Toxicology Letters 225 (2014) 158–166
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Dichlorodiphenyltrichloroethane exposure induces the growth of hepatocellular carcinoma via Wnt/-catenin pathway Xiao-Ting Jin a,1 , Li Song a,b,1 , Jun-Yu Zhao a , Zhuo-Yu Li a,c,∗ , Mei-Rong Zhao d , Wei-Ping Liu b a Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of National Ministry of Education, Shanxi University, Taiyuan 030006, China b MOE Key Lab of Environmental Remediation and Ecosystem Health, College of Environmental and Resource Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, China c College of Life Science, Zhejiang Chinese Medical University, Hangzhou 310053, China d Research Center of Environmental Science, Zhejiang University of Technology, Hangzhou 310032, China
h i g h l i g h t s
g r a p h i c a l
• Low doses p,p -DDT exposure pro-
p -DDT exposed to hepatocellular carcinoma, first activates ROS and stimulates the oxidative stress. Next, it promotes phosphorylation of GSK3at Ser 9. Afterwards, active -catenin enters the nucleus and binds to the transcription factor TCF, which regulates expression of its downstream target genes (c-Myc and CyclinD1). These downstream targets are associated with proliferation, thus promoting the growth of hepatocellular carcinoma.
motes the proliferation of HepG2 cells. • Both oxidative stress and Wnt/catenin pathway act as pivotal player in p,p -DDT-induced proliferation of HepG2 cells. • The stimulation of Wnt/-catenin pathway is mediated by oxidative stress. • p,p -DDT treatment increased the growth of tumor in nude mice.
a r t i c l e
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Article history: Received 30 October 2013 Received in revised form 29 November 2013 Accepted 6 December 2013 Available online 17 December 2013 Keywords: p,p -DDT Hepatocellular carcinoma Proliferation Wnt/-catenin pathway ROS
a b s t r a c t
a b s t r a c t Dichlorodiphenyltrichloroethane (DDT) is a persistent organic pollutant, involved in the progression of many cancers, including liver cancer. However, the underlying mechanism(s) of DDT, especially how low doses DDT cause liver cancer, is poorly understood. In this study, we evaluated the impact of p,p DDT on the growth of hepatocellular carcinoma using both in vitro and in vivo models. The present data indicated that the proliferation of HepG2 cells was strikingly promoted after exposed to p,p -DDT for 4 days. In addition, reactive oxygen species (ROS) content was significantly elevated, accompanied with inhibitions of ␥-glutamylcysteine synthetase (␥-GCS) and superoxide dismutase (SOD) activities. Interestingly, the levels of -catenin and its downstream target genes (c-Myc and CyclinD1) were significantly up-regulated, and co-treatment of NAC, the ROS inhibitor, inhibited these over-expressed proteins. Moreover, the p,p -DDT-stimulated proliferation of HepG2 cells could be reversed after NAC or -catenin siRNA co-treatment. Likewise, p,p -DDT treatment increased the growth of tumor in nude mice, stimulated oxidative stress and Wnt/-catenin pathway. Our study indicates that low doses p,p -DDT exposure
∗ Corresponding author at: Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of National Ministry of Education, Shanxi University, Taiyuan 030006, PR China. Tel.: +86 351 7018268; fax: +86 351 7018268. E-mail address: [email protected] (Z.-Y. Li). 1 These authors contributed equally to this work. 0378-4274/$ – see front matter. Crown Copyright © 2013 Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2013.12.006
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promote the growth of hepatocellular carcinoma via Wnt/-catenin pathway which is activated by oxidative stress. The finding suggests an association between low dose DDT exposure and liver cancer growth. Crown Copyright © 2013 Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Hepatocellular carcinoma (HCC), the major primary malignant tumor of liver, is the sixth most common cancer and the thirdleading cause of cancer-related deaths worldwide (Finn, 2013; Yang et al., 2005). Epidemiological investigations have shown that main causes of liver cancer are viral hepatitis, alcohol-induced cirrhosis, diet, and other environmental factors. It is noteworthy that the diet and environmental factors are more closely associated with the incidence of liver cancer (Knowles et al., 1980; Shyu et al., 2013). Thus, carcinogens present in diet and the environment have been an increasing concern (ManningáSandanger, 2007; Zhang et al., 2012). DDT, dichlorodiphenyltrichloroethane, is one of the carcinogens and persistent organic pollutants present in diet and the environment. It was originally synthesized in 1874 and widely used in agriculture as an effective insecticide (Beard, 2006). However, many studies showed that DDT was hazardous to human health causing many developed countries to ban the insecticide by the early 1970s, with China prohibiting its use in 1983 (Qiu et al., 2005). However, due to its long-term existence, lipophilicity, difficult degradation, and bio-accumulative properties through direct contact and food chains, DDT still persists in environment for decades after it bans, and its residues cause a few health problems in humans, such as cancer, and endocrine and immunological disorders (Glynn et al., 2007; Mrema et al., 2013; Qiu et al., 2005). Hence, research of the link between DDT and human health, especially its link with cancer, are drawing more and more people’s attention. Both animal and human studies have suggested an association between DDT and liver cancer. For example, laboratory animals exposed to DDT exhibit a dose-related increase in liver tumors and rodents exposed to p,p -DDT increase the risk of HCC (Beard, 2006; Persson et al., 2012). Human exposure to DDT have also been reported to elevate the rate of liver cancer (Cocco et al., 1997; Figàtalamanca et al., 1993). Moreover, a recent ecological study has reported a statistically significant correlation between levels of DDE (a metabolite of DDT) in adipose tissue and mortality rates of liver cancer (Cocco et al., 2000). McGlynn KA et al. have also reported that there is a significant correlation between DDT serum concentration and incidence of liver cancer in humans (McGlynn et al., 2006). In view of DDT possibly increasing the risk for hepatocellular carcinoma, the cytotoxic effects of DDT on hepatocellular carcinoma are of concern. Numerous epidemiological studies have suggested that DDT exposure may increase the risk of liver cancer, but the molecular mechanisms for how DDT promotes the progression of liver cancer remains unclear. The pathogenesis of hepatocellular carcinoma is complicated, in which cell signaling pathways play a vital. One key signaling pathway coordinating liver cancer is the Wnt/-catenin signaling pathway (Monga, 2011; Thompson and Monga, 2007). The Wnt/-catenin signaling pathway is commonly deregulated in hepatocellular carcinoma (HCC) and aberrations in this pathway have been established to be critical contributors toward hepatocarcinogenesis (Lee et al., 2005; Takigawa and Brown, 2008). In the absence of Wnt ligands, Wnt/-catenin signaling pathway is not activated. The serine/threonine kinase glycogen synthase kinase-3 (GSK3) phosphorylates -catenin, targeting it to the proteasome for degradation. It has been shown that -catenin becomes stable in the cytosol followed by its eventual nuclear translocation when the pathway is stimulated. -catenin binds to transcription factor TCF, then regulates target gene transcriptions, which affect cell fate
