Midori Hashimoto

Preventive Effects of Isoflavones, Genistein and Daidzein, on Estradiol-17β-related Endometrial Carcinogenesis in Mice

The effects of isoflavones (genistein and daidzein) on endometrial carcinogenesis in mice were investigated in two experiments. In the short‐term experiment (2 weeks), single subcutaneous (s.c.) administration of genistein [1 mg/30 g body weight (b.w.)] significantly decreased the levels of estradiol‐l7β (E2) (5 ppm in diet)‐induced expression of c‐jun, interleukin‐lα (IL‐lα) and tumor necrosis factor‐a (TNF‐a) mRNAs in the uteri of ovariectomized mice (P<0.005, P<0.05 and P<0.01, respectively). Daidzein significantly inhibited E2‐induced expression of c‐fos and IL‐lα (P<0.01, P<0.01 respectively). In the long‐term experiment (30 weeks), 140 female ICR mice were given N‐methyl‐N‐nitrosourea‐containing solution (1 mg/100 g b.w.) and normal saline (as controls) into their left and right uterine corpora, respectively. They were divided into six groups; group 1 was given E2 (in diet) alone. Group 2 was given E2 and genistein (1 mg/30 g b.w., s.c., every four weeks). Group 3 was exposed to E2 and daidzein (1 mg/30 g b.w., s.c., every four weeks). Groups 4 and 5 respectively received genistein and daidzein, and were kept on the basal diet. Group 6 was kept on the basal diet and served as a control. At the termination of the experiment, incidences of endometrial adenocarcinoma and atypical endometrial hyperplasia of the group given E2 and genistein or daidzein were significantly lower than of the group with E2 alone (P<0.01 and P<0.05, respectively). It is suggested that both genistein and daidzein have an inhibitory effect on estrogen‐related endometrial carcinogenesis in mice, possibly by suppressing expression of estrogen‐induced estrogen‐related genes c‐fos and c‐jun, and internal cytokines IL‐lα and TNF‐α through a cytokine and estrogen receptor‐mediated pathway.

Effects of tamoxifen on endometrial carcinogenesis in mice

Two experiments were conducted to determine the effect of tamoxifen (TAM) in mouse endometrium in comparison with that of 17β‐estradiol (E2). In a medium‐term assay, TAM as well as E2 treatment semi‐dose‐dependently increased the levels of fos/jun mRNA and their oncoproteins (Fos/Jun). The long‐term effect of TAM on mouse endometrial carcinogenesis was also examined in the following model. A total of 150 female ICR mice, 12–13 weeks of age, were used. Of these, 125 mice received an injection of N‐methyl‐N‐nitrosourea (MNU) solution (1 mg/100 g body weight) into their left uterine tube and saline into the right. One week later, they were divided into four groups: groups 1 (35 mice) and 2 (30 mice) were given 25 ppm and 5 ppm E2‐containing diet, respectively, while group 3 (30 mice) was fed 5 ppm TAM‐containing diet. Group 5 (30 mice) was fed basal diet alone. The remaining 25 mice (group 4) received 5 ppm TAM‐containing diet alone. At the termination of the experiment (30 weeks), endometrial carcinomas were confirmed to be present in the groups exposed to MNU. TAM increased the incidence of preneoplastic lesions of the endometrium, while E2 enhanced the occurrence of the carcinoma. No carcinomas were found in the group given TAM alone. In the ovaries, corpora lutea were lacking in most of the mice exposed to TAM, suggesting that the animals were not cycling. Such findings indicate that TAM has an enhancing effect on endometrial carcinogenesis in mice, probably via a mechanism involving overexpression of Fos/Jun proteins.

