Aromatase inhibitors as potential cancer chemopreventives.

Epidemiological and experimental evidence strongly supports a role for estrogens in the development and growth of breast tumors. A role for estrogen in prostate neoplasia has also been postulated. Therefore, one chemopreventive strategy for breast and prostate cancers is to decrease estrogen production. This can be accomplished by inhibiting aromatase, the enzyme that catalyzes the final, rate-limiting step in estrogen biosynthesis. The use of aromatase inhibitors is of clinical interest for cancer therapy, and selective, potent aromatase inhibitors have been developed. Several of these agents have demonstrated chemopreventive efficacy in animal models. The rationale for the use of aromatase inhibitors as chemopreventives and identification of inhibitors to serve as potential chemopreventive agents are the subjects of this review. After background information regarding aromatase is presented, the data for each inhibitor are summarized separately. The discussion focuses on those inhibitors that are clinically available or in clinical trials, including: aminoglutethimide (Cytadren), rogletimide, fadrozole hydrochloride, liarozole hydrochloride, anastrozole (Arimidex), letrozole, vorozole, formestane, exemestane, and atamestane. On the basis of results from preclinical studies, aromatase inhibitors may be promising agents for clinical trials in populations at high risk for developing estrogen-dependent cancers. Total suppression of aromatase may have adverse effects, as is evident in postmenopausal women (increased osteoporosis, cardiovascular disease, and urogenital atrophy). However, on the basis of preclinical studies of chemopreventive efficacy and chemotherapeutic applications of aromatase inhibitors showing dose-response efficacy, it may be possible to obtain chemopreventive effects without total suppression of aromatase and circulating estrogen levels. Suppressing local estrogen production may be an alternative strategy, as suggested by the discovery of a unique transcriptional promoter of aromatase gene expression, I.4, in breast adipose tissue. The development of drugs that target this promoter region may be possible.

Telomerase activity in female and male rat germ cells undergoing meiosis and in early embryos.

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA at the ends of eukaryotic chromosomes. It has been hypothesized that telomerase activity is necessary for cellular immortalization and that telomerase activity is present in cells of germline origin. The objective of the present study was to determine the level of telomerase activity in the following rat cells: 1) oocytes from follicles at different stages of development, 2) spermatogenic cells, and 3) early embryos. Telomerase activity was quantitated using a recently developed, sensitive polymerase chain reaction-based assay and a human kidney cell line (293) as a standard. Telomerase activity was found in oocytes from early antral and preovulatory follicles, as well as in ovulated oocytes. The level of enzyme activity in early antral and preovulatory follicles was comparable to that of the 293 cells, while levels in ovulated oocytes were 50-fold lower. Telomerase activity was present in even lower levels in pachytene spermatocytes and round spermatids, and no telomerase activity was detected in spermatozoa from either the caput or the cauda epididymis. After fertilization, telomerase activity was present in 4-cell embryos. Telomerase activity was also detected in several rat somatic tissues. These data demonstrate that telomerase activity is present in germ cells at several stages of differentiation, with the exception of spermatozoa, and suggest that telomerase activity may be important during meiosis. The high levels of telomerase activity in individual oocytes may serve as a marker for monitoring the effects of hormonal agents, aging, and toxins on oocyte quality.

Tumor necrosis factor-alpha and its second messenger, ceramide, stimulate apoptosis in cultured ovarian follicles.

