HK Inhibits Angiogenesis/Peritoneal Dissemination Figure 6. Effect of honokiol on STAT-3 phosphorylation in gastric cancer cells and HUVECs. AGS (A) and MKN45 (B) quiescent human gastric cancer cells and HUVECs (C) were treated with and without honokiol (HK, 5–40 mM) for the indicated times. Whole cell lysates were prepared and analyzed by Western blotting for detection of phosphorylated STAT-3 (pSTAT-3, Tyr705). The blottings were reprobed with anti-STAT-3 antibodies for normalization. The results are representative of at least five independent experiments. doi:10.1371/journal.pone.0043711.g006 using the methods of co-immunoprecipitation and Western blotting. As shown in Figure 11C, SHP-1 was specifically associated with STAT-3 in AGS cells and HUVECs in the presence of honokiol as compared with IgG control. Honokiol Altered ER Morphology, Induced Calpain-II- regulated SHP-1-induced STAT-3 Dephosphorylation, and Decreased VEGF Generation Previous studies have suggested that ER stress plays an important role in angiogenesis [20,21]. We next examined the effect of honokiol on ER microstructure by transmission electron microscopy. As shown in Figure 12A, ER dilation and fragmen- tation were exhibited in honokiol-treated SCM-1 gastric cancer cells and HUVECs. We also wanted to determine whether calpain-II activation is required for SHP-1 activity in honokiol-treated cells. Pharmaco- logical calpain inhibitor and calpain-II siRNA transfection effectively reduced honokiol-enhanced SHP-1 activity in AGS cells and HUVECs (Fig. 12B). Cells treated with recombinant calpain-II, as a positive control, showed a 4 to 5-fold increase in SHP-1 activity (Fig. 12B). VEGF is known to contain the STAT-3 promoter binding site. We next examined whether honokiol can regulate VEGF expression. Honokiol significantly reduced the VEGF generation in AGS cells and HUVECs, and this effect was Figure 7. Quantification of honokiol-inhibited STAT-3 phos- phorylation in gastric cancer cells and HUVECs. AGS (A) and MKN45 (B) quiescent human gastric cancer cells and HUVECs (C) were treated with and without honokiol (HK, 5–40 mM) for the indicated times. Whole cell lysates were prepared and analyzed by Western blotting for detection of phosphorylated STAT-3 (pSTAT-3, Tyr705). The blottings were reprobed with anti-STAT-3 antibodies for normalization. All data are presented as mean 6 SEM of five independent experiments. *p,0.05 as compared with control. doi:10.1371/journal.pone.0043711.g007 reversed by SHP-1 siRNA transfection (Fig. 12C). In addition, transfection of mutant-STAT-3 plasmid into cells also inhibited the VEGF generation. The combination of honokiol with mutant- STAT-3 plasmid transfection in cells synergistically reduced the VEGF production as compared with either treatment alone (Fig. 12C). To further confirm the interaction between calpain-II and SHP-1, co-immunoprecipitation and Western blotting were performed in gastric cancer cells. As shown in Figure 12D, PLOS ONE | www.plosone.org 8 August 2012 | Volume 7 | Issue 8 | e43711