Laura Berta

The receptor-mediated action of sex steroid binding protein (SBP, SHBG): Accumulation of cAMP in MCF-7 cells under SBP and estradiol treatment

The interaction of sex steroid binding protein (SBP) with its specific receptor in MCF-7 cell (estrogen-sensitive human breast cancer cells), followed by the binding of estradiol (E2) to the complex SBP-receptor, induced a significant accumulation of intracellular cAMP. SBP alone as well as E2 alone did not elicit any modification of the nucleotide. The maximal increase in cAMP was observed with 1 nM SBP + 1 nM E2. Increasing doses of both SBP and E2, even raising cAMP levels with respect to basal, did not give any higher response. Both testosterone and dihydrotestosterone, used instead of E2, were not able to induce any significant modification of cAMP. E2-induced MCF-7 cell proliferation was significantly reduced by 8Br-cAMP. MDA-MB 231 cells (estrogen-insensitive breast cancer cells) were not shown to bind SBP, or to respond to SBP + E2 as far as both their proliferation and cAMP content are concerned. In summary, the present study provides evidence that the SBP receptor is part of the G-protein receptor family, and that SBP can act as modulator of E2 action at cell site through the second messenger cAMP.

Extracorporeal shock waves enhance normal fibroblast proliferation in vitro and activate mRNA expression for TGF-beta1 and for collagen types I and III.

Background and purpose Extracorporeal shock waves (ESWs) are used to good effect in the treatment of soft tissue injuries, but the underlying mechanisms are still unknown. We therefore determined the effects of ESWs on normal fibroblasts in vitro, in order to assess treatment-induced cell response. Methods A normal human fibroblast cell line (NHDF-12519) was treated with ESWs generated by a piezoelectric device (Piezoson 100; Richard Wolfe) using different protocols of impulses (300, 1,000, or 2,000 shots) and energy (0.11 or 0.22 mJ/mm2). Untreated controls and treated cells were cultivated for 12 days following a single shock-wave treatment. Viability, growth rate, and expression of mRNA for TGFβ -1 and collagen types I and III were evaluated at days 3, 6, 9, and 12. Results 1 hour after shock-wave treatment, cell viability showed a decrease related mainly to impulse numbers applied. Fibroblasts treated with energy of 0.22 mJ/mm2 subsequently showed an increase in proliferation from day 6 to day 9 that was higher than in untreated controls, without interference with the normal cell kinetic profile. mRNA expression was also higher in treated fibroblasts than in untreated controls for TGFβ -1 on day 6 and day 9, for collagen type I on day 6, and for collagen type III on day 9. Interpretation These in vitro data confirm that the main factors involved in the repair process of connective tissues are activated by ESWs. The study gives the rationale for, and may provide schedules for, ESW treatment of tendonopathies.

Altered expression of androgen-receptor isoforms in human colon-cancer tissues

Many groups have examined of androgen the effects on normal and neoplastic colon tissues, but no clear picture has hitherto emerged. In particular, the presence and the function of the androgen receptor (AR) has only partially been investigated in the past. The present study reports analysis of expression of the AR gene as messenger RNA and as protein in surgical samples of neoplastic colon mucosa and of corresponding healthy surrounding tissue. Specific binding for DHT, demonstrating the presence of AR, was observed in almost all the samples (2 samples out of 12 were negative). No significant difference was observed between healthy and neoplastic mucosa, or between male and female patients. A further characterization of AR was performed with Western blot, using 2 different primary antibodies. Both AR isoforms, AR‐B and AR‐A, were detected in healthy mucosa, while only AR‐A, resolving at 87 kDa, was observed in neoplastic mucosa. RT‐PCR analysis revealed the transcript for AR in both healthy and neoplastic mucosa in 10 samples; no message was detectable in 2 samples (negative also for binding); 2 additional samples presented AR mRNA only in healthy colon mucosa, 2 others only in neoplastic mucosa. In addition, a variant AR messenger RNA, probabily derived from alternative splicing, was observed. We found that AR is expressed both in healthy and in neoplastic colon mucosa, either as mRNA or as protein. Neoplastic colon tissue shows a characteristic loss of expression of the AR‐B isoform, while AR‐A expression is maintained. These findings underscore the possible role of androgen and its receptor in colon carcinogenesis. Int. J. Cancer 86:325–330, 2000.

