the evaluation of p,p′-ddt exposure on cell adhesion of hepatocellular carcinoma

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ARTICLE IN PRESSG ModelOX 51385 1–10

Toxicology xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Toxicology

j ourna l ho me page: www.elsev ier .com/ locate / tox ico l

he evaluation of p,p′-DDT exposure on cell adhesion ofepatocellular carcinoma

iaoting Jina,1, Meilan Chena,1, Li Songa, Hanqing Li c, Zhuoyu Lia,b,∗

Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of National Ministry of Education,hanxi University, Taiyuan 030006, ChinaCollege of Life Science, Zhejiang Chinese Medical University, Hangzhou 310053, ChinaCollege of Life Science, Shanxi University, Taiyuan 030006, China

r t i c l e i n f o

rticle history:eceived 26 March 2014eceived in revised form 22 April 2014ccepted 4 May 2014vailable online xxx

eywords:,p′-DDTepatocellular carcinomaell adhesion

AK/STAT3 pathwayxidative stress

a b s t r a c t

Many studies have found a positive association between the progression of hepatocellular carcinomaand DDT exposure. These studies mainly focus on the effect of DDT exposure on cell proliferation andepithelial to mesenchymal transition (EMT) promotion. However, the influence of DDT on cell adhesionof hepatocellular carcinoma remains to be unclear. The aim of our study was to determine the effect ofp,p′-DDT on cell adhesion of hepatocellular carcinoma in vitro and in vivo. The data showed that p,p′-DDT, exposing HepG2 cells for 6 days, decreased cell–cell adhesion and elevated cell–matrix adhesion.Strikingly, p,p′-DDT increased reactive oxygen species (ROS) content, and this was accompanied by theactivation of JAK/STAT3 pathway. Moreover, ROS inhibitor supplement reversed these effects signifi-cantly. However, the addition of ER inhibitor, ICI, had no effect on the p,p′-DDT-induced effects. p,p′-DDTaltered the mRNA levels of related adhesion molecules, including inhibition of E-cadherin and promotionof N-cadherin along with CD29. Interestingly, the p,p′-DDT-altered adhesion molecules could be reversed

′
with JAK inhibitor or STAT3 inhibitor. Likewise, p,p -DDT stimulated the JAK/STAT3 pathway in nude mice,as well as altered the mRNA levels of E-cadherin, N-cadherin, and CD29. Taken together, these resultsindicate that p,p′-DDT profoundly promotes the adhesion process by decreasing cell–cell adhesion andinducing cell–matrix adhesion via the ROS-mediated JAK/STAT3 pathway. All these events account forthe carcinogenic potential of p,p′-DDT in liver.
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. Introduction

DDT, dichlorodiphenyltrichloroethane, one of the probable car-inogens and persistent organic pollutants, presents in diet andhe environment, banned by the early 1970s and prohibited itsse in 1983 in China (Qiu et al., 2005). However, DDT still per-ists in the environment for decades due to its long-term existence,ipophilicity, difficult degradation, and bio-accumulative proper-ies. Its residues cause a few health problems in humans, such asancer, endocrine and immunological disorders (Glynn et al., 2007;

Please cite this article in press as: Jin, X., et al., The evaluation of pToxicology (2014), http://dx.doi.org/10.1016/j.tox.2014.05.002

rema et al., 2013). Hepatocellular carcinoma (HCC) is the fifthost common malignancy worldwide and HCC-associated annualortality ranks third among all tumors (El-Serag and Rudolph,

∗ Corresponding author at: Institute of Biotechnology, Key Laboratory of Chem-cal Biology and Molecular Engineering of National Ministry of Education, Shanxiniversity, Taiyuan 030006, China. Fax: +86 351 7018268.

E-mail addresses: [email protected] (X. Jin), [email protected] (Z. Li).1 These authors contributed equally to this work.

ttp://dx.doi.org/10.1016/j.tox.2014.05.002300-483X/© 2014 Published by Elsevier Ireland Ltd.

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© 2014 Published by Elsevier Ireland Ltd.

2007). Accumulating ecological studies have reported a statisti-cally significant correlation between DDT serum concentration andincidence of liver cancer in humans (McGlynn et al., 2006; Perssonet al., 2012). The association between liver cancer incidence andexposure to DDT suggests that this pesticide may have an etiologicrole in this process. The liver is the most sensitive tissue to xenobi-otic exposure and constitutes the main target of DDT toxicity. Wepreviously reported the tumorigenesis of human liver cancer cellsafter exposure to DDT, as well as the promotion of liver cancer cellproliferation, but the adhesion involved in the progression is notknown (Jin et al., 2014).

