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Transforming growth factor beta enhances endometrial epithelial cell invasion by transforming growth factor beta receptor III signaling
P-370 Wednesday, October 27, 2010
P-372 Wednesday, October 27, 2010
ASSOCIATION OF WT1 AND INDUCTION OF APOPTOSIS IN ENDOMETRIOTIC CELL LINES TREATED WITH MIS. S. F. Karipcin, L. Fangxian, M. Borahay, I. Tekedereli, B. Gurates, S. G. Kilic. Obstetrics and Gynecology, UTMB in Galveston, Galveston, TX; Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX; Department of Obstetrics & Gynecology, Firat University, Elazig, Turkey.
TRANSFORMING GROWTH FACTOR BETA ENHANCES ENDOMETRIAL EPITHELIAL CELL INVASION BY TRANSFORMING GROWTH FACTOR BETA RECEPTOR III SIGNALING. J. F. Knudtson, P. A. Binkley, R. S. Schenken, N. B. Kirma. Obstetrics and Gynecology, University of Texas Health Science Center at San Antonio, San Antonio, TX.
OBJECTIVE: Products of steroidogenic factor-1 (SF-1) and Wilms’ tumor-1(WT1) genes are essential for mammalian gonadogenesis prior to sexual differentiation. Prior studies demonstrated WT1 inhibits cAMP SF-1 pathway dependent p450arom expression in cultured endometriotic and endometrial stromal cells. Mullerian inhibiting substance is first identified as a factor that causes regression of Mullerian ducts in developing male embryos. Recent research has revealed MIS causes cell-cycle arrest in ovarian, cervical and endometrial cancer cell lines in vitro. In our unpublished data, we showed MIS induces apoptosis and inhibits basal autophagy in ectopic endometrial cells. We hypothesized that WT1 and cAMP SF-1 dependent p450arom plays a role in the effect of MIS on endometriotic cell lines. In this study we aimed to explain the association of WT1 and induction of apoptosis with MIS treatment in endometriosis cell lines. DESIGN: We studied WT1 protein expression in endometriotic cell lines treated with MIS compared to no treatment. MATERIALS AND METHODS: Cultured CRL-7566 endometriosis cell lines from ovarian cyst (ATCC) were harvested at 80% confluency and placed in wells. Each well was treated with 5mcg/ml human recombinant MIS twice 4 days apart or with PBS as control. WT1 protein expression was determined using Western analysis and normalized to b-actin. RESULTS: MIS treatment significantly induced WT1 expression on endometriotic stromal cells(p<0.05) compared to the sample treated with control. CONCLUSION: MIS induces apoptosis, inhibits cellular growth and basal autophagy in endometriosis cell lines. MIS treatment induces WT1 expression which may partially explain the pathway that leads to apoptosis. Supported by: UTMB Chairman’s Career Development Grant.
OBJECTIVE: To investigate the involvement of transforming growth factor beta receptor III (TGFBRIII) in transforming growth factor beta (TGF-b) enhancement of transmesothelial invasion in endometrial cells, an essential step in endometriotic lesion development. DESIGN: TGFBRIII deficient and control immortalized endometrial cells were introduced into a three dimensional in vitro system modeling the peritoneum with and without the addition of TGF-b. MATERIALS AND METHODS: Specific siRNA transfection was used to knockdown expression of TGFBRIII in EM 42 cells. Receptor expression was assessed with Real Time PCR and Western blot analysis. An established three dimensional in vitro system modeling the peritoneum was used for invasion assays. Differences were assessed by one-way ANOVA. RESULTS: TGF-b1 significantly increased invasion of control immortalized endometrial epithelial cells containing scrambled non-silencing siRNA through an in vitro transmesothelial membrane (p¼ 0.004). Invasion of TGFBRIII knockdown immortalized endometrial cells following treatment with TGF-b1 was significantly less than controls treated with TGF-b1 (p¼0.004). CONCLUSION: TGFBRIII mediates TGF-b1 enhanced endometrial epithelial cell invasiveness by TGF-b. Combined with our previous studies showing that TGFBRI antagonists are potent inhibitors of TGF-b1 induced transmesothelial invasion, our data suggest that drugs targeting TGFBRIII pathways maybe useful in the treatment of endometriosis.
