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. 2012 Feb 8;86(5):160. doi: 10.1095/biolreprod.111.097295

Mono-(2-Ethylhexyl) Phthalate (MEHP) Promotes Invasion and Migration of Human Testicular Embryonal Carcinoma Cells1

Pei-Li Yao 1,3, Yi-Chen Lin 1, John H Richburg 1,2
PMCID: PMC3364929  PMID: 22321834

ABSTRACT

Testicular dysgenesis syndrome refers to a collection of diseases in men, including testicular cancer, that arise as a result of abnormal testicular development. Phthalates are a class of chemicals used widely in the production of plastic products and other consumer goods. Unfortunately, phthalate exposure has been linked to reproductive dysfunction and has been shown to adversely affect normal germ cell development. In this study, we show that mono-(2-ethylhexyl) phthalate (MEHP) induces matrix metalloproteinase 2 (MMP2) expression in testicular embryonal carcinoma NT2/D1 cells but has no significant effect on MMP9 expression. NT2/D1 cells also have higher levels of MYC expression following MEHP treatment. It is widely recognized that activation of MMP2 and MYC is tightly associated with tumor metastasis and tumor progression. Gelatin zymographic analysis indicates that MEHP strongly activates MMP2 in NT2/D1 cells. Addition of the MMP2-specific inhibitor SB-3CT inhibited MEHP-enhanced cell invasion and migration, demonstrating that MMP2 plays a functional role in promoting testicular embryonal carcinoma progression in response to MEHP exposure. Furthermore, we investigated genome-wide gene expression profiles of NT2/D1 cells following MEHP exposure at 0, 3, and 24 h. Microarray analysis and semiquantitative RT-PCR revealed that MEHP exposure primarily influenced genes in cell adhesion and transcription in NT2/D1 cells. Gap junction protein-alpha 1, vinculin, and inhibitor of DNA-binding protein-1 were significantly down-regulated by MEHP treatment, while claudin-6 and beta 1-catenin expression levels were up-regulated. This study provides insight into mechanisms that may account for modulating testicular cancer progression following phthalate exposure.

Keywords: embryonal carcinoma, invasion, MEHP, migration, MMP2, testis

MEHP, a phthalate metabolite found widely in the environment, promotes tumor metastasis and progression via the activation of MMP2 and dysregulation of cell adhesion molecules in testicular embryonal carcinoma cells.

INTRODUCTION

Testicular cancer is most prevalent in young adult men ages 15–40 yr and is approximately 5 times more common in Caucasians than in other races [1]. This disease has been reported to be correlated with other common testicular disorders that likely arise from shared mechanisms, such as poor semen quality and reduced sperm numbers, cryptorchidism, and hypospadias [2]. The incidence of these diseases has dramatically increased over only the last 50 yr, and it has been hypothesized that environmental influences rather than genetic defects account for this increase [2–7]. Together, these diseases are suggested to comprise testicular dysgenesis syndrome (TDS). Therefore, gaining insights into the mechanisms that underlie testicular cancer development and testicular tumor progression induced by environmental toxicants may also provide an understanding of the etiology of other testicular diseases.

Phthalates are synthetic chemicals that are widely used as plasticizers in order to provide flexibility in plastics [8, 9]. Phthalates are not covalently bound to plastic products and therefore can easily leach into the environment. Human populations can be exposed to phthalates through direct contact or general environmental contamination [10]. Phthalate exposure has been linked to reproductive dysfunction and may affect human health [11, 12]. Although it has been shown that phthalates induce hepatotoxicity and cause hepatocellular tumorigenesis through several mechanisms [13–15], little is known about the incidence of testicular cancer initiation and the mechanism(s) that accounts for tumor progression triggered by environmental stimuli. We previously discovered that mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of di(2-ehtylhexyl)phthalate (DE-HP), induces the activation of matrix metalloproteinase 2 (MMP2) in the seminiferous tubule [16], leading to impaired spermatogenesis. The ratio between MMP2 and its endogenous inhibitor, tissue inhibitor of matrix metalloproteinase 2 (TIMP2), is critical for maintaining normal germ cell development [16, 17] and also influencing the tumor metastasis in different types of tumors [18, 19]. Interestingly, it has been shown that the serum level of MMP2 is increased in patients with a diagnosis of testicular germ cell tumors [20]. It has been shown that in utero exposure to dibutyl phthalate (DBP) in rats has pathologic conditions similar to those in human TDS, such as cryptorchidism, hypospadias, infertility, reduced spermatogenesis, and multinucleated gonocytes [21, 22]. MEHP exposure also results in a decrease in testosterone levels and insulin-like factor 3 expression, which are directly associated with clinical features of TDS [23, 24]. These findings led us to test whether MEHP-activated MMP2 in the testis plays a role in regulating testicular cancer metastasis. We had also previously shown that MEHP-induced MYC expression is partially responsible for the down-regulation of TIMP2 in the testis [25]. It has been widely appreciated that MYC functions as a switch in tumor promotion by regulating proliferation or tumor suppression by modulating apoptosis [26]. Therefore, the study of the functional significance of MYC in testicular cancer progression in response to phthalate is of interests.

In this study, we investigated the effects of MEHP on stimulation of cell invasion and migration in testicular embryonal carcinoma cells. One mechanism that may account for the increase in invasion and migration following MEHP exposure is activation of MMP2. Microarray analysis demonstrated that cell adhesion molecules and transcription factors may also be responsible for tumor progression in the testis after MEHP exposure.

MATERIALS AND METHODS

Cell Line

The human testicular embryonal carcinoma cell line NTERA-2 cl. D1 (NT2/D1) was purchased from American Type Culture Collection (ATCC, Manassas, VA) and cultured in Dulbecco modified Eagle medium (ATCC) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (all from Invitrogen, Carlsbad, CA) at 37°C in 5% CO2.

MEHP Treatment

NT2/D1 cells were treated with designated concentrations of MEHP (Wako Chemicals USA, Inc., Richmond, VA) diluted in dimethyl sulfoxide (DMSO) for various time periods. Cells treated with DMSO were used as control groups.

Western Blot Analysis

Total cellular proteins were extracted from NT2/D1 cells treated with MEHP or DMSO. Protein concentrations in samples were determined by Lowry protein assay using DC protein assay kit (Bio-Rad, Hercules, CA). Proteins (30 μg) were loaded into 4%–12% bis-Tris gradient gel (Novex, Invitrogen), separated at 150 V for 1 h and transferred to polyvinylidene fluoride membrane (Invitrogen) with NuPage transfer buffer (Invitrogen). Membranes were incubated with blocking buffer (5% skim milk in Tris-buffered saline plus Tween-20 [TBST]) at room temperature for 30 min and washed in TBST buffer (20 mM Tris-HCl, pH7.5, 150 mM NaCl, 0.1% Tween-20). Membranes were then incubated with primary antibodies against MYC, MMP2, MMP9 (1:1000 dilution; Abcam Inc., Cambridge, MA) and beta-actin (ACTB; 1:500 dilution; Santa Cruz Biotechnology Inc., Santa Cruz, CA) at 4°C overnight, after being washed twice with TBST buffer at room temperature for 10 min each and incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000 dilution; Santa Cruz Biotechnology Inc.) at room temperature for 2 h. ECL substrate (Amersham Bioscience, Piscataway, NJ) was used as the detection reagent, and ACTB was used as a loading control. Images of Western blots were captured with a Kodak Gel Logic 100 imaging system (Kodak, Rochester, NY). Densitometry for bands on Western blots was determined by ImageJ software (National Institute of Mental Health, Bethesda, MD). The relative expression level of each protein was normalized by the value of ACTB expression.