Adhesion, including cell–cell and cell–matrix adhesive, is animportant process in tumor progression. Deregulated cell adhe-sion is frequently observed in a number of pathologic conditionsincluding cancer progression, while the regulation of cell–celland cell–matrix adhesion is strictly controlled in normal cells

,p′-DDT exposure on cell adhesion of hepatocellular carcinoma.

(Bourboulia and Stetler-Stevenson, 2010). The molecular mecha-nism of adhesion is implicated in many pathological processes, suchas carcinogenesis and metastasis. It plays key roles in many dif-ferent aspects of cell invasion and migration, and has been used

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dx.doi.org/10.1016/j.tox.2014.05.002

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o assess the aggressive and malignant phenotypes of the cellsSchmidmaier and Baumann, 2008).

The initiation and progression of adhesion require transduc-ion of cell signals. Accumulated evidences have indicated that thebnormal activation of JAK (Janus kinase)/STAT (signal transducersnd activators of transcription) signaling pathway played a criti-al role in hepatocellular carcinoma (HCC) oncogenesis (Schindler,002). After cytokines combine to receptors, JAK, a tyrosine proteininase, is activated by phosphorylation. The activated JAK raisesonomer STAT3 of cytoplasm and forms p-STAT3. p-STATs release

rom receptors in a dimmer form. Then, it enters into the nucleus,here it binds to special DNA sites, and regulates the transcrip-

ion of downstream target genes, thus promoting cell malignantransformation and tumorigenesis (Ahamed et al., 2013; Li et al.,006; Sanchez et al., 2003). Additionally, several lines of evidencesave supported that stress response may induce activation of the

AK/STAT3 pathway (Rivat et al., 2004; Wei et al., 2011).Adhesion molecules act as pivotal players in cell adhesion,

ncluding cadherins and Integrin. Cadherins, which are calcium-ependent cell adhesion receptors, have important roles in thealignant progression of various human cancers, such as cell adhe-

ion, aggregation, cell polarity, and morphogenesis (Jeanes et al.,008). As a key component of adherens junctions, cadherins play

crucial role in the cell–cell adhesion (Hazan et al., 2004). Sev-ral studies have suggested that E-cadherin and N-cadherin aremportant epithelial adhesion molecules in normal epithelium as arerequisite for normal cell function and the preservation of tissue

ntegrity (Cavallaro and Christofori, 2004). CD29 (Integrin beta-), an integrin unit associated with very late antigen receptors, ishe beta subunit of an integrin family of molecules expressed oniverse cell types, which function as a key molecule in the processf cell–matrix adhesion (Guo and Giancotti, 2004).

In the present study, we investigated the cellular and molecu-ar effects of DDT in human liver cancer cells using the HepG2 celline. Human hepatocarcinoma (HepG2) cells have been used as aurrogate for human hepatocytes and retain many cellular func-ions allowing the study of several molecular processes (Jan et al.,008). This study aimed to elucidate the mechanism of p,p′-DDTction on the adhesion of hepatocellular carcinoma using in vitrond in vivo models. The result unveils, for the first time, that p,p′-DT exposure alters adhesion of hepatocellular carcinoma. Thisrocess is characterized by the repression of E-cadherin expres-ion, along with the elevation of N-cadherin and CD29 expressions.hese effects are due to p,p′-DDT’s ability to initiate the activationf JAK/STAT3 pathway mediated by oxidative stress. The presentnvestigation provides molecular evidence that p,p′-DDT could haven adverse impact on human health and contribute to liver cancerevelopment.

. Materials and methods

.1. Materials

Antibodies for p-JAK1 (Tyr 1022), STAT3, and p-STAT3 (Ser 727) were pur-hased from BBI (Sangon Biotech, Shanghai, China). �-Tubulin was purchased fromigma (St. Louis, MO, USA). Antibodies for LaminB were obtained from Bioworld.,p′-DDT (Sigma) was dissolved into dimethyl sulfoxide (DMSO) as stock solu-ions. The equal concentration of DMSO was added to medium for the control cells.-acetyl-l-cysteine (NAC, the scavenger of ROS) and 2′ ,7′-dichlorofluorescein diac-tate (DCFH-DA) were obtained from Beyotime Institute of Biotechnology (Nan tong,hina). ER inhibitor ICI 182780 was purchased from Sigma (St. Louis, MO, USA). JAK

nhibitor Ruxolitinib and STAT3 inhibitor WP1066 were purchased from CaymanSan Diego, California, UAS).

