Calpain/SHP-1 Interaction by Honokiol Dampening Peritoneal Dissemination of Gastric Cancer in nu/nu Mice Shing Hwa Liu1., Keh Bin Wang2,9, Keng Hsin Lan 3 , Wen Jane Lee4, Hung Chuan Pan4,9, Sheng Mao Wu 9 , Yen Chun Peng 5 , Yi Ching Chen 9 , Chin Chang Shen6,7., Hsu Chen Cheng10, Ko Kaung Liao 8 , Meei Ling Sheu4,9* 1 Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan, 2 Department of Nuclear Medicine, Kuang Tien General Hospital, Taichung, Taiwan, 3 Division of Gastroenterology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan, 4 Department of Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan, 5 Division of Gastroenterology, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan, 6 Department of Chemistry, National Taiwan University, Taipei, Taiwan, 7 Institute of Nuclear Energy Research, Atomic Energy Council, Taoyuan, Taiwan, 8 Department of Anatomy, Chung Shan Medical University, Taichung, Taiwan, 9 Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan, 10 Department of life Sciences, National Chung Hsing University, Taichung, Taiwan Abstract Background: Honokiol, a small-molecular weight natural product, has previously been reported to activate apoptosis and inhibit gastric tumorigenesis. Whether honokiol inhibits the angiogenesis and metastasis of gastric cancer cells remains unknown. Methodology/Principal Findings: We tested the effects of honokiol on angiogenic activity and peritoneal dissemination using in vivo, ex vivo and in vitro assay systems. The signaling responses in human gastric cancer cells, human umbilical vascular endothelial cells (HUVECs), and isolated tumors were detected and analyzed. In a xenograft gastric tumor mouse model, honokiol significantly inhibited the peritoneal dissemination detected by PET/CT technique. Honokiol also effectively attenuated the angiogenesis detected by chick chorioallantoic membrane assay, mouse matrigel plug assay, rat aortic ring endothelial cell sprouting assay, and endothelial cell tube formation assay. Furthermore, honokiol effectively enhanced signal transducer and activator of transcription (STAT-3) dephosphorylation and inhibited STAT-3 DNA binding activity in human gastric cancer cells and HUVECs, which was correlated with the up-regulation of the activity and protein expression of Src homology 2 (SH2)-containing tyrosine phosphatase-1 (SHP-1). Calpain-II inhibitor and siRNA transfection significantly reversed the honokiol-induced SHP-1 activity. The decreased STAT-3 phosphorylation and increased SHP-1 expression were also shown in isolated peritoneal metastatic tumors. Honokiol was also capable of inhibiting VEGF generation, which could be reversed by SHP-1 siRNA transfection. Conclusions/Significance: Honokiol increases expression and activity of SPH-1 that further deactivates STAT3 pathway. These findings also suggest that honokiol is a novel and potent inhibitor of angiogenesis and peritoneal dissemination of gastric cancer cells, providing support for the application potential of honokiol in gastric cancer therapy. Citation: Liu SH, Wang KB, Lan KH, Lee WJ, Pan HC, et al. (2012) Calpain/SHP-1 Interaction by Honokiol Dampening Peritoneal Dissemination of Gastric Cancer in nu/nu Mice. PLoS ONE 7(8): e43711. doi:10.1371/journal.pone.0043711 Editor: Henrik Einwaechter, Klinikum rechts der Isar der TU Mu¨ nchen, Germany Received January 17, 2012; Accepted July 24, 2012; Published August 24, 2012 Copyright: 2012 Liu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by research grants from Taichung Veterans General Hospital, Taiwan (TCVGH-997314C, TCVGH-1007313C) and the National Science Council of Taiwan (NSC99-2320-B-005-003-MY3). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mlsheu@nchu.edu.tw . These authors contributed equally to this work. Introduction The majority of patients (,60%) with gastric cancer are diagnosed with late stage disease. Gastric cancer is the second most common cause of global cancer mortality in developed nations and exhibits metastatic disease at the time of diagnosis [1]. Surgery and combination chemotherapies for gastric cancer have been shown to confer only modest survival benefits in advanced cases, and nearly 50% of patients still die after recurrence [2,3]. A major form of recurrence is peritoneal dissemination. Cancer growth and peritoneal metastasis are angiogenesis dependent, a process which involves several angiogenic factors including vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor, prostaglandin E2, interleukin-8, chemo- kine (C-X-C motif) ligand 1, and the matrix metalloproteinase family [4–6]. Several molecular signals, such as signal transducer and activator of transcription-3 (STAT-3), nuclear factor-eˆB, Akt, mitogen-activated protein kinases, cyclooxygenase-2, lipoxygen- ase, inducible nitric-oxide synthase, tumor necrosis factor and others, have also been shown to be involved in tumor progression and angiogenesis [7–9]. However, the cellular and molecular PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e43711