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1.
Mol Reprod Dev ; 89(4): 165-174, 2022 04.
Article in English | MEDLINE | ID: mdl-35384116

ABSTRACT

Nuclear receptor coactivator 6 (Ncoa6), a modulator of several nuclear receptors and transcription factors, is essential for the decidualization of endometrial stromal cells in mice. However, the function of Ncoa6 in the human endometrium remains unclear. We investigated its function in the decidualization of human endometrial stromal cells (HESCs) isolated from resected uteri. Knockdown of Ncoa6 was performed using two independent small interfering RNAs. Decidualization was induced in vitro via medroxyprogesterone and cyclic adenosine monophosphate. We compared decidualized cellular morphology between the Ncoa6 knockdown cells and control cells. Messenger RNA (mRNA) sequencing was performed to determine the Ncoa6 target genes in undecidualized HESCs. We found that the knockdown of Ncoa6 caused the failure of morphological changes in decidualized HESCs compared to that in the control cells. mRNA sequencing revealed that Ncoa6 regulates the expression of genes associated with the regulation of actin fibers. Ncoa6 knockdown cells failed to reorganize actin fibers during the decidualization of HESCs. Ncoa6 was shown to play an essential role in decidualization via the appropriate regulation of actin fiber regulation in HESCs. Herein, our in vitro studies revealed a part of the mechanisms involved in endometrial decidualization. Future research is needed to investigate these mechanisms in women with implantation defects.


Subject(s)
Actins , Decidua , Nuclear Receptor Coactivators/metabolism , Actins/metabolism , Animals , Cells, Cultured , Endometrium/metabolism , Female , Humans , Mice , Nuclear Receptor Coactivators/genetics , RNA, Messenger/metabolism , Stromal Cells/metabolism
2.
Mol Hum Reprod ; 26(8): 567-575, 2020 08 01.
Article in English | MEDLINE | ID: mdl-32514562

ABSTRACT

Endoplasmic reticulum (ER) stress is associated with several aging-related diseases; however, the mechanism underlying age-related deterioration of oocyte quality is unclear. Here, we used post-ovulatory, in vivo aged mouse oocytes as a model. Super-ovulated oocytes harvested from the oviduct at 14 h and 20 h post-hCG injection were designated as 'fresh' and 'aged', respectively. Embryo development following IVF was compared between fresh, aged and ER stress-induced oocytes. Expression of the ER stress marker GRP78 was examined at each stage. To evaluate the effect of salubrinal, an ER stress suppressor, on embryo development following IVF, expression levels of GRP78 and phospho-eukaryotic initiation factor 2 alpha were compared between aged and salubrinal-treated aged oocytes. Embryo transfer of salubrinal-treated aged oocytes was performed to examine the safety of salubrinal. Similar to aged oocytes, ER stress-induced oocytes showed lower fertilization rates and poor embryo development. Following IVF, expression of GRP78 decreased with embryo development. GRP78 expression was significantly higher in aged oocytes than in fresh oocytes. Salubrinal lowered GRP78 levels and improved embryo development. No adverse effect of salubrinal treatment was found on the birth weight of pups or on organogenesis in mice. The limitation of this study was that protein kinase-like ER kinase was the only ER stress pathway examined; the role of IRE1 and ATF6 pathways was not considered. Nevertheless, salubrinal can significantly improve embryo development in in vivo aged oocytes undergoing ER stress. Hence, regulation of ER stress might represent a promising therapeutic strategy to overcome poor oocyte quality.


Subject(s)
Endoplasmic Reticulum Stress/physiology , Oocytes/metabolism , Animals , Apoptosis/physiology , Cinnamates/metabolism , Endoplasmic Reticulum Chaperone BiP , Endoplasmic Reticulum Stress/genetics , Female , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Male , Mice , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Thiourea/analogs & derivatives , Thiourea/metabolism , eIF-2 Kinase/genetics , eIF-2 Kinase/metabolism
3.
Reprod Biomed Online ; 40(3): 374-380, 2020 Mar.
Article in English | MEDLINE | ID: mdl-32033910

ABSTRACT

RESEARCH QUESTION: What is the prevalence of triplet and quadruplet pregnancies after single embryo transfer (SET) in Japan. DESIGN: A retrospective observational study was conducted on 274,605 pregnancies after 937,848 SET cycles in registered assisted reproductive technology (ART) data from the Japanese ART national registry database between 2007 and 2014. A questionnaire survey of ART centres was also conducted. Data on pregnancies with embryo division into three or more after SET were analysed. RESULTS: According to the Japanese ART national registry database, SET resulted in 109 triplet pregnancies (0.04% of pregnancies), and the questionnaire reports from 31 centres revealed 33 triplet and one quadruplet pregnancies. After exclusion of 20 duplicated cases, 122 triplet and one quadruplet pregnancies included 46 monochorionic (one gestational sac [37.4%]), 18 dichorionic (two gestational sacs [14.6%]) and 59 trichorionic pregnancies (three gestational sacs [48.0%]). Compared with singleton pregnancies, patients with monozygotic triplet or quadruplet pregnancies were less frequently diagnosed with unexplained infertility (P = 0.004), more often received gonadotrophin injections for ovarian stimulation in 39 cases with information available (P = 0.021) and underwent more blastocyst transfers and assisted hatching (P = 0.002 and P < 0.001, respectively). The proportion of live birth, defined as at least one baby born, excluding induced abortion, was 64.6% (73/116 pregnancies) of monozygotic triplet or quadruplet pregnancies. CONCLUSIONS: Combined Japanese ART national registry and survey data revealed 122 triplet and one quadruplet pregnancies, the majority after cryopreserved embryo transfer. Most were conceived after blastocyst transfer and often after assisted hatching, which are potential risk factors for zygotic splitting.