.2. Cell culture and drug treatments


Human hepatoma cells (HepG2) were maintained in Dulbecco’s modifiedagle’s medium (DMEM) (HyClone) supplemented with 10% FBS (Boster), 1%peni-illin/streptomycin (Solarbio) at 37 ◦C in a 5% CO2 humidified tissue culturencubator. To observe the toxicity of p,p′-DDT in HepG2 cells, cells were exposed

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to p,p′-DDT at different doses (from 10−11 to 10−7 mol/L) over a 6 days period. Cellswith the treatments were then assayed for cell adhesion assay. For all experimentalconditions, FBS was reduced to 2% in DMEM medium and cells were treated for theindicated time with p,p′-DDT prepared as DMSO stock solution.

2.3. Nude mice assay

All animal experiments were approved by the Committee of Animal Care atChinese Institute for Radiation Protection. As described in our previous study (Jinet al., 2014), HepG2 cells were washed twice and re-suspended in physiologicalsaline at a concentration of 5 × 107 cells/mL. A 200 �L cell suspension of HepG2was then injected subcutaneously into the left armpit of SPF-free male BALB/c-numice. After three days, one group of mice received intra-peritoneal injections ofp,p′-DDT diluted in DMSO (5 nmol/kg). Control mice received DMSO-diluted PBS.p,p′-DDT was administered only at the beginning of the tumor experiment. Tumorspecimens were collected at 7 weeks after injections and split. Six independentexperiments were performed. Tumor tissues were collected for western blot andRT-PCR analysis.

2.4. Cell–matrix adhesion assay

HepG2 cells were digested with 0.05% trypsin and re-suspended at1 × 104 cells/100 �L in DMEM media. Cells (100 �L) were added to 96-well plate andallowed to incubate at 37 ◦C for 30 min, 60 min, 90 min, and 120 min, respectively.Following incubation, non-adherent cells were removed by PBS washing, and adher-ent cells were fixed with 50 �L 4% paraformaldehyde solution for 10 min at roomtemperature. Then, the wells were washed three times and stained with 1% crystalviolet for 20 min at room temperature. Excess dye was removed, and intracellularstain was solubilized by the addition of 50 �L 1% SDS. Absorbances at 570 nm weredetermined using a microplate reader. Calculation of cell–matrix adhesion ratio wasusing the formula: (experiment group A570/control group A570) × 100.

2.5. Cell–cell adhesion assay

The cells were re-suspended at 1 × 104 per 100 �L with DMEM media, and100 �L cell suspension was seeded in each well of 96-well plates. The plates wereplaced in a 37 ◦C shaker and rotated at 80 rpm for 1 h. The wells were then gentlystirred. The number of aggregates and single cells were counted with a hemacytome-ter. The cell–cell adhesion ratio was calculated using the formula: Nd/Nc. WhereNc is the number of group cells (single cells forming aggregations) in control, andNd is the number of group cells detected in cultures at various time points afterincubation. Each value is the average of at least three independent experiments.

2.6. Western blot analysis

Extracted proteins from cells and tissues were resolved by 10% SDS–PAGEand transferred onto nitrocellulose membranes for western blotting. The blotswere blocked for 1 h in PBS containing 5% non-fat dry milk (w/v) and incubatedat 4 ◦C overnight, then probed with antibody for 1 h at room temperature orovernight at 4 ◦C. After washing, membranes were incubated at 37 ◦C for 1 h withthe appropriate horseradish peroxidase-conjugated secondary antibody (diluted at1:1000, Invitrogen). Protein loading was controlled by probing the membranes for�-tubulin protein. Immune-reactive proteins were detected using ECL western blot-ting detection system. For measurement STAT3 in cells and tissues, the cytoplasmicprotein and the nuclear protein were extracted according to the instructions of theNuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotech Inc., Nantong,China).

2.7. Measurement of ROS generation

DCFH-DA is a cell-permeable, nonfluorescent probe that is cleaved by intracel-lular esterases and turns into a highly fluorescent dichlorofluorescein upon reactionwith H2O2. After treatment with p,p′-DDT (10−9 mol/L) for 6 days with or withoutNAC (5 mmol) co-treatment for the last 3 days, the cells were stained with 10 �mol/LDCFH-DA for 30 min at 37 ◦C. H2O2 generation was determined by dichlorofluores-cein fluorescence. Cells were collected and the fluorescence intensity in the cellswas measured using a fluorescence microplate reader (Thermo Scientific varioskanflash) with excitation 488 nm and emission 525 nm.

2.8. Determinations of oxidative stress-related parameters

SOD activity and GSH content were determined in cells using a commercialdetermination kit (Nanjing Jiancheng Bioengineering Institute). Cells were plated


onto 6-well dishes (1 × 106 cells/well) and exposed to p,p′-DDT (10−9 mol/L) for 6days with or without NAC (5 mmol) co-treatment for the last 3 days. Scraped cellswere dissolved in physiological saline, followed by cell disruption using ultrasoundequipment. After being centrifuged at 6000 rpm for 10 min, the supernatants wereused to measure enzyme activities. The data were normalized to protein content.