P-373 Wednesday, October 27, 2010 P-371 Wednesday, October 27, 2010 ABERRANT EXPRESSION OF IAP FAMILY IN ENDOMETRIOTIC CELLS MAY CAUSE RESISTANCE TO APOPTOSIS. F. Taniguchi, M. Izawa, T. Iwabe, N. Terakawa, T. Harada. Ob/Gyn, Tottori University Faculty of Medicine, Yonago, Japan; Biosignaling, Tottori University Faculty of Medicine, Yonago, Japan. OBJECTIVE: Inhibitor of apoptosis protein (IAP) family may be closely linked to the development of endometriosis. We sought to determine the difference of IAP family expression and the susceptibility to apoptosis between ovarian endometriomas and eutopic endometrial tissues. DESIGN: Experimental study. MATERIALS AND METHODS: Ectopic endometrial tissue from ovarian endometriomas and matched eutopic endometrial tissue samples were collected from premenopausal women (n¼22). Eutopic endometrial tissues (n¼20) from infertile women with benign gynecologic diseases and no evidence of endometriosis were used as control. Ectopic or eutopic endometrial stromal cells (ESCs) were isolated from these tissues.Gene and protein expression patterns for IAP family, cIAP-1 (birc2), cIAP-2 (birc3), XIAP (birc4), and survivin (birc5) were evaluated by real-time RT-PCR and immunohistochemical staining. Number of surviving cells and activation of caspases were assessed by WST-8 assay, Annexin-V staining and immunoblotting. RESULTS: All 4 IAP family mRNAs were more intensively expressed in endometriotic tissues derived from ovarian endometrioma compared with those of eutopic endometrial tissues. Survivin mRNA expression in endometriotic tissues showed 2.4-fold increase than the control. IAP family mRNA expression in eutopic endometrial tissues of women with ovarian endometriomas was higher than the control. Both epithelial and stromal cells of ovarian endometriotic tissues exhibited higher protein expression of these 4 IAPs than eutopic endometrial tissues. After induction of apoptosis by staurosporine (SS), 55% of eutopic ESCs survived versus 70% of ectopic ESCs. Procaspase-3 or -7 was more activated by SS in eutopic than in ectopic ESCs. Survivin gene silencing in SS-treated ectopic ESCs led to activation of caspases and increase of apoptotic cells. CONCLUSION: Aberrant expression of IAP family genes in ovarian endometriomas may sustain their abnormal survival in ectopic sites. Supported by: Ministry of Education, Science, Culture, Sports and technology of Japan.
FERTILITY & STERILITYÒ
EPITHELIAL-MESENCHYMAL TRANSITION MIGHT BE MICROENVIRONMENT-DEPENDENT DURING THE EVOLUTION OF ENDOMETRIOTIC IMPLANTS. S. Matsuzaki, C. Darcha, E. Maleysson, M. Canis, G. Mage. CHU Clermont-Ferrand, Clermont-Ferrand, Auvergne, France. OBJECTIVE: Our previous study demonstrated that expression levels of E-cadherin were distinctive among different forms of endometriosis. We further investigated several markers of epithelial-mesenchymal transition (EMT) (N-cadherin, vimentin, S100A4, dephosphorylated form of beta-catenin) in endometriosis. DESIGN: Prospective study. MATERIALS AND METHODS: Samples of deep infiltrating endometriosis (n¼ 61), ovarian endometriosis (n¼48) and superficial peritoneal endometriosis (red lesions: n¼30, black lesions: n ¼ 46) were collected during laparoscopic surgery. In addition, samples of menstrual endometrium, abdominal wall endometriosis, other benign ovarian cysts, carcinoma arising in ovarian endometriosis, colon endometriosis and scar endometriosis, were included in the present study for comparison. Immunohistochemical staining was performed on paraffin sections and quantitation of immunostained cells (percentage of immunostained surface mean staining intensity) was obtained using a computerized image analysis system. RESULTS: Vimentin expression was detected in the menstrual endometrium, whereas no expression of N-cadherin, S100A4 and dephosphorylated beta-catenin was detected. S100A4 expression was significantly lower in deep infiltrating endometriosis compared to that of ovarian endometriosis and red peritoneal lesions. Vimentin expression was significantly lower in ovarian endometriosis compared to that of deep infiltrating endometriosis, red and black peritoneal lesions. In addition, vimentin expression in deep infiltrating endometriosis, red and black peritoneal lesions was significantly higher than that of menstrual endometrium. Dephosphorylated beta-catenin expression was significantly higher in deep infiltrating endometriosis compared to that of ovarian endometriosis, red and black peritoneal lesions. CONCLUSION: The present findings suggested that markers of EMT might be gained depending on the microenvironment during the evolution of endometriotic implants. Supported by: Karl Storz Endoscopy & GmbH (Tuttlingen, Germany).
