RU486, a progestin antagonist, binds to progesterone receptors in a human endometrial cancer cell line and reverses the growth inhibition by progestins

The human endometrial cancer cell line, IK-90 cells, contains estrogen-independent progesterone receptors (PR) and is progestin sensitive. Accumulation of glycogen in the cytoplasm of IK-90 cells as well as growth inhibition of the cells in response to progestins are observed. In the present study, the effects of RU486, a progestin antagonist, on IK-90 cells were investigated in a serum-supplemented medium. Scatchard plot analysis of cytoplasmic binding data in the cells revealed a high affinity binding site for RU486 (Kd, 2.6 nM) with maximum binding sites of 169 fmol/mg protein. However, the binding ability to DNA-cellulose of heat activated [3H]RU486-PR complexes was lower when compared with that of the progestin agonist [3H]R5020-PR complexes, suggesting a decrease in progestin activity of RU486 in IK-90 cells. The addition of 1 microM RU486 to culture medium produced periodic acid-Schiff-positive granules in the cytoplasm of the cells. On the other hand, RU486 (1 nM-1 microM) did not significantly inhibit the growth of cells. However, RU486 (0.1-1 microM) totally prevented the growth-inhibitory effect of R5020 (0.1-1 microM) on IK-90 cells. In conclusion, RU486, an antiprogestin, had a dual activity both a progestin antagonist and weak agonist in human endometrial cancer cells, which was not mediated through the estrogen receptor system.     

Roles of antiestrogen binding sites in human endometrial cancer cells

Estrogen-noncompatible antiestrogen binding sites (AEBS) as well as estrogen receptors (ER), and the growth-inhibitory effect of tamoxifen were investigated in two human endometrial cancer cell lines, IK-90 and HEC-IA cells. IK-90 cells contained specific AEBS, but no ER was found in these cells. Scatchard plot analysis of AEBS in 12,000 g supernatant from IK-90 cells showed a high affinity binding site for tamoxifen (Kd:5.6 +/- 1.0 nM) with the maximum binding site of 457 +/- 47 fmol/mg protein. However, no measurable ER or AEBS was found in HEC-IA cells. The effect of tamoxifen on the growth of cells was found to be identical in both cell lines; the addition of 10 microM tamoxifen to culture medium was cytocidal whereas tamoxifen at lower concentrations (1 nM-1 microM) did not significantly affect the growth of both IK-90 and HEC-IA cells. These results demonstrate for the first time the presence of AEBS in human endometrial cancer cells. The present results also suggest that AEBS does not play a fundamental role in mediating the growth-inhibitory effect of tamoxifen in endometrial cancer cells.     

Inhibitory effects of danazol and medroxyprogesterone acetate on [3H]thymidine incorporation in human endometrial cancer cells

Effects of medroxyprogesterone acetate (MPA) and danazol (1 nM-10 microM) on cultured cancer cells from human endometrial adenocarcinomas obtained by hysterectomy were simultaneously investigated. Of twenty-four endometrial adenocarcinomas examined, five tumors were successfully maintained in primary cell culture. The addition of MPA as well as danazol in culture of cells from the five tumors resulted in a significant inhibition of [3H]thymidine incorporation in cancer cells from three tumors having progesterone receptors (PR). The minimum effective concentrations of MPA and danazol for the inhibition of [3H]thymidine incorporation were found to be 10 and 100 nM, respectively. The difference in effective concentration could be explained by a higher affinity of MPA to PR than that of danazol in cancer cells. On the other hand, neither danazol nor MPA affected [3H]thymidine incorporation in cultured cells from the remaining two tumors, in which PR was absent in one but present in the other. These findings, together with our previous findings that danazol inhibited the growth of a human endometrial cancer cell line with PR, suggest that a growth-inhibitory effect of danazol on human endometrial cancer cells is mediated through PR in the cells.     

