Molecular Medicine ReportsMolecular Medicine Reports1791-29971791-3004D.A. Spandidos10.3892/mmr.2014.1932mmr-09-04-1371ArticlesInterleukin-11 induces the expression of matrix metalloproteinase 13 in gastric cancer SCH cells partly via the PI3K-AKT and JAK-STAT3 pathwaysYANGGONGLI*MAFENG*ZHONGMUXIAOFANGLINPENGYAOXINXIAOMINGZHONGJIETAOZHUWEIZHANGYALIGuangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Baiyun, Guangzhou 510515, P.R. ChinaCorrespondence to: Professor Wei Zhu or Professor Yali Zhang, Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, 1838 Guangzhou North Avenue, Baiyun, Guangzhou 510515, P.R. China, E-mail: chnz_w@126.com, E-mail: sygznfyy@163.com
Interleukin (IL)-11 is expressed in the majority of gastric carcinomas and has been associated with an aggressive phenotype and poor prognosis of gastric adenocarcinoma. Matrix metalloproteinase (MMP)-13 has been detected in numerous invasive malignant tumor types and exhibits a broad spectrum of activities on connective tissue components. In this study, we investigated whether IL-11 affects the expression of MMP-13 in human gastric cancer cells, as well as the underlying mechanism. Using western blot assays, we investigated the effect of recombinant human (rh) IL-11 on the expression of MMP-13 in gastric carcinoma cell lines. Using the PI3K inhibitor wortmannin and RNA interference to target the STAT3 gene, we investigated the effects of PI3K inhibition and/or STAT3 depletion on the expression of the MMP-13 protein. Results showed that IL-11 induced MMP-13 expression in a time- and concentration-dependent manner in SCH cells. IL-11 activated PI3K-AKT and JAK-STAT3 signal transduction. Wortmannin and depletion of STAT3 by means of small interfering RNA (siRNA) synergistically reduced the expression of MMP-13. These findings suggested that IL-11 induces the expression of MMP-13 in gastric cancer SCH cells partly via the PI3K-AKT and JAK-STAT3 pathways.
Gastric carcinoma, the fourth most common malignancy and the second most frequent cause of cancer death, is the result of accumulated genomic damage, affecting cell functions essential for cancer development (1). Despite recent advances in combined chemotherapies (2), the outcome on unresectable gastric cancer remains poor. Tumor metastasis is the most common cause of treatment failure in cancer patients (3); in gastric cancer, mortality depends on the metastatic spread of the primary adenocarcinoma (4). However, the mechanism of invasion and metastasis of gastric carcinoma is not fully understood.
A considerable body of evidence suggests that the inflammatory cells and cytokines in the tumor microenvironment are major factors that determine the behavior of malignant cells. Interleukin (IL)-11 is a member of the IL-6 family of cytokines, which mediate signaling via the common signal-transducing component gp130 and a cytokine-specific subunit (5). IL-11 was originally cloned as a mediator of proliferation of plasmacytoma cells and was later found to exhibit a wide variety of biological effects in neural cells as well as in the hematopoietic and immune systems (6,7). IL-11 was also found to be involved in chronic inflammation and associated tumorigenesis in experimental models (8,9). IL-11 is expressed in the majority of gastric carcinoma cells and has been associated with an aggressive phenotype and poor prognosis of gastric adenocarcinoma (10,11). However, whether and via which mechansim(s) IL-11 may contribute to tumor progression remains unknown.
Tumor invasion and metastasis require proteolytic degradation of the basement membrane and the extracellular matrix (ECM). Matrix metalloproteinases (MMPs) are primarily involved in the dissemination of cancer cells by breaking down the ECM and creating an environment that supports the initiation and maintenance of tumor growth. The ~26 members of the MMP family display a conserved structure and enzymatic mechanism (12). Human collagenase-3 (MMP-13) is one such member, which was originally cloned from breast carcinoma (13). It has also been detected in other invasive malignant tumor types, i.e., squamous cell carcinomas (SCCs) of the head and neck (14,15), the vulva (16), chondrosarcomas (17), malignant melanomas (18), and gastric cancer (19). Furthermore, expression of MMP-13 has been linked to increased tumor aggressiveness and poor prognosis (19).
