Figure 7. CP690550 inhibits cell viability of AsPC-1 and PANC-1 cells. (A), AsPC-1 cells were seeded in 96 well plates at 46104 cells per well and treated with CP690550 (0–15 mM) for 72 h. Cell viability was measured by XTT assay. Data represent mean 6 SD. * = significantly different from control, P,0.05. (B), PANC-1 cells were seeded in 96 well plates at 46104 cells per well and treated with CP690550 (0–15 mM) for 72 h. Cell viability was measured by XTT assay. Data represent mean 6 SD. * = significantly different from control, P,0.05. doi:10.1371/journal.pone.0031067.g007 Previous studies have shown that STAT3 can regulate the expression of many gene products involved in proliferation, cell survival, angiogenesis and anti-apoptosis [7]. For instance, VEGF, Bcl-XL, c-Myc, Survivin and Cyclin D1 are regulated by STAT3 activation [17,36]. As illustrated in Fig. 3B, STAT3-regulated genes, c-Myc, Survivin and Cyclin D1 were inhibited by EGCG in AsPC-1 and PANC-1 cells. EGCG also decreased the expression of VEGF in AsPC-1 cells, and Bcl-XL in PANC-1 cells. These data demonstrate that EGCG can inhibit the expression of STAT3- regulated genes in pancreatic cancer cells. These STAT-3 target genes have been shown to regulate cell proliferation, cell cycle, apoptosis and angiogenesis. Inhibition of STAT3 enhances the inhibitory effects of EGCG on cell motility and viability in pancreatic cancer cells In this work, STAT3 shRNA was used to silence STAT3 gene expression in AsPC-1 and PANC-1 cells. STAT3 shRNA inhibited the expression of STAT3 in both AsPC-1 and PANC-1 cells as demonstrated by the Western blot analysis (Fig. 4A). To examine the effects of EGCG on pancreatic cancer cells, scratch and cell viability assays were performed using both scrambled and STAT3 shRNA cells. In scratch assay, the inhibitory effects of EGCG on the migration of AsPC-1 cells were enhanced by inhibiting STAT3 gene expression (Fig. 4B). EGCG and STAT3 shRNA alone reduced percent of viable AsPC-1 and PANC-1 cells in a dose- dependent manner (Fig. 4C). Interestingly, the inhibitory effects of EGCG on cell viability were further enhanced by STAT3 shRNA in both the cell lines. STAT3 Signaling in Pancreatic Cancer We next examined the effects of STAT3 shRNA on the regulation of STAT3-target genes by EGCG. EGCG inhibited the expression of STAT3-regulated gene Cyclin D1 in both AsPC- 1/scrambled and PANC-1/scrambled cells (Fig. 5 and B). STAT3 shRNA completely inhibited the expression of cyclin D1 in both pancreatic cancer cell lines in the presence or absence of EGCG. EGCG also inhibited he expression of Bcl-XL and c-Myc in PANC- 1/scrambled cells. Bcl-XL and c-Myc were also inhibited in PANC- 1/STAT3 shRNA cells compared to PANC-1/Scrambled cells. However, EGCG was unable to further inhibit the expression of Bcl-XL and c-Myc in PANC-1/STAT3 shRNA cells. These data suggest that EGCG can regulate pancreatic cancer cell motility and viability which are associated with STAT3 pathway. Gemcitabine synergizes with EGCG to inhibit cell viability and induce apoptosis in pancreatic cancer cells Gemcitabine has emerged as a popular chemotherapeutic agent in the treatment of advanced and metastatic pancreatic cancer, and the benefit of this single-agent is small but significant in the improvement of median overall survival [19]. To test the effects of gemcitabine with EGCG on cell viability, pancreatic cancer cells were treated with gemcitabine (0.5 mM) with or without increasing concentrations of EGCG (0–60 mM) for 72 hours. As shown in Fig. 5A, cell viability was inhibited by EGCG and gemcitabine alone, and the inhibitory effects of gemcitabine on cell viability were further enhanced by EGCG in these two cell lines (Fig. 6A). We next examined the interactive effects of EGCG with gemcitabine on apoptosis in both AsPC-1 and PANC-1 cell lines (Fig. 6B). EGCG induced apoptosis in both the cell lines in a dose- dependent manner. Similarly, gemcitabine induced apoptosis in both AsPC-1 and PANC-1 cells. All the doses of EGCG further enhanced the effects of gemcitabine on apoptosis. These data suggest that EGCG can be combined with gemcitabine to treat pancreatic cancer patients. We next examined the effects of EGCG and gemcitabine on the expression of c-Myc and cyclin D1 in AsPC-1 and PANC-1 cells (Fig. 6C). EGCG and gemcitabine alone inhibited the expression of VEGF, c-Myc, survivin and cyclin D1was in both the cell lines. The combination of EGCG and gemcitabine had additive effects on these target genes. These data suggest that EGCG can enhance the therapeutic potential of gemcitabine in pancreatic cancer cells by inhibiting STAT3. JAK3 inhibitor CP690550 inhibits cell viability in pancreatic cancer cells CP690550 is a novel JAK3 inhibitor and expected to target JAK3, which is expressed generally only in immune cells and is only bound by gamma-chain-bearing cytokine receptors involved in the JAK/STAT signaling pathway [37]. We next examined the effects of CP690550 on cell viability in AsPC-1 and PANC-1 cells (Fig. 7). CP690550 inhibited cell viability in both the cell lines in a dose- dependent manner. These data suggest that CP690550 can be a potential anticancer drug for the treatment of pancreatic cancer. CP690550 synergizes with EGCG to inhibit cell viability in pancreatic cancer cells Since CP690550 induced apoptosis in pancreatic cancer cells, we next sought to examine the interactive effects of CP690550 and EGCG on cell viability and apoptosis of pancreatic cancer cells (Fig. 8). To test the effects of CP690550 with EGCG on cell viability and apoptosis, pancreatic cancer cells were treated with CP690550 (0.5 mM) and increasing concentrations of EGCG (0– 60 mM) for 72 hours. As shown in Fig. 8A and B, EGCG and PLoS ONE | www.plosone.org 8 February 2012 | Volume 7 | Issue 2 | e31067