Apoptosis induction and inhibition of hyperplasia formation by 2-[piperidinoethoxyphenyl]-3-[4-hydroxyphenyl]-2H-benzo(b)pyran in rat uterus.

OBJECTIVE: The study was undertaken to explore the antiproliferative mechanism of action of 2-[piperidinoethoxyphenyl]-3-[4-hydroxyphenyl]-2H-benzo(b)pyran (K-1) in estradiol-induced rat uterine hyperplasia. STUDY DESIGN: Adult ovariectomized rats received vehicle or estradiol alone (20 µg/kg) or estradiol along with K-1 (100 or 200 µg/kg) for 14 days. Uterine histomorphometric analysis and immunoblotting were performed. Caspase-3 activity and terminal deoxynucleotidyl transferase-mediated nick end-labeling staining were performed to analyze the apoptotic potential of compound. RESULTS: Compound inhibited estradiol-induced uterine weight and histomorphometric changes pertaining to endometrial growth and down-regulated the expression of estrogen response element and activator protein-1 regulated genes and transcription factors. The compound significantly induced apoptosis, interfered with Akt activation, decreased X-linked inhibitor of apoptosis protein expression leading to an increased cleavage of caspase-9, caspase-3, poly(adenosine diphosphate-ribose) polymerase, increased Bax/Bcl2 ratio, and caspase-3 activity. CONCLUSION: K-1 inhibits endometrial proliferation via nonclassical estrogen receptor signaling mechanisms. It interfered with Akt activation and induced apoptosis via the intrinsic pathway and inhibited estradiol-induced hyperplasia formation in rat uterus.

Anti-implantation effect of 2-[piperidinoethoxyphenyl]-3-[4-hydroxyphenyl]-2H-benzo(b)pyran, a potent antiestrogenic agent in rats.

OBJECTIVE: To investigate the anti-implantation effect and hormonal profile of 2-[piperidinoethoxyphenyl]-3-[4-hydroxyphenyl]-2H-benzo(b)pyran (K-1) in rats. DESIGN: In vivo assays for anti-implantation activity were performed in pregnant rats. Assays for estrogenicity/antiesrogenicity were performed in immature ovariectomized female rats. In vitro competitive binding of K-1 to human recombinant ERα, transient transfection assay using ERE-luciferase reporter, and alkaline phosphatase (ALP) activity as a measure of estrogenicity and/antiestrogenicity in human endometrial carcinoma cells were performed. SETTING: Research laboratory. ANIMAL(S): Adult female rats for anti-implantation activity, immature ovariectomized female rats, and immature castrated/intact male rats. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Number of implantations, uterine growth, luciferase reporter activity, ER binding affinity, and ALP activity. RESULT(S): Compound K-1 given orally for 1-7 days post coitum at the dose of 100 µg/kg body weight prevented pregnancy in 100% of rats. K-1 was a potent antiestrogenic, and at 50 µg/kg, it could inhibit the effect of 1 µg E(2) in immature rats. Compound was devoid of uterotrophic, androgenic, or antigonadotropic activity. A high affinity binding to ERα was displayed by K-1, with a relative binding affinity of 5% of E(2). In human endometrial carcinoma cells, K-1 did not induce ERα-mediated transcriptional activation that is measured as luciferase reporter activity. K-1 antagonized the E-induced transcriptional activation significantly. K-1 also antagonized E-induced ALP activity in human endometrial cells. CONCLUSION(S): K-1 appeared to exert its antifertility action by virtue of its strong antiestrogenic activity.

Synthesis and biological evaluation of 2,3,4-triarylbenzopyran derivatives as SERM and therapeutic agent for breast cancer.

A novel class of 2,3,4-triarylbenzopyrans has been synthesized and were evaluated for their selective estrogen receptor modulation activity and as a therapeutic agent for breast cancer. Among the compounds synthesized, compounds 11a and 12c exhibited 73.91% and 69.24% inhibition as estrogen antagonistic activity, respectively. Compound 12a showed the lowest IC(50) at 6.97 microM against MCF-7 and 11f showed the lowest IC(50) value of 5.6 microM against MDA-MB-231 cell line in spite of their low receptor binding affinity implicating these compounds probably act through ER independent mechanism.

Expression of alphaVbeta3 integrin in rat endometrial epithelial cells and its functional role during implantation.

The alpha(V)beta(3) integrin as a marker of endometrial receptivity has been well established in human and other mammalian species; however, its expression is still not known in rats. Our objective was to establish the expression of alpha(V)beta(3) integrin as a marker of endometrial receptivity in rat and to further prove its role in implantation by function-blocking studies in this species. Immunocytochemical, immunohistochemical and flow-cytometric studies were performed in rat endometrial epithelial cells (EEC) to demonstrate the expression of alpha(V)beta(3) integrin during non-receptive, pre-receptive and receptive phases of the uterus. Results revealed positive immunocytochemical staining for alpha(v) and beta(3) subunits on the surface of EEC of days 4 and 5p.c. (post-coitum), but the intensity was higher in cells of day 5p.c. Flow-cytometric study revealed higher level of alpha(V)beta(3) on day 5p.c. as compared to day 4p.c. and non-pregnant animals. Immunohistochemical analysis of uterine tissue also revealed that the alpha(V)beta(3) expression in LE was higher on day 5p.c. morning as compared to that observed on day 4p.c. In addition, the expression of beta(3) subunit was not evident in rats receiving ormeloxifene, an agent known to inhibit the uterine receptivity. Immunoblotting experiments also revealed higher expression of uterine beta(3) on day 5p.c. On day 6, expression of beta(3) was high in implantation sites than on inter-implantation sites. In immature ovariectomized rats, alpha(V)beta(3) was up-regulated by progesterone and by a combination of estrogen and progesterone. The expression of alpha(V)beta(3) was also up-regulated in EEC co-cultured with blastocysts. All the agents used for function-blocking studies showed significant reduction in the number of implantation sites in treated horn as compared to sham control horn. The present study has successfully demonstrated the expression of alpha(V)beta(3) in rat EEC as a marker of endometrial receptivity and showed that this molecule is indispensable for the process of implantation in this species.