Cell Motility Facilitated by Mono(2-ethylhexyl) Phthalate via Activation

Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Cell Motility Facilitated by Mono(2-ethylhexyl) Phthalate via Activation of the AKT−β-Catenin−IL‑8 Axis in Colorectal Cancer Chi-Wen Luo,† I-Ling Hsiao,‡ Jaw-Yuan Wang,‡,§ Chun-Chieh Wu,⊥ Wen-Chun Hung,¶ Yu-Han Lin,‡ Tzu-Yi Chen,‡ Yin-Chou Hsu,‡,□ Tian-Lu Cheng,■,○,● and Mei-Ren Pan*,‡ †

Division of Cardiology, Chang Gung Memorial Hospital, Kaohsiung Medical Center, Kaohsiung 833, Taiwan Graduate Institute of Clinical Medicine, Kaohsiung Medical University, Number 100, Tzyou First Road, Kaohsiung 807, Taiwan § Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan ⊥ Department of Pathology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 807, Taiwan ¶ National Institute of Cancer Research, National Health Research Institutes, Tainan 704, Taiwan □ Department of Emergency Medicine, E-Da Hospital, I-Shou University, Kaohsiung 824, Taiwan ■ Center for Biomarkers and Biotech Drugs, Kaohsiung Medical University, Kaohsiung 807, Taiwan ○ Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung 807, Taiwan ● Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung 804, Taiwan

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S Supporting Information *

ABSTRACT: Di(2-ethylhexyl) phthalate (DEHP) is a common plasticizer that is widely used in many consumer products and medical devices. Humans can be exposed to DEHP through ingestion, inhalation, or dermal absorption. Previous studies on DEHP have focused on its role as an endocrine-disrupting chemical leading to endocrine-related diseases. However, the correlation between DEHP exposure and the progression of colorectal cancer (CRC) is largely unknown. The aim of this study was to investigate the effects of mono(2-ethylhexyl) phthalate (MEHP), an active metabolite of DEHP, on the progression of CRC. Our results showed that treatment with MEHP enriched the population of cancer-stem-cell (CSC)-like cells and upregulated IL-8 expression by inducing the AKT−β-catenin−TCF4 signaling pathway. Blocking β-catenin−TCF4-mediated IL-8 expression reversed the MEHP-induced migration and enrichment of CSC-like cells. Consistent with the in vitro data, DEHP treatment increased the levels of nuclear β-catenin, polyp formation, and invasive adenocarcinoma in a mouse model. Our results suggest that MEHP facilitates the progression of CRC through AKT−β-catenin signaling. KEYWORDS: MEHP, cancer stem cells, β-catenin, colorectal cancer, IL-8, AKT

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INTRODUCTION

revealed that DEHP exposure causes a malignant phenotype for breast cancer. For example, DEHP upregulates the proliferation of MCF-7 and MDA-MB-231 breast-cancer cells.11 During metabolism, DEHP is rapidly hydrolyzed to mono(2-ethylhexyl) phthalate (MEHP) by esterases and lipase in the small intestine and liver. MEHP is further metabolized by cytochrome P450 and UDP-glucuronosyl transferases into secondary metabolites, which are quickly excreted from the body.12−14 Results from an in vitro study show that DEHP exposure modulates the progression of cancers associated with the digestive system and liver. For example, DEHP enhances proliferation, apoptosis, anchorage-independent growth, migration, and invasion of hepatocellular-carcinoma cells.15 DEHP has also been shown to increase Helicobacter pylori cytotoxicity and induce gastric-epithelial-cell apoptosis; therefore, coexposure to DEHP and H. pylori might disrupt the integrity of

