Akitoshi Tsuno

Application of the histone deacetylase inhibitors for the treatment of endometriosis: histone modifications as pathogenesis and novel therapeutic target

BACKGROUND Accumulating evidence suggests that various epigenetic aberrations play definite roles in the pathogenesis of endometriosis. We investigated the histone acetylation status in endometriosis and the application of the histone deacetylase inhibitors (HDACIs) for the treatment of endometriosis. METHODS The levels of acetylated histones in the endometriotic cyst stromal cells (ECSCs) and normal endometrial stromal cells (NESCs) were evaluated. The effects of the HDACIs on cell proliferation, the cell cycle, apoptosis of ECSCs and NESCs, and the expression of genes related to these cellular events were investigated. The effects of HDACIs on histone acetylation in chromatin of the promoter region of the cell cycle regulatory genes in ECSCs were also investigated. RESULTS The acetylated histone levels were significantly lower in ECSCs than in NESCs (P < 0.025). HDACIs inhibited cell proliferation and induced cell cycle arrest and apoptosis of ECSCs. The effects of HDACIs on NESCs were marginal or weak. These HDACIs induced an accumulation of acetylated histones in total cellular chromatin and in the promoter regions of the p16(INK4a), p21(Waf1/Cip1), p27(Kip1) and cycle checkpoint kinase 2 genes in ECSCs. HDACIs induced the protein expression of these cell cycle regulators and suppressed the protein expression of Bcl-2 and Bcl-X(L) in ECSCs. CONCLUSIONS The present findings demonstrated that aberrant histone modifications are present in endometriosis and that HDACIs reactivated epigenetically silenced genes, resulting in the suppression of cell proliferation, induction of cell cycle arrest and apoptosis of ECSCs. HDACIs are therefore promising agents for the treatment of endometriosis.

Simvastatin inhibits the proliferation and the contractility of human endometriotic stromal cells: a promising agent for the treatment of endometriosis

Simvastatin significantly inhibited the proliferation of endometriotic stromal cells, attenuated the collagen gel contraction mediated by these cells, and suppressed endometriotic stromal cell attachment to collagen fibers. Simvastatin is considered to be a promising agent for the treatment of endometriosis-associated fibrosis, which is among the major pathologies caused by endometriosis.

Attachment to extracellular matrices is enhanced in human endometriotic stromal cells: a possible mechanism underlying the pathogenesis of endometriosis

OBJECTIVE Endometriosis is characterized by the ectopic growth of endometrial tissue. One of the first steps to the spread of endometriosis in the peritoneal cavity is the attachment of endometriotic cells to peritoneal surfaces after they have been released into the peritoneal fluid from pre-existing endometriotic lesions. The increased adhesive and proliferative potential of endometriotic cells in response to specific extracellular matrix (ECM) components has been suggested to contribute to the pathogenesis of endometriosis. STUDY DESIGN Adhesive properties of endometriotic stromal cells (ECSC) and normal eutopic endometrial cells (NESC) to various extracellular matrix proteins were investigated by in vitro cell adhesion assays. The expression levels of integrins in these cells were also examined by Western blot analysis. RESULTS Both ECSC and NESC significantly adhered to collagen type I and collagen type IV. ECSC revealed higher adhesive properties to these ECM proteins than NESC did. ECSC, but not NESC, adhered to fibronectin and laminin. Higher levels integrin of α1, α2, αv, β1, and β3 protein expression were observed in ECSC than in NESC. On the other hand, the levels of integrin α3 and αL proteins were lower in ECSC than in NESC. CONCLUSIONS The results suggest that endometriotic cells possess stronger adhesion to ECM proteins, and that increase may be mediated, in part, through integrins. These findings may elucidate one of the mechanisms underlying the formation of peritoneal endometriotic lesions.

Aberrant expression of apoptosis-related molecules in endometriosis: a possible mechanism underlying the pathogenesis of endometriosis.

Endometriosis, a disease affecting 3% to 10% of women of reproductive age, is characterized by the ectopic growth of endometrial tissue under the influence of estrogen. It is also becoming recognized as a condition in which ectopic endometrial cells exhibit abnormal proliferative and apoptotic regulation in response to appropriate stimuli. Apoptosis plays a critical role in maintaining tissue homeostasis and represents a normal function to eliminate excess or dysfunctional cells. Accumulated evidence suggests that, in healthy women, endometrial cells expelled during menstruation do not survive in ectopic locations because of programmed cell death, while decreased apoptosis may lead to the ectopic survival and implantation of these cells, resulting in the development of endometriosis. Both the inability of endometrial cells to transmit a “death” signal and the ability of endometrial cells to avoid cell death have been associated with increased expression of antiapoptotic factors and decreased expression of preapoptotic factors. Further investigations may elucidate the role of apoptosis-associated molecules in the pathogenesis of endometriosis. Medical treatment with apoptosis-inducing agents may be novel and promising therapeutic strategy for endometriosis.

