Chronic exposure to estrogen and tamoxifen regulates synaptophysin and phosphorylated cAMP response element-binding (CREB) protein expression in CA1 of ovariectomized rat hippocampus.

We report here the in vivo effects of estrogen (E2) on modulation of synaptic plasticity and the agonistic (estrogen-like) role of selective estrogen receptor modulator (SERM), tamoxifen (TAM) in the CA1 of the rat hippocampus. Effects on synaptophysin (SYP), a presynaptic vesicular protein, and phosphorylated cyclic AMP responsive element-binding (p-CREB) protein, a signal transduction pathway molecule, were studied using the ovariectomized (OVX) experimental rat model. Bilateral ovariectomy was performed on 40 rats and these were divided into 4 groups based on the treatment they received (at 2 weeks post-ovariectomy, a subcutaneous injection daily for 4 weeks) viz., OVX+E2 (0.1 mg/kg body weight), OVX+TAM (0.05 mg/kg body weight), OVX+vehicle and one group served as OVX control. An additional 10 animals served as the ovary intact control group. At the end of the treatment schedule, five animals/group were used for immunohistochemical staining of SYP and p-CREB using specific antibodies with peroxidase anti-peroxidase technique on paraformaldehyde-fixed cryostat sections. Protein estimation and Western blot analysis coupled with densitometric analysis (using gel-documentation system and image analysis software) were performed on unfixed hippocampus collected from rest of the five animals/group. Serum estradiol levels were estimated with radioimmunoassay prior to sacrifice. The results revealed that ovariectomy reduced SYP and p-CREB expression whereas E2 or TAM administration resulted in their upregulation. Serum estradiol levels of E2 administered animals were comparable with the ovary intact group whereas those of TAM administered group persisted in the range of OVX controls. To conclude, long-term estrogen therapy modulates the synaptic plasticity of hippocampal neurons and presumably, the agonist biocharacter of TAM as observed in the present investigations, may in the long run have a potential in the treatment and prevention of various estrogen-related disorders.

Estrogen receptor alpha and beta immunoreactive neurons in normal adult and aged female rat hippocampus: a qualitative and quantitative study.

We have studied the distribution pattern and levels of expression of two estrogen receptor (ER) subtypes, ERalpha and ERbeta, in the normal adult (n = 10) and the aged (n = 10) female rat hippocampus with the objective to establish baseline data and the changes that occur during aging. Techniques including immunohistochemical localization, co-localization with double immunofluorescence and confocal microscopy, image analysis including neuronal counts/mm(2) area and measurements of optical density (OD) of immunoreactivity in immunoreactive neurons and Western blot analysis have been used. The results revealed ERalpha and ERbeta positive neurons in all subfields of the hippocampus with maximum presence in the stratum pyramidale of CA3. Some stained neurons in CA3 exhibited pyramidal neuron like morphological characteristics; such neurons were not found in CA1. All other immunoreactive neurons showed non-pyramidal neuron like morphological characteristics. Neuronal counts revealed a significant decrease in the number of immunoreactive neurons in CA3-CA1 of aged hippocampus. The percent decrease in counts of the immunoreactive neurons/mm(2) area in the aged rat (compared to the adult) was 78% for the ERalpha and 88% for the ERbeta (P < 0.001) in CA3. In CA1, it was 56% (P < 0.001) and 41% (P < 0.01) respectively. The OD of immunoreactivity was significantly decreased (P < 0.01) in CA3 but increased (P < 0.01) in the CA1 immunoreactive neurons. Western blot analysis also showed a significant decline (P < 0.01) in the levels of the ERalpha and ERbeta proteins in the aged hippocampus. Co-localization revealed that the two ER subtypes do co-exist in the same hippocampal neurons.

Do the progesterone receptors have a role to play in gallbladder cancer?

BACKGROUND: Gallstone disease as well as gallbladder cancer are more common in women and female sex hormones may be involved in their etiology. AIM AND METHODS: To determine whether female sex hormones have a role in the pathogenesis, of gallbladder carcinoma and in its prognosis, we estimated, by enzyme immunoassay, the estrogen and progesterone receptors (ER and PgR) in the gallbladders of 21 patients with gallbladder cancer, 19 patients with cholelithiasis, and 6 patients who underwent incidental removal of essentially normal gallbladder as a component of wider resection. RESULTS: ER were present in the gallbladder mucosa in all the three groups in proportions which were not significantly different (9/21 in carcinoma, 4/19 in gallstones, and 1/6 normal), whereas the expression of PgR was greater in carcinomas (13/18), less in cholelithiasis (4/12), and absent in normal gallbladders. PgR expression was higher in tumors of lower stage (7/7) and lower in advanced disease stage IV tumors (6/11). PgR expression was associated with better disease stage (p=0.05) and significantly longer overall survival (median survival of 301 d vs 54 d) as well as better survival within the same stage (269 d vs 54 d for stage IV disease, p=0.011). Cox's regression analysis showed that PgR was an independent risk factor (R=0.2283, p=0.0035). CONCLUSIONS: Our findings suggest that the female sex hormones may have a role in the pathogenesis of gallbladder cancer and that PgR expression has a prognostic significance. We believe that when this relationship is reaffirmed by larger studies, gallbladder cancer may be treated with appropriate sex hormonal manipulation.

