CI = 0.71 to 0.95, Ptrend = .01). The inverse associations between phytoestrogen intakes and postmenopausal breast cancer risk were limited to ER- and PR-positive disease (e.g., RR for highest versus lowest quartiles of total plant lignan intake = 0.72, 95% CI = 0.58 to 0.88, Ptrend = .01, 174 versus 214 cases per 100 000 person-years, and RR for highest versus lowest quartiles of total enterolignan level = 0.77, 95% CI = 0.62 to 0.95, Ptrend = .01, 164 versus 204 cases per 100 000 person-years).

Conclusions

High dietary intakes of plant lignans and high exposure to enterolignans were associated with reduced risks of ER- and PR-positive postmenopausal breast cancer in a Western population that does not consume a diet rich in soy.

The two main pathways for metabolizing estrogen are via 16alpha-hydroxylation and 2-hydroxylation. The 16alpha-hydroxy metabolites are biologically active; the 2-hydroxy metabolites are not. It is suggested that women who metabolize a larger proportion of their endogenous estrogen via the 16alpha-hydroxy pathway may be at significantly elevated risk of breast cancer compared with women who metabolize proportionally more estrogen via the 2-hydroxy pathway. In particular, it is suggested that the ratio of urinary 2-hydroxyestrone (2-OHE1) to 16alpha-hydroxyestrone (16alpha-OHE1) is an index of reduced breast cancer risk. This pilot study compared this ratio in postmenopausal women diagnosed with breast cancer to those of healthy controls. Urinary concentrations of estrone (E1), 17beta-estradiol (E2) and estriol (E3) were also quantified. White women who were subjects in a previous breast cancer case-control study at our institution were eligible for inclusion. All participants provided a sample of their first morning urine. The results from the first 25 cases and 23 controls are presented here. The ratio of 2-OHE1 to 16alpha-OHE1 was 12% lower in the cases (p=0.58). However, urinary E1 was 30% higher (p=0.10), E2 was 58% higher (p=0.07), E3 was 15% higher (p=0.48), and the sum of E1, E2, and E3 was 22% higher (p=0.16) in the cases. These preliminary results do not support the hypothesis that the ratio of the two hydroxylation metabolites (2-OHE1/16alpha-OHE1) is an important risk factor for breast cancer or that it is a better predictor of breast cancer risk than levels of E1, E2 and E3 measured in urine.

Tamoxifen, a selective estrogen receptor modulator, is the standard of care for premenopausal women with estrogen or progesterone receptor-positive breast cancer and a valid option for treating post-menopausal women. However, a substantial number of tamoxifen-treated patients relapse following surgical resection, while remain disease-free for many years. It appears that the primary effectors of tamoxifen activity are its active metabolites, rather than tamoxifen itself. Cytochrome P450 (CYP) enzymes, CYP2D6 in particular, play a major role in the metabolism of tamoxifen to active metabolites. More than 75 germline CYP2D6 variants have been identified.

A test predicting lack of response to tamoxifen could supplement information used by clinicians and patients in treatment decision-making. For example, physicians and patients may opt to switch to an alternative therapy upfront.

Tamoxifen, a selective estrogen receptor modulator, is the standard of care for premenopausal women with estrogen or progesterone receptor-positive breast cancer and a valid option for treating post-menopausal women. However, a substantial number of tamoxifen-treated patients relapse following surgical resection, while others remain disease-free for many years. It appears that the primary effectors of tamoxifen activity are its active metabolites, rather than tamoxifen itself. Cytochrome P450 (CYP) enzymes, CYP2D6 in particular, play a major role in the metabolism of tamoxifen to active metabolites. More than 75 germline CYP2D6 variants have been identified.

A test predicting lack of response to tamoxifen could supplement information used by clinicians and patients in treatment decision-making. For example, physicians and patients may opt to switch to an alternative therapy upfront.