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.9. Reverse transcription-quantitative polymerase chain reaction (RT-PCR)

To identify E-cadherin, N-cadherin, and CD29 RNA transcripts, quantitative realime PCR (qRT-PCR) was performed using an Applied Bio-systems platform. In brief,DNA was generated from 500 ng of total RNA using PrimeScript RT Master Mix. Real-ime PCR was performed in a total volume of 15 �L containing 1.5 �L RT products,.2 �L primers and 12.3 �L SYBR Green PCR Master Mix. GAPDH mRNA amplifiedrom the same samples served as an internal control. The relative expression of eachargeted gene was normalized by subtracting the corresponding GAPDH thresh-ld cycle (Ct) values using the ��Ct comparative method. PCR amplification waserformed using the following primers:

E-cadherin-Fw: 5′AGGACCAGGTGACCACCCTAGA3′

E-cadherin-Rw: 5′ATGCCCAAGATGGCAGGAAC3′

N-cadherin-Fw: 5′TCCATGTGCCGGATAGC3′

N-cadherin-Rw: 5′AGTTCAGTCATCACCTCCACCATACA3′

CD29-Fw: 5′AATGAAGGGCGTGTTGGTAG3′

CD29-Rw: 5′CTGCCAGTGTAGTTGGGGTT3′

GAPDH-Fw: 5′GCACCGTCAAGGCTGAGAAC3′

GAPDH-Rw: 5′TGGTGAAGACGCCAGTGGA3′


.10. Statistical methods

Statistical analysis was carried out using the SPSS 17.0 software program.ata, derived from three or four independent experiments, were presented as theean ± SD. Differences among groups were tested by one-way analysis of variance

ig. 1. Effects of p,p′-DDT exposure on the cell–cell adhesion and cell–matrix adhesion offrom 10−11 to 10−7 mol/L) for 6 days. (A) Morphological photo of cell–cell adhesion, (B)

hown. Values are representative of at least three biologically independent experiments wompared to controls.

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(ANOVA) followed by Tukey’s post hoc test. Comparisons between two groups wereevaluated using Student’s t-test. A value of p < 0.05 was considered statisticallysignificant.

3. Results

3.1. p,p′-DDT disrupts cell–cell adhesion and cell–matrixadhesion in HepG2 cells

To assess the effect of p,p′-DDT on cell adhesion of hepatomacells, HepG2 cells were treated with increased concentrationsof p,p′-DDT from 10−11 to 10−7 mol/L. As shown in Fig. 1A, thecell–cell adhesion was reduced after exposure to p,p′-DDT. Quan-titative analysis of cell–cell adhesion is represented in Fig. 1B withhistograms showing the standard error, indicating that attachedcells were significantly reduced to about 30% in 10−9 mol/L p,p′-DDT-treated cells compared with the vehicle control (P < 0.01).Simultaneously, cells had relatively quick adhesion with various


time periods in p,p′-DDT-treated group compared with control(P < 0.05) (Fig. 1C). At four different time points, relative cell–matrixadhesion ratios (normalized with respect to control) were 242, 254,172 and 204 in 10−9 mol/L p,p′-DDT-treated cells respectively and

HepG2 cells. Cell adhesion was investigated after HepG2 cells exposed to p,p′-DDTquantitative analysis of cell–cell adhesion, and (C) cell–matrix adhesion ratios areith similar results. Asterisks (*) indicate significant differences (*p < 0.05, **p < 0.01)

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Fig. 2. The stimulation of oxidative stress in p,p′-DDT-treated HepG2 cells. HepG2cells were exposed to p,p′-DDT (10−9 mol/L) for 6 days with or without NAC (5 mmol)co-treatment for the last 3 days, (A) ROS levels, (B) SOD activity, and (C) GSH contentwtc

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ere assessed as described in Section 2. The values are shown as means ± SD ofriplicate determinations. An asterisk (*) represents a significant difference fromontrols (*p < 0.05, **p < 0.01).

ell–matrix adhesion ratios in DMSO-treated cells were set to 100.reatment with 10−9 mol/L p,p′-DDT resulted in the biggest effectn cell adhesion (P < 0.01). Taken together, it suggested that p,p′-DT affected cell adhesion of HepG2 cells.

