Department of Physiology, St George's Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: estradiol/genistein/human granulosa luteal cells/progesterone/tyrphostin
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
An alternative target for the action of genistein is on steroidogenic enzymes, and in this respect genistein has been shown to inhibit the reduction of estrone to the more active estradiol by inhibiting 17ß-hydroxysteroid dehydrogenase (17ß-HSD) type 1 (Mäkelä et al., 1995). It could therefore exert anti-estrogenic effects by inhibiting the production of the most potent estrogen, estradiol. More recently, it has been reported that genistein can inhibit the reductive/oxidative activity of 17ß-HSD type 5, thereby inhibiting the conversion of androstenedione to testosterone and androstenediol to androsterone respectively (Krazeisen et al., 2001
). Since both testosterone and androstenedione are substrates for the action of aromatase, which converts testosterone to estradiol and androstenedione to estrone, genistein may also affect the local conversion of these androgen precursors to the active estradiol or estrone. Indeed, it has been shown that genistein has a weak inhibitory activity on aromatase (Pelissero et al., 1996
).
Genistein is, however, also a potent inhibitor of tyrosine kinase activity (Akiyama et al., 1987). Whilst the classical stimulatory pathway for steroidogenesis is via a cyclic AMP-stimulated signalling system, there is evidence of cross talk between adenylate cyclase- and tyrosine kinase (TK)-dependent signalling pathways in the control of steroidogenesis (Andreis et al., 2000
).
Recent experiments in our laboratory, concerning the effects of a variety of endocrine disrupting chemicals on steroidogenesis, have shown that genistein and the non-estrogenic TK inhibitor lavendustin A can potently and dose-dependently inhibit progesterone production in cultured rat granulosa luteal (GL) cells (Whitehead and Lacey, 2000). In light of these studies, and the evidence that local conversion of relatively inactive steroid metabolites to the highly active estradiol, may be important in the aetiology of breast cancer (Labrie et al., 2000
), we hypothesized that the protective effect of genistein on breast cancer could be exerted by inhibiting the activity of the enzymes that convert steroid precursors to estradiol. We further hypothesized that such effects were induced by the inhibitory effect of genistein on protein TKs rather than its ability to bind weakly to estrogen receptors. Thus the present studies were undertaken to compare the effects of three TK inhibitorsgenistein, lavendustin A and tyrphostin A23on progesterone and estradiol production in human GL cells. Subsequently, we aimed to identify which enzymes might be targeted by genistein and tyrphostin A23 by using pregnenolone and androstenedione, testosterone or estrone as substrates for progesterone and estradiol synthesis respectively.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The second series of experiments were carried out to investigate which steroidogenic enzymes might be affected by TK inhibitors by exposing the cultures to steroid substratespregnenolone, androstenedione, estrone or testeronefor 4 h at the end of two culture periods with subsequent measurement of progesterone or estradiol.
GL cells were cultured for 48 h with fetal calf serum, for 24 h in serum-free medium but in the presence of hFSH or rHCG and, after changing the medium, for a further 4 h in the presence of drugs and the appropriate steroid substrate. After removal of the medium for the first steroid measurements, cells were washed and cultured for a further 24 h in the presence of the same TK inhibitors. Subsequently, in fresh medium, cells were exposed to the same steroid substrates for a final 4 h to determine enzyme activity. In this way the same samples could be used to investigate the possible acute action of TK inhibitors on steroidogenic enzymes as well as their `chronic' effects over a 24 h period.
At the end of all experiments, media samples were taken and stored at 20°C and viability of cells was tested either with the Trypan Blue exclusion test or by measuring cellular dehydrogenases with methyl thiazolyl tetrazolium (MTT) (Shakil and Whitehead, 1994).
