Valérie Leblanc

X-linked Inhibitor of Apoptosis Protein (XIAP) Regulates PTEN Ubiquitination, Content, and Compartmentalization

Apoptotic cell death plays a normal role in various physiological processes, and deregulated apoptosis is a hallmark of several diseases, including cancer. Cell fate is dictated by the balance between pro- and antiapoptotic factors. Akt is one of these antiapoptotic factors, which must be activated through phosphorylation. The phosphorylation of Akt has previously been shown to be promoted by X-linked inhibitor of apoptosis protein (XIAP), another antiapoptotic protein dictating the fate of normal and cancer cells. However, the underlying mechanisms are poorly understood. We have observed that XIAP associates with PTEN (phosphatase and tensin homolog deleted on chromosome ten), the best characterized negative regulator of Akt phosphorylation, in vitro and in vivo. XIAP knockdown reduces constitutive mono- and polyubiquitination of PTEN, increases PTEN protein levels, and prevents nuclear accumulation of PTEN. Overexpression of XIAP induces polyubiquitination of PTEN and proteasome-dependent decrease of PTEN protein levels. RNA interference experiments showed that XIAP-induced regulation of Akt phosphorylation is PTEN-dependent. Additional experiments confirmed that XIAP also regulates PTEN in vivo; primary mouse embryonic fibroblasts derived from XIAP−/− mice contain higher levels of PTEN protein, less mono- and polyubiquitinated PTEN, and less nuclear PTEN than primary mouse embryonic fibroblasts derived from XIAP+/+ mice. Finally, we found that XIAP can directly ubiquitinate PTEN in vitro. We thus propose that XIAP acts as an E3 ubiquitin ligase for PTEN and promotes Akt activity by regulating PTEN content and compartmentalization.

TGF-beta expression during rat pregnancy and activity on decidual cell survival

BackgroundDuring early rat pregnancy, trophoblast of the tiny embryo joins with the endometrium and epithelial cells undergo apoptosis. Near the end of pregnancy, regression of the decidua basalis (DB) is also observed (from day 14 to 20). However, little is known about the intra-cellular and molecular mechanisms involved in apoptosis regulation in the uterus during pregnancy. The objective of the present study was to investigate the presence and the developmental expression of transforming growth factor-beta isoforms (TGF-beta well known differentiation factor) in the rat endometrium throughout pregnancy and its action in vitro using cultured endometrial stromal cells.MethodsIn vivo: Rats were killed at different days of pregnancy (days 2–20) and uteri removed to collect endometrial protein extracts or the uteri were fixed, embedded and sectioned for immunohistochemistry (IHC) and in situ cell death analyses using TdT-mediated dUTP nick end labeling (TUNEL). In vitro: Rats were ovariectomized and decidualization was induced using sex steroids. Endometrial stromal decidual cells were then collected and cultured.ResultsAn increase of apoptosis in the DB on days 14, 16 and 18 was observed. Cleaved caspase-3 was clearly detected during regression of the DB by Western analysis and immunofluorescence. Western analyses using endometrial protein extracts demonstrated that TGF-beta1, TGF-beta2 and TGF-beta3 were highly expressed at the time of DB regression (day 14). During early pregnancy, TGF-beta1 and -beta2 expressions raised at days 5.5 to 6.5. TGF-beta3 protein was not detected during early pregnancy. IHC analyses revealed that TGF-beta1 and -2 were found surrounding both epithelium (luminal and glandular) in the stroma compartment at the implantation site, and TGF-beta3 was mainly located surrounding endometrial epithelium in the stroma compartment. Smad2 phosphorylation was increased at the time of DB regression. In vitro studies using decidual endometrial stromal cells revealed that TGF-beta1 induced apoptosis and Smad2 phosphorylation. Moreover, TGF-beta1 reduced both Akt (a well known survival factor) phosphorylation and XIAP (X-linked inhibitor of apoptosis protein) expression in decidual endometrial stromal cells in vitro.ConclusionTaken together, these results suggest that TGF-beta isoforms are regulated differently during pregnancy and may have an important role in the control of apoptosis and cell survival at specific stages during pregnancy.

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.

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

BackgroundCyclooxygenases (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.MethodsUteri 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).ResultsCOX-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.ConclusionsTaken 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.

