Niina Saarinen

Altered Structure and Function of Reproductive Organs in Transgenic Male Mice Overexpressing Human Aromatase1

Aromatization of androgens is a key step in estrogen production, and it regulates the delicate balance between estrogens and androgens in the gonads and sex steroid target tissues. In the present study, we generated transgenic mice (AROM(+)) bearing the human ubiquitin C promoter/human P450 aromatase fusion gene. AROM(+) male mice are characterized by an imbalance in sex hormone metabolism, resulting in elevated serum E(2) concentrations, combined with significantly reduced testosterone and FSH levels, and elevated levels of PRL and corticosterone. AROM(+) males present a multitude of severe structural and functional alterations in the reproductive organs, such as cryptorchidism associated with Leydig cell hyperplasia, dysmorphic seminiferous tubules, and disrupted spermatogenesis. The males also have small or rudimentary accessory sex glands with abnormal morphology; a prominent prostatic utricle with squamous epithelial metaplasia, and edema in the ejaculatory ducts and vas deferens. In addition, the abdominal muscle wall is thin, and the adrenal glands are enlarged, with cortical hyperplasia. Some of the abnormalities, such as undescended testes and undeveloped prostate, resemble those observed in animals exposed perinatally to high levels of exogenous estrogen, indicating that the elevated aromatase activity results in excessive estrogen exposure during early phases of development. Some of the disorders in the reproductive organs, furthermore, can be explained by the fact that AROM(+) males are hypoandrogenic, and have elevated levels of serum PRL and corticosterone. Thus, the AROM(+) mouse model provides a novel tool to investigate the consequences of a prolonged increase in conversion of androgens to estrogens which results in complex hormonal disturbances altering the structure and function of various male reproductive organs.

Hydroxymatairesinol, a Novel Enterolactone Precursor With Antitumor Properties From Coniferous Tree (Picea abies)

Abstract: The potential for the extraction of the plant lignan hydroxymatairesinol (HMR) in large scale from Norway spruce (Picea abies) has given us the opportunity to study the metabolism and biological actions of HMR in animals. HMR, the most abundant single component of spruce lignans, was metabolized to enterolactone (ENL) as the major metabolite in rats after oral administration. The amounts of urinary ENL increased with the dose of HMR (from 3 to 50 mg/kg), and only minor amounts of unmetabolized HMR isomers and other lignans were found in urine. HMR (15 mg/kg body wt po) given for 51 days decreased the number of growing tumors and increased the proportion of regressing and stabilized tumors in the rat dimethylbenz[a]anthracene-induced mammary tumor model. HMR (50 mg/kg body wt) did not exert estrogenic or antiestrogenic activity in the uterine growth test in immature rats. HMR also showed no antiandrogenic responses in the growth of accessory sex glands in adult male rats. Neither ENL nor enterodiol showed estrogenic or antiestrogenic activity via a classical a- or b-type estrogen receptor-mediated pathway in vitro at <1.0 mM. HMR was an effective antioxidant in vitro.

The role of early life genistein exposures in modifying breast cancer risk

Review of the existing literature suggests that consumption of soy foods or an exposure to a soy isoflavone genistein during childhood and adolescence in women, and before puberty onset in animals, reduces later mammary cancer risk. In animal studies, an exposure that is limited to the fetal period or adult life does not appear to have the same protective effect. A meta-analysis of human studies indicates a modest reduction in pre- and postmenopausal risk when dietary intakes are assessed during adult life. These findings concur with emerging evidence indicating that timing may be vitally important in determining the effects of various dietary exposures on the susceptibility to develop breast cancer. In this review, we address the mechanisms that might mediate the effects of an early life exposure to genistein on the mammary gland. The focus is on changes in gene expression, such as those involving BRCA1 and PTEN. It will be debated whether mammary stem cells are the targets of genistein-induced alterations and also whether the alterations are epigenetic. We propose that the effects on mammary gland morphology and signalling pathways induced by pubertal exposure to genistein mimic those induced by the oestrogenic environment of early first pregnancy.

Dietary phytoestrogens and their role in hormonally dependent disease

Epidemiological studies suggest that diets rich in phytoestrogens (plant estrogens), particularly soy and unrefined grain products, may be associated with low risk of breast and prostate cancer. It has also been proposed that dietary phytoestrogens could play a role in the prevention of other estrogen-related conditions, namely cardiovascular disease, menopausal symptoms and post-menopausal osteoporosis. However, there is no direct evidence for the beneficial effects of phytoestrogens in humans. All information is based on consumption of phytoestrogen-rich diets, and the causal relationship and the mechanisms of phytoestrogen action in humans still remain to be demonstrated. In addition, the possible adverse effects of phytoestrogens have not been evaluated. It is plausible that phytoestrogens, as any exogenous hormonally active agent, might also cause adverse effects in the endocrine system, i.e. act as endocrine disrupters.