Gelatin Zymography

MMP2 and MMP9 gelatinolytic activities in medium derived from NT2/D1 cells were assayed by gelatin zymography with minor modifications [16]. Briefly, 12 h after MEHP (200 μM) was added to 2 × 106 NT2/D1 cells, samples of conditioned medium were collected and directly applied to a 10% Zymogram gel (Invitrogen). After electrophoresis, the gels were washed twice for 15 min with 2.5% Triton X-100 in 50 mM Tris-HCl (pH 7.5) to remove the SDS and restore the enzyme activity and then incubated in developing buffer (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 10 mM CaCl2 and 0.02% sodium azide) overnight at 37°C. Gels were then stained with 30% methanol/10% acetic acid containing 0.5% (w/v) Coomassie Brilliant Blue R-250 for 30 min and destained in the same solution without dye. Clear bands shown on the blue background indicate enzyme activity.

Measurement of Soluble MMP2 Levels

NT2/D1 cells (2 × 106) were exposed to MEHP for various time periods. Culture medium samples were collected and centrifuged to remove cellular debris. The supernatant was rapidly frozen at −80°C until assayed for MMP2 by ELISA (R&D Systems, Inc.) following the manufacturer's protocol. The number of cells was counted to normalize the protein expression found by ELISA. According to the manufacturer's specifications, the limit of soluble MMP2 assay sensitivity was 0.165 ng/ml. Inter- and intra-assay precision coefficients of variation were 12.65% and 6.33%, respectively.

Invasion Assay

The in vitro invasion assay was performed using Transwell chambers with 8-μm-pore polycarbonate filters (Costar, Cambridge, MA) as previously described [27]. Transwell membranes were coated with Matrigel diluted in serum-free medium (1:3 dilution; BD Biosciences, San Diego, CA). Nontreated or MEHP-treated NT2/D1 (1 × 105) cells were seeded on Matrigel and incubated at 37°C for 18 h. Membranes coated with Matrigel were wiped with cotton swabs, fixed with methanol, and stained with 20% Giemsa solution (Sigma, St. Louis, MO). Cells attached to the lower surface of the filters were then counted using light microscopy (at 200× magnification) and photographed with a Canon 5D digital camera attached to the microscope.

Migration Assay

Cell migration was estimated by wound-healing test in NT2/D1 cells. Nontreated or MEHP-treated NT2/D1 (5 × 105) cells were seeded into 6-cm culture plates and grown to almost confluent cell monolayer. A pipette tip was used to scratch the cell monolayer to generate the cell-free zone in the middle of culture plates. Cell debris was removed by washing with PBS. Cells were then incubated at 37°C for up to 36 h. The number of cells that migrated to the cell-free zone was counted under light microscopy (200× magnification) and photographed with a Canon 5D digital camera attached to the microscope.

Gelatinase Inhibitor SB-3CT Treatment In Vitro

It has been shown that SB-3CT is able to inhibit the activity of endogenous gelatinases, including MMP2 [28]. NT2/D1 (5 × 105) cells were treated with 10 μM SB-3CT (Chemicon, Temecula, CA) in the presence of MEHP (200 μM) for 12 h. Treated cells were then used for invasion and migration assays.

Microarray and Data Analysis

Microarray analysis was performed using human whole-genome OneArray version 5 (Phalanx Biotech Group, Belmont, CA), carrying 29 187 human genome probes and 1088 experimental control probes. Total RNA from NT2/D1 cells treated with MEHP for 0, 3, and 24 h was isolated using Qiagen RNeasy kit (Qiagen, Valencia, CA). Array hybridization, image processing, signal intensity acquisition, and data normalization were performed by Phalanx Biotech Service Group. Three experimental sets were independently harvested and analyzed. Normalized values for each data point were averaged using these three biological replicates, and the fold change for individual gene expression levels among three time points was determined. Significant difference in expression level was defined as a P value of <0.05, and genes with a value greater than 2-fold change were selected by Cluster software (Stanford University and Massachusetts Institute of Technology). Selected genes were grouped according to their biological function and clustered using a hierarchical cluster method (TreeView, Stanford University and Massachusetts Institute of Technology).

Semi-Quantitative RT-PCR

To confirm the results derived from microarray analysis, we randomly selected 11 differentially expressed genes from the cluster analysis and measured their mRNA levels using semiquantitative RT-PCR. First-strand cDNA was prepared using 5 μg of total RNA with Superscript II reverse transcriptase and oligo(dT) primer (all Invitrogen). The primers used to amplify differentially expressed genes are listed in Table 1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was quantified as an internal control. PCR assay was performed using 28 cycles of 92°C for 30 sec, 54°C for 1 min, and 72°C for 30 sec, followed by 72°C for 5 min with 1 unit of Taq DNA polymerase. PCR products were separated on a 1.5% agarose gel, and images were captured with a Kodak Gel Logic 100 imaging system. Densitometry for bands on PCR products was determined using ImageJ software. The relative expression level of each gene was normalized according to the value for GAPDH.

TABLE 1. .

Nucleotide sequences of RT-PCR primers designed to generate MEHP-regulated genes.

graphic file with name i0006-3363-86-5-160-t01.jpg

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a 

F, forward; R, reverse.

Statistical Analysis

All experiments were performed in triplicate and repeated using three independent sets of cell preparations. Data were subjected to Student t-test or a parametric one-way ANOVA followed by the Tukey test for post hoc comparisons. Statistical significance was considered to be achieved when P was <0.05.

RESULTS

MEHP Treatment Up-Regulates MMP2 Expression and Activity in NT2/D1 Cells

Changes in expression levels and activities of both MMP2 and MMP9 in response to MEHP exposure were determined by several approaches. The MMP2 protein level in NT2/D1 cells was significantly increased at 3 h after MEHP exposure and then remained steady until 24 h of incubation (2.53-fold compared to that of the nontreated group) (Fig. 1A, left panel). Lower doses of MEHP treatment showed no significant effects on MMP2 expression, while 200 μM MEHP strongly induced MMP2 protein levels after 12 h of incubation (2.38-fold compared to that of nontreated group) (Fig. 1A, right panel). No significant change in MMP9 expression was observed after MEHP exposure. The amount of soluble MMP2 secreted from NT2/D1 cells was measured by ELISA. Figure 1B shows the time-dependent increase in soluble MMP2 level after MEHP exposure (4.23 ± 0.02 ng/ml at 0 h; 18.87 ± 1.06 ng/ml at 24 h of incubation). Higher doses of MEHP treatment were found to stimulate a significant production of soluble MMP2 (3.25 ± 0.17 ng/ml at 0 μM; 13.41 ± 2.32 ng/ml at 200 μM) (Fig. 1B), even though soluble MMP2 production was decreased at a dose of 400 μM (7.48 ± 0.11 ng/ml). The activities of MMP2 and MMP9 in vitro, as determined by gelatin zymography (Fig. 1C), indicated that MMP2 was time- and dose-dependently activated by MEHP treatment. MMP9 level was relatively low compared to that of MMP2 and was slightly increased by MEHP exposure, suggesting that MEHP exposure has a major effect on MMP2 activity but not on MMP9.