Subject(s)
Pregnancy, Quadruplet/statistics & numerical data , Pregnancy, Triplet/statistics & numerical data , Single Embryo Transfer/statistics & numerical data , Adult , Female , Humans , Japan , Pregnancy , Pregnancy Outcome , Registries , Reproductive Techniques, Assisted/statistics & numerical data , Retrospective Studies
5.
Dev Cell ; 23(4): 858-65, 2012 Oct 16.
Article in English | MEDLINE | ID: mdl-23079602

ABSTRACT

Uterine receptivity to embryo implantation is coordinately regulated by 17ß-estradiol (E(2)) and progesterone (P(4)). Although increased E(2) sensitivity causes infertility, the mechanisms underlying the modulation of E(2) sensitivity are unknown. We show that nuclear receptor coactivator-6 (NCOA6), a reported coactivator for estrogen receptor α (ERα), actually attenuates E(2) sensitivity to determine uterine receptivity to embryo implantation under normal physiological conditions. Specifically, conditional knockout of Ncoa6 in uterine epithelial and stromal cells does not decrease, but rather markedly increases E(2) sensitivity, which disrupts embryo implantation and inhibits P(4)-regulated genes and decidual response. NCOA6 enhances ERα ubiquitination and accelerates its degradation, while loss of NCOA6 causes ERα accumulation in stromal cells during the preimplantation period. During the same period, NCOA6 deficiency also caused a failure in downregulation of steroid receptor coactivator-3 (SRC-3), a potent ERα coactivator. Therefore, NCOA6 controls E(2) sensitivity and uterine receptivity by regulating multiple E(2)-signaling components.


Subject(s)
Embryo Implantation , Estradiol/metabolism , Nuclear Receptor Coactivators/metabolism , Uterus/metabolism , Animals , Female , Male , Mice , Mice, Knockout , Nuclear Receptor Coactivators/deficiency , Nuclear Receptor Coactivators/genetics , RNA, Messenger/genetics , RNA, Messenger/isolation & purification , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Uterus/cytology , Uterus/embryology
6.
Mol Cell Neurosci ; 48(3): 217-24, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21864685

ABSTRACT

Estrogen (E2) has direct in vivo and in vitro effects, such as inducing neurite outgrowth, on neurons. We investigated the morphological changes and intracellular signaling pathway induced by E2 in neuroblastoma (SH-SY5Y) cells. The effect of medroxyprogesterone acetate (MPA) or progesterone (P4) on the E2-induced neurite outgrowth was also examined using SH-SY5Y cells. Neurite outgrowth was induced by E2 in association with the phosphorylation of Akt, and these effects of E2 were abolished by MPA but not by P4. Progesterone receptor antagonist RU486 blocked the inhibitory effects of MPA. Estrogen receptor antagonist ICI 182,780 and phosphatidylinositol 3-kinase inhibitor LY294002 inhibited the E2-induced neurite outgrowth. Because the Rho family of small GTPases has been shown to be involved in the regulation of neurite outgrowth, we examined the cross-talk among Rac1, Cdc42 and RhoA in the E2-induced neurite outgrowth. E2 immediately increased the Rac1 and Cdc42 activity and decreased the RhoA activity. E2-induced neurite outgrowth was attenuated in cells expressing dominant-negative mutants for Rac1 or Cdc42. These results suggest that regulation of Rho family GTPase activity by E2 is important for the neurite outgrowth in neuroblastoma cells, and that MPA may have an antagonistic effect against E2.


Subject(s)
Estradiol/pharmacology , Neurites/drug effects , Neurons/drug effects , rho GTP-Binding Proteins/metabolism , Cell Line, Tumor , Humans , Medroxyprogesterone Acetate/pharmacology , Mifepristone/pharmacology , Neurites/metabolism , Neurons/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation/drug effects , Progesterone/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects , Tumor Cells, Cultured
7.
Biol Reprod ; 80(3): 493-502, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19038861