Tamoxifen, flaxseed and the lignan enterolactone increase stroma and cancer cell derived IL-1Ra and decrease tumor angiogenesis in estrogen dependent breast cancer

The proinflammatory cytokines IL-1α and IL-1β promote tumor angiogenesis that might be counteracted by the IL-1 receptor antagonist (IL-1Ra), anakinra, a clinically approved agent. A diet with high amounts of phytoestrogens, such as flaxseed (Flax), genistein (GEN), and the mammalian lignan enterolactone (ENL), may affect breast cancer progression in a similar fashion as the antiestrogen tamoxifen. Both cancer cells and tumor stroma may be targets for cancer therapy. By using microdialysis in a model of human breast cancers in nude mice, we could perform species-specific analyses of released proteins in the microenvironment. We show that tumors treated with tamoxifen and fed Flax or ENL exhibited decreased in vivo release of IL-1β derived from the murine stroma and decreased microvessel density whereas dietary GEN had no effects. Cancer cell-released IL-1Ra were approximately 5 times higher than stroma-derived IL-1Ra. Tamoxifen, Flax, and ENL increased IL-1Ra levels significantly whereas GEN did not. The tumor stroma contained macrophages, which expressed the estrogen receptor. In vitro, estradiol decreased IL-1Ra released from breast cancer cells and from cultured macrophages. IL-1Ra decreased endothelial cell proliferation significantly in vitro whereas breast cancer cell proliferation was unaffected in presence of estradiol. Finally, IL-1Ra therapy of tumor-bearing mice opposed estrogen-dependent breast cancer growth and decreased angiogenesis. We conclude that the release of IL-1s both by cancer cells and the stroma, where macrophages are a key component, may offer feasible targets for antiestrogen therapy and dietary interventions against breast cancer.

Anticancer effects of a plant lignan 7-hydroxymatairesinol on a prostate cancer model in vivo

Clinical intervention studies and experimental studies with lignan-rich diets suggest that lignans may have inhibitory effects on prostate cancer, but no clinical or experimental studies with purified lignans have been published. The purpose of this study was to investigate the effect of a plant lignan 7-hydroxymatairesinol (HMR) on LNCaP human prostate cancer xenografts in athymic mice. Athymic nude male mice were injected subcutaneously with LNCaP cells. Starting 3 days after tumor cell injections, a control diet or a control diet supplemented with 0.15% or 0.30% of HMR was administered to mice and the tumor take rate and growth was observed for 9 weeks. HMR diet inhibited the growth of LNCaP tumors. Mice treated with HMR had smaller tumor volume, lower tumor take rate, increased proportion of nongrowing tumors, and higher tumor cell apoptotic index compared with controls. Furthermore, the cell proliferation index was reduced in mice receiving the 0.30% HMR diet compared with mice receiving the control diet. Our results suggest that dietary HMR started at the early phase of the tumor development inhibits the growth of the LNCaP human prostate cancer xenografts in athymic male mice.

Uptake and Metabolism of Hydroxymatairesinol in Relation to Its Anticarcinogenicity in DMBA-Induced Rat Mammary Carcinoma Model

The chemopreventive effects of hydroxymatairesinol (HMR), a lignan extracted from Norway spruce (Picea abies), on the development of mammary carcinoma induced by 7,12-dimethylbenz[a]anthracene (DMBA) was studied in rats. HMR administered via diet in an average daily dose of 4.7 mg/kg body wt starting before DMBA induction reduced tumor volume and tumor growth, but no significant reduction in tumor multiplicity (number of tumors/rat) was observed. The predominant histological type in the control group was type B (well-differentiated adenocarcinoma, 78%). The proportion of type B tumors decreased to 35% in the HMR group, while the type A (poorly differentiated) and type C (atrophic) tumor proportions increased. Anticarcinogenic effects of dietary HMR (4.7 mg/kg) were also evident when the administration started after DMBA induction and was seen as growth inhibition of established tumors. Dietary HMR supplementation significantly increased serum and urinary enterolactone and HMR concentrations but had no significant effect on the uterine weight, suggesting that HMR or its major metabolite enterolactone did not have an antiestrogenic effect. Further studies are warranted to further clarify and verify HMR action and the associated mechanisms in mammary tumorigenesis.