Drugs and chemicals
The following drugs were used in these experiments: androstenedione, genistein (4',5,7-trihydroxyisoflavone), lavendustin A (5-amino-[N-2,5-dihydroxybenzyl-N'-2-hydroxybenzyl] salicylic acid), estrone, pregnenolone, testosterone, tyrphostin A23 ([3,4-dihydroxybenzylidene] malono-nitrile, -cyano-[3,4-dihydroxy] cinnamonitrile), hFSH and rHCG. With the exception of hFSH and rHCG, kindly supplied by The National Hormone and Pituitary Agency, Torrance, CA, USA, and lavendustin A and tyrphostin A 23 supplied by the Alexis Corporation, Nottingham, UK, all other drugs, chemicals and culture media were supplied by Sigma, Dorset, UK.
Steroids, genistein and tyrphostin A23 were initially dissolved in ethanol (methanol for lavendustin A) and diluted appropriately with culture medium before they were stored as stock solutions. Stock solutions were then diluted appropriately with medium and added to cultures in 10 µl volumes to give the desired final concentration. Controls were performed to ensure that the maximum volume (1 µl) of ethanol/methanol diluent did not affect cellular responses.
Steroid assays
Progesterone and estradiol concentrations in the culture medium were measured in duplicate by direct radioimmunoassay kits (ICN Pharmaceuticals Ltd, Basingstoke, UK) according to the manufacturer's instructions. All drugs used in these experiments were tested for their possible cross reactivity with the anti-sera used, but with the exception of estrone, none was detected. The cross reactivity of the progesterone anti-serum with 20-dihydroprogesterone, 17
-hydroxyprogesterone and pregnenolone was 5.4, 0.6 and 0.4% respectively and the estradiol anti-serum with estrone, estriol, progesterone and testosterone was 20, 1.5, <0.01 and <0.01%. Inter- and intra-assay coefficients of variation were 6.2 and 3.0% for the progesterone assay and 9.1 and 5.2% for the estradiol assay respectively. In view of the high cross reactivity of the estradiol antiserum with estrone, in each experiment 10-7 mol/l estrone was incubated in culture medium without cells for 4 h. The mean concentration of estradiol in the `blank' triplicate wells was subsequently subtracted from the estradiol concentrations measured in the cell cultures that had been exposed to 10-7 mol/l estrone.
Statistical analysis
Data shown represent mean ± SEM of triplicate cultures obtained from at least three independent experiments and n = the total number of observations. Comparisons were only made with paired controls within each experiment and doseresponses were obtained in the same experiments. This was to ensure that inter-experimental variability did not bias the analysis. Statistical differences in the doseresponses to the different drugs were compared with an analysis of variance followed by Gabriel's test, which is suitable for groups of unequal size. A Student's t-test was used to test significance between two groups of data.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Genistein, lavendustin A and tyrphostin A23 all significantly inhibited basal and rHCG-induced progesterone synthesis when GL cells were exposed to these drugs over a 48 h period (Figure 1AC). Whilst significant stimulation of progesterone production by rHCG alone was seen in those experiments investigating the effects of genistein and, to a lesser extent, lavendustin A, no effect of this gonadotrophin was seen in the experiments using tyrphostin A23. There is no obvious explanation for this discrepancy, except that the initial experiments comparing the action of genistein with lavendustin A were carried out on GL cells obtained exclusively from St George's Hospital. However, the inhibitory effects of all these compounds were dose-dependent, though genistein induced a significant inhibition at 1 µmol/l, lavendustin A at 1050 µmol/l and tyrphostin A23 at 50100 µmol/l. Since both lavendustin A and tyrphostin A23 had similar effects on progesterone production and tyrphostin has been more widely used to investigate the effects of TK inhibition on follicular steroidogenesis, our subsequent experiments only compared the effects of genistein and tyrphostin A23 on steroidogenesis.
|
|
|
|
Routine monitoring of cell viability showed that neither lavendustin A, genistein or tyrphostin A23 had any effects on cell viability compared with controls (Table I) and thus even the change in effect of tyrphostin A23 from stimulatory to inhibitory that occurred between the short-term (4 h) and long-term (48 h) exposure of GL cells to this drug could not be attributed to loss of cell viability.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
After a 24 h exposure of GL cells to genistein there was a generalized inhibition of enzyme activity (Figure 4b), as was seen after 48 h. These results suggest that genistein may have a direct, acute effect on the activity of specific enzymes in the steroidogenic pathway, but that inhibition of other enzymes requires suppression of enzyme expression or other genomic effects.