determination and cancer development (Chien et al., 2009; Moon et al., 2004). Main molecular targets, which DDT exposure involves, include estrogen receptors (ERs) and reactive oxygen species (ROS) (Hardell et al., 2004; Radice et al., 2006; Tebourbi et al., 2011). However, p,p -DDT, which we concentrate on, is one main isomer of DDT; the other is o,p -DDT. The binding ability of o,p -DDT to ERs is 100fold greater than that of p,p -DDT (Kojima et al., 2004). Therefore, we focused on the ROS, which is generally involved in toxicological mechanisms of environmental contaminants (Kojima et al., 2004). In the present study, we aim to elucidate the mechanism of p,p DDT action on the growth of hepatocellular carcinoma using both in vitro and in vivo models. In conclusion, our study unveils for the first time that p,p -DDT exposure stimulates proliferation of hepatocellular carcinoma, leading to the progression of hepatocellular carcinoma. These effects are due to p,p -DDT’s ability to initiate the Wnt/-catenin signaling pathway mediated by oxidative stress. The present investigation provides the molecular evidences that persistent pesticides could have an adverse impact on human health and contribute to liver cancer development. 2. Materials and methods 2.1. Antibodies and agents Antibodies for c-Myc and CyclinD1 were purchased from Bioworld, p-GSK3 (Ser 9) was purchased from Cell Signaling Technology, ki-67 was purchased from ZSGB-BIO and ␣-tubulin was purchased from Sigma (St. Louis, MO, USA). Antibodies for -catenin and PCNA were obtained from Abmart (USA). p,p DDT (Sigma) was dissolved into dimethyl sulfoxide (DMSO) as stock solutions. The equal concentration of DMSO was added to medium for the control cells. N-Acetyl-L-cysteine (NAC, the scavenger of ROS) and 2 ,7 -dichlorofluorescein diacetate (DCFH-DA) were obtained from Beyotime Biotechnology. 2.2. Cell culture and treatments Human hepatoma cells (HepG2) and human normal liver cells (HL-7702) were maintained in RPMI-1640 medium (HyClone) supplemented with 10% FBS (Boster), 1%penicillin/streptomycin (Solarbio) at 37 ◦ C in a 5% CO2 humidified tissue culture incubator. To observe the toxicity of p,p -DDT in HepG2 cells, cells were exposed to p,p -DDT at different doses (from 10−12 to 10−7 mol/L) over a 4 day period. Cells with the treatments were then assayed for cellular viability assay. 2.3. Cell viability assay To investigate viability of HepG2 and HL-7702 cells, the MTT assay was performed. 100 L of HepG2 or HL-7702 cells in suspension (1 × 103 cells) were briefly plated in a 96-well plate. After treatment with p,p -DDT for 4 days, viable cells were stained with 20 L MTT. The medium was then removed, and formazan crystals produced in the wells were dissolved by addition of dimethyl sulfoxide (DMSO). Absorbance was measured at 490 nm using a microplate reader. Cell viability was defined relative to untreated control [i.e. viability (% control) = 100 × (absorbance of treated sample − absorbance of control/absorbance of control)].
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2.4. Nude mice assay All animal experiments were approved by the Committee of Animal Care at Chinese Institute for Radiation Protection. HepG2 cells were washed twice and resuspended in physiological saline at a concentration of 5 × 107 cells/mL. A 200 L cell suspension of HepG2 was then injected subcutaneously into the left armpit of SPF-free male BALB/c-nu mice. After three days, one group of mice received intraperitoneal injections of p,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. Mice were weighed and tumor diameters were checked every 7 days. Tumor volume was calculated according to 0.4ab2 /2 (a > b; a = maximum length, b = maximum width).Tumor specimens were collected at 7 weeks after injections and split. Three independent experiments were performed and yielded similar results. 2.5. Western blot analysis Cells extracted proteins were resolved by 10% SDS-PAGE and transferred onto nitrocellulose membranes for Western blotting. The blots were blocked for 1 h in PBS containing 5% non-fat dry milk (w/v) and incubated at 4 ◦ C overnight, then probed with antibody for 1 h at room temperature or overnight at 4 ◦ C. After washing, membranes were incubated at 37 ◦ C for 1 h with the appropriate horseradish peroxidase-conjugated secondary antibody (diluted at 1:1000, Invitrogen). Protein loading was controlled by probing the membranes for ␣-tubulin protein. Immune-reactive proteins were detected using ECL western blotting detection system. For measurement of -catenin, the cytoplasmic protein and the nuclear protein were extracted according to the instructions of the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotech Inc., Nantong, China). 2.6. RNA interference The small interference RNA (siRNA) targeting -catenin and non-silencing siRNA were synthesized by Genepharma (Shanghai, China). About 1 × 106 cells grown in 6-well plates were transfected with 100 nM siRNA using lipofectamine 2000. After transfection for 4 days, cells were lysed. The lysis was used for western blot analysis.
disruption using ultrasound equipment. After being centrifuged at 6000 rpm for 10 min, the supernatants were used to measure enzyme activities. The data were normalized to protein content. 2.9. Immuno-histochemical analysis Tumors were fixed in 10% formalin over 24 h. The tissues were dehydrated and then embedded in paraffin wax. The sections were stained with antibody to Ki-67, -catenin, c-Myc and CyclinD1. After washing, these sections were stained with horseradish peroxidase-conjugated secondary antibody (ZSGB-BIO, China) and then incubated with streptavidin–horseradish peroxidase complex. The sections finally were stained with diaminobenzidine (DAB) and counterstained with hematoxylin. 2.10. Statistical methods Statistical analysis was carried out using the SPSS software program. Data, derived from three or four independent experiments, were presented as the mean ± SD. Differences among groups were tested by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. Comparisons between two groups were evaluated using Student’s t-test. A value of p < 0.05 was considered statistically significant. 3. Results 3.1. Low doses of p,p -DDT exposure promote the proliferation of HepG2 cells We used a MTT assay to measure the cell viability after treatment with p,p -DDT from 10−12 to 10−7 mol/L for 4 days. Compared with the vehicle control (DMSO, 0.1%), cell viabilities, observed after 4 days exposure at concentrations of 10−12 , 10−11 , 10−10 , 10−9 , 10−8 and 10−7 mol/L, were 15.26%, 33.14%, 55.31%, 66.83%, 49.67% and 17.89% respectively (Fig. 1). These results indicate that p,p -DDT promotes the proliferation of HepG2 cells. However, p,p DDT displayed a negligible effect on HL-7702 cell proliferation. The doses of p,p -DDT, measured by its concentrations in human blood, were about 5.36 × 10−9 to 5.07 × 10−6 mol/L (Röllin et al., 2009; Sholtz et al., 2011). Treatment with 10−9 mol/L p,p -DDT resulted
Targeting -catenin (5 -CAGUUGUGGUUAAGCUCUUdTdT-3 ). Non-silencing siRNA (5 -TTCTCCGAACGTGTCACGT-3 ). 2.7. Measurement of ROS generation DCFH-DA is a cell-permeable, nonfluorescent probe that is cleaved by intracellular esterases and turns into a highly fluorescent dichlorofluorescein upon reaction with H2 O2 . After treatment with p,p -DDT (10−9 mol/L) with or without NAC(1 mmol) for 4 days, the cells were stained with 10 mol/L DCFH-DA for 30 min at 37 ◦ C. H2 O2 generation was determined by dichlorofluorescein fluorescence. Cells were collected and the fluorescence intensity in the cells was measured using a fluorescence microplate reader (Thermo Scientific varioskan flash) with excitation 488 nm and emission 525 nm. 2.8. Determinations of oxidative stress-related parameters ␥-GCS and SOD activities as well as GSH content were determined in cell and tissues using a commercial determination kit (Nanjing Jiancheng Bioenginneering Institute). Cells were plated onto 6-well dishes (1 × 106 cells/well) and were exposed to p,p DDT (10−9 mol/L) with or without NAC (1 mmol) for 4 days. Scraped cells were dissolved in physiological saline, followed by cell
Fig. 1. p,p -DDT exposure promotes the proliferation of HepG2 cells and has no significant effect on HL7702. Cells viability was measured using a MTT assay after HepG2 and HL7702 cells exposed to p,p -DDT (from 10−12 to 10−7 mol/L) for 4 days. Values were representative of at least three biologically independent experiments with similar results. Error bars represent the SD. Asterisks (*) indicate significant differences (*p < 0.05, **p < 0.01) compared to controls.
