Ikuya Shimizu

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.

Danazol binds to progesterone receptors and inhibits the growth of human endometrial cancer cells in vitro

Based on our recent findings that danazol, an isoxazol derivative of ethinyltestosterone, has a profound growth-inhibitory effect on an established human endometrial adenocarcinoma cell line, the effects of danazol on cancer cells from human endometrial adenocarcinomas obtained by hysterectomy were investigated in the present study. Of the 22 uterine adenocarcinomas, estrogen, progesterone, and androgen receptors were found in 12, 14, and 4 tumors, respectively. Competitive binding studies showed that danazol specifically binds to progesterone and androgen receptors but not to estrogen receptors. Of the five cancer cells from five patients succeeded in primary cell culture, a marked inhibition of cell growth was demonstrated by addition of danazol in two cancer cells having progesterone but not androgen receptors. However, danazol did not affect the growth of the remaining three cancer cells lacking progesterone receptors. These results strongly suggest that danazol has a significant growth-inhibitory effect on human endometrial adenocarcinoma cells, possibly through progesterone receptors in the cells.

Preliminary Report on the Use of Danazol in the Treatment of Endometrial Adenomatous Hyperplasia

Since the authors recently reported that danazol inhibits the growth of human endometrial cancer cells in vitro, a clinical trial was initiated to examine whether danazol can regress adenomatous hyperplasia, a precursor of endometrial adenocarcinoma. Danazol was used for 3 months in the treatment of five patients with a history of hypermenorrhea and irregular uterine bleeding. Within a 9‐month follow‐up period, all patients were symptom‐free, and none of the specimens obtained by endometrial biopsy showed the presence of adenomatous hyperplasia. These findings suggest that danazol has a potential application in the treatment of patients with endometrial adenomatous hyperplasia. A possible mechanism of action of the compound in adenomatous hyperplasia is also discussed.

Growth inhibition by danazol in a human endometrial cancer cell line with estrogen-independent progesterone receptors

Since we recently found that danazol, an isoxazol derivative of ethinyltestosterone, has a growth-inhibitory effect on human endometrial cancer cells in primary culture, the effects of danazol on a human endometrial cancer cell line (IK-90 cells), which contains estrogen-independent progesterone receptors (PR), were investigated in the present study. The addition of danazol (1 nM-1 microM) in culture medium caused a decrease in the growth of IK-90 cells in a dose-dependent manner. Competitive binding studies showed that danazol effectively binds to PR in IK-90 cells, and the binding affinity for PR was estimated to be 6.0% of that of R5020. The addition of 1 microM danazol in culture medium resulted in a rapid and significant increase in nuclear PR with a concomitant decrease in cytoplasmic PR in the cells. These findings suggest that danazol has a growth-inhibitory effect on human endometrial adenocarcinoma cells directly through PR system in the cells.

4-Hydroxytamoxifen binds to estrogen receptors and inhibits the growth of human endometrial cancer cells in vitro

Effects of 4‐hydroxytamoxifen, a major metabolite of tamoxifen, on the proliferation of cancer cells from human endometrial adenocarcinomas obtained by hysterectomy were investigated in primary culture. Competitive binding studies showed that 4‐hydroxytamoxifen effectively binds to cytoplasmic estrogen receptors (ER) in uterine adenocarcinomas. Of 20 endometrial adenocarcinomas examined, five tumors were successfully grown in primary cell culture. The addition of 4‐hydroxytamoxifen (1 nmol/l to 1 μmol/1) in a medium supplemented with estrogen‐free serum resulted in a dose‐dependent inhibition of the growth of cancer cells in two tumors having ER. However, 4‐hydroxytamoxifen did not affect the growth in the culture system of the remaining three tumors, in which ER were absent in two tumors but were present in one. These results strongly suggest that tamoxifen has a direct growth‐inhibitory effect on human endometrial adenocarcinoma possibly through ER in the tumor.

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.