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Endocrinology Vol. 148, No. 2 896-902
Copyright © 2007 by The Endocrine Society

Histone Deacetylase Inhibitors Stimulate Cell Migration in Human Endometrial Adenocarcinoma Cells through Up-Regulation of Glycodelin

Hiroshi Uchida, Tetsuo Maruyama, Masanori Ono, Kuniaki Ohta, Takashi Kajitani, Hirotaka Masuda, Takashi Nagashima, Toru Arase, Hironori Asada and Yasunori Yoshimura

Department of Obstetrics and Gynecology, Keio University School of Medicine, Shinjuku, Tokyo 160-8582, Japan

Address all correspondence and requests for reprints to: Tetsuo Maruyama, M.D., Ph.D., Department of Obstetrics and Gynecology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan. E-mail: tetsuo{at}sc.itc.keio.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Histone deacetylase inhibitors (HDACIs) have recently emerged as promising anticancer drugs to induce cell cycle arrest, cytodifferentiation, and apoptosis. It is suggested, however, that HDACIs promote cell migration and invasion depending on the cell type. We have reported previously that treatment with HDACIs, including trichostatin A and suberoylanilide hydroxamic acid (SAHA) or progesterone in combination with estrogen, can induce cytodifferentiation of endometrial adenocarcinoma Ishikawa cells through up-regulation of glycodelin, a progesterone-induced endometrial glycoprotein. Given the reported role of glycodelin in cell motility and the migration-modulating potential of HDACIs, we investigated using wound healing assay and transwell migration assay whether ovarian steroid hormones, trichostatin A, or SAHA affects cell migration in endometrial cancer cell lines, Ishikawa and RL95-2. Treatment with ovarian steroid hormones, trichostatin A, and SAHA enhanced cell migration together with up-regulation of glycodelin. SAHA-augmented cell migration was almost completely blocked by gene silencing of glycodelin. Furthermore, overexpression of gycodelin alone resulted in increased cell motility in Ishikawa cells. Our results collectively indicate that glycodelin positively regulates cell motility acting as a mediator of HDACI-enhanced endometrial cell migration, suggesting the involvement of glycodelin in the dynamic endometrial gland morphogenesis during menstrual cycle. Our results raise a possibility that the use of HDACIs in the therapy for glycodelin-inducible endometrial and presumably other gynecological cancers may enhance invasion in cases in which the HDACIs fail to exert differentiation-inducing and/or antiproliferative effects.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ACETYLATION AND DEACETYLATION of nucleosomal histones play an important role in the modulation of chromatin structure/function and the regulation of gene expression. Histone acetyltransferases and histone deacetylases are two opposing classes of enzymes that tightly control the acetylation status of histones. Deregulation of histone acetylation is associated with carcinogenesis and cancer progression (1). Histone deacetylase inhibitors (HDACIs) are a novel class of anticancer drugs that mainly lead to an accumulation of acetylated proteins, thereby inducing cell cycle arrest, differentiation, and apoptosis in cancer cells and transformed cells (2). These characteristics are directly relevant for their use as anticancer drugs. Several clinical trials have been instituted to test the efficacy of HDACIs for the treatment of different malignant tumors (3).

In addition to the antiproliferative and apoptosis-inducing effects, HDACIs also can inhibit cell migration and invasion in many types of cells, including human lung cancer cells, endothelial cells, hepatic satellite cells, and v-Fos-transformed cells (4, 5, 6, 7, 8, 9), which further substantiates the potential of HDACIs as anticancer drugs. The inhibitory effects of HDACIs on cell motility involves various molecular events such as down-regulation of endothelial nitric oxide synthetase (7), suppression of nuclear factor-{kappa}B activity (9), or inhibition of matrix metalloproteinase 2 activation through up-regulation of RECK, a negative regulator of matrix metalloproteinases (5). Several studies, however, have reported the contradictory activities of HDACIs by which they potentiate cell migration, for instance, in hepatocellular carcinoma cells and melanoma cells through up-regulation of integrins and chemokine receptors, respectively (10, 11, 12, 13). The effect of HDACIs on cell migration and invasion is thought to depend on the type of targeted cells and/or HDACIs used. It should be, therefore, kept in mind that the use of HDACIs as cancer therapy may paradoxically promote invasion through enhancement of cancer cell migration.

