STAT3 Signaling in Pancreatic Cancer Figure 1. Effects of EGCG on pancreatic cancer cells. (A), Transwell migration assay. AsPC-1 and PANC-1 cells were plated in the top chamber of the transwell and treated with EGCG (0–60 mM) for 24 h. Cells migrated to the lower chambered were fixed with methanol, stained with crystal violet and counted. Data represent mean 6 SD. * or ** = significantly different from respective controls, P,0.05. (B) Matrigel invasion assay. AsPC-1 and PANC-1 cells were plated onto the Matrigel-coated membrane in the top chamber of the transwell and treated with EGCG (0–60 mM) for 48 h. Cells invaded to the lower chamber were fixed with methanol, stained with crystal violet and counted. Data represent mean 6 SD. * or ** = significantly different from respective controls, P,0.05. (C), Caspase-3 activity. AsPC-1 and PANC-1 cells were treated with EGCG (0–40 mM) for 48 h, and the caspase-3 activity was measured as per manufacturer’s instructions (Invitrogen). Data represent mean 6 SD. * = significantly different from respective controls, P,0.05. (D), AsPC-1 and PANC-1 cells were treated with EGCG (0–60 mM) with or without gemcitabine (0.5 mM) for 48 h. Cells were harvested and the Western blot analysis was performed to examine the expression of PARP and caspase-3. b-actin was used as a loading control. PARP antibody recognizes cleaved PARP, and caspase-3 antibody recognizes cleaved/active caspase-3. doi:10.1371/journal.pone.0031067.g001 members JAK1, JAK2, JAK3 and tyrosine kinase 2 [13,14]. Additionally, the STAT3 phosphorylation can also be mediated by crosstalk with c-Src kinase [13,14,15]. The major phosphorylation sites in STAT3 include tyrosine and serine residues at positions Tyr705 and Ser727, respectively, located in the transactivation domain. The activation of STAT3 results in expression of many target genes required for tumor cell survival (e.g. Bcl-XL, Mcl-1 and survivin), proliferation (e.g. cyclin D1 and c-myc) and angiogenesis [e.g. vascular endothelial growth factor (VEGF)] as well as metastasis [16]. Thus, STAT3-signaling pathway has been a favorite therapeutic target for drug development [17,18]. Gemcitabine (a nucleoside analog) showed more clinical benefit on pancreatic cancer patients compared with the conventional medications [19]. Some potent and selective JAK3 inhibitors, e.g. CP690550, demonstrated significant clinical activity in cancer [20,21]. CP690550 represents only a starting point in the search for a safer small molecule immunosuppressant, and that an isozyme-selective JAK3 inhibitor identified by rational drug design might be substantially safer. In recent years, many new insights have been gained into the investigation on a variety of purified compounds from natural products. For instance, EGCG is the major catechin from green tea and has been recognized as an important chemopreventive agent and as modulators of tumor cell response to chemotherapy [22,23,24]. It has been shown to inhibit cell proliferation [25], induce apoptosis [26] in tumor cells, prevent angiogenesis [27], modulate the invasion and migration of cancers, and interfere with multiple signaling pathways, including the nuclear factor-kB signaling pathway [28], epidermal growth factor-mediated pathway [29], insulin-like growth factor-I signal- ing pathway [30], mitogen-activated protein kinase-dependent pathway [31], and proteasome degradation pathway [32]. In this paper, we examined the effects of EGCG on STAT3 signaling in human pancreatic cancer cells, and also assessed the interactive effects of EGCG with gemcitabine or JAK3 inhibitor CP690550 on their therapeutic potential. We found that EGCG inhibited the expression of JAK3 and STAT3 (phospho and total), STAT transcription and activation, and the expression of STAT3- regulated genes, resulting in the inhibition of cell motility, migration and invasion, and the induction of caspase-3 and PARP cleavages. Inhibition of STAT3 by shRNA in pancreatic cancer PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e31067