Responsiveness and mechanisms of action of steroid hormones in human endometrial adenocarcinoma cells

The responsiveness and action mechanisms of steroid hormones and epidermal growth factor on human endometrial carcinoma cells are analyzed by using in vitro culture system. 1) The Ishikawa cells, derived from a well differentiated endometrial adenocarcinoma and possess ER and PR, are shown to respond to estrogens by increasing a variety of parameters, viz cell proliferation, PR levels, ALP and DNA polymerase activities. 2) ER and PR of those cells are localized in the nuclei by immunocytochemical staining using the monoclonal antibodies against to ER and PR, confirming the correctness of Gorski and Greene's one step theory involving the action mechanisms of steroid hormones. 3) Progestins reduced the ER level and stimulate E2DH activities and glycogen content, which are completely abolished by anti-progestin (RU486), suggesting that PR of those cells should be functional. 4) These responses to steroid hormones of Ishikawa cells are synergistically enhanced or appeared earlier by addition of EGF. 5) The main metabolite of E2 incubated with Ishikawa cells is E2-3-sulfate instead of E1, indicate that the higher estrogenic status may be persisted in endometrial cancer tissues.     

Can vitamin A modify the activity of docetaxel in MCF-7 breast cancer cells?

Docetaxel is one of the most effective chemotherapeutic agents in the treatment of breast cancer. On the other hand, the vitamin A family compounds play the essential roles in many biological processes in mammary gland. The aim of our study was to investigate the effect of all-trans retinol, carotenoids (beta-carotene, lycopene) and retinoids (9-cis, 13-cis and all-trans retinoic acid) on the activity of docetaxel and to compare these effects with the estradiol and tamoxifen actions on human ER(+) MCF-7 breast cancer cell line. The evaluation was based on [3H] thymidine incorporation and the proliferative activity of PCNA and Ki 67 positive cells. In our study, the incorporation of [3H] thymidine into cancer cells was inhibited to 50% by 0.2, 0.5 and 1 microM of docetaxel in the 24-hour culture and addition of estradiol (0.001 microM) didn't influence the results. However, addition of tamoxifen caused a statistically significant decrease of the percentage of the proliferating cells in the culture medium with 0.2 and 0.5 microM of docetaxel (38.99 +/- 2.84%, p<0.01 and 40.67 +/- 5.62%, p<0.01) in comparison to the docetaxel only group. The above-mentioned observations were also confirmed with the use of the immunohistochemical investigations. Among the examined vitamin A family compounds, the simultaneous application of beta-carotene (0.1 microM) and docetaxel (0.2 microM) resulted in a statistically significant reduction in the percentage of proliferating cells (40.25 +/- 14.62%, p<0.01). Lycopene (0.1 microM), which stimulates the growth of breast cancer cells in a 24-hour culture, had an inhibitory effect (42.97 +/- 9.58%, p<0.01) when combined with docetaxel (0.2 microM). Although, beta-carotene and lycopene belong to the different chemical groups, they surprisingly had a similar inhibitory influence on both growth and proliferation of MCF-7 breast cancer cells when combined with docetaxel. The application of docetaxel either with beta-carotene or lycopene had comparable inhibitory effect on breast cells growth and proliferation as tamoxifen. Therefore, it may suggest a possible important role of these carotenoids in the breast cancer therapy in women especially when docetaxel is applied.     

Inhibitory effects of dispersed human amnion cells on production rates of prostaglandin E and F by endometrial cells

Dispersed cells were prepared from amniotic membranes obtained either by caesarian section near term before labos (CS) or after spontaneous vaginal delivery (SL) and from human endometrial curettings. The cells were maintained separately in primary culture for about 18 h. Production rates (PR) of PGE and PGF during incubation for 1 h in defined medium were determined whent the cell-types were separate (n=60) or combined (n=27) and when endometrial cells were incubated in medium conditioned by CS amnion cells (n=13) or SL amnion cells (n=12). The PR of PGE by CS amnion cells was five times greater than that of PGF and there was a two-fold increase (p <0.01) in PGE but not PGF by SL cells. Co-incubation was associated with a 25–32% fall in PR of both PGE and PGF (p <0.01) compared to the sum from separately incubated cells when CS cells were used whereas values for co-incubated SL cells did not differ from controls. Conditioned medium from CS but not SL cells inhibited PGE and PGF output by 30% and 40% (p <0.01) respectively. These findings suggest that human amnion cells release an inhibitor of prostaglandin synthesis in endometrial cells.     