In the mammalian ovary, only a small fraction of follicles fully mature and ovulate, while most of them die via apoptosis. Multiple factors promoting follicle survival have been identified, but intraovarian mediators of apoptosis are poorly known. Tumor necrosis factor-alpha (TNF alpha) is a cytokine capable of inducing apoptosis in diverse cell types, and the apoptotic effect of TNF alpha is, partially, coupled to the sphingomyelin signaling pathway with ceramide as a second messenger. Because TNF alpha has been localized in the rat ovary, and TNF alpha treatment increases granulosa cell ceramide production, we studied the effect of treatment with TNF alpha and ceramide on follicle apoptosis. Immature rats were implanted with diethylstilbestrol to stimulate the development of early antral follicles. Follicles were isolated and cultured in a serum-free medium for 24 h with or without hormone treatments. During culture, spontaneous follicle apoptosis occurred (10-fold increase in DNA fragmentation), which was partially blocked by 100 ng/ml FSH (60% suppression). The effect of FSH was counteracted by TNF alpha in a dose-dependent manner, with the maximal effect at 100 ng/ml TNF alpha (90% reversal of FSH action). In situ analysis indicated that the granulosa cell is the follicle cell type undergoing DNA fragmentation. A membrane-permeable ceramide analog, C2-ceramide N-acetyl sphingosine, mimicked the effect of TNF alpha and was able to completely abolish the action of FSH at 50 microM. In contrast, another ceramide analog, C2-dihydroceramide N-acetyl dihydrosphingosine, did not alter the effect of FSH, verifying the specificity of ceramide action. To study the mechanism of TNF alpha and ceramide action, the effect of sodium aurathiomalate (ATM), an inhibitor of interleukin-1 beta-converting enzyme/ced-3-related cystine proteases known to be essential in the execution of mammalian cell apoptosis, was studied. Treatment with ATM (1 mM) prevented the apoptosis-inducing effect of both TNF alpha and ceramide, suggesting a role for cysteine proteases in mediating follicle apoptosis. Treatment with either TNF alpha or ceramide increased both basal and FSH-stimulated progesterone production by cultured follicles. Concomitant treatment by ATM did not alter the stimulatory effect of TNF alpha or ceramide on progesterone production, ruling out nonspecific toxic effect of the inhibitor and indicating that the apoptotic and steroidogenic pathways are independent. In summary, treatment with TNF alpha or its second messenger, ceramide, stimulates apoptosis of early antral follicles in culture, suggesting a potential role for TNF alpha as an intraovarian regulator of follicle atresia by acting through the ceramide signaling pathway.

Hormonal regulation of apoptosis in early antral follicles: follicle-stimulating hormone as a major survival factor.

Hormonal regulation of apoptosis has been studied in cultured preovulatory follicles. Because early antral follicles are most vulnerable to undergo atretic degeneration under physiological conditions in vivo, the present studies were designed to investigate the hormonal regulation of apoptosis using in vitro culture of early antral follicles. Rats were implanted with diethylstilbestrol at 24 days of age to stimulate the development of early antral follicles, and ovaries were collected at day 27 of age. Early antral follicles were dissected and cultured (four per vial) for 24 h with or without hormonal treatments. After culture, DNA was extracted from follicles, and the degree of apoptotic DNA fragmentation was determined using 3'-end labeling and gel electrophoresis. In situ analysis of apoptotic DNA fragmentation revealed that granulosa cells in these follicles are the main cell type undergoing apoptosis. Follicles cultured in the absence of hormones showed a 12-fold increase in the level of apoptotic DNA fragmentation which was prevented by treatment with FSH in a dose-dependent manner (60% maximal suppression and apparent ED50 of 30 ng/ml). Similarly, treatment with (Bu)2cAMP also suppressed follicle apoptosis. Treatment with LH or human CG, however, minimally suppressed apoptotic DNA fragmentation (35% maximal suppression). Insulin-like growth factor-I (IGF-I) also suppressed apoptosis by 45%. Moreover, the suppressive effect of FSH on apoptosis was partially reversed by coincubation with IGF-binding protein-3, suggesting a potential mediatory role of endogenous IGF-I. However, recombinant bovine GH had no effect on follicle apoptosis despite its ability to stimulate IGF-I messenger RNA (mRNA) levels. Incubation of follicles with epidermal growth factor (EGF) and basic fibroblast growth factor maximally suppressed follicle apoptosis by only 32% and 42%, respectively. Ligand binding analysis indicated the minimal effectiveness of EGF on apoptosis in early antral follicles, as compared with its potent action in preovulatory follicles reported earlier, may be due to a 3.5 fold increase in EGF receptor concentration in the mature follicles. High doses (150 or 500 ng/ml) of interleukin-1beta also suppressed apoptosis by 48% whereas treatment with an NO generator, sodium nitroprusside, or a cyclic GMP analog suppressed apoptosis as effectively as that of FSH. Furthermore, treatment with activin resulted in a dose-related suppression of follicle apoptosis, reaching a maximal 40% suppression. In contrast, cotreatment of activin with its binding protein, follistatin, abolished this effect. Collectively, these data demonstrated a stage-dependent difference in the hormonal regulation of follicle apoptosis. Although FSH, LH/human CG, GH, IGF-I, EGF, basic fibroblast growth factor, and interleukin-1beta are all effective survival factors for preovulatory follicles, FSH is a major survival factor for early antral follicles, the stage during which a majority of follicle undergo atresia under physiological conditions.