FIG. 1. .

FIG. 1. 

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MMP2 protein expression and activity in NT2/D1 cells are increased by MEHP exposure. A) Total protein from NT2/D1 cells treated with or without MEHP were analyzed by Western blot analysis. Time- and dose-dependent induction of MMP2 were detected following MEHP exposure (left and right panel, respectively). However, MMP9 expression was not altered by MEHP treatment. ACTB serves as the loading control. The quantified relative protein levels of MMP2 and MMP9 are shown under the blot images. Values are means ± SEM. Asterisks denote significant differences between the treated and control groups (n = 9; *P < 0.05; Student t-test). B) The amount of soluble MMP2 released from NT2/D1 cells was measured by ELISA. Time- and dose-dependent inductions of soluble MMP2 were detected in response to MEHP exposure (upper and lower panels, respectively). Values are means ± SEM. Asterisks denote significant differences between the treatment and control groups (n = 9; *P < 0.05; **P < 0.01; Student t-test). C) Gelatinolytic activities of MMP2 and MMP9 in medium were determined by gelatin zymography. Clear bands the blue background indicate enzyme activity. MEHP treatment caused an increase in MMP2 activity in a time- and dose-dependent manner. MMP9 activity was relatively weaker than that of MMP2, while it was increased by MEHP exposure as well.

MEHP Induces MYC Expression in NT2/D1 Cells

Western blot analysis of MYC protein in NT2/D1 cells showed that its expression was up-regulated shortly after treatment with 200 μM MEHP (1.52-fold compared to that in the nontreated group), decreased at 3 h, and then increased after 12 h of incubation (Fig. 1A, left panel). In addition, MEHP treatment significantly enhanced MYC levels as concentration increased (3.22-fold at 200 μM compared to that in the nontreated group) (Fig. 1A, right panel), indicating that the induction of MYC by MEHP exposure is dose-dependent.

SB-3CT Suppressed MEHP-Induced MMP2 Activation in NT2/D1 Cells

The specific gelatinase inhibitor SB-3CT and MEHP were applied to NT2/D1 cells. After 12 h of incubation, samples of conditioned medium were collected, and MMP2 activity was further analyzed. ELISA results indicated that a low dose of SB-3CT treatment (5 μM) significantly reduced the amount of soluble MMP2 compared to that of MEHP treatment alone (10.27 ± 1.73 ng/ml compared to control group 4.79 ± 0.02 ng/ml, respectively) (Fig. 2A). Figure 2B shows that MMP2 gelatinase activity was enhanced by MEHP but decreased dose-dependently when SB-3CT was added.

FIG. 2. .

FIG. 2. 

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SB-3CT inhibited MEHP-induced MMP2 activation in NT2/D1 cells. NT2/D1 cells were treated with various doses of SB-3CT and 200 μM of MEHP for 12 h, and conditioned medium was collected. A) The amount of soluble MMP2 released from NT2/D1 cells was measured by ELISA. SB-3CT treatment slightly decreased the amount of free MMP2. Values are means ± SEM. Asterisks denote significant differences between the treatment and control groups (n = 9; *P < 0.05; **P < 0.01; Student t-test). B) Gelatinolytic activities of MMP2 and MMP9 in the medium were determined by gelatin zymography. MMP2 activity was reduced by SB-3CT and MEHP cotreatment compared to that with MEHP treatment only in a dose-dependent manner. No change in MMP9 activity was observed.

MEHP Exposure Promotes Invasive Activity and Migration Capability of NT2/D1 Cells

In order to estimate the effect of MEHP treatment on cell invasion, nontreated or MEHP-treated NT2/D1 cells were tested by Transwell invasion assay. Interestingly, after MEHP exposure, NT2/D1 cells had a more robust ability to digest and pass through Matrigel. Figure 3A shows that the number of invasive cells was significantly increased in the MEHP-treated group compared to that in the nontreated group (3.23-fold changes from 3.97 ± 1.04 to 12.83 ± 1.62 cells per view, respectively). In addition to accounting for cells' invasive ability, we also counted the number of cells that migrated into the cell-free zone. More cells are observed in the cell-free zone after MEHP treatment (1.59-fold at 36 h compared to that in the control group; actual cell numbers changed from 90.14 ± 14.50 to 143.33 ± 16.86 cells per view, respectively), indicating that MEHP facilitated the migration of NT2/D1 cells (Fig. 3B).

FIG. 3. .

FIG. 3. 

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That invasion and migration activities of NT2/D1 cells were enhanced by MEHP exposure and suppressed by SB-3CT shows the compensatory effect on MEHP treatment. A) In vitro invasion assays were performed using Transwell chambers coated with Matrigel. Nontreated, MEHP-treated, and SB-3CT/MEHP cotreated cells were seeded on Matrigel and incubated overnight. Invasive cells were stained with Giemsa solution. Cells attached to the lower surface of the filter were observed using light microscopy (original magnification, ×200). The quantified numbers of cells penetrating Matrigel were counted. Values are means ± SEM. Asterisks denote significant differences between the treatment and control groups (n = 9; **P < 0.01; Student t-test). B) Wound-healing migration assay was performed to determine the change in migration rate of NT2/D1 cells after MEHP treatment or SB-3CT/MEHP cotreatment. Cells were observed using a light microscope (original magnification, ×200). The number of cells migrating to the cell-free zone was evaluated. Values are means ± SEM. Asterisks denote significant differences between the treatment and control groups (n = 9; *P < 0.05; Student t-test).

SB-3CT Shows Inhibitory Effects on MEHP-Enhanced Invasion and Migration in NT2/D1 Cells

To determine whether the activated MMP2 was necessary for MEHP-enhanced cell invasion and migration, SB-3CT was applied to NT2/D1 cells in the presence of MEHP. The addition of SB-3CT and MEHP cotreatment suppressed the number of invasive cells (down to 0.68-fold compared to that of MEHP-treated groups; cell number changes from 12.83 ± 1.6 to 8.72 ± 2.33 per view) (Fig. 3A). SB-3CT treatment was also able to decrease the number of cells that migrated in the presence of MEHP (down to 0.81-fold compared to that of MEHP-treated group; actual cell numbers changed from 143.33 ± 16.86 to 116.10 ± 10.97 per view) (Fig. 3B). These results suggest that gelatinases play an essential role in testicular cancer invasion and migration, stimulated by MEHP exposure.