Value of serum CEA and AFP in the diagnosis and prognosis of carcinoma gallbladder.

BACKGROUND/OBJECTIVE: The poor prognosis of carcinoma of the gallbladder (CAGB) is attributable to delayed presentation in the absence of specific clinical findings in the early stages. To ascertain whether the commonly available serum tumour markers (carcino-embryonic antigen-CEA and alpha foeto protein-AFP) could be used for distinguishing CAGB from other biliary disorders and in assessing the prognosis of patients with CAGB, serum levels of these markers in patients with CAGB and those with cholelithiasis were studied. METHODS: Estimation of serum CEA in 28 patients with CAGB and 30 patients with cholelithiasis and AFP in some of these cases was done by enzyme immunoassay. RESULTS: The mean values of CEA and AFP were 15.1 ng/ml and 166.5 ng/ml respectively for the CAGB group and 12.6 ng/ml and 166.5 ng/ml respectively for the cholelithiasis group. There was no statistical difference between the groups (p > 0.05). These markers did not show any statistically significant correlation with the stage of disease or length of survival in the patients with CAGB. CONCLUSION: Serum levels of CEA and AFP do not have any diagnostic or prognostic significance in the management of CAGB.

Effect of hormones and antihormones on phospholipase A2 activity in human endometrial stromal cells.

Phospholipase A2 activity was studied in isolated human endometrial predecidual cells, and in human endometrium collected from day 19-23 of the menstrual cycle, by performing a radiochemical assay. Phospholipase A2 activity on day 20 was significantly higher than other days (P < 0.001), and the activity was found to gradually decrease after day 20 of the menstrual cycle. The effects of the hormones estradiol and progesterone, and antihormones tamoxifen and RU 486, were studied on the phospholipase A2 activity in isolated predecidual stromal cells. Estradiol produced a significant stimulatory effect (P < 0.001) on phospholipase A2 activity in predecidual cells, and this effect was antagonized by tamoxifen. The combination of estradiol and tamoxifen was significantly different from estradiol alone (P < 0.001), but not from tamoxifen alone. RU 486 alone significantly increased (P < 0.001) phospholipase A2 activity in predecidual stromal cells. However, progesterone had no effect on phospholipase A2 activity in predecidual stromal cells.

Effect of antiprogestin RU 486 on the proliferation and metabolic activity of stromal cells separated from human endometrium.

The effects of RU 486 on the proliferation and metabolic activity of human endometrial stromal cells in culture were studied. RU 486 at 10(-7) M/well significantly stimulated (P < 0.001) the growth as well as metabolic activity in the culture system. Interestingly, RU 486 at 10(-6) M/well did not stimulate metabolic activity in the culture. Progesterone, in combination with RU 486 at 10(-7) M/well, caused a significant increase in proliferation (assessed by thymidine incorporation) (P < 0.001) over control and P4 alone, but not significantly different from RU 486 at 10(-7) M/well alone. The same pattern was observed for metabolic functions (assessed by uridine incorporation) when RU 486 at 10(-7) M/well, along with P4, was added to the culture. Interestingly, RU 486 at 10(-6) M/well with P4 had no effect on RNA synthesis in the culture. The relevance of these findings is discussed.

Tamoxifen increases the proliferation of human endometrial stromal cells in in vitro. A model for evaluation of endometrial hyperplasia.

Tamoxifen given for breast cancer therapy, has a complex and an unclear action on the endometrium. A large number of literatures has attributed the proliferous changes in the endometrium caused by tamoxifen (Tam). No report has appeared on the endometrial cellular changes induced by Tam. The present study shows a significant (P < 0.001) increase in the proliferative activity due to Tam in endometrial stromal cells over control and estradiol (E2). This in vitro model is useful for the study of the hyperplasic effect of Tam at the cellular level.

Effect of tamoxifen, estradiol 17 beta on coenzymes NAD, NADPH and the metabolism of estradiol and estrone in rabbit uterus in vivo.

Biotransformation of estradiol (E2) and estrone (E1) and the concentrations of NAD, NADPH and 17 beta-estradiol dehydrogenase (E2DH) were measured in the uterus of rabbits treated with tamoxifen (Tam) in two doses; 100 micrograms/day, (Tam 100) and 500 micrograms/day, (Tam 500), E2 (10 micrograms/day) and combination of E2 + Tam 500 for 4 days. The concentration of NAD in Tam 500 treated group was significantly higher than E2, low dose Tam and E2 + Tam 500 treated groups (P < 0.01). The concentration of NAD in E2+ Tam 500 uteri was also significantly higher than E2 treated uteri. The concentration of NADPH was not significantly different from each other amongst the various treatment groups. The studies have shown that E2DH in E2 treated uteri was less than control and Tam 500 treated groups. A significant rise in the enzyme estradiol oxidoreductase (E2OR) activity (P < 0.02) was observed in E2 + Tam 500 treated uteri over control and other treated groups whereas high dose Tam decreased the E2OR activity significantly over the E2 treated group. The rate of conversion of E1 to E2 in Tam 500 treated group was significantly less than the other treatment groups except E2 + Tam 500 treated group (P < 0.04). This study showed that E2 decreases the uterine biosynthesis of NAD and E2DH and the biotransformation of E2 to E1, while high dose Tam increases uterine NAD, E2DH activity and E2 to E1 conversion.