ABSTRACT

We examined whether impairment of intracellular Ca(2+) homeostasis is related to poor embryo development in in vitro-aged oocytes. We found that in vitro aging of mouse oocytes affected the patterns of Ca(2+) oscillations at fertilization: these Ca(2+) oscillations were lower in amplitude and higher in frequency compared with oocytes without in vitro aging. We also observed that the intracellular Ca(2+) store was decreased in in vitro-aged oocytes. A decrease in the Ca(2+) store induced by thapsigargin, a specific endoplasmic reticulum (ER) membrane Ca(2+)-ATPase inhibitor, resulted in a lower fertilization rate and in poorer embryo development. The frequency of Ca(2+) oscillations was significantly increased at fertilization, whereas their amplitude was decreased in thapsigargin-treated oocytes. These results suggest that impairment of intracellular Ca(2+) homeostasis (such as a decrease in the ER Ca(2+) store) caused an alteration in Ca(2+) oscillations and the poor embryo development in in vitro-aged oocytes. Because embryo fragmentation is closely related to apoptosis, we examined expression of BAX (a proapototic protein) and BCL2 (an antiapoptotic protein) in in vitro-aged oocytes. Although BCL2 was strongly expressed in oocytes without in vitro aging, expression of BCL2 was significantly reduced in oocytes of other culture conditions and treatments such as those in in vitro aging and those that were pretreated with H(2)O(2) or thapsigargin. Acting together, alteration in Ca(2+) oscillations and decrease in BCL2 expression in in vitro-aged oocytes may lead to poor embryo development.


Subject(s)
Calcium/metabolism , Cellular Senescence/physiology , Embryonic Development/physiology , Homeostasis/physiology , Oocytes/metabolism , Animals , Cells, Cultured , Embryonic Development/drug effects , Endoplasmic Reticulum/metabolism , Enzyme Inhibitors/pharmacology , Female , Fertilization in Vitro/methods , Hydrogen Peroxide/pharmacology , In Vitro Techniques , Mice , Oocytes/cytology , Oocytes/drug effects , Oxidants/pharmacology , Oxidative Stress/physiology , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2 , Thapsigargin/pharmacology
8.
Mol Cancer Ther ; 7(6): 1551-61, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18566226

ABSTRACT

Hypoxic response of endothelial cells (EC) is an important component of tumor angiogenesis. Especially, hypoxia-inducible factor-1 (HIF-1)-dependent EC-specific mechanism is an essential component of tumor angiogenesis. Recently, the Rho/Rho-associated kinase (ROCK) signaling has been shown to play a key role in HIF-1alpha induction in renal cell carcinoma and trophoblast. The present study was designed to investigate whether low oxygen conditions might modulate HIF-1alpha expression through the Rho/ROCK signaling in human umbilical vascular ECs (HUVEC). Pull-down assay showed that hypoxia stimulated RhoA activity. Under hypoxic conditions, HUVECs transfected with small interfering RNA of RhoA and ROCK2 exhibited decreased levels of HIF-1alpha protein compared with nontargeted small interfering RNA transfectants, whereas HIF-1alpha mRNA levels were not altered. One of ROCK inhibitors, fasudil, inhibited hypoxia-induced HIF-1alpha expression without altering HIF-1alpha mRNA expression. Furthermore, proteasome inhibitor prevented the effect of fasudil on HIF-1alpha expression, and polyubiquitination was enhanced by fasudil. These results suggested that hypoxia-induced HIF-1alpha expression is through preventing HIF-1alpha degradation by activating the Rho/ROCK signaling in ECs. Furthermore, hypoxia induced both vascular endothelial growth factor (VEGF) and VEGF receptor-2 expression through the Rho/ROCK/HIF-1alpha signaling in HUVECs. Thus, augmented VEGF/VEGF receptor-2 autocrine mechanism stimulated HUVEC migration under hypoxic conditions. In summary, the Rho/ROCK/HIF-1alpha signaling is an essential mechanism for hypoxia-driven, VEGF-mediated autocrine loop in ECs. Therefore, fasudil might have the antimigratory effect against ECs in tumor angiogenesis.


Subject(s)
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/analogs & derivatives , Autocrine Communication/drug effects , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Protein Processing, Post-Translational/drug effects , Vascular Endothelial Growth Factor A/metabolism , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Amides/pharmacology , Cell Hypoxia/drug effects , Cell Movement/drug effects , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Drug Screening Assays, Antitumor , Endothelial Cells/enzymology , Endothelial Cells/pathology , Enzyme Activation/drug effects , Gene Expression Regulation/drug effects , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Leupeptins/pharmacology , Myosin Light Chains/metabolism , Neovascularization, Pathologic/pathology , Phosphorylation/drug effects , Protein Transport/drug effects , Pyridines/pharmacology , Ubiquitination/drug effects , Vascular Endothelial Growth Factor Receptor-2/metabolism , rho-Associated Kinases/metabolism
9.
Endocrinology ; 148(12): 6092-9, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17872375