Microarray Analysis of Gene Expression in NT2/D1 Cells Following MEHP Exposure

Of nearly 30 000 putative probes, 1289 genes in the testicular embryonic carcinoma NT2/D1 cell line were significantly altered by MEHP exposure. Forty-five genes displayed more than 2-fold changes in mRNA levels following MEHP exposure (Fig. 4A) and were grouped into seven categories on the basis of their biological functions, including adhesion molecules, metabolism modifiers, transcription factors, translation components, cell-cycle regulators, signal transduction mediators, and miscellanea (Fig. 4B). Among these categories, the cell adhesion molecule (9 of 45 genes, 19%) appears to be the group most strongly altered in response to MEHP treatment. This group of genes includes pleckstrin homology domain containing family O member 1 (PLEKHO1), vinculin (VCL), gap junction protein alpha 1 (GJA1), WD repeat domain 1 (WDR1), leucine rich repeat containing 8 family member A (LRRC8A), N-cadherin (CDH2), claudin-6 (CLDN6), beta 1-catenin (CTNNB1), and MMP2. Seven genes (16%) were grouped as transcription factors, including inhibitor of DNA binding 1 (ID1), inhibitor of DNA binding 2 (ID2), inhibitor of DNA binding 3 (ID3), TEA domain family member 4 (TEAD4), polyhomeotic homolog 1 (PHC1), zinc finger protein 429 (ZHF429), and DNA damage-inducible transcript 3 (DDIT3). Seven genes (16%) were grouped as metabolism modifiers, including neurotensin (NTS), methionine adenosyltransferase II alpha (MAT2A), 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1), phosphoglucomutase 1 (PGM1), phosphoserine aminotransferase 1 (PSAT1), phosphatidylinositol-specific phospholipase C, X domain containing 1 (PLCXD1), and phosphoserine phosphatase (PSPH). Four genes (9%) are components of the translation process, including ribosomal protein L36a (RPL36A), methionyl-tRNA synthetase (MARS), NOP58 ribonucleoprotein homolog (NOP58), and eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1). Seven genes (16%) are related to cell cycle, including cyclin D1 (CCND1), fibroblast growth factor receptor 3 (FGFR3), transmembrane protein 158 (TMEM158), growth differentiation factor 3 (GDF3), WEE1 homolog (WEE1), growth arrest and DNA damage-inducible beta (GADD45B), and sestrin 2 (SESN2). Six genes are involved in signal transduction, including LFNG O-fucosylpeptide 3-beta-N-acetylglucosaminyltransferase (LFNG), interleukin 17 receptor D (IL17RD), myosin light chain 12A (MYL12A), BMP and activin membrane-bound inhibitor homolog (BAMBI), perilipin 2 (PLIN2), and transgelin (TAGLN). The remaining five genes were grouped as miscellanea, including immunoglobulin superfamily, DCC subclass, member 3 (IGDCC3); and splicing factor proline/glutamine-rich (SFPQ), ribonucleotide reductase M2 (RRM2), solute carrier family 3 member 2 (SLC3A1), and TRK-fused gene (TFG).

FIG. 4. .

FIG. 4. 

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Microarray analysis of the gene expression profile in NT2/D1 cells treated or untreated with MEHP. Microarray analysis was performed using human whole-genome OneArray chips version 5 (Phalanx Biotech Group), carrying 29 187 human genome probes and 1088 experimental control probes. NT2/D1 cells were treated with MEHP for 0, 3, and 24 h. A) Forty-five genes differentially expressed (more than 2-fold) in response to MEHP exposure are identified, and their mRNA levels are shown as hierarchical clustering. Relative expression levels of these genes are color coded on the bottom. B) Forty-five differentially expressed genes were grouped into seven categories on the basis of biological functions. C) Semiquantitative RT-PCR was performed in order to confirm the results derived from the microarray analysis. Eleven randomly selected genes were assayed, and GAPDH expression serves as the control. Values means ± SEM. Significant difference among groups are denoted by bars with different letters (n = 9; *P < 0.05; ANOVA).

Semiquantitative RT-PCR was performed to verify mRNA expression of 11 MEHP-regulated genes (6 genes for down-regulation and 5 for up-regulation), which were randomly selected from microarray results, including NTS, ID1, VCL, GJA1, LFNG, IL17RD, CLDN6, CTNNB1, MARS, MMP2, and DDIT3 (Fig. 4C). Among adhesion molecules, semiquantitative RT-PCR confirmed the fact that GJA1 was significantly down-regulated by MEHP treatment (0.54-fold at 24 h compared with the 0-h group), while CLDN6 and CTNNB1 were up-regulated (2.77- and 1.38-fold at 24 h). RT-PCR confirmed the fact that VCL expression was reduced by MEHP at 3 h (0.79-fold), while no changes were observed at 24 h, which is not consistent with microarray data. Microarray analysis and RT-PCR data both revealed that MMP2 mRNA expression in NT2/D1 cells was significantly increased at 24 h after MEHP stimulation (2.87- and 1.61-fold, respectively). Even though RT-PCR showed that MMP2 level was strongly induced at 3 h of incubation, microarray analysis indicated that MMP2 levels were not induced at 3 h. Among transcription factors, RT-PCR results showed that ID1 and PHC were decreased after MEHP treatment (0.45- and 0.78-fold at 3 h), and DDIT3 was increased (1.81-fold at 3 h). However, NTS expression pattern was not consistent according to microarray and RT-PCR results. Down-regulation of LFNG and IL17RD expression were consistent in both microarray and RT-PCR data (down to 0.65- and 0.72-fold, respectively, at 24 h compared with 0-h group). Microarray results revealed that MARS mRNA level was elevated at 24 h after MEHP exposure, while RT-PCR data indicated that MEHP-induced MARS mRNA expression occurred as early as 3 h of exposure.

DISCUSSION

We previously identified several important molecular factors that are critical in controlling normal germ cell development, including MYC and MMP2, which are altered following phthalate exposure [16, 25] and have been shown to be tightly associated with tumor metastasis and tumor progression [29–31]. Because MYC controls cell proliferation, cell differentiation, and self-renewal [32, 33], up-regulation of MYC seen in testicular embryonal carcinoma following MEHP exposure may suggest that phthalates affect both normal cell differentiation and tumor cell development. Exposure to a high level of DE-HP results in liver lesions and an increased incidence of hepatocellular carcinoma in rodents [34, 35], and those studies also suggest that DE-HP is a potential tumor promoter but is not genotoxic. A recent study indicates that DBP exposure promoted the vulnerability of fetal germ cells and was possibly related to testicular germ cell tumor formation [36]. In addition, it has been suggested that DBP decreases the methylation of MYC gene in rat livers, resulting in the activation of the MYC gene [37]. Therefore, the effect of phthalate exposure on the regulation of testicular cancer progression and further investigation of epigenetic regulation of MYC pathway in the testis are of interests.

