Growth regulation of ovarian cancer cells by epidermal growth factor and tranforming growth factors α and β1

Regulation of ovarian cancer growth is poorly understood. In this study, the effects of EGT, TGFα and TGFβ1 on two ovarian cancer cell lines (OVCAR-3 and CAOV-3) were investigated. The results showed that EGF/TGFα stimulated cell growth and DNA synthesis in OVCAR-3 cell, but inhibited cell proliferation and DNA synthesis in CAOV-3 cells. TGFβ1 invariably inhibited cell proliferation and DNA synthesis in both cell lines. These efefects on growth factors are dose dependent. The interaction of TGFβ1 and EGF/TGFα was antagonistic in OVCAR-3 cells. In contrast, EGF/TGFα and TGFβ1 had an additive inhibitory effect on CAOV-3 cells. Our results demonstrated that mature and functional EGF receptors are present in both cell lines and that they are capable of ligand binding, internalization, processing and ligand-enhanced autophosphorylation. Both high- and low-affinity binding are present in these cell lines, with CAOV-3 cells having about 2–3 fold higher total receptors than OVCAR-3 cells. These results together with those from our previous studies show that these cells express TGFα, TGFβ1 and EGF receptors and that cell growth may be modulated by these growth factors in an autocrine can paracrine manner. This report presents evidence supporting the important roles of growth factors in ovarian cancer growth and provides a foundation for futher study into the mechanism of growth regulation by growth factors in these cell lines.     

Estradiol 17β-dehydrogenase and 20α-hydroxysteroid dehydrogenase from human placental cytosol: one enzyme with two activities?

The soluble enzyme estradiol 17β-dehydrogenase (17β-ED) from human term placental cytosol is reported to be a stereospecific oxidoreductase for estrogen substrates. A published purification scheme (heat treatment and affinity chromatography) yielded a homogeneous protein which had the reported characteristics of pure 17β-ED and also had 20α-hydroxysteroid dehydrogenase (20α-HSD) activity. Spectrophotometric assay when the buffer contained albumin, 8 mg/ml, masked the 20α-HSD activity observed in albumin-free conditions and may explain why this bifunctional activity has gone unrecognized. In human placenta, one enzyme may catalyze stereospecific oxidation/reduction of both estrogen and progesterone.     

Ovarian metabolic pathways of steroid biosynthesis in the European eel (Anguilla anguilla L.) at the silver stage

Ovarian slices of the European eel at the silver stage were incubated with 4 tritiated precursors (pregnenolone, progesterone, androstenedione, testosterone) in the presence or not of an inhibitor of 11β-hydroxylase activity, metopirone. Ether extracts were submitted to a gradient elution chromatography on celite columns. Isolated peaks were identified by isopolarity on TLC, microchemical reactions and recrystallization to constant specific activity. Interpretation of the results shows that the ovary of the European eel contains the following enzymes: a 3β-hydroxysteroid dehydrogenase, 5→4-ene-isomerase complex, a 17α-hydroxylase, a C₂₁-C₁₉ desmolase, a 17β-hydroxysteroid oxidoreductase, a 5α-reductase, a 3β-hydroxysteroid oxidoreductase and an aromatase complex. Metopirone effect indicates the presence of an 11β-hydroxylase activity. At this stage, 5β-reductase, 20β-reductase and 21-hydroxylase activities are not detected in the ovary. Water-soluble steroids were formed from all the precursors used. It appears that the ovarian biosynthesis is orientated towards the production of 5α-reduced compounds and that this might limit the production of 17β-estradiol by lowering the amount of disposable endogenous precursors.     

Expression and regulation of aromatase and 17β-hydroxysteroid dehydrogenase type 4 in human THP 1 leukemia cells