We have previously reported that the differentiation of primary cultured human endometrial stromal cells is enhanced by treatment with trichostatin A (TSA) in combination with 17ß-estradiol (E2) and progesterone (P4) (14) and that TSA and suberoylanilide hydroxamic acid (SAHA) both belong to the hydroxamate group of HDACIs and induce morphological and functional differentiation in the human endometrial adenocarcinoma cell line, Ishikawa (derived from epithelial origin) (15). Takai et al. (16) have demonstrated the antiproliferative and apoptosis-inducing effects of HDACIs on endometrial cancer cell lines. Taken together, these data indicate the therapeutic potential of HDACIs for endometrial cancer; however, whether HDACIs affect endometrial cancer cell migration and invasion remains to be clarified. To address this question, we examined, using wound healing and transwell assays, whether ovarian steroids and/or HDACIs have a potential to regulate cell motility of human endometrial adenocarcinoma. Unexpectedly, we found that HDACIs, in particular SAHA, enhanced single and collective cell migration and demonstrated that HDACI-induced glycodelin, a progesterone-induced endometrial glycoprotein, played an essential and sufficient role in promotion of Ishikawa cell migration.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Phenol red-free MEM, DMEM/F12, and fetal bovine serum were purchased from Invitrogen (Tokyo, Japan). TSA and SAHA were obtained from Wako (Osaka, Japan) and BIOMOL (Plymouth Meeting, PA), respectively. Antibodies against glycodelin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and horseradish peroxidase or Cy3-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) were purchased from commercial sources. Unless indicated otherwise, all other chemicals were obtained from Sigma-Aldrich (St. Louis, MO) and Wako.

Cell culture
Ishikawa (clone 3-H-12) (17), the human endometrial adenocarcinoma cell line of epithelial origin positive for estrogen receptor (ER){alpha} and P4 receptor (PR), was a gift from Dr. M. Nishida (National Kasumigaura Hospital, Ibaragi, Japan). RL95-2 cells, moderately differentiated human endometrial adenosquamous carcinoma cell line, were purchased from American Type Cell Culture (Rockville, MD). Ishikawa cells and RL95-2 cells were cultured in phenol-red free MEM or phenol-red free DMEM/F12 supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml of penicillin, and 100 mg/ml of streptomycin. Ishikawa cells were used within 10 passages according to the provider’s recommendation to avoid changes of the cell characters, including down-regulation of ER{alpha} and PR expression.

Small interference RNA (siRNA) and transfection
siRNA-targeting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was obtained from Ambion (Austin, TX). The target sequence in the glycodelin gene was 5' CGTGGCCCTGGTCTGTGGTGT-3'. Sense RNA (5'-UGGCCCUGGUCUGUGGUGUUU-3') and antisense RNA (5'-ACACCACAGACCAGGGCCACG-3') were independently synthesized, annealed, and then used as glycodelin siRNA (QIAGEN, Hilden, Germany). Either GAPDH or glycodelin siRNA was transfected into Ishikawa cells using RNAiFect Transfection Reagent (QIAGEN).

Wound healing assay
Confluent monolayer cells were transfected without or with glycodelin or GAPDH siRNA, scratched by yellow tip (0 h), and then cultured in the absence or presence of 10 nM E2 in combination with 1 µM P4 (E2P4) or various concentrations of HDACIs for 24 h. At 0 h and 24 h, the scratched monolayer cultures were photographed using an inverted microscope (Leica Microsystems, Heidelberg, Germany). Quantification was done by measuring the number of pixels in each wound closure area using Adobe Photoshop. Data are expressed as percentage wound closure relative to the wound closure area in the control medium. The wound closure area of the control cells was set at 100%.

