Resistin-induced stromal cell-derived factor-1 expression through Toll-like receptor 4 and activation of p38 MAPK/ NFκB signaling pathway in gastric cancer cells

Background Stromal cell-derived factor-1 (SDF-1) (CXC chemokine ligand-12)/CXC chemokine receptor 4 (CXCR4) is involved in the carcinogenesis of human gastric cancer, where it stimulates angiogenesis and favors metastasis of tumor cells to distant organs. In addition, resistin is suggested to be an important link between obesity and the development of gastric cancer. Resistin has identified as an important player in inflammatory responses, and emerged as a mediator in inflammation-associated cancer. A limited number of studies have investigated the association of resistin and SDF-1 with gastric cancer. Herein, we investigated the molecular mechanisms by which resistin influences the expression of SDF-1 in gastric carcinoma cells. Results Human gastric cancer cell lines were exposed to doses of resistin; SDF-1 expression and secretion levels were then determined. Real-time polymerase chain reaction and western blotting analyses were performed to clarify molecular changes. Inhibition of Toll-like receptor 4 (TLR4) by a competitive antagonist inhibited resistin-induced SDF-1 expression. Pharmacological inhibitors and small interfering RNA (siRNA) demonstrated that activation of the p38 mitogen-activated protein kinase (MAPK) pathway is critical for resistin-induced SDF-1 expression mediated by TLR4. The promoter activity and transcription factor enzyme-linked immunosorbent assay revealed that resistin induced expression of SDF-1 mediated by NF-κB in gastric cancer cells. Inhibition of p38 MARK activation blocked the SDF-1-induced expression and the SDF-1 promoter activity in the cancer gastric cells. Chromatin immunoprecipitation assay revealed that inhibition of p38 MARK activation also blocked the resistin-increased NF-κB-DNA-binding activity. Conclusions Resistin-induced SDF-1 upregulation by activation of TLR4, p38 MARK and NF-κB may explain a new role of resistin in the link of obesity and gastric cancer.


Background
Gastric cancer ranks as the fourth most frequent of malignant tumors [1]. Although it is the second or third most frequent cause of cancer death worldwide [2], its etiology is not well understood. The disease is often the result of long-term interactions of many factors relating to individual habits, diet, environment, and genetics, as well as chronic gastritis, gastric polyps, gastric dysplasia, post-surgery gastric remnants, and long-term infection by Helicobacter pylori [3]. H. pylori is recognized as a causative factor of chronic gastritis, gastroduodenal ulcers, gastric cancer, and mucosa-associated lymphatic tissue lymphoma. In previous study, chronic gastritis related human diseases, including gastroduodenal ulcers, gastric cancer, and mucosa-associated lymphatic tissue lymphoma, were found to result from an excessive formation of epithelial cells or gastric mucin injury and inflammation caused by chronic H. pylori infection [4]. Several studies have indicated that the typical LPSs recognized by the Toll-like receptor 4 (TLR4) complex expressed on host cells, contributing to activation of the p38 mitogen-activated protein kinase (MARK) pathway [5,6].
Obesity is a risk factor for gastric cancer mainly because obesity enhances the incidence of gastroesophageal reflux, which may damage the mucosa around the gastric cardia, resulting in an increased likelihood of cancer. Obesity is defined as a very high ratio of body fat to other tissue [7]. Too much body fat has a significant impact on health, particularly in terms of insulin resistance. The proteins secreted by the fat tissues play a very important role in regulating metabolism [8]. The regulation of resistin in carbohydrate metabolism is considered to be associated with insulin resistance. Resistin is capable of regulating the insulin-stimulated metabolism of carbohydrates in many insulin target organs [9]. Since the discovery of resistin, most studies have focused on the relationship of resistin to obesity and diabetes. An increasing number of studies have focused on the role of resistin in cancer development, and the impact of resistin on inflammation is the focus of several current academic studies [10,11]. These studies have found that resistin is negatively correlated with high density lipoproteins in diabetic patients; however, resistin is positively correlated with C-reactive protein (CRP), an indicator of inflammation [12]. This association indicates that higher levels of the resistin may be related to inflammatory responses. In addition, studies have found that adipose tissue is not the exclusive source of resistin; large amounts of resistin and resistin-like molecules are also found in nonadipose tissues under inflammation [13]. Inflammatory response can release interleukin (IL)-6, IL-8, IL-1β, and tumor necrosis factor alpha (TNFα) through the NF-κB pathway [11]. Thus far, no study has investigated the association of resistin and any known receptor to activate downstream MAPK kinase that further activate nuclear factor-κB (NF-κB p50/p65) in human gastric cancer.
Chemoattractant proteins are a group of small proteins of molecular weight ranging from 8 to 12 kDa that can be induced by inflammatory substances to release into the extracellular environment. More than 40 types of human cell chemoattractant proteins have been identified [14]. Chemoattractant proteins have a number of functions such as inducing the movement, growth, and differentiation of white blood cells. These inflammatory responses are closely related to gastric cancer [15]. One of the causative factors of inflammatory responses is the production and induction of chemoattractant proteins. Previous studies have found that the stromal cell-derived factor-1 (SDF-1) can regulate cancerous cell movement and blood vessel regeneration via its specific receptors CXCR4 and CXCR7 [16]. Gastric inflammation is an integral step in gastric cancer development; therefore, factors inducing and regulating responses to inflammation may play a key role in gastric cancer prognoses [17]. From this viewpoint, because chemokines have certain roles in microbial immune and inflammation responses, the resistin-induced secretion of SDF-1 may be correlated to the control of gastric cancer.
Gastric cancer can be correlated with obesity. Researchers have pointed out that resistin is the blood biological indicator of gastric cancer and is related to patient prognosis [18]. Moreover, SDF-1 acts in cancerous cells as a growth and survival factor; however, the implication of resistin stimulation by the chemoattractant SDF-1 has not been studied. In the present study, we investigated whether resistin stimulates the expression of SDF-1 by activating the p38 MAPK intracellular signaling cascades and the transcription factors NF-κB and p50. Our findings provide evidence of the molecular mechanisms of SDF-1 expression and its secretion by resistin via a TLR4-dependent pathway in gastric cancer cells.