ABSTRACT

Diverse estrogen actions are controlled via estrogen receptors (ERs). Mechanisms of action of ERs are modulated by various factors such as ER subtypes, conformation of the ER-ligand complex, and recruitment of coregulator complexes to a target gene promoter. Estrogen exerts divergent actions on vascular cells; namely it increases endothelial cell and inhibits smooth muscle cell growth, resulting in a vasoprotective action. We particularly focused on these divergent effects and examined the mechanisms. The effects of raloxifene, which shows estrogen-like vasoprotective actions, were also examined. To examine the effects of 17beta-estradiol (E(2)) and raloxifene on human aortic smooth muscle cells (HASMCs) and human umbilical venous endothelial cells (HUVECs), we evaluated the effect of E(2) and raloxifene on transcriptional activity, recruitment of the coregulator complex to a target gene promoter, and acetylation of histone of both the IGF-I and COX-2 genes. Treatment with E(2) or raloxifene increased both IGF-I and cyclooxygenase (COX)-2 mRNA expression in HUVECs, whereas they attenuated the serum-induced increase of these genes in HASMCs. Treatment by E(2) and raloxifene induced recruitment of coactivator complex and histone acetylation at both the IGF-I and COX-2 gene promoter in HUVECs. In contrast, in HASMCs, E(2), and raloxifene attenuated the serum-induced recruitment of coactivator complexes and histone acetylation at both the IGF-I and COX-2 gene promoters. Estrogen and raloxifene exert divergent transcriptional regulation on both mRNA expression and the remodeling of IGF-I and COX-2 gene promoters in HUVECs vs. HASMCs.


Subject(s)
Cell Proliferation/drug effects , Endothelial Cells/drug effects , Estrogens/pharmacology , Myocytes, Smooth Muscle/drug effects , Acetylation/drug effects , Aorta/cytology , Blotting, Western , Cells, Cultured , Cyclooxygenase 2/genetics , Endothelial Cells/cytology , Endothelial Cells/metabolism , Estradiol/pharmacology , Histone Acetyltransferases/genetics , Histones/metabolism , Humans , Insulin-Like Growth Factor I/genetics , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/metabolism , Nuclear Receptor Coactivator 3 , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , Raloxifene Hydrochloride/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Trans-Activators/genetics , Transcription, Genetic/drug effects , Transfection , Umbilical Cord/cytology
10.
Anticancer Res ; 27(4C): 2685-7, 2007.
Article in English | MEDLINE | ID: mdl-17695433

ABSTRACT

BACKGROUND: Small cell carcinoma of the ovary is a rare type of ovarian carcinoma with a very poor prognosis. CASE REPORT: We report here a case of a 55-year-old woman with small cell carcinoma of the left ovary. The patient underwent cytoreductive surgery with residual tumors of 6 cm at the cul-de-sac and was found to have stage IIIc disease. After six courses of irinotecan (CPT-11) and cisplatin (CDDP) combination therapy, secondary cytoreductive surgery was performed. The patient showed no evidence of residual tumors. After an additional three courses of chemotherapy, the patient is still alive and well without evidence of disease. CONCLUSION: CPT-11 and CDDP combination chemotherapy may be effective and safe for patients with small cell carcinoma of the ovary.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carcinoma, Small Cell/drug therapy , Ovarian Neoplasms/drug therapy , Camptothecin/administration & dosage , Camptothecin/analogs & derivatives , Cisplatin/administration & dosage , Combined Modality Therapy , Female , Humans , Irinotecan , Middle Aged , Ovarian Neoplasms/surgery , Remission Induction
11.
Mol Cancer Ther ; 6(5): 1517-25, 2007 May.
Article in English | MEDLINE | ID: mdl-17513600

ABSTRACT

Vascular endothelial growth factor (VEGF)-induced endothelial cell migration is an important component of tumor angiogenesis. Rho and Rho-associated kinase (ROCK) are key regulators of focal adhesion, stress fiber formation, and thus cell motility. Inhibitors of this pathway have been shown to inhibit endothelial cell motility and angiogenesis. In this study, we investigated the antiangiogenic effect of fasudil, one of the ROCK inhibitors. Fasudil inhibited VEGF-induced endothelial cell migration, viability, and tube formation in vitro in human umbilical vein endothelial cells. VEGF-induced endothelial cell migration was reduced by fasudil associated with loss of stress fiber formation, focal adhesion assembly, and with the suppression of tyrosine phosphorylation of focal adhesion proteins. Furthermore, fasudil inhibited VEGF-induced phosphorylation of myosin light chain, which is one of the main substrates of ROCK. Therefore, the effect of fasudil was suggested to be ROCK dependent. Fasudil not only inhibited VEGF-induced cell proliferation but also reversed the protective effect of VEGF on apoptosis, which resulted in the decrease of cell viability. Moreover, fasudil inhibited VEGF-induced angiogenesis in a directed in vivo angiogenesis assay. These data are the first demonstration that fasudil has antiangiogenic properties. Therefore, fasudil might be useful for the treatment of angiogenesis-related diseases, especially cancer.