Sex steroid binding protein (SBP) receptors in estrogen sensitive tissues

Since the discovery of a specific membrane binding site for sex steroid binding protein (SBP) in human decidual endometrium and in hyperplastic prostate numerous speculations have been raised on the existence of an additional non-receptor-mediated system for steroid hormone action. In the present work SBP cell membrane binding was investigated in human estrogen target tissues other than those previously studied either in the absence of steroids or in the presence of varying amounts (10(-10)-10(-6) M) of estradiol, testosterone and dihydrotestosterone, respectively. Plasma membranes obtained by differential centrifugation from homogenized samples of pre-menopausal endometrium, endometrium adenocarcinoma, normal liver and post-menopausal breast showed a specific binding of highly purified [125I]SBP: a major displacement of labeled SBP was elicited by radioinert SBP, while no significant displacement occurred when other human plasma proteins were used as cold competitors (molar excess ranging 500-10,000-fold). A specific, time-dependent binding of [125I]SBP was also observed in MCF-7 and in Hep-G2 cell lines. The different patterns of specific binding, observed in membranes from different tissues when SBP was liganded with different sex steroid molecules, leads us to consider the tissue individuality of the receptor as a further entity in the membrane recognition system for SBP.

Estradiol induction of cAMP in breast cancer cells is mediated by foetal calf serum (FCS) and sex hormone-binding globulin (SHBG).

Plasma sex hormone-binding globulin (SHBG or SBP), the specific carrier for estradiol and androgens, after binding to its membrane receptor (SHBG-R), causes a significant increase of cAMP in the presence of estradiol, in both breast (MCF-7) and prostate (LNCaP) cancer cells maintained in serum-free medium. On the other hand, it has been proposed that estrogens, in addition to the well-known nuclear receptor pathway, exert their biological effect inducing cAMP, as a consequence of a direct membrane action, in breast cancer and uterine cells. The aim of the present study was to clarify this controversial issue by verifying if the cAMP increase in MCF-7 cells was a direct effect of estradiol, or if it was mediated by FCS proteins, such as bovine sex hormone-binding globulin; and to reevaluate the effect of human SHBG on cAMP induction in the presence of FCS. MCF-7 cells were maintained in DCC-FCS (treated with DCC to remove steroids), in SHBG-FREE/DCC-FCS (treated with DCC and with a specific affinity chromatography to remove bovine sex hormone-binding globulin), or in serum-free medium (SFM). It was observed that estradiol determined a significant time-dependent increase of cAMP only in MCF-7 cells maintained in 10% DCC-FCS. When cells were maintained in 10% SHBG-FREE/DCC-FCS, estradiol had no detectable effect. However, its ability to increase cAMP was observed again after the addition of human SHBG, in doses ranging from 5 to 50 nM. Moreover, in the presence of 10% SHBG-FREE/DCC-FCS, SHBG, even in the absence of estradiol, caused a significant increase of cAMP. In conclusion, the data reported in the present study suggest that the ability of estradiol to induce cAMP in MCF-7 cells is not due to a direct membrane effect of the hormone, but rather it is mediated by FCS. SHBG is one of the serum factors mediating estradiol action. Lastly, it was proven that SHBG triggers the cAMP pathway in MCF-7 cells in a physiologic culture condition and at physiologic concentrations.

Sex steroid-binding protein interacts with a specific receptor on human premenopausal endometrium membrane: modulating effect of estradiol☆

Sex steroid-binding protein receptor was detected on membranes prepared from human premenopausal endometrium. The binding of sex steroid-binding protein to membranes was specific, saturable, and high affinity. Scatchard analysis showed the presence of two binding sites at different affinities. The addition of estradiol (10(-8) M) did not produce any inhibition of binding; indeed, it resulted in a modification of binding characteristics. The demonstration of sex steroid-binding protein receptor on membranes of human premenopausal endometrium indicates that the expression of receptor on membranes is not an effect of estrogen over stimulation on target tissues. Estradiol could act as a modulating factor of the binding, probably reflecting the sensitivity of tissues to different steroids.