Estradiol is active in proliferation and differentiation of sex-related tissues like ovary and breast. Glandular steroid metabolism was for a long time believed to dominate the estrogenic milieu around any cell of the organism. Recent reports verified the expression of estrogen receptors in “non-target” tissues as well as the extraglandular expression of steroid metabolizing enzymes. Extraglandular steroid metabolism proved to be important in the brain, skin and in stromal cells of hormone responsive tumors. Aromatase converts testosterone into estradiol and androstenedione into estrone, thereby activating estrogen precursors. The group of 17β-hydroxysteroid dehydrogenases catalyzes the oxidation and/or reduction of the forementioned compounds, e.g. estradiol/estrone, thereby either activating or inactivating estradiol. Aromatase is expressed and regulated in the human THP 1 myeloid leukemia cell line after vitamin D/GMCSF-propagated differentiation. Aromatase expression is stimulated by dexamethasone, phorbolesters and granulocyte/macrophage stimulating factor (GMCSF). Exons I.2 and I.4 are expressed in PMA-stimulated cells only, exon I.3 in both PMA- and dexamethasone-stimulated cells. Vitamin D-differentiated THP 1 cells produce a net excess of estradiol in culture supernatants, if testosterone is given as aromatase substrate. In contrast, the 17β-hydroxysteroid dehydrogenase type 4 (17β-HSD 4) is abundantly expressed in unstimulated THP 1 cells and is further stimulated by glucocorticoids (2-fold). The expression is unchanged after vitamin D/GMCSF-propagated differentiation. 17β-HSD 4 expression is not altered by phorbolester treatment in undifferentiated cells but is abolished after vitamin D-propagated differentiation along with downregulation of β-action. Protein kinase C activation therefore appears to dissociate the expression of aromatase and 17β-HSD 4 in this differentiation stage along the monocyte/phagocyte pathway of THP 1 myeloid cells. The expression of steroid metabolizing enzymes in myeloid cells is able to create a microenvironment which is uncoupled from dominating systemic estrogens. These findings may be relevant in the autocrine, paracrine or iuxtacrine cellular crosstalk of myeloid cells in their respective states of terminal differentiation, e.g. in bone metabolism and inflammation.     

Rosiglitazone stimulates peroxisome proliferator-activated receptor gamma expression and directly affects in vitro steroidogenesis in porcine ovarian follicles

Rosiglitazone is a peroxisome proliferator-activated receptor gamma (PPARγ) synthetic activator from the group of thiazolidinediones often used in the treatment of chronic diseases such as type 2 diabetes and other forms of insulin resistance. The present in vitro study assessed the direct effects of rosiglitazone at 25 and 50 μM doses on PPARγ gene expression, steroid secretion (progesterone [P4], androstenedione [A4], testosterone [T], and estradiol), and protein expression of PPARγ, 3βHSD, CYP17, 17βHSD, CYP19 by porcine ovarian follicles from prepubertal and cycling animals. We analyzed also steroid enzymatic activity by conversion of pregnen-3β-ol-20-one to P4, P4 to A4, and A4 to T. Our results indicated that rosiglitazone increased significantly PPARγ expression, P4 secretion, 3βHSD activity, and protein expression. Rosiglitazone decreased A4 and T secretion by reducing the expression and activity of CYP17 and 17βHSD and did not change estradiol secretion and CYP19. Similarly results was observed both in prepubertal and cycling pigs. Our results indicate that these direct effects of rosiglitazone on ovarian steroidogenesis provide a framework for testing several potential new mechanisms of PPAR-γ actions on porcine ovarian function.     

Early steroid metabolism by chick blastoderm invitro

Yolk free blastoderms of chick embryo were incubated 3 or 22 hours with labeled pregnenolone, progesterone, 17-hydroxyprogesterone, dehydro-epiandrosterone, androstenedione, testosterone and estradiol-17β. Metabolites and unconverted substrates were found both in the incubation medium and in the cells. Enzymes responsible for identified conversions were: 17α-hydroxylase, 17-20-desmolase, Δ⁵3β- and 3α-hydroxysteroid dehydrogenase, 17β-hydroxysteroid dehydrogenase and 5α- and 5β-reductase. The results suggest that the steroid metabolizing enzyme activities found may reflect a more general ability of early embryonic cells.     