Decidualization Attenuates the Contractility of Eutopic and Ectopic Endometrial Stromal Cells: Implications for Hormone Therapy of Endometriosis

CONTEXT Decidualization of the endometrium involves the morphological and biochemical reprogramming of the estrogen-primed proliferative endometrial stromal compartment under the continuing influence of progesterone. OBJECTIVES The aim of this study was to evaluate the involvement of the extracellular matrix contractility of eutopic and ectopic endometrial stromal cells during the tissue remodeling processes associated with decidualization. DESIGN The effect of decidualization on the contractile profile of the endometriotic cyst stromal cells and eutopic endometrial stromal cells with or without endometriosis in the three-dimensional collagen gel culture was investigated using laser scanning microscopy, collagen gel contraction assays, and Western blot analysis. RESULTS Decidualized ectopic and eutopic endometrial stromal cells in the three-dimensional collagen gel culture mimicked the morphology of decidual tissue in vivo. In vitro decidualization inhibited the contractility of these eutopic and ectopic endometrial stromal cells. Down-regulation of integrin alpha1beta1 and alpha2beta1 expression, suppression of Ras homology A (Rho A), Rho-associated coiled-coil-forming protein kinase (ROCK)-I and ROCK-II expression, inhibition of the differentiation into the myofibroblastic phenotype, and induction of differentiation into epithelioid decidual phenotype were observed in these cells during decidualization. CONCLUSIONS It is suggested that the attenuation of eutopic endometrial stromal cell-mediated contractility by decidualization is a novel and integral mechanism of the physiological endometrial tissue remodeling process during menstrual cycles. Although ectopic endometrial stromal cells have enhanced contractile profile, decidualization can attenuate the contractility of these cells. These findings may be one of the action mechanisms by which oral contraceptives and progestins ameliorate endometriosis.

Novel target genes responsive to the anti-growth activity of triptolide in endometrial and ovarian cancer cells

Triptolide (TPL), a bioactive component of the Chinese medicinal herb Tripterygium wilfordii Hook F, induces apoptosis in some lines of human tumor cells. However, the effect of TPL on gynecologic cancer cells has not yet been well-described. We investigated the effects of TPL on cell growth, cell cycle, and apoptosis in endometrial and ovarian cancer cell lines. Furthermore, we examined global changes in gene expression after treatment with TPL. By using a list of 20 differentially expressed genes, Western blot analyses were performed on five endometrial and ovarian cancer cell lines. All cell lines were sensitive to the growth-inhibitory effect of TPL. TPL increased the proportion of cells in the S-phase of the cell cycle and induced apoptosis. cDNA microarray assay demonstrated that the treatment with TPL changed the expression of cell cycle regulators, apoptosis-related factors and cell proliferation markers. Of the gene expression changes induced by TPL treatment, up-regulation of LRAP, CDH4, and SFRP1 and down-regulation of cystatin, TNNT 1, and L1-CAM were confirmed using Western blot analysis in all the cell lines examined. We found a strong anticancer activity of TPL and identified some potential target genes of this drug, raising hopes that TPL may become a useful therapy for endometrial and ovarian cancers.

Fasudil Inhibits the Proliferation and Contractility and Induces Cell Cycle Arrest and Apoptosis of Human Endometriotic Stromal Cells: A Promising Agent for the Treatment of Endometriosis

CONTEXT During the development of endometriotic lesions, excess fibrosis may lead to scarring and to the alterations of tissue function that are the characteristic features of this disease. Enhanced extracellular matrix contractility of endometriotic stromal cells (ECSC) mediated by the mevalonate-Ras homology (Rho)/Rho-associated coiled-coil-forming protein kinase (ROCK) pathway has been shown to contribute to the pathogenesis of endometriosis. DESIGN To assess the use of fasudil, a selective ROCK inhibitor, for the medical treatment of endometriosis-associated fibrosis, the effects of this agent on the cell proliferation, apoptosis, cell cycle, morphology, cell density, and contractility of ECSC were investigated. The effects of fasudil on the expression of contractility-related, apoptosis-related, and cell cycle-related molecules in ECSC were also evaluated. RESULTS Fasudil significantly inhibited the proliferation and contractility of ECSC and induced the cell cycle arrest in the G2/M phase and apoptosis of these cells. Morphological observation revealed the suppression of ECSC attachment to collagen fibers and decrease of cell density by fasudil. The expression of α-smooth muscle actin, RhoA, ROCK-I, and ROCK-II proteins was inhibited by fasudil administration. The expression of the antiapoptotic factors, Bcl-2 and Bcl-X(L), in two-dimensional cultured ECSC were down-regulated by the addition of fasudil, whereas, the expression of p16(INK4a) and p21(Waf1/Cip1) was up-regulated by the addition of fasudil. CONCLUSIONS The present findings suggest that fasudil is a promising agent for the treatment of endometriosis. The inhibition of cell proliferation, contractility, and myofibroblastic differentiation, the attenuation of attachment to collagen fibers, the decrease of cell density, and the induction of cell cycle arrest and apoptosis of ECSC are involved in the active mechanisms of fasudil.