Phyto-oestrogens are plant compounds structurally similar to oestradiol, which have been proposed to have protective effects against breast cancer. The main class of phyto-oestrogens in the Western diet is lignans. Literature reports on the effect of lignans in breast cancer risk have been conflicting. We performed three separate meta-analyses to examine the relationships between (i) plant lignan intake, (ii) enterolignan exposure and (iii) blood enterolactone levels and breast cancer risk. Medline, BIOSIS and EMBASE databases were searched for publications up to 30 September 2008, and 23 studies were included in the random effects meta-analyses. Overall, there was little association between high plant lignan intake and breast cancer risk (11 studies, combined odds ratio (OR): 0.93, 95% confidence interval (95% CI): 0.83–1.03, P=0.15), but this association was subjected to marked heterogeneity (I2=44%). Restricting the analysis to post-menopausal women, high levels of plant lignan intake were associated with reduced breast cancer risk (7 studies, combined OR: 0.85, 95% CI: 0.78, 0.93, P<0.001) and heterogeneity was markedly reduced (I2=0%). High enterolignan exposure was also associated with breast cancer (5 studies, combined OR: 0.73, 95% CI: 0.57, 0.92, P=0.009) but, again, there was marked heterogeneity (I2=63%). No association was found with blood enterolactone levels (combined OR: 0.82, 95% CI: 0.59–1.14, P=0.24). In conclusion, plant lignans may be associated with a small reduction in post-menopausal breast cancer risk, but further studies are required to confirm these results.

Estrogen deficiency is a major risk factor for osteoporosis in postmenopausal women. Although hormone replacement therapy (HRT) has been rampantly used to recompense for the bone loss, but the procedure is coupled with severe adverse effects. Hence, there is a boost in the production of newer synthetic products to ward off the effects of menopause-related osteoporosis. As of today, there are several prescription products available for the treatment of postmenopause osteoporosis; most of these are estrogenic agents and combination products. Nevertheless, in view of the lack of effect and/or toxicity of these products, majority of the postmenopausal women are now fascinated by highly publicized natural products. This is an offshoot of the generalized consensus that these products are more effective and free from any adverse effects. Recently, certain plant-derived natural products, mostly phytoestrogens (isoflavones, lignans, coumestanes, stilbenes, flavonoids) and many more novel estrogen-like compounds in plants have been immensely used to prevent menopause-related depletion in bone mineral density (BMD). Although, a number of papers are published on menopause-related general symptoms, sexual dysfunction, cardiovascular diseases, Alzheimer's disease, diabetes, colon, and breast cancers, there is paucity of literature on the accompanying osteoporosis and its treatment. In view of the controversies on synthetic hormones and drugs and drift of a major population of patients toward natural drugs, it was found worthwhile to investigate if these drugs are suitable to be used in the treatment of postmenopausal osteoporosis. Preparation of this paper is an attempt to review the (a) epidemiology of postmenopausal osteoporosis, (b) treatment modalities of postmenopausal osteoporosis by hormones and synthetic drugs and the associated drawbacks and adverse effects, and (c) prevention and treatment of postmenopausal osteoporosis by phytoestrogens, their drawbacks and toxicity. It is apparent that both the categories of treatment are useful and both have adverse effects, but the plant products are nonscientific and hence are not advised to be used till more studies are undertaken to ensure that the benefits clearly outweigh the risk, in addition to recognition by Food and Drug Administration.

BACKGROUND

Hormonal effects of soy and isoflavones have been investigated in numerous trials with equivocal findings. We aimed to systematically assess the effects of soy and isoflavones on circulating estrogen and other hormones in pre- and post-menopausal women.

METHODS

The Cochrane Library, MEDLINE and EMBASE (plus reviews and experts) were searched to December 2007. Inclusion of randomized or residential crossover trials of soy or isoflavones for 4 or more weeks on estrogens, SHBG, FSH, LH, progesterone and thyroid hormones in women was assessed independently in duplicate. Six percent of papers assessed were included. Data concerning participants, interventions, outcomes, potential effect modifiers and trial quality characteristics were extracted independently in duplicate.

RESULTS

Forty-seven studies (11 of pre-, 35 of post- and 1 of perimenopausal women) were included. In premenopausal women, meta-analysis suggested that soy or isoflavone consumption did not affect primary outcomes estradiol, estrone or SHBG concentrations, but significantly reduced secondary outcomes FSH and LH [by ∼20% using standardized mean difference (SMD), P = 0.01 and 0.05, respectively]. Menstrual cycle length was increased by 1.05 days (95% CI 0.13, 1.97, 10 studies). In post-menopausal women, there were no statistically significant effects on estradiol, estrone, SHBG, FSH or LH, although there was a small statistically non-significant increase in total estradiol with soy or isoflavones (∼14%, SMD, P = 0.07, 21 studies).

CONCLUSIONS

Isoflavone-rich soy products decrease FSH and LH in premenopausal women and may increase estradiol in post-menopausal women. The clinical implications of these modest hormonal changes remain to be determined.