The time course of the effects of tyrphostin A23 were very different. An acute 4 h exposure of this drug tended to reduce progesterone synthesis, but stimulated estradiol production when androstenedione or testosterone were used as substrates. No effects were observed in the presence of estrone (Figure 4a,b). These data suggest that tyrphostin might stimulate the activity of aromatase. After GL cells had been exposed to tyrphostin A23 for 24 h, all steroidogenic responses were similar to controls and it was not until cells has been exposed to this inhibitor for 48 h that both progesterone and estradiol were inhibited.
The present results agree with our recent studies on the effects of genistein and lavendustin A on progesterone production in rat granulosa cells (Whitehead and Lacey, 2000), although in these studies acute effects on enzyme activity were not investigated. Similarly Haynes-Johnson et al., found that the TK inhibitor RG 50810 and genistein inhibited FSH-stimulated estradiol and progesterone production in rat granulosa cells (Haynes-Johnson et al., 1999
). Gregoraszczuk et al., also reported that genistein, tyrphostin and herbimycin inhibited prolactin-stimulated progesterone production by porcine thecal and luteal cells, tyrphostin being least potent in this respect (Gregoraszczuk et al., 1999
). In fetal and post-natal human adrenal cortical cells, genistein and another phytoestrogen, diadzein, suppressed cortisol synthesis but not androgen synthesis (Mesiano et al., 1999
). There was no reduction in the expression of any steroidogenic enzyme, only a reduced activity of 21-hydroxylase.
In line with the current experiments, Mäkelä et al., reported that genistein, as well as several other phytoestrogens, including apigenin and coumesterol, inhibited 17ß-HSD type 1 (Mäkelä et al., 1995). This may have important clinical implications. Expression of 17ß-HSD type 1 has not only been found in the human ovary, but also in breast tissue, and its expression is sometimes high in the stromal cells of malignant tissue (Poutanen et al., l995
). The growth-promoting effects of estradiol on breast cancer cells can only be mimicked by estrone when cells are cultured in the presence of 17ß-HSD type 1, but not in the absence of this enzyme (Miettinen et al., 1996
). It is possible that the inhibition of this enzyme by genistein could explain the association between dietary soy intake and the lower incidence of breast cancer.
Another enzyme which has been shown to be targeted by genistein is 17-ßHSD type 5, which reduces androstenedione to testosterone. Krazeisen et al., demonstrated that, in a cell-free system, genistein inhibited the activity of recombinant human 17-ßHSD type 5 with an IC50 >20 µmol/l (Krazeisenet al., 2001). However, they also found that a wide range of other phytoestrogens inhibited the activity of this enzyme, many of them at lower IC50 values. For example, biochanin A and coumesterol were inhibitory at <15 µmol/l and zearalenone at <5 µmol/l. Thus, genistein had relatively weak inhibitory activity in this model. The lack of any acute effects of genistein on the conversion of androstenedione to estradiol observed in the present experiments could be explained by the relatively low dose of genistein (50 µmol/l) used in a cell culture that could convert genistein to inactive metabolites.
