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

Abstract

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 Capan-2 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 MMP-2 genes in pancreatic cancer cells. The Stat3 signaling pathway may provide a novel therapeutic target for treatment of pancreatic cancer.

Figure 1

Pancreatic cancer cell growth was detected by MTT assay. SW1990 and Capan-2 cells growing in 96-well plates were treated with AG490 and interleukin-6 (IL-6), respectively, for 24, 48 and 72 hours. Incubation with 20 μM/L AG490 for 72 hours markedly reduced proliferation of SW1990 cells (P = 0.000), but incubation with 20 μM/L AG490 for 24, 48 hours did not reduce proliferation of SW1990 cells (P = 0.051, P = 0.060). Incubation with 100 ng/ml IL-6 for 48 and 72 hours increased proliferation of Capan-2 cells significantly (P = 0.001, P = 0.000) , but incubation with 100 ng/ml IL-6 for for 24 hours did not increase proliferation of SW1990 cells (P = 0.073). Data are mean ± SD of 8 wells. A = Absorbance.

Figure 2

VEGF and MMP-2 mRNA levels in SW1990 and Capan-2 cells were detected by real time PCR. The extracted total RNA was reverse-transcribed into single-stranded cDNA, and real-time PCR was performed. Interleukin-6 (IL-6) markedly increased MMP-2 and VEGF mRNA expression in Capan-2 cells(P = 0.000, P = 0.000). AG490 significantly decreased MMP-2 and VEGF mRNA expression in SW1990 cells(P = 0.008, P = 0.000). β-actin was used as an endogenous control. * P < 0.01, versus Capan-2 cell group; #P < 0.01, versus SW1990 cell group.

Figure 3

p-Stat3 protein expression was detected by immunocytochemistry. Immunocytochemical staining showed that p-Stat3 was mainly expressed in the nucleus and weakly expressed in the cytoplasm of SW990 cells and Capan-2 cells. Expression of p-Stat3 protein in Capan-2 cells (A) and SW1990 cells (C). After treatment with interleukin-6 (IL-6) for 24 hours on Capan-2 cells (B), we observed that the intensity of p-Stat3 expression increased(P = 0.012). After treatment with AG490 for 24 hours on SW1990 cells (D), we observed that the intensity of p-Stat3 expression decreased (P = 0.006) (original magnification, ×400). (E) Integrated optical density of every group. Bars indicate mean ± SD. * P < 0.01, versus Capan-2 cell group; # P < 0.01, versus SW1990 cell group.

Figure 4

Stat3, p-Stat3, MMP-2 and VEGF protein expression in SW1990 and Capan-2 cells were detected by Western blotting. Protein samples extracted from SW1990 and Capan-2 cells treated for 24 hours with AG490 and interleukin-6 (IL-6), respectively, were subjected to western blotting for Stat3, p-Stat3, MMP-2, VEGF and β-actin proteins. AG490 and IL-6 did not affect total Stat3 protein levels. AG490 decreased p-Stat3, MMP-2 and VEGF protein expression in SW1990 cells(P = 0.010, P = 0.000, P = 0.009). IL-6 markedly increased p-Stat3, MMP-2 and VEGF protein expression in Capan-2 cells(P = 0.000, P = 0.011, P = 0.005). The levels of β-actin expression were determined as a control for equivalent protein loading.

Figure 5

The invasion assay was performed using a specialized invasion chamber. The invasion chamber included a 24-well tissue-culture plate with 12 cell-culture inserts. The blue-stained cells are those that invaded the basement membrane matrix (ECMatrix) and migrated through the polycarbonate membrane to the lower surface of the membrane. The invasion assay indicated that interleukin-6 (IL-6) significantly increased the invasion ability of Capan-2 cells (A, B) (P = 0.004), and AG490 markedly reduced invasion of SW1990 cells (C, D) (P = 0.010) (original magnification ×200). (E) Effects of AG490 and IL-6 on invasion ability of pancreatic cancer cells. Bars indicate mean ± SD. * P < 0.05, versus Capan-2 cell group; #P < 0.01, versus SW1990 cell group.