The development of colorectal cancer (CRC) is a wellunderstood, multistep process that involves alterations in the expression of oncogenes and tumor-suppressor genes.1−3 Early detection and diagnosis have been shown to decrease CRCrelated mortality. Owing to the high incidence of CRC in Taiwan,4 the identification of risk factors associated with CRC may be useful for developing effective prevention strategies and improving patient outcomes. For example, several foods and nutrients, including red meat, fried foods, and excess fat, have been shown to be associated with an increased risk of colon cancer;5−7 thus, avoiding these foods may reduce the risk of developing CRC. Di(2-ethylhexyl) phthalate (DEHP) is a common class of phthalate esters and is often used as a plasticizer. Humans may be exposed to DEHP through food intake, inhalation, or skin contact. It has been reported that DEHP may act as an endocrine-disrupting chemical (EDC), leading to the disturbance of androgen-dependent development and semen quality in males, a decrease of female fertility, and an increase in spontaneous abortions.8−10 With respect to the effect of DEHP on cancer, recent studies using animal models have © XXXX American Chemical Society

Received: July 7, 2018 Revised: August 26, 2018 Accepted: August 26, 2018

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DOI: 10.1021/acs.jafc.8b03558 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 1. Levels of epithelial−mesenchymal-transition (EMT)-associated and stemness-associated gene expression in colorectal cancer (CRC) cells upregulated by mono(2-ethylhexyl) phthalate (MEHP). (A) Cell viability of HCT116 cells treated with ethanol (EtOH, control) or the indicated dose of MEHP for 72 h, examined using MTT assays. Data shown are the means ± SD of three independent experiments. (B) Size of spheres in HCT116 cells seeded in low-attachment plates, determined after 14 days. Results shown are the means ± SD from three independent assays, *P < 0.05. The original magnification is 40×, and the scale bar is 100 μm. (C) mRNA levels in HCT116 cells treated with EtOH (control) or the indicated dose of MEHP for 72 h, analyzed by qPCR and normalized to GAPDH. Results shown are the means ± SD from three independent assays, *P < 0.05. (D,E) Protein levels of E-cadherin and N-cadherin in (D) SW48 and (E) HCT116 cells treated with 1 nM MEHP for 72 h, analyzed by Western blotting. Tubulin was used as an internal control. The original magnification is 20×, and the scale bar is 100 μm.

the gastric mucosa and promote gastric carcinogenesis.16 In addition, previous studies indicated that exposure to a chemical closely related to DEHP, diethyl phthalate (DEP), may also play a role in gut microbiota modification and result in coloncancer formation.17,18 Daily oral gavage of DEHP leads to an increased number of aberrant crypt foci (ACF) or tumors through the upregulation of β-catenin and cyclin D1.19 Details regarding the mechanisms associated with accumulated DEHP exposure and CRC progression are largely unexplored. Many previous studies have indicated that cancer stem cells (CSCs) are involved in the tumorigenesis, metastasis and chemoresistance.20−22 Disturbance of the homeostatic control of normal stem cells in colon epithelia is a critical factor leading to the development of CRC. Therefore, the aim of the current study was to investigate the role of MEHP in CSC-mediated CRC progression.

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obtained from Sigma (St. Louis, MO). Cells were treated with 1 and 2 nM MEHP for 72 h for further analysis. Anti-GAPDH and antitubulin antibodies were purchased from GeneTex (San Antonio, TX). Anti-β-catenin, anti-TCF4, anti-phospho-GSK3β, anti-GSK3β, antiAKT, and anti-phospho-AKT antibodies were purchased from Cell Signaling Technology (Danvers, MA). The TCF4 dominant-negative (DN)-mutant construct has a DNA-binding domain but lacks the Nterminus required for β-catenin binding and was kindly provided by Professor Wen-Chun Hung (NHRI, Tainan, Taiwan). Immunoblotting and Immunofluorescence. Cells were lysed in modified RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% nonidet P-40, 150 nM NaCl, 1 mM EDTA, 0.25% sodium deoxycholate, 1 mM PMSF, and protease inhibitors), and cellular debris was removed by centrifugation. Proteins were separated on an SDS−polyacrylamide gel and then transferred to nitrocellulose. Immunoblotting was performed with appropriate antibodies. For immunofluorescence, cells were washed twice with PBS and permeabilized with 0.1% Triton X100 for 10 min. After permeabilization, cells were incubated with 0.05% BSA to block nonspecific binding. Then, an anti-β-catenin antibody was added and incubated at room temperature for 1 h. Cell nuclei were stained with DAPI, and the fluorescent image was observed under a fluorescence microscope. Quantitative Real-time PCR. Total RNA was extracted using the RNeasy mini kit (Qiagen, Valencia, CA). RT-PCR was performed