Cell culture
The gastric carcinoma cell line TSGH 9201 and AGS cells was purchased from the Bioresources Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a CO 2 incubator at 37°C.

ELISA
CXCL12/SDF-1 expression on the cancer cell surface was measured by ELISA as previously described [19]. Release of SDF-1 into culture media was analyzed using commercially available ELISA kit purchased from Cell Sciences (Canton, MA). The assays and data calculations were performed according to the manufacturer's instructions.

Chromatin immunoprecipitation assay (ChIP)
The ChIP assay was carried out as previously described and ChIP assay kit used was from Upstate Biotechnology (Lake Placid, NY) [19]. Cells were fixed with 1% formaldehyde, washed, then harvested in SDS lysis buffer. After sonication, lysates containing soluble chromatin were immunoprecipitated using 2 μg of antibody against p50. DNA was purified with a PCR Purification Kit (QIA-GEN, Venlo, The Netherlands). The resulting DNA was used for PCR analysis, and the amplified DNA fragments were visualized on an agarose gel. PCR was performed with the following primers that amplify the parts of the human SDF-1 promoter that contain the p50 binding sites from −669~−569: 5'-GTTTCCACAGGCGAATGG -3' and 5'-GGACCTCACAGCCTCAAGTC -3'.

Statistical analysis
The experiments were performed in triplicate independent experiments, and data were presented as three repeats from one independent experiment. Data were reported as the mean ± standard deviation or standard error of the mean and evaluated by one-way analysis of variance. SPSS version 16.0 (SPSS, Inc., Chicago, IL) was used for all statistical analyses. Significant differences were established at P < 0.05.

Effect of resistin on expression of SDF-1 in gastric carcinoma TSGH 9201 and AGS cells
To determine whether SDF-1 is induced by resistin, we exposed the human gastric cancer cell lines TSGH 9201 and AGS to a range of resistin doses and performed experimental assays. Cells were exposed to a 25 ng/mL dose of resistin for the indicated times. The changes in SDF-1 mRNA expression were analyzed by real-time PCR; SDF-1 secretion in conditioned media was detected by ELISA. The SDF-1 mRNA reached its highest level at 4 h of resistin stimulation ( Figure 1A). The secretion of SDF-1 protein began to increase after resistin treatment and reached its highest level at 6 h ( Figure 1B). In addition, the resistin-induced SDF-1 mRNA expression and protein secretion in TSGH 9201 cells was dose dependent ( Figure 1C-D). The results demonstrate that resistin significantly induced gene expression. Based on our results, it is possible that in gastric carcinoma cell, resistin induced pathway-related proteins may be studied as potential markers in terms of the prediction of response to treatment or prognosis. Further investigation, we used TSGH 9201 Cell to evaluate the effect of resistin on other pro-tumoral CXC chemokines gene expression. Our data demonstrate that resistin significantly induced-related gene expression, such as GRO, ENA78, GCP-2 or IL-8 (Additional file 1: Figure S1).