Estrogen metabolism may play an important role in mammary carcinogenesis in postmenopausal women. We evaluated the effects of prior oral contraceptive (OC) treatment and current soy isoflavone consumption on endogenous estrogen metabolite concentration and biomarkers of tissue estrogen exposure in a monkey model. One hundred eighty-one female cynomolgus macaques were randomized to receive OC or placebo for 26 months premenopausally, then ovariectomized and randomized to one of three diets for 36 months: an isoflavone-depleted soy protein isolate (Soy−) diet, a diet containing soy protein isolate with a human equivalent of 129 mg isoflavone/d (Soy+), or a Soy− diet supplemented with conjugated equine estrogens (CEE+) at a human equivalent dose of 0.625 mg/d. Reverse-phase high-performance liquid chromatography directly coupled with tandem mass spectrometry was used to measure the concentrations of estrogen species in urine samples. Generally, prior OC treatment was associated with significantly reduced urinary estrogen metabolites (25–55% reduction; P < 0.05 for each versus OC−). Animals that consumed isoflavones postmenopausally had increased urinary 2-hydroxyestrone and 16α-hydroxyestrone (50% and 56% increases, respectively), but reduced levels of 2-hydroxyestradiol, 2-methoxyestradiol, and 17-epiestriol (92%, 63%, and 66%, respectively), compared with animals fed a Soy− diet. Isoflavones did not have widespread effects on uterine or mammary proliferation biomarkers, whereas prior OC significantly reduced two of three proliferation end points in the endometrium. Premenopausal OCs may have long-term systemic effects on response to estrogen and its metabolism whereas postmenopausal dietary isoflavones may alter endogenous estrogen metabolism in a modest but selective manner.

The link between estrogen and the development and proliferation of breast cancer is well documented. Estrogen stimulates growth and inhibits apoptosis through estrogen receptor-mediated mechanisms in many cell types. Interestingly, there is strong evidence that estrogen induces apoptosis in breast cancer and other cell types. Forty years ago, before the development of tamoxifen, high-dose estrogen was used to induce tumor regression of hormone-dependent breast cancer in post-menopausal women. While the mechanisms by which estrogen induces apoptosis were not completely known, recent evidence from our laboratory and others demonstrates the involvement of the extrinsic (Fas/FasL) and the intrinsic (mitochondria) pathways in this process. We discuss the different apoptotic signaling pathways involved in E2 (17β-estradiol)-induced apoptosis, including the intrinsic and extrinsic apoptosis pathways, the NF-κB (nuclear factor-kappa-B)-mediated survival pathway as well as the PI3K (phosphoinositide 3-kinase)/Akt signaling pathway. Breast cancer cells can also be sensitized to estrogen-induced apoptosis through suppression of glutathione by BSO (L-buthionine sulfoximine). This finding has implications for the control of breast cancer with low-dose estrogen and other targeted therapeutic drugs.

Background

Estrogens are recognized causal factors in breast cancer. Interindividual variation in estrogen metabolism may also influence the risk of breast cancer and could provide clues to mechanisms of breast carcinogenesis. Long-standing hypotheses about how estrogen metabolism might influence breast cancer have not been adequately evaluated in epidemiological studies because of the lack of accurate, reproducible, and high-throughput assays for estrogen metabolites.

Methods

We conducted a prospective case–control study nested within the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO). Participants included 277 women who developed invasive breast cancer (case subjects) and 423 matched control subjects; at PLCO baseline, all subjects were aged 55–74 years, postmenopausal and not using hormone therapy, and provided a blood sample. Liquid chromatography–tandem mass spectrometry was used to measure serum concentrations of 15 estrogens and estrogen metabolites, in unconjugated and conjugated forms, including the parent estrogens, estrone and estradiol, and estrogen metabolites in pathways defined by irreversible hydroxylation at the C-2, C-4, or C-16 positions of the steroid ring. We calculated hazard ratios (HRs) approximating risk in highest vs lowest deciles of individual estrogens and estrogen metabolites, estrogens and estrogen metabolites grouped by metabolic pathways, and metabolic pathway ratios using multivariable Cox proportional hazards models. All statistical tests were two-sided.

Results

Nearly all estrogens, estrogen metabolites, and metabolic pathway groups were associated with an increased risk of breast cancer; the serum concentration of unconjugated estradiol was strongly associated with the risk of breast cancer (HR = 2.07, 95% confidence interval [CI] = 1.19 to 3.62). No estrogen, estrogen metabolite, or metabolic pathway group remained statistically significantly associated with the risk of breast cancer after adjusting for unconjugated estradiol. The ratio of the 2-hydroxylation pathway to parent estrogens (HR = 0.66, 95% CI = 0.51 to 0.87) and the ratio of 4-hydroxylation pathway catechols to 4-hydroxylation pathway methylated catechols (HR = 1.34, 95% CI = 1.04 to 1.72) were statistically significantly associated with the risk of breast cancer and remained so after adjustment for unconjugated estradiol.

