Regulation of the PI3K/Akt pathway during decidualization of endometrial stromal cells.

Infertility is constantly increasing in Canada, where 16% of Canadian couples are experiencing difficulty conceiving. It is thought that infertility can emanate from the dysregulated communication between the embryo and the maternal endometrium. In order to allow for this window of implantation to be open at the right moment, endometrial stromal cells proliferate and differentiate by a mechanism called decidualization. Intracellular and molecular mechanisms involved in the regulation of apoptosis and cell proliferation during decidualization of the endometrium are yet to be fully understood. It has been well demonstrated previously that Akt is importantly involved in cell survival and glycogen synthesis. Akt1, Akt2 and Akt3 isoforms have distinct physiological roles; this could also be the case during decidualization and pregnancy. The aim of this study is to investigate the regulation of PI3K/Akt pathway during the decidualization process of endometrial stromal cells. Expression of Akt isoforms, Akt activity (phospho-Akt), pIκB and substrates of Akt during decidualization were measured. To our knowledge, these results are the first to suggest a decrease in levels of Akt isoforms as well as a downregulation of Akt activity in the process of decidualization of human endometrial stromal cells. We also uncovered that decidualization induced nuclear localization of p65 through the phosphorylation of IκB, its inhibitory subunit; however, Par-4, a recently uncovered regulator of cell differentiation, was displaced from the nucleus upon decidualization. Our results also suggest that HIESC cells exhibit decreased motility during decidualization and that PI3K pathway inhibition could be involved in this process. Finally, we demonstrate that specific Akt isoforms present unique effects on the successful induction of decidualization. Further analyses will involve investigations to understand the precise signaling mechanisms by which this pathway is regulated.

Exploring the synthesis and anticancer potential of L-tyrosine-platinum(II) hybrid molecules.

The search for new specific chemotherapeutic drugs designed to minimize the toxic side effects resulting from chemotherapy is still a subject of intense research. The objective of the current study was to design a non-steroidal-platinum(II) derivative that would target the estrogen receptor alpha (ERα) without triggering estrogenic cell proliferation. For this purpose, the amino acid L-tyrosine was modified and attached to a cisplatin analog. Hence, the L-tyrosine portion of the molecule could possibly act as a transporter to target the ERα protein and, by doing so concentrate the cytotoxic moiety to hormone-dependent breast cancer cells. Herein, we describe three different alternative methodologies that were used to make these new anticancer molecules. The L-tyrosine-Pt(II) hybrid 5b was made in four steps with 36% overall yield by the first method, in six steps with 11% overall yield by the second method and, in four steps with 23% overall yield by the third method. Preliminary biological activity on breast cancer cell lines indicated that the final hybrids (5a and 5b) were unfortunately inactive but their platinum(II) precursors (14a and 14b) showed activity similar to that of cisplatin.

New testosterone derivatives as semi-synthetic anticancer agents against prostate cancer: synthesis and preliminary biological evaluation.

Prostate cancer (PC) is a major health issue in the world. Treatments of localized PC are quite efficient and usually involve surgery, radiotherapy and/or hormonal therapy. Metastatic PC is however rarely curable to this day. Treatments of metastatic PC involve radiotherapy, chemotherapy and hormonal treatment such as orchiectomy, antiandrogens and luteinizing hormone-releasing hormone agonists. The suppression of tumor growth by hormonal treatment is efficient but overtime resistance still occurs and the disease progresses. Thus, more urgently than ever there is a need for discovery of new treatment options for castration-resistant PC (CRPC). Hence, we designed and tested a series of amide derivatives located at position 7α of testosterone as prospective "natural" or "semisynthetic" anticancer agents against CRPC with the goal of discovering therapeutic alternatives for the disease. This manuscript describes an efficient path towards the target molecules that are made in only 6 or 7 chemical steps from testosterone in good overall yields. This strategy can be used to make several compounds of interest that present higher biological activity than the classic antiandrogen; cyproterone acetate (3). The best testosterone-7α-amide was the N-2-pyridylethylamide (25) which was as active as the antiandrogen cyproterone acetate (3) on androgen-dependent LNCaP cells and 2.7 times more active on androgen-independent PC3 prostate cancer cells. The results obtained show the synthetic feasibility and the potential for future development of this unique class of semi-synthetic anticancer agents that offer the premise of new treatment modalities for patients afflicted with CRPC.

