Role of estrogens in development of prostate cancer

Estrogens have previously been extensively used in prostate cancer treatment. Serious side effects, primarily in cardiovascular system have, however, limited their use. The therapeutic effect of estrogen in preventing prostate cancer growth was mainly obtained indirectly by feedback inhibition of the hypothalamic release of LRH leading to lowered serum androgen levels and castration like effects. Prostate tissue is also most probably a target for direct regulation by estrogens. Prostate contains estrogen receptor alpha (ERalpha) and beta (ERbeta), which are localized characteristically in stroma and epithelium, respectively. The physiological function of these receptors is not known but there is evidence of the role of estrogens in prostatic carcinogenesis. Developing prostate seems particularly sensitive to increased level of endogenous and/or exogenous estrogens. Perinatal or neonatal exposure of rats and mice to estrogens leads to "imprinting" of prostate associated with increased proliferation, inflammation and dysplastic epithelial changes later in life. Prolonged treatment of adult rodents with estrogens along with androgens also leads to epithelial metaplasia, PIN-like lesions and even adenocarcinoma of prostate speaking for the role of estrogen in prostate cancer development. Recent results concerning antiestrogen inhibition of prostate cancer development beyond PIN-type lesions in transgenic mouse models further suggests a role for estrogens in prostate cancer progression. These results also suggest that direct inhibition of estrogen action at the level of prostate tissue may provide an important novel principle of development of prostate cancer therapies.     

The androgenic regulation of the activities of enzymes engaged in the synthesis of deoxyribonucleic acid in rat ventral prostate gland.

The restoration of mitosis and growth of the prostate gland of castrated animals by androgens provides a favourable experimental system for studying the hormonal regulation of enzymes engaged in DNA replication. 2. Many DNA polymerase activities were identified in the prostate gland, but only a 9S form with a particular preference for denatured DNA as template was conspicuously enhanced by androgenic stimulation. 3. Thymidine kinase also provided a sensitive indicator of the hormonal regulation of DNA replication, and on electrophoretic criteria, one discrete form of the enzyme appeared precisely with the onset of mitoris. 4. Evidence is presented to support the view that DNA ligase activity is intimately associated in the process of DNA replication in the prostate gland. 5. A spectrum of deoxyribonuclease activities is present in the prostate gland, but only one form (pI7.0) can safely be said to be implicated in the process of DNA replication. 6. Androgenic stimulation of the prostate gland leads to the appearance of a component capable of denaturing or unwinding prostate DNA. This component is seemingly distinct from RNA or DNA polymerase activities on the basis of several distince physicochemical characteristics. 7. The conspicuous feature of all the changes in enzyme activities evoked by androgens in the prostate gland is their acute tissue- and steroid-specificity. Such changes could not be mimicked in liver or spleen and the regulatory role of androgens could not be simulated by other classes of steroid hormones. Particularly on the basis of studies with the anti-androgen cyproterone acetate, it is concluded that the changes are initially mediated by the androgen-receptor system and the high-affinity binding of 5alpha-dihydrotestosterone in the prostate gland. 8. The results are discussed in the context of the mechanism of action of androgens.     

Effects of the aromatase inhibitor fadrozole on plasma sex steroid secretion,spermatogenesis and epididymis morphology in the lizard,Podarcis sicula

Recently, increasing importance has been attached to the role of estrogens and their receptors in male reproduction, since they have been found to be abundant in the male reproductive tract. In the lizard, Podarcis sicula, a seasonal breeder, estrogens seem to be involved in the regulation of testicular activity. Particularly, it has been hypothesized that the block of spermatogenesis and the complete regression of the epididymis and other secondary sexual characters (SSCs) in autumn might be due to high estrogen levels. To investigate the role of estrogens in the reproductive process of male lizards, we utilized Fadrozole ((AI) [4-(5,6,7,8-tetrahydroimidazole [1,5-a] pyridin-5-yl)-benzonitrile monohydrochloride] (CGS 16949A)), a nonsteroidal inhibitor of aromatase, the enzyme involved in the aromatization of androgens to estrogens, evaluating its effects on plasma sex-hormone release, spermatogenesis and epididymis morphology. For this purpose, adult male lizards, captured during the autumnal recrudescence, were intraperitoneally injected with 0.5 microg and 5 microg/g/body weight of AI for 15 and 30 days. In the animals treated with the higher AI dose, estrogen levels decreased if compared to the control groups, whereas androgen levels increased. Furthermore, histologic sections of testes and epididymes showed that the 30-day treatment with AI-induced spermatogenesis resumption with release of sperms into the large lumen of the seminiferous tubules, and the epididymes appeared more developed with moderately secreting columnar canal cells. Therefore, it is proposed that failure of spermatogenesis in autumn might be due to high estrogen levels.     