The tumor-promoting activity of phthalates has been reported in liver and breast cancer [38–40]. Matrigel invasion and wound-healing migration assays are well-developed methods used to observe the ability of tumor cells to metastasize and are strongly related to MMP2 and MMP9 activities [41–43]. In the current study, after MEHP exposure, testicular embryonal carcinoma NT2/D1 cells have the stronger ability to digest and pass through the Matrigel. In addition, our data show that MEHP promotes the migration of cancer cells. These results suggest that MEHP exposure may enhance the potential of testicular cancer cells to become more metastatic and malignant. It is then reasonable to conclude that the molecular mechanism that accounts for this alteration can be explained by the activation of MMP2. Using specific MMP2 inhibitor, SB-3CT, we were able to suppress endogenous MMP2 activity stimulated by MEHP, both in vitro and in vivo [16, 17]. We found that the addition of SB-3CT to NT2/D1 cells partially inhibited MEHP-induced invasion and migration. Therefore, MEHP-induced MMP2 activation appears to be important in regulating the metastatic status of testicular germ cell tumor.

In addition to analyzing MMP2 and MYC activation, we performed microarray analysis in order to examine the gene expression pattern altered by MEHP exposure on a genome-wide basis. Phthalate exposure has been demonstrated to impair junctional connections in the seminiferous tubule by altering junctional protein expression [17, 44]. Interestingly, MEHP influences primarily cell adhesion molecules and transcription factors, which are also closely related to tumorigenesis. Hence, we further investigated the functional significance of distinct genes in testicular cancers following toxicant exposure.

Gap junctional intercellular communication controls cell growth, development, and differentiation [45]. Inhibition of gap junctional communication has been proposed to be related to tumor promotion [46, 47]. The GJA1 gene encodes the connexin 43 (Cx43) protein, forming the main structure of the gap junction. Mutation or deficiency in the GJA1 gene leads to a variety of human diseases or disorders [48, 49]. Interestingly, it has been shown that the reduction of Cx43 enhances the invasion capacity of human glioblastoma cells [50]. In the mouse testis, a decrease in Cx43 protein level and the change in Cx43 localization have been observed after DE-HP exposure [44]. Similar results also demonstrate that DE-HP treatment inhibits gap junctional communication in vitro [51]. Our current results indicate that MEHP exposure leads to a decrease in GJA1 mRNA expression in NT2/D1 cells. The reduction of GJA1 expression following MEHP exposure may partially explain the mechanism of MEHP-enhanced invasion in NT2/D1 cells. On the other hand, although DE-HP-induced tumorigenesis in liver is strongly correlated with the inhibition of gap junctional communication in rodents, it appears that this phenomenon is species-specific [14, 52]. Therefore, the role of GJA1 suppression in human testicular cancer progression by phthalates remains an open question.

The vocal adhesion molecule VCL is important for maintaining the structure of adherens junction through interaction with actin filament and integrin [53, 54]. We previously showed that MEHP exposure breaks down laminin/integrin complex at the ectoplasmic specialization [17]. In this study, we further found that MEHP treatment reduces vinculin mRNA level in testicular cancer cells, suggesting that phthalate impairs primarily the junctional connection in the testis. Overexpression of VCL has been shown to suppress tumorigenesis [55]. Immunostaining results also demonstrated that vinculin expression is negatively correlated with the metastasis of squamous cell tumor [56], indicating that reduced cellular adhesion is strongly associated with the metastasis of malignant tumor. Therefore, MEHP-decreased VCL may be involved in regulating the invasive and migration ability of testicular cancer cells in parallel with the activation of MMP2.

CTNNB1, an oncogene, has been demonstrated to be involved in various biological processes, including cell adhesion, cell growth, and tumorigenesis. Accumulation of CTNNB1 in germ cell nuclei, associated with abnormal OCT3/4 expression, leads to the malignant transformation of seminoma [57]. Activation of CTNNB1 signaling may cause Sertoli cell tumor formation [58]. DE-HP induces the up-regulation of alpha-catenin and N-cadherin in rat testes [59]. Fetal exposure to DE-HP in mice causes the delay in liver development and the alteration of glycogen metabolism through the activation of WNT/CTNNB1 signaling [60]. Here we show that CTNNB1 mRNA level is up-regulated by MEHP in NT2/D1 cells, demonstrating that phthalate has the potential to promote tumor progression by up-regulation of oncogenes. In addition, MYC expression can be regulated by the WNT/CTNNB1/TCF signaling pathway, resulting in colorectal and skin carcinogenesis [61, 62]. Therefore, we speculate that MEHP-induced CTNNB1 up-regulation may play a role in modulating the differentiation status of testicular cells by influencing MYC expression.

CLDN6, belonging to claudin superfamily, is a key component of tight junctions. Several studies had suggested that claudin-6 functions as a tumor suppressor in breast cancer, accompanied by the alteration of MMP activity [63–65]. The frequency of epigenetic silencing in the CLDN6 gene by methylation is increased in esophageal squamous cell carcinomas [66]. However, overexpression of claudin-6 increases the invasiveness, migration, and proliferation rate of human gastric adenocarcinoma cell line [67]. CLDN6 is also potentially a diagnostic marker for atypical teratoid/rhabdoid tumor (AT/RT) [68]. These observation demonstrate that the function of claudin-6 in promoting or suppressing tumor progression may be cell-specific. In testicular embryonal carcinoma, we reveal that MEHP exposure increases the mRNA level of CLDN6. Interestingly, CLDN6 expression appears to be limited in some testicular embryonal carcinoma and immature epithelial teratocarcinoma [69] and may developmentally regulate adipogenesis and epithelialization of embryos [70–72]. Therefore, claudin-6 not only serves as a basis of tight junction structure but also is important for cell differentiation that may be MYC-dependent.

ID proteins are transcription factors, which are important for cell cycle, cell development, and tumorigenesis [73, 74]. Surprisingly, MEHP exposure significantly reduces the mRNA level of three ID genes in testicular embryonal carcinoma, demonstrating that phthalates influence the dynamic expression of transcriptomes in the tumor. In the murine testis, ID3 expression is regulated by follicle-stimulating hormone (FSH) [75]. MEHP has been shown to disrupt FSH signaling [76, 77]. Hence, impaired FSH signaling may be responsible for the decreases in ID genes after MEHP exposure.

Findings of the current study indicate that cellular mechanisms triggered by MEHP exposure act to enhance tumor progression/metastasis in testicular embryonal carcinoma cells (NT2/D1). We observed that (1) NT2/D1 cells had the increased ability to digest and migrate into Matrigel matrix following MEHP exposure and that this was dependent on the increased activity of MMP2, as the enhancing effects of MEHP exposure can be partially inhibited by addition of the specific MMP2 inhibitor SB-3CT. (2) We showed that MEHP not only increases the activation of MMP2 but also down-regulates the expression of both the tight-junction (GJA1) and adherens junction (VCL) genes, which could further aid in the capacity for invasion and migration of NT2/D1 cells. Finally, (3) we describe the activation of the MYC transcription factor after MEHP exposure. Because MYC plays a role in promoting the expression of CLDN6 and inhibits TIMP2 expression, disruption of MYC expression could provide an underlying mechanism to account for MMP2 activation after MEHP treatment. Taken together, these novel findings provide important mechanistic insights by which exposure to environmental toxicants, such as the phthalates, can enhance testicular cancer metastasis and invasion.