.2. Cell–cell adhesion and cell–matrix adhesion impacted by,p′-DDT are mediated by ROS not ER

To investigate the potential mechanism underlying the cell′


dhesion affected by p,p -DDT, we measured the changes in intra-ellular ROS levels, total GSH content and SOD activity. Notably,,p′-DDT exposure increased intracellular ROS levels (Fig. 2A),educed SOD activity (Fig. 2B) and GSH content (Fig. 2C), indicating


that p,p′-DDT activates the oxidative stress of HepG2 cells. Todemonstrate the functional role of ROS and ER in the adhesion ofp,p′-DDT-affected cells, NAC (ROS inhibitor) and ICI (ER inhibitor)were conducted in p,p′-DDT-exposed cells. Based upon our mor-phological findings (Fig. 3A) and quantitative analysis (Fig. 3B andC), we found p,p′-DDT affected cell adhesion depending on ROSrather not ER. Taken together, these findings demonstrate thatp,p′-DDT disrupts cell–cell adhesion and cell–matrix adhesion inHepG2 cells, probably via inducing the oxidative stress.

3.3. p,p′-DDT stimulated JAK/STAT3 signal pathway in HepG2cells

The initiation and progression of cell adhesion require transduc-tion of cell signals. It has been known that the JAK/STAT pathwayis closely related to the occurrence of liver cancer development.We therefore investigated whether the JAK/STAT3 signal pathwayplayed a role in the adhesion regulation of p,p′-DDT-exposed cells.As shown in Fig. 4A, compared to the control group, the expressionsof p-JAK, p-STAT3, and STAT3 were strikingly up-regulated in p,p′-DDT-exposed HepG2 cells. The right bars, which were grayscalescans of western blot lines, showed that the expressions of pro-teins were about 2.6–3.2 folds in 10−9 mol/L p,p′-DDT-treated cellswith respect to control cells. Concomitantly, we observed a nucleartranslocation of STAT3 into the nucleus of HepG2 cells treated withp,p′-DDT (Fig. 4B). It suggests that the JAK/STAT pathway plays animportant role in mediating p,p′-DDT-affected cell adhesion eventsof liver cancer cells.

3.4. p,p′-DDT-stimulated JAK/STAT3 signal pathway viaup-regulated ROS

The above experiments showed oxidative stress and theJAK/STAT3 signal pathway were both activated in HepG2 cells afterp,p′-DDT exposure. We wonder if there is the correlation betweenoxidative stress and the JAK/STAT3 pathway with p,p′-DDT treat-ment in HepG2 cells. Western blot was performed to determineJAK and STAT3 expressions after co-treating with NAC. Interest-ingly, supplement NAC reduced the boosted expression of p-JAK,p-STAT3, and total STAT3 (Fig. 5A). However, co-treatment with ICIhad no influence in the expressions of p-JAK, p-STAT3, and STAT3caused by p,p′-DDT treatment (Fig. 5B). The results indicate a newrole for the p,p′-DDT-inducible JAK/STAT3 signal pathway mediatedby ROS but not ER in HepG2 cells.

3.5. p,p′-DDT regulates adhesion molecules via activating theJAK/STAT3 signal pathway

To further understand the underlying mechanism of p,p′-DDT-induced cell adhesion, we investigated the expressions of keymolecules in the process of cell–cell adhesion (E-cadherin andN-cadherin) and cell–matrix adhesion (CD29) by quantitative real-time PCR (qPCR). Among these, E-cadherin was found to be moststrikingly down-regulated in p,p′-DDT-exposed cells compared tocontrol cells. N-cadherin was moderately up-regulated in thesecells. Moreover, CD29 was significantly increased approximately3.5 times after p,p′-DDT treatment for 6 days (Fig. 6A). Addition-ally, we observed the effect of inhibiting ROS or ER on expressionsof cell adhesion molecules. As shown in Fig. 6B, inhibition of ROS,but not ER, reversed the alteration of cell adhesion molecules inmRNA levels impacted by p,p′-DDT exposure. These data suggestedthat cell adhesion molecule expressions are affected in HepG2 cells


after p,p -DDT exposure, which is mediated by ROS but not ER.To better understand the mechanisms of JAK/STAT3 signal path-

way on HepG2 cells adhesion, we tested the effect of Ruxolitinib orWP1066, which is a specific JAK or STAT3 inhibitor respectively, on

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Fig. 3. Cell–cell adhesion and cell–matrix adhesion affected by p,p′-DDT are mediated by ROS not ER. After co-treatment with NAC or ICI, inhibiting ROS or ER, cell adhesionaffected by p,p′-DDT was observed. (A) Morphological images of cell–cell adhesion, (B) quantitative analysis of cell–cell adhesion, and (C) quantitative analysis of cell–matrixa dent es

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dhesion are shown. Values are representative of at least three biologically indepenignificant differences (*p < 0.05, **p < 0.01) compared to controls.

ell adhesion regulatory molecules altered by p,p′-DDT. As shownn Fig. 6C, the p,p′-DDT-induced p-JAK expression was decreased byuxolitinib co-treatment, and p-STAT3 was reduced by Ruxolitinib10 �M) or WP1066 (5 �M) co-treatment. Compared to p,p′-DDT-reatment alone, the mRNA levels of adhesion molecules altered by,p′-DDT were remarkably reversed by Ruxolitinib or WP1066 co-reatment, increasing E-cadherin, and decreasing N-cadherin alongith CD29 (Fig. 6D). Statistically significant alterations are note-orthy. Our data suggests that activation of the JAK/STAT3 pathway

s responsible for the alterations of adhesion molecules.