Estrogen-induced angiogenic factors derived from stromal and cancer cells are differently regulated by enterolactone and genistein in human breast cancer in vivo

Angiogenesis is a key in cancer progression and its regulators are released both by the tumor cells and the stroma. Dietary phytoestrogens, such as the lignan enterolactone (ENL) and the isoflavone genistein (GEN), may differently affect breast cancer growth. In this study, human breast cancer cells (MCF‐7) were established in mice creating a tumor with species‐specific cancer and stroma cells. Ovariectomized athymic mice supplemented with estradiol (E2) were fed basal AIN‐93G diet (BD) or BD supplemented with 100 mg/kg ENL, 100 mg/kg GEN or their combination (ENL+GEN). We show that ENL and ENL+GEN inhibited E2‐induced cancer growth and angiogenesis, whereas GEN alone did not. Microdialysis was used to sample extracellular proteins in tumors in vivo. ENL and ENL+GEN decreased both stroma‐ and cancer cell‐derived VEGF, whereas cancer cell‐derived PlGF increased. In subcutaneous Matrigel plugs in mice, ENL and ENL+GEN decreased E2‐induced endothelial cell infiltration, whereas GEN alone did not. In endothelial cells, ENL inhibited E2‐induced VEGFR‐2 expression, whereas GEN did not. These results suggest that ENL has potent effects on breast cancer growth, even in combination with GEN, by downregulating E2‐stimulated angiogenic factors derived both from the stroma and the cancer cells, whereas dietary GEN does not possess any antiestrogenic effects.

Dietary lariciresinol attenuates mammary tumor growth and reduces blood vessel density in human MCF-7 breast cancer xenografts and carcinogen-induced mammary tumors in rats

Lariciresinol is a dietary lignan that accounts for a significant portion of the total phytoestrogen intake from Western foods. Recent epidemiological studies suggest that high dietary intake of lignans and lariciresinol is associated with reduced breast cancer risk. However, no causal relationship between lariciresinol intake and breast cancer development has been established. In this study, we investigated for the first time the effects and possible mechanisms of action of lariciresinol on hormone responsive mammary cancer in vivo in dimethylbenz[a]anthracene induced mammary cancer in rats, and in human MCF‐7 breast cancer xenografts in athymic mice. For tumor bearing rats, lariciresinol (3 or 15 mg/kg of body weight) or vehicle was administered p.o. daily for 9 weeks. For E2‐maintained ovariectomized athymic mice bearing orthotopic MCF‐7 tumors, control diet (AIN‐93G) or lariciresinol containing diet (AIN‐93G supplemented with 20 or 100 mg of lariciresinol/kg of diet) was administered for 5 weeks. In both models, lariciresinol administration inhibited the tumor growth and tumor angiogenesis. In MCF‐7 cells, enterolactone significantly inhibited the E2‐stimulated VEGF secretion. Moreover, in MCF‐7 xenografts, lariciresinol administration enhanced tumor cell apoptosis and increased estrogen receptor beta expression. Lariciresinol and its further metabolites secoisolariciresinol, enterodiol and enterolactone were found in serum of both rats and athymic mice confirming a similar lignan metabolism pattern as in humans. These findings indicate conceivable importance of dietary lignan lariciresinol in inhibition of breast cancer development.

Structural determinants of plant lignans for growth of mammary tumors and hormonal responses in vivo.

Low risk of breast cancer (BC) has been proposed to be associated with high intake of lignans. Some plant lignans are converted to mammalian lignans, e.g., enterolactone (ENL), suggested to be the biologically active lignan forms. Until now, little attention has been paid to the possible biological activities of plant lignans, even though some plant lignans are absorbed and present in serum and urine. In this study, we have investigated the antitumorigenic and endocrine-modulatory activities of different plant lignans in order to clarify the structure-activity relationships. 7-Hydroxymatairesinol (HMR) is [corrected] converted to ENL, and both HMR and ENL inhibit the growth of 7,12-dimethylbenz[a]-anthracene (DMBA)-induced mammary cancer. Nortrachelogenin (NTG) resembles HMR, but has a hydroxyl group at C-8 instead of C-7 and is not converted to ENL. In DMBA-model, NTG showed no inhibition of tumor growth, but increased the uterine weight. Furthermore, life-long exposure to NTG increased uterine weight in immature females and ventral prostate weight in adult males. In contrast, life-long exposure to HMR had no effects on uterine or prostate weights at any age. Our results indicate that a difference in the position of one hydroxyl group results in distinct biological responses in vivo, as well as different lignan metabolite profiles.