IL-11 and MMP-13 have been independently reported to be upregulated in gastric cancer cells, which indicates a potential link between expression patterns of these two proteins. Howlett et al (20) found that gene knock-out mice gp130757F/F/IL-6−/− develop gastric antrum cancer and show 10- to 20-fold higher submucosal tumor invasion rates and higher rates of IL-11 and MMP-13 protein synthesis compared to gp130757F/F mice. They also found that treatment with recombinant IL-11 stimulated the expression of MMP-13 and MMP-9 in stomachs of wild-type mice. However, the expression of MMP-13 was largely restricted to tumor-associated stroma and was not detected in epithelial cells. It is unclear whether IL-11 can increase the expression of MMP-13 in human gastric cancer cells and via which mechanism. In this study, we employed a gastric cancer cell model to investigate the relationship between IL-11 and MMP-13.
Materials and methodsReagents and cell culture
Gastric cancer cell lines (BGC823, MGC803, SGC7901 and MKN45) were obtained from the China Center for Type Culture Collection (CCTCC, Wuhan, China). The gastric cancer cell line SCH was obtained from the Human Health Resources Bank (Osaka, Japan). All the cells were maintained in RPMI-1640 medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum at 37°C in 5% CO2.
To elucidate the effect of IL-11 on the regulation of MMP-13, we treated the cells with different concentrations (10, 50, 100 and 200 ng/ml) of recombinant human (rh) IL-11 (R&D Systems, Minneapolis, MN, USA) after starving them in medium with reduced serum (OPTI-MEM-I; Invitrogen Gaithersburg, MD, USA) for 24 h. In the time-course assays, cells treated with 100 ng/ml of rh IL-11 were harvested at 24, 48 and 72 h post-treatment and were used to extract proteins as described below.
To elucidate the mechanism by which IL-11 regulates the expression of MMP-13, we used the PI3K inhibitor wortmannin and RNA interference experiments targeting the STAT3 gene. The cells were classified into three groups: cells treated with wortmannin, cells transfected with small interfering RNA (siRNA) targeting STAT3, and cells treated with both wortmannin and siRNA-STAT3. The cells were first starved in medium with reduced serum for 24 h prior to treatment with rh IL-11. Wortmannin (Sigma-Aldrich, St. Louis, MO, USA) was added 30 min prior to IL-11 at a concentration of 100 nM. The siRNA-STAT3-treated group was transfected with the siRNA-STAT3 for 24 h (as described below), and then treated with 100 ng/ml IL-11. The cells were collected at 24, 48 and 72 h post-treatment and used to extract proteins as described below.
siRNA-mediated gene silencing of STAT3
The STAT3-targeting siRNA construct was designed by Shanghai GenePharma Co., Ltd. (Shanghai, China). The sequence of the sense strand primer for specific targeting of the STAT3 gene was 5′-AACAUCU GCCUAGAUCGGCUATT-3′ and the antisense strand sequence was 5′-UAGCCGAUCUAGGCAGAUGTT-3′. The primers for the negative control double-strand (ds) RNA were as follows: 5′-UUCUCCGAACGUGUCACGUTT-3′ (forward) and 5′-ACG UGACACGUUCGGAGAATT-3′ (reverse). Transfection of cells with siRNA-STAT3 was achieved with Lipofectamine® 2000 (Invitrogen) in 6-well plates, following the manufacturer’s instructions. Cells that had reached the exponential growth phase were plated in 6-well plates at a density of 2×105 cells/ml, cultured for 24 h and transfected with 1 μg of siRNA in reduced serum medium at 30–50% confluence, according to the manufacturer’s protocol.