Gonadal cell apoptosis: hormone-regulated cell demise.

It has become evident that apoptosis, an active form of cell 'suicide', plays an important role in the normal function of all tissues. A balance of cell proliferation and apoptosis is maintained in a healthy individual and any imbalance of the two processes could lead to pathological changes. In both sexes, massive apoptosis accounts for the demise of a majority of gonadal cells (ovarian granulosa cells and male germ cells) during reproductive life. Recent studies have indicated the important role of gonadotrophins as survival factors in both the ovary and the testis. Furthermore, intra-gonadal survival. factors in the ovary (oestrogens, insulin-like growth factor I, epidermal growth factor, basic fibroblast growth factor, interleukin-1 beta, nitric oxide, etc.) and testis (androgens) have been shown to act in concert with the gonadotrophins. In contrast, several apoptotic factors (androgens, gonadotrophin-releasing hormone-like peptide and interleukin-6) may be important in inducing the demise of ovarian follicles. Understanding of the hormonal and cellular mechanisms responsible for gonadal cell apoptosis will provide new approaches for the treatment of gonadal degenerative conditions such as premature ovarian failure and cryptorchidism, as well as for the design of new contraceptive approaches.

Gonadal cell apoptosis.

Apoptosis is an important cellular process by which superfluous or unwanted cells are deleted from an organism during tissue remodeling and differentiation. Recent studies have demonstrated the role of this programmed cell death or "controlled cell suicide" in the physiological function of an organism. Suppression of apoptosis increases the susceptibility of an individual to malignancy whereas uncontrolled cell death is associated with degenerative diseases. Normal development of both female and male gonads is characterized by massive cell death. More than 99% of ovarian follicles endowed at early life are destined to undergo apoptosis and the exhaustion of these follicles serves as a "clock" for female reproductive senescence. In the testis, up to 75% of male germ cells also undergo apoptosis, perhaps as a mechanism to delete superfluous or defective germ cells. Gonadal cell apoptosis provides valuable models to study hormonal regulation of apoptosis. In the ovary, gonadotropins, estrogens, growth hormone, growth factors (IGFI, EGF/TGF-alpha, basic FGF), cytokine (interleukin-1 beta) and nitric oxide act in concert to ensure the survival of preovulatory follicles. In contrast, androgens, interleukin-6 and gonadal GnRH-like peptide are apoptotic factors. Developmental studies further indicate that fractions of endowed follicles are recruited throughout the reproductive life whereas most of the primordial follicles are "arrested" at the initial stage of development for a prolonged time. Because a transcriptional factor WT1 is expressed in high levels in follicles at early stages of development and because WT1 over-expression represses the promoter activity of inhibin-alpha gene, this nuclear protein may be important in the maintenance of follicles at early stages of development. Once a cohort of follicles is recruited to grow, it is destined to undergo apoptosis unless rescued by survival factors. After puberty onset and under gonadotropin stimulation, some of the growing antral follicles are "selected" to continue their final maturation and secrete high levels of estrogens to trigger ovulation. Following repeated cycles of recruitment, atresia or ovulation, the follicle reserve is exhausted, thus signaling the onset of reproductive senescence. Although the somatic granulosa cell is the major cell type undergoing apoptosis in the ovary, the germ cells in the testis also exhibit signs of apoptotic cell demise. In the testis, gonadotropins and androgens act as survival factors whereas exposure to elevated temperature in cryptorchid testes increases apoptosis. In the seasonally breeding hamster model, photoperiod-entrained regression and recrudescence of testis tissue serves as a unique natural model of apoptosis. With recent advances in our understanding of the cellular mechanism of apoptosis, including the elucidation of the Ced9/bc12 and Ced3/ICE family of proteins, further investigation of gonadal apoptosis may lead to a better understanding of gonadal degenerative disorders (such as premature ovarian failure and oligospermia), reproductive senescence and tumorigenesis. The gonadal model should also be valuable in studying the regulation of intracellular apoptosis genes by external hormonal signals.