Effect of antiestrogens and aromatase inhibitor on basal growth of the human breast cancer cell line MCF-7 in serum-free medium

Antiestrogens are efficient inhibitors of estrogen-mediated growth of human breast cancer. Besides inhibiting estradiol-stimulated growth, antiestrogens may have a direct growth-inhibitory effect on estrogen receptor (ER) positive cells and thus be more efficient than aromatase inhibitors, which will only abrogate estrogen-dependent tumor growth. To address this issue, we have used the human breast cancer cell line MCF-7/S9 as a model system which is maintained in a chemically defined medium without serum and estrogen. The addition of estradiol results in an increase in cell growth rate. Thus, the MCF-7/S9 cell line is estrogen-responsive but not estrogen-dependent. Three different types of antiestrogens, namely tamoxifen, ICI 182,780 and EM-652 were found to exert a significant and dose-dependent inhibition of basal growth of MCF-7/S9 cells. The growth-inhibitory effect of the three antiestrogens was prevented by simultaneous estradiol treatment. Antiestrogen treatment also reduced the basal pS2 mRNA expression level, thus indicating spontaneous estrogenic activity in the cells. However, treatment with the aromatase inhibitor had no effect on basal cell growth, excluding that endogenous estrogen synthesis is involved in basal growth. These data demonstrate that in addition to their estrogen antagonistic effect, antiestrogens have a direct growth-inhibitory effect which is ER-mediated. Consequently, in the subset of ER positive breast cancer patients with estrogen-independent tumor growth, antiestrogen therapy may be superior to treatment with aromatase inhibitors which only inhibit estrogen formation but do not affect cancer cell growth in the absence of estrogens.     

Selective growth-inhibitory, cell-cycle deregulatory and apoptotic response of apigenin in normal versus human prostate carcinoma cells

Agents that are capable of inducing selective apoptosis of cancer cells are receiving considerable attention in developing novel cancer-preventive approaches. In the present study, employing normal human prostate epithelial cells (NHPE), virally transformed normal human prostate epithelial cells (PZ-HPV-7), and human prostate adenocarcinoma (CA-HPV-10) cells, we evaluated the growth-inhibitory effects of apigenin, a flavonoid abundantly present in fruits and vegetables. Apigenin treatment to NHPE and PZ-HPV-7 resulted in almost similar growth inhibitory responses of low magnitude. In sharp contrast, apigenin treatment resulted in a significant decrease in cell viability of CA-HPV-10 cells. Similar selective growth inhibitory effects were also observed for human epidermoid carcinoma A431 cells compared to normal human epidermal keratinocytes. Apigenin treatment resulted in significant apoptosis of CA-HPV-10 cells as evident from (i) DNA ladder assay, (ii) fluorescence microscopy, and (iii) TUNEL assay, whereas the NHPE and PZ-HPV-7 cells did not undergo apoptosis but showed exclusive necrotic staining only at a high dose of 40 microM. Apigenin (1-10 microM) also resulted in a dose-dependent G2-M phase cell cycle arrest of CA-HPV-10 cells but not of PZ-HPV-7 cells. The growth-inhibitory and apoptotic potential of apigenin was also observed in a variety of prostate carcinoma cells representing different stage and androgen responsiveness. Apigenin may be developed as a promising chemopreventive and/or chemotherapeutic agent against prostate cancer.     

Characterization and functional properties of the A and B forms of human progesterone receptors synthesized in a baculovirus system.

