HK Inhibits Angiogenesis/Peritoneal Dissemination Figure 11. Honokiol enhanced the phosphatase SHP-1 activity and the interaction with STAT-3 in gastric cancer cells and endothelial cells and the SHP-1 protein expression in tumors. Cells were treated with honokiol (HK) for various time courses as indicated. (A) Cells were treated with honokiol (HUVECs, 20 mM; SVECs, 20 mM; AGS, 20 mM; SCM-1, 40 mM) at the indicated times, and then the SHP-1 activities were measured. Data are presented as mean6SEM (n = 7). *P,0.05 as compared with control. (B) Immunohistochemistry for SHP-1 expression in metastatic tumors isolated from mice with or without HK treatment is shown. The sections were stained with anti-SHP-1 antibody. (C) Interaction of STAT-3 and SHP-1 was detected in AGS cells or HUVECs. Immunoprecipitated proteins were collected and subjected to SDS-PAGE and immunoblotting with anti-STAT-3 or anti-SHP-1 antibodies. In A and B, data are presented as mean 6 SEM (n = 5). *p,0.05 as compared with control. In C, the results shown are representative of at least four independent experiments. doi:10.1371/journal.pone.0043711.g011 Materials and Methods Cells and Culture Human gastric cancer cell lines including a Caucasian cell line (AGS, moderately/poorly (mixed) differentiated adenocarcinoma cells) and Asian cell lines (MKN45 and SCM-1, undifferentiated adenocarcinoma cells) were obtained from the cell bank of Taipei Veterans General Hospital (Taiwan). Cells were maintained in RPMI1640 medium containing 10% FCS and streptomycin/ penicillin in a humidified 5% CO2 atmosphere. HUVECs were obtained from umbilical cord veins by collagenase treatment as described previously [41]. This study conforms to the principles for use of human tissues outlined in the Declaration of Helsinki. The study protocol was approved by the Ethics Review Board of Taichung Veterans General Hospital, Taichung, Taiwan (approved document TCVGH-C0062-1). HUVECs at the third passage were used. The efficiency of transfection (.80%) by Lipofectin reagent (Invitrogen) was determined at the third passage. In some experiments, SV-40 immortalized mouse microvascular endothelial cells (SVECs) cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. SVEC cell line was kindly gifted by Dr. KH Lan (Cancer Center, Taipei Veterans General Hospital, Taiwan). Honokiol was obtained from Wako Chemical Company (Osaka, Japan), and it was determined to be at least 99% pure by HPLC. Different concentrations of honokiol were used in this study depending on the drug sensitivity indicated by IC50 values (20 mM in AGS, 40 mM in MKN45 and SCM-1). MKN45 and SCM-1 cells, but not AGS cells, have been shown to effectively induce tumors in a xenograft tumor mouse model. Animals All animal care and experimental procedures were approved and conducted by the Committee for Animal Experiments, National Chung Hsing University, Taichung, Taiwan (approved document NCHU-100-26). Four- to six-week-old male BALB/c nude mice were purchased from NLAC (Taipei, Taiwan). The mice were bred and maintained under specific pathogen-free conditions, provided with sterilized food and water ad libitum and housed in a barrier facility with a 12 h light/dark cycle. Xenograft Tumor Mouse Model To evaluate the peritoneal metastasis, cultured human gastric cancer cells (4,56106 cells) were inoculated into the peritoneal cavity of BALB/c nude mice [42]. Peritoneal tumors in nude mice were established for 7 days after the injection of gastric cancer cells, in which the tumors were checked by PET/CT surveillance. Then, mice were injected intraperitoneally with honokiol (5 mg/ kg/twice per week) for 28 days. The effect of honokiol on peritoneal dissemination was evaluated by PET/CT. A statement/ picture of the metastasis situation was described in Figure 1. Then, the mice were sacrificed under anesthesia (pentobarbital) and examined macroscopically for the presence of peritoneal metas- tasis. The tumors were excised, cut into blocks, fixed in 10% formalin, and embedded in paraffin blocks or snap-frozen in liquid nitrogen. Positron Emission Tomography/Computed Tomography (PET/CT) Scanning After at least 6 h of fasting, the mice were given a 7.4 MBq (0.2 mCi) dose of 18F-FDG orally before flushing with 1 mL of water. The mice were anesthetized with an isoflurane vaporizer before each scan. Experiments for small animal imaging were performed with a combined PET/CT scanner (Discovery ST; GE Medical Systems). A multidetector row helical CT scanner was used. Technical parameters used in the CT portion of PET/CT were as follows: CT scan type with a 0.5-second full helical scan, a detector row configuration of 1661.25 mm, an interval space of 2.75 mm, a slice thickness of 1.25 mm, a pitch of 1.75:1 (high quality mode), a speed of 17.5 mm per rotation, a large field-of-view (FOV), voltage of 120 kVp and current of 200 mA. Technical parameters used for the PET portion of PET/CT were as follows: 10.0 min in each bed, the FOV chosen for imaging reconstruction was 20 cm and the PET resolution was about 4.5 mm full width at half maximum (FWHM). The reconstructive parameters were type 3D iteration as 21 subsets and 2 iterations. To evaluate the quantification ability of the PET/CT scanner in small animal imaging, a region of interest (ROI) was placed on smaller FOV transaxial PET images to completely surround the areas of FDG uptake in the observed tissues while avoiding nearby tissues. The mean of each pixel’s activity value within each ROI was recorded and expressed as Bq/ml. Chick Chorioallantoic Membrane Assay (CAM Assay) To investigate the in vivo angiogenic activity, a modified chick whole animal CAM assay was carried out as described previously [43]. Fertilized chick eggs were incubated in a continuously humidified egg breeder at 37uC. At 3 days of incubation, chick embryos were transferred to a culture dish. Twenty mL of honokiol entrapped in type I collagen mixture was applied onto coating disks and polymerized by warming. The disks were loaded onto the CAM of 6-day-old embryos. After 24-h incubation, the area around the loaded disk was photographed with a Nikon digital camera, and the number of newly formed vessels was counted by two observers in a double-blind manner. Assays for each test sample were carried out using 10–13 eggs. Matrigel Plug Assay Nude mice or C57BL/6 mice were injected subcutaneously with 0.6 mL of matrigel containing the indicated amount of capsaicin, 100 ng of VEGF, and 10 units of heparin. The injected matrigel rapidly formed a single, solid gel plug. After 7–21 days, the skin of the mouse was easily pulled back to expose the matrigel plug, which remained intact. Hemoglobin was measured using the Drabkin method and a Drabkin reagent kit 525 (Sigma, St Louis, MO) for the quantification of blood vessel formation. The concentration of hemoglobin was calculated from a known PLOS ONE | www.plosone.org 14 August 2012 | Volume 7 | Issue 8 | e43711