There is increasing evidence that TKs can interact with other cell signalling pathways and in this way alter the activity/expression of steroidogenic enzymes. For example tyrphostins, as well as phytoestrogens such as genistein, can inhibit the degradation of cAMP by inhibiting certain phosphodiesterase isozymes (Nichols and Morimoto, 2000) and Andreis et al., demonstrated that tyrphostin A23 enhanced basal and ACTH-induced steroid secretion from dispersed human and rat adrenocortical cells (Andreis et al., 2000
). Their results suggested that this effect was mediated by TK inhibition of phosphdiesterase and they concluded that the observed increase in intracellular cAMP concentrations increased PKA activation and hence steroid hormone production. Along a similar line of reasoning, the acute stimulatory effects of tyrphostin A23 seen in these experiments could be due to increased FSH-induced cAMP accumulation in the GL cells. It would appear that residual stimulatory effects are also observed after a 24 h exposure of GL cells to tyrphostin A23 (Figure 4b
) but by 48 h, only inhibition was observed (Figure 2B
).
Overall, the present study shows that TK inhibitors suppress steroidogenesis in human GL cells, although it has not addressed the mechanism of such suppression. There is clearly a divergence between the acute effects of genistein and tyrphostin A23, but when cells are exposed to these drugs for a period of 48 h, both progesterone and estradiol production are dose-dependently inhibited. Such parallel effects of three different TK inhibitors on steroidogenesis suggest that TKs are important in the expression of steroidogenic enzymes. The observed acute effects of these drugs suggest a direct inhibition of 3ß-HSD and 17ß-HSD type 1 activity by genistein, but an increase of aromatase activity by tyrphostin A23, perhaps mediated by sustaining increased intracellular concentrations of cAMP induced by FSH. Whether or not any of the effects of genistein are mediated by its ability to bind to estrogen receptors remains to be determined. In this respect, there is emerging evidence that genistein may act on transcriptional processes and can even alter proliferation of cells which do not express estrogen receptors (Barnes et al., 2000).
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Andreis, P.G., Neri, G., Tortorella, C., Gottardo, L. and Nussdorfer, GG. (2000) Tyrphostin-23 enhances steroid hormone secretion from dispersed human and rat adrenocortical cells. Endocrinol. Res., 26, 319332.[ISI][Medline]
Awoniyi, C.A., Roberts, D., Veeramachaneni, D.N. and Hurst, B.S. (1998) Reproductive sequelae in female rats after in utero and neonatal exposure to the phytoestrogen genistein. Fertil. Steril., 70, 440447.[ISI][Medline]
Barnes, S., Kim, H., Darley-Usmar, V., Patel, R., Xu, J., Lavalleur, J., Phipps, W.R. and Kurzer, M.S. (2000) Beyond ER and ERß: estrogen receptor binding is only part of the isoflavone story. J. Nutr., 130, 656S657S.[ISI][Medline]
Dixon-Shanies, D. and Shaikh, N. (1999) Growth inhibition of human breast cancer cells by herbs and phytoestrogens. Oncol. Rep., 6, 13831387.[ISI][Medline]
Duncan, A.M., Underhill, K.E., Xu, X. Lavalleur, J., Phipps, W.R. and Kurzer, M.S. (1999) Modest hormonal effects of soy isoflavones in postmenopausal women. J. Clin. Endocrinol. Metab., 84, 34793484.
Gregoraszczuk, E., Slomczynska, M. and Stoklosowa, S. (1999) Effect of genistein, tyrphostin and herbimycin on prolactin-stimulated progesterone reproduction by procine thecal and luteal cells. J. Physiol. Pharmacol., 50, 477484.[ISI][Medline]
Haynes-Johnson, D., Lai, M.T., Campen, C. and Palmer, S. (1999) Diverse effects of tyrosine kinase inhibitors on follicle-stimulating hormone-stimulated estradiol and progesterone production from rat granulosa cells in serum-containing epidermal growth factor. Biol. Reprod., 61, 147153.
Knight, D.C. and Eden, J.A. (1996) A review of the clinical effects of phytoestrogens. Obstet. Gynecol., 87, 897904.