Regulation of Akt expression and phosphorylation by 17β-estradiol in the rat uterus during estrous cycle

Molecular and intra-cellular mechanisms involved in the regulation of apoptosis processes in endometrial cells are poorly understood and documented. We have investigated the possibility that Akt survival pathway might be involved in the regulation of apoptosis in the uterus during the estrous cycle. Rats with regular estrous cycle (4 days) were killed at different days of estrous cycle (diestrus, proestrus, estrus and metestrus). Uteri were collected and fixed for immunohistochemical staining (IHC) and apoptotic cell death detection by [TdT]-mediated deoxyuridinetriphosphate nick end-labelling (TUNEL) or endometrial protein extracts collected for Western analysis. TUNEL analysis revealed that apoptosis was mainly found at estrus compared to other day of estrous cycle. TUNEL positive cells were apparent in luminal epithelial cells only. No apoptotic cells were observed at proestrus. In contrast, proliferation was maximal at proestrus as confirmed with the expression of CDC47/MCM7 (a cell proliferation marker). Intact form of caspase-3 was maximal at proestrus and was reduced only at estrus. Likewise, presence of a specific cleaved caspase-3 fragment was observed only at estrus and IHC revealed that cleaved caspase-3 signal was found in luminal epithelial cells. PTEN protein, a phosphatase involved in the regulation of Akt phosphorylation, was present at all days of estrous cycle and showed no significant regulation in relation to cycle. Expression of phospho-Akt (the activated form of Akt) was present at metestrus, diestrus, and proestrus but decreased significantly at estrus. Akt protein expression was maximal at estrus. IHC revealed that Akt expression was high in both stromal and epithelial cells at estrus. Further studies using ovariectomized rats demonstrated that 17β-estradiol increased endometrial cell proliferation which was accompanied by an increase of both Akt expression and phosphorylation. These results suggest that increased Akt expression and activity in response to estradiol may be an important mechanism to protect endometrial cells from apoptotic triggering and to induce endometrial cell proliferation, whereas inhibition of Akt activity leads to caspase-3 activation and apoptosis in endometrial cells.

Improved synthesis of unique estradiol-linked platinum(II) complexes showing potent cytocidal activity and affinity for the estrogen receptor alpha and beta

We have recently reported the synthesis of a platinum(II) complex, made of estradiol, the female sex hormone, and a cisplatin analog, an anticancer drug, linked together by an eleven carbon atoms chain. The novel estradiol-Pt(II) hybrid molecule was synthesized in nine chemical steps with 10% overall yield. This new compound has been tested in vitro on estrogen-dependent (MCF-7) and -independent (MDA-MD-231) (ER(+) and ER(-)) cell lines. Interestingly, the biological activity was quite significant, more potent than that of cisplatin, the compound currently used in chemotherapy. The estrogen receptor binding affinity (ERBA) of this compound was very similar to that of 17beta-estradiol (E(2)) on both estrogen receptors (ERs), alpha and beta. In order to further study this type of molecule, we have decided to synthesize several analogs with the same estrogenic scaffold but with various chain lengths separating the estradiol from the toxic part of the molecule. This was planned in order to study the effect of the length of the linking chain on the biological activity of the hybrids. Four E(2)-Pt(II) hybrid molecules having 6-14 carbon atoms linking chain have been synthesized using a new synthetic methodology. They are synthesized in only eight chemical steps with 21% overall yield. The 17beta-estradiol-linked platinum(II) complexes have been tested for their receptor binding affinity as well as for their cytocidal activity on several breast cancer cell lines. The synthesis and biological results are reported herein.

VP-128, a novel oestradiol-platinum(II) hybrid with selective anti-tumour activity towards hormone-dependent breast cancer cells in vivo.

We have previously reported the synthesis of VP-128, a new 17beta-oestradiol (E(2))-linked platinum(II) hybrid with high affinity for oestrogen receptor alpha (ERalpha). In the present study, we have investigated the anti-tumour activity of VP-128 towards breast cancer cells in vitro and in vivo. We used human ERalpha-positive (MCF-7) and -negative (MDA-MB-468) cells as a model for treatment with increasing doses of VP-128, cisplatin or E(2) in vitro and for xenograft experiments in nude mice in vivo. Compared with cisplatin, VP-128 showed markedly improved in vitro and in vivo anti-tumour activity towards ERalpha-positive MCF-7 breast cancer cells, without increased systemic toxicity. In these caspase-3-deficient cells, treatment with VP-128 overcame weak cellular sensitivity to cisplatin in vitro and in vivo. In these cells, only the hybrid induced apoptosis in an ERalpha-dependent manner, inactivated both X-linked inhibitor of apoptosis protein and Akt, and induced selective nuclear accumulation of ERalpha and the expression of ER-regulated genes c-myc and tff1, which was blocked by ERalpha-specific antagonist ICI 282 780. In the case of ERalpha-negative MDA-MB-468 cells, VP-128, but not cisplatin, induced nuclear accumulation of apoptosis-inducing factor and inhibited c-myc expression. However, VP-128 did not show enhanced in vivo anti-tumour activity compared with cisplatin. These results reveal two different modes of action for VP-128 in ERalpha-positive and -negative breast cancer cells, and highlight the promising therapeutic value of this unique E(2)-platinum hybrid for selective targeting of hormone-dependent cancers.