.6. p,p′-DDT stimulated JAK/STAT3 signal pathway and disruptedhe expressions of cell adhesion molecules in nude mice models

The above results indicate that low dose p,p′-DDT affected celldhesion of HepG2 cells through the JAK/STAT3 pathway medi-ted by oxidative stress in vitro. To validate the findings, an in vivoude mice assay was performed to further determine the adhesionapability in tumor tissues induced by p,p′-DDT. After exposure


o 5 nmol/kg p,p′-DDT for 7 weeks, we measured the JAK/STAT3athway in tumor tissues in response to p,p′-DDT treatment. Ashown in Fig. 7A, p,p′-DDT significantly increased p-JAK, p-STAT3,nd total STAT3 protein levels. At the same time, STAT3 protein

xperiments with similar results. Error bars represent the ±SD. Asterisks (*) indicate

accumulation in the nucleus was dramatically elevated in p,p′-DDT-treated tumors compared to vehicle treatment (Fig. 7B). Thedata suggests that the JAK/STAT3 pathway is activated in p,p′-DDT-treated tumors. Additionally, the mRNA levels of relevant celladhesion molecules in tumor tissues were examined, showing thatE-cadherin was inhibited, but N-cadherin as well as CD29 were ele-vated (Fig. 7C). These results coincide with the data obtained byin vitro experiments.

4. Discussion

Numerous epidemiological studies have suggested that DDTexposure is likely to contribute to the increase of liver cancer(McGlynn et al., 2006; Persson et al., 2012), and we previouslyreported the correlation between liver cancer and DDT exposurein vitro and in vivo (Jin et al., 2014), but the cell adhesion involvedin the progression of liver cancer and the molecular mechanismsunderlying the carcinogenic effects of DDT remains poorly under-stood. In view of p,p′-DDT’s possible risk for human health, the


cytotoxic effects of p,p′-DDT are of concern. In the present study,we further investigated the cell adhesion responses of p,p′-DDTto human liver cancer using via in vitro and in vivo models. Thestudy unveiled, for the first time, that p,p′-DDT exposure alters cell

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Please cite this article in press as: Jin, X., et al., The evaluation of p,p′-DDT exposure on cell adhesion of hepatocellular carcinoma.Toxicology (2014), http://dx.doi.org/10.1016/j.tox.2014.05.002


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Fig. 4. The activation of JAK/STAT3 signal pathway in p,p′-DDT-exposed HepG2 cells. (A) Western blots were applied to check relevant protein expressions of HepG2 cells afterp,p′-DDT treatment, including p-JAK, p-STAT3, and STAT3. (B) Expression of STAT3 in cytoplasm and nucleus of HepG2 cells was determined. The right bars were grayscalescans of western blot lines. The above blots and data are representative of at least three independent experiments with similar results. An asterisk (*) represents a significantdifference from controls (*p < 0.05, **p < 0.01).

Fig. 5. p,p′-DDT-stimulated the JAK/STAT3 signal pathway via up-regulated ROS. (A) After co-treatment with NAC, inhibiting ROS, western blots were carried out to checkrelevant protein expressions of HepG2 after p,p′-DDT (10−9 mol/L) treatment, including p-JAK, p-STAT3, and STAT3. (B) After co-treatment with ICI, inhibiting ER, proteinexpressions of p-JAK, p-STAT3, and STAT3 were determined in HepG2 cells. The right bars were grayscale scans of western blot lines. The above blots and data are representativeof at least three independent experiments with similar results. An asterisk (*) represents a significant difference from controls (*p < 0.05, **p < 0.01).

dx.doi.org/10.1016/j.tox.2014.05.002



Fig. 6. Cell adhesion molecules affected by p,p′-DDT were regulated by the JAK/STAT3 signal pathway. (A) Cell adhesion molecules mRNA levels were assessed by quantitativereal-time PCR after 6 days of p,p′-DDT treatment, including E-cadherin, N-cadherin, and CD29. (B) After co-treatment with NAC or ICI, inhibiting ROS or ER, relative mRNAexpression levels (normalized with respect to GAPDH) were determined and mRNA levels in DMSO-treated cells were set to 1. (C) Western blots were performed to checkr ) or WD cules

