STAT3 Signaling in Pancreatic Cancer Figure 6. EGCG and gemcitabine inhibit cell viability and STAT3 target genes. (A), AsPC-1 and PANC-1 cells were treated with EGCG (0, 20, 40, 60 mM) with or without gemcitabine (0.5 mM) for 72 h. Cell viability was measured by XTT assay. Data represent mean 6 SD. * or ** = significantly different from respective controls, P,0.05. (B), AsPC-1 and PANC-1 cells were treated with EGCG (0, 20, 40, 60 mM) with or without gemcitabine (0.5 mM) for 72 h. Apoptosis was measured by TUNEL assay. Data represent mean 6 SD. * or ** = significantly different from respective controls, P,0.05. (C), Inhibition of STAT3 target genes by EGCG and gemcitabine. AsPC-1 and PANC-1 cells were treated with EGCG (20 mM) or gemcitabine (0.5 mM) for 48 h. The expression of VEGF, c-Myc, survivin and cyclin D1was was measured by qRT-PCR. Data represent mean 6 SD. * = significantly different from respective controls, P,0.05. doi:10.1371/journal.pone.0031067.g006 lines. Furthermore, the combination of EGCG with gemcitabine induced significantly more caspase-3 cleavage than single agent alone (Fig. 1D). EGCG and gemcitabine alone showed PARP cleavage in AsPC-1 and PANC-1 cells. By comparison, the combination of EGCG with gemcitabine resulted in an enhanced PARP cleavage. These data suggest that EGCG can induce apoptosis by caspase-3 activation and PARP cleavage. EGCG inhibits JAK3/STAT3 pathway in pancreatic cancer We next examined the effects of EGCG on expression of STAT3, phosphorylation of STAT3 and JAK3, and nuclear expression of phospho-STAT3 in AsPC-1 and PANC-1 cells. To examine the effects of EGCG on STAT3 expression, we performed qRT-PCR analysis (Fig. 2A). EGCG inhibited the expression of STAT3 mRNA in both AsPC-1 and PANC-1 cells. We next measured the expression of total and phosphorylated JAK3 and STAT3 by the Western blot analysis (Fig. 2B). EGCG inhibited the phosphorylation of both JAK3 and STAT3 in a dose-dependent manner in AsPC-1 and PANC-1 cells. Surprisingly, the expression of both JAK3 and STAT3 was also inhibited by EGCG. These data suggest that EGCG can inhibit the expression of JAK3 and STAT3, as well as their post-translation modification. Since STAT3 is constitutively active in pancreatic cancer cells, we next examined the effects EGCG on STAT3 expression by immunofluorescence. As shown in Fig. 2C, EGCG inhibited the expression of STAT3 (presence of the green color) in a dose- dependent manner in both the cell lines. These data suggest that inhibition of apoptosis by EGCG is associated with suppression of JAK3/STAT3 pathway. EGCG inhibits STAT3 transcription and expression of STAT3-regulated genes STAT3 is considered as an oncogene because it is correlated with tumorigenesis [7]. Therefore, it is necessary to determine the effect of EGCG on STAT transcriptional activity. Pancreatic cancer cells were transfected with pGreen fire1-STAT3 reporter plasmid and treated with EGCG (0–80 mM). After incubation of 24 h, luciferase activity was determined by reporter assay. As shown in Fig. 3A, EGCG inhibited STAT3 transcriptional activity in a dose-dependent manner in pancreatic cancer AsPC-1 and PANC-1 cells. PLoS ONE | www.plosone.org 7 February 2012 | Volume 7 | Issue 2 | e31067