Non-invasive repeated measurement of urinary progesterone, 17beta-estradiol, and testosterone in developing, cycling, pregnant, and postpartum female mice

Excretory samples from adult female mice were collected non-invasively during development, estrous cycling, pregnancy, and postpartum. In initial studies, urinary measures were statistically more dynamic over days than were fecal measures; thus subsequent studies focused on urine. Higher 17beta-estradiol levels were present in isolated females than in those exposed to males. In cycling females, urinary 17beta-estradiol was more variable than were measures of testosterone or progesterone, showing peaks with an approximate 5-day periodicity. When urinary estradiol and progesterone were monitored in conjunction with vaginal smear cell counts, patterns were idiosyncratic; most females showed distinct peaks in urinary steroids, not in clear synchrony with vaginal cell cornification. Levels of progesterone rose markedly during the first 10 days of pregnancy, then declined before birth. Estradiol showed a substantial peak on days 7-8 of gestation in all females measured. Urinary testosterone was not dynamic during pregnancy, but rose in immediate prenatal and postpartum measures. During post-weaning, pre-pubertal development, urinary levels of progesterone remained constant but levels of estradiol rose substantially over time.     

Ovarian steroidogenic enzyme activities during the rat estrous cycle

We have correlated the concentrations of serum LH, estradiol and progesterone with the activities of 2 ovarian steroid biosynthetic enzymes during the rat estrous cycle. Ovarian 3 β-hydroxysteroid dehydrogenase isomerase (3-βHSD) activity decreased from 29 ± 6 nmol/mg protein/ min (mean ± SEM) in diestrus, to 7 ± 0.4 nmol/mg protein/min in late proestrus (p < 0.005), and subsequently increased to 36 ± 9 nmol/mg protein/min in metestrus (p < 0.01). Ovarian 17-hydroxylase (17-OH) activity decreased from early to late proestrus (3.3 ± 0.2 vs 2.2 ± 0.2 nmol/mg protein/min, p <0.0025), and subsequently increased to 3.9 ± 0.2 in metestrus (p<0.001). Serum LH, estradiol and progesterone peaked during proestrus, and reached a nadir during estrus. We conclude that the activities of 3-βHSD and 17-OH in the rat ovary vary markedly during the estrous cycle. These changes may underlie the pattern of steroid secretion characteristic of this process.     

In the absence of protein, estradiol suppresses meiosis of porcine oocytes in vitro

The effect of estradiol on the spontaneous maturation of porcine oocytes was investigated. Cumulus-enclosed (intact) and cumulus-free (denuded) oocytes were cultured in the presence of estradiol-17 beta (0 to 10 microgram/ml) in a chemically defined bicarbonate-buffered medium that contained either dextran or BSA, or in a complex Hepes-buffered medium that was supplemented with serum. After 24 hr, chromatin spreads were prepared and meiotic maturation was scored. The biochemical integrities of the cumulus cells were assessed by determination of the estradiol and progesterone content of spent media after culture of intact oocytes in the presence of 0.5 X 10(-6) M testosterone and 10 microgram/ml follicle-stimulating hormone. Estradiol did not significantly affect the onset of maturation of either intact or denuded oocytes that were cultured in medium containing either BSA or serum. In serum-supplemented medium, however, the progression of maturation beyond metaphase I was significantly affected by the steroid in a dose-dependent manner. The steroid significantly inhibited the release from meiotic arrest of both types of oocyte cultured in medium supplemented with dextran. Supplementation of all media with testosterone and FSH significantly stimulated the synthesis of estradiol by the cumulus cells, compared with that of control groups. The synthesis of progesterone, however, was significantly stimulated by testosterone and FSH only in the BSA and serum-supplemented media. It is concluded that exogenous estradiol has the capacity to arrest meiosis in vitro but that this capacity can only be expressed if no exogenous protein(s) is present. In the absence of exogenous protein, progesterone synthesis by the adherent cumulus cells is minimal.     

Steroid metabolism in the hormone dependent MCF-7 human breast carcinoma cell line and its two hormone resistant subpopulations MCF-7/LCC1 and MCF-7/LCC2