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.     

Sex steroid hormone metabolism and prostate cancer

The growth and function of the prostate is dependent on androgens. The two predominant androgens are testosterone, which is formed in the testis from androstenedione and 5alpha-dihydrotestosterone, which is formed from testosterone by 5alpha-reductases and is the most active androgen in the prostate. Prostate cancer is one of the most common cancers among men and androgens are involved in controlling the growth of androgen-sensitive malignant prostatic cells. The endocrine therapy used to treat prostate cancer aims to eliminate androgenic activity from the prostatic tissue. Most prostate cancers are initially responsive to androgen withdrawal but become later refractory to the therapy and begin to grow androgen-independently. Using LNCaP prostate cancer cell line we have developed a cell model to study the progression of prostate cancer. In the model androgen-sensitive LNCaP cells are transformed in culture conditions into more aggressive, androgen-independent cells. The model was used to study androgen and estrogen metabolism during the transformation process. Our results indicate that substantial changes in androgen and estrogen metabolism occur in the cells during the process. A remarkable decrease in the oxidative 17beta-hydroxysteroid dehydrogenase activity was seen whereas the reductive activity seemed to increase. The changes suggest that during transformation estrogen influence is increasing in the cells. This is supported by the cDNA microarray screening results which showed over-expression of several genes up-regulated by estrogens in the LNCaP cells line representing progressive prostate cancer. Since local steroid metabolism controls the bioavailability of active steroid hormones in the prostate, the variations in steroid-metabolizing enzymes during cancer progression may be crucial in the regulation of the growth and function of the organ.     

Evidence for a role of progesterone in the control of uterine ornithine decarboxylase in the pregnant hamster

Changes in uterine ornithine decarboxylase (ODC) activity throughout pregnancy and the importance of progesterone in the regulation of this enzyme during the early post-implantation period have been studied in the hamster. Soon after implantation, from day 5 to day 6 of pregnancy, ODC activity rapidly increased. It reached a plateau on day 7, then abruptly fell on day 8 and remained low until the end of pregnancy. DL-alpha-difluoromethyl-ornithine (DFMO), an irreversible inhibitor of ODC, induced pregnancy termination as a consequence of the reduction of uterine ODC activity. When pregnancy arrest was induced by removing endogenous progesterone by administration of prostaglandin F2 alpha (PGF2 alpha) or by ovariectomy, the ODC rise was completely abolished, and exogenous progesterone was able to entirely counteract this effect on the enzyme activity and the termination of pregnancy. These results suggest that progesterone play a significant role in the rise of uterine ODC activity, which appears to be essential for the early post-implantation events needed for pregnancy maintenance.     

Regulation of aromatase and 5alpha-reductase by 25-hydroxyvitamin D(3), 1alpha,25-dihydroxyvitamin D(3), dexamethasone and progesterone in prostate cancer cells

Estrogens and androgens are proposed to play a role in the pathogenesis of prostate cancer. The effective metabolites, estradiol and 5alpha-dihydrotestosterone are produced from testosterone by aromatase and 5alpha-reductase, respectively. Metabolites of vitamin D have shown to inhibit the growth of prostate cancer cells. The aim of the present study was to verify whether 25-hydroxyvitamin D(3) (25OHD(3)), 1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)], dexamethasone, and progesterone regulate the expression of aromatase and 5alpha-reductase in human prostate cancer cells. LNCaP and PC3 cells were treated with 25OHD(3), 1alpha,25-(OH)(2)D(3), dexamethasone, or progesterone. Aromatase and 5alpha-reductase mRNA was quantified by real-time RT-PCR and aromatase enzyme activity was measured by the [(3)H] water assay. Aromatase enzyme activity in LNCaP and PC3 cells was increased by both 10nM dexamethasone, 1-100 nM 1alpha,25-(OH)(2)D(3) and 100 nM-10 microM progesterone. The induction was enhanced when hormones were used synergistically. Real-time RT-PCR analysis showed no regulation of the expression of aromatase mRNA by any steroids tested in either LNCaP or PC3 cells. The expression of 5alpha-reductase type I mRNA was not regulated by 1alpha,25-(OH)(2)D(3) and no expression of 5alpha-reductase type II was detected in LNCaP.     