Western blot analysis
The cells were suspended in RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 0.05% SDS, pH 7.4). The supernatant was collected, and the protein concentration was quantified using a protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). After boiling, the proteins (25 μg) were separated by polyacrylamide gel electrophoresis (PAGE) under denaturing conditions, and transferred to a Hybond enhanced chemiluminescence (ECL) nitrocellulose membrane (Amersham Biosciences, Arlington Height, IL, USA). The membranes were blocked with 5% low-fat dried milk in TBS containing 0.1% Tween-20 (TBS-T), and then incubated for 1 h at room temperature with 1:500 dilutions of the anti-human anti-MMP-13, -p-Akt1/2/3, -p-p38 MAPK, -p-STAT3, -p-ERK1/2 and -IL-11 antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). We used the anti-human antibody for β-actin (N-21; Santa Cruz Biotechnology, Inc.) as an indicator of the loaded amounts of protein. The membranes were incubated for 1 h with a 1:1,000 dilution of horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin (sc-45106; Santa Cruz Biotechnology, Inc.). The membranes were developed with a horseradish peroxidase chemiluminescence detection reagent (ECL Plus system), and exposed on Hyperfilm ECL (both from Amersham Biosciences). Experiments were performed in triplicate.
Statistical analysis
The statistical software SPSS 13.0 (SPSS Inc., Chicago, IL, USA) was used for data analysis. Numerical results for different groups were expressed as mean ± standard deviation (SD). Statistical analysis was performed using a one-way ANOVA and Fisher’s least significant difference (LSD) tests to compare individual groups. P<0.05 was considered to indicate statistically significant differences.
Resultsrh IL-11 increases the expression of MMP-13 in the gastric carcinoma cell line SCH
The gastric cancer cell SCH was selected since IL-11 was expressed at a relatively low level in this line compared to the other gastric cancer cell lines (Fig. 1). Following treatment of SCH cells with four different concentrations of rh IL-11 (10, 50, 100 and 200 ng/ml), the MMP-13 level significantly increased in a concentration-dependent manner (Fig. 2). After treatment of SCH cells with 100 ng/ml of rh IL-11 at three different time-points (24, 48 and 72 h), MMP-13 protein expression was again found significantly increased in a time-dependent manner (P<0.05) (Fig. 3).
p-STAT3 levels are increased upon rh IL-11 stimulation
In SCH cells, the p38 MAPK protein was not phosphorylated in unstimulated cells, and rh IL-11 did not influence its phosphorylation state. ERK1/2/3 was detected in phosphorylated form in unstimulated cells, and rh IL-11 did not influence its phosphorylation state (Fig. 4). The AKT1/2/3 and STAT3 proteins were detected in phosphorylated form from the first 15 min of stimulation with rh IL-11, and for at least 1 h.
Wortmannin and siRNA-STAT3 synergistically reduce the expression of MMP-13 induced by rh IL-11
Wortmannin, a PI3K inhibitor, was added to the SCH cell culture prior to rh IL-11 addition. The MMP-13 protein level was semi-quantitatively assessed by western blot analysis at three time-points post-treatment (24, 48 and 72 h). Wortmannin reduced the expression of MMP-13 in all time-points. Similarly, following transfection with siRNA-STAT3, the expression of MMP-13 was reduced at each time-point. Notably, when used in combination with siRNA-STAT3, wortmannin reduced the expression of MMP-13 up to ~80% (Figs. 5 and 6).
Discussion
In the present study, we have demonstrated that exogenous IL-11 directly affects the expression of the MMP-13 in the SCH cell line. The expression of MMP-13 was inhibited by siRNA-STAT3 and wortmannin, suggesting that PI3K-AKT and JAK-STAT3 signal transduction pathways may be involved in the increased expression of MMP-13 following rh IL-11 treatment.