Footnotes

1

Supported in part by grants ES016591 to J.H.R. and T32 ES07247 to Y.L. from the National Institute of Environmental Health Sciences/National Institutes of Health and from the Center for Molecular and Cellular Toxicology to Y.L.

REFERENCES

  1. Holmes L, Jr, Escalante C, Garrison O, Foldi BX, Ogungbade GO, Essien EJ, Ward D. Testicular cancer incidence trends in the USA (1975–2004): plateau or shifting racial paradigm? Public Health 2008; 122: 862 872 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Skakkebaek NE. Rajpert-De Meyts E, Main KM. Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects. Hum Reprod 2001; 16: 972 978 [DOI] [PubMed] [Google Scholar]
  3. Cook MB, Trabert B, McGlynn KA. Organochlorine compounds and testicular dysgenesis syndrome: human data. Int J Androl 2011; 34: e68 e84 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Jorgensen N, Meyts ER, Main KM, Skakkebaek NE. Testicular dysgenesis syndrome comprises some but not all cases of hypospadias and impaired spermatogenesis. Int J Androl 2010; 33: 298 303 [DOI] [PubMed] [Google Scholar]
  5. Akre O, Richiardi L. Does a testicular dysgenesis syndrome exist? Hum Reprod 2009; 24: 2053 2060 [DOI] [PubMed] [Google Scholar]
  6. Wohlfahrt-Veje C, Main KM, Skakkebaek NE. Testicular dysgenesis syndrome: foetal origin of adult reproductive problems. Clin Endocrinol (Oxf) 2009; 71: 459 465 [DOI] [PubMed] [Google Scholar]
  7. Sharpe RM, Skakkebaek NE. Testicular dysgenesis syndrome: mechanistic insights and potential new downstream effects. Fertil Steril 2008; 89: e33 38 [DOI] [PubMed] [Google Scholar]
  8. Blount BC, Silva MJ, Caudill SP, Needham LL, Pirkle JL, Sampson EJ, Lucier GW, Jackson RJ, Brock JW. Levels of seven urinary phthalate metabolites in a human reference population. Environ Health Perspect 2000; 108: 979 982 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Heudorf U, Mersch-Sundermann V, Angerer J. Phthalates: toxicology and exposure. Int J Hyg Environ Health 2007; 210: 623 634 [DOI] [PubMed] [Google Scholar]
  10. Chou K, Wright RO. Phthalates in food and medical devices. J Med Toxicol 2006; 2: 126 135 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Swan SH. Environmental phthalate exposure in relation to reproductive outcomes and other health endpoints in humans. Environ Res 2008; 108: 177 184 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jurewicz J, Hanke W. Exposure to phthalates: reproductive outcome and children health. A review of epidemiological studies. Int J Occup Med Environ Health 2011; 24: 115 141 [DOI] [PubMed] [Google Scholar]
  13. Salvatore M, Lorenzetti S, Maranghi F, Mantovani A, Taruscio D. Molecular link(s) between hepatoblastoma pathogenesis and exposure to di-(2-ethylhexyl)phthalate: a hypothesis. Folia Med (Plovdiv) 2008; 50: 17 23 [PubMed] [Google Scholar]
  14. McKee RH. The role of inhibition of gap junctional intercellular communication in rodent liver tumor induction by phthalates: review of data on selected phthalates and the potential relevance to man. Regul Toxicol Pharmacol 2000; 32: 51 55 [DOI] [PubMed] [Google Scholar]
  15. Ren H, Aleksunes LM, Wood C, Vallanat B, George MH, Klaassen CD, Corton JC. Characterization of peroxisome proliferator-activated receptor alpha–independent effects of PPARalpha activators in the rodent liver: di-(2-ethylhexyl) phthalate also activates the constitutive-activated receptor. Toxicol Sci 2010; 113: 45 59 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Yao PL, Lin YC, Richburg JH. TNF alpha-mediated disruption of spermatogenesis in response to sertoli cell injury in rodents is partially regulated by MMP2. Biol Reprod 2009; 80: 581 589 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Yao PL, Lin YC, Richburg JH. Mono-(2-ethylhexyl) phthalate-induced disruption of junctional complexes in the seminiferous epithelium of the rodent testis is mediated by MMP2. Biol Reprod 2010; 82: 516 527 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Theret N, Musso O, Turlin B, Lotrian D, Bioulac-Sage P, Campion JP, Boudjema K, Clement B. Increased extracellular matrix remodeling is associated with tumor progression in human hepatocellular carcinomas. Hepatology 2001; 34: 82 88 [DOI] [PubMed] [Google Scholar]
  19. Ara T, Kusafuka T, Inoue M, Kuroda S, Fukuzawa M, Okada A. Determination of imbalance between MMP-2 and TIMP-2 in human neuroblastoma by reverse-transcription polymerase chain reaction and its correlation with tumor progression. J Pediatr Surg 2000; 35: 432 437 [DOI] [PubMed] [Google Scholar]
  20. Diamantopoulos N, Boutis AL, Koratzis I, Andreadis C, Galaktidou G, Mouratidou D, Kortsaris A. Matrix metalloproteinases and proangiogenic factors in testicular germ cell tumors. J Buon 2010; 15: 116 121 [PubMed] [Google Scholar]
  21. Fisher JS, Macpherson S, Marchetti N, Sharpe RM. Human “testicular dysgenesis syndrome”: a possible model using in-utero exposure of the rat to dibutyl phthalate. Hum Reprod 2003; 18: 1383 1394 [DOI] [PubMed] [Google Scholar]
  22. Hutchison GR, Scott HM, Walker M, McKinnell C, Ferrara D, Mahood IK, Sharpe RM. Sertoli cell development and function in an animal model of testicular dysgenesis syndrome. Biol Reprod 2008; 78: 352 360 [DOI] [PubMed] [Google Scholar]
  23. Ge RS, Chen GR, Tanrikut C, Hardy MP. Phthalate ester toxicity in Leydig cells: developmental timing and dosage considerations. Reprod Toxicol 2007; 23: 366 373 [DOI] [PubMed] [Google Scholar]
  24. Hu GX, Lian QQ, Ge RS, Hardy DO, Li XK. Phthalate-induced testicular dysgenesis syndrome: Leydig cell influence. Trends Endocrinol Metab 2009; 20: 139 145 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Yao PL, Lin YC, Richburg JH. Transcriptional suppression of Sertoli cell Timp2 in rodents following mono-(2-ethylhexyl) phthalate exposure is regulated by CEBPA and MYC. Biol Reprod 2011; 85: 1203 1215 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Larsson LG, Henriksson MA. The Yin and Yang functions of the Myc oncoprotein in cancer development and as targets for therapy. Exp Cell Res 2010; 316: 1429 1437 [DOI] [PubMed] [Google Scholar]