Androgen and estrogen metabolism was investigated in the hormone-dependent human breast cancer cell line MCF-7 and its two hormone-resistant sublines MCF-7/LCC1 and MCF-7/LCC2. Using the product isolation method, the activity of aromatase, 5-reductase, 3/β-hydroxysteroid oxidoreductase and 17β-hydroxysteroid oxidoreductase were investigated isolating the following steroids: estriol (E₃), estradiol (E₂), estrone (E₁), 3/β-androstanediol (A-diol), testosterone (T), dihydrotestosterone (DHT), androsterone (AND), androstenedion (4-AD) and androstanedione (A-dion). For all experiments, cells were preincubated with cortisol and subsequently incubated with [¹⁴C]T or [¹⁴C]4-AD as the substrate in medium without phenol red and with serum charcoal stripped of steroids. The results showed no aromatase activity in any of the cell lines under the experimental conditions used, and preincubation with cortisol had no effect on the enzyme activity. With [¹⁴C]T as the substrate, the metabolized level of DHT was very similar in the three cell lines, though MCF-7/LCC1 and MCF-7/LCC2 utilized the substrate to a much lesser extent. The amount of DHT and 4-AD produced were comparable in the two hormone-resistant cell lines, while the amount of 4-AD was significantly higher in MCF-7 cells. No differences in enzyme activity were found in the three cell lines when [¹⁴C]4-AD was used as the substrate. This study showed an altered androgen metabolism in the MCF-7/LCC1 and MCF-7/LCC2 sublines compared to the parent MCF-7. However, since treatment with DHT and T inhibited cell growth equally well in all three tumor cell lines, it is unlikely that the found differences in steroid metabolism was involved in the acquisition of the endocrine resistance of the two MCF-7 sublines.     

Oestradiol inhibits spontaneous and cisplatin-induced apoptosis in epithelial ovarian cancer cells: relationship to DNA repair capacity

A prospective role of sex steroid hormones in the pathogenesis of common epithelial ovarian cancer remains equivocal. We hypothesized that oestradiol can protect ovarian cells from apoptosis by augmenting their DNA repair capacity. Two established oestrogen receptor-positive human cancer cell lines of ovarian surface epithelial origin (OVCAR-3, SKOV-3) were studied during short-term (24 h) subculture in the absence or presence of oestradiol-17β and/or the DNA-damaging chemotherapeutic agent cisplatin. Apoptosis was monitored among individual cells by in situ DNA fragmentation analysis. Basal rates of apoptosis were diminished by exposure to oestradiol (progesterone or testosterone were without effect). Oestradiol also suppressed apoptosis induced by cisplatin and enhanced the repair of a cisplatin-damaged reporter chloramphenicol-O-acetyltransferase gene transfected into ovarian cells. The ability of oestrogen-responsive ovarian cancer cells to efficiently repair DNA and thereby avoid apoptosis may be related to propensity for clonal expansion and drug resistance. This revised version was published online in November 2006 with corrections to the Cover Date.     

Growth regulation of ovarian cancer cells by epidermal growth factor and transforming growth factors alpha and beta 1.

Regulation of ovarian cancer growth is poorly understood. In this study, the effects of EGF, TGF alpha and TGF beta 1 on two ovarian cancer cell lines (OVCAR-3 and CAOV-3) were investigated. The results showed that EGF/TGF alpha stimulated cell growth and DNA synthesis in OVCAR-3 cells, but inhibited cell proliferation and DNA synthesis in CAOV-3 cells. TGF beta 1 invariably inhibited cell proliferation and DNA synthesis in both cell lines. These effects on growth factors are dose dependent. The interaction of TGF beta 1 and EGF/TGF alpha was antagonistic in OVCAR-3 cells. In contrast, EGF/TGF alpha and TGF beta 1 had an additive inhibitory effect on CAOV-3 cells. Our results demonstrated that mature and functional EGF receptors are present in both cell lines and that they are capable of ligand binding, internalization, processing and ligand-enhanced autophosphorylation. Both high- and low-affinity binding are present in these cell lines, with CAOV-3 cells having about 2-3-fold higher total receptors than OVCAR-3 cells. These results together with those from our previous studies show that these cells express TGF alpha, TGF beta 1 and EGF receptors and that cell growth may be modulated by these growth factors in an autocrine and paracrine manner. This report presents evidence supporting the important roles of growth factors in ovarian cancer growth and provides a foundation for further study into the mechanism of growth regulation by growth factors in these cell lines.     

Characterization of two new enzymatic activities of the rat ventral prostate: 5 alpha-androstane-3 beta, 17 beta-diol 6 alpha-hydroxylase and 5 alpha-androstane-3 beta, 17 beta-diol 7 alpha-hydroxylase.

