A proposed sequence of hormones controlling the induction of luteal 20alpha-hydroxy steroid dehydrogenase and progesterone withdrawal in the late-pregnant rat.
1. The previously reported induction of luteal 20alpha-hydroxy steroid dehydrogenase by administration of aminoglutethimide to late-pregnant rats was shown to be unaffected by prior removal of the foetuses. Aminoglutethimide therefore does not act via the foetuses in this context. 2. The ability of injected oestrogen to prevent the above induction was lost by delaying the injection for 12h after aminoglutethimide, although the increase in enzyme activity begins only after 24h. 3. Induction of 20alpha-hydroxy steroid dehydrogenase by foetoplacental removal on day 18 of pregnancy was inhibited by human choriogonadotropin, lutropin (luteinizing hormone) and pregnant-mare serum gonadotropin, but not by somatotropin (growth hormone), thyrotropin or follitropin (follicle-stimulating hormone) 4. Indomethacin blocked the normal induction of 20alpha-hydroxy steroid dehydrogenase in late pregnancy and that caused by aminoglutethimide. It partially blocked that caused by human choriogonadotropin given on days 19-20 and that caused by 2-bromo-alpha-ergocryptine on days 5-6, but failed to block that caused by human choriogonadotropin on days 15-16 or by foetoplacental removal on day 18 of pregnancy. 5. These findings, and the control of progesterone synthesis in late pregnancy, are interpreted in terms of a sequence of hormonal or enzymic syntheses, each of which is inhibited by the product of the preceding synthesis. (+info)
Product of side-chain cleavage of cholesterol, isocaproaldehyde, is an endogenous specific substrate of mouse vas deferens protein, an aldose reductase-like protein in adrenocortical cells.
Mouse vas deferens protein (MVDP) is an aldose reductase-like protein that is highly expressed in the vas deferens and adrenal glands and whose physiological functions were unknown. We hereby describe the enzymatic characteristics of MVDP and its role in murine adrenocortical Y1 cells. The murine aldose reductase (AR) and MVDP cDNAs were expressed in bacteria to obtain recombinant proteins and to compare their enzymatic activities. Recombinant MVDP was functional and displayed kinetic properties distinct from those of murine AR toward various substrates, a preference for NADH, and insensitivity to AR inhibitors. For MVDP, isocaproaldehyde, a product of side-chain cleavage of cholesterol generated during steroidogenesis, is the best natural substrate identified so far. In Y1 cells, we found that NADH-linked isocaproaldehyde reductase (ICR) activity was much higher than NADPH-linked ICR activity and was not abolished by AR inhibitors. We demonstrate that in Y1 cells, forskolin-induced MVDP expression enhanced NADH-linked ICR activity by 5-6-fold, whereas no variation in ICR-linked NADPH activity was observed in the same experiment. In cells stably transfected with MVDP antisense cDNA, NADH-linked ICR activity was abolished even in the presence of forskolin, and the isocaproaldehyde toxicity was increased compared with that of intact Y1 cells, as measured by isocaproaldehyde LD(50). In Y1 cells transfected with MVDP antisense cDNA, forskolin-induced toxicity was abolished by aminoglutethimide. These results indicate that in adrenocortical cells, MVDP is responsible for detoxifying isocaproaldehyde generated by steroidogenesis. (+info)
Decreased progesterone levels and progesterone receptor antagonists promote apoptotic cell death in bovine luteal cells.
We tested the hypothesis that progesterone (P(4)) acts at a local level to inhibit luteal apoptosis. Initial experiments employed aminoglutethimide, a P450 cholesterol side-chain cleavage inhibitor, to inhibit steroid synthesis. Cultured bovine luteal cells were treated with aminoglutethimide (0.15 mM) +/- P(4) (500 ng/ml) for 48 h. Luteal cells were recovered and snap frozen for isolation and analysis of oligonucleosomal DNA fragmentation or fixed for morphological analysis. Medium was collected for analysis of P(4) levels by RIA. Aminoglutethimide inhibited P(4) synthesis by > 95% and increased the level of apoptosis as evidenced by (32)P-labeled oligonucleosomal DNA fragmentation (> 40%). P(4) supplementation inhibited the onset of apoptosis that was induced by aminoglutethimide. These data were further supported by morphological assessment of apoptotic cells utilizing a Hoechst staining technique and together strongly suggest that P(4) has anti-apoptotic capacity. Using reverse transcription-polymerase chain reaction, we were able to isolate a 380-base pair cDNA from the bovine corpus luteum (CL) that was 100% homologous to the progesterone receptor (PR) previously found in bovine oviductal tissue. Furthermore, PR transcripts were present in large and small luteal cells. Immunohistochemistry also revealed that PR protein was present in both large and small luteal cells. To determine whether the anti-apoptotic effect of P(4) was regulated at the receptor level, luteal cells were cultured in the presence of PR antagonists, RU-486 and onapristone, for 48 h. Both antagonists caused approximately a 40% increase in (32)P-labeled oligonucleosomal DNA fragmentation. Interestingly, there was no difference (P >/= 0.05) in P(4) levels after treatment with PR antagonists. These observations support the concept that P(4) represses the onset of apoptosis in the CL by a PR-dependent mechanism. (+info)
Status of aromatase inhibitors in relation to other breast cancer treatment modalities.