Resistin-induced SDF-1 expression in gastric cancer is mediated by p38 MAPK
To clarify the events of resistin-induced SDF-1 expression, we analyzed specific MAPK siRNAs to determine the signaling pathways associated with resistin-induced SDF-1 expression in TSGH 9201 cells ( Figure 1A). As shown in Figure 2B and C, the mRNA level and secretion of SDF-1 were increased by the resistin stimulation, and they were significantly inhibited by SB203580, but not by PD98059 or SP600125. To further confirm the involvement of p38 MAPK, but not ERK and JNK, in the modulation of the SDF-1 expression by resistin induction, we examined the effects of specific MAPK siRNAs of these signaling pathways on resistin-induced SDF-1 expression in TSGH 9201 cells (Figure 2A). The resistininduced SDF-1 mRNA expression ( Figure 2B) and SDF-1 secretion ( Figure 2C) were inhibited by transfection with p38-siRNA, but not by transfection with ERK-, JNK-, and control siRNAs (100 mmol/ml for each). These data suggest that the p38 MAPK pathway is involved in regulating the resistin-induced SDF-1 expression in gastric cancer cells. To determine the effect of resistin on the activation of the kinase signaling pathway, we assessed whole cell lysates from resistin-treated TSGH 9201 cells by Western blotting analysis using antibodies against activated Phospho-p38 MAPK and p38 MAPK. As shown in Figure 2D, the treatment of TSGH 9201 cells with resistin resulted in the timedependent phosphorylation of p38 MAPK within 2 h. SDF-1 expression analysis revealed that the resistin induction is mediated by the p38 MAPK-dependent pathway in TSGH 9201 cells.  Figure 3B). To evaluate whether the SDF-1 expression by TLR4 involved the MAPK signaling pathway at the transcriptional level, we compared control cells to those stimulated with resistin for 30 min. LPS-RS significantly inhibited the resistininduced phosphorylation of p38 MAPK after 2 h ( Figure 3C). Furthermore, TSGH 9201 cells were transfected with the TLR4 siRNA, and the phosphorylation of p38 MAPK and the SDF-1 expression were then examined. Figure 3D indicates the effectiveness of TLR4 siRNA on p38 MAPK and SDF-1expression after resistin stimulation.

NF-κB is necessary for resistin induction of human SDF-1 promoter activity
The human SDF-1 gene promoter contains multiple transcription binding sites. To determine the cis-acting elements in the SDF-1 gene promoter that mediate resistin-induced SDF-1 transcription, a luciferase assay was applied using the p1010-Luc plasmid and several deletion promoter constructs ( Figure 4A). To clarify the binding region of the SDF-1 promoter, we constructed and analyzed a series of 5′-deletion mutants.
To investigate whether p50 binds the SDF-1 promoter region in TSGH 9201 cells, we performed quantitative analysis to determine the binding activity of NF-κB p50 using TF ELISA kits ( Figure 5A). The results showed that treating TSGH 9201 cells with resistin raised the binding activity of p50 DNA within 2 h. To confirm these results, ChIP analysis was performed in vitro. Immunoprecipitated chromosomal DNA with anti-p50 antibody was subjected to PCR using primers designed to amplify the SDF-1 promoter region (−1010 to −430) harboring the p50 binding sites. NF-κB p50, but not the control antibody, did indeed bind to the SDF-1 promoter region ( Figure 5B). These data suggest that these sequences were indeed p50 binding sites. We used double labeling of p50 and DAPI to evaluate the effect of resistin in TSGH 9201 cells at 12 h. Representative immunoreactivity for phase contrast microscopy, DAPI (blue), p50 (green), and overlays in the TSGH cells ( Figure 5C).

MAPK signaling pathways are involved in resistin-induced SDF-1 promoter activity
Members of the MAPK family have been implicated in the regulation of gene expression by resistin [22]. To evaluate the induction of SDF-1 expression by MAPK signaling pathways through the transcriptional level, TSGH 9201 cells were incubated with a specific inhibitor of p38 MAPK (SB203580) for 1 h before and during stimulation with resistin, and the SDF-1 promoter activity and ChIP were analyzed. The data clearly demonstrated that pretreatment of cells with SB203580 resulted in marked inhibition of the resistin-induced SDF-1 promoter activity ( Figure 6A). In addition, SB203580 significantly inhibited both resistininduced p50 activation ( Figure 6B) and NF-κB p50-DNA binding activity ( Figure 6C). We have used TSGH 9201 cells to evaluate the effect of resistin on phosphorylation of IκBβ as well as on p50 nuclear translocation. Our data demonstrate that resistin significantly induced p50 expression in TSGH 9201 cells via p38 MAPK. Taken together, these results showed that p38 MARK signaling pathway are involved in the resistin-induced SDF-1 expression ( Figure 6D).