Short term treatment of tamoxifen and estradiol on estradiol and progesterone receptors in rabbit uterus.

Effects of tamoxifen (Tam) on cytosolic estradiol (E2) receptors (ERc), progesterone (P4) receptors (PRc), nuclear estradiol (ERn) and progesterone receptors (PRn) were studied in adult normal rabbit uterine tissue. The ratio of cytosol: nuclear estradiol receptors (ER) was greater in rabbits treated with Tam than E2 or control uterine tissues. Rabbit uterine progesterone receptors (PR) in E2 treated animal were greater than Tam-treated animals. Tam caused nuclear accumulation of estradiol receptor, and simultaneous administration of E2 + Tam 500, estradiol could not revert Tam mediated accumulation of ER. The results suggest that Tam has an essentially antagonist action in the rabbit uterine tissue.

Progestin and antiprogestin interactions with progesterone receptors in human myomas.

Progesterone receptors (PRs) in human fibromyomata (myomas) and normal myometrium were characterized by gel filtration, sucrose gradient sedimentation analysis, isoelectrofocusing and ligand specificity. The PR population in myoma tissues, 1242 +/- 505 fmol/mg protein, was 3-fold higher than normal myometrial tissues under similar hormonal milieu. The increased PR concentration in myomas appears to be of potential importance in physiopathology of fibromyoma.

Functional estrogen receptors as biochemical marker for the detection of hormone-dependent breast cancer.

The translocatable receptors categorized as functional receptors were quantitated in a cross-incubation study of breast cancer nuclei with receptor-rich uterine cytosol. Data demonstrated that tumors that contained cytosolic estrogen receptor (ER) but had translocation defect might not be hormone dependent, whereas tumors with low ER but intact nuclear translocation step will respond to antiestrogen therapy. Cytosolic ER was estimated in 114 primary breast cancer tissues and ten metastatic axillary lymph nodes; 58% of postmenopausal and 54% of premenopausal breast cancer tissues were ER+ with a cutoff value of 10 fmoles and 3 fmoles/mg protein, respectively. Of tumors in the premenopausal and postmenopausal state, 62% and 57%, respectively, were positive for nuclear ER, with a cutoff value of 10 fmoles/100 micrograms DNA. This study suggested that evaluation of functional ER level would reduce the number of false-negative and false-positive tumors.

Effect of progestins, estradiol, and coenzymes NAD and NADPH on the interconversion of estradiol and estrone in rabbit uterus in vitro.

The biotransformation of estradiol (E2) and estrone (E1) in the uterus of rabbits treated with norgestrel (NG), norethindrone (NET), norethindrone acetate (NETA), progesterone (P4), and E2 either by subcutaneous injection in oil or by intrauterine steroid-releasing silastic implants was carried out under an in vitro short-term incubation system. The studies have shown that E2 stimulates 17 beta-hydroxysteroid dehydrogenase (17 beta-OHSD) much more than P4 as compared to untreated controls. The kinetic studies on E2 metabolism in the presence of added coenzyme NAD showed an initial rapid estrone formation and a gradual reconversion of E1 to E2. The addition of NADPH, ATP, and glucose-6-phosphate facilitates the reconversion of E1 to E2. The interconversion of E2 and estrone in the presence of coenzymes was five- to ten-fold higher in the endometrium than in the myometrium per milligram protein. Both E2 and progestins stimulate the uterine 17 beta-OHSD activity in rabbit uterus. This study further suggested that the hormone-induced metabolism of estradiol and estrone in the rabbit uterus is essentially modulated by the availability of coenzymes.

Interaction of antiprogestins with progesterone receptors in rat uterus.

Cytosolic and nuclear progesterone receptors (PRc and PRn) under antiprogestin treatment were measured in rat deciduoma and compared with values for contralateral (nondeciduomatous) rat uterine tissue. Uterine PRc and PRn of the progesterone treated group were 101 +/- 8.7 and 4770 +/- 590 fmol/mg DNA respectively. After treatment with antiprogestins STS-557, 5 alpha-DNE, (5 alpha-dihydronorethisterone), 5 alpha-DNG (5 alpha-dihydronorgestrel), RU-22092 and RU-16556, PRc in the nondeciduomatous control horn ranged from 127 to 377 fmol/mg DNA and PRn from 2785 to 17925 fmol/mg DNA. In the decidual tissue, PRc decreased significantly (4.6 +/- 0.8 fmol/mg DNA) on 5 alpha-DNG treatment as compared with the progesterone alone treatment group (147 +/- 3.8). PRn in decidual tissue also decreased maximally on 5 alpha-DNG treatment. These results suggest that the interaction of antiprogestins may not be identical in control uterine tissue and in deciduoma.