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elevant protein expressions of HepG2 cells after p,p′-DDT and Ruxolitinib (10 �MDT and Ruxolitinib or WP1066 co-treatment, mRNA levels of cell adhesion moleeterminations from three independent experiments. An asterisk (*) represents a s

dhesion of hepatocellular carcinoma. This process is caused by theepression of E-cadherin expression, and the enhancement of N-adherin as well as CD29. The alterations of cell adhesion moleculexpressions are due to the activation of the JAK/STAT3 pathwayediated by oxidative stress (Fig. 8).The present study implied that cell adhesion was affected

ith DDT treatment. The implied doses of p,p′-DDT were about.36 × 10−9–5.07 × 10−6 mol/L, which were similar to its concen-rations in human blood, (Röllin et al., 2009; Sholtz et al., 2011).


ur previous study in human hepatocarcinoma (HepG2) cells sug-ested that p,p′-DDT could act through the Wnt/�-catenin pathwaynd promote cell prolifeation (Jin et al., 2014). Nathalie Zucchini-ascal etal. (2012) found that DDT exposure promoted the epithelial

P1066 (5 �M) co-treatment, including p-JAK, p-STAT3, and STAT3. (D) After p,p′-in HepG2 cells were investigated. Error bars indicate the means ± SD of triplicateant difference from controls (*p < 0.05, **p < 0.01).

to mesenchymal transition (EMT) in primary cultured humanhepatocytes. Besides, many studies have demonstrated that pes-ticides can lead to the disruption of cell adhesion. For example,Endosulfan, an organochlorine pesticide, induces cell detachmentand promotes cell adhesion of HepG2 liver cells (Peyre et al.,2012). Chlorpyrifos (CPF), a widely used organophosphorus insec-ticide, disrupts multiple cellular pathways in neonatal forebrain,in particular cell adhesion, contributing to the developmental neu-rotoxicity potential of this pesticide (Ray et al., 2010). Parathion,


an organophosphorous pesticide, affects human breast cell adhe-sion changes via increasing the expression of related cell adhesionproteins (Calaf and Roy, 2008). Malathion, an organophosphorouspesticide, induces cell adhesion of cultured breast carcinoma cells

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Fig. 7. p,p′-DDT stimulated the JAK/STAT3 signal pathway and disrupted the expressions of cell adhesion molecules in nude mice models. HepG2 cells were injected intothe nude mice. After 3 days, one group of mice received intraperitoneal (i.p.) injections of p,p′-DDT (5 nmol/kg). Control mice received vehicle-diluted PBS. After 7 weeksp ove top vels ofa

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ost-injection, mice were killed. (A) Western blots were performed as mentioned abrotein in cytoplasm and nuclear were expressed in tumor tissues. (C) The mRNA lere representative of at least three independent experiments with similar results.

Cabello et al., 2003). Consistently, this studies showed that p,p′-DT disrupted cell adhesion of HepG2 cells, including the decreasef cell–cell adhesion and the promotion of cell–matrix adhesion.

Main molecular targets, which DDT exposure involves, includestrogen receptors (ERs) and reactive oxygen species (ROS) (Hardellt al., 2004; Radice et al., 2006; Tebourbi et al., 2011). However,,p′-DDT, which we concentrate on, is one main isomer of DDT; thether is o,p′-DDT. The binding ability of o,p′-DDT to ERs is 100-foldreater than that of p,p′-DDT (Kojima et al., 2004). Therefore, weocused on the ROS, which is generally involved in toxicological

echanisms of environmental contaminants (Monks et al., 1992).ased upon our morphological findings and quantitative analysis,


e found p,p′-DDT affected cell adhesion depending on ROS not ER.The study has shown here that p,p′-DDT activated the JAK/STAT3

athway which is mediated by oxidative stress. Consistent withur conclusions, Shen and Novak (1997) showed that DDT at

examine p-JAK, p-STAT3, and STAT3 protein levels of tumor tissues, and (B) STAT3 cell adhesion molecules in tumor tissues were examined. The above blots and data

physiologically relevant concentrations (i.e. 10 nM) elevated STAT1phosphorylation. The JAK/STAT pathway is activated in DDT-exposed mosquito Aedes aegypti (Behura et al., 2011). In addition,STAT3 is involved in the regulation of cellular adhesion, leading toincreased tumorigenic potential in nude mice. For example, inhibi-tion of STAT3 phosphorylation is responsible for the loss of cell–cellcontacts and spreading in human colorectal HCT8/S11 cancer cells(Rivat et al., 2004). Wooten et al. (2000) suggests that STAT3 mayplay an important role in mediating cytokine-dependent cell adhe-sion events in myeloid progenitor cells. ROS has shown to induceactivation of the JAK/STAT3 pathway (Cheng et al., 2013; Lee et al.,2013; Mantel et al., 2012; Zheng et al., 2010).


