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.

Weight gain and inflammation regulate aromatase expression in male adipose tissue, as evidenced by reporter gene activity.

Obesity and white adipose tissue (WAT) inflammation are associated with enhanced aromatization in women, but little is known about the regulation of aromatase (CYP19A1) gene expression in male WAT. We investigated the impact of weight gain and WAT inflammation on the regulation of CYP19A1 in males, by utilizing the hARO-Luc aromatase reporter mouse model containing a >100-kb 5'-region of the human CYP19A1 gene. We show that hARO-Luc reporter activity is enhanced in WAT of mice with increased adiposity and inflammation. Dexamethasone and TNFα, as well as forskolin and phorbol 12-myristate 13-acetate, upregulate hARO-Luc activity, suggesting the involvement of promoters I.4 and I.3/II. Furthermore, we show that diet enriched with antioxidative plant polyphenols attenuates WAT inflammation and hARO-Luc activity in obese males. In conclusion, our data suggest that obesity-associated WAT inflammation leads to increased peripheral CYP19A1 expression in males, and that polyphenol-enriched diet may have the potential to attenuate excessive aromatization in WAT of obese men.

Differential regulation of carbonic anhydrase II by androgen and estrogen in dorsal and lateral prostate of the rat.

Hormone regulation of carbonic anhydrase II (CA II) was studied in rat dorsal and lateral prostate. CA II is a major soluble protein in these accessory sex glands. The immunoelectronmicroscopy showed that CA II is expressed in their epithelial cells only. For studies on hormone regulation, adult male rats were castrated for 2 or 7 days. Groups of 7-day castrates and normal rats were treated daily either with testosterone or 17-beta-estradiol for 6 days and 2-day castrates for 1 day. CA II protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and quantified by RIA. The levels of CA II mRNA were studied by Northern blotting and hybridization of total RNA with a 32P-labeled mouse CA II cDNA clone. Castration of the rats decreased the concentration of CA II in lateral prostate but increased in dorsal prostate. These changes were reversed in both prostatic lobes by testosterone treatment. Estrogen treatment of castrated rats enhanced CA II concentration in lateral prostate but no effects were seen in the dorsal prostate of the same animals. In normal rats estrogen increased CA II concentration of dorsal prostate but there was no change in lateral prostate. Corresponding changes were observed in the levels of CA II mRNA in both tissues. The morphometric analyses showed that the castration- and hormone-induced changes of the mRNA and protein levels of the exclusively epithelial CA II could not be explained by any alterations in the proportions of epithelial and stromal components of the glands after hormone manipulations. The results demonstrate the differential steroid regulation of CAII in two prostatic lobes. Androgen regulates the expression of CAII at messenger RNA level, but the responses of CAII to testosterone are opposite in dorsal and lateral prostate. Estrogen increases CA II expression in lateral prostate but in dorsal prostate the castration-like effects of estrogen on CAII expression are probably indirect.

Estrogen and prolactin regulation of rat dorsal and lateral prostate in organ culture.

Besides androgens, estrogen (E) and PRL are thought to have important roles in the regulation of the growth and function of the prostate. We have established organ cultures of rat dorsolateral prostate for the analysis of the multiple hormone actions. Explants of dorsal prostate (DP) and lateral prostate (LP) were cultured in a serum-free basal medium containing insulin and corticosterone with or without the hormones studied. The viability and overall integrity of the tissues were maintained for at least 14 days. The morphology of the explants showed castration-like changes in the basal medium, but the addition of testosterone (T) prevented them. Androgen receptors in the prostate cultured with T were demonstrated by immunohistochemistry. When the explants were grown with E the epithelium became stratified, and the cells were flat. The epithelium was also layered when the explants were grown with PRL, but the epithelial cells were hypersecretory and large. The glandular morphology of the cultured prostate was, however, best preserved if T was added along with E or PRL. The wet weights and DNA contents of the explants declined during the culture, but they were better maintained if T, E, or PRL were added to the medium. The rate of DNA labeling with [3H]thymidine was activated in the cultured explants, but it was higher in those grown with T, E, or PRL than in those grown in the basal medium. The tissue specific functions were evaluated by measuring the expression of the genes RWB and M-40.3 encoding androgen-regulated secretory proteins. The steady state levels of RWB and M-40.3 mRNA were low in the explants grown in the basal medium but in the presence of T they were high. E and PRL also increased the expression of RWB and M-40.3 messenger RNA, although the responses in DP and LP were somewhat different. The antihormones cyproterone and toremifene opposed the increase of M-40.3 messenger RNA by T and E, respectively. The results show that the cultured DP and LP of the rat maintain the androgen responsiveness and tissue-specific functions in vitro. In addition, E and PRL have androgen-independent, direct effects in them. Rat dorsolateral prostate in culture thus provides a useful model for the studies on the mechanisms of hormone regulation of the prostate.