MATERIALS AND METHODS

Cell Cultures and Reagents. HCT116 (BCRC, HsinChu, Taiwan), SW480 (ATCC, Manassas, VA), and SW48 (ATCC, Manassas, VA) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum. MEHP was B


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Figure 2. Mono(2-ethylhexyl) phthalate (MEHP) triggering the accumulation of nuclear β-catenin through a post-transcriptional mechanism. (A) Levels of β-catenin analyzed by Western blotting and quantified. Tubulin or actin was used as the internal control. (B) β-catenin mRNA levels analyzed by qPCR and normalized to those of GAPDH. (C) β-Catenin in HCT116 cells treated with MEHP for 72 h. The cells were fixed, and βcatenin was detected by immunofluorescence microscopy. Nuclei (blue) were labeled with DAPI. The original magnification is 40×, and the scale bar is 10 μm. (D) Western blotting of subcellular fractions of HCT116 cells performed to determine the location of β-catenin. Tubulin was used an internal control for the cytoplasmic fraction. ORC2 was used an internal control for the nuclear fraction. using the following primer pairs: β-catenin (forward) 5′-TCTGA GGA CAAGCCACAAGATTACA-3′ and (reverse) 5′TGGGCACCAATATCAAGTC CAA-3′, IL-8 (forward) 5′CACCGGAAGGAACCATCTCACT-3′ and (reverse) 5′-T CAG CCCTCTTCAAAAACTTCTCC-3′, Nanog (forward) 5′CA AAGGCAAACA ACCCACTT-3 ′ a n d (r e v e r s e ) 5 ′TCTGCTGGAGGCTGAGGTAT-3′, SOX2 (forward) 5′-A CACCAATCCCATCCACACT-3′ and (reverse) 5′GCAAACTTCCTGCAAA GCTC-3′, Oct4 (forward) 5′GTACTCCTCGGTCCCTTTCC-3′ and (reverse) 5′-CA AAAACCCTGGCACAAACT-3′, IL-6 (forward) 5′GACAGCCACTCACCTCTTC A-3′ and (reverse) 5′ TGCAGGAACTGGATCAGGAC-3′, and TNF-α (forward) 5′-GG AGAAGGGTGACCGACTCA-3′ and (reverse) 5′-CTGCCCAGACTCGGCAA-3′. Quantitative PCR was performed in an ABI 7900 realtime PCR instrument. Chromatin-Immunoprecipitation Assay. Cells were harvested, and a chromatin-immunoprecipitation assay was performed as described previously.23 DNA fragments were recovered and subjected to PCR amplification using IL-8-promoter-specific primers to detect the −350 to −174 bp region, which contains the acetylation sites. The sequences of the primers were 5′-AGAAAATCATCCATGATCTTGTT-3′ (forward) and 5′-AATACGGAGTATGAC GAAAGTT-3′ (reverse). DNA fragments were amplified using a PCR machine. Sphere-Formation Assay. A sphere-formation assay was performed as previously described.24 In brief, cells were collected and suspended in serum-free DMEM/F12 supplemented with 100

IU/mL penicillin, 100 mg/mL streptomycin, 20 ng/mL human recombinant epidermal-growth factor (hrEGF), 10 ng/mL human recombinant basic fibroblast-growth factor (hrbFGF), 2% B27 supplement without vitamin A, and 1% N2 supplement (Invitrogen, Carlsbad, CA). Subsequently, the cells were cultured in six-well ultralow-attachment plates (Corning, Inc., Corning, NY) at a density of 5 × 103 cells/well. After 14 days, the numbers and sizes of the spheres were analyzed by using ImageJ software. Animals. C57BL/6J-APCMin/+ mice were purchased from Jackson Laboratories (Bar Harbor, ME). At 6 weeks of age, eight mice of each group were administered a daily oral gavage of corn oil and DEHP (100 mg/kg body weight, 99% pure, CAS no. 117-81-7) for 6 weeks. After DEHP exposure, the animals were weighed and anesthetized using CO2. Paraffin-embedded tumor samples were stained with βcatenin, IL-8, and SOX2 primary antibodies, appropriate secondary antibodies, and Envision system (Dako, Glostrup, Denmark). Finally, sections were counterstained with hematoxylin and analyzed with a microscope. Statistical Analysis. All experiments were performed in triplicate. Data are expressed as means ± SD and were used to compare the differences between experimental groups. Multiple comparisons were evaluated and calculated by one-way ANOVA.