Synthesis and preliminary in vitro biological evaluation of 7α-testosterone-chlorambucil hybrid designed for the treatment of prostate cancer.

The synthesis of 7α-testosterone-chlorambucil hybrid is reported. This compound is made from testosterone in a 6 step reaction sequence and with 23% overall yield. An alternative convergent reaction sequence yielded the same hybrid through a Grubbs metathesis reaction between chlorambucil allyl ester and 7α-allyltestosterone with 35% overall yield. MTT assays showed that the hybrid is selective towards hormone-dependent prostate cancer cell line (LNCaP (AR+)) and shows similar activity than the parent drug, chlorambucil. Thus, the new hybrid shows promising potential for drug targeting of hormone-dependent prostate cancer through its capacity of delivering chlorambucil directly to the site of treatment. This could extend the use of chlorambucil to prostate cancer in the future.

Regulation of the PI3-K/Akt survival pathway in the rat endometrium.

The occurrence of apoptosis and cell survival in the receptive uterus is intimately involved in the embryo implantation process in order to facilitate embryo attachment to the maternal endometrium. The initial stimulus leading to successful implantation might be triggered by the conceptus itself. By the end of rat embryo implantation, decidualization begins, followed by the regression of the decidua basalis on Day 14. The phosphatidylinositol 3-kinase (PI3-K) survival pathway and TGF-beta have been thought to play a role in this process. The objective of the present study was to investigate the regulation of the PI3-K/PTEN/Akt pathway in rat endometrium during pregnancy. Rats were killed on different days of pregnancy (Day 1-22 and postpartum) or pseudopregnancy (Day 1-9), and uteri were removed to collect endometrial tissues. The active form of Akt (pAkt) was increased at Day 5 of pregnancy and at Day 3 of pseudopregnancy as well as at Day 12 of pregnancy and at Day 1 postpartum. Of the three Akt isoforms (Akt1, Akt2, and Akt3), Akt3 was the only isoform phosphorylated at Day 5 during the implantation process and at postpartum as demonstrated by immunoprecipitation studies. PI3-K inhibition in vivo blocked Akt phosphorylation, reduced Smad2 phosphorylation, and reduced both TGF-beta2 and XIAP expression. PI3-K inhibition in cultured decidual cells led to inhibition of pAkt and decrease XIAP expression. These results suggest that Akt and XIAP may be important surviving signaling molecules by which apoptosis is regulated in the rat endometrium during pregnancy and that TGF-beta could be linked to this process.

Involvement of Akt isoforms in chemoresistance of endometrial carcinoma cells.

OBJECTIVE: In tumors, upstream regulation of Akt is affected by oncogenic events which lead to its constitutive activation and promote cell survival. Since studies have demonstrated that the three Akt isoforms exhibit different physiological functions, Akt isoforms may contribute differently in chemoresistance. The objective of the study was to determine the role of each Akt isoforms in chemoresistance. METHODS: We stably transfected the chemoresistant KLE endometrial carcinoma cells with specific shRNAs for Akt1, Akt2 or Akt3. Alternatively, we stably transfected the chemosensitive Hec-1-A endometrial carcinoma cells, in which no Akt activity is detected, with constitutively active Akt expression vectors for each isoform. RESULTS: We demonstrated that Akt1 and Akt2 downregulation by RNAi highly sensitizes KLE cells to cisplatin by inducing the activation of pro-apoptotic factors such as the cleavage of caspases-3, -6, -9 and PARP; downregulation of all Akt isoforms leads to increased sensitivity to doxorubicin while only Akt1-2 downregulation increases taxol sensitivity. Proliferation of Akt1, and mostly Akt2 deficient cells was affected by cisplatin treatment. Constitutive Akt1 or Akt2 expression led to an increased resistance to apoptosis. Akt isoforms have been shown to influence migration in other cancer cells. We showed that Akt2 blocks cell motility, while Akt1-3 had less effect on our endometrial cancer cell models. CONCLUSION: Our findings highlight the contribution of Akt1 and Akt2 in the molecular mechanisms that govern chemoresistance of endometrial carcinomas. Furthermore, Akt isoform-specific transfectants will provide a strong model to determine the involvement of each Akt isoform in tumor progression and metastasis.