Subject(s)
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/analogs & derivatives , Angiogenesis Inhibitors/pharmacology , Neovascularization, Pathologic , Protein Kinase Inhibitors/pharmacology , Vascular Endothelial Growth Factor A/metabolism , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Animals , Apoptosis , Cell Movement , Cell Proliferation , Cell Survival , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Umbilical Veins , rho-Associated Kinases
12.
Menopause ; 14(4): 656-61, 2007.
Article in English | MEDLINE | ID: mdl-17242632

ABSTRACT

OBJECTIVE: To examine the effect of raloxifene on the endothelial dysfunction caused by surgical menopause. DESIGN: Ten premenopausal women who underwent gynecological surgery with ovariectomy were divided into two groups. Five participants used raloxifene (60 mg/d) for 7 days staring 1 week after the surgery, and the other five participants did not use raloxifene. We examined the changes in flow-mediated dilatation (FMD) of the brachial artery using ultrasonography. Vasodilation in response to nitroglycerin was also studied. We also measured the brachial-ankle pulse wave velocity to examine the change in arterial stiffness in these participants before and after surgical menopause. RESULTS: In both the raloxifene and control groups, a significant decrease in FMD was observed 1 week after the surgery. Although no further changes in FMD were observed in the control group at 2 weeks after surgery, FMD was significantly increased in the raloxifene group. No remarkable changes in nitroglycerin or brachial-ankle pulse wave velocity were observed after surgery in either group. CONCLUSIONS: Raloxifene rapidly restored FMD that was impaired after surgical menopause. Therefore, raloxifene may be effective for ameliorating and maintaining endothelial function in premenopausal women who undergo ovariectomy.


Subject(s)
Endothelium, Vascular/drug effects , Hot Flashes/drug therapy , Raloxifene Hydrochloride/therapeutic use , Selective Estrogen Receptor Modulators/therapeutic use , Vasodilation/drug effects , Adult , Ankle/blood supply , Blood Flow Velocity , Brachial Artery/diagnostic imaging , Brachial Artery/physiology , Female , Hot Flashes/pathology , Humans , Menopause , Middle Aged , Ovariectomy , Pulsatile Flow , Raloxifene Hydrochloride/administration & dosage , Raloxifene Hydrochloride/pharmacology , Selective Estrogen Receptor Modulators/administration & dosage , Selective Estrogen Receptor Modulators/pharmacology , Treatment Outcome , Ultrasonography
13.
J Biol Chem ; 281(34): 24270-8, 2006 Aug 25.
Article in English | MEDLINE | ID: mdl-16798746

ABSTRACT

Vascular endothelial senescence is involved in human atherosclerosis. Telomerase activity is known to be critical in cellular senescence and its level is modulated by regulation of telomerase catalytic subunit (telomerase reverse transcriptase (TERT)) at both the transcriptional and post-transcriptional levels. Since the cardioprotective effect of estrogen itself has not been ruled out, we examined that of raloxifene, which has been classified as a selective estrogen receptor modulator, on the proliferation and telomerase activity of human umbilical vein endothelial cells (HUVECs). Raloxifene, like estrogen, clearly induced the telomerase activity and human TERT (hTERT) expression via estrogen receptor (ER) alpha and ERbeta. Treatment with raloxifene for 5 days significantly induced cell growth, and either cotreatment with a telomerase inhibitor, 3'-azido-3'-deoxythymidine, or transfection with hTERT-specific small interfering RNA significantly attenuated the raloxifene-induced cell growth. Raloxifene also induced the phosphorylation of Akt, and pretreatment with a phosphatidylinositol 3-kinase inhibitor, LY294002, significantly attenuated the raloxifene-induced telomerase activity. In addition, raloxifene induced both the phosphorylation of hTERT and IkappaB. Moreover, cotreatment with an IkappaBalpha phosphorylation inhibitor, BAY-11-7082, or a specific NFkappaB nuclear translocation inhibitor, SN50, significantly attenuated the raloxifene-induced telomerase activity and the association of NFkappaB with hTERT. These results show that raloxifene induced the up-regulation of telomerase activity not only by the transcriptional regulation of hTERT but also by post-translational regulation of the phosphorylation of Akt and hTERT and the association of hTERT with NFkappaB in HUVECs. Thus, the up-regulation of telomerase activity in vascular endothelial cells might be one mechanism contributing to the potential atheroprotective effect of raloxifene.


Subject(s)
DNA-Binding Proteins/metabolism , Endothelium, Vascular/drug effects , Raloxifene Hydrochloride/pharmacology , Telomerase/metabolism , Cell Proliferation/drug effects , Cells, Cultured , Chromones/pharmacology , DNA-Binding Proteins/antagonists & inhibitors , Endothelium, Vascular/cytology , Endothelium, Vascular/enzymology , Estrogen Receptor alpha/metabolism , Estrogen Receptor beta/metabolism , Humans , Morpholines/pharmacology , Phosphorylation , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , RNA, Small Interfering , Signal Transduction/drug effects , Telomerase/antagonists & inhibitors , Up-Regulation/drug effects , Zidovudine/pharmacology
14.
Endocrinology ; 147(4): 1761-9, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16396982