Krazeisen, A., Breitling, R., Möller, G. and Adamski, J. (2001) Phytoestrogens inhibit human 17ß-hydroxysteroid dehydrogenase type 5. Mol. Cell. Endocrinol., 171, 151162.[ISI][Medline]
Kuiper, G.G.J.M., Lemmen, J.G., Carlson, B., Corton J.C., Safe, S.H., van der Saag, P.T., van der Burg, B. and Gustafsson, J.A. (1998) Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor ß. Endocrinology, 139, 42524563.
Kurzer, M.S. and Xu, X. (1997) Dietary phytoestrogens. Ann. Rev. Nutr., 17, 353381.[ISI][Medline]
Labrie, F., Luu-The, V., Lin, S.X., Simard, J., Labrie, C., El-Alfy, M., Pelletier, G. and Bélanger, A. (2000) Intracrinology: role of the family of 17ß-hydroxysteroid dehydrogenases in human physiology and disease. J. Mol. Endocrinol., 25, 116.
Mäkelä, S., Poutanen, M., Lehtimäki, J., Kostain, M.L., Santti, R. and Vihko, R. (1995) Estrogen specific 17ß-hydroxysteroid oxidoreductase type 1 (E.C.1.1.1.62) as a possible target for the action of phytoestrogens. Proc. Soc. Exp. Biol. Med., 208, 5159.[Abstract]
Mesiano, S., Katz, S.L., Lee, J.Y. and Jaffe, R.B. (1999) Phytoestrogens alter adrenocortical functions: genistein and daidzein suppress glucocorticoid and stimulate androgen production by cultured adrenal cortical cells. J. Clin. Endocrinol. Metab., 84, 24432448.
Miettinen, M., Poutanen, M.H. and Vihko, R.K. (1996) Characterization of estrogen-dependent growth of cultured MCF-7 human breast cancer cells expressing 17ß-hydroxysteroid dehydrogenase type 1. Int. J. Cancer, 68, 600604.[ISI][Medline]
Miksicek, R.J. (1995) Estrogenic flavonoids: structural requirements for biological activity. Proc. Soc. Exp. Biol. Med., 208, 4450.[Abstract]
Morito, K., Hirose, T., Kinjo, J., Hirakawa, T., Okawa, S., Inoue, S., Mura, M. and Masamune, Y. (2001) Interaction of phytoestrogens with estrogen receptors and ß. Biol. Pharm. Bull., 24, 351356.[ISI][Medline]
Nichols, M.R. and Morimoto, B.H. (2000) Differential inhibition of multiple cAMP phosphodiesterase isozymes by isoflavones and tyrphostins. Mol. Pharmacol., 57, 738745.
Pelissero, C., Lenczowski, M.J. Chinzi, D., Davail-Cuisset, B., Sumpter, J.P. and Fostier, A. (1996) Effects of flavonoids on aromatase activity, an in vitro study. J. Steroid. Biochem. Mol. Biol., 57, 215223.[ISI][Medline]
Poutanen, M., Isomaa, V., Peltoketo, H. and Vihko, R. (1995) Role of 17ß-hydroxysteroid dehydrogenase type 1 in endocrine and intracrine estradiol biosynthesis. J. Steroid Biochem. Mol. Biol., 55, 525532.[ISI][Medline]
Shakil, T. and Whitehead, S.A. (1994). Inhibitory action of peritoneal macrophages on progesterone secretion from co-cultured rat granulosa cells. Biol. Reprod., 50, 11831189.[Abstract]
Wang, C. and Kurzer, M.S. (1998) Effects of phytoestrogens on DNA synthesis in MCF- 7 cells in the presence of estradiol or growth factors. Nutr. Cancer, 31, 90100.[ISI][Medline]
Whitehead, S.A. and Lacey, M. (2000) Protein tyrosine kinase activity of the phytoestrogen genistein and lavendustin A on progesterone synthesis in cultured ovarian cells of the rat. Fertil. Steril., 73, 613619.[ISI][Medline]
Submitted on July 20, 2001; accepted on October 31, 2001.