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RESULTS CRC Migration and the Number of CSC-like Cells Increased by MEHP. MEHP is the most common metabolite of DEHP.25 We first examined the effect of MEHP on cell C


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Figure 3. Treatment of CRC cell lines with mono(2-ethylhexyl) phthalate (MEHP) overcoming AKT IV mediated β-catenin degradation. (A) Levels of the indicated proteins analyzed by Western blotting and quantified. After treatment of HCT116, SW48, and SW480 cells with EtOH (control) or the indicated dose of MEHP for 72 h, the indicated proteins were analyzed by Western blotting. Tubulin was used as an internal control. (B) Levels of β-catenin in SW480 cells pretreated with AKT IV (1 μM) for 24 h and then exposed to MEHP at the indicated doses for 48 h. (C) β-Catenin protein levels in SW480 cells pretreated with either AKT IV (1 μM) or 1 nM MEHP or a combination for 72 h followed by the addition of cycloheximide (CHX, 40 mg/mL) for the indicated times. Densitometry was used to quantify the β-catenin protein levels after normalization to the actin control in order to obtain the percentage of β-catenin degradation (means ± SD, n = 3). Error bars indicate SD, *P < 0.05.

viability and found that MEHP at concentrations from 10−9 to 10−6 M had no detectable effect on cell viability (Figure 1A). To investigate the effects of low-dose long-term exposure to MEHP on the progression of CRC, we used MEHP at nanomole concentrations. To determine the role of DEHP during the early stages of colon-cancer carcinogenesis, we examined cells treated with MEHP for two malignant phenotype markers: stem-like properties and mobility. As shown in Figure 1B, cells treated with MEHP formed larger spheres. In addition, several stemness-associated genes, including SOX2, Nanog, and Oct4, were induced in the MEHP-treated cells (Figure 1C). Next, we determined whether MEHP could promote metastasis of colon-cancer cells. As shown in Figure 1D,E, an increase in cell migration was observed in MEHP-treated SW48 and HCT116 cells. In addition, E-cadherin levels had decreased after exposure to MEHP, in contrast to N-cadherin levels, which increased. These data suggested that MEHP promoted CSC formation, and long-term exposure may be correlated with cell migration. Level and Nuclear Translocation of β-Catenin Enhanced by MEHP. The formation of polyps by normal colon epithelial cells is the earliest step in colon-cancer progression. Adenomatous polyposis coli (APC) is a well-

documented tumor suppressor that functions as part of the natural defense against the development of colon cancer.26−28 In mouse models, the loss of APC-function results in the formation of colonic polyps.29 APC mutations or loss of APC expression leads to the accumulation of β-catenin and recruitment of the TCF−LEF transcription-factor complex to cell-proliferation- and stemness-associated genes, promoting their expression.30−32 To further evaluate the MEHP-mediated malignant phenotype of colon-cancer cells, we determined whether the levels of β-catenin were altered in cells exposed to MEHP. As shown in Figure 2A, MEHP treatment significantly increased the levels of β-catenin in HCT116, SW48, and SW480 cells. This increase was post-transcription-dependent (Figure 2B). Results from immunofluorescence assays demonstrated that the nuclear localization of β-catenin was increased in the cells treated with MEHP (Figure 2C). Consistent with the in situ staining, an increase in the amount of β-catenin in the nuclear fraction of cell lysates was also observed by Western-blot analysis (Figure 2D). In summary, MEHP promoted a malignant phenotype of CRC cells by increasing the expression and nuclear localization of β-catenin. MEHP-Treatment Attenuation of the Half-Life of βCatenin through the Activation of AKT. It is known that D