Design, synthesis, cytocidal activity and estrogen receptor α affinity of doxorubicin conjugates at 16α-position of estrogen for site-specific treatment of estrogen receptor positive breast cancer.

Doxorubicin (DOX) is an important medicine for the treatment of breast cancer, which is the most frequently diagnosed and the most lethal cancer in women worldwide. However, the clinical use of DOX is impeded by serious toxic effects such as cardiomyopathy and congestive heart failure. Covalently linking DOX to estrogen to selectively deliver the drug to estrogen receptor-positive (ER(+)) cancer tissues is one of the strategies under investigation for improving the efficacy and decreasing the cardiac toxicity of DOX. However, conjugation of drug performed until now was at 3- or 17-position of estrogen, which is not ideal since the hydroxyl groups at this position are important for receptor binding affinity. In this study, we designed, prepared and evaluated in vitro the first estrogen-doxorubicin conjugates at 16α-position of estradiol termed E-DOXs (8a-d). DOX was conjugated using a 3-9 carbon atoms alkylamide linking arm. E-DOXs were prepared from estrone using a seven-step procedure to afford the desired conjugates in low to moderate yields. The antiproliferative activities of the E-DOX 8a conjugate through a 3-carbon spacer chain on ER(+) MCF7 and HT-29 are in the micromolar range while inactive on M21 and the ER(-) MDA-MB-231 cells (>50 µM). Compound 8a exhibits a selectivity ratio (ER(+)/ER(-) cell lines) of >3.5. Compounds 8b-8d bearing alkylamide linking arms ranging from 5 to 9 carbon atoms were inactive at the concentrations tested (>50 µM). Interestingly, compounds 8a-8c exhibited affinity for the estrogen receptor α (ERα) in the nanomolar range (72-100 nM) whereas compound 8d exhibited no affinity at concentrations up to 215 nM. These results indicate that a short alkylamide spacer is required to maintain both antiproliferative activity toward ER(+) MCF7 and affinity for the ERα of the E-DOX conjugates. Compound 8a is potentially a promising conjugate to target ER(+) breast cancer and might be useful also for the design of more potent E-DOX conjugates.

Tub bathing improves thermoregulation of the late preterm infant.

OBJECTIVE: To compare body temperature of the late preterm infant after 24 hours of life at three time points before and after immersion tub bathing or sponge bathing. We hypothesized that late preterm infants achieve significantly improved thermoregulation when bathed by immersion tub bath compared to traditional sponge bathing. DESIGN: This study was a randomized controlled trial. SETTING: A large metropolitan teaching hospital in the northeastern United States. POPULATION: Late preterm infants (100) born between 35 and 36 6/7 weeks gestation, bathed in the well-baby nursery of a 30-bed mother/baby unit. METHODS: Infant participants were identified and informed consent was obtained from the parent. Infants were randomized into two groups: 50 bathed by sponge and 50 bathed by immersion tub. Infant body temperature was measured at three time points: 10 minutes prior to bathing, 10 minutes following bathing, and 30 minutes following bathing. RESULTS: Infants who were tub bathed experienced significantly less variability in body temperature and overall were warmer 10 minutes and 30 minutes following the bath compared to infants who were sponge bathed (p = .024). CONCLUSION: The study findings support the hypothesis that late preterm infants who are tub bathed experience significantly less body temperature variability and an overall higher body temperature following the bathing procedure.

Design of novel tyrosine-nitrogen mustard hybrid molecules active against uterine, ovarian and breast cancer cell lines.

L-para-Tyrosine was linked to ortho-hydroxyaniline, meta-hydroxyaniline and para-hydroxyaniline giving three distinct tyrosinamide molecules. The new extended amino acid derivatives were constructed to imitate, in part, the estradiol (E(2), the natural female sex hormone) nucleus. The resulting tyrosinamides were then linked to chlorambucil either directly, or via a 5 and 10 carbon atoms spacer chain. This was done in an attempt to target cancerous cells expressing the estrogen receptor alpha (ERα) and to obtain a more specific chemotherapeutic agent. The tyrosinamide-chlorambucil molecules were designed and synthesized in good yields, according to two different approaches. The novel compounds were evaluated for their anticancer efficacy in hormone-dependent and hormone-independent (ER+; MCF-7 and ER-; MDA-MB-231) breast cancer cell lines. Interestingly, the meta-hydroxyphenyl-tyrosinamide-chlorambucil derivatives were more active than the ortho- and para- analogs. The molecules bearing a 5 carbon atoms spacer were selected for additional biological study using a panel of female cancerous cells; breast (ZR-75-1, MDA-MB-436, MDA-MB-468), ovarian (OVCAR-3, A2780) and uterine (Ishikawa, HEC-1A). It was discovered that for breast cancer cells, the new compounds were up to 4.2 times more active than chlorambucil itself.