S201
P-372 Wednesday, October 27, 2010
ASSOCIATION OF WT1 AND INDUCTION OF APOPTOSIS IN ENDOMETRIOTIC CELL LINES TREATED WITH MIS. S. F. Karipcin, L. Fangxian, M. Borahay, I. Tekedereli, B. Gurates, S. G. Kilic. Obstetrics and Gynecology, UTMB in Galveston, Galveston, TX; Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX; Department of Obstetrics & Gynecology, Firat University, Elazig, Turkey.
TRANSFORMING GROWTH FACTOR BETA ENHANCES ENDOMETRIAL EPITHELIAL CELL INVASION BY TRANSFORMING GROWTH FACTOR BETA RECEPTOR III SIGNALING. J. F. Knudtson, P. A. Binkley, R. S. Schenken, N. B. Kirma. Obstetrics and Gynecology, University of Texas Health Science Center at San Antonio, San Antonio, TX.
OBJECTIVE: Products of steroidogenic factor-1 (SF-1) and Wilms’ tumor-1(WT1) genes are essential for mammalian gonadogenesis prior to sexual differentiation. Prior studies demonstrated WT1 inhibits cAMP SF-1 pathway dependent p450arom expression in cultured endometriotic and endometrial stromal cells. Mullerian inhibiting substance is first identified as a factor that causes regression of Mullerian ducts in developing male embryos. Recent research has revealed MIS causes cell-cycle arrest in ovarian, cervical and endometrial cancer cell lines in vitro. In our unpublished data, we showed MIS induces apoptosis and inhibits basal autophagy in ectopic endometrial cells. We hypothesized that WT1 and cAMP SF-1 dependent p450arom plays a role in the effect of MIS on endometriotic cell lines. In this study we aimed to explain the association of WT1 and induction of apoptosis with MIS treatment in endometriosis cell lines. DESIGN: We studied WT1 protein expression in endometriotic cell lines treated with MIS compared to no treatment. MATERIALS AND METHODS: Cultured CRL-7566 endometriosis cell lines from ovarian cyst (ATCC) were harvested at 80% confluency and placed in wells. Each well was treated with 5mcg/ml human recombinant MIS twice 4 days apart or with PBS as control. WT1 protein expression was determined using Western analysis and normalized to b-actin. RESULTS: MIS treatment significantly induced WT1 expression on endometriotic stromal cells(p<0.05) compared to the sample treated with control. CONCLUSION: MIS induces apoptosis, inhibits cellular growth and basal autophagy in endometriosis cell lines. MIS treatment induces WT1 expression which may partially explain the pathway that leads to apoptosis. Supported by: UTMB Chairman’s Career Development Grant.
OBJECTIVE: To investigate the involvement of transforming growth factor beta receptor III (TGFBRIII) in transforming growth factor beta (TGF-b) enhancement of transmesothelial invasion in endometrial cells, an essential step in endometriotic lesion development. DESIGN: TGFBRIII deficient and control immortalized endometrial cells were introduced into a three dimensional in vitro system modeling the peritoneum with and without the addition of TGF-b. MATERIALS AND METHODS: Specific siRNA transfection was used to knockdown expression of TGFBRIII in EM 42 cells. Receptor expression was assessed with Real Time PCR and Western blot analysis. An established three dimensional in vitro system modeling the peritoneum was used for invasion assays. Differences were assessed by one-way ANOVA. RESULTS: TGF-b1 significantly increased invasion of control immortalized endometrial epithelial cells containing scrambled non-silencing siRNA through an in vitro transmesothelial membrane (p¼ 0.004). Invasion of TGFBRIII knockdown immortalized endometrial cells following treatment with TGF-b1 was significantly less than controls treated with TGF-b1 (p¼0.004). CONCLUSION: TGFBRIII mediates TGF-b1 enhanced endometrial epithelial cell invasiveness by TGF-b. Combined with our previous studies showing that TGFBRI antagonists are potent inhibitors of TGF-b1 induced transmesothelial invasion, our data suggest that drugs targeting TGFBRIII pathways maybe useful in the treatment of endometriosis.