Transwell assay
Transwell cell migration assays were performed by using modified Boyden chambers (tissue culture treated, 6.5-mm diameter, 10-µm thickness, 8-µm pores, Transwell; Corning, Corning, NY) with the underside of the membranes coated with 10 µg/ml of collagen type I (Upstate Biotechnology, Lake Placid, NY), fibronectin, vitronectin, laminin-5, or BSA as previously described (18). In brief, cells with or without transfection of GAPDH or glycodelin siRNA were trypsinized, washed, and suspended in the control medium at 2–10 x 105 cells/ml. Each 100 µl of cells was then applied onto the upper migration chambers and allowed to migrate for 24 h. At the bottom side, medium without or with ovarian steroid hormone(s) or HDACIs was prepared. Cells migrated to the underside of the chamber were fixed with 3.7% paraformaldehyde in PBS. After swabbing of nonmigrating cells at the upper side of chamber, the removed membranes were mounted with VECTASHIELD Mounting Medium with 4',6-diamidino-2-phenylindole (DAPI) (Vector Laboratories Inc., Burlingame, CA). Migrating cells were counted using a fluorescent microscope (Leica Microsystems). The numbers of applied cells were decided by our prior study. Relative migrated cells of parental cells set at 100%.

Immunofluorescence
Confluent Ishikawa cells were cultured for 24 h in the indicated medium after scratching with yellow tip, fixed with 3.7% paraformaldehyde, permeabilized with 0.2% Triton X-100 in PBS, and then incubated with antiglycodelin antibody for 1 h at room temperature followed by incubation with Cy3-conjugated appropriate secondary antibody. Confocal images were acquired using a Leica TCS SP2 confocal microscopy system with a Leica DMIRE2 inverted microscope (Leica Microsystems). Nonspecific background fluorescence was determined by staining the cells with irrelevant antibodies coupled with the appropriate dye conjugated secondary antibodies.

Immunoprecipitation and immunoblotting
Confluent Ishikawa or RL95-2 cells transfected without or with siRNA were cultured in the indicated medium for 3 d and lysed on ice with RIPA buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1 mM EDTA; 1% Na-deoxycholate; 0.1% sodium dodecyl sulfate; 1 mM Na3VO4; 50 mM NaF; 1 mM Na2MoO4) containing protease inhibitor cocktail (Roche, Basel, Switzerland). Protein concentrations were determined using DC protein assay kit (Bio-Rad Laboratories, Hercules, CA) with BSA as a standard. Each 250 µg of protein was subjected to immunoprecipitation with antiglycodelin antibody and protein G-Sepharose beads (Amersham Biosciences, Piscataway, NJ) for 3 h at 4 C. Immunoprecipiates were separated by electrophoresis on a 15% SDS-PAGE gel and transferred onto polyvinylidene difluoride membrane. After incubation with antiglycodelin antibody, followed by horseradish peroxidase-conjugated secondary antibody, the immunoreactive proteins were detected by the enhanced chemiluminescence method (Amersham Biosciences).

Establishment of stable clones expressing enhanced green fluorescent protein (EGFP)-fused glycodelin
Semiconfluent Ishikawa cells were transfected with pEGFP-N3 (TaKaRa, Tokyo, Japan) or EGFP-tagged glycodelin expression vector, named pcGd1-EGFP (15), using LipofectAmine (Invitrogen, Carlsbad, CA). Transfected Ishikawa cells were cultured in the medium containing 500 µg/ml geneticin for more than 2 wk to select cells stably expressing EGFP or EGFP-tagged glycodelin. The expression levels of the exogenous proteins in geneticin-resistant subcloned Ishikawa cells were analyzed with immunoblotting using anti-GFP and antiglycodelin antibodies.