Estrogen and prostate cancer: an eclipsed truth in an androgen-dominated scenario

Prostate cancer is the commonest non-skin cancer in men. Incidence and mortality rates of this tumor vary strikingly throughout the world. Although several factors have been implicated to explain this remarkable variation, lifestyle and dietary factors may play a dominant role, with sex hormones behaving as intermediaries between exogenous factors and molecular targets in development and progression of prostate cancer. Human prostate cancer is generally considered a paradigm of androgen-dependent tumor; however, estrogen role in both normal and malignant prostate appears to be equally important. The association between plasma androgens and prostate cancer remains contradictory and mostly not compatible with the androgen hypothesis. Similar evidence apply to estrogens, although the ratio of androgen to estrogen in plasma declines with age. Apart from methodological problems, a major issue is to what extent circulating hormones can be considered representative of their intraprostatic levels. Both nontumoral and malignant human prostate tissues and cells are endowed with key enzymes of steroid metabolism, including 17betahydroxysteroid dehydrogenase (17betaHSD), 5beta-reductase, 3alpha/3betaHSD, and aromatase. A divergent expression and/or activity of these enzymes may eventually lead to a differential prostate accumulation of steroid derivatives having distinct biological activities, as it occurs for hydroxylated estrogens in the human breast. Locally produced or metabolically transformed estrogens may differently affect proliferative activity of prostate cancer cells. Aberrant aromatase expression and activity has been reported in prostate tumor tissues and cells, implying that androgen aromatization to estrogens may play a role in prostate carcinogenesis or tumor progression. Interestingly, many genes encoding for steroid enzymes are polymorphic, although only a few studies have supported their relation with risk of prostate cancer. In animal model systems estrogens, combined with androgens, appear to be required for the malignant transformation of prostate epithelial cells. Although the mechanisms underlying the hormonal induction of prostate cancer in experimental animals remain uncertain, there is however evidence to support the assumption that long term administration of androgens and estrogens results in an estrogenic milieu in rat prostates and in the ensuing development of dysplasia and cancer. Both androgen and estrogen have been reported to stimulate proliferation of cultured prostate cancer cells, primarily through receptor-mediated effects. As for estrogens, the two major receptor types, ERalpha and ERbeta, are expressed in both normal and diseased human prostate, though with a different cellular localization. Since these two receptors are different in terms of ligand binding, heterodimerization, transactivation, and estrogen response element activity, it is likely that an imbalance of their expression may be critical to determine the ultimate estrogen effects on prostate cancer cells. In prostate cancer, ERbeta activation appears to limit cell proliferation directly or through ERalpha inhibition, and loss of ERbeta has been consistently associated with tumor progression. Several splicing variants of both ERalpha and ERbeta exist. Little is known about their expression and function in the human prostate, although reciprocal regulation and interaction with gene promoter both warrant further investigation. In summary, although multiple consistent evidence suggests that estrogens are critical players in human prostate cancer, their role has been only recently reconsidered, being eclipsed for years by an androgen-dominated interest.     

Comparable amounts of sex steroids are made outside the gonads in men and women: Strong lesson for hormone therapy of prostate and breast cancer

The objective of this study was comparison of circulating androgens and their metabolites as well as estrogens measured for the first time by a validated mass spectrometry technology in 60–80-year-old men and women of comparable age.Castration in men (n = 34) reduces the total androgen pool by only about 60% as indicated by the decrease in the serum levels of the glucuronide metabolites of androgens compared to intact men (n = 1302). Such data are in agreement with the 50 to 75% decrease in intraprostatic dihydrotestosterone (DHT) concentration after castration. Most interestingly, the same amounts of androgens and estrogens are found in postmenopausal women (n = 369) and castrated men of comparable age.The most significant therapeutic implication of these findings is the absolute need to add a pure (nonsteroidal) antiandrogen to castration in men with prostate cancer in order to block the action of the 25 to 50% DHT left in the prostate after castration. Not adding an antiandrogen to castration in men treated for prostate cancer is equivalent to not prescribing a blocker of estrogens in women suffering from breast cancer because they are postmenopausal and have low circulating estradiol.     