Aromatase is one of the key enzymes possibly linked with the perpetuation or even initiation of breast cancer. Modulation of its activity by the new generation inhibitors has resulted in increased responses and improved therapeutic ratio compared with those of parent aromatase inhibitors. More recent trials have shown promising results with regard to improved therapeutic ratio compared with what is seen with presently accepted second-line hormonal approaches. Present data and laboratory research indicate that new aromatase inhibitors have the potential to play an important role as adjuvants, and possibly in the prevention of human breast cancer. It is probable that it may be as adjuvants that their real therapeutic strength in terms of a beneficial impact on survival may be realized. The absence of estrogen agonist activity of new aromatase inhibitors on lipid and bone metabolism calls for more clinical studies having late mortality in breast cancer survivors as the ultimate outcome objective; in this regard, interaction of new aromatase inhibitors with new selective estrogen receptor modulators looks promising. Achievement of these outcomes, and understanding of interactions with other therapies, await the termination of present trials and the start of new initiatives. (+info)
Use of aromatase inhibitors in breast carcinoma.
Aromatase, a cytochrome P-450 enzyme that catalyzes the conversion of androgens to estrogens, is the major mechanism of estrogen synthesis in the post-menopausal woman. We review some of the recent scientific advances which shed light on the biologic significance, physiology, expression and regulation of aromatase in breast tissue. Inhibition of aromatase, the terminal step in estrogen biosynthesis, provides a way of treating hormone-dependent breast cancer in older patients. Aminoglutethimide was the first widely used aromatase inhibitor but had several clinical drawbacks. Newer agents are considerably more selective, more potent, less toxic and easier to use in the clinical setting. This article reviews the clinical data supporting the use of the potent, oral competitive aromatase inhibitors anastrozole, letrozole and vorozole and the irreversible inhibitors 4-OH androstenedione and exemestane. The more potent compounds inhibit both peripheral and intra-tumoral aromatase. We discuss the evidence supporting the notion that aromatase inhibitors lack cross-resistance with antiestrogens and suggest that the newer, more potent compounds may have a particular application in breast cancer treatment in a setting of adaptive hypersensitivity to estrogens. Currently available aromatase inhibitors are safe and effective in the management of hormone-dependent breast cancer in post-menopausal women failing antiestrogen therapy and should now be used before progestational agents. There is abundant evidence to support testing these compounds as first-line hormonal therapy for metastatic breast cancer as well as part of adjuvant regimens in older patients and quite possibly in chemoprevention trials of breast cancer. (+info)
Lipoproteins regulate expression of the steroidogenic acute regulatory protein (StAR) in mouse adrenocortical cells.
The steroidogenic acute regulatory protein (StAR) is required for the movement of cholesterol from the outer to the inner mitochondrial membrane, the site of cholesterol side chain cleavage. Here we describe a novel form of regulation of StAR gene expression in steroidogenic cells. Treatment of Y-1 BS1 adrenocortical cells with either low density lipoprotein (LDL) or high density lipoprotein (HDL) increases expression of endogenous StAR mRNA and protein in a dose-dependent manner. Induction of StAR mRNA by lipoprotein requires basal cAMP-dependent protein kinase, since the inhibitor, R(p)-8-Br-cAMP, inhibited induction of StAR protein by LDL. Likewise, basal StAR expression or LDL induction of StAR protein was not detectable in Y-1 kin-8 cells which are deficient in cAMP-dependent protein kinase. Aminoglutethimide and ketoconazole were used to determine if side chain cleavage of lipoprotein-derived cholesterol is required for induction of StAR mRNA. Treatment with either drug alone induced StAR mRNA expression 1.5-3-fold, while induction of StAR in cells treated with either drug plus LDL, was equal to, or greater than, induction seen with either agent alone, suggesting that lipoprotein does not regulate StAR via generation of an oxysterol intermediate. Both LDL and HDL increased expression of a mouse -966 StAR promoter-reporter construct 1.5-2.5-fold, indicating that regulation occurs at the level of transcription. In contrast, neither lipoprotein was able to induce transcription from a -966 StAR promoter in which the steroidogenic factor-1 site at -135 was abolished, indicating that regulation of StAR transcription by lipoproteins requires steroidogenic factor-1. The regulation of StAR gene expression by lipoproteins may represent a positive feedback circuit which links cholesterol availability with steroidogenic output. (+info)
Contribution of progesterone, follicle stimulating hormone and glucocorticoids in survival of serum-free cultured granulosa cell explants.