The present study showed that E-cadherin expression wasrepressed after p,p′-DDT exposure. It has been reported that E-cadherin, which plays an essential role in maintaining cell–celladhesion of epithelial cells, locates at the adherent junction and is a

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Fig. 8. Proposed mechanism of p,p′-DDT disrupts cell adhesion of hepatocellular carcinoma. Exposure of p,p′-DDT firstly activates ROS and stimulates the oxidative stress.Next, it promotes phosphorylation of JAK1. Afterwards, actived STAT3 enters the nucleus and binds to the transcription factor, which regulates expression of cell adhesionm pact

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olecules (E-cadherin, N-cadherin, and CD29). These adhesion molecules further im

egative regulator of cell–cell adhesion (Cavallaro and Christofori,004). The link between the loss of E-cadherin function and theccurrence of adhesion in liver disease is well documented. Forxample, decreased E-cadherin expression has been observed inpproximatively 40% of hepatocellular carcinoma samples (Yangt al., 2009). Liver fibrosis is accompanied by the loss of E-cadherin,hich promotes the process of adhesion. Loss of E-cadherin expres-

ion is a crucial step and fundamental event of adhesion in cancerrogression. In epithelial cells, E-cadherin is very important forompact association of the cells in epithelial sheets, and in thisapability, E-cadherin might function as a suppressor of inva-iveness and metastasis of epithelial tumors. The present resulthowed that p,p′-DDT elevated N-cadherin and CD29 expressions.-cadherin is one of junction proteins and promotes cell–cell adhe-

ion, which is usually over-expressed in cancer cells. CD29 (Integrineta-1) is an integrin unit associated with very late antigen recep-ors. It is the beta subunit of an integrin family of moleculesxpressed on diverse cell types which function as the major recep-ors for extracellular matrix and as cell–cell adhesion molecules18]. Increased evidences suggest that the inhibition of E-cadherinxpression is mediated by the JAK/STAT3 signaling pathway, andTAT3 has been identified as potentially down-regulateing E-


adherin in cancers (Yadav et al., 2011). Liang et al. (2013) proposedhat knockdown of STAT3 significantly increased E-cadherin, butecreased N-cadherin, indicating that STAT3 involved in the regu-

ation of E-cadherin and N-cadherin.

cell adhesion of hepatocellular carcinoma.

Compared with the control group, the expressions of p-JAK,p-STAT3, and STAT3 were strikingly up-regulated in p,p′-DDT-exposed HepG2 cells. However, it was not clear that the effectof p,p′-DDT on STAT3 was direct or indirect. We therefore testedthe effect of Ruxolitinib or WP1066, which is a specific JAKor STAT3 inhibitor respectively, on JAK/STAT3 pathway and celladhesion regulatory molecules altered by p,p′-DDT. Ruxolitinib(INCB018424, INC424) is highly effective in the clinical manage-ment of patients and is currently the only JAK inhibitor that caneffectively inhibit JAK1 and JAK2 with very low affinity for non-JAK targets (Verstovsek et al., 2010; Yadav et al., 2011). WP1066, aspecific and potent STAT3 inhibitor and a cell-permeable analog ofAG490, potently prevented the phosphorylation of STAT3 but hasno effect on the regulation of STAT3 mRNA or protein levels at earlytime-points after stimulation (Iwamaru et al., 2007). The resultssuggested that p,p′-DDT altered the expressions of cell adhesionregulatory molecules via both directly and indirectly activating theJAK/STAT3 pathway.

Exposure to p,p′-DDT from environmental sources remains seri-ous public health risks. Elucidating the effect of p,p′-DDT on celladhesion of liver cancer is therefore critical for understanding itsassociation risk. The results indicated that the presence of DDT


induced the majority of changes related to cell adhesion, anddemonstrated that DDT exposure can result in cancers with variouspathways. Such experiments could account for the enhanced car-cinogenic potential of DDT. The present investigation provides the

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0 X. Jin et al. / Toxico

olecular evidence that p,p′-DDT has an adverse impact on humanealth and contribute to liver cancer progression.

onflict of interest

The authors declare that there are no conflicts of interest.

ransparency document

The Transparency document associated with this article can beound in the online version.

cknowledgments

This work was supported by the National Natural Sciencesoundation of China (Nos. 31271516, 21207084, 31201072),esearch Fund for the Doctoral Program of Higher Educationf China (20111401110011), China Postdoctoral Science Foun-ation (2012M521178), Natural Science Foundation of Shanxi2009021035-2), and Research Fund for the Doctoral Program ofigher Education of China (20111401110011).

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