Role of fibroblast growth factor 8 in growth and progression of hormonal cancer

Hormonal cancers such as breast and prostate cancer arise from steroid hormone-regulated tissues. In addition to breast and prostate cancer hormonal regulation has also a role in endometrial, ovarian, testis and thyroid carcinomas. The effects of estrogens, androgens and progestagens on tumor growth are largely mediated by paracrine and autocrine target molecules which include growth factors and growth factor receptors. During cancer progression the hormonal growth regulation is often lost or overcome by an inappropriate activation of growth factor signaling cascades. One of the growth factors which have been associated with the regulation of growth and progression of hormonal cancer is fibroblast growth factor 8 (FGF8) which has also been recognized as an oncogene. FGF8 is widely expressed during embryonic development. It has been shown to mediate embryonic epithelial-mesenchymal transition and to have a crucial role in gastrulation and early organization and differentiation of midbrain/hindbrain, pharyngeal, cardiac, urogenital and limb structures. During adulthood FGF8 expression is much more restricted but in hormonal cancers it becomes frequently activated. High level of FGF8 expression in tumors is associated with a poor prognosis at least in prostate cancer. In experimental models FGF8 induces and facilitates prostate tumorigenesis and increases growth and angiogenesis of tumors. Several lines of evidence for autocrine and paracrine loops in the growth regulation of breast, prostate and ovarian cancer by FGF8 have been suggested.     

Carcinogenicity of catechol estrogens in Syrian hamsters

Estradiol and other estrogens induce renal carcinoma in male Syrian hamsters. The mechanism of carcinogenesis still remains unclear. Activation of estrogens to catechol metabolites has in the past been postulated to play a role in estrogen-induced carcinogenesis. Therefore, the carcinogenic activity of catechol estrogens was investigated. After 175 days of treatment, 4-hydroxyestradiol was found to be as carcinogenic as estradiol in male Syrian hamsters (4/5 and 4/5 animals with kidney tumors, respectively). Animals treated with 2-hydroxyestradiol (0/5) or 2-methoxyestradiol (0/6) did not develop renal carcinoma. The catechol estrogens failed to be mutagenic in the Ames test (reversions of his- S. typhimurium to histidine prototrophy in the TA 100 strain). The lack of carcinogenic activity of 2-hydroxyestradiol was not due to a failure to stimulate estrogen-dependent tumor growth. Growth of H-301 cells, an estrogen-dependent hamster kidney tumor cell line, was supported in vivo by estrogens in the following order: estradiol greater than 4-hydroxyestradiol greater than 2-hydroxyestradiol. Stimulation of tumor growth by 2-methoxyestradiol was not detected. It was concluded that the carcinogenic activity of 4-hydroxyestradiol was consistent with a role of catechol metabolites in estrogen-induced carcinogenesis. However, the intrinsic carcinogenic or hormonal activity of 2-hydroxyestradiol probably can not be assessed accurately in vivo because of its rapid methylation and metabolic clearance.     

Inhibition of hepatic 17 beta- and 3 alpha-hydroxysteroid dehydrogenases by antiinflammatory drugs and nonsteroidal estrogens

Antiinflammatory agents and estrogens have been tested as inhibitors of two isozymes of guinea pig liver testosterone 17 beta-dehydrogenase (NADP) 1.1.1.64) and rat liver 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50). Antiinflammatory steroids and estradiols were highly inhibitory to 3 alpha-hydroxysteroid dehydrogenase and one isozyme of testosterone 17 beta-dehydrogenase, respectively, but nonsteroidal antiinflammatory agents and nonsteroidal estrogens such as hexestrol, dienstrol, diethylstilbestrol and zearalenone showed potent inhibitions on all the enzymes. Although the inhibitory potency of indomethacin for one isozymes of testosterone 17 beta-dehydrogenase and 3 alpha-hydroxysteroid dehydrogenase decreased with changing pH from 9.7 to 7.0, that of the nonsteroidal estrogens for all the enzymes was little affected by pH. No additive effect in double inhibitor experiments with indomethacin and the nonsteroidal estrogens was observed, and the compounds were all competitive inhibitors with respect to steroidal substrate. The results suggest that there is a very similar region in substrate binding sites of the enzymes.     