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Fig. 2. Oxidative stress plays an important role in p,p -DDT-mediated proliferation. HepG2 cells were treated with 0.1% DMSO, 10−9 mol/L p,p -DDT, and/or 1 mmol NAC for 4 days, (A) ROS levels, (B) SOD activity, (C) ␥-GCS activity, and (D) cell viability were assessed as described in Section 2. The values were showed as means ± SD of triplicate determinations. An asterisk (*) represents a significant difference from controls (*p < 0.05, **p < 0.01).
in the biggest increase in cell viability (about 66.83%). Therefore, 10−9 mol/L was selected as exposure concentration of p,p -DDT to test its mechanism on the proliferation of HepG2 cells. 3.2. Oxidative stress acts as a pivotal player in p,p -DDT-induced proliferation Because the main target of p,p -DDT was ROS, as stated in Section 1, we hypothesized that production of ROS by p,p -DDT may promote the proliferation of HepG2 cells. We found that the level of ROS was elevated in p,p -DDT stimulated cells (Fig. 2A). ROS is one of the most important components in oxidative stress. Thus, we further investigated other indexes of oxidative stress: SOD and ␥-GCS, which are important antioxidant enzymes. As shown in Fig. 2B and C, SOD and ␥-GCS activities were significantly decreased after p,p DDT exposure, demonstrating that p,p -DDT induced the oxidative stress of HepG2 cells. To determine the role of oxidative stress in p,p -DDT-induced proliferation, we selected NAC, an ROS inhibitor, to reduce oxidative stress, and then accessed the cell viability. We found that co-treatment of NAC (1 mmol) with p,p -DDT decreased ROS content as well as elevated SOD and ␥-GCS activities, indicating that oxidative stress was inhibited. Next, we measured the cell viability and found that compared with p,p -DDT exposure alone, cell viability was reduced with NAC co-treatment (Fig. 2D). These results indicate that oxidative stress play an important role in the stimulation of proliferation in HepG2 cells induced by p,p -DDT. 3.3. p,p -DDT stimulates Wnt/ˇ-catenin pathway mediated by oxidative stress Previous studies showed that the Wnt/-catenin signaling pathway was abnormally activated in hepatic carcinoma (Zeng et al.,
2007; Zucman-Rossi et al., 2006). Hence, we took one further step to explore the relationship between the Wnt/-catenin pathway and p,p -DDT-mediated proliferation. As shown in Fig. 3A, p,p -DDT induced the over-expression of -catenin and p-GSK3 (Ser 9) proteins. Furthermore, the protein levels of downstream CyclinD1 and c-Myc were also strongly accumulated. These results reflect that Wnt/-catenin signaling pathway is activated by p,p -DDT. However, p,p -DDT did not stimulated Wnt/-catenin signaling pathway in HL7702 cells. A hallmark event of activated Wnt/-catenin signaling is the nucleus input of -catenin (Polakis, 2000). Notably, p,p -DDT exposure caused accumulation of -catenin protein in the nucleu and cytoplasm compared to the vehicle treatment (Fig. 3B). Over the past decades, evidence has shown that the Wnt/catenin pathway could be stimulated through the induction of ROS (Bowerman, 2005; Essers et al., 2005). The above data illustrates that both ROS and Wnt/-catenin pathway are involved in p,p DDT-induced proliferation in HepG2 cells. Hence, we speculated that Wnt/-catenin pathway activated by p,p -DDT was mediated by ROS. To verify this hypothesis, HepG2 cells were treated with 10−9 mol/L p,p -DDT and/or 1 mmol NAC for 4 days. As shown in Fig. 3A, NAC dramatically disrupted accumulations of -catenin, pGSK3 (Ser 9), and downstream CyclinD1, as well as c-Myc protein levels induced by p,p -DDT. Consistently the increase of -catenin in the nucleu and cytoplasm was disrupted by NAC co-treatment (Fig. 3B). The results demonstrate that the Wnt/-catenin pathway activated by p,p -DDT is mediated by ROS. To further examine the role of Wnt/-catenin signaling, -catenin siRNA was performed to reduce -catenin expression and thereby inhibited Wnt/-catenin pathway in HepG2 cells (Fig. 4A). Fig. 4B showed that -catenin siRNA inhibited the effect of p,p -DDT on proliferation of HepG2, similar to the study that shown proliferation of liver cancer cells was associated with the Wnt/-catenin pathway (Zeng et al., 2007).
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Fig. 3. p,p -DDT exposure activates the Wnt/-catenin pathway mediated by oxidative stress. (A) Western blots were applied to check relevant protein expressions of HepG2 and HL7702 cells after p,p -DDT treatment alone or NAC co-treatment, including -catenin, p-GSK and downstream target genes (c-Myc and CyclinD1). (B) Expression of -catenin in cytoplasm and nucleus of HepG2 cells was determined. The above blots and data were representative of at least three independent experiments with similar results.
Fig. 4. Wnt/-catenin pathway plays an vital role in p,p -DDT-mediated proliferation. (A) HepG2 cells were transfected with -catenin siRNA and treated with p,p -DDT (10−9 mol/L) for 4 days and (B) cell viability of HepG2 cells were examined. The above blots and data were representative of at least three independent experiments with similar results.
Fig. 5. p,p -DDT increases the growth of tumor in nude mice model. HepG2 cells were injected into the 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 weeks post-injection, mice were killed. (A) Tumor volume, (B) tumor size, and (C) the average weight of tumor were determined. Value shown was given as the mean ± SD of 5 animals in each group. An asterisk (*) indicates that the data are significantly different comparing with control mice (*p < 0.05, **p < 0.01).
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3.4. p,p -DDT promotes tumor growth in nude mice models The above results indicate that low dose p,p -DDT stimulated proliferation of HepG2 cells through the Wnt/-catenin pathway mediated by oxidative stress in vitro. To validate our findings, in vivo nude mice assay was performed to further determine the proliferative capability in tumor tissues induced by p,p -DDT. We measured tumor growth for 7 weeks after exposure to 5 nmol/kg p,p -DDT (Fig. 5B). Tumor volume and weight in p,p -DDT-treated group were notably increased compared with control tumors (Fig. 5A and C). This data indicates that the p,p -DDT markedly elevated the formation of tumors. 3.5. Oxidative stress mediates the stimulation of Wnt/ˇ-catenin pathway by p,p -DDT in nude mice model Oxidative stress, which was evaluated by measuring SOD activity, ␥-GCS activity, and GSH content in tumor tissues, were further analyzed after exposure to p,p -DDT for 7 weeks. As shown in Fig. 6A and B, significant decreases in SOD activity along with ␥-GCS activity were found in p,p -DDT-treated tumors. We also found that GSH content was attenuated in p,p -DDT-treated tumors (Fig. 6C). These Fig. 7. Wnt/-catenin pathway is activated by p,p -DDT exposure in nude mice model. (A) Western blots were performed as mentioned above to examine -catenin protein levels of tumor tissues in cytoplasm and nuclear and (B) -catenin and c-Myc protein were expressed in tumor tissues. The above blots and data were representative of at least three independent experiments with similar results.