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Figure 4. Overexpression of dominant-negative TCF4 blocking the mono(2-ethylhexyl) phthalate (MEHP)-mediated increase of IL-8 expression. (A) mRNA levels of IL-8, IL-6, and TNF-α in HCT116 cells treated with MEHP for 72 h, detected by qPCR and normalized to that of GAPDH. Results shown are the means ± SD of three independent assays, *P < 0.05. (B) Secretion of IL-8 analyzed by ELISA. Results shown are the means ± SD of three independent assays, *P < 0.05. (C) IL-8 mRNA expression in cells transfected with a DN-TCF4-expression plasmid and treated with MEHP for 72 h. IL-8 mRNA expression was detected by qPCR and normalized to that of GAPDH. Results shown are the means ± SD of three independent assays, *P < 0.05. (D) Secretion of IL-8 assessed in DN-TCF4-overexpressing cells by ELISA. HCT116 cells were treated with EtOH (control) or MEHP for 72 h. (E,F) Chromatin-immunoprecipitation−quantitative-polymerase-chain-reaction (ChIP-qPCR) analysis performed to evaluate (E) β-catenin and (F) TCF4 binding to the IL-8 promoter. The experiments were repeated three times, *P < 0.05.

Axin, APC, and GSK-3β interact with β-catenin and consequently trigger the ubiquitination and degradation of βcatenin. Inhibitory Ser-9 phosphorylation of GSK3β by the AKT pathway promotes β-catenin−TCF-complex-coordinated gene expression, which is positively correlated with poor outcomes in colon-cancer patients.33,34 We analyzed whether the AKT−GSK3β axis played a role in MEHP-mediated stabilization of β-catenin. Exposure of HCT116, SW48, and SW480 cells to MEHP significantly upregulated the levels of Ser9-phosphorylated GSK3β and AKT activation (Figure 3A). These data suggested that exposure to MEHP enhanced the AKT-mediated phosphorylation of GSK3β and the accumulation of β-catenin. Consistent with this hypothesis, treatment of the cells with the AKT inhibitor AKT IV significantly reduced the basal level of β-catenin and blocked the MEHPmediated expression of β-catenin (Figure 3B). To address the function of MEHP in AKT-mediated βcatenin stabilization, we performed protein-stability assays to assess the effects of MEHP on the cellular response to the inhibition of AKT. As shown in Figure 3C, β-catenin stabilization was affected by the presence of MEHP during treatment with AKT IV. MEHP decelerated the degradation of β-catenin in cells, combating the AKT IV treatment (Figure 3C). Together, our data suggested that MEHP functioned as a regulator of AKT-mediated β-catenin expression as it contributed to tumor progression.

MEHP-Mediated IL-8 Transcription and Secretion Abolished by Blockage of TCF4. It is well-known that βcatenin functions as a transcriptional activator and is involved in TCF−LEF-mediated downstream-gene expression in CRC. The defined target genes of β-catenin−TCF are those encoding c-myc; cyclin D1; and several proinflammatory cytokines, including IL-6, IL-8, and TNF-α.35,36 Evaluation by qPCR revealed that IL-8 mRNA levels were significantly upregulated in cells exposed to MEHP compared with the transcript levels of IL-6 and TNF-α (Figure 4A). Therefore, we used IL-8 as a model for MEHP-mediated β-catenin−TCFcoordinated gene expression. Consistent with the increased transcript levels, MEHP-mediated upregulation of IL-8 secretion was also detected by ELISA (Figure 4B). These findings were contrasted by the overexpression of DN-TCF4, which abolished the MEHP-mediated upregulation of IL-8 mRNA and secreted-protein levels (Figure 4C,D). To further examine the role of the β-catenin−TCF complex at the IL-8 promoter, we performed a chromatin-immunoprecipitation (ChIP) experiment. As shown in Figure 4E,F, the results of the ChIP−quantitative-polymerase-chain-reaction (ChIP-qPCR) assay demonstrated that MEHP treatment increased the binding of β-catenin to the IL-8 promoter (Figure 4E). Similarly, MEHP-treatment also induced an increase in binding to the IL-8 promoter (Figure 4F). Our results suggested that MEHP increased IL-8 expression by E