Statistical analysis
All experimental data of bioassays represent the results obtained from three independent experiments of each duplicate or triplicate assay and expressed as mean ± SEM. Statistical analysis was performed using ANOVA followed by a Bonferroni test. Immunoprecipitation and immunoblotting studies were repeated three times and the representative images were shown.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
HDACIs stimulate collective cell migration in Ishikawa cells
To examine the effect of ovarian steroid hormones and HDACIs on collective cell migration, we first performed wound healing assays using Ishikawa cells. Confluent monolayers of Ishikawa cells were scratched to form wound and cultured in the absence or presence of E2P4 or various concentrations of SAHA and TSA for 24 h. Phase contrast images were photographed and the wound area was measured (Fig. 1AGo). Although E2 is known to stimulate cell proliferation in Ishikawa cells (19), it did not enhance but rather suppressed the wound healing (Fig. 1BGo), indicating that wound closure reflected collective cell migration but not cell proliferation. There was a tendency that E2P4 augmented the wound closure (Fig. 1BGo); however, the enhancing effect was not statistically significant. In contrast, SAHA or TSA alone significantly up-regulated collective cell migration in a dose-dependent manner (Fig. 1BGo).


Figure 1
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  		FIG. 1. HDACIs promote collective cell migration of Ishikawa cells in a dose-dependent fashion. A, Confluent monolayers of Ishikawa cells were wounded with a uniform scratch, washed to remove cell debris, and cultured for 24 h in the absence (control) or presence of E2P4, 2 µM SAHA, or 250 nM TSA. Phase-contrast images of cell cultures were captured 0 h (upper four panels) and 24 h (lower four panels) after scratching. Leading edges of the wounds are indicated by solid (0 h) and broken lines (24 h). Scale bar, 100 µm. B, Each bar indicates mean ± SEM of relative wound closure of Ishikawa cells treated without (control) or with E2, E2P4, or the indicated concentrations of SAHA or TSA for 24 h. Data are expressed as percentage wound closure relative to the width of control wounds photographed at time zero. The wound closure area of the control cells was set at 100%. Asterisks show significant differences compared with control (P < 0.05).

 
HDACIs enhance fibronectin-oriented single cell migration in Ishikawa cells
We next investigated using modified Boyden chamber assay whether ovarian steroid hormones or HDACIs influence single cell migration. In this assay, Ishikawa cells were seeded on the top side of the membrane and were allowed to migrate into the bottom side coated with BSA or various extracellular matrices (ECMs). The nuclei of the cells at the bottom side were then stained with DAPI, photographed, and counted. Because cell type-specific expression of adhesion molecules determines the preference for ECMs, we first examined which ECMs preferentially drive single cell migration of Ishikawa cells. Modified Boyden chamber assays using various types of ECMs revealed that Ishikawa cell motility was significantly enhanced when the bottom membrane was coated with fibronectin, collagen type I, or vitronectin compared with BSA and laminin (Fig. 2AGo).


Figure 2
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  		FIG. 2. Single cell migration is enhanced by treatment with E2P4 or HDACIs. A, Each bar indicates mean ± SEM of cell migration activity as determined by transwell assays using Boyden chamber coated with BSA, collagen type I (Col), fibronectin (FN), vitronectin (VN), or laminin 5 (LN) at the bottom side. The number of Ishikawa cells migrating onto the BSA-coated membrane was set at 100%. Asterisks show significant differences compared with BSA (P < 0.05). B, Each bar indicates mean ± SEM of the FN-oriented migration activity of Ishikawa cells treated without (control) or with E2, E2P4, or various concentrations of HDACIs for 24 h. The number of control migrating cells was set at 100%. Asterisks show significant differences compared with control cells (P < 0.05). C, Ishikawa cells were allowed for 24 h to migrate into FN-coated membrane in the absence (control) or presence of E2P4 or 2 µM SAHA. After removal of the cells from the top side of the membrane by swabbing, migrating cells present at the bottom side were then fixed, stained with DAPI, and the immunofluorescence images were photographed. Scale bar, 20 µm.