IL-11 is upregulated in mouse and human gastric cancer cells and has been associated with an aggressive phenotype and poor prognosis in gastric adenocarcinoma (10,21). However, whether and via which mechanism(s) IL-11 may contribute to tumor progression is not known. MMPs play an important role in the dissemination of cancer cells by breaking down the ECM and creating an environment that supports the initiation and maintenance of tumor growth. MMP-13 is detected in numerous invasive malignant tumor types and exhibits a broad spectrum of activities on connective tissue components. In addition to fibrillar collagens and gelatin, MMP-13 degrades type IV, IX, X and XIV collagens, the large tenascin C isoform, fibronectin, laminin, aggrecan, core protein, fibrillin-1 and serine proteinase inhibitors (22,23). Since MMP-13 widely degrades components of the basement membrane and tumor cells enveloping connective tissue, it is likely to play crucial roles in modulating extracellular matrix degradation and cell matrix interactions involved in metastasis. In this study, rh IL-11 increased the expression of MMP-13 in a concentration- and time-dependent manner, suggesting that IL-11 promotes gastric cancer progression partly by induction of MMP-13.
The signaling pathway(s) triggered by IL-11 are activated by its receptor, IL-11RA, which utilizes gp130 receptor as a common subunit such as IL-6, leukemia inhibitory factor to determine signaling. Signal transduction is dependent on co-expression of IL-11RA and the gp130 receptor common subunit (24). The major signaling pathways that are activated upon IL-11 stimulation are JAK-STAT3, Ras-MAPK and PI3K-AKT (6,25). We hypothesize that these pathways are responsible for IL-11-induced gastrointestinal cancer growth and metastasis, involving angiogenesis and ECM degradation. MMP-13 is regulated via the PI3K-AKT pathway in chondrosarcoma (26) and breast cancer cells (27), but not in laryngeal and hypopharyngeal squamous cells (28). MMP-13 is also regulated by p38 MAPK (29), MAPK-ERK (28) and SMAD (30) in different cancer cells. In this study, the PI3K inhibitor wortmannin reduced the expression of MMP-13 that was induced by IL-11. Similarly, siRNA-STAT3 reduced MMP-13 expression, suggesting that PI3K-AKT and JAK-STAT3 pathways are involved in signal transduction triggered by IL-11. Notably, siRNA-STAT3 and wortmannin acted synergistically and almost completely eliminated MMP-13 expression. By contrast, p38 MAPK was not detected in SCH gastric cancer cells treated or not treated with rh IL-11. We can thus not conclude on its involvement in MMP-13 regulation.
In summary, IL-11 significantly induced MMP-13 expression. This induction may be mediated by the PI3K-AKT and JAK-STAT3 signal transduction pathways, and therefore, these pathways may be considered for therapeutic treatment of metastatic gastric cancer.
Acknowledgements
This study was supported by grants from the Guangdong Provincial Science & Technology Projects (nos. 2010B031600243, 2011B05040009 and 2012B050600020) and the National Natural Science Foundation of China (no. 81272761).
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Levels of endogenous interleukin (IL)-11 in five gastric cancer cell lines.
Effects of different concentrations of recombinant human interleukin (rh IL)-11 on the protein expression of matrix metalloproteinase-13 (MMP-13) in SCH cells.
Effects of 100 ng/ml of recombinant human interleukin (rh IL)-11 on the protein expression of matrix metalloproteinase-13 (MMP-13) in SCH cells at three different time-points (24, 48 and 72 h) post-treatment.
Effects of treatment of gastric carcinoma cell lines with recombinant human interleukin (rh IL)-11 on different signaling pathways. The cells were treated with 100 ng/ml of rh IL-11 for 5 min. AKT1/2/3 and STAT3 proteins were detected in phosphorylated form for 1 h.
In vitro silencing of STAT3 reduces the expression of matrix metalloproteinase 13 (MMP-13) induced by recombinant human interleukin (rh IL)-11.
Wortmannin reduces the expression of matrix metalloproteinase-13 (MMP-13), induced by recombinant human interleukin (rh IL)-11.