Fig. 6. Oxidative stress is stimulated by p,p -DDT exposure in nude mice model. The oxidative stress-related parameters in tumor tissues, including (A) SOD activity, (B) ␥-GCS activity, and (C) GSH levels, were determined as described in Section 2. An asterisk (*) indicates that the data are statistically significantly different from controls (*p < 0.05, **p < 0.01).
Fig. 8. Immunohistochemistry analysis of the expression of -catenin, c-Myc, CyclinD1, and Ki-67 in liver cancer tissues by p,p -DDT-treatment or without. Matched sections of control (left panel) and p,p -DDT-treated tumors were stained with -catenin, c-Myc, CyclinD1, and Ki-67. There were significant differences between control and p,p -DDT-treated group. Magnification ×40.
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Fig. 9. Proposed mechanism of p,p -DDT-promoted progression of hepatocellular carcinoma. p,p -DDT exposed to hepatocellular carcinoma, first activates ROS and stimulates the oxidative stress. Next, it promotes phosphorylation of GSK3 at Ser 9. Afterwards, active -catenin enters the nucleus and binds to the transcription factor TCF, which regulates expression of its downstream target genes (c-Myc and CyclinD1). These downstream targets are associated with proliferation, thus promoting the growth of hepatocellular carcinoma.
alterations of oxidative stress-related parameters confirm that p,p DDT exposure elicited a significant stimulation of oxidative stress. We also measured -catenin protein levels in tumor tissues in response to p,p -DDT treatment. As shown in Fig. 7A, -catenin accumulation in the nucleu was dramatically elevated in p,p -DDTtreated tumors compared with vehicle treatment. At the same time, p,p -DDT significantly increased -catenin and c-Myc protein levels (Fig. 7B). The data suggests that the Wnt/-catenin pathway is activated in p,p -DDT-treated tumors. These results coincide with the data obtained in our in vitro experiments. Furthermore, histological examination revealed that p,p -DDT-treated tumors had a histological appearance with massive grown, as indexed by the significant increase of -catenin, c-Myc, CyclinD1, and Ki-67 (Fig. 8). 4. Discussion Hepatocellular carcinoma is the third most common malignancy worldwide and has a poor long-term survival rate (Jemal, 2011). p,p -DDT is a persistent organic pollutants (POPs), which are hazardous chemicals present in our food chain that have been internationally regulated to protect public health. Although recent studies have highlighted an unexpected implication of POPs in the development of hepatocellular carcinoma (Arrebola et al., 2012; Jemal, 2011; Ruzzin, 2012; Zhao et al., 2011), the mechanisms underlying how p,p -DDT causes liver cancer remains elusive. Elucidation of these mechanisms is a major goal since it could lead to the elimination of p,p -DDT induced liver cancer. Accumulated studies have shown that Wnt/-catenin activation plays important roles in a variety of cancers, including
hepatocallular carcinoma (Monga, 2011; Takigawa and Brown, 2008). Notably, Zhang et al. recently identified a role of -catenin as a “potential” tumor suppressor. This study reported a paradoxical rise in the susceptibility of -catenin knockout mice to DEN-induced carcinogenesis. The data indicated PDGFR␣ was upregulated with -catenin KO livers, which enhanced the tumorigenesis through PDGFR␣/PIK3CA pathway. This study suggests an addition important role of -catenin in maintaining redox homeostasis in the liver (Zhang et al., 2010). In this research, we indicated a mechanism for the p,p -DDT-promoted growth of hepatocellular carcinoma. p,p -DDT exposed hepatocellular carcinoma, first activates ROS and stimulates the oxidative stress. It then promotes phosphorylation of GSK3 at Ser 9. Active -catenin was accumulates in the cytoplasm and enters the nucleus where it binds to the transcription factor TCF. Binding to TCF allows regulation of the expressions of its downstream target genes (c-Myc and CyclinD1), which are associated with proliferation, thus promoting progression of hepatocellular carcinoma (Fig. 9). These data, in conjunction with published reports, elucidate a direct interaction between the oxidative stress and Wnt/-catenin pathway in the development of liver cancer induced by p,p -DDT (Chen et al., 2010; Wang et al., 2012). The results from both in vitro and in vivo models implicate that p,p -DDT markedly induced proliferation of hepatocellular carcinoma. In contrast to the proliferation observed with low doses, high-dose of p,p -DDT exposure resulted in apoptosis of liver cancer cells (Buchmann et al., 1999). Previous studies have reported that DDT can lead to the hepatocellular carcinoma, which coincides with our results (Arrebola et al., 2012).
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The mechanism of proliferation stimulated by DDT in liver cancer has been reported in previous studies. For example, Kazantseva et al. have recently determined that DDT treatment may result in cell cycle progression and apoptosis inhibition in mouse livers, leading to hepatocarcinogenesis (Kazantseva et al., 2013). In addition, Harada et al. have reported that oxidative stress could be a key factor in hepatocarcinogenesis by DDT exposure (Harada et al., 2003). Similarly, our present study also found that oxidative stress was activated after p,p -DDT exposure. p,p -DDT elevated the ROS content along with decrease of SOD activity. And it also decreased the activity of ␥-GCS with a concomitant reduction in GSH content, thus reducing the cell’s ability to scavenge oxygen free radicals. These alterations of enzymes were consistent with previous reports (Lin and Yang, 2007; Walsh et al., 2001; Yoshida et al., 1995). Moreover, NAC reduced the proliferation induced by p,p -DDT. Results presented here strongly suggested that p,p -DDT-stimulated proliferation of HepG2 cells is ROS dependent. Moreover, accumulation of nuclear and cellular -catenin has been observed in 18–67% of HCC tumors. It has also been observed that the Wnt/-catenin pathway is involved in cancer as an early regulatory factor, which is prematurely activated by ROS (Devereux et al., 2001; Korswagen, 2006). Kazantseva et al. reported that DDT treatment could induce liver responses through CAR- and ER␣ activation, and it was independent of Wnt signaling pathway in mouse livers (Kazantseva et al., 2013). Notably, our results showed that Wnt signaling and -catenin were not detected in human normal liver cell HL7702 after exposing to DDT for 4 days in our studies (Fig. 3A). By contrast, it showed significant changes in live cancer cell, HepG2. In addition, the differences of applied doses of DDT and exposure time to samples need to be concerned as well. Our results indicate, for the first time, that p,p -DDT increases ROS generation, which then stimulates the Wnt/-catenin pathway in HepG2 cells. We also demonstrate that p,p -DDT elevates the expression of -catenin and that its effect on proliferation is inhibited by -catenin siRNA. These results support a role for -catenin in promotion of proliferation and indicates a mechanism by which p,p -DDT increases proliferation. Even though previous studies have shown that p,p -DDT promoted proliferation in liver cancer, the mechanism(s) of p,p -DDT, especially how low dose of p,p -DDT causes liver cancer, is poorly unknown. Taken together, this paper unveils that p,p -DDT stimulates the proliferation of liver cancer cells via oxidative stress and Wnt/-catenin signaling pathway. Thus, p,p -DDT exposure leads to the deterioration of hepatocellular carcinoma. The present data provides a molecular mechanism regarding p,p -DDT’s distinct effects on hepatocellular carcinoma. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments This work was supported by the National Natural Sciences Foundation of China (Nos. 31271516, 21207084, 31201072), Research Fund for the Doctoral Program of Higher Education of China (20111401110011), China Postdoctoral Science Foundation (2012M521178), Natural Science Foundation of Shanxi (2009021035-2), and Research Fund for the Doctoral Program of Higher Education of China (20111401110011).We thank Dr. Enmin Zou of the Department of Biological Sciences of Nicholls State University, USA, for his help in improving the English of this manuscript.
