Effects of IL-6 and AG490 on regulation of Stat3 signaling pathway and invasion of human pancreatic cancer cells in vitro

Journal of Experimental & Clinical Cancer Research REefsefaercch ts of IL-6 and AG490 on regulation of Stat3 signaling pathway and invasion of human pancreatic cancer cells in vitro Chen Huang 0 Guang Yang 1 Tao Jiang 0 Kejian Huang 0 Jun Cao 0 Zhengjun Qiu 0 0 Department of General Surgery, Affiliated First People's Hospital, Shanghai Jiao Tong University , Shanghai, 200080 , PR China 1 Department of General Surgery, Taian central hospital , Shandong, 27100 PR China Background: Signal transducer and activator of transcription 3 (Stat3) is a member of the Janus-activated kinase(Jak)/ Stat signaling pathway. Abnormal activation of Stat3 plays a critical role in metastasis and invasion in varieties of human tumors including pancreatic cancer. This study aimed to investigate the mechanisms of activation and blocking of the Stat3 signaling pathway and its effects on invasion and metastasis of human pancreatic cancer cells. Methods: The Jak inhibitor AG490 and interleukin-6 (IL-6) were added to the culture media of human pancreatic cancer cells SW1990 and Capan-2, respectively. Cell growth was measured by MTT assays. Western blotting and immunocytochemistry were performed to detect phosphorylated Stat3 (p-Stat3) protein, while VEGF and MMP-2 mRNA and protein expression were examined with fluorescence quantitative polymerase chain reaction and Western blotting, respectively. The invasion ability of SW1990 and Capan-2 cells was determined by cell invasion assay. Results: Stat3 was activated by IL-6 in Capan-2 cells; protein expression of p-Stat3 was increased significantly in Capan2 cells. IL-6 remarkably promoted the growth of Capan-2 cells (P < 0.05), and VEGF and MMP-2 mRNA and protein expression were increased significantly. Also, IL-6 increased the invasion ability of Capan-2 cells. AG490 inhibited Stat3 activation in SW1990 cells. Western blotting and immunocytochemistry analysis showed that p-Stat3 protein expression was decreased significantly with AG490 treatment in SW1990 cells. AG490 remarkably inhibited the growth of Capan-2 cells (P < 0.05), and VEGF and MMP-2 mRNA and protein expression was decreased significantly. And AG490 decreased the invasion ability of SW1990 cells. Conclusions: Abnormal activation of Stat3 plays an important role in the invasion and metastasis of pancreatic cancer. Activation and blocking of the Stat3 signaling pathway can affect invasion ability and expression of the VEGF and MMP2 genes in pancreatic cancer cells. The Stat3 signaling pathway may provide a novel therapeutic target for treatment of pancreatic cancer. - Introduction Pancreatic cancer is one of the most virulent malignances, with an overall 5-year survival rate of only 3-5% and a median survival time after diagnosis of less than 6 months[1]. This highly lethal disease is usually diagnosed in an advanced stage, when there are few or no effective therapies[2]. Even among patients undergoing a potentially curative resection, the long-term outcome remains unsatisfactory because of early recurrence and metastatic disease[3]. Despite the immensity of the clinical problem, the biology of pancreatic cancer remains only poorly understood. Signal transducer and activator of transcription (Stat) proteins were initially described in the context of regulating physiological cell signaling. An increasing number of studies have implicated Stat protein activation, particularly Stat3, in transformation and tumor progression[4]. Activated Stat3 has been shown to promote cell proliferation, metastasis, and angiogenesis, as well as protect tumor cells from apoptosis by regulating associated genes, such as Bcl-xL, Mcl-1, Bcl-2, Fas, cyclin D1, survivin, c-Myc, VEGF, MMP-2, and MMP-9[5-7]. Recently, accumulating evidence has indicated that abnormalities in the Stat3 pathway are involved in the oncogenesis of several cancers. For example, Scholz [8] and coworkers reported that activation of the Stat3 signaling pathway plays an important role in the progression of pancreatic cancer, and constitutive activation of Stat3 correlates with cell proliferation in stomach adenocarcinoma[9], prostate cancer[10], breast carcinoma[11], and non-small cell lung cancer[12] and also inhibits apoptosis[13,14]. Conversely, inhibition of the Stat pathway suppresses cancer cell growth and invasion and induces apoptosis in various cancers[8,11,15,16]. Jak is responsible for the tyrosine phosphorylation of Stat3 in response to extracellular signals and oncogenes. The newly described Jak inhibitor AG490 blocks the constitutive activation of Stat3[17]. AG490 was used to selectively block the Jak/Stat3 signaling pathway and inhibit activation of Stat3 in colorectal cancer cells[18]. The pleiotropic cytokine interleukin-6 (IL-6) is a major activator of Stat3; IL-6 stimulates the formation of tyrosine-phosphorylated Stat3 (p-Stat3) in cancer cells[19,20]. Through the Jak/Stat3 signaling pathway, IL6 plays an important role in cell proliferation, apoptosis, metastasis, and other biological activities [21]. In the present study, we used AG490 to deplete Stat3 protein in the human pancreatic cancer cell line SW1990 and IL-6 to activate Stat3 protein in the human pancreatic cancer cell line Capan-2; we then investigated the changes in cell proliferation and invasion. We also examined the expression of Stat3 and its active phosphorylated form in human pancreatic cancer cell lines. In addition, we evaluated the changes in matrix metalloproteinase 2 (MMP-2) and vascular endothelial growth factor (VEGF) mRNA and protein expression. Our aim was to demonstrate that the Stat3 signaling pathway may be critical for the invasive behavior of pancreatic tumors. Inhibition of this pathway may offer a novel strategy for pancreatic cancer treatment. The human pancreatic cancer cell lines SW1990 and Capan-2 were obtained from the American Type Culture Collection. Tumor cells were maintained in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal calf serum, 100 units/ml penicillin and 100 g/ mL streptomycin, in a humidified incubator with an atmosphere of 5% CO2 and 95% air at 37C. AG490 (Sigma, St Louis, MO, USA) was dissolved in ethanol, 5 mg/ml, and then diluted with the culture medium for experiments. SW1990 cells were treated with 20 M AG490 for 24 hours. Recombinant IL-6 (Peprotech, Princeton, NJ, USA) was dissolved in 5-10 mmol/L acetic acid to a concentration of 0.1-0.5 mg/ml and then diluted with the culture medium for experiments. Capan-2 cells were treated with 100 ng/mL IL-6 for 24 hours. Cell viability was determined by 3-(4,5-dimethylthiazole2-yl)-2.5-diphenyltetrazolium bromide (MTT) assay. Pancreatic cancer cells were seeded in 96-well culture plates in culture medium. After 24 hours, the medium was changed to fresh culture medium containing either 20 M/L AG490 or 100 ng/ml IL-6. MTT assays were performed 24, 48, and 72 hours after AG490 and IL-6 treatment. At the time of the assay, the cells wer (...truncated)