STAT3 Signaling in Pancreatic Cancer Figure 8. EGCG and CP690550 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 CP690550 (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 CP690550 (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 CP690550. AsPC-1 and PANC-1 cells were untreated or treated with EGCG (20 mM) or CP690550 (0.5 mM) for 48 h. The expression of VEGF, c-Myc, survivin and cyclin D1 was measured by qRT-PCR. Data represent mean 6 SD. * = significantly different from respective controls, P,0.05. doi:10.1371/journal.pone.0031067.g008 CP690550 inhibited cell viability and induced apoptosis in both AsPC-1 and PANC-1 cell lines. Interestingly, EGCG further enhanced the effects of CP690550 on cell viability and apoptosis in both the cell lines. These data suggest that CP690550 can be combined with EGCG to target pancreatic cancer cells. Since EGCG and CP690550 inhibited cell viability and induced apoptosis, we next sought to examine their effects on STAT3 target genes. EGCG and CP690550 inhibited the expression VEGF, c-Myc, survivin and cyclin D1was in both the cell lines (Fig. 8C). The combination of EGCG and CP690550 had additive effects on these target genes. These data suggest that EGCG can enhance the therapeutic potential of CP690550 in pancreatic cancer cells by inhibiting STAT3. Discussion Pancreatic cancer belongs to the group of extremely aggressive human cancers; conventional treatments have little impact. In 2010, it accounts for only 3% of new cancer cases in the United States, and the fourth leading cause of cancer death. Only 6 percent of patients will survive 5 years after diagnosis [38]. Various factors, which include its aggressive nature, lack of early screening, absence of therapeutic targets, and lack of effective treatments, make the pancreatic cancer become one of the most difficult cancers to treat. In recent years, the clinicians and cancer scientists have made some significant advances into the management of the disease, especially discovery and development of targeted therapeutics [39]. Many oncogenic molecular pathways including EGF/EGFR, Ras-Raf-MEK, PI3K/Akt, JAK/STAT, p16INK4A/retinoblas- toma, Smad4/TGF-b, and hedgehog signaling pathways, have been reported to be involved in the pathogenesis of pancreatic cancer [40,41,42,43,44,45]. Among them, STAT3 is thought by many researchers as a suitable therapeutic target for drug discovery because constitutive activation of STAT3 alone is sufficient to induce the relevant disease, the inhibition of STAT3 signaling could suppress and reverse the development of relevant disease, and the molecular mechanism of tumorigenesis caused by STAT3 pathway has been well defined. Chemoprevention was first defined by Sporn in 1976 and refers to the use of natural or synthetic agents to reverse, suppress or PLoS ONE | www.plosone.org 9 February 2012 | Volume 7 | Issue 2 | e31067