Strategy and planning for chemopreventive drug development: clinical development plans II.

This is the second publication of Clinical Development Plans from the National Cancer Institute, Division of Cancer Prevention and Control, Chemoprevention Branch and Agent Development Committee. The Clinical Development Plans summarize the status of promising chemopreventive agents regarding evidence for safety and chemopreventive efficacy in preclinical and clinical studies. They also contain the strategy for further development of these drugs, addressing pharmacodynamics, drug effect measurements, intermediate biomarkers for monitoring efficacy, toxicity, supply and formulation, regulatory approval, and proposed clinical trials. Sixteen new Clinical Development Plans are presented here: curcumin, dehydroepiandrosterone, folic acid, genistein, indole-3-carbinol, perillyl alcohol, phenethyl isothiocyanate, 9-cis-retinoic acid, 13-cis-retinoic acid, l-selenomethionine and 1, 4-phenylenebis(methylene)selenocyanate, sulindac sulfone, tea, ursodiol, vitamin A, and (+)-vorozole. The objective of publishing these plans is to stimulate interest and thinking among the scientific community on the prospects for developing these and future generations of chemopreventive drugs.

Interleukin-1 beta suppresses apoptosis in rat ovarian follicles by increasing nitric oxide production.

A growing body of evidence suggests that intraovarian interleukin-1 beta (IL-1 beta) may play an intermediary role in the ovulatory process. Furthermore, induction of nitric oxide (NO) by IL-1 beta has been reported in a wide variety of tissues. As the majority of ovarian follicles undergo an atretic degeneration process involving apoptotic cell death, we set out to determine whether IL-1 beta rescues follicles from apoptosis and the possible involvement of NO. Preovulatory follicles obtained from PMSG-primed rats were cultured for 24 h in serum-free medium with or without hormone treatments. After culture, follicular apoptotic DNA fragmentation was analyzed by autoradiography of size-fractionated DNA labeled at 3'-ends with [32P]dideoxy-ATP. Follicular NO production was also determined by a colorimetric method. Treatment with IL-1 beta dose-dependently suppressed the spontaneous onset of apoptosis in cultured follicles, but stimulated NO production. In contrast, the addition of IL-1 receptor antagonist eliminated both effects of IL-1 beta, confirming receptor mediation. Follicles treated with sodium nitroprusside, a NO generator or an analog of cGMP, the second messenger for NO, also showed decreased follicle apoptosis. Moreover, the addition of NG-monomethyl-L-arginine, a NO synthase inhibitor, reversed both IL-1 beta stimulation of NO production and suppression of apoptosis, suggesting a mediatory role of NO in these IL-1 beta effects. Gonadotropins also prevent follicle apoptosis. Of interest, treatment with hCG stimulated NO production, and the hCG suppression of follicle apoptosis and stimulation of NO production were partially blocked by cotreatment with IL-1 receptor antagonist, indicating the mediation of endogenous IL-1 beta. Treatment with IL-1 beta also stimulated a small increase in the production of cAMP, estrogen, and progesterone. Taken together, these findings suggest that IL-1 beta is a survival factor for ovarian follicles, and its action is partially mediated via NO and cGMP generation. Moreover, part of the suppressive action of gonadotropins on follicle apoptosis is mediated by endogenously produced IL-1 beta.

Growth hormone suppression of apoptosis in preovulatory rat follicles and partial neutralization by insulin-like growth factor binding protein.