Synthesis, antiproliferative activity and estrogen receptor α affinity of novel estradiol-linked platinum(II) complex analogs to carboplatin and oxaliplatin. Potential vector complexes to target estrogen-dependent tissues.

In the course of efforts to develop 17ß-estradiol-linked to anticancer agents targeting estrogen-dependent tissue, we identified three estradiol-linked platinum(II) complex analogs to cisplatin (E-CDDP) derivatives namely: VP-128 (1), CD-38 (2) and JMP-39 (3) that exhibit potent in vitro and in vivo (for derivative VP-128) activity along with interaction with the estrogen receptor α (ERα). In this study, we prepared and biologically evaluated two novel classes of estradiol-linked platinum(II) complex analogs to carboplatin (E-CarboP, 1a-3a) and oxaliplatin (E-OxaP, 1b-3b). E-CarboP and E-OxaP were designed and based on the estradiol-linker scaffold of E-CDDP derivatives previously identified. Consequently, we assessed the importance of the nature of platinum(II) salt on the antiproliferative activity on MCF-7 and MDA-MB-231 human mammary carcinoma cell lines together with affinity for the ERα by replacing the dichloroplatinum(II) moiety by a cyclobutane-1,1-dicarboxylateplatinum(II) or an oxalateplatinum(II) moiety. Except for compound 3b which is inactive at the concentration tested, the antiproliferative activity of all compounds on both human mammary carcinomas cell lines are in micromolar range and are more active than carboplatin and oxaliplatin alone but less active that their E-CDDP counterparts (1-3). In addition, E-CarboP derivatives 1a-3a show very low affinity for ERα whereas E-OxaPs 1b and 2b show higher affinity for ERα than their parents E-CDDPs (1-2), suggesting that the nature of the platinum(II) salt involved in the vector complexes is extremely important to both retain significant antiproliferative activity and selectivity for the ERα and possibility to target estrogen-dependent tissues. Finally, E-OxaPs 1b and 2b are potentially promising alternatives vector complexes to target estrogen-dependent tissues.

Cisplatin increases B-cell-lymphoma-2 expression via activation of protein kinase C and Akt2 in endometrial cancer cells.

Human carcinomas often show resistance to cisplatin and Bcl-2 is associated with resistance to cisplatin. However, Bcl-2 regulation on cisplatin treatment in human cancers is unknown. Here, we show a novel mechanism by which cisplatin treatment promotes resistance by increasing the expression of Bcl-2 mRNA. Bcl-2 mRNA and protein expression was increased in cisplatin-resistant endometrial cancer cell lines (KLE and HEC-1-A), but not in cisplatin-sensitive cell line (Ishikawa). Cisplatin-mediated increase in Bcl-2 expression was blocked by combination with either actinomycin D or cycloheximide. In addition, Bcl-2 inhibition by HA14-1 led to increased cisplatin-induced apoptosis in KLE and HEC-1-A, whereas Bcl-2 overexpression in Ishikawa led to decreased cisplatin-induced apoptosis. Inhibition of protein kinase C (PKC) activity prevented cisplatin-dependant increase in Bcl-2 mRNA, and induced apoptosis in KLE cells. Furthermore, PKC inhibition was associated with decreased Akt and NF-κB activity. Cells stably expressing shRNA for Akt isoforms revealed that Akt2 was involved in cisplatin-dependant increase in Bcl-2 and apoptosis. Overexpression of Akt2 in Akt2-deficient cells led to increased Bcl-2 expression on cisplatin treatment. Our data suggest a novel regulation pathway of Bcl-2 by cisplatin, via the activation of PKC and Akt2, which has a profound impact on resistance to cisplatin-induced apoptosis in endometrial cancer cells.