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Figure 5. Blocking the AKT−β-catenin axis to abolish mono(2-ethylhexyl) phthalate (MEHP)-mediated increases in gene expression associated with cell migration and stemness. (A) Cell migration evaluated by cell-motility assays performed after treatment of the cells with MEHP alone or in combination with AKT IV (1 μM) for 24 h. (B) Cell migration evaluated by cell-motility assays performed after treatment of the cells with MEHP alone or in combination with FH535 (10 μM) for 24 h. (C) Cell migration in control and TCF4-overexpressing cells analyzed after treatment with MEHP. (D) Cell migration analyzed in cells pretreated with a neutralizing anti-IL-8 antibody prior to MEHP treatment. (E) Number and size of the spheres in cells cultured in low-attachment plates, analyzed after 14 days of culturing. Results are shown from three independent assays and expressed as the means ± SE, *P < 0.05. (A−D) The original magnification is 20×, and the scale bar is 100 μm. (E) The original magnification is 40×, and the scale bar is 20 μm.

modulating the recruitment of the β-catenin−TCF complex to the promoter. MEHP-Induced CSC Migration and Population Increase Blocked by the Neutralization of IL-8 Antibody and AKT IV. To further define the role of MEHP in the formation and migration of CSCs, we examined the effect of blocking AKT and the β-catenin−TCF complex on the MEHP-mediated increase in sphere formation and CSC migration. First, we found that the ability for cell migration was markedly suppressed in SW480 cells cotreated with AKT IV and MEHP compared with that of cells treated with only MEHP (Figure 5A). This phenomenon was similar to what was observed for cells treated with the β-catenin inhibitor FH535 (Figure 5B). Next, the overexpression of DN-TCF4 reduced the MEHPinduced expression of stemness-associated genes (Supplementary Figure 1) and cell-migration ability (Figure 5C). Furthermore, MEHP-induced migration was inhibited by treatment of the cells with a neutralizing anti-IL-8 antibody (Figure 5D). Finally, we evaluated the capacity of sphere formation, to provide additional support for the role of MEHP in the AKT-mediated phenotype of CSCs. Treatment with AKT IV resulted in significantly fewer spheres, suggesting that AKT was required for MEHP-mediated CSC sphere formation (Figure 5E). Taken together, these data indicated that the β-

catenin−TCF complex played an important role as a regulator of the MEHP-mediated malignant phenotype of CRC cells. IL-8 Expression and Levels of Nuclear β-Catenin Increased by MEHP Treatment in Vivo. The C57BL/6JAPCMin/+ (Min) mouse carries an APC mutation and is a useful model for studying intestinal neoplasms.37,38 We found that treatment of these mice with DEHP increased the number of intestinal polyps that formed (Figure 6A). To verify the involvement of the β-catenin−TCF axis in the MEHP-mediated increase in IL-8 expression, we determined the nuclear localization of β-catenin using immunohistochemical staining of mouse intestines. We observed nuclear staining of β-catenin in the adenomatous lesions of DEHP-treated Min mice. This suggested that the β-catenin pathways were activated following DEHP exposure (Figure 6B). Consistent with the in vitro data, DEHP treatment led to increased expression of IL-8 and SOX2. These results indicated that DEHP exposure may have triggered an aggressive phenotype of the CRC tumors in Min mice through the activation of the β-catenin−TCF complex by IL-8. CRC tumors in Min mice are typically benign adenomas that lack an aggressive invasive phenotype. Therefore, we evaluated the effect of MEHP on the histopathologic features of the tumors in these mice. As shown in Figure 6C, MEHP-treated Min mice developed invasive adenocarcinoma after 12 weeks of age, whereas Min mice F


