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Angiogenesis is regulated by hyperglycemic conditions, which can induce cellular stress responses, reactive oxygen species (ROS), and anti-oxidant defenses that modulate intracellular signaling to prevent oxidative damage. The RUNX2 DNA-binding transcription factor is activated by a glucose-mediated intracellular pathway, plays an important role in endothelial cell (EC) function and angiogenesis, and is a target of oxidative stress. RUNX2 DNA-binding and EC differentiation in response to glucose were conserved in ECs from different tissues and inhibited by hyperglycemia, which stimulated ROS production through the aldose reductase glucose-utilization pathway. Furthermore, the redox status of cysteine and methionine residues regulated RUNX2 DNA-binding and reversal of oxidative inhibition was consistent with an endogenous Methionine sulfoxide reductase-A (MsrA) activity. Low molecular weight MsrA substrates and sulfoxide scavengers were potent inhibitors of RUNX2 DNA binding in the absence of oxidative stress, but acted as antioxidants to increase DNA binding in the presence of oxidants. MsrA was associated with RUNX2:DNA complexes, as measured by a sensitive, quantitative DNA-binding ELISA. The related RUNX2 protein family member, RUNX1, which contains an identical DNA-binding domain, was a catalytic substrate of recombinant MsrA. These findings define novel redox pathways involving aldose reductase and MsrA that regulate RUNX2 transcription factor activity and biological function in ECs. Targeting of these pathways could result in more effective strategies to alleviate the vascular dysfunction associated with diabetes or cancer.

// De-Wei Lai 1 , Shing-Hwa Liu 2,* , Anna Isabella Karlsson 3 , Wen-Jane Lee 4,* , Keh-Bin Wang 5 , Yi-Ching Chen 5 , Chin-Chang Shen 6 , Sheng-Mao Wu 1 , Chia-Yu Liu 1 , Hsing-Ru Tien 1 , Yen-Chun Peng 7 , Yee-Jee Jan 8 , Te-Hsin Chao 9 , Keng-Hsin Lan 10,11 , Jack L. Arbiser 3 and Meei-Ling Sheu 1,4,12 1 Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan 2 Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan 3 Department of Dermatology, Emory University School of Medicine, Winship Cancer Institute, Atlanta Veterans Administration Health Center, Atlanta, Georgia, USA 4 Department of Medical Research, Taichung Veterans General Hospital, Taichung, Taiwan 5 Department of Nuclear Medicine, Kuang Tien General Hospital, Taichung, Taiwan 6 Institute of Nuclear Energy Research, Atomic Energy Council, Longtan, Taoyua, Taiwan 7 Division of Gastroenterology, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan 8 Department of Pathology and Laboratory Medicine, Taichung Veterans General Hospital, Taichung, Taiwan 9 Division of Colorectal Surgery, Department of Surgery, Taichung Veterans General Hospital, Taichung, Taiwan 10 Department and Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan 11 Division of Gastroenterology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan 12 Rong Hsing Research Center for Translational Medicine, National Chung Hsing University, Taichung, Taiwan * These authors contributed equally to first author Correspondence: Meei-Ling Sheu, email: // Jack L. Arbiser, email: // Keywords : ER stress, Calpain-10, AhR, Snail, EMT Received : April 23, 2014 Accepted : August 03, 2014 Published : August 04, 2014 Abstract Biseugenol (Eug) is known to antiproliferative of cancer cells; however, to date, the antiperitoneal dissemination effects have not been studied in any mouse cancer model. In this study, Aryl hydrocarbon receptor (AhR) expression was associated with lymph node and distant metastasis in patients with gastric cancer and was correlated with clinicolpathological pattern. We evaluated the antiperitoneal dissemination potential of knockdown AhR and Biseugenol in cancer mouse model and assessed mesenchymal characteristics. Our results demonstrate that tumor growth, peritoneal dissemination and peritoneum or organ metastasis implanted MKN45 cells were significantly decreased in shAhR and Biseugenol-treated mice and that endoplasmic reticulum (ER) stress was caused. Biseugenol-exposure tumors showed acquired epithelial features such as phosphorylation of E-cadherin, cytokeratin-18 and loss mesenchymal signature Snail, but not vimentin regulation. Snail expression, through AhR activation, is an epithelial-to-mesenchymal transition (EMT) determinant. Moreover, Biseugenol enhanced Calpain-10 (Calp-10) and AhR interaction results in Snail downregulation. The effect of shCalpain-10 in cancer cells was associated with inactivation of AhR/Snail promoter binding activity. Inhibition of Calpain-10 in gastric cancer cells by short hairpin RNA or pharmacological inhibitor was found to effectively reduced growth ability and vessel density in vivo . Importantly, knockdown of AhR completed abrogated peritoneal dissemination. Herein, Biseugenol targeting ER stress provokes Calpain-10 activity, sequentially induces reversal of EMT and apoptosis via AhR may involve the paralleling processes. Taken together, these data suggest that Calpain-10 activation and AhR inhibition by Biseugenol impedes both gastric tumor growth and peritoneal dissemination by inducing ER stress and inhibiting EMT.