SAR study of tyrosine-chlorambucil hybrid regioisomers; synthesis and biological evaluation against breast cancer cell lines.

Amino acids were transformed and coupled to chlorambucil, a well-known chemotherapeutic agent, in an attempt to create new anticancer drugs with selectivity for breast cancer cells. Among the amino acids available, tyrosine was selected to act as an estrogenic ligand. It is hypothesized that tyrosine, which shows some structural similitude with estradiol, could possibly mimic the natural hormone and, subsequently, bind to the estrogen receptor. In this exploratory study, several tyrosine-drug conjugates have been designed. Thus, ortho-, meta- and para-tyrosine-chlorambucil analogs were synthesized in order to generate new anticancer drugs with structural diversity, more specifically in regards to the phenol group location. These new analogs were produced in good yield following efficient synthetic methodology. All the tyrosine-chlorambucil hybrids were more effective than the parent drug, chlorambucil. In vitro biological evaluation on estrogen receptor positive and estrogen receptor negative (ER(+) and ER(-)) breast cancer cell lines revealed an enhanced cytotoxic activity for compounds with the phenol function located at position meta. Molecular docking calculations were performed for the pure L-ortho, L-meta- and L-para-tyrosine phenolic regioisomers. The synthesis of all tyrosine-chlorambucil hybrid regioisomers and their biological activity are reported herein. Possible orientations within the targeted protein [estrogen receptor alpha (ERα)] are discussed in relation to the biological activity.

Oxytocin increases invasive properties of endometrial cancer cells through phosphatidylinositol 3-kinase/AKT-dependent up-regulation of cyclooxygenase-1, -2, and X-linked inhibitor of apoptosis protein.

Traditionally, oxytocin (OT) is well known to play a crucial role in the regulation of cyclic changes in the uterus, implantation of the embryo, and parturition. Recently, an additional role for OT has been identified in several types of cancer cells in which OT acts as a growth regulator. In endometrial cancer cells, OT is known to efficiently inhibit cellular proliferation. In the present study, we show that OT increases invasiveness of human endometrial carcinoma (HEC) cells, which are otherwise resistant to the growth-inhibiting effects of OT. Using pharmacological inhibitors, invasion assay, RNA interference, and immunofluorescence, we found that OT enhances the invasive properties of HEC cells through up-regulation of X-linked inhibitor of apoptosis protein (XIAP), matrix-metalloproteinase 2 (MMP2), and matrix-metalloproteinase 14 (MMP14). In addition, we show that OT-mediated invasion is both cyclooxygenase 1 (PTGS1) and cyclooxygenase-2 (PTGS2) dependent via the phosphatidylinositol 3-kinase/AKT (PIK3/AKT) pathway. PTGS2 knockdown by shRNA resulted in XIAP down-regulation. We also show that OT receptor is overexpressed in grade I to III endometrial cancer. Taken together, our results describe for the first time a novel role for OT in endometrial cancer cell invasion.

Resveratrol modulates the expression of PTGS2 and cellular proliferation in the normal rat endometrium in an AKT-dependent manner.

Resveratrol (trans-3,4N-trihydroxystilbene), a phytoalexin present in grapes and red wine is emerging as a natural compound with anticancer properties. However, the physiological and molecular effects of resveratrol on normal uterine cells are poorly understood. In the present study we evaluated the effects of resveratrol on normal uterine cells and the mechanisms involved in vivo. Healthy immature rats were treated s.c. with resveratrol (0, 0.5, 5, and 50 mg/kg body weight) for 7 consecutive days and euthanized on the eighth day. Uteri were collected and weighed, and endometrium was recovered for total protein extraction, followed by Western blot analysis. Estrogen receptor alpha 1 (ESR1) and beta 2 (ESR2) affinity and activation by resveratrol were also determined by in vitro ESR-binding assays. Immunohistochemistry (IHC) studies were performed to visualize the proliferation marker, proliferating cell nuclear antigen (PCNA), and immunofluorescence (IF) studies were done to study the localization of PTGS2. The results showed that resveratrol increased uterine wet weight and uterine body weight ratios significantly. This local cellular proliferation in terms of the thickening of the columnar epithelial cells and an increase in the number of glands was accompanied by an increase of AKT 16 phosphorylation and PTGS2 and XIAP protein expression. These results were further supported by IF and IHC analyses. Total AKT, ESR1, and ESR2 protein expression levels were not modulated by the treatment; however, resveratrol showed moderate estrogenicity for both ESR isoforms. Expression of progesterone receptor A (PGR) was induced in the presence of resveratrol. These data support the hypothesis that resveratrol can act in a prosurvival or antiapoptotic way through AKT, XIAP, and PTGS2 regulation in the endometrium and could positively affect the outcome of pregnancy and favor fertility.