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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Dichlorodiphenyltrichloroethane exposure induces the growth of hepatocellular carcinoma via Wnt/-catenin pathway Xiao-Ting Jin a,1 , Li Song a,b,1 , Jun-Yu Zhao a , Zhuo-Yu Li a,c,∗ , Mei-Rong Zhao d , Wei-Ping Liu b a Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of National Ministry of Education, Shanxi University, Taiyuan 030006, China b MOE Key Lab of Environmental Remediation and Ecosystem Health, College of Environmental and Resource Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, China c College of Life Science, Zhejiang Chinese Medical University, Hangzhou 310053, China d Research Center of Environmental Science, Zhejiang University of Technology, Hangzhou 310032, China
h i g h l i g h t s
g r a p h i c a l
• Low doses p,p -DDT exposure pro-
p -DDT exposed to hepatocellular carcinoma, first activates ROS and stimulates the oxidative stress. Next, it promotes phosphorylation of GSK3at Ser 9. Afterwards, active -catenin enters the nucleus and binds to the transcription factor TCF, which regulates expression of its downstream target genes (c-Myc and CyclinD1). These downstream targets are associated with proliferation, thus promoting the growth of hepatocellular carcinoma.
motes the proliferation of HepG2 cells. • Both oxidative stress and Wnt/catenin pathway act as pivotal player in p,p -DDT-induced proliferation of HepG2 cells. • The stimulation of Wnt/-catenin pathway is mediated by oxidative stress. • p,p -DDT treatment increased the growth of tumor in nude mice.
a r t i c l e
i n f o
Article history: Received 30 October 2013 Received in revised form 29 November 2013 Accepted 6 December 2013 Available online 17 December 2013 Keywords: p,p -DDT Hepatocellular carcinoma Proliferation Wnt/-catenin pathway ROS
a b s t r a c t
a b s t r a c t Dichlorodiphenyltrichloroethane (DDT) is a persistent organic pollutant, involved in the progression of many cancers, including liver cancer. However, the underlying mechanism(s) of DDT, especially how low doses DDT cause liver cancer, is poorly understood. In this study, we evaluated the impact of p,p DDT on the growth of hepatocellular carcinoma using both in vitro and in vivo models. The present data indicated that the proliferation of HepG2 cells was strikingly promoted after exposed to p,p -DDT for 4 days. In addition, reactive oxygen species (ROS) content was significantly elevated, accompanied with inhibitions of ␥-glutamylcysteine synthetase (␥-GCS) and superoxide dismutase (SOD) activities. Interestingly, the levels of -catenin and its downstream target genes (c-Myc and CyclinD1) were significantly up-regulated, and co-treatment of NAC, the ROS inhibitor, inhibited these over-expressed proteins. Moreover, the p,p -DDT-stimulated proliferation of HepG2 cells could be reversed after NAC or -catenin siRNA co-treatment. Likewise, p,p -DDT treatment increased the growth of tumor in nude mice, stimulated oxidative stress and Wnt/-catenin pathway. Our study indicates that low doses p,p -DDT exposure
∗ Corresponding author at: Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of National Ministry of Education, Shanxi University, Taiyuan 030006, PR China. Tel.: +86 351 7018268; fax: +86 351 7018268. E-mail address: [email protected] (Z.-Y. Li). 1 These authors contributed equally to this work. 0378-4274/$ – see front matter. Crown Copyright © 2013 Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2013.12.006
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promote the growth of hepatocellular carcinoma via Wnt/-catenin pathway which is activated by oxidative stress. The finding suggests an association between low dose DDT exposure and liver cancer growth. Crown Copyright © 2013 Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Hepatocellular carcinoma (HCC), the major primary malignant tumor of liver, is the sixth most common cancer and the thirdleading cause of cancer-related deaths worldwide (Finn, 2013; Yang et al., 2005). Epidemiological investigations have shown that main causes of liver cancer are viral hepatitis, alcohol-induced cirrhosis, diet, and other environmental factors. It is noteworthy that the diet and environmental factors are more closely associated with the incidence of liver cancer (Knowles et al., 1980; Shyu et al., 2013). Thus, carcinogens present in diet and the environment have been an increasing concern (ManningáSandanger, 2007; Zhang et al., 2012). DDT, dichlorodiphenyltrichloroethane, is one of the carcinogens and persistent organic pollutants present in diet and the environment. It was originally synthesized in 1874 and widely used in agriculture as an effective insecticide (Beard, 2006). However, many studies showed that DDT was hazardous to human health causing many developed countries to ban the insecticide by the early 1970s, with China prohibiting its use in 1983 (Qiu et al., 2005). However, due to its long-term existence, lipophilicity, difficult degradation, and bio-accumulative properties through direct contact and food chains, DDT still persists in environment for decades after it bans, and its residues cause a few health problems in humans, such as cancer, and endocrine and immunological disorders (Glynn et al., 2007; Mrema et al., 2013; Qiu et al., 2005). Hence, research of the link between DDT and human health, especially its link with cancer, are drawing more and more people’s attention. Both animal and human studies have suggested an association between DDT and liver cancer. For example, laboratory animals exposed to DDT exhibit a dose-related increase in liver tumors and rodents exposed to p,p -DDT increase the risk of HCC (Beard, 2006; Persson et al., 2012). Human exposure to DDT have also been reported to elevate the rate of liver cancer (Cocco et al., 1997; Figàtalamanca et al., 1993). Moreover, a recent ecological study has reported a statistically significant correlation between levels of DDE (a metabolite of DDT) in adipose tissue and mortality rates of liver cancer (Cocco et al., 2000). McGlynn KA et al. have also reported that there is a significant correlation between DDT serum concentration and incidence of liver cancer in humans (McGlynn et al., 2006). In view of DDT possibly increasing the risk for hepatocellular carcinoma, the cytotoxic effects of DDT on hepatocellular carcinoma are of concern. Numerous epidemiological studies have suggested that DDT exposure may increase the risk of liver cancer, but the molecular mechanisms for how DDT promotes the progression of liver cancer remains unclear. The pathogenesis of hepatocellular carcinoma is complicated, in which cell signaling pathways play a vital. One key signaling pathway coordinating liver cancer is the Wnt/-catenin signaling pathway (Monga, 2011; Thompson and Monga, 2007). The Wnt/-catenin signaling pathway is commonly deregulated in hepatocellular carcinoma (HCC) and aberrations in this pathway have been established to be critical contributors toward hepatocarcinogenesis (Lee et al., 2005; Takigawa and Brown, 2008). In the absence of Wnt ligands, Wnt/-catenin signaling pathway is not activated. The serine/threonine kinase glycogen synthase kinase-3 (GSK3) phosphorylates -catenin, targeting it to the proteasome for degradation. It has been shown that -catenin becomes stable in the cytosol followed by its eventual nuclear translocation when the pathway is stimulated. -catenin binds to transcription factor TCF, then regulates target gene transcriptions, which affect cell fate