To investigate the role of progesterone (P4) as a survival factor in quail granulosa cell explants, P4 content was determined under various conditions and correlated with apoptotic indexes (AIs) evaluated by 2',6'-diamidino-2-phenylindole (DAPI)-staining. Analysis of serum-free cultures from 24 to 96 h shows decreased P4 levels in the medium paralleled by increasing AI. Inhibiting apoptosis by gonadotropic support (FSH, 100 ng/ml) stimulates a 3-fold increase of the P4 level in the medium (83.49+/-8.69 vs 26.31+/-1.61 ng/ml in serum-free controls) together with a significant decrease in AI from 8.81+/-1.06% in serum-free controls to 3.50+/-0.72%. Substantial evidence for P4 as an autocrine/paracrine survival factor can be inferred from experiments with aminoglutethimide (AG, 1 mM) and RU486 (20 microM). Blocking P4 synthesis by AG causes a 2-fold increase in apoptosis from 6.08+/-0.67% in serum-free controls to 12.53+/-1.60%. Blocking P4 receptors by RU486 causes a similar increase in AI (3.02+/-0.98% in serum-free controls to 17.07+/-3.20%) and about a 50% decrease in P4. The effect of RU486 could be attenuated by exogenous P4 but not by dexamethasone indicating selective binding of P4 to the progesterone receptor. Dexamethasone treatment promotes survival without affecting P4 levels. In further support of an autocrine/paracrine action for P4 in the granulosa cells, both the A and B form of the avian P4 receptor (PR) are identified in vivo and in vitro by Western blotting. Exogenous administration of P4 only affects survival when endogenous P4 synthesis is blocked or after 48 h of serum-free culture when endogenous P4 production is very low. Because FSH also affects survival when its stimulatory effect on P4 synthesis is blocked by AG (AI decrease from 6.08+/-0.67% in serum-free controls to 1.64+/-0.71% in FSH+AG treated) it is proposed that (1) P4 is an autocrine/paracrine survival factor in the preovulatory granulosa and (2) FSH mediates both P4-dependent and P4-independent survival pathways. (+info)
Aromatase inhibition by an 11,13-dihydroderivative of a sesquiterpene lactone.
Compounds that inhibit aromatase activity are used for the treatment of breast cancer. A group of sesquiterpene lactones inhibit aromatase activity and also exert cytotoxicity through their reactive alpha-methylene-gamma-lactone group. To synthesize sesquiterpene lactones with greater specificity for aromatase inhibition and lower cytotoxicity, we chemically reduced the alpha-methylene-gamma-lactone group in the active aromatase inhibitor 10-epi-8-deoxycumambrin B (compound 1), to obtain the new compound 11betaH,13-dihydro-10-epi-8-deoxycumambrin B (compound 2). Reduction of the alpha-methylene-gamma-lactone group abrogated the cytotoxic activity of compound 1 against the JEG-3, HeLa, and COS-7 cell lines. Compound 2 had higher aromatase inhibitory activity than compound 1 (IC(50) = 2 +/- 0.5 microM versus 7 +/- 0.5 microM, K(i) = 1.5 microM versus 4.0 microM) and was a more potent type II ligand to the heme iron present in the cytochrome P450(arom) active site. Compound 2 inhibited aromatase activity in JEG-3 cells in a comparable manner to the inhibitor aminoglutethimide (AG) used clinically for the treatment of breast cancer. Additionally, compound 2 inhibited androstenedione-induced uterine hypertrophy in sexually immature mice (41% of uterine weight suppression for compound 2 versus 51% for AG). We conclude that the anti-aromatase activity of sesquiterpene lactones does not depend on the presence of the highly reactive alpha-methylene-gamma-lactone group, whereas their cytotoxicity does. These findings may facilitate the development of safer agents for breast cancer therapy. (+info)