Design, synthesis and biological evaluation of estradiol-PEG-linked platinum(II) hybrid molecules: comparative molecular modeling study of three distinct families of hybrids.

The synthesis of a series of 17ß-estradiol-platinum(II) hybrid molecules is reported. The hybrids are made of a PEG linking chain of various length and a 2-(2'-aminoethyl)pyridine ligand. They are prepared from estrone in only 5 chemical steps with an overall yield of 22%. The length of the PEG chain does not influence the solubility of the compounds as it remains relatively constant throughout the series. MTT assays showed that the derivative with the longest PEG chain showed the best activity against two human breast cancer cell lines (MCF-7 and MDA-MB-231). The novel PEG-hybrids are also compared in terms of activities with two other families of 17ß-estradiol-platinum(II) hybrids that we reported in previous studies. Molecular modeling study performed on a representative member of each family of hybrids reveals distinct molecular interactions with the estrogen receptor α which further corroborates their notably contrasting cytocidal activities on breast cancer cell lines. This study also shows that lipophilicity and the orientation of the tether chain between the estrogenic portion and the platinum(II) core contribute markedly to the biological activity of the various families of hybrids. The most active hybrids are those possessing an alkyl tether chain at position 16ß of the steroid nucleus. For example, derivative 3 (p=6) is about 16 times more potent on MCF-7 breast cancer cells than the corresponding 16α-PEG-hybrids (2b) made in this study.

Synthesis of D- and L-tyrosine-chlorambucil analogs active against breast cancer cell lines.

A series of D- and L-tyrosine-chlorambucil analogs was synthesized as anticancer drugs for chemotherapy of breast cancer. The novel compounds were synthesized in good yields through efficient modifications of D- and L-tyrosine. The newly synthesized compounds were evaluated for their anticancer efficacy in different hormone-dependent and hormone-independent (ER+ and ER-) breast cancer cell lines. The novel analogs showed significant in vitro anticancer activity when compared to chlorambucil. Structure-activity relationship (SAR) reveals both, the influence of the length of the spacer chain and the stereochemistry of the tyrosine moiety. Interestingly, the D- and L-tyrosinol-chlorambucil derivatives with 10 carbon atoms spacer are selective towards MCF-7 (ER+) breast cancer cell line.

Expression of COX-1 and COX-2 in the endometrium of cyclic, pregnant and in a model of pseudopregnant rats and their regulation by sex steroids.

BACKGROUND: Cyclooxygenases (COXs) are the rate limiting enzymes in the process of prostaglandins (PGs) synthesis, which are critical regulators of a number of reproductive processes, including ovulation, implantation, decidualization and parturition. The aim of the present study was to investigate the expression and regulation of COX-1 and COX-2 and levels of prostaglandins during rat pregnancy, in a model of pseudopregnancy and estrous cycle. METHODS: Uteri were collected from the cyclic rats on each day of estrous cycle, after every two days for pregnant (days 2 to 22) and pseudopregnant rats (days 1 to 9). In vitro primary endometrial stromal cells were cultured in the presence of steroid hormones and their respective inhibitors for the possible modulation of COX-1 and COX-2. Endometrial protein extracts were used for western blot analysis and tissue sections were prepared for protein localization using immunofluorescence. Measurements of PGF2alpha and PGE2 metabolites in serum were performed by enzyme immunoassay (EIA). RESULTS: COX-1 expression was found to be elevated during implantation and parturition, however, the levels of COX-1 decreased during decidualization periods. COX-2 was detected during early pregnancy from day 2 to 5, increased during decidual regression, and was also expressed at the time of parturition. COX-2 protein expression was found to be increased at estrus phase in cyclic rats. Both enzymes were found to be modulated in the endometrium of pseudopregnant rats, suggesting that they are regulated by 17beta-estradiol and progesterone. A significant increase in PGE2 metabolite levels was observed on day 10, 12 and 14 of pregnancy. However, an increase in PGF2alpha metabolite levels was observed only on day 14. The concentration of both these metabolites changed during pseudopregnancy and maximum levels were observed at day 7. Significant increase in PGE2 metabolite was observed at proestrus phase, on the other hand, PGF2alpha metabolite was significantly increased at proestrus and metestrus phase. COX-2 protein was regulated by 17beta-estradiol in cultured endometrial stromal cells which was blocked in the presence of ICI-182,780. CONCLUSIONS: Taken together, these results suggest that COX-1 and COX-2 could be differentially regulated by steroid hormones and might be the key factors involved in embryo implantation, decidualization, decidua basalis regression and parturition in rats.