Smoking Effects on the Hormonal Balance of Fertile Women

We evaluated serum pituitary hormones (prolactin, follicle-stimulating hormone, luteinizing hormone), gonadal hormones (estrone, estradiol, progesterone), sex steroid binding protein (SBP) and urine estrogens in 684 healthy fertile women, subdivided into smokers (n = 237) and nonsmokers (n = 447). The aim of the work was to elucidate whether smoking habits can affect hormonal balance. Smoking interference of estrogen metabolism has been postulated, but no unequivocal data have been reported. A protective role against breast cancer has even been suggested on the basis of a reduced estrogenic activity found in smokers. Our data showed a considerable interference of smoking on PRL secretion, probably related to a direct inhibiting activity of nicotine. Estrogen catabolism could also be involved, and a catabolic shift of 16 alpha-hydroxylation in favour of 2 alpha-hydroxylated catabolites, via the hepatic cytochrome P-450 system could be hypothesized.

The membrane receptor for sex steroid binding protein is not ubiquitous

The tissue distribution of the membrane receptor for the Sex Steroid Binding Protein (SBP) has been studied, either in estrogen/androgen dependent tissues and in tissues not strictly sex steroid dependent. A specific interaction of SBP with cell mem-branes has been observed to occur only in estro gen/androgen dependent tissues, some of them had been previously shown by our laboratory and by other authors to possess a specific receptor for the protein. Thus, the sex steroid dependence of the tissue is likely to be determinant for the expression of the membrane receptor for Sex Steroid Binding Protein.

High energy shock waves (HESW) enhance paclitaxel cytotoxicity in MCF-7 cells.

High energy shock waves (HESW) produced by a piezoelectric generator were studied for their effect on human breast cancer cell (MCF-7) viability and sensitivity to paclitaxel. A dose-dependent impairment of cell viability was observed after HESW treatment (250–2000 shock waves, rate = 4/s, energy flux density = 0.25 mJ/mm2). Single treatment with shock waves produced no significant growth inhibition. Combined exposure to paclitaxel (ranging 0.1 nM to 20µM) and shock waves (100, 500 and 1000 shots, respectively) resulted in a significant reduction of MCF-7 cell proliferation at day 3 after treatment in respect with cells treated with paclitaxel alone. Notably, a cell viability reduction of about 50% was obtained after combined treatment with HESW and 10 nM paclitaxel, in front of a reduction of only 40% using 10 µM paclitaxel alone. Moreover, an earlier induction as well as an enhancement of apoptotis was observed in cells subjected to combined treatment with shock waves and paclitaxel (200 nM; 20 µM). In conclusion, HESW can enhance paclitaxel cytotoxicity in MCF-7 cells, thus allowing the treatment with lower doses of drug.

The receptor for human sex steroid binding protein (SBP) is expressed on membranes of neoplastic endometrium.

Sex steroid binding protein (SBP) receptor was detected on cell membranes obtained from human endometrium adenocarcinoma. The binding of SBP was proved to be highly specific, saturable, and at high affinity. It was, additionally, shown to occur at two sites at different affinities, as previously described for other human tissues. SBP was, therefore, demonstrated to recognized a specific receptor on endometrium adenocarcinoma membranes. The effect of steroid hormones on SBP-receptor interaction was also evaluated. Both dihydrotestosterone and estradiol were shown to inhibit the binding of SBP to its specific receptor on neoplastic membranes. Testosterone at a dose of 10(-9) M was shown not to interfere to a significant extent with SBP-receptor binding. The sensitivity for estradiol we had previously observed in normal premenopausal endometrium was completely lost in postmenopausal neoplastic tissue. These observations suggest that the SBP-membrane recognition system is still present in neoplastic postmenopausal endometrium, but it has been modified either by the postmenopausal endogenous milieu or by the neoplastic transformation.