 
Because the migration was most evident with fibronectin (Fig. 2AGo), we next tested the effects of ovarian steroid hormones, SAHA, and TSA on the fibronectin-oriented cell motility. Treatment with E2P4, but not E2 alone, significantly enhanced single cell migration in Ishikawa cells (Fig. 2BGo). Furthermore, even in the absence of ovarian steroid hormones, SAHA or TSA alone dramatically up-regulated single cell migration in a dose-dependent manner (Fig. 2BGo). Figure 2CGo shows the representative immunofluorescence images of migrating Ishikawa cells treated without or with E2P4 or 2 µM SAHA.

HDACIs-induced glycodelin mediates single and collective cell migration
We have previously shown that HDACIs up-regulate glycodelin mRNA and protein (15). Consistently, immunostaining using antiglycodelin antibody revealed that treatment with E2P4 and SAHA increased the fluorescence intensity in Ishikawa cells compared with the control treatment (Fig. 3AGo). Reported roles of glycodelin in cell motility (20, 21) prompted us to address the question of whether HDACI-induced glycodelin per se functionally contributes to the stimulation of Ishikawa cell migration. Taking advantage of glycodelin siRNA as described previously (15), we examined the effect of glycodelin knockdown on HDACI-induced collective and single cell migration. As shown in Fig. 3BGo (lower panels), glycodelin siRNA, but not GAPDH siRNA, decreased the basal glycodelin level and further dramatically inhibited the E2P4- or SAHA-mediated induction of glycodelin, indicating the validity of the gene silencing effect of the siRNA used in this study. Consistent with the magnitude of the inhibitory effect on glycodelin expression, glycodelin siRNA, but not GAPDH siRNA, almost completely abrogated E2P4- or SAHA-induced collective and single cell migration as determined by wound healing assay (Fig. 3BGo, upper panel) and transwell assay (Fig. 3CGo), respectively.


Figure 3
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  		FIG. 3. Glycodelin mediates HDACI-induced migration in Ishikawa cells. A, Ishikawa cells were cultured without or with E2P4 or 2 µM SAHA for 24 h, fixed, and immunostained with antiglycodelin antibody followed by Cy3-conjugated antigoat secondary antibody. Scale bar, 20 µm. B and C, Ishikawa cells untransfected (no siRNA) or transiently transfected with GAPDH siRNA or glycodelin siRNA were subjected to wound healing assays for 24 h or transwell assays for 24 h in the absence (control) or presence of E2P4 or 2 µM SAHA. Each bar indicates mean ± SEM of (B) relative wound closure or (C) cell migration activity. The expression of glycodelin protein in the cells undergoing each treatment was detected by immunoprecipitation and immunoblotting (B, lower panels). Asterisks (B) and double asterisks (C) show significant differences compared with untransfected or GAPDH siRNA-transfected cells in the same treatment group (P < 0.05).

 
SAHA up-regulates collective and single cell migration in RL95-2 cells
SAHA and TSA stimulated both single and collective cell migration in Ishikawa cells. To address that the migration-enhancing effect of SAHA is not specific for Ishikawa cells but rather common in endometrial cancer cells, we examined the effect of SAHA on another endometrial cancer cell line, RL95-2, which was originated from moderately differentiated human endometrial cancer. Because SAHA was more toxic to RL95-2 cells than Ishikawa cells, we stimulated RL95-2 cells with a lower concentration of SAHA (0.5 µM). In a similar way to Ishikawa cells, E2P4 and SAHA significantly up-regulated collective cell migration in RL95-2 cells as determined by would healing assay (Fig. 4AGo). In transwell migration assay, we found that RL95-2 cells migrated most efficiently when collagen type I was used as a coating for ECM. Single cell migration activity of RL95-2 cells was also significantly enhanced by the stimulation with E2P4 and 0.5 µM SAHA (Fig. 4BGo). RL95-2 cells exhibited a low induction of glycodelin levels in response to E2P4 and SAHA (Fig. 4CGo), which appears to be consistent with the relatively weak migration. These results collectively suggest that the migration-promoting and glycodelin-inducing effects of HDACIs may be commonly observed in human endometrial cancer cells in general.