determination and cancer development (Chien et al., 2009; Moon et al., 2004). Main molecular targets, which DDT exposure involves, include estrogen receptors (ERs) and reactive oxygen species (ROS) (Hardell et al., 2004; Radice et al., 2006; Tebourbi et al., 2011). However, p,p -DDT, which we concentrate on, is one main isomer of DDT; the other is o,p -DDT. The binding ability of o,p -DDT to ERs is 100fold greater than that of p,p -DDT (Kojima et al., 2004). Therefore, we focused on the ROS, which is generally involved in toxicological mechanisms of environmental contaminants (Kojima et al., 2004). In the present study, we aim to elucidate the mechanism of p,p DDT action on the growth of hepatocellular carcinoma using both in vitro and in vivo models. In conclusion, our study unveils for the first time that p,p -DDT exposure stimulates proliferation of hepatocellular carcinoma, leading to the progression of hepatocellular carcinoma. These effects are due to p,p -DDT’s ability to initiate the Wnt/-catenin signaling pathway mediated by oxidative stress. The present investigation provides the molecular evidences that persistent pesticides could have an adverse impact on human health and contribute to liver cancer development. 2. Materials and methods 2.1. Antibodies and agents Antibodies for c-Myc and CyclinD1 were purchased from Bioworld, p-GSK3 (Ser 9) was purchased from Cell Signaling Technology, ki-67 was purchased from ZSGB-BIO and ␣-tubulin was purchased from Sigma (St. Louis, MO, USA). Antibodies for -catenin and PCNA were obtained from Abmart (USA). p,p DDT (Sigma) was dissolved into dimethyl sulfoxide (DMSO) as stock solutions. The equal concentration of DMSO was added to medium for the control cells. N-Acetyl-L-cysteine (NAC, the scavenger of ROS) and 2 ,7 -dichlorofluorescein diacetate (DCFH-DA) were obtained from Beyotime Biotechnology. 2.2. Cell culture and treatments Human hepatoma cells (HepG2) and human normal liver cells (HL-7702) were maintained in RPMI-1640 medium (HyClone) supplemented with 10% FBS (Boster), 1%penicillin/streptomycin (Solarbio) at 37 ◦ C in a 5% CO2 humidified tissue culture incubator. To observe the toxicity of p,p -DDT in HepG2 cells, cells were exposed to p,p -DDT at different doses (from 10−12 to 10−7 mol/L) over a 4 day period. Cells with the treatments were then assayed for cellular viability assay. 2.3. Cell viability assay To investigate viability of HepG2 and HL-7702 cells, the MTT assay was performed. 100 L of HepG2 or HL-7702 cells in suspension (1 × 103 cells) were briefly plated in a 96-well plate. After treatment with p,p -DDT for 4 days, viable cells were stained with 20 L MTT. The medium was then removed, and formazan crystals produced in the wells were dissolved by addition of dimethyl sulfoxide (DMSO). Absorbance was measured at 490 nm using a microplate reader. Cell viability was defined relative to untreated control [i.e. viability (% control) = 100 × (absorbance of treated sample − absorbance of control/absorbance of control)].
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2.4. Nude mice assay All animal experiments were approved by the Committee of Animal Care at Chinese Institute for Radiation Protection. HepG2 cells were washed twice and resuspended in physiological saline at a concentration of 5 × 107 cells/mL. A 200 L cell suspension of HepG2 was then injected subcutaneously into the left armpit of SPF-free male BALB/c-nu mice. After three days, one group of mice received intraperitoneal injections of p,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. Mice were weighed and tumor diameters were checked every 7 days. Tumor volume was calculated according to 0.4ab2 /2 (a > b; a = maximum length, b = maximum width).Tumor specimens were collected at 7 weeks after injections and split. Three independent experiments were performed and yielded similar results. 2.5. Western blot analysis Cells extracted proteins were resolved by 10% SDS-PAGE and transferred onto nitrocellulose membranes for Western blotting. The blots were blocked for 1 h in PBS containing 5% non-fat dry milk (w/v) and incubated at 4 ◦ C overnight, then probed with antibody for 1 h at room temperature or overnight at 4 ◦ C. After washing, membranes were incubated at 37 ◦ C for 1 h with the appropriate horseradish peroxidase-conjugated secondary antibody (diluted at 1:1000, Invitrogen). Protein loading was controlled by probing the membranes for ␣-tubulin protein. Immune-reactive proteins were detected using ECL western blotting detection system. For measurement of -catenin, the cytoplasmic protein and the nuclear protein were extracted according to the instructions of the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotech Inc., Nantong, China). 2.6. RNA interference The small interference RNA (siRNA) targeting -catenin and non-silencing siRNA were synthesized by Genepharma (Shanghai, China). About 1 × 106 cells grown in 6-well plates were transfected with 100 nM siRNA using lipofectamine 2000. After transfection for 4 days, cells were lysed. The lysis was used for western blot analysis.
disruption using ultrasound equipment. After being centrifuged at 6000 rpm for 10 min, the supernatants were used to measure enzyme activities. The data were normalized to protein content. 2.9. Immuno-histochemical analysis Tumors were fixed in 10% formalin over 24 h. The tissues were dehydrated and then embedded in paraffin wax. The sections were stained with antibody to Ki-67, -catenin, c-Myc and CyclinD1. After washing, these sections were stained with horseradish peroxidase-conjugated secondary antibody (ZSGB-BIO, China) and then incubated with streptavidin–horseradish peroxidase complex. The sections finally were stained with diaminobenzidine (DAB) and counterstained with hematoxylin. 2.10. Statistical methods Statistical analysis was carried out using the SPSS software program. Data, derived from three or four independent experiments, were presented as the mean ± SD. Differences among groups were tested by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. Comparisons between two groups were evaluated using Student’s t-test. A value of p < 0.05 was considered statistically significant. 3. Results 3.1. Low doses of p,p -DDT exposure promote the proliferation of HepG2 cells We used a MTT assay to measure the cell viability after treatment with p,p -DDT from 10−12 to 10−7 mol/L for 4 days. Compared with the vehicle control (DMSO, 0.1%), cell viabilities, observed after 4 days exposure at concentrations of 10−12 , 10−11 , 10−10 , 10−9 , 10−8 and 10−7 mol/L, were 15.26%, 33.14%, 55.31%, 66.83%, 49.67% and 17.89% respectively (Fig. 1). These results indicate that p,p -DDT promotes the proliferation of HepG2 cells. However, p,p DDT displayed a negligible effect on HL-7702 cell proliferation. The doses of p,p -DDT, measured by its concentrations in human blood, were about 5.36 × 10−9 to 5.07 × 10−6 mol/L (Röllin et al., 2009; Sholtz et al., 2011). Treatment with 10−9 mol/L p,p -DDT resulted
Targeting -catenin (5 -CAGUUGUGGUUAAGCUCUUdTdT-3 ). Non-silencing siRNA (5 -TTCTCCGAACGTGTCACGT-3 ). 2.7. Measurement of ROS generation DCFH-DA is a cell-permeable, nonfluorescent probe that is cleaved by intracellular esterases and turns into a highly fluorescent dichlorofluorescein upon reaction with H2 O2 . After treatment with p,p -DDT (10−9 mol/L) with or without NAC(1 mmol) for 4 days, the cells were stained with 10 mol/L DCFH-DA for 30 min at 37 ◦ C. H2 O2 generation was determined by dichlorofluorescein fluorescence. Cells were collected and the fluorescence intensity in the cells was measured using a fluorescence microplate reader (Thermo Scientific varioskan flash) with excitation 488 nm and emission 525 nm. 2.8. Determinations of oxidative stress-related parameters ␥-GCS and SOD activities as well as GSH content were determined in cell and tissues using a commercial determination kit (Nanjing Jiancheng Bioenginneering Institute). Cells were plated onto 6-well dishes (1 × 106 cells/well) and were exposed to p,p DDT (10−9 mol/L) with or without NAC (1 mmol) for 4 days. Scraped cells were dissolved in physiological saline, followed by cell
Fig. 1. p,p -DDT exposure promotes the proliferation of HepG2 cells and has no significant effect on HL7702. Cells viability was measured using a MTT assay after HepG2 and HL7702 cells exposed to p,p -DDT (from 10−12 to 10−7 mol/L) for 4 days. Values were representative of at least three biologically independent experiments with similar results. Error bars represent the SD. Asterisks (*) indicate significant differences (*p < 0.05, **p < 0.01) compared to controls.