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Figure 6. Colorectal cancer (CRC) tumors in di(2-ethylhexyl) phthalate (DEHP)-treated APCMin/+ mice showing an accumulation of nuclear βcatenin and a more aggressive phenotype. (A) Representative photographs of APCMin/+ mouse intestines (upper panel) and number of polyps per mouse in the control APCMin/+ mice and DEHP-treated APCMin/+ mice at 90 days (lower panel). The scale bar is 1 cm. (B) Levels of β-catenin, IL8, and SOX2 in DEHP-treated APCMin/+ mice assessed by immunohistochemistry. The original magnification is 20×, and scale bar is 20 μm. (C) Histopathological features of polyps from DEHP-treated APCMin/+ mice. Invasive adenocarcinoma (black star, left panel), intramucosal adenocarcinoma (white star, middle panel), and submucosal adenocarcinoma (black star, right panel) were collected from DEHP-treated APCMin/+ mice. The original magnification is 20×, and the scale bar is 100 μm.

Figure 7. Proposed model of mono(2-ethylhexyl) phthalate (MEHP) signaling involving AKT-mediated nuclear β-catenin accumulation in colorectal-cancer (CRC) progression. MEHP-treatment enhances AKT signaling, which leads to increased recruitment of the β-catenin−TCF complex to the IL-8 promoter and the promotion of a malignant phenotype in CRC.

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treated with corn oil failed to demonstrate this invasive phenotype. Collectively, these data suggested that DEHP exposure triggered more aggressive lesions in the Min mice through the AKT−β-catenin−IL-8 axis(Figure 7).

DISCUSSION

DEHP is commonly used in many plastics. Although there is no direct evidence that exposure to DEHP at the levels found in the environment causes harmful health effects in humans, in G


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associated genes (e.g., increased expression of N-cadherin and decreased expression of E-cadherin) and stemness-associated genes (e.g., SOX2, Oct4, and Nanog). It is known that mutations in β-catenin in SW48 cells (C-to-A change in codon 33) and HCT116 cells (deletion of codon 45), results in the loss of targets for GSK3β. We found that the levels of β-catenin were significantly increased in SW480 cells compared with those in HCT116 and SW48 cells. In addition, the levels of GSK3β serine-9 phosphorylation in HCT116 and SW480 cells were enhanced following MEHP exposure. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor. Evidence indicates that AhR plays major roles in toxicant metabolism and the regulation of gastrointestinal function.50,51 Recent studies have further shown that signaling pathways upregulated by the phthalate−AhR axis regulate CSCs in breast cancer and liver cancer.52,53 However, a previous study using AhR-null mice as a model showed increased numbers of colorectal tumors and intestinal inflammation compared with in control mice.54−56 Therefore, the precise role of AhR-associated activity in DEHP-mediated malignancy of CRC requires further investigation. As an illustration of oncogenic signaling, a recent study indicated that MEHP mediates the induction of AKT activation in cervical cancer through the G-protein-coupled estrogen receptor (GPER).57 Our results, suggesting that the effects of the MEHP−AKT axis on CRC metastasis are mediated by the regulation of CSC-like cells through βcatenin−TCF and complex−IL-8 signaling pathways, are consistent with this previous finding. The blocking of IL-8 expression and suppressing of AKT signaling inhibited MEHPmediated cancer malignancy. Consistent with these in vivo results, MEHP-treatment induced the accumulation of nuclear β-catenin, and the MEHP-activated β-catenin−TCF complex increased IL-8 transcription, which promoted the development of metastatic CSC. Overall, data from the current study provides evidence that DEHP affects CSC signaling pathways in the colon and may help elucidate the effect of DEHP exposure on cancer malignancy. The findings are consistent with phthalate exposure being a risk factor for the progression of CRC. The current study identified new research avenues regarding environmental exposure to DEHP and the molecular pathways that are active during CRC pathogenesis.