A growing body of evidence suggests that growth hormone (GH) plays a role in regulating ovarian function by augmenting gonadotropin stimulation of granulosa cell differentiation and folliculogenesis. The majority of follicles in the mammalian ovary do not ovulate, but instead undergo a degenerative process (atresia) involving apoptotic cell death. The objective of the present study was to investigate the role of GH in regulating follicle apoptosis and to determine whether or not insulin-like growth factor-I (IGF-I) mediates GH action in this process. Preovulatory follicles obtained from eCG-primed rats were cultured for 24 h in serum-free conditions with or without hormone treatments. After culture, follicular apoptotic DNA fragmentation was analyzed by autoradiography of size-fractionated DNA labeled at 3' ends with [32P]dideoxy-ATP. Culture of preovulatory follicles resulted in a spontaneous onset of apoptotic DNA fragmentation that was suppressed by ovine GH (oGH) in a dose-dependent manner, reaching a maximum of 65% suppression. To rule out the effect of residual gonadotropin in the oGH preparation, follicles were also cultured with recombinant bovine growth hormone (rbGH). Like oGH, rbGH suppressed apoptotic DNA fragmentation. Our earlier study indicated that hCG and FSH treatment also suppress apoptosis in the present model system, but no additive effect of GH and either hCG or FSH on the suppression of apoptosis was observed. To determine whether the observed effect of GH action on follicle apoptosis is mediated by IGF-I, three types of studies were carried out.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of lipoprotein, equine luteinizing hormone, equine follicle-stimulating hormone, and equine prolactin on equine testicular steroidogenesis in vitro.

A stallion testicular cell incubation system was developed and used to investigate the regulation of steroidogenesis in stallion testes. Cells isolated from testes of 2- to 4-year-old stallions (n = 6) were cultured for 12 hours in a defined medium with and without varying doses of lipoprotein, equine luteinizing hormone (eLH), human chorionic gonadotropin (hCG), equine follicle-stimulating hormone (eFSH), and/or equine prolactin (ePRL). Estrogen conjugate (EC), testosterone (T), and estradiol-17 beta (E2) production were determined by RIA. Increasing doses of lipoprotein significantly (P < 0.001) increased basal, hCG- and eLH-stimulated EC production, resulting in a maximal fourfold increase in each case. A maximal dose of lipoprotein (3 mg/ml) significantly (P < 0.001) increased basal T production threefold, whereas hCG- and eLH-stimulated T production were increased 76- and 30-fold, respectively. In the presence of 0.5 mg/ml of lipoprotein, increasing doses of eLH significantly (P < 0.001) stimulated EC, T, and E2 production. The increase in T production (5.6-fold) at a physiological dose of eLH (5 ng/ml) was significantly (P < 0.05) greater than the increase in EC or E2 production (2.1- and 2.3-fold, respectively). However, the total mass of EC produced was significantly greater (P < 0.05) than the total amount of T produced at both basal (15 ng vs. 148 pg) or hormone-stimulated (48 ng vs. 2,427 pg at 5 ng/ml eLH) levels. hCG significantly (P < 0.001) stimulated EC and T production and was 82-fold more active in stimulating EC production and 41-fold more active in stimulating T production than was eLH. FSH had no significant effect on steroidogenesis either alone or in the presence of eLH, except at the highest dose tested (50 ng/ml), which was above the physiological level of circulating FSH (4-7 ng/ml) in the stallion. PRL (1-50 ng/ml) had no significant effect on steroidogenesis either alone or in the presence of eLH. These data suggest that in the postpubertal stallion, both estrogen and T production are regulated by LH, and this regulation appears to be dependent on the availability of lipoprotein-derived cholesterol. Furthermore, the observation that testicular cells produced a larger mass of EC than T, but responded to eLH with a larger relative increase in T production, suggests that production of these two steroids may be independently regulated.

Localization of aromatase in equine Leydig cells.

Stallion testes secrete large amounts of estrogens, but the cellular location of the enzyme that converts androgens to estrogens, cytochrome P450 aromatase, has not been determined. The goal of the present study was to immunocytochemically localize stallion testicular aromatase using a polyclonal antibody generated against human placental cytochrome P450 aromatase. Testes were obtained from 12 stallions from 2 to 23 years of age, during both the breeding and non-breeding seasons. Immunoreactivity was confined to the Leydig cells in all testes examined. No immunostaining was observed in the Sertoli or germ cells. Heterogeneity in the level of immunostaining among individual Leydig cells was observed. The results of this study indicate that in postpubertal, adult, and aged stallions, testicular aromatase is located in Leydig cells.