Estrogen-mediated up-regulation of the Ca-dependent constitutive nitric oxide synthase in the rat aorta and heart

The role of endogenous estrogens has been studied in the regulation of the Ca-dependent constitutive nitric oxide synthase (cNOS) enzyme activity in aortic and cardiac tissues of the rat. The activity of cNOS enzyme was measured by the citrulline assay in the abdominal aorta and in the left ventricle of the heart obtained from male, sham-operated female and ovariectomized female Wistar rats. Estrogen replacement therapy (17-beta-estradiol, 20-100 microg/kg/day, s.c.) has been performed in ovariectomized rats over two weeks. We found that cNOS activity was higher in the aorta and heart of female rats compared to males. Ovariectomy decreased cNOS activity in both tissues to that level what could be observed in males. Estrogen supplementation caused a dose-dependent elevation of cNOS enzyme activity in cardiac and aortic tissues, where the higher dose (100 microg) completely restored cNOS enzyme activity to the levels found in females. We concluded that endogenous estrogens up-regulate the activity of the cNOS isoenzymes in the rat aorta and heart.     

The role of estrogens in the regulation of lactate dehydrogenase activity and its submolecular organization in rat anterior pituitary

Estrogens increase LDH activity and decrease H/M subunit ratio in rat anterior pituitary in both the experimental circumstances and the physiological conditions. The cellular messengers mediating estrogenic effect are structure- and stereospecific. The activity increasing and subunit ratio decreasing potency of the three tested estrogens was of following decreasing order: 17 beta-estradiol, estrone, and estriol. 17 alpha-estradiol did not affect activity parameters and submolecular organisation of the enzyme. The estrogen induced activity increase is consequence of the enhanced de novo enzyme protein synthesis which could be inhibited by Actinomycin-D. The lack of adrenocorticoids did not involve the alteration of LDH activity and H/M ratio in female rat anterior pituitary. Accordingly, these steroids do not mediate estrogenic action. 17 beta-estradiol had a substantial increasing effect on LDH activity in the subrenal implanted pituitary homografts and decreased H/M subunit ratio. Pituitary LDH activity in androgenized female rats decreased only after the removal of the polycystic ovaries. The two latter observations suggest that hypothalamic hormones are not involved in the regulation of pituitary LDH activity and its submolecular organization. De novo synthesis of LDH enzyme protein and the regulation its submolecular organization is induced by the direct action on anterior pituitary cells of the estrogens.     

Effects of sulpiride on mRNA levels of steroid 5alpha-reductase isozymes in prostate of adult rats

Prolactin (PRL) is implicated in prostate growth and in the development and regulation of benign prostatic hypertrophy (BPH) and prostate cancer (PCa). PRL may exert its effects on prostate in synergism with androgens. The most active androgen in the prostate is the 5alpha-dihydrotestosterone (DHT) obtained from testosterone by the 5alpha-reductase (5alpha-R) enzyme, which is expressed in the prostate as two isozymes, 5alpha-R1 and 5alpha-R2. In this study, sulpiride, a prolactin-secretion inductor, was administered to male rats. mRNA levels of 5alpha-R1 and 5alpha-R2 were measured in prostate of controls and sulpiride-treated rats, using one-step quantitative RT-PCR coupled with laser-induced fluorescence capillary electrophoresis (LIF-CE). Results demonstrated that sulpiride-induced hyperprolactinemia is associated with an increase in mRNA levels of both 5alpha-R1 and 5alpha-R2 in prostate of adult rats. Although a direct effect of sulpiride on prostate gland cannot be ruled out, hyperprolactinemia may be a factor to be considered in aging males, in whom prostatic diseases such as BPH and PCa are more frequent.