Figure 4
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  		FIG. 4. SAHA and ovarian steroid hormones up-regulate collective and single cell migration in RL95-2 cells. A, Each bar indicates mean ± SEM of relative wound closure of RL95-2 cells treated without (control) or with E2P4 or the 0.5 µM SAHA for 24 h. Data are expressed as percentage wound closure relative to the width of control wounds photographed at time zero. The wound closure area of the control cells was set at 100%. Asterisks show significant differences compared with control (P < 0.05). B, Each bar indicates mean ± SEM of the Col-oriented migration activity of RL95-2 cells treated without (control) or with E2P4 or 0.5 µM SAHA for 24 h. The number of control migrating cells was set at 100%. Asterisks show significant differences compared with control cells (P < 0.05). C, The glycodelin protein in RL95-2 cells undergoing each treatment was detected by immunoprecipitation and immunoblotting.

 
Overexpression of glycodelin alone enhances single and collective cell migration
Lastly, to address the potential of glycodelin to promote cell migration, we examined using transfectants stably expressing EGFP-fused glycodelin (cGd1-EGFP) whether simple overexpression of glycodelin enhances single and collective cell migration. Immunoblot analysis with anti-GFP and antiglycodelin antibodies revealed that two stable clones generated from Ishikawa cells exhibited different levels of cGd1-EGFP expression (Fig. 5AGo). Both clones displayed more potent migration ability than MOCK or an EGFP-expressing clone as determined by wound healing assay (Fig. 5BGo, left panel) and transwell assay (Fig. 5BGo, right panel). The magnitude of the migration potentials, however, did not appear to correlate with the expression level of cGd1-EGFP (Fig. 5BGo), suggesting that an optimal level of glycodelin may be required for eliciting the maximal single cell motility in Ishikawa cells.


Figure 5
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  		FIG. 5. Overexpression of glycodelin alone enhances single and collective cell migration in Ishikawa cells. A, Total cell lysate (25 µg protein/lane) obtained from a permanent transfectant of pEGFP-N3 (EGFP-N3) or two different stable clones expressing EGFP-fused glycodelin (cGd1-EGFP 1 and 2) and total cell lysates from Ishikawa cells transiently transfected with pcGd1-EGFP (transient, 5 µG protein/lane) were separated on 12% SDS-PAGE, transferred, and immunoblotted with anti-GFP or antiglycodelin antibodies. Arrowheads indicated positive bands for EGFP alone (black) and EGFP-tagged glycodelin (white). B, Untransfected Ishikawa cells (MOCK), the permanent EGFP transfectant (EGFP-N3), and the two stable cGd1-EGFP clones were subjected to wound healing assay or cell migration assay for 24 h. Each bar indicates mean ± SEM of relative wound closure (left panel) or cell migration activity (right panel). Asterisks (left panel) and double asterisks (right panel) show significant differences compared with MOCK and EGFP-N3 (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Glycodelin is the progesterone-induced glycoprotein secreted into uterine luminal cavity by secretory/decidualized endometrial glands (22). It inhibits human sperm-egg binding (23) and has immunosuppressive activity through inactivation of natural killer cells and suppression of T lymphocyte proliferation (24, 25), presumably functioning to avoid multiple pregnancy and to protect the embryonic semiallograft from maternal immune attack. Extracellular glycodelin also has a potential for positive or negative regulation of cell motility via an as-yet-unidentified glycodelin receptor: for instance, suppression of cAMP-activated U937 cell migration (21) and enhancement of HUVEC cell migration (20). In contrast, its intracellular action on cell migration has not been elucidated. Given that Ishikawa cells alone are unable to detect secretion of glycodelin (26), we demonstrated for the first time that intracellular glycodelin, whichever primarily expressed or induced, promoted cell migration, being required for HDACI-enhanced cell motility in endometrial adenocarcinoma Ishikawa cells.