Heparin is a promising agent for the treatment of endometriosis-associated fibrosis

OBJECTIVE To assess the use of heparin for the medical treatment of endometriosis-associated fibrosis. DESIGN The effects of heparin on the endometriotic stromal cells (ECSCs)-mediated contractility were investigated. SETTING Research laboratory at a medical school. PATIENT(S) Endometriotic tissues from nine patients were used. INTERVENTION(S) Endometriotic stromal cells were cultured three dimensionally in the presence of heparin. MAIN OUTCOME MEASURE(S) The contractility of ECSCs was assessed by collagen gel contraction assay. Heparin-induced morphological changes of ECSCs were evaluated by laser scanning microscopy. The expression of contractility-related molecules in ECSCs was examined by Western blot analysis. RESULT(S) In the presence of 10% fetal bovine serum, treated ECSCs showed significant collagen gel contractility (75.9% decrease in surface area after 48 hour vs. 0 hour controls). Endometriotic stromal cell-mediated gel contraction was significantly attenuated in the presence of heparin in a dose-dependent manner (55.7% reduction of the gel contraction at a concentration of 100 microg/mL of heparin sodium versus untreated controls after 48 hours). Heparin suppressed the ECSC attachment to collagen fibers. The expression of alpha-smooth muscle actin, Ras homology (Rho) A, Rho-associated coiled-coil-forming protein kinase (ROCK)-I, and ROCK-II was down-regulated by heparin administration. CONCLUSION(S) The present study suggests that heparin is a promising agent for the treatment of endometriosis-associated fibrosis. The inhibition of myofibroblastic differentiation, the attenuation of attachment to collagen fibers, and the suppression of Rho-ROCK-mediated pathway activation in ECSCs are involved in the action mechanisms of heparin.

Mevalonate-Ras Homology (Rho)/Rho-Associated Coiled-Coil-Forming Protein Kinase (ROCK)-Mediated Signaling Pathway as a Therapeutic Target for the Treatment of Endometriosis-Associated Fibrosis

a disease affecting 3-10% of women of reproductive age, is characterized by the ectopic growth of endometrial tissue. Histologically, this disease is characterized by endometrial gland and stroma with surrounding dense fibrous tissue. During the development of endometriotic lesions, excess fibrosis may lead to scarring and to altera- tion of the tissue function. It has been suggested that type I collagen is a major contributor to endometriosis-associated fibrosis. One approach to understanding the pathogenesis of endometriosis is to investigate the mechanisms underlying fibrogenesis associated with this disease. Using three-dimensional collagen gel culture model, we have evaluated the extracellular matrix contractility and myofibroblastic differentiation of endometriotic stromal cells. Endometriotic stromal cells showed enhanced extracellular matrix contractility in comparison with normal endometrial stromal cells. Activation of the Ras homology (Rho)/Rho- associated coiled-coil-forming protein kinase (ROCK)-mediated signaling pathway with simultaneously enhanced myofi- broblastic differentiation is involved in this mechanism. We have proposed the mevalonate-Rho/ROCK-mediated signal- ing pathways as possible therapeutic targets for the treatment of endometriosis-associated fibrosis. This paper is a review of the recent information concerning the mechanism of endometriosis-associated fibrosis, focusing on the contractility of endometriotic stromal cells. The modulators of mevalonate-Rho/ROCK signaling pathways were also evaluated as promising agents for the treatment and prevention of endometriosis-associated fibrosis.

Effects of parity and gestational age on second-trimester induction–abortion interval in combination with osmotic dilators and gemeprost

BACKGROUND The true prognostic factors for induced medical abortion are unknown. We sought to investigate the effects of a patients obstetric parameters on the induction-abortion interval in second-trimester medical abortion. STUDY DESIGN We studied 216 consecutive women. Pregnancy was terminated with cervical preparation using osmotic dilators followed by 1 mg vaginal gemeprost administered every 3 h for a maximum of five doses in the first 24 h. All variables are expressed in categorical form (parity, gestational age, maternal age and body mass index) and analyzed by the Cox proportional hazards model. RESULTS Parity ≥ 3 was associated with a shorter duration of the induction-abortion interval (adjusted hazards ratio 1.96; 95% confidence interval 1.13-3.40). A gestational age ≥ 16 weeks was associated with a longer duration of the induction-abortion interval (0.71; 0.52-0.98). No significant association was found in maternal age and body mass index. CONCLUSIONS In combination with osmotic dilators and gemeprost, gestational age and parity are independent factors that affected the induction to abortion interval of second-trimester medical abortion.