ABSTRACT

The phosphatidylinositol 3-kinase (PI3K)/Akt cascade has an important role in the resistance of ovarian cancer cells to cisplatin in vitro; however, there have been no reports about whether blocking the PI3K/Akt cascade enhances the sensitivity to cisplatin in vivo. We investigated whether inhibition of PI3K increased the efficacy of cisplatin in an in vivo ovarian cancer model. Blocking the PI3K/Akt cascade with a PI3K inhibitor (wortmannin) increased the efficacy of cisplatin-induced inhibition of intraabdominal dissemination and production of ascites in athymic nude mice inoculated ip with the Caov-3 human ovarian cancer cell line. In addition, wortmannin increased the efficacy of cisplatin-induced apoptosis in tumors cells. There were no detectable side effects in mice treated with wortmannin. Moreover, the antitumor effect of cisplatin detected in mice inoculated with Caov-3 cells stably transfected with empty vector was significantly attenuated, compared with mice inoculated with Caov-3 cells stably transfected with a dominant-negative Akt, K179M-Akt. We confirmed that wortmannin blocked Akt phosphorylation and the downstream targets of the PI3K/Akt cascade, such as BAD (Bcl-2-associated death protein) and nuclear factor-kappaB in vivo by immunohistochemical staining and Western blotting. In accordance with the previously reported in vitro results, these in vivo results support the idea that combination therapy with cisplatin and a PI3K inhibitor would increase the therapeutic efficacy of cisplatin.


Subject(s)
Antineoplastic Agents/therapeutic use , Cisplatin/therapeutic use , Ovarian Neoplasms/drug therapy , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/pharmacology , Androstadienes/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , Female , Humans , NF-kappa B/metabolism , Ovarian Neoplasms/pathology , Phosphorylation , Wortmannin , bcl-Associated Death Protein/metabolism
15.
Endocrinology ; 146(11): 4917-25, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16123159

ABSTRACT

The mechanism of medroxyprogesterone acetate (MPA)-induced cell proliferation in human breast cancer cells remains elusive. We examined the mechanism by which MPA affects the cyclin D1 expression in progesterone receptor (PR)-positive T47D human breast cancer cells. MPA (10 nM) treatment for 48 h induced proliferation of the cells (1.6-fold induction). MPA induced cyclin D1 expression (3.3-fold induction), and RU486, a selective PR antagonist, blocked the MPA-induced cell proliferation and cyclin D1 expression (23% inhibition). MPA increased both the protein level (2.2-fold induction) and promoter activity (2.7-fold induction) of cyclin D1 in MCF-7 cells transfected with PRB but not with PRA. Although MPA transcriptionally activated cyclin D1 expression, cyclin D1 promoter does not have progesterone-responsive element-related sequence. We further examined the mechanism for the regulation of the cyclin D1 expression. Because the cyclin D1 promoter contains three putative nuclear factor-kappaB (NFkappaB)-binding motifs and NFkappaB is a substrate of Akt, we investigated the effect of the phosphatidylinositol 3-kinase (PI3K)/Akt/NFkappaB cascade on the responses of cyclin D1 to MPA. MPA induced the transient phosphorylation of Akt (2.7-fold induction at 5 min), and treatment with PI3K inhibitor (wortmannin) attenuated the MPA-induced up-regulation of cyclin D1 expression (40% inhibition) and cell proliferation (40% inhibition). MPA also induced phosphorylation of inhibitor of NFkappaBalpha (IkappaBalpha) (2.3-fold induction), and treatment with wortmannin attenuated the MPA-induced IkappaBalpha phosphorylation (60% inhibition). Treatment with an IkappaBalpha phosphorylation inhibitor (BAY 11-7085) or a specific NFkappaB nuclear translocation inhibitor (SN-50) attenuated the MPA-induced up-regulation of both cyclin D1 expression (80 and 50% inhibition, respectively) and cell proliferation (55 and 34% inhibition, respectively). Because MPA induced a transient phosphorylation of Akt and the cyclin D1 promoter contains no progesterone-responsive element-related sequence, the MPA-induced cell proliferation through PRB by up-regulation of cyclin D1 expression via the PI3K/Akt/NFkappaB cascade may be a nongenomic mechanism.


Subject(s)
Antineoplastic Agents, Hormonal/pharmacology , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cyclin D1/metabolism , Medroxyprogesterone Acetate/pharmacology , NF-kappa B/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Humans , Phosphorylation/drug effects , Up-Regulation
16.
Arch Gynecol Obstet ; 272(4): 278-82, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16012777

ABSTRACT

OBJECTIVE: Since ovarian borderline tumor with invasive implant behaves as carcinoma, it should be managed like carcinoma. Since its characteristic features have not been reported, its preoperative diagnosis was thought to be impossible. We evaluated the features of MRI and macroscopic appearance in two cases of ovarian borderline tumor with invasive implant. METHODS: Borderline tumor with invasive implant was evaluated in two patients by MRI and macroscopic examination. RESULTS: In these patients, MRI revealed profuse papillary projections. Although the lesion showed high signal intensity on contrast-enhanced T1-weighted images compared with that on T1-weighted ones, most of the signal intensity on T2-weighted images was high, suggesting that the lesion is an assembly of vesicles and an obvious solid part is absent. The macroscopic appearance of the tumor showed profuse papillary projections consisting of many vesicles perforating and extending far beyond the ovarian capsule, without a solid part. The histological findings indicated serous borderline tumors with invasive implant. CONCLUSION: In these two cases, we found the characteristic features of serous borderline tumor with invasive implant by MRI and macroscopic examination. Our findings may be of clinical value since the preoperative information about the possibility of invasive implant may be quite important for the management of borderline tumor with invasive implant, especially for young patients wishing to bear children.