Discussion
Obesity has been associated with lower rates of survival in patients with gastric cancer [23]. Adipocytokines such as TNFα, IL-6, adiponectin, leptin, visfatin, and resistin are cytokines secreted primarily by visceral adipose tissue and are thought to be involved in the positive correlation between obesity and the increased risk of gastric cancer [24]. On the other hand, several observers have suggested that resistin mediates the induction of inflammation in both adipose and non-adipose tissue [25]. The elevation of resistin and its role in inflammation in the intestine has resulted in the release of cytokines through the TLR4/ NF-κB pathway [26][27][28]. Recent studies have demonstrated the essential role of the resistin cascade, and a higher expression of resistin was evident in intestinal-type gastric carcinomas with tumor differentiation, tumor invasion, and lymph node metastasis [13]. The essential role of resistin, as well as its association with gastric cancer, make it a factor of concern as well as a potential a biomarker for gastric cancer progression [18]; therefore, it is clinically relevant to study the mechanism by which resistin influences tumor cells. In this study, we evaluated the molecular mechanisms underlying the roles of resistin in controlling SDF-1 expression in gastric cancer cells. SDF-1 was upregulated by resistin stimulation in TSGH 9201 cells. Resistin-induced expression of SDF-1 was mediated by the p38 MAPK and NF-κB pathways, and interaction between resistin and TLR4 was required for resistin-induced intracellular signaling and SDF-1 expression.
SDF-1 also promotes tumor development by stimulating angiogenesis and by processing the metastasis of CXCR4-positive tumor cells to distant organs producing SDF-1 [27]. Studies have shown that the level of plasma SDF-1 was higher in the high-incidence cancer group. In addition, SDF-1 modulates the angiogenic process directly (by binding to receptors CXCR4 expressed on endothelial cells) or indirectly (by inducing the secretion of matrix-metalloproteases or angiogenic factors) [29]. It has been suggested that SDF-1 is produced by gastric tumor cells themselves and can act on the tumor cells in a paracrine or autocrine fashion [30]. In summary, our study demonstrated that the effect of resistin on TSGH 9201 cells was mediated by the activation of p38 MAPK and that NF-κB transcriptional activation plays an important role in the expression of SDF-1. TF ELISA and luciferase assays demonstrated a resistin-induced increase in the NF-κB-and DNA-binding activity. In contrast, the inhibition of NF-κB and p38 MAPK activation in TSGH 9201 cells after treatment with NF-κB and p38 MAPK inhibitors and transfection with specific p50 and p38 siRNAs prohibited the resistin-induced expression and secretion of SDF-1.
The promoter region of the SDF-1 gene has several transcriptional factor binding sites [21]. This study demonstrated the mechanism by which resistin induces SDF-1 gene expression of gastric cancer cells. The important findings are as follows: the expression of SDF-1 is mediated by the NF-κB p50 pathway. Construction and analyses of 5′-deletions in the −1010 to −430 region of the SDF-1 promoter showed that the activity decreased to 30% and was nearly abolished. ChIP DNA with anti-p50 antibody that was subjected to PCR analysis showed the SDF-1 promoter region (−1010 to −430) harboring the NF-κB p50 binding sites. NF-κB proteins are members of a superfamily of transcription factors whose activities play a crucial role in cellular activation, proliferation, and apoptosis, which can be triggered through the MAPK pathway in gastric cancer cells [31]. During the early stages of invasion and metastasis of carcinoma cells, p38 MAPK plays a key role [5,32,33]. In our present study, we found that the gastric cell line, TSGH 9201, persisted in expressing activated p38 MAPK after exposure to resistin and high levels of this kinase are associated with an increased capacity to induce the binding of NF-κB p50 to the promoter region of SDF-1.
Previous data suggest that regulation of TLR receptors in gastric carcinogenesis might go beyond H. pylori infection, and is thought to be associated with tumor cancers [34]. Resistin has been reported to be significantly correlated with stage progression of gastric cancer [18,35]. We investigated the role of resistin signaling factors downstream of the p38 MARK and NF-κB activation sites that lead to SDF-1 transcriptional activation in TSGH 9201, and the pathophysiological implication of the role of resistin in gastric cancer should be further explored.

Conclusion
Taken together, our data suggest the mechanism by which resistin induces SDF-1 expression in gastric cancer cells. We found that treatment of gastric cancer cells with resistin resulted in the activation of signaling pathways mediated by TLR4. Further studies are required to explore the potential role of the resistin/ TLR4 axis as an effective therapeutic agent against gastric cancer.