Human progesterone receptors (PR) were overexpressed in Spodoptera frugiperda (Sf9) insect cells using a recombinant baculovirus system. Recombinant viruses were constructed that produced either full-length A (94K) or B (120K) forms of human PR, and each was expressed as a functional protein. Steroid and DNA binding activities were found to be indistinguishable from that of endogenous human PR in T47D breast cancer cells. Moreover, as analyzed by gel-mobility shift, recombinant PR-A and PR-B each bound to specific progesterone response elements in a strictly hormone-dependent manner. Native receptors expressed in Sf9 cells also exhibited structural properties similar to that of endogenous PR. Cytosolic PR (PR-A or PR-B), prepared in low salt buffer, sedimented on density gradients as an 8S oligomeric complex that was converted largely to 4S by treatment with 0.4 M NaCl. Immune isolation of the 8S cytosol PR complex and analysis of protein composition revealed the presence of two specific copurifying proteins of approximately 90K and 70K. The 90-K component was identified immunologically as heat shock protein 90. The 70-K component was not identified but is likely to be the insect equivalent of heat shock protein 70. Immune isolation of PR from Sf9 cells metabolically labeled with [32Pi], revealed that expressed PR was capable of being phosphorylated in insect cells. Hormone addition to Sf9 cells, however, did not stimulate the same increase in PR phosphorylation or upshift in mobility on sodium dodecyl sulfate gels that occurs with endogenous receptors in T47D cells. Thus some, but not all, phosphorylations occur with human PR expressed in Sf9 cells. These phosphorylation data, together with the fact that expressed PR required hormone for DNA binding, indicate that the hormone-dependent phosphorylation step responsible for PR upshifts on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is not required for receptor binding to DNA. The baculovirus expression system, therefore, may prove valuable in dissecting the functional role(s) for both hormone-dependent and hormone-independent PR phosphorylation.     

Progestins, progesterone receptor modulators, and progesterone antagonists change VEGF release of endometrial cells in culture

The influences of the synthetic progestin, medroxyprogesterone acetate (MPA), the progesterone receptor modulator J867, and the antagonist ZK137316 were studied in vitro on isolated endometrial epithelial cells, as well as endometrial fibroblasts. We evaluated the expression of estrogen receptor alpha (ER) and the progesterone receptor (PR) by RT-PCR. ER and PR were strongly expressed in the fibroblasts and epithelial cells under treatment with 10(-8) M 17beta-estradiol (E(2)). Treatment with 10(-6) M J867 or ZK137316 upregulated the PR expression as did E(2), in contrast to treatment with 10(-6) M MPA, which caused a downregulation of PR in epithelial cells, but not in fibroblasts. In addition, the vascular endothelial growth factor (VEGF) release into the cell culture medium was analyzed by a VEGF-ELISA. VEGF which plays an important role in angiogenesis, is regulated by steroid hormones as well as hypoxia. E(2) stimulates VEGF release into the medium in both cell types. MPA reduces VEGF release significantly in the fibroblast cell culture, but increases it in the epithelial cell culture. ZK137316, in the presence or absence of E(2), reduces VEGF release in fibroblast cell culture. J867 increases the VEGF production in fibroblasts only in the presence of E(2). Both compounds show no significant effects, compared to E(2), in epithelial cell culture. The different results for the epithelial cells and fibroblasts indicate that the pharmacological effects of PR modulators (PRMs) and progesterone antagonists (PAs) may be cell specific and depend on the presence or absence of partial progestagenic agonistic activities. This observation opens up new perspectives for various clinical applications.     