Subject(s)
Cystadenocarcinoma, Serous/pathology , Ovarian Neoplasms/pathology , Adult , Contrast Media , Cystadenocarcinoma, Serous/diagnosis , Cystadenocarcinoma, Serous/surgery , Female , Gadolinium DTPA , Humans , Magnetic Resonance Imaging , Neoplasm Invasiveness , Ovarian Neoplasms/diagnosis , Ovarian Neoplasms/surgery
17.
Endocrinology ; 146(9): 4082-9, 2005 Sep.
Article in English | MEDLINE | ID: mdl-15947004

ABSTRACT

In the present study, to examine the dynamic changes in the localization of nuclear estrogen receptor (ER)alpha induced by growth factors, we used time-lapse confocal microscopy to directly visualized ERalpha fused with green fluorescent protein (GFP-ERalpha) in single living cells treated with epidermal growth factor (EGF) or IGF-I. We observed that 17beta-estradiol (E2) changed the normally diffuse distribution of GFP-ERalpha throughout the nucleoplasm to a hyperspeckled distribution within 10 min. Both EGF and IGF-I also changed the nuclear distribution of GFP-ERalpha, similarly to E2 treatment. However, the time courses of the nuclear redistribution of GFP-ERalpha induced by EGF or IGF-I were different from that induced by E2 treatment. In the EGF-treated cells, the GFP-ERalpha nuclear redistribution was observed at 30 min and reached a maximum at 60 min, whereas in the IGF-I-treated cells, the GFP-ERalpha nuclear redistribution was observed at 60 min and reached a maximum at 90 min. The EGF-induced redistribution of GFP-ERalpha was blocked by pretreatment with a MAPK cascade inhibitor, PD98059, whereas the IGF-I-induced redistribution of GFP-ERalpha was blocked by pretreatment with a phosphatidylinositol 3-kinase inhibitor, LY294002. Analysis using an activation function-2 domain deletion mutant of GFP-ERalpha showed that the change in the distribution of GFP-ERalpha was not induced by E2, EGF, or IGF-I treatment. These data suggest that MAPK and phosphatidylinositol 3-kinase cascades are involved in the nuclear redistribution of ERalpha by EGF and IGF-I, respectively, and that the activation function-2 domain of ERalpha may be needed for the nuclear redistribution of ERalpha.


Subject(s)
Breast Neoplasms , Epidermal Growth Factor/pharmacology , Estrogen Receptor alpha/metabolism , MAP Kinase Signaling System/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Animals , COS Cells , Cell Line, Tumor , Cell Nucleus/metabolism , Chlorocebus aethiops , Estradiol/pharmacology , Estrogen Receptor alpha/genetics , Green Fluorescent Proteins/genetics , Humans , Insulin-Like Growth Factor I/pharmacology , MAP Kinase Signaling System/physiology , Mutagenesis , Transcriptional Activation/drug effects , Transcriptional Activation/physiology
18.
Gynecol Obstet Invest ; 60(3): 162-6, 2005.
Article in English | MEDLINE | ID: mdl-15980658

ABSTRACT

BACKGROUND/AIMS: Aortic stiffness, determined by pulse wave velocity (PWV), is an independent marker of cardiovascular risk. PWV is mainly influenced by age-associated alterations in arterial wall structure and blood pressure. The present study was conducted to assess the impact of menopause on the brachial-ankle PWV (baPWV) in healthy women. METHODS: Fifty premenopausal women aged 22-54 years and 40 postmenopausal women aged 40-73 years were recruited for this study. Subjects with hypertension, diabetes, and hyperlipidemia were strictly excluded. The results of baPWV were analyzed chronologically by 10- or 5-year age intervals. RESULTS: There was no significant difference in baPWV between premenopausal and postmenopausal women in their 40s and 50s. The baPWV of postmenopausal women aged over 60 years was significantly higher than that of postmenopausal women in their 50s. To clarify the age-dependent elevation in baPWV in detail, women their 50s and 60s were divided into subgroups by 5-year age intervals. There was no significant difference in baPWV among the 50-54-, 55-59- and 60-64-year subgroups. baPWV significantly increased in the 65-69- year subgroup (p< 0.05). There was a significant relationship between baPWV and age in premenopausal (r = 0.452, p = 0.001) and postmenopausal (r = 0.581, p < 0.0001) women. The slope of the regression line for baPWV plotted against age was steeper in postmenopausal than in premenopausal women. CONCLUSIONS: This study produces suggestive evidence that menopause amplifies the age-dependent increase in arterial stiffness.