Preventive Effects of Glycyrrhizae radix Extract on Estrogen-related Endometrial Carcinogenesis in Mice

Short‐ and long‐term experiments were conducted to examine the effects of Glycyrrhizae radix (Gl radix) extract on mouse endometrial carcinogenesis. Gl radix treatment (2 weeks) decreased the levels of c‐fos/jun mRNA and the corresponding oncoproteins induced by estradiol‐17β(E2) in castrated mice uteri, as determined by reverse transcription‐polymerase chain reaction and Southern blot analysis, and immunohistochemical methods, respectively. For the long‐term assays, 98 female ICR mice were given N‐methyl‐N‐nitrosourea (MNU) solution (1 mg/100 g body wt.) and normal saline (as controls) into their left and right uterine corpora, respectively. They were divided into four groups as follows: group 1 was given 0.625%Gl radix‐ and 5 ppm E2–containing diet; group 2, 5 ppm E2–containing diet; group 3, 0.625%Gl radix‐containing diet; and group 4, the basal diet alone. Gl radix treatment significantly decreased uterine weights and the incidences of uterine endometrial atypical hyperplastic and malignant lesions. It is suggested that Gl radix has inhibitory effects on E2‐related endometrial carcinogenesis in mice, through suppression of estrogen‐induced c‐fos/jun‐expressions.

Shimotsu-to is the agent in Juzen-taiho-to responsible for the prevention of endometrial carcinogenesis in mice

We have found that Juzen-taiho-to has a preventive effect on endometrial carcinogenesis in mice (Carcinogenesis 22 (2001) 587). In the present study, the constituents of Juzen-taiho-to responsible for this effect were explored using a short-term experiment. Thirty female ICR mice were divided into five groups: Group 1 was given a diet containing 0.2% of Juzen-taiho-to and 5ppm estradiol-17beta (E(2)); Group 2 was given a diet containing Shimotsu-to (0.07%) and E(2) (5ppm); Group 3 received Shikunshi-to (0.08%) and E(2) (5ppm) in the diet; Group 4 was given 5ppm E(2) in the diet; and Group 5 served as a control. Exposure of Juzen-taiho-to or Shimotsu-to decreased E(2)-stimulated expression of estrogen-related gene c-fos mRNA (P<0.05), and the cytokines interleukin-1alpha mRNA and tumor necrosis factor alpha mRNA P<0.01). A similar trend was not found upon treatment with Shikunshi-to. These findings suggest that Shimotsu-to is responsible for the inhibitory effects of Juzen-taiho-to on the estrogen-related endometrial carcinogenesis in mice.

Preventive effects of danazol on endometrial carcinogenesis in mice

Short and long-term experiments were designed to determine effects of danazol on estrogen-related endometrial carcinogenesis in mice. The short-term assays showed that danazol decreased expression levels of c-fos/jun mRNA and their oncoproteins induced by estradiol-17beta (E2). For the long-term assay, 85 female ICR mice were given N-methyl-N-nitrsourea solution into their uterine corpora. The animals were divided into three groups as follows: Group 1, E2-diet (5 ppm) plus danazol (2 mg/body (s.c.), every 4 weeks); Group 2, E2-diet alone, Group 3, basal diet alone. At 30 weeks, incidences of atypical and complex endometrial hyperplasia were significantly decreased by danazol-treatment. These results suggest that danazol has preventive effects on estrogen-related endometrial carcinogenesis in mice, through the suppression of estrogen-induced c-fos/jun-expression.

Conservative Therapy for Endometrial Carcinoma in Young Women Treated with Repeated Curettage and Progestogen

Three women less than 35 years old, who wanted to preserve their fertility, were conservatively treated with repeated endometrial curettage and medroxyprogesterone acetate (600mg/d) for endometrial carcinoma. Treatments were given once a month for 6 to 8 months. No patient had evidence of recurrence for at least 16 months after the treatment, and 1 patient subsequently delivered 2 full-term babies. This conservative treatment by endometrial curettage with progestogens may be considered for young patients with early endometrial lesions who wish to preserve their fertility.

Effects of sex steroids on silver stained proteins of nucleolar organizer regions (Ag-NOR) in the rabbit uterus.