This study has characterized two new enzymatic hydroxylase activities specific for 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol) in the rat ventral prostate: 5 alpha-androstane-3 beta, 17 beta-diol 6 alpha-hydroxylase (6 alpha-hydroxylase) and 5 alpha-androstane-3 beta, 17 beta-diol 7 alpha-hydroxylase (7 alpha-hydroxylase). Both of these irreversible hydroxylase activities require NADPH and are localized in the microsomal fraction of the prostate. The apparent Km for 3 beta-diol is 2.5 microM for both the 6 alpha- and 7 alpha-hydroxylase activities. The apparent Km for NADPH is 7.6 microM for the 6 alpha-hydroxylase and 7.0 microM for the 7 alpha-hydroxylase. The pH optimum for both activities is 7.4. Several steroid inhibitors of these hydroxylase activities in vitro were identified including cholesterol, progesterone, and estradiol. Estradiol was found in vitro to be a noncompetitive inhibitor (Ki = 5 microM). Injection of estradiol into intact male rats, simultaneously receiving exogenous testosterone, also produced a significant lowering of the 6 alpha-plus 7 alpha-hydroxylase activities. Both the 6 alpha- and 7 alpha-hydroxylase were found to be androgen sensitive. Following castration there is a rapid decrease in both activities.     

Studies of the 3beta-hydroxysteroid oxidoreductase activity in rat ventral prostate

The reduction of ³H-androstanolone into 3β-androstanediol was used to study the 3β-hydroxysteroid oxidoreductase activity of rat ventral prostate. This activity is present only in the cytosol and has a pH optimum of 8.5 with NADH as cofactor. The 3β-hydroxysteroid oxidoreductase activity , as compared to 3α-hydroxysteroid oxidoreductase activity, is much lower in the present study than was observed previously during prostate perfusion $\text{in vivo}$ or prostate organ culture superfusion.     

The effects of steroid hormones and gonadotropins on in vitro placental conversion of pregnenolone to progesterone

Using human term placental mitochondrial preparations, optimal conversion of [3H]pregnenolone to [3H]progesterone was obtained at 30 min incubation and with a mitochondrial protein content of 2.5-3.5 mg/ml. Estradiol, estrone, progesterone and testosterone in a dose range of 0.03-8.66 mumol inhibited the in vitro conversion of [3H]pregnenolone to [3H]progesterone by placental homogenates. All four steroids inhibited the pregnenolone to progesterone conversion in a dose-dependent manner. The ID50 (dose required to inhibit conversion of pregnenolone to progesterone by 50%) was 0.04 mumol for estradiol, 0.13 mumol for testosterone, 0.3 mumol for progesterone and 1.0 mumol for estriol. Neither gonadotropin releasing hormone (50-1000 ng) nor human chorionic gonadotropin (5-500 IU) affected the placental basal conversion rate of pregnenolone to progesterone in vitro. Our findings indicate that steroid hormones such as estradiol, estrone, testosterone and progesterone can inhibit local placental progesterone biosynthesis through inhibition of the enzyme complex 5-ene-3 beta-hydroxysteroid dehydrogenase.     

Androgenic and estrogenic 17beta-hydroxysteroid dehydrogenase/17-ketosteroid reductase in human ovarian epithelial tumors: evidence for the type 1, 2 and 5 isoforms

17β-Hydroxysteroid dehydrogenase/17-ketosteroid reductases (17HSD/KSR) play a key role in regulating steroid receptor occupancy in normal tissues and tumors. Although 17HSD/KSR activity has been detected in ovarian epithelial tumors, our understanding of which isoforms are present and their potential for steroid metabolism is limited. In this investigation, 17HSD/KSR activity from a series of ovarian epithelial tumors was assayed in cytosol and microsomes under conditions which differentiate between isoforms. Inhibition studies were used to further characterize the steroid specificities of isoforms in the two subcellular fractions. Activity varied widely between tumors of the same histopathologic classification. The highest levels of activity were observed in mucinous tumors. Michaelis constants, maximum velocities, estradiol-17β/testosterone (E₂/T) activity ratios and inhibition patterns were consistent with a predominance of microsomal 17HSD/KSR2 and cytosolic 17HSD/KSR5, isoforms reactive with both E₂ and T, with evidence of estrogenic 17HSD/KSR1 in cytosol from some samples. In tumors where activity and mRNA expression were both characterized, Northern blots, PCR and sequence analysis indicated 17HSD/KSR5 was the predominant isoform. The presence of 17HSD/KSR5, which also has both 3-HSD/KSR and 20HSD/KSR activity, and 17HSD/KSR2 which also has 20-HSD activity, could influence not only estrogen and androgen binding but progesterone receptor occupancy, as well, in receptor-containing tumors.     