vivo models show that high levels of DEHP can damage the liver and kidneys and affect the reproductive abilities of mice and rats.8−10,39,40 In addition, experiments using an in vivo model of chronic carcinogenicity shows lung and liver degeneration, shrinkage of the testes, and the development of liver cancer following DEHP exposure.41 Several in vitro studies have shown that exposure to DEHP enhances the proliferation, migration, invasion, and chemoresistance of several cancer types, including breast cancer, gastric cancer, hepatocellular carcinoma, and glioblastoma.11,15,16,42 We note that heavy exposure to DEHP may result from intravenous fluid delivery through DEHP-containing plastic tubing and from ingesting contaminated foods or water. Esterases, lipases, cytochrome P450, and UDP-glucuronosyl transferases in the digestive system and liver are major enzymes involved in the conversion of DEHP to MEHP.43,44 The prevalence of patients with CRC in Taiwan is high, and the incidence is increasing each year. Extensive use of plastics is common in Taiwan.45,46 Therefore, it is important to determine whether DEHP exposure is related to CRC carcinogenesis. To identify the effects of environmental exposure to DEHP on CRC progression, we used APCMin/+ mice as a model to clarify the effect of DEHP on the malignant phenotype of CRC. We found that APCMin/+ mice treated with DEHP at a dose of 100 mg/kg per day demonstrated enhanced polyp formation and developed invasive adenocarcinoma more frequently compared with untreated APCMin/+ mice. MEHP was the primary breakdown product of DEHP. Therefore, we then verified the role of MEHP in the development of advanced CRC in vitro. Several lines of evidence have shown that CSCs are a relatively small population of cells involved in the carcinogenesis and malignancy of CRC.20−22 Therefore, studying metastasis-initiating CSCs may provide insight into potential fundamental strategies for the development of anticancer agents for CRC. However, CRC is a heterogeneous disease with three common molecular phenotypes that reflect different genome instabilities, including a phenotype associated with the chromosomal-instability pathway (CIN), microsatellite-instability (MSI) phenotype, and the CpG-island methylator phenotype (CIMP). CRCs are associated with high mutation rates in TP53, KRAS, PIK3CA, PTEN, APC, and the β-catenin gene.47 The β-catenin−TCF axis functions as an important oncogenic signaling pathway in the digestive system. Loss of APC−GSK3β expression induces β-catenin−TCF-complextargeted transcription signaling to promote cancer malignancy.48 In the current study, we studied the role of MEHP with respect to the function of the APC−β-catenin axis in CRC cells. Analysis of 21 cell lines from 19 patients revealed that only four of the cell lines (SW 48, HCT116, LS174T, and HCA 7) contained wild-type (WT) APC protein.49 In order to identify the role of phthalate in the progression of colon cancer mediated by the APC−β-catenin axis, we used three CRC cell lines: SW480 (APC mutant, β-catenin WT), HCT116 (APC WT, β-catenin mutant), and SW48 (APC WT, β-catenin mutant). Specifically, the cell lines were used to determine whether MEHP induced CRC cell migration, epithelial− mesenchymal transition (EMT), and stemness. Detailed characteristics of the three cell lines are shown in the Supplementary Table. We found that exposure to MEHP significantly promoted cell migration and sphere formation by regulating EMT-

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b03558. Expression levels of SOX2, Oct4, and Nanog and characteristics of the three CRC cell lines used in the study (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +886-7-3121101-509234. Fax:+886-7-3218309. ORCID

Tian-Lu Cheng: 0000-0001-6424-4731 Mei-Ren Pan: 0000-0003-2039-7570 Author Contributions

C.-W.L., J.-Y.W., W.-C.H., T.-L.C., and M.-R.P. conceived and designed the experiments; I-L.H., C.-W.L., Y.-H.L., T.-Y.C., H


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and Y.-C.H. performed the experiments; I-L.H., C.-C.W., and Y.-H.L. analyzed the data; C.-W.L. and M.-R.P. wrote the paper; C.-W.L. and M.-R.P. helped to complete the manuscript. All authors read and approved the final manuscript. Funding

This study was supported by grants from the Ministry of Science and Technology of the Republic of China (106-2314B-037-049-MY3); the Research Center for Environmental Medicine, Kaohsiung Medical University (KMU-TP105A15); Kaohsiung Medical University (106CM-KMU-08); KMUKMUH Co-Project of Key Research, grant nos. KMUDK107013 and KMU-DK108011; the Taiwan Ministry of Health and Welfare (MOHW106-TDU-B-212-144007); and the Health and Welfare surcharge of tobacco products. Notes

The authors declare no competing financial interest.

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