XIAP gene expression and function is regulated by autocrine and paracrine TGF-beta signaling.

BACKGROUND: X-linked inhibitor of apoptosis protein (XIAP) is often overexpressed in cancer cells, where it plays a key role in survival and also promotes invasiveness. To date however, the extracellular signals and intracellular pathways regulating its expression and activity remain incompletely understood. We have previously showed that exposure to each of the three TGF-beta (transforming growth factor beta) isoforms upregulates XIAP protein content in endometrial carcinoma cells in vitro. In the present study, we have investigated the clinical relevance of TGF-beta isoforms in endometrial tumours and the mechanisms through which TGF-beta isoforms regulate XIAP content in uterine cancer cells. METHODS: TGF-beta isoforms immunoreactivity in clinical samples from endometrial tumours was assessed using immunofluorescence. Two model cancer cell lines (KLE endometrial carcinoma cells and HeLa cervical cancer cells) and pharmacological inhibitors were used to investigate the signalling pathways regulating XIAP expression and activity in response to autocrine and paracrine TGF-beta in cancer cell. RESULTS: We have found immunoreactivity for each TGF-beta isoform in clinical samples from endometrial tumours, localizing to both stromal and epithelial/cancer cells. Blockade of autocrine TGF-beta signaling in KLE endometrial carcinoma cells and HeLa cervical cancer cells reduced endogenous XIAP mRNA and protein levels. In addition, each TGF-beta isoform upregulated XIAP gene expression when given exogenously, in a Smad/NF-kappaB dependent manner. This resulted in increased polyubiquitination of PTEN (phosphatase and tensin homolog on chromosome ten), a newly identified substrate for XIAP E3 ligase activity, and in a XIAP-dependent decrease of PTEN protein levels. Although each TGF-beta isoform decreased PTEN content in a XIAP- and a Smad-dependent manner, decrease of PTEN levels in response to only one isoform, TGF-beta3, was blocked by PI3-K inhibitor LY294002. CONCLUSIONS: XIAP gene expression and function is positively regulated by exposure to the three TGF-beta isoforms in a Smad-dependent manner, similar to constitutive XIAP gene expression which depends on autocrine TGF-beta/Smad signalling.

First synthesis of separable isomeric testosterone dimers showing differential activities on prostate cancer cells.

The synthesis of two separable isomeric testosterone dimers is reported. The dimers are made from testosterone in a 5 step sequence and with 36% overall yield. The key dimerization step was performed using Hoveyda-Grubb's metathesis catalysts on 7alpha-allyltestosterone with 75% yield. The synthesis led to separable isomeric dimers (trans and cis, 2:1). X-ray diffraction crystallography, performed on monocrystal of the minor isomer, confirms the cis geometry of the double bound between the two testosterone units. MTT assays showed that the cis dimer has the highest activity against prostate cancer cell lines. The novel cis dimer is more active than the antiandrogen cyproterone acetate indicating the possible therapeutic value of this molecule.

Design, synthesis and biological evaluation of estradiol-chlorambucil hybrids as anticancer agents.

A series of estradiol-chlorambucil hybrids was synthesized as anticancer drugs for site-directed chemotherapy of breast cancer. The novel compounds were synthesized in good yields through efficient modifications of estrone at position 16alpha of the steroid nucleus. The newly synthesized compounds were evaluated for their anticancer efficacy in different hormone-dependent and hormone-independent breast cancer cell lines. The novel hybrids showed significant in vitro anticancer activity when compared to chlorambucil. Structure-activity relationship (SAR) reveals the influence of the length of the spacer chain between carrier and drug molecule.