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Fig. 2. Oxidative stress plays an important role in p,p -DDT-mediated proliferation. HepG2 cells were treated with 0.1% DMSO, 10−9 mol/L p,p -DDT, and/or 1 mmol NAC for 4 days, (A) ROS levels, (B) SOD activity, (C) ␥-GCS activity, and (D) cell viability were assessed as described in Section 2. The values were showed as means ± SD of triplicate determinations. An asterisk (*) represents a significant difference from controls (*p < 0.05, **p < 0.01).
in the biggest increase in cell viability (about 66.83%). Therefore, 10−9 mol/L was selected as exposure concentration of p,p -DDT to test its mechanism on the proliferation of HepG2 cells. 3.2. Oxidative stress acts as a pivotal player in p,p -DDT-induced proliferation Because the main target of p,p -DDT was ROS, as stated in Section 1, we hypothesized that production of ROS by p,p -DDT may promote the proliferation of HepG2 cells. We found that the level of ROS was elevated in p,p -DDT stimulated cells (Fig. 2A). ROS is one of the most important components in oxidative stress. Thus, we further investigated other indexes of oxidative stress: SOD and ␥-GCS, which are important antioxidant enzymes. As shown in Fig. 2B and C, SOD and ␥-GCS activities were significantly decreased after p,p DDT exposure, demonstrating that p,p -DDT induced the oxidative stress of HepG2 cells. To determine the role of oxidative stress in p,p -DDT-induced proliferation, we selected NAC, an ROS inhibitor, to reduce oxidative stress, and then accessed the cell viability. We found that co-treatment of NAC (1 mmol) with p,p -DDT decreased ROS content as well as elevated SOD and ␥-GCS activities, indicating that oxidative stress was inhibited. Next, we measured the cell viability and found that compared with p,p -DDT exposure alone, cell viability was reduced with NAC co-treatment (Fig. 2D). These results indicate that oxidative stress play an important role in the stimulation of proliferation in HepG2 cells induced by p,p -DDT. 3.3. p,p -DDT stimulates Wnt/ˇ-catenin pathway mediated by oxidative stress Previous studies showed that the Wnt/-catenin signaling pathway was abnormally activated in hepatic carcinoma (Zeng et al.,
2007; Zucman-Rossi et al., 2006). Hence, we took one further step to explore the relationship between the Wnt/-catenin pathway and p,p -DDT-mediated proliferation. As shown in Fig. 3A, p,p -DDT induced the over-expression of -catenin and p-GSK3 (Ser 9) proteins. Furthermore, the protein levels of downstream CyclinD1 and c-Myc were also strongly accumulated. These results reflect that Wnt/-catenin signaling pathway is activated by p,p -DDT. However, p,p -DDT did not stimulated Wnt/-catenin signaling pathway in HL7702 cells. A hallmark event of activated Wnt/-catenin signaling is the nucleus input of -catenin (Polakis, 2000). Notably, p,p -DDT exposure caused accumulation of -catenin protein in the nucleu and cytoplasm compared to the vehicle treatment (Fig. 3B). Over the past decades, evidence has shown that the Wnt/catenin pathway could be stimulated through the induction of ROS (Bowerman, 2005; Essers et al., 2005). The above data illustrates that both ROS and Wnt/-catenin pathway are involved in p,p DDT-induced proliferation in HepG2 cells. Hence, we speculated that Wnt/-catenin pathway activated by p,p -DDT was mediated by ROS. To verify this hypothesis, HepG2 cells were treated with 10−9 mol/L p,p -DDT and/or 1 mmol NAC for 4 days. As shown in Fig. 3A, NAC dramatically disrupted accumulations of -catenin, pGSK3 (Ser 9), and downstream CyclinD1, as well as c-Myc protein levels induced by p,p -DDT. Consistently the increase of -catenin in the nucleu and cytoplasm was disrupted by NAC co-treatment (Fig. 3B). The results demonstrate that the Wnt/-catenin pathway activated by p,p -DDT is mediated by ROS. To further examine the role of Wnt/-catenin signaling, -catenin siRNA was performed to reduce -catenin expression and thereby inhibited Wnt/-catenin pathway in HepG2 cells (Fig. 4A). Fig. 4B showed that -catenin siRNA inhibited the effect of p,p -DDT on proliferation of HepG2, similar to the study that shown proliferation of liver cancer cells was associated with the Wnt/-catenin pathway (Zeng et al., 2007).
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Fig. 3. p,p -DDT exposure activates the Wnt/-catenin pathway mediated by oxidative stress. (A) Western blots were applied to check relevant protein expressions of HepG2 and HL7702 cells after p,p -DDT treatment alone or NAC co-treatment, including -catenin, p-GSK and downstream target genes (c-Myc and CyclinD1). (B) Expression of -catenin in cytoplasm and nucleus of HepG2 cells was determined. The above blots and data were representative of at least three independent experiments with similar results.
Fig. 4. Wnt/-catenin pathway plays an vital role in p,p -DDT-mediated proliferation. (A) HepG2 cells were transfected with -catenin siRNA and treated with p,p -DDT (10−9 mol/L) for 4 days and (B) cell viability of HepG2 cells were examined. The above blots and data were representative of at least three independent experiments with similar results.
Fig. 5. p,p -DDT increases the growth of tumor in nude mice model. HepG2 cells were injected into the 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 weeks post-injection, mice were killed. (A) Tumor volume, (B) tumor size, and (C) the average weight of tumor were determined. Value shown was given as the mean ± SD of 5 animals in each group. An asterisk (*) indicates that the data are significantly different comparing with control mice (*p < 0.05, **p < 0.01).
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3.4. p,p -DDT promotes tumor growth in nude mice models The above results indicate that low dose p,p -DDT stimulated proliferation of HepG2 cells through the Wnt/-catenin pathway mediated by oxidative stress in vitro. To validate our findings, in vivo nude mice assay was performed to further determine the proliferative capability in tumor tissues induced by p,p -DDT. We measured tumor growth for 7 weeks after exposure to 5 nmol/kg p,p -DDT (Fig. 5B). Tumor volume and weight in p,p -DDT-treated group were notably increased compared with control tumors (Fig. 5A and C). This data indicates that the p,p -DDT markedly elevated the formation of tumors. 3.5. Oxidative stress mediates the stimulation of Wnt/ˇ-catenin pathway by p,p -DDT in nude mice model Oxidative stress, which was evaluated by measuring SOD activity, ␥-GCS activity, and GSH content in tumor tissues, were further analyzed after exposure to p,p -DDT for 7 weeks. As shown in Fig. 6A and B, significant decreases in SOD activity along with ␥-GCS activity were found in p,p -DDT-treated tumors. We also found that GSH content was attenuated in p,p -DDT-treated tumors (Fig. 6C). These Fig. 7. Wnt/-catenin pathway is activated by p,p -DDT exposure in nude mice model. (A) Western blots were performed as mentioned above to examine -catenin protein levels of tumor tissues in cytoplasm and nuclear and (B) -catenin and c-Myc protein were expressed in tumor tissues. The above blots and data were representative of at least three independent experiments with similar results.