Silver staining of argyrophilic proteins of nucleolar organizer regions (Ag-NOR) has been used to evaluate a hyperactive state of cells. We studied the effects of sex steroids on the Ag-NOR proteins in the rabbit uterus. Estradiol-17beta (E2) caused uterine hypertrophy and increased the number of Ag-NOR dots in epithelial and stromal cells of the endometrium and smooth muscles cells in a dose dependent manner without proliferative change in the endometrium. Progestins including progesterone, medroxyprogesterone acetate (MPA), and norethindrone following E2 caused progestational proliferation of the endometrium with an increase in the Ag-NOR number to various extents, whereas methyltestosterone, a synthetic androgen, caused only a minimal proliferative change in the endometrium without an increase in the Ag-NOR number. Western blot analysis revealed that progesterone and MPA increased the amounts of a 100 kDa protein (nucleolin) and a 37 kDa protein (B23 protein) in the endometium and a marked proliferative change, whereas E2 did not. These results suggest that although the Ag-NOR number was enhanced by both progestin and estrogen, only progestins substantially increase the Ag-NOR protein in the rabbit uterus. This implies that an increase in the amount of nucleolin and B23 protein is associated with the proliferative potential of the cells.

Inhibitory Effects of Toremifene on N-Methyl-N-nitrosourea and Estradiol-17β-induced Endometrial Carcinogenesis in Mice

Short‐ and long‐term experiments were designed to determine the effects of toremifene (TOR) on estrogen‐related endometrial carcinogenesis in mice. In the short‐term experiment, a single low dose of TOR (0.2 mg/30 g body weight) decreased expression of c‐fos, interleukin (IL)‐1α, estrogen receptor (ER)‐α mRNAs and corresponding proteins induced by estradiol‐l7β (E2), in the uteri of the ovariectomized mice. Expression of ER‐β mRNA was increased by the TOR treatment, compared with the control. In the long‐term experiment, 106 female ICR mice were given N‐methyl N‐nitrosourea (MNU) into their uterine corpora. The animals were divided into four groups as follows: group 1, E2 diet (5 ppm) plus TOR (0.2 mg/30 g body weight, subcutaneously, every four weeks); group 2, E2 diet alone; group 3, basal diet plus TOR. Group 4 served as the control. TOR treatment decreased the incidence of MNU and E2‐induced endometrial adenocarcinoma and atypical hyperplasia at the termination of the experiment (30 weeks after the start). These results suggest that TOR exerts preventive effects against estrogen‐related endometrial carcinogenesis in mice, through the suppression of c‐fos as well as IL‐1α expression induced by E2. Such suppressive effects of TOR may be related to the decreased ER‐α and increased ER‐β expressions.

Introduction of p21Waf1/Cip1 gene into a carcinoma cell line of the uterine cervix with inactivated p53

In carcinomas of the uterine cervix of which p53 is frequently inactivated by papilloma viruses, gene transfer of p21, effector of p53, is an alternative tool to suppress cell growth. We introduced the p21 gene into HeLa cells. The transfectant with p21 showed a significant growth retardation by blockage of G1 to S transfer of the cell cycle. This cell line showed significantly reduced anchorage-independent growth as well as attenuated telomerase activity. These data suggest that gene transfer of p21 is an effective tool to lead carcinoma cells with inactivated p53 into less malignant phenotype.

Silver-stained nucleolar organizer regions in the uterine myomatous tumors

The numbers of silver-stained nucleolar organizer regions (AgNORs) were counted in uterine myomatous tumors, and compared with those in corresponding normal myometria. The mean number of AgNORs in myomatous tumors tended to increase according to the neoplastic changes. The mean number of AgNORs in leiomyosarcoma (6.4 +/- 0.9) was significantly higher than that in cellular leiomyoma (4.5 +/- 0.7, P < 0.01). or leiomyoma (3.0 +/- 0.5, P < 0.001). In the normal myometria, the mean number of AgNORs (3.3 +/- 0.4) in the premenopausal women showed a higher tendency than that in the post-menopausal women (2.4 +/- 0.5). These results therefore suggest that AgNOR counts may be useful for diagnosis of cellular activity in uterine myomatous tumors.