mechanisms of the development, progression, and metastasis of gastric cancer still remain to be clarified. The Janus-activated kinase (Jak)/STAT signaling pathway plays an important role in the regulation of cell growth, angiogenesis, differentiation, migration, and metastasis [10]. Constitutive activation of STAT pathways, particularly STAT-3, is associated with a wide variety of human malignancies. Persistent STAT-3 phosphorylation has been observed in various human cancers, such as solid tumors of the stomach, colon, liver, prostate, breast, lung, and head and neck as well as blood malignancies [8,10]. Previous study has shown that phosphorylation of STAT-3 and VEGF protein expression are increased in human gastric cancer tissue, which in turn elevate the angiogenic phenotype and contribute to gastric cancer development and progression [11]. On the other hand, certain phosphatases are known to be tumor suppressors and may play an important role in the inhibition or control of cancer growth [12–15]. Protein tyrosine phosphatases (PTPs), including SH2 domain-containing tyrosine phosphatase (SHP)-1 and SHP-2, are able to negatively regulate STAT signaling by the tyrosine dephosphorylation of several components in the related signaling pathways [13–15]. The continuous activation of STAT-3 in the tumors might be facilitated at least in part by loss of function of these phosphatases. Calpain II has been shown to play a role in endoplasmic reticulum (ER) stress- regulated tumorigenesis, which is involved in the mechanism of honokiol-inhibited gastric tumorigenesis [16]. In addition, pre- vious study has also indicated that SHP-1 is an endogenous substrate for calpain following A23187-induced platelet activation [17]. Therefore, a calpain/SHP-1-regulated STAT-3 and VEGF pathway may be involved in the angiogenesis, growth, and peritoneal dissemination of gastric cancer cells. Honokiol, a small-molecular weight natural product, is a major active biphenolic compound of Magnolia officinalis, which is known to ameliorate microbial infection, inflammation and gastrointes- tinal disorders in traditional Asian medicinal systems [18]. Our previous study demonstrated that honokiol inhibits gastric tumorigenesis by activation of 15-lipoxygenase-1 and consequent inhibition of peroxisome proliferator-activated receptor-a˜ and COX-2-dependent signals [19]. Honokiol has been shown to induce ER stress and trigger calpain-II-mediated glucose-regulated protein-94 cleavage and apoptosis in human gastric cancer cells [16]. It has been demonstrated that VEGF expression could be responsive to nutrient deprivation conditions, which cause ER stress [20,21]. Moreover, the ER stress activator tunicamycin has been found to markedly prevent microvasculature development, suggesting that this ER stress inducer may have a potential role in breast tumor treatment [21]. The effects of honokiol on ER stress- correlated angiogenesis and gastric tumor metastasis are still unclear. Here, we hypothesized that honokiol inhibits the angiogenesis and peritoneal dissemination of gastric cancer cells through a calpain/SHP-1-regulated STAT-3 and VEGF pathway. The results showed that honokiol markedly inhibited angiogenesis and peritoneal dissemination of gastric cancer cells via a calpain/ SHP-1 interaction-activated STAT-3 dephosphorylation and VEGF down-regulation pathway. Results Honokiol Blocked Peritoneal Metastasis of Gastric Cancer in vivo Cancer is frequently characterized by the increased uptake of [18F]-fluoro-2-deoxy-D-glucose (FDG), and [18F]-FDG/PET can serve as a surrogate measure of therapeutic efficacy. The functional possibility of small animal imaging using a clinical HK Inhibits Angiogenesis/Peritoneal Dissemination PET/CT scanner with FDG was evaluated. Four groups of mice for peritoneal metastasis experiments were investigated. A statement/picture of the metastasis situation was shown in Figure 1. We utilized [18F]-FDG-PET/CT to detect peritoneal metastases in mice inoculated with human gastric cancer cells (MKN45 or SCM-1) with or without honokiol treatment. As shown in Figure 2, the maximum intensity projection was generated from typical representative mice. The peritoneal metastasis was marked in the control mice (left panel), and it was effectively reversed by the honokiol treatment (right panel). These images clearly showed that noninvasive [18F]-FDG uptakes in peritoneal metastatic tumors of control mice were much higher than those in the honokiol-treated mice. The quantification of intensity is shown in Figure 3A. Intraperitoneal injection of honokiol (5 mg/kg, twice/weekly) significantly reduced the estimated radioactivity counts and specific uptake values (SUV) determined by FDG-PET/CT in mice inoculated with gastric cancer cells (Fig. 3). Moreover, many metastatic nodules were found in the peritoneal cavity (mesentery) of the control mice inoculated with MKN45 and SCM-1 (Fig. 2) cells. In contrast, peritoneal tumor nodules were observed sporadically in mice treated with honokiol. Quantification of nodules per field is shown in Figure 3B. Honokiol Inhibited Angiogenesis in vivo, ex vivo, and in vitro Endothelial cells are critical for the angiogenic process, which is necessary for tumor growth and metastasis. We next investigated the angiogenic effect of honokiol using the chick chorioallantoic membrane assay (CAM assay), matrigel plug assay, aortic ring sprouting assay, and endothelial cell tube formation. As shown in Figure 4A, honokiol effectively inhibited the neo-vascular forma- tion in the CAM assay without any visible effect on the pre-existing blood vessels. Quantitative analysis revealed that honokiol caused a 2.5-fold decrease in the number of newly formed blood vessels as compared with that of medium control. In the matrigel plug assay, matrigel containing VEGF (100 ng/ ml gel) with or without honokiol (10 mg/ml) was subcutaneously implanted into nude mice. After 21 days of implantation, the formed matrigel plugs were excised and photographed. Plugs with VEGF alone were markedly streaky, vascular and red in color. Plugs containing VEGF and honokiol were pallid, indicating no or less blood vessel formation (Fig. 4B-a). We also examined the vessel density and vessel morphology in the plug sections by H & E staining and immunohistochemical staining with an antibody against CD31, an endothelial cell marker. The vascularization in the honokiol+VEGF group was significantly reduced as compared with the VEGF alone group (Fig. 4B-b). Quantification of the vascularization by counting the vessels and presented endothelial cells revealed that the vascular density in the honokiol-treated group was significantly decreased (Figs. 4B-c and 4B-d). Next, endothelial cells were induced to sprout from the isolated rat aortic rings in the presence of matrigel and ECGM (endothelial cell growth medium) containing angiogenic cytokines, such as VEGF and basic fibroblast growth factor. Extensive endothelial cell outgrowth from rat aorta ring explants was observed in the control group (Fig. 5A). Honokiol treatment resulted in a signifi- cant (,five-fold) reduction of endothelial outgrowth and sprouting from aortic rings (Figs. 5A and 5C). Furthermore, the morpho- logical differentiation of endothelial cell tube formation was investigated using a two-dimensional matrigel method. As shown in Figure 5B, seeding of HUVECs in matrigel led to vascular tube- like structure formation. Honokiol effectively inhibited the PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e43711