Steroid metabolism in granulosa and theca interna cells from preovulatory follicles of domestic hen (Gallus domesticus)

The capability of granulosa and theca interna cells, from preovulatory follicles of the domestic hen, to metabolize steroid precursors was evaluated. Granulosa and theca interna cells were isolated from ovarian preovulatory follicles at three different developmental stages: F1, F3 and F5. Tritiated pregnenolone (P5), progesterone (P4), dehydroepiandrosterone (DHEA), androstenedione (A4) and testosterone (T) were employed as precursors and their metabolic products were evaluated. The major metabolite of P5 by granulosa cells was P4, but we also observed low amounts of 5β-pregnandione. DHEA metabolism by granulosa cells yielded mainly A4, and minute quantities of 5β-androstan-3,17-dione (5β-dione) were detected. The only significant metabolite obtained in granulosa cells from A4 was 5β-dione, whereas T was only transformed into A4. On the other hand, P5 metabolism by theca interna cells yielded A4 as the main product, also P4, 17α-OHP4, 17α-OHP5, 5β-pregnandione, and DHEA, were found. When DHEA was the precursor A4 was produced in higher amounts than 5β-dione. A4 was mainly transformed into 5β-dione. In similar conditions, T was transformed into A4. These results show that granulosa cells have enzymatic activities of 3β-hydroxysteroid dehydrogenase/5-4 isomerase (3β-HSD from P5 and DHEA), 17β-hydroxysteroid dehydrogenase (17β-HSD from T) and 5β-reductase (from P5, DHEA and A4). Whereas theca interna cells have enzymatic activities of cytochrome P450c17 (from P5 and P4), 3β-HSD (from P5 and DHEA), 17β-HSD (from T) and 5β-reductase (from P4, DHEA and A4). These data support the concept that theca interna cells have the ability to synthesize androgens from progestins produced in granulosa cells. In addition, since theca interna cells did not show the capacity to aromatize androgens suggests that interaction between theca interna and theca externa cells occurs in vivo, thus confirming the three cell model for estrogen production. Furthermore, the fact that other metabolites were produced both in granulosa and theca interna cells, but in a different extent, suggests that complex mechanisms are participating in the regulation of steroid synthesis in avian ovary follicles.     

Reactivity of the epithelial cells of the bovine oviduct in vitro on the exogenic gonadotropic and steroid hormones. Part I: The effect of gonadotropic and steroid hormones on the amount of lipids and activity of dehydrogenases.

Effects of 17 beta-estradiol, testosterone, progesterone, luteinizing hormone and a combined effect of estradiol and progesterone on the epithelial cells of the bovine oviduct cultured in vitro were investigated. It was found that these cells may transform under the effect of hormones. The effect of the applied hormones on the amount of lipids and activity of the dehydrogenases delta 5 3 beta-OH-SDH and G6P-DH was evident. Cells in vitro most intensely reacted on testosterone and estradiol: these hormones caused an increase of lipids and of enzymatic activity. The cells also reacted to progesterone and the luteinizing hormone which in turn decreased both activity and accumulation of lipids in cells.     

The subcellular localization of 17β-hydroxysteroid dehydrogenase type 4 and its interaction with actin

The porcine 17β-hydroxysteroid dehydrogenase type 4 is the key enzyme for the inactivation of estradiol. Its localization in peroxisomes was proven by immunogold electron microscopy. Interactions of the 17β-hydroxysteroid dehydrogenase with cytoskeletal proteins might be mandatory for a topical assignment of enzymatic activity to defined subcellular compartments.     

Curcumin derivatives inhibit testicular 17β-hydroxysteroid dehydrogenase 3

Non-steroidal compounds that inhibit 17β-hydroxysteroid dehydrogenase isoform 3 (17β-HSD3), an enzyme catalyzing the final step in testosterone biosynthesis in Leydig cells, are under development for male contraceptive or treatment of androgen dependent diseases including prostate cancer. A series of curcumin analogues with more stable chemical structures were compared to curcumin as inhibitors of 17β-HSD3 in rat intact Leydig cells as well as rat and human testis microsomes.