HK Inhibits Angiogenesis/Peritoneal Dissemination Figure 9. Effects of honokiol on p-STAT-3 expression in tumors and STAT-3 DNA binding activity in gastric cancer cells and HUVECs. (A) Western blotting for detection of p-STAT-3 (Tyr705) proteins in metastatic tumors isolated from mice with or without honokiol (HK) treatment is shown. (B) EMSA was performed for detection of STAT-3 DNA binding activity in AGS, SCM-1 (left panel), and HUVECs (right panel) with or without honokiol treatment. Cells were treated with honokiol for various time courses as indicated. VEGF (20 ng/ml) enhanced the STAT-3 DNA binding activity in HUVECs, and this enhancement was reversed by honokiol (10 mM). 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.g009 calpain-II was specifically associated with SHP-1 in AGS cells in the presence of honokiol (20 mM) as compared with IgG control. Similarly, the honokiol-induced calpain/SHP-1 interaction was also found in HUVECs (data not shown). Discussion Honokiol is a major component of Magnolia officinalis, which is a transitional medicine in Asia [18]. Honokiol possesses anti- oxidative and anti-inflammatory effects in vitro and in vivo [22–25]. Honokiol has been shown to exhibit potent anti-proliferative activity against endothelial cells in vitro and anti-tumor effects against angiosarcoma in nude mice [26]. In this study, we demonstrated for the first time that honokiol is a potent inhibitor of angiogenesis and peritoneal metastasis of gastric cancer. Our research has focused on the effects and possible mechanisms of honokiol on peritoneal metastasis and angiogenesis using PET/ CT, CAM assay, matrigel plug assay, aortic ring endothelial cell sprouting assay, endothelial cell tube formation assay, and in vitro cell experiments. The results showed that honokiol can suppress angiogenesis and peritoneal dissemination through a calpain-II- activated SHP-1 activation signaling cascade. Activation of phosphatase SHP-1 by honokiol further led to the down-regulation of STAT-3 activation and VEGF production, resulting in the inhibition of angiogenesis and peritoneal metastasis. However, the inhibition of angiogenesis and inhibition of metastatic potential of cancer cells by honokiol may be the separate aspects, one being an effect of honokiol on angiogenesis directly and another effect of honokiol on cancer cells. During the past several years, a number of studies have indicated that constitutive activation of the STAT family, especially STAT-3, is associated with cell proliferation, angiogen- esis and metastasis [10,11,27–30]. Increased activation of STAT-3 expression has been found in various tumor-derived cells and samples from human cancer tissues. Deregulated STAT-3 activation has been suggested to play an important role in overexpression of VEGF and increased angiogenic phenotypes in gastric cancer, which may contribute to gastric cancer de- velopment and progression [11]. In the present study, we found that honokiol can effectively inhibit the phosphorylation of STAT- 3 in HUVECs (both Tyr705 and Serine727 sites), human gastric cancer cells (Tyr705 site), and peritoneal metastatic tumors (Tyr705 site). As the oncogenic activation of tyrosine kinases is a common feature in cancers, we focused on the role of Tyr705 phosphorylation of STAT-3. SHP-1 possesses a potential tumor suppressor function and is a negative regulator of the JAK/STAT signaling pathway [15]. It has also been shown that SHP-1 phosphatase can bind to the phosphorylated Y1173 domain, which leads to EGFR dephos- phorylation [31]. Y429 in the cytoplasmic domain of the erythropoietin receptor has been suggested to be the binding site for protein tyrosine phosphatase SH-PTP1, which may play a major role in terminating proliferative signals [32]. SHP-1 has also been shown to be an antagonist of growth-factor signaling in epithelial and hematopoietic cells [15]. SHP-1 was particularly highlighted as a potential physiological antagonist of VEGFR signaling [33]. In endothelial cells, SHP-1 is physically associated with VEGFR2 and is also required for both TNFa-mediated and tissue-inhibitor of metalloproteinases (TIMP)-mediated inhibition of angiogenesis [31]. Thus, SHP-1 activation may have important consequences for the regulation of proliferation and angiogenesis. In this study, we found that honokiol specifically enhanced the expression of SHP-1, but not SHP-2, in gastric cancer cells, endothelial cells, and peritoneal metastatic tumors. These findings suggest that honokiol may inhibit endothelial cell angiogenesis and cancer cell metastasis, which may be through a SHP-1-induced STAT-3 down-regulation pathway. Previous studies have proposed an important role for ER stress in the molecular mechanism of angiogenesis and cancer cell growth [16,20,21]. Koyama and colleagues have also suggested that ER stress-induced calcium disruption may be involved in the induction of amyloid b accumulation and angiogenic factor expression in the retinal pigment epithelium [34]. A recent study has shown that ER stress-regulated IRE-1a signaling possesses an essential function in placental development and embryonic viability, highlighting the relationship of ER stress, and angiogen- esis in the placenta during pregnancy [35]. Kim and colleagues have shown that both SHP-1 and SHP-2 are endogenous substrates for calpain, which is involved in the context of Entamoeba histolytica-induced host cell death [36]. Notably, calpains become active when the intracellular calcium ([Ca2+]i) concentration is elevated; m-calpain (calpain II) requires [Ca2+]i at a millimolar range, but micromolar concentrations of [Ca2+]i are enough for the activation of m-calpain (calpain I) [37,38]. It has been shown that a high level of [Ca2+]i hinders protein tyrosine kinase activity as a result of calpain activation during stimulation by ionophore in platelets [39]. Honokiol was capable of inducing [Ca2+]i mobili- zation in neuronal cells [40]. Recently, tunicamycin has been reported to inhibit angiogenesis in vivo. Our previous study has shown that honokiol induces human gastric cancer cell apoptosis and inhibits tumorigenesis through a calpain-mediated glucose- regulated protein-94 cleavage [16]. In the present study, we straightforwardly demonstrated that the ER were dilated and fragmented in honokiol-treated gastric cancer cells and HUVECs. Moreover, pharmacological inhibitor of calpain and calpain-II siRNA transfection effectively reduced honokiol-enhanced SHP-1 activity in gastric cancer cells and HUVECs. Notably, our results for the first time demonstrated that calpain/SHP-1 interaction is directly triggered by honokiol inhibition of STAT-3 phosphory- lation in gastric cancer cells. These results indicate that honokiol can induce ER stress and trigger the calpain-II-activated SHP-1 activity in gastric cancer cells and endothelial cells. In conclusion, the results of the present study provide in vivo, ex vivo and in vitro evidence for inhibition of angiogenesis and peritoneal dissemination of gastric cancer cells by honokiol and a molecular basis for its effects. These data highlight the involvement of ER calpain and protein tyrosine phosphatase SHP-1 signaling in honokiol-inhibited STAT-3 activation and VEGF production in gastric cancer cells and endothelial cells (Fig. 13). These findings also suggest that the down-regulation of STAT-3 and VEGF signaling induced by calpain/SHP-1 may be a promising therapeutic strategy for gastric cancer treatment. PLOS ONE | www.plosone.org 11 August 2012 | Volume 7 | Issue 8 | e43711