Fig. 6. Oxidative stress is stimulated by p,p -DDT exposure in nude mice model. The oxidative stress-related parameters in tumor tissues, including (A) SOD activity, (B) ␥-GCS activity, and (C) GSH levels, were determined as described in Section 2. An asterisk (*) indicates that the data are statistically significantly different from controls (*p < 0.05, **p < 0.01).
Fig. 8. Immunohistochemistry analysis of the expression of -catenin, c-Myc, CyclinD1, and Ki-67 in liver cancer tissues by p,p -DDT-treatment or without. Matched sections of control (left panel) and p,p -DDT-treated tumors were stained with -catenin, c-Myc, CyclinD1, and Ki-67. There were significant differences between control and p,p -DDT-treated group. Magnification ×40.
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Fig. 9. Proposed mechanism of p,p -DDT-promoted progression of hepatocellular carcinoma. p,p -DDT exposed to hepatocellular carcinoma, first activates ROS and stimulates the oxidative stress. Next, it promotes phosphorylation of GSK3 at Ser 9. Afterwards, active -catenin enters the nucleus and binds to the transcription factor TCF, which regulates expression of its downstream target genes (c-Myc and CyclinD1). These downstream targets are associated with proliferation, thus promoting the growth of hepatocellular carcinoma.
alterations of oxidative stress-related parameters confirm that p,p DDT exposure elicited a significant stimulation of oxidative stress. We also measured -catenin protein levels in tumor tissues in response to p,p -DDT treatment. As shown in Fig. 7A, -catenin accumulation in the nucleu was dramatically elevated in p,p -DDTtreated tumors compared with vehicle treatment. At the same time, p,p -DDT significantly increased -catenin and c-Myc protein levels (Fig. 7B). The data suggests that the Wnt/-catenin pathway is activated in p,p -DDT-treated tumors. These results coincide with the data obtained in our in vitro experiments. Furthermore, histological examination revealed that p,p -DDT-treated tumors had a histological appearance with massive grown, as indexed by the significant increase of -catenin, c-Myc, CyclinD1, and Ki-67 (Fig. 8). 4. Discussion Hepatocellular carcinoma is the third most common malignancy worldwide and has a poor long-term survival rate (Jemal, 2011). p,p -DDT is a persistent organic pollutants (POPs), which are hazardous chemicals present in our food chain that have been internationally regulated to protect public health. Although recent studies have highlighted an unexpected implication of POPs in the development of hepatocellular carcinoma (Arrebola et al., 2012; Jemal, 2011; Ruzzin, 2012; Zhao et al., 2011), the mechanisms underlying how p,p -DDT causes liver cancer remains elusive. Elucidation of these mechanisms is a major goal since it could lead to the elimination of p,p -DDT induced liver cancer. Accumulated studies have shown that Wnt/-catenin activation plays important roles in a variety of cancers, including
hepatocallular carcinoma (Monga, 2011; Takigawa and Brown, 2008). Notably, Zhang et al. recently identified a role of -catenin as a “potential” tumor suppressor. This study reported a paradoxical rise in the susceptibility of -catenin knockout mice to DEN-induced carcinogenesis. The data indicated PDGFR␣ was upregulated with -catenin KO livers, which enhanced the tumorigenesis through PDGFR␣/PIK3CA pathway. This study suggests an addition important role of -catenin in maintaining redox homeostasis in the liver (Zhang et al., 2010). In this research, we indicated a mechanism for the p,p -DDT-promoted growth of hepatocellular carcinoma. p,p -DDT exposed hepatocellular carcinoma, first activates ROS and stimulates the oxidative stress. It then promotes phosphorylation of GSK3 at Ser 9. Active -catenin was accumulates in the cytoplasm and enters the nucleus where it binds to the transcription factor TCF. Binding to TCF allows regulation of the expressions of its downstream target genes (c-Myc and CyclinD1), which are associated with proliferation, thus promoting progression of hepatocellular carcinoma (Fig. 9). These data, in conjunction with published reports, elucidate a direct interaction between the oxidative stress and Wnt/-catenin pathway in the development of liver cancer induced by p,p -DDT (Chen et al., 2010; Wang et al., 2012). The results from both in vitro and in vivo models implicate that p,p -DDT markedly induced proliferation of hepatocellular carcinoma. In contrast to the proliferation observed with low doses, high-dose of p,p -DDT exposure resulted in apoptosis of liver cancer cells (Buchmann et al., 1999). Previous studies have reported that DDT can lead to the hepatocellular carcinoma, which coincides with our results (Arrebola et al., 2012).
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The mechanism of proliferation stimulated by DDT in liver cancer has been reported in previous studies. For example, Kazantseva et al. have recently determined that DDT treatment may result in cell cycle progression and apoptosis inhibition in mouse livers, leading to hepatocarcinogenesis (Kazantseva et al., 2013). In addition, Harada et al. have reported that oxidative stress could be a key factor in hepatocarcinogenesis by DDT exposure (Harada et al., 2003). Similarly, our present study also found that oxidative stress was activated after p,p -DDT exposure. p,p -DDT elevated the ROS content along with decrease of SOD activity. And it also decreased the activity of ␥-GCS with a concomitant reduction in GSH content, thus reducing the cell’s ability to scavenge oxygen free radicals. These alterations of enzymes were consistent with previous reports (Lin and Yang, 2007; Walsh et al., 2001; Yoshida et al., 1995). Moreover, NAC reduced the proliferation induced by p,p -DDT. Results presented here strongly suggested that p,p -DDT-stimulated proliferation of HepG2 cells is ROS dependent. Moreover, accumulation of nuclear and cellular -catenin has been observed in 18–67% of HCC tumors. It has also been observed that the Wnt/-catenin pathway is involved in cancer as an early regulatory factor, which is prematurely activated by ROS (Devereux et al., 2001; Korswagen, 2006). Kazantseva et al. reported that DDT treatment could induce liver responses through CAR- and ER␣ activation, and it was independent of Wnt signaling pathway in mouse livers (Kazantseva et al., 2013). Notably, our results showed that Wnt signaling and -catenin were not detected in human normal liver cell HL7702 after exposing to DDT for 4 days in our studies (Fig. 3A). By contrast, it showed significant changes in live cancer cell, HepG2. In addition, the differences of applied doses of DDT and exposure time to samples need to be concerned as well. Our results indicate, for the first time, that p,p -DDT increases ROS generation, which then stimulates the Wnt/-catenin pathway in HepG2 cells. We also demonstrate that p,p -DDT elevates the expression of -catenin and that its effect on proliferation is inhibited by -catenin siRNA. These results support a role for -catenin in promotion of proliferation and indicates a mechanism by which p,p -DDT increases proliferation. Even though previous studies have shown that p,p -DDT promoted proliferation in liver cancer, the mechanism(s) of p,p -DDT, especially how low dose of p,p -DDT causes liver cancer, is poorly unknown. Taken together, this paper unveils that p,p -DDT stimulates the proliferation of liver cancer cells via oxidative stress and Wnt/-catenin signaling pathway. Thus, p,p -DDT exposure leads to the deterioration of hepatocellular carcinoma. The present data provides a molecular mechanism regarding p,p -DDT’s distinct effects on hepatocellular carcinoma. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments This work was supported by the National Natural Sciences Foundation of China (Nos. 31271516, 21207084, 31201072), Research Fund for the Doctoral Program of Higher Education of China (20111401110011), China Postdoctoral Science Foundation (2012M521178), Natural Science Foundation of Shanxi (2009021035-2), and Research Fund for the Doctoral Program of Higher Education of China (20111401110011).We thank Dr. Enmin Zou of the Department of Biological Sciences of Nicholls State University, USA, for his help in improving the English of this manuscript.
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