Transforming growth factor beta isoforms regulation of Akt activity and XIAP levels in rat endometrium during estrous cycle, in a model of pseudopregnancy and in cultured decidual cells.

BackgroundDuring the estrous cycle, the rat uterine endometrium undergoes many changes such as cell proliferation and apoptosis. If implantation occurs, stromal cells differentiate into decidual cells and near the end of pregnancy, a second wave of apoptosis occurs. This process called decidual regression, is tightly regulated as is it crucial for successful pregnancy. We have previously shown that TGF-beta1, TGF-beta2 and TGF-beta3 are expressed in the endometrium during decidual basalis regression, but although we had demonstrated that TGF- beta1 was involved in the regulation of apoptosis in decidual cells, the ability of TGF- beta2 and TGF-beta3 isoforms to trigger apoptotic mechanisms in these cells remains unknown. Moreover, we hypothesized that the TGF-betas were also present and regulated in the non-pregnant endometrium during the estrous cycle. The aim of the present study was to determine and compare the specific effect of each TGF-β isoform in the regulation of apoptosis in sensitized endometrial stromal cells in vitro, and to investigate the regulation of TGF-beta isoforms in the endometrium during the estrous cycle in vivo.MethodsRats with regular estrous cycle (4 days) were killed at different days of estrous cycle (diestrus, proestrus, estrus and metestrus). Pseudopregnancy was induced with sex steroids in ovariectomized rats and rats were killed at different days (days 1–9). Uteri were collected and either fixed for immunohistochemical staining (IHC) or processed for RT-PCR and Western analyses. For the in vitro part of the study, rats were ovariectomized and decidualization was induced using sex steroids. Endometrial stromal decidual cells were purified, cultured and treated with different concentrations of TGF-beta isoforms.ResultsOur results showed that all three TGF-beta isoforms are present, but are localized differently in the endometrium during the estrous cycle and their expression is regulated differently during pseudopregnancy. In cultured stromal cells, we found that TGF-beta3 isoform induced Smad2 phosphorylation, indicating that the Smad pathway is activated by TGF-beta3 in these cells. Furthermore, TGF-beta2 and TGF-beta3 induced a dose-dependant increase of apoptosis in cultured stromal cells, as demonstrated by Hoechst nuclear staining. Noteworthy, TGF-beta2 and TGF-beta3 reduced the level of the anti-apoptotic XIAP protein, as well as the level of phosphorylated/active Akt, a well known survival protein, in a dose-dependent manner.ConclusionThose results suggest that TGF-beta might play an important role in the remodelling endometrium during the estrous cycle and in the regulation of apoptosis in rat decidual cells, in which inhibition of Akt survival pathway might be an important mechanism involved in the regulation of apoptosis.

Synthesis and cytotoxic activity of benzopyran-based platinum(II) complexes.

A series of benzopyran-based platinum complexes of types 4 and 5 were synthesized as potential anticancer agents. The novel compounds were synthesized in several steps using simple and efficient chemistry. The newly synthesized compounds were evaluated for their biological efficacy and showed significant in vitro cytotoxic activity in different hormone-dependent and -independent breast cancer cell lines. Docking and other molecular modeling experiments were also performed for one of the potent compounds, 5f, which showed that both the possible enantiomeric forms (5f with 3R,4R and 5f with 3S,4S) of the molecule have comparable lowest energy (for 5f with 3R,4R, -31.953 kcal/mol and for 5f with 3S,4S, -31.944 kcal/mol). The 3D QSAR was examined for the derivatives of both enantiomeric forms and a novel relationship for the 3S,4S derivatives is discussed.

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.