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

Opposite regulation of XIAP and Smac/DIABLO in the rat endometrium in response to 17β-estradiol at estrus

During rat estrous cycle, the endometrium proliferates in response to sex steroids and specific endometrial epithelial cells undergo apoptosis in absence of embryonic factors. The central executioner of apoptosis is a family of aspartic acid-specific cysteine proteases known as caspases. Smac/DIABLO is released from the mitochondria during apoptosis and its stimulation promotes caspases activation by neutralizing members of the inhibitor of apoptosis proteins (IAPs) family, such as X-linked inhibitor of apoptosis protein (XIAP). The aim of this study was to investigate the involvement of Smac/DIABLO and XIAP in the control of caspases activation in endometrium of cycling rats. Polyoestrus female rats were sacrificed at each stage of estrous cycle (diestrus, proestrus, estrus, and metestrus). Endometrial protein extracts were collected to perform Western Blot analysis. Alternatively, uterine horns were sectioned for immunohistochemistry (IHC). We and others showed previously the presence of apoptosis at estrus in rat uterine epithelium. In the present study, cleaved caspase-3, -6, and -7 fragments were detected at estrus. IHC confirmed that caspase-3 was present only in luminal and glandular epithelium at estrus. XIAP was highly expressed at estrus in both epithelial and stromal cells. In contrast, expression of Smac/DIABLO was elevated at diestrus, proestrus and metestrus but was minimal at estrus. Treatment of ovariectomized rats with 17β-estradiol induced XIAP expression and inhibited Smac/DIABLO protein expression in the endometrium. Cleaved caspase-3, -6, and -7 fragments increased in endometrial protein extracts following 17β-estradiol treatment. Expression of NF-κB and IκB proteins, and IκB phosphorylation status were detected in the endometrium but were not influenced by the estrous cycle. These findings suggest that Smac/DIABLO and XIAP are regulated differently and may play important roles in the regulation of endometrial cell fate. Moreover, this study confirms a key role for executioner caspases in the control of apoptotic processes at estrus in the rat uterus

Characterization of EN-1078D, a poorly differentiated human endometrial carcinoma cell line: a novel tool to study endometrial invasion in vitro

<p>Abstract</p> <p>Background</p> <p>To date, tools to study metastasis in endometrial cancers are insufficiently developed. The aim of this study was to characterize the cell line EN-1078D, a new endometrial carcinoma cell line derived from a metastasis to the ovary.</p> <p>Methods and Results</p> <p>Cells were characterized using cytology, transmission electron microscopy, karyotyping and morphological appearance in culture. Molecular features were determined by RT-PCR, Western Blot, FISH and sequencing. MTT proliferation assays were performed to investigate the sensitivity of EN-1078D to anticancer agents such as cisplatin and doxorubicin. Also, subcutaneous and intravenous injections in nude mice were done to test the tumorigenic and metastatic properties of EN-1078D cells. Our results indicate that EN-1078D cells express both oestrogen receptors isoforms (ER alpha and ER beta) and also low levels of progesterone receptor B (PR-B). In addition, this cell line expresses high levels of MMP-2 and MMP-14 mRNA, low levels of TIMP-1 and TIMP-2 transcripts and no detectable levels of MMP-9 mRNA. Moreover, all nude mice developed tumors by subcutaneous injections and cell invasion was observed in vitro in response to TGF-beta 3. Her-2/neu was not overamplified but mutations in the C-2 domain of PTEN gene as well as codon 12 of the K-Ras gene were found. Finally, EN-1078D shows sensitivity to drugs commonly used in chemotherapy such as cisplatin and doxorubicin: IC50 of 2.8 μM of cisplatin after 72 hours of exposure and 0.54 μM of doxorubicin after 48 hours.</p> <p>Conclusion</p> <p>Taken together, these results suggest that EN-1078D will be an excellent tool to study the properties of metastatic endometrial cancer cells in vitro and their regulation by sex steroids.</p

A) Phase contrast features of the monolayer-cultured EN-1078D cells revealing a sheet of polygonal cells with a pavement-like arrangement

<p><b>Copyright information:</b></p><p>Taken from "Characterization of EN-1078D, a poorly differentiated human endometrial carcinoma cell line: a novel tool to study endometrial invasion in vitro"</p><p>http://www.rbej.com/content/5/1/38</p><p>Reproductive biology and endocrinology : RB&E 2007;5():38-38.</p><p>Published online 25 Sep 2007</p><p>PMCID:PMC2092433.</p><p></p> B) Transmission electron microscopy showing the tendency of cells to pile up. C) Ultrastructural aspects of EN-1078D cell line by transmission electron microscopy. Cultured cells show a high nucleus-cytoplasmic ratio, euchromatic nuclei, prominent nucleoli and well-developed microvilli. D) Higher magnification shows intracytoplasmic lipid droplets (L), and exhibits intricate cytoplasmic interdigitations but no desmosomes were observed

Determination of EN-1078D sensitivity to cisplatin (left panel) and doxorubicin (right panel)

<p><b>Copyright information:</b></p><p>Taken from "Characterization of EN-1078D, a poorly differentiated human endometrial carcinoma cell line: a novel tool to study endometrial invasion in vitro"</p><p>http://www.rbej.com/content/5/1/38</p><p>Reproductive biology and endocrinology : RB&E 2007;5():38-38.</p><p>Published online 25 Sep 2007</p><p>PMCID:PMC2092433.</p><p></p> A, D) 24 hours of exposure to compounds B, E) 48 hours and C, F) 72 hours. Both Hela and EN-1078D are sensitive to cisplatin after 24 hours in contrast to KLE, which is chemoresistant. The three cell lines seem not to be affected by doxorubicin after exposure of 24 hours. KLE is more resistant than other cells lines for both 48 and 72 hours. Data shown are representative of results obtained from three independent assays