P-373 Wednesday, October 27, 2010 P-371 Wednesday, October 27, 2010 ABERRANT EXPRESSION OF IAP FAMILY IN ENDOMETRIOTIC CELLS MAY CAUSE RESISTANCE TO APOPTOSIS. F. Taniguchi, M. Izawa, T. Iwabe, N. Terakawa, T. Harada. Ob/Gyn, Tottori University Faculty of Medicine, Yonago, Japan; Biosignaling, Tottori University Faculty of Medicine, Yonago, Japan. OBJECTIVE: Inhibitor of apoptosis protein (IAP) family may be closely linked to the development of endometriosis. We sought to determine the difference of IAP family expression and the susceptibility to apoptosis between ovarian endometriomas and eutopic endometrial tissues. DESIGN: Experimental study. MATERIALS AND METHODS: Ectopic endometrial tissue from ovarian endometriomas and matched eutopic endometrial tissue samples were collected from premenopausal women (n¼22). Eutopic endometrial tissues (n¼20) from infertile women with benign gynecologic diseases and no evidence of endometriosis were used as control. Ectopic or eutopic endometrial stromal cells (ESCs) were isolated from these tissues.Gene and protein expression patterns for IAP family, cIAP-1 (birc2), cIAP-2 (birc3), XIAP (birc4), and survivin (birc5) were evaluated by real-time RT-PCR and immunohistochemical staining. Number of surviving cells and activation of caspases were assessed by WST-8 assay, Annexin-V staining and immunoblotting. RESULTS: All 4 IAP family mRNAs were more intensively expressed in endometriotic tissues derived from ovarian endometrioma compared with those of eutopic endometrial tissues. Survivin mRNA expression in endometriotic tissues showed 2.4-fold increase than the control. IAP family mRNA expression in eutopic endometrial tissues of women with ovarian endometriomas was higher than the control. Both epithelial and stromal cells of ovarian endometriotic tissues exhibited higher protein expression of these 4 IAPs than eutopic endometrial tissues. After induction of apoptosis by staurosporine (SS), 55% of eutopic ESCs survived versus 70% of ectopic ESCs. Procaspase-3 or -7 was more activated by SS in eutopic than in ectopic ESCs. Survivin gene silencing in SS-treated ectopic ESCs led to activation of caspases and increase of apoptotic cells. CONCLUSION: Aberrant expression of IAP family genes in ovarian endometriomas may sustain their abnormal survival in ectopic sites. Supported by: Ministry of Education, Science, Culture, Sports and technology of Japan.
FERTILITY & STERILITYÒ
EPITHELIAL-MESENCHYMAL TRANSITION MIGHT BE MICROENVIRONMENT-DEPENDENT DURING THE EVOLUTION OF ENDOMETRIOTIC IMPLANTS. S. Matsuzaki, C. Darcha, E. Maleysson, M. Canis, G. Mage. CHU Clermont-Ferrand, Clermont-Ferrand, Auvergne, France. OBJECTIVE: Our previous study demonstrated that expression levels of E-cadherin were distinctive among different forms of endometriosis. We further investigated several markers of epithelial-mesenchymal transition (EMT) (N-cadherin, vimentin, S100A4, dephosphorylated form of beta-catenin) in endometriosis. DESIGN: Prospective study. MATERIALS AND METHODS: Samples of deep infiltrating endometriosis (n¼ 61), ovarian endometriosis (n¼48) and superficial peritoneal endometriosis (red lesions: n¼30, black lesions: n ¼ 46) were collected during laparoscopic surgery. In addition, samples of menstrual endometrium, abdominal wall endometriosis, other benign ovarian cysts, carcinoma arising in ovarian endometriosis, colon endometriosis and scar endometriosis, were included in the present study for comparison. Immunohistochemical staining was performed on paraffin sections and quantitation of immunostained cells (percentage of immunostained surface mean staining intensity) was obtained using a computerized image analysis system. RESULTS: Vimentin expression was detected in the menstrual endometrium, whereas no expression of N-cadherin, S100A4 and dephosphorylated beta-catenin was detected. S100A4 expression was significantly lower in deep infiltrating endometriosis compared to that of ovarian endometriosis and red peritoneal lesions. Vimentin expression was significantly lower in ovarian endometriosis compared to that of deep infiltrating endometriosis, red and black peritoneal lesions. In addition, vimentin expression in deep infiltrating endometriosis, red and black peritoneal lesions was significantly higher than that of menstrual endometrium. Dephosphorylated beta-catenin expression was significantly higher in deep infiltrating endometriosis compared to that of ovarian endometriosis, red and black peritoneal lesions. CONCLUSION: The present findings suggested that markers of EMT might be gained depending on the microenvironment during the evolution of endometriotic implants. Supported by: Karl Storz Endoscopy & GmbH (Tuttlingen, Germany).
S201