Neonatal exposure to diethylstilbestrol permanently alters the basal and 17 beta-estradiol induced expression of c-fos proto-oncogene in mouse urethroprostatic complex.

Perinatal estrogen exposure induces permanent structural and functional changes in the male reproductive tract. We have studied the effect of neonatal estrogenization on the estrogen-responsive c-fos proto-oncogene expression in mouse prostate. Fos is involved in growth and differentiation, and may play a central role in regulating diverse estrogen-related cellular differentiation. In adult control mouse prostate, basal c-fos mRNA expression is very low. Neonatal treatment with diethylstilbestrol on days 1-3 (neoDES) results in permanently increased fos expression in the prostatic urethra and all prostatic lobes. In adult castrated animals, estradiol induces a rapid transient increase in c-fos expression in the prostatic urethra, with maximum induction being higher in neoDES animals. In situ hybridization and immunohistochemistry show that in neoDES mice fos transcripts and protein are localized primarily in the epithelium of posterior periurethral prostatic collecting ducts. These are the sites previously reported to show the most pronounced morphological changes after estrogen treatment. Our results indicate that neonatal estrogenization affects both basal and estrogen stimulated c-fos mRNA levels in the prostate of mature mice, which supports the hypothesis that estrogen-induced morphological changes in mouse prostate may involve altered c-fos expression.

Dietary soybean may be antiestrogenic in male mice.

We tested whether dietary soybeans alter prostatic growth and development of prostatic dysplasia in mice that were treated with a synthetic nonsteroidal estrogen, diethylstilbestrol during the first 3 d after birth. Soybeans were chosen because they contain substantial amounts of isoflavonic estrogens. The presence of estrogenic isoflavonoids in soybean-containing feed was confirmed by measuring the excretion of seven different plant estrogens in the urine of normal adult male mice. Estrogenicity of dietary soybean was confirmed by the growth response in uteruses of immature mice. In addition to their estrogenic effect, antiestrogenic properties of soybeans on uterine growth were observed in the presence of a more potent estrogenic growth stimulator, diethylstilbestrol in feed. In neonatally estrogenized male mice, soybean feeding reduced the prostatic growth inhibition due to diethylstilbestrol and, in preliminary experiments, delayed the development of dysplastic changes in the prostate. The number of animals showing severe dysplasia in prostatic epithelium was significantly lower in 9-mo-old animals given soybean-containing feed from fertilization onwards, but in 12 mo-old animals the difference was less obvious and was not significant. Our findings suggest an antiestrogenic action for dietary soybean in male mice, which may be important for the hormonal regulation of normal as well as neoplastic prostatic growth.

Interaction of male and female sex hormones in cultured rat prostate.

The organ culture of the rat ventral prostate was chosen as a model to determine whether any of the estrogen effects in vivo on the prostate are direct and expressed at the hormone concentrations normally found in the male. During 2 weeks of culture, estradiol at the high concentration of 10(-5) M blocked the androgenic activation of [3H]thymidine incorporation into DNA. The inhibition was localized in epithelium. Protein content of testosterone-treated explants and the accumulation of prostatein in the medium were considerably decreased, indicating inhibition of secretion. Antiandrogenic effects were not seen in morphology of estrogen-treated explants. The lower concentrations (from 10(-9) M to 10(-6) M) of estradiol increased the volume density of epithelium from day 7 onwards. The height of epithelium was concomitantly increased. The volume density of epithelium as well as the percentage of acini with metaplastic changes were significantly increased. These epithelial changes were less pronounced in the presence of androgen, suggesting that physiological concentrations of androgen prevent the expression of estrogen action in the morphology of the prostate. A change in staining with peanut (PNA)- and wheat germ agglutinin (WGA)-lectins indicated defective secretory capacity in metaplastic epithelium. In spite of the increased protein content in the explants, no constant pattern of the changes in prostatein accumulation could be recorded. Although the concentrations of estrogen required to induce squamous metaplasia were still unphysiological, the occurrence of this abnormal differentiation of the prostatic epithelium suggests that the cooperative action of estrogen is involved in androgen-dependent normal epithelial growth and possibly also in promoting growth of prostatic neoplasia.

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.

Hydroxymatairesinol, a novel enterolactone precursor with antitumor properties from coniferous tree (Picea abies).

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 alpha- or beta-type estrogen receptor-mediated pathway in vitro at < 1.0 microM. HMR was an effective antioxidant in vitro.