Conclusions

More extensive 2-hydroxylation of parent estrogens is associated with lower risk, and less extensive methylation of potentially genotoxic 4-hydroxylation pathway catechols is associated with higher risk of postmenopausal breast cancer.

Evidence suggests dietary phytoestrogens may reduce the risk of certain hormonal cancers (e.g. breast and prostate). There is a paucity of data regarding phytoestrogens and colorectal cancer risk. Phytoestrogens are plant compounds with estrogen-like activities. Main classes include isoflavones (found in legumes such as soy) and lignans (found in grains, seeds, nuts, fruits, and vegetables). Although isoflavones have dominated phytoestrogen cancer research, lignans may be more relevant to North American diets. Food questionnaires and analytic databases have recently been modified to incorporate some lignan information. We conducted a case-control study to evaluate the association between phytoestrogen intake and colorectal cancer risk. Colorectal cancer cases were diagnosed in 1997–2000, aged 20–74 y, identified through the population-based Ontario Cancer Registry, and recruited by the Ontario Familial Colorectal Cancer Registry. Controls were a sex and age-group matched random sample of the population of Ontario. Epidemiologic and food frequency questionnaires were completed by 1095 cases and 1890 control subjects. Multivariate logistic regression analysis was used to obtain adjusted odds ratio (OR) estimates. Dietary lignan intake was associated with a significant reduction in colorectal cancer risk [OR (T3 vs. T1) = 0.73; 95% CI: 0.56, 0.94], as was isoflavone intake [OR (T3 vs. T1) = 0.71; 95% CI: 0.58, 0.86]. We evaluated interactions between polymorphic genes that encode enzymes possibly involved in metabolism of phytoestrogens (CYPs, catechol O-methyl transferase, GSTs, and UGTs) and found no significant effect modification with respect to phytoestrogen intake. This finding that phytoestrogen intake may reduce colorectal cancer risk is important, because dietary intake is potentially modifiable.

Phytoestrogens have been shown to exert anti-estrogenic and estrogenic effects in some tissues, including the breast. However, only a few studies have evaluated their role in endometrial cancer risk. We evaluated this association in a population-based case-control study in New Jersey. A total of 424 cases and 398 controls completed an interview, including a food frequency questionnaire with supplemental questions for phytoestrogen foods. Risk estimates were derived using unconditional logistic regression, adjusting for major risk factors for endometrial cancer. There was some suggestion of a decreased risk with quercetin intake (OR: 0.65; 95% CI: 0.41–1.01 for the highest compared to the lowest quartile; p for trend: 0.02). We found limited evidence of an association with any of the lignans evaluated, total lignans, coumestrol, individual isoflavones, total isoflavones, or total phytoestrogens. However, there was some suggestion of an inverse association with total isoflavone intake limited to lean women (BMI <25) (OR for the highest tertile: 0.50, 95% CI: 0.25–0.98) and those with a waist-to-hip ratio ≤0.85 (OR: 0.59; 95% CI: 0.33–1.05). There was no evidence of effect modification by HRT use. This study suggests a reduction in endometrial cancer risk with quercetin intake and with isoflavone intake in lean women.

We have established in human breast cancer cells that indole-3-carbinol (I3C), a promising anti-cancer phytochemical from Brassica vegetables, ablates ERα expression by stimulating the Rbx-1 E3 ligase mediated degradation of ERα protein and disruption of a cross-regulatory positive feedback loop involving ERα and the GATA3 transcription factor.

Estrogen receptor (ER)α is a critical target of therapeutic strategies to control the proliferation of hormone-dependent breast cancers. Preferred clinical options have significant adverse side effects that can lead to treatment resistance due to the persistence of active estrogen receptors. We have established the cellular mechanism by which indole-3-carbinol (I3C), a promising anticancer phytochemical from Brassica vegetables, ablates ERα expression, and we have uncovered a critical role for the GATA3 transcription factor in this indole-regulated cascade. I3C-dependent activation of the aryl hydrocarbon receptor (AhR) initiates Rbx-1 E3 ligase-mediated ubiquitination and proteasomal degradation of ERα protein. I3C inhibits endogenous binding of ERα with the 3′-enhancer region of GATA3 and disrupts endogenous GATA3 interactions with the ERα promoter, leading to a loss of GATA3 and ERα expression. Ectopic expression of GATA3 has no effect on I3C-induced ERα protein degradation but does prevent I3C inhibition of ERα promoter activity, demonstrating the importance of GATA3 in this I3C-triggered cascade. Our preclinical results implicate I3C as a novel anticancer agent in human cancers that coexpress ERα, GATA3, and AhR, a combination found in a large percentage of breast cancers but not in other critical ERα target tissues essential to patient health.