  27. Chen JJ, Lin YC, Yao PL, Yuan A, Chen HY, Shun CT, Tsai MF, Chen CH, Yang PC. Tumor-associated macrophages: the double-edged sword in cancer progression. J Clin Oncol 2005; 23: 953 964 [DOI] [PubMed] [Google Scholar]
  28. Tao P, Fisher JF, Mobashery S, Schlegel HB. DFT studies of the ring-opening mechanism of SB-3CT, a potent inhibitor of matrix metalloproteinase 2. Org Lett 2009; 11: 2559 2562 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Jezierska A, Motyl T. Matrix metalloproteinase-2 involvement in breast cancer progression: a mini-review. Med Sci Monit 2009; 15: RA32 40 [PubMed] [Google Scholar]
  30. Wolfer A, Ramaswamy S. MYC. and metastasis. Cancer Res 2011; 71: 2034 2037 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mook OR, Frederiks WM, Van Noorden CJ. The role of gelatinases in colorectal cancer progression and metastasis. Biochim Biophys Acta 2004; 1705: 69 89 [DOI] [PubMed] [Google Scholar]
  32. Dony C, Kessel M, Gruss P. Post-transcriptional control of myc and p53 expression during differentiation of the embryonal carcinoma cell line F9. Nature 1985; 317: 636 639 [DOI] [PubMed] [Google Scholar]
  33. Sejersen T, Jin P, Rahm M, Ringertz NR. Changes in c-onc expression during embryonal carcinoma cell differentiation. Environ Health Perspect 1989; 80: 247 256 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Doull J, Cattley R, Elcombe C, Lake BG, Swenberg J, Wilkinson C, Williams G, van Gemert M. A cancer risk assessment of di(2-ethylhexyl)phthalate: application of the new U.S. EPA Risk Assessment Guidelines. Regul Toxicol Pharmacol 1999; 29: 327 357 [DOI] [PubMed] [Google Scholar]
  35. Ashby J, Brady A, Elcombe CR, Elliott BM, Ishmael J, Odum J, Tugwood JD, Kettle S, Purchase IF. Mechanistically-based human hazard assessment of peroxisome proliferator-induced hepatocarcinogenesis. Hum Exp Toxicol 1994; 13 (suppl 2): S1 S117 [DOI] [PubMed] [Google Scholar]
  36. Jobling MS, Hutchison GR, van den Driesche S, Sharpe RM. Effects of di(n-butyl) phthalate exposure on foetal rat germ-cell number and differentiation: identification of age-specific windows of vulnerability. Int J Androl 2011; 34: e386 e396 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Kostka G, Urbanek-Olejnik K, Wiadrowska B. Di-butyl phthalate-induced hypomethylation of the c-myc gene in rat liver. Toxicol Ind Health 2010; 26: 407 416 [DOI] [PubMed] [Google Scholar]
  38. Ward JM, Diwan BA, Ohshima M, Hu H, Schuller HM, Rice JM. Tumor-initiating and promoting activities of di(2-ethylhexyl) phthalate in vivo and in vitro. Environ Health Perspect 1986; 65: 279 291 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Diwan BA, Ward JM, Rice JM, Colburn NH, Spangler EF. Tumor-promoting effects of di(2-ethylhexyl)phthalate in JB6 mouse epidermal cells and mouse skin. Carcinogenesis 1985; 6: 343 347 [DOI] [PubMed] [Google Scholar]
  40. Hsieh TH, Tsai CF, Hsu CY, Kuo PL, Lee JN, Chai CY, Wang SC, Tsai EM. Phthalates induce proliferation and invasiveness of estrogen receptor-negative breast cancer through the AhR/HDAC6/c-Myc signaling pathway. FASEB J 2012; 26: 778 787 [DOI] [PubMed] [Google Scholar]
  41. Wang CC, Tsai MF, Hong TM, Chang GC, Chen CY, Yang WM, Chen JJ, Yang PC. The transcriptional factor YY1 upregulates the novel invasion suppressor HLJ1 expression and inhibits cancer cell invasion. Oncogene 2005; 24: 4081 4093 [DOI] [PubMed] [Google Scholar]
  42. Chen HW, Yu SL, Chen JJ, Li HN, Lin YC, Yao PL, Chou HY, Chien CT, Chen WJ, Lee YT, Yang PC. Anti-invasive gene expression profile of curcumin in lung adenocarcinoma based on a high throughput microarray analysis. Mol Pharmacol 2004; 65: 99 110 [DOI] [PubMed] [Google Scholar]
  43. Tsai MF, Wang CC, Chang GC, Chen CY, Chen HY, Cheng CL, Yang YP, Wu CY, Shih FY, Liu CC, Lin HP, Jou YS. et al. A new tumor suppressor DnaJ-like heat shock protein, HLJ1, and survival of patients with non-small-cell lung carcinoma. J Natl Cancer Inst 2006; 98: 825 838 [DOI] [PubMed] [Google Scholar]
  44. Sobarzo CM, Lustig L, Ponzio R, Suescun MO, Denduchis B. Effects of di(2-ethylhexyl) phthalate on gap and tight junction protein expression in the testis of prepubertal rats. Microsc Res Tech 2009; 72: 868 877 [DOI] [PubMed] [Google Scholar]
  45. Weider K, Bergmann M, Brehm R. Connexin 43: its regulatory role in testicular junction dynamics and spermatogenesis. Histol Histopathol 2011; 26: 1343 1352 [DOI] [PubMed] [Google Scholar]
  46. Kandouz M, Batist G. Gap junctions and connexins as therapeutic targets in cancer. Expert Opin Ther Targets 2010; 14: 681 692 [DOI] [PubMed] [Google Scholar]
  47. Leithe E, Sirnes S, Omori Y, Rivedal E. Downregulation of gap junctions in cancer cells. Crit Rev Oncog 2006; 12: 225 256 [DOI] [PubMed] [Google Scholar]
  48. Paznekas WA, Karczeski B, Vermeer S, Lowry RB, Delatycki M, Laurence F, Koivisto PA, Van Maldergem L, Boyadjiev SA, Bodurtha JN, Jabs EW. GJA1 mutations, variants, and connexin 43 dysfunction as it relates to the oculodentodigital dysplasia phenotype. Hum Mutat 2009; 30: 724 733 [DOI] [PubMed] [Google Scholar]
  49. Song YN, Zhang H, Zhao JY, Guo XL. Connexin 43, a new therapeutic target for cardiovascular diseases. Pharmazie 2009; 64: 291 295 [PubMed] [Google Scholar]
  50. Strale PO, Clarhaut J, Lamiche C, Cronier L, Mesnil M, Defamie N. Down-regulation of connexin43 expression reveals the involvement of caveolin-1 containing lipid rafts in human U251 glioblastoma cell invasion. Mol Carcinog 2012; (in press) Published online ahead of print 31 August 2011; DOI: 10.1002/mc.20853 [DOI] [PubMed]
  51. Kang KS, Lee YS, Kim HS, Kim SH. Di-(2-ethylhexyl) phthalate-induced cell proliferation is involved in the inhibition of gap junctional intercellular communication and blockage of apoptosis in mouse Sertoli cells. J Toxicol Environ Health A 2002; 65: 447 459 [DOI] [PubMed] [Google Scholar]
