STAT3 Signaling in Pancreatic Cancer Figure 5. Effects of STAT3 shRNA on the regulation of cyclin D1, Bcl-XL and c-Myc by EGCG. (A), AsPC-1/scrambled and AsPC-1/STAT3 shRNA cells were treated with or without EGCG (60 mM) for 48 h. The expression of cyclin D1 was measured by q-RT-PCR. Data represent mean 6 SD. * = significantly different from respective controls, P,0.05. (B), PANC-1/scrambled and PANC-1/STAT3 shRNA cells were treated with or without EGCG (60 mM) for 48 h. The expression of cyclin D1 was measured by q-RT-PCR. Data represent mean 6 SD. * = significantly different from respective controls, P,0.05. (C), PANC-1/scrambled and PANC 1/STAT3 shRNA cells were treated with or without EGCG (60 mM) for 48 h. The expression of Bcl-XL was measured by qRT-PCR. Data represent mean 6 SD. * = significantly different from respective controls, P,0.05. (D), PANC-1/scrambled and PANC- 1/STAT3 shRNA cells were treated with or without EGCG (60 mM) for 48 h. The expression of c-Myc was measured by q-RT-PCR. Data represent mean 6 SD. * = significantly different from respective controls, P,0.05. doi:10.1371/journal.pone.0031067.g005 Statistical analysis The mean and SD were calculated for each experimental group. Differences between groups were analyzed by one or two way ANOVA using PRISM statistical analysis software (GrafPad Software, Inc., San Diego, CA). Significant differences among groups were calculated at P,0.05. Results EGCG inhibits migration and invasion of pancreatic cancer cells, and induces caspase3 activity It has been demonstrated that STAT3 plays an important role in regulating cell movement by controlling cytoskeleton reorganization and cell adhesion properties [33]. Due to the correlation between STAT3 and cell movement, we examined the effects of EGCG on the migration and invasion of AsPC-1 and PANC-1. Cells were plated in the top of chamber onto the noncoated membrane and the Matrigel coated membrane for migration and invasion detection, respectively. After the treatments of EGCG, the migrated and invaded cells were stained and counted. The results show that the migrated and invaded pancreatic cells reduced in a dose-dependent manner (Fig. 1A and B). These data suggested that EGCG can inhibit the migration and invasion of pancreatic cancer cells. Caspase-3 is a member of the cysteine-aspartic acid protease (caspase) family and activated in the apoptotic cell both by extrinsic (death ligand) and intrinsic (mitochondrial) pathways [34]. EGCG induced caspase-3 activity in a dose dependent manner in both AsPC-1 and PANC-1 cells, as measured by fluorometric assay (Fig. 1C). These data suggest that EGCG can induce apoptosis by activating caspase-3. EGCG enhances gemcitabine-induced cleavage of caspase3 and PARP in pancreatic cancer cells PARP is normally involved in DNA repair, DNA stability, and other cellular events, and cleaved by members of the caspase family during early apoptosis; therefore, it is a substrate for caspase activity and a reliable marker of apoptosis [35]. We next examined whether EGCG and gemcitabine interact together to cleave caspase-3 and PARP in AsPC-1 and PANC-1 cells. EGCG and gemcitabine alone induced cleavage of caspase-3 in both the cell PLoS ONE | www.plosone.org 6 February 2012 | Volume 7 | Issue 2 | e31067