Despite the migration-promoting potential of glycodelin, its expression in cancer is positively associated with good prognosis (27, 28). Chemotherapy-treated patients with glycodelin-expressing serous ovarian carcinoma have longer survival times than those with glycodelin-negative tumors with the same differentiation grade and clinical stage (27). Furthermore, the patients with glycodelin-positive breast tumors had better prognoses compared with glycodelin-negative subjects (27, 28). Relatively good prognoses of patients with glycodelin-positive cancers can be partially accounted by its cytodifferentiation-inducing (15, 29), antiproliferative (29, 30), and/or apoptosis-inducing properties (30). These inhibitory effects of glycodelin on tumor progression may usually overcome its migration-promoting action. However, it is possible that the aberrant induction of glycodelin by HDACIs may provoke imbalance between its negative and positive effects, and eventually cancer cells may acquire the invasive potential. Thus, the cell motility-enhancing potential of HDACIs and glycodelin should be noted when HDACIs are developed and clinically applied as an anticancer drug for glycodelin-inducible gynecological cancer cells.

We showed that treatment with ovarian steroid hormones could significantly enhance cell motility in Ishikawa cells, although the magnitude was less dramatic than that with HDACIs. Because Ishikawa is a well-differentiated endometrial cancer cell line of human glandular epithelial origin and expresses functioning ER{alpha} and PR, it has been widely used for studies on the physiopathology of human endometrial glandular cells (17). Given that Ishikawa cells possess some phenotypes similar to normal endometrial glandular cells (17), it is possible that the promotion of cell motility by ovarian steroid hormones presented herein may be relevant to the endometrial physiology, in particular, the morphogenesis and repair of the endometrial gland, rather than endometrial carcinogenesis. Enhancement of cell migration by E2P4 together with its abrogation by glycodelin siRNA suggests that endometrial epithelial cells may become more motile through up-regulation of glycodelin during the progesterone-dominated secretory phase than those during the estrogen-dominated proliferative phase. In the secretory phase, glandular epithelial cells are thought to exhibit functional and morphological differentiation concomitant with migration into the stroma to develop well-branched glands. Thus, the ovarian steroid hormone-augmented cell motility may contribute to the morphogenesis and development of the glands during the secretory phase. Alternatively, epithelial sheet migration may be required for the repair of epithelial surface defects caused by embryo implantation.

In summary, ovarian steroid hormones and HDACIs enhanced the motility of endometrial cancer Ishikawa cells through up-regulation of glycodelin. The migration-enhancing potential of glycodelin suggests its possible involvement in the endometrial gland morphogenesis during the menstrual cycle. HDACIs have several anticancer potentials, including inhibition of cell proliferation and induction of cytodifferentiation and apoptosis. It is possible that the migration-promoting effects of HDACIs as shown here may overcome these anticancer actions in assistance with glycodelin. In the case that HDACIs fail to exert those anticancer effects, the clinical use of HDACIs might enhance invasion of endometrial cancers through up-regulation of glycodelin.


   Acknowledgments
 
We are grateful to Ms. Rei Sakurai for her technical assistance and Ms. Shino Kuwabara for her secretarial work. We also thank Dr. Masato Nishida (National Kasumigaura Hospital, Ibaragi, Japan) for the gift of Ishikawa cells.


   Footnotes
 
This work was supported in part by grants-in-aid for scientific research C (2) 16591683, C 18591812 (to H.U.) and C (2) 13671743 (to T.M.) from the Ministry of Education, Science, Sports, and Culture of Japan; and grants from Keio University for Encouragement of Young Medical Scientists (to H.U.), Keio Medical Association, and Mitsukoshi Health and Welfare Foundation (to T.M.).

First Published Online October 26, 2006

Abbreviations: DAPI, 4',6-Diamidino-2-phenylindole; E2, 17ß-estradiol; ECM, extracellular matrix; EGFP, enhanced green fluorescent protein; ER, estrogen receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDACI, histone deacetylase inhibitor; P4, progesterone; PR, P4 receptor; SAHA, suberoylanilide hydroxamic acid; siRNA, small interference RNA; TSA, trichostatin A.

Received July 5, 2006.

Accepted for publication October 16, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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