Subject(s)
Aging , Ankle/blood supply , Postmenopause , Premenopause , Adult , Age Factors , Aged , Blood Flow Velocity , Blood Pressure , Brachial Artery/physiopathology , Female , Humans , Middle Aged , Pulsatile Flow , Reference Values
19.
J Endocrinol ; 183(3): 605-15, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15590986

ABSTRACT

Although estrogen is known to protect against beta-amyloid (Abeta)-induced neurotoxicity, the mechanisms responsible for this effect are only beginning to be elucidated. In addition, the effect of raloxifene on Abeta-induced neuro-toxicity remains unknown. Here we investigated whether raloxifene exhibits similar neuro-protective effects to estrogen against Abeta-induced neurotoxicity and the mechanism of the effects of these agents in PC12 cells transfected with the full-length human estrogen receptor (ER) alpha gene (PCER). Raloxifene, like 17beta-estradiol (E2), significantly inhibited Abeta-induced apoptosis in PCER cells, but not in a control line of cells transfected with vector DNA alone (PCCON). Since telomerase activity, the level of which is modulated by regulation of telomerase catalytic subunit (TERT) at both the transcriptional and post-transcriptional levels, is known to be involved in suppressing apoptosis in neurons, we examined the effect of E2 and raloxifene on telomerase activity. Although both E2 and raloxifene induced telomerase activity in PCER cells, but not in PCCON cells, treated with Abeta, they had no effect on the level of TERT expression. These results suggest that neither E2 nor raloxifene affects the telomerase activity at the transcriptional level. We therefore studied the mechanism by which E2 and raloxifene induce the telomerase activity at the post-transcriptional level. Both E2 and raloxifene induced the phosphorylation of Akt, and pre-treatment with a phosphatidylinositol 3-kinase inhibitor, LY294002, attenuated both E2- and raloxifene-induced activation of the telomerase activity. Moreover, both E2 and raloxifene induced both the phosphorylation of TERT at a putative Akt phosphorylation site and the association of nuclear factor kappaB with TERT. Our findings suggest that and raloxifene exert neuroprotective effects by E2 telomerase activation via a post-transcriptional cascade in an experimental model relevant to Alzheimer's disease.


Subject(s)
Amyloid beta-Peptides/metabolism , Brain/metabolism , Estrogens/pharmacology , Neurodegenerative Diseases/prevention & control , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Raloxifene Hydrochloride/pharmacology , Selective Estrogen Receptor Modulators/pharmacology , Blotting, Western/methods , Brain/drug effects , Cell Line, Tumor , Enzyme Activation , Humans , Neurodegenerative Diseases/metabolism , Pheochromocytoma , Proto-Oncogene Proteins c-akt , Receptors, Estrogen/genetics , Reverse Transcriptase Polymerase Chain Reaction , Telomerase/metabolism , Transfection/methods
20.
Clin Cancer Res ; 10(22): 7645-54, 2004 Nov 15.
Article in English | MEDLINE | ID: mdl-15569997

ABSTRACT

We investigated whether inhibition of nuclear factor-kappaB (NFkappaB) increases the efficacy of paclitaxel in in vitro and in vivo ovarian cancer models. Treatment of paclitaxel-sensitive Caov-3 cells with paclitaxel transiently activated the phosphorylation of Akt, the phosphorylation of IkappaB kinase (IKK), and the phosphorylation of inhibitor of NFkappaB (IkappaBalpha). Paclitaxel also caused a transient increase in NFkappaB activity, followed by a decrease in NFkappaB activity. We show an association between Akt and IKK and show that the phosphorylation of IKK induced by paclitaxel is blocked by treatment with a phosphatidylinositol 3-kinase inhibitor (wortmannin or LY294002). Furthermore, interference of the Akt signaling cascade inhibits the transient induction of IkappaBalpha phosphorylation and NFkappaB activity by paclitaxel. Inhibition of NFkappaB activity by treatment with an IkappaBalpha phosphorylation inhibitor (BAY 11-7085) attenuated both basal and transient induction of IkappaBalpha phosphorylation by paclitaxel. Treatment with BAY 11-7085 also enhanced the inhibition of NFkappaB activity by paclitaxel for up to 24 hours. In addition, treatment with BAY 11-7085 decreased the viability of cells treated with paclitaxel. Moreover, treatment with BAY 11-7085 increased the efficacy of paclitaxel-induced inhibition of intraabdominal dissemination and production of ascites in athymic nude mice inoculated intraperitoneally with Caov-3 cells. These results suggest that paclitaxel transiently induces NFkappaB activity via the phosphatidylinositol 3-kinase/Akt cascade and that combination therapy with paclitaxel and an NFkappaB inhibitor would increase the therapeutic efficacy of paclitaxel.


Subject(s)
NF-kappa B/antagonists & inhibitors , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/pathology , Paclitaxel/pharmacology , Androstadienes/pharmacology , Animals , Anti-Infective Agents/pharmacology , Antineoplastic Agents/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Blotting, Western , Cell Line, Tumor , Cell Proliferation , Chromones/pharmacology , Collagen/pharmacology , Dose-Response Relationship, Drug , Drug Combinations , Drug Synergism , Enzyme Inhibitors/pharmacology , Female , Humans , I-kappa B Kinase , Laminin/pharmacology , Mice , Mice, Nude , Morpholines/pharmacology , NF-kappa B/metabolism , Nitriles , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Plasmids/metabolism , Protein Serine-Threonine Kinases/metabolism , Proteoglycans/pharmacology , Signal Transduction , Sulfones , Time Factors , Transcriptional Activation , Wortmannin
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