  52. Isenberg JS, Kamendulis LM, Smith JH, Ackley DC, Pugh G, Jr, Lington AW, Klaunig JE. Effects of Di-2-ethylhexyl phthalate (DEHP) on gap-junctional intercellular communication (GJIC), DNA synthesis, and peroxisomal beta oxidation (PBOX) in rat, mouse, and hamster liver. Toxicol Sci 2000; 56: 73 85 [DOI] [PubMed] [Google Scholar]
  53. Ziegler WH, Liddington RC, Critchley DR. The structure and regulation of vinculin. Trends Cell Biol 2006; 16: 453 460 [DOI] [PubMed] [Google Scholar]
  54. Carisey A, Ballestrem C. Vinculin, an adapter protein in control of cell adhesion signalling. Eur J Cell Biol 2011; 90: 157 163 [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Rodriguez Fernandez JL, Geiger B, Salomon D, Sabanay I, Zoller M, Ben-Ze'ev A Suppression of tumorigenicity in transformed cells after transfection with vinculin cDNA. J Cell Biol 1992; 119: 427 438 [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Lifschitz-Mercer B, Czernobilsky B, Feldberg E, Geiger B. Expression of the adherens junction protein vinculin in human basal and squamous cell tumors: relationship to invasiveness and metastatic potential. Hum Pathol 1997; 28: 1230 1236 [DOI] [PubMed] [Google Scholar]
  57. Palma I, Pena RY, Contreras A, Ceballos-Reyes G, Coyote N, Erana L, Kofman-Alfaro S, Queipo G. Participation of OCT3/4 and beta-catenin during dysgenetic gonadal malignant transformation. Cancer Lett 2008; 263: 204 211 [DOI] [PubMed] [Google Scholar]
  58. Chang H, Guillou F, Taketo MM, Behringer RR. Overactive beta-catenin signaling causes testicular sertoli cell tumor development in the mouse. Biol Reprod 2009; 81: 842 849 [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Sobarzo CM, Lustig L, Ponzio R, Denduchis B. Effect of di-(2-ethylhexyl) phthalate on N-cadherin and catenin protein expression in rat testis. Reprod Toxicol 2006; 22: 77 86 [DOI] [PubMed] [Google Scholar]
  60. Maranghi F, Lorenzetti S, Tassinari R, Moracci G, Tassinari V, Marcoccia D, Di Virgilio A, Eusepi A, Romeo A, Magrelli A, Salvatore M, Tosto F. et al. In utero exposure to di-(2-ethylhexyl) phthalate affects liver morphology and metabolism in post-natal CD-1 mice. Reprod Toxicol 2010; 29: 427 432 [DOI] [PubMed] [Google Scholar]
  61. Bhatia N, Spiegelman VS. Activation of Wnt/beta-catenin/Tcf signaling in mouse skin carcinogenesis. Mol Carcinog 2005; 42: 213 221 [DOI] [PubMed] [Google Scholar]
  62. Yochum GS, Sherrick CM, Macpartlin M, Goodman RH. A beta-catenin/TCF-coordinated chromatin loop at MYC integrates 5′ and 3′ Wnt responsive enhancers. Proc Natl Acad Sci U S A 2010; 107: 145 150 [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Osanai M, Murata M, Chiba H, Kojima T, Sawada N. Epigenetic silencing of claudin-6 promotes anchorage-independent growth of breast carcinoma cells. Cancer Sci 2007; 98: 1557 1562 [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Wu Q, Liu Y, Ren Y, Xu X, Yu L, Li Y, Quan C. Tight junction protein, claudin-6, downregulates the malignant phenotype of breast carcinoma. Eur J Cancer Prev 2010; 19: 186 194 [DOI] [PubMed] [Google Scholar]
  65. Yafang L, Qiong W, Yue R, Xiaoming X, Lina Y, Mingzi Z, Ting Z, Yulin L, Chengshi Q. Role of estrogen receptor-alpha in the regulation of claudin-6 expression in breast cancer cells. J Breast Cancer 2011; 14: 20 27 [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Oka D, Yamashita S, Tomioka T, Nakanishi Y, Kato H, Kaminishi M, Ushijima T. The presence of aberrant DNA methylation in noncancerous esophageal mucosae in association with smoking history: a target for risk diagnosis and prevention of esophageal cancers. Cancer 2009; 115: 3412 3426 [DOI] [PubMed] [Google Scholar]
  67. Zavala-Zendejas VE, Torres-Martinez AC, Salas-Morales B, Fortoul TI, Montano LF, Rendon-Huerta EP. Claudin-6, 7, or 9 overexpression in the human gastric adenocarcinoma cell line AGS increases its invasiveness, migration, and proliferation rate. Cancer Invest 2011; 29: 1 11 [DOI] [PubMed] [Google Scholar]
  68. Birks DK, Kleinschmidt-DeMasters BK, Donson AM, Barton VN, McNatt SA, Foreman NK, Handler MH. Claudin 6 is a positive marker for atypical teratoid/rhabdoid tumors. Brain Pathol 2010; 20: 140 150 [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Väre P, Soini Y. Twist is inversely associated with claudins in germ cell tumors of the testis. APMIS 2010; 118: 640 647 [DOI] [PubMed] [Google Scholar]
  70. Turksen K, Troy TC. Permeability barrier dysfunction in transgenic mice overexpressing claudin 6. Development 2002; 129: 1775 1784 [DOI] [PubMed] [Google Scholar]
  71. Hong YH, Hishikawa D, Miyahara H, Nishimura Y, Tsuzuki H, Gotoh C, Iga T, Suzuki Y, Song SH, Choi KC, Lee HG, Sasaki S. et al. Up-regulation of the claudin-6 gene in adipogenesis. Biosci Biotechnol Biochem 2005; 69: 2117 2121 [DOI] [PubMed] [Google Scholar]
  72. Turksen K, Troy TC. Claudin-6: a novel tight junction molecule is developmentally regulated in mouse embryonic epithelium. Dev Dyn 2001; 222: 292 300 [DOI] [PubMed] [Google Scholar]
  73. Ruzinova MB, Benezra R. Id proteins in development, cell cycle and cancer. Trends Cell Biol 2003; 13: 410 418 [DOI] [PubMed] [Google Scholar]
  74. Perk J, Iavarone A, Benezra R. Id family of helix-loop-helix proteins in cancer. Nat Rev Cancer 2005; 5: 603 614 [DOI] [PubMed] [Google Scholar]
  75. Sadate-Ngatchou PI, Pouchnik DJ, Griswold MD. Follicle-stimulating hormone induced changes in gene expression of murine testis. Mol Endocrinol 2004; 18: 2805 2816 [DOI] [PubMed] [Google Scholar]
  76. Grasso P, Heindel JJ, Powell CJ, Reichert LE., Jr Effects of mono(2-ethylhexyl) phthalate, a testicular toxicant, on follicle-stimulating hormone binding to membranes from cultured rat Sertoli cells. Biol Reprod 1993; 48: 454 459 [DOI] [PubMed] [Google Scholar]
  77. Treinen KA, Dodson WC, Heindel JJ. Inhibition of FSH-stimulated cAMP accumulation and progesterone production by mono(2-ethylhexyl) phthalate in rat granulosa cell cultures. Toxicol Appl Pharmacol 1990; 106: 334 340 [DOI] [PubMed] [Google Scholar]

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