Exposure to exogenous (oral contraceptives, post-menopausal hormone therapy) and endogenous (number of ovulatory cycles, adiposity) steroid hormones is associated with breast cancer risk. Breast cancer risk associated with these exposures could hypothetically be modified by genes in the steroid hormone synthesis, metabolism, and signaling pathways. Estrogen receptors are the first step along the path of signaling cell growth and development upon stimulation with estrogens. The National Cancer Institute Breast and Prostate Cancer Cohort Consortium has systematically selected haplotype tagging SNPs in genes along the steroid hormone synthesis, metabolism, and binding pathways, including the estrogen receptor beta (ESR2) gene. Four htSNPs tag the six major (> 5% frequency) haplotypes of the ESR2 gene. These polymorphisms have been genotyped in 5,789 breast cancer cases and 7,761 controls nested within the American Cancer Society Cancer Prevention Study II, European Prospective Investigation into Cancer and Nutrition, Multiethnic Cohort, Nurses’ Health Study, and Women’s Health Study cohorts. None of the SNPs were independently associated with breast cancer risk. One haplotype of the ESR2 gene was associated with breast cancer risk before correction for multiple testing (OR 1.17, 95% CI 1.07–1.28, p=0.0007). This haplotype remained associated with breast cancer risk after adjustment for multiple testing using a permutation procedure. There was no statistically significant heterogeneity in SNP or haplotype odds ratios across cohorts. These data suggest that inherited variants in ESR2, while possibly conferring a small increased risk of breast cancer, are not associated with appreciable (OR > 1.2) changes in breast cancer risk among Caucasian women.

During the course of oncogenesis and tumor progression, cancer cells constitutively upregulate signaling pathways relevant to cell proliferation and survival as a strategy to overcome genomic instability and acquire resistance phenotype to chemotherapeutic agents. In light of this clinical and molecular heterogeneity of human cancers, it is desirable to concomitantly target these genetic abnormalities by using an agent with pleiotropic mode of action. Indole-3-carbinol and its metabolite 3,3’-diindoylmethane (DIM) target multiple aspects of cancer cell cycle regulation and survival including Akt-NFκB signaling, caspase activation, cyclin-dependent kinase activities, estrogen metabolism, estrogen receptor signaling, endoplasmic reticulum stress, and BRCA gene expression. This broad spectrum of antitumor activities in conjunction with low toxicity underscores the translational value of indole-3-carbinol and its metabolites in cancer prevention/therapy. Furthermore, novel antitumor agents with overlapping underlying mechanisms have emerged via structural optimization of indole-3-carbinol and DIM, which may provide considerable therapeutic advantages over the parental compounds with respect to chemical stability and anti-tumor potency. Together, these agents might foster new strategies for cancer prevention and therapy.

The authors further analyzed results from the Women's Health Initiative randomized trials (1993–2004) of conjugated equine estrogens, with or without medroxyprogesterone acetate, focusing on health benefits versus risks among women who initiated hormone therapy soon after menopause. Data from the Women's Health Initiative observational study (1993–2004) were included in some analyses for additional precision. Results are presented here for incident coronary heart disease, stroke, venous thromboembolism, breast cancer, colorectal cancer, endometrial cancer, or hip fracture; death from other causes; a summary global index; total cancer; and total mortality. Hazard ratios for breast cancer and total cancer were comparatively higher (P < 0.05) among women who initiated hormone therapy soon after menopause, for both regimens. Among these women, use of conjugated equine estrogens appeared to produce elevations in venous thromboembolism and stroke and a reduction in hip fracture. Estrogen plus progestin results among women who initiated use soon after menopause were similar for venous thromboembolism, stroke, and hip fracture but also included evidence of longer-term elevations in breast cancer, total cancer, and the global index. These analyses provide little support for the hypothesis of favorable effects among women who initiate postmenopausal estrogen use soon after menopause, either for coronary heart disease or for health benefits versus risk indices considered.