Evaluation of sphinganine and sphingosine as human breast cancer chemotherapeutic and chemopreventive agents

No comparative study of the effects of sphingolipid metabolites on proliferation and differentiation in normal human breast epithelial cells versus stem cells and tumorigenic cells has been reported. The purpose of this study was to evaluate the chemotherapeutic and chemopreventive potential of sphingoid bases (sphingosine and sphinganine) using a novel cell culture system of normal human breast epithelial cells (HBEC) developed from breast tissues of healthy women obtained during reduction mammoplasty (Type I HBEC with stem cell characteristics and Type II HBEC with basal epithelial cell phenotypes) and transformed tumorigenic Type I HBEC. The results show that sphinganine inhibited the growth and induced apoptosis of transformed tumorigenic Type I HBEC more potently than sphingosine (IC(50) for sphinganine 4 microM; sphingosine 6.4 microM). Both sphinganine and sphingosine at high concentrations (8-10 lM) arrested the cell cycle at G(2)/M. Sphinganine inhibited the growth and caused death of Type I HBEC more strongly than sphingosine. In comparison, Type II HBEC (normal differentiated cells) were less sensitive to the growth-inhibitory effects of sphingoid bases than Type I HBEC (stem cells) or transformed tumorigenic Type I HBEC, suggesting that sphingoid bases may serve as chemotherapeutic agents. At concentrations (0.05, 0.1, and 0.5 microM) that are below the growth-inhibitory range, sphingoid bases induced differentiation of Type I HBEC to Type II HBEC, as detected morphologically and via expression of a tumor suppressor protein, maspin, which is a marker of Type II HBEC. Thus, sphingoid bases may function as chemotherapeutic as well as chemopreventive agents by preferentially inhibiting cancer cells and eliminating stem cells from which most breast cancer cells arise.     

Direct effect of danazol on endometrial hyperplasia in adenomyotic women: treatment with danazol containing intrauterine device

It is well known that danazol has a direct effect on endometriosis tissue and cell. We have been treating adenomyotic women with danazol containing intrauterine device (D-IUD) from June 1993 to August 2000 and significant decrease of dysmenorrhea and serum CA-125 levels were observed. Of fifty-nine adenomyotic women, eight women were also diagnosed by endometrial biopsy as endometrial hyperplasia and one woman was diagnosed as atypical endometrial hyperplasia. In these endometrial hyperplastic patients, endometrial tissues were obtained before insertion and at the time of removal or exchange of D-IUD and examined pathologically. In all of the 9 women, histopathological findings of endometrial hyperplasia disappeared after D-IUD treatment. In particular, in one patient, findings of atypical endometrial hyperplasia also disappeared after D-IUD treatment. She is now closely observed at our clinic using D-IUD. By these evidences, we postulate that D-IUD is one of the treatment choices of endometrial hyperplasia given exposure of the endometrium to such an extraordinary high concentration of danazol released by D-IUD and avoidance of adverse effects of oral danazol or general administration of GnRH and progesterone. In particular, in atypical endometrial hyperplasia case, its mechanisms might give great benefit to patient. However, mechanisms of direct effect of danazol on endometrial hyperplasia remain to be elucidated in the future study.     

The effect of progesterone and human interferon alpha-2 on the release of PGF2 alpha and PGE from epithelial cells of human proliferative endometrium.

Progesterone and interferon-like trophoblastic proteins modulate prostaglandin (PG) synthesis from endometrium in early ovine and bovine pregnancy. Enriched epithelial cells were prepared from human endometrium removed in the proliferative phase of menstrual cycle (n = 8). Progesterone at a concentration of 1 microM suppressed PGE release from the cells during the first 24 hours in culture. After 48 hours in culture progesterone at a dose of 100 nM and 1 microM suppressed both the release of PGF2 alpha and PGE from the cells and this suppression was maintained for a further two days. Addition of exogenous 30 microM arachidonic acid (AA) abolished this effect of progesterone on both PGF2 alpha and PGE release. Interferon alpha-2 did not suppress the basal release of PGF2 alpha nor PGE. In the presence of progesterone, interferon alpha-2 attenuated the progesterone mediated suppression of PGF2 alpha but not PGE release from endometrial cells. These findings suggest that progesterone suppresses the basal release of PGs from human endometrium, but unlike the sheep, interferon alpha-2 does not exert this action on human endometrium.