Tsung-Teng Huang1, Ying-Wei Lan2, Yun-Fei Ko3, Chuan-Mu Chen4, Hsin-Chih Lai5, David M Ojcius6, Jan Martel7, John D Young8, Kowit-Yu Chong9. 1. Department of Medical Biotechnology and Laboratory Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Graduate Institute of Biomedical Sciences, Division of Biotechnology, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Center for Molecular and Clinical Immunology, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan. 2. Department of Medical Biotechnology and Laboratory Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Graduate Institute of Biomedical Sciences, Division of Biotechnology, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan. 3. Chang Gung Biotechnology Corporation, Taipei 10508, Taiwan; Biochemical Engineering Research Center, Ming Chi University of Technology, New Taipei City 24301, Taiwan; Chang Gung Immunology Consortium, Chang Gung Memorial Hospital, Linkou, Taoyuan 33305, Taiwan. 4. Department of Life Sciences, Agricultural Biotechnology Center, National Chung Hsing University, Taichung 402, Taiwan; Rong Hsing Research Center for Translational Medicine, National Chung Hsing University, Taichung 402, Taiwan; Center for Integrative Evolutionary Galliformes Genomics, National Chung Hsing University, Taichung 402, Taiwan. 5. Department of Medical Biotechnology and Laboratory Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Center for Molecular and Clinical Immunology, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Chang Gung Immunology Consortium, Chang Gung Memorial Hospital, Linkou, Taoyuan 33305, Taiwan; Research Center of Bacterial Pathogenesis, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Department of Laboratory Medicine, Chang Gung Memorial Hospital, Linkou, Taoyuan 33305, Taiwan; Research Center for Chinese Herbal Medicine, College of Human Ecology, Chang Gung University of Science and Technology, Taoyuan 33303, Taiwan; Research Center for Food and Cosmetic Safety, College of Human Ecology, Chang Gung University of Science and Technology, Taoyuan 33303, Taiwan. 6. Center for Molecular and Clinical Immunology, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Chang Gung Immunology Consortium, Chang Gung Memorial Hospital, Linkou, Taoyuan 33305, Taiwan; Department of Biomedical Sciences, University of the Pacific, Arthur Dugoni School of Dentistry, San Francisco, CA 94103, USA. 7. Center for Molecular and Clinical Immunology, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Chang Gung Immunology Consortium, Chang Gung Memorial Hospital, Linkou, Taoyuan 33305, Taiwan; Laboratory of Nanomaterials, Chang Gung University, Taoyuan 33302, Taiwan. 8. Center for Molecular and Clinical Immunology, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Chang Gung Biotechnology Corporation, Taipei 10508, Taiwan; Biochemical Engineering Research Center, Ming Chi University of Technology, New Taipei City 24301, Taiwan; Chang Gung Immunology Consortium, Chang Gung Memorial Hospital, Linkou, Taoyuan 33305, Taiwan; Laboratory of Nanomaterials, Chang Gung University, Taoyuan 33302, Taiwan; Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, NY 10021, USA. Electronic address: jdyoung@mail.cgu.edu.tw. 9. Department of Medical Biotechnology and Laboratory Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Graduate Institute of Biomedical Sciences, Division of Biotechnology, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Department of Thoracic Medicine, Chang Gung Memorial Hospital, Linkou, Taoyuan 33305, Taiwan. Electronic address: kchong@mail.cgu.edu.tw.
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE: The medicinal mushroom Antrodia cinnamomea has been used to treat cancer but its anti-angiogenic effects have not been studied in detail. AIM OF THE STUDY: The main objective of this study was to determine the molecular mechanism of activity underlying the anti-angiogenic effects of A. cinnamomea. MATERIALS AND METHODS: The effects of an A. cinnamomea ethanol extract (ACEE) on cell migration and microvessel formation were investigated in endothelial cells in vitro and Matrigel plugs implanted into mice in vivo. Activation of intracellular signaling pathways was examined using Western blotting. Protein expression was assessed using immunohistochemistry in a mouse model of lung metastasis. RESULTS: We show that treatment with ACEE inhibits cell migration and tube formation in human umbilical vein endothelial cells (HUVECs). ACEE suppresses phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) and expression of pro-angiogenic kinases in vascular endothelial growth factor (VEGF)-treated HUVECs, in addition to reducing expression of Janus kinase 2 (JAK2) and phosphorylation of signal transducer and activator of transcription 3 (STAT3). ACEE treatment inhibits VEGF-induced microvessel formation in Matrigel plugs in vivo. In addition, ACEE significantly reduces VEGFR2 expression in Lewis lung carcinoma cells and downregulates the expression of cluster of differentiation 31 (CD31) and VEGFR2 in murine lung metastases. CONCLUSION: These results indicate that A. cinnamomea produces anti-angiogenic effects by inhibiting the VEGFR2 signaling pathway.
ETHNOPHARMACOLOGICAL RELEVANCE: The medicinal mushroomAntrodia cinnamomea has been used to treat cancer but its anti-angiogenic effects have not been studied in detail. AIM OF THE STUDY: The main objective of this study was to determine the molecular mechanism of activity underlying the anti-angiogenic effects of A. cinnamomea. MATERIALS AND METHODS: The effects of an A. cinnamomeaethanol extract (ACEE) on cell migration and microvessel formation were investigated in endothelial cells in vitro and Matrigel plugs implanted into mice in vivo. Activation of intracellular signaling pathways was examined using Western blotting. Protein expression was assessed using immunohistochemistry in a mouse model of lung metastasis. RESULTS: We show that treatment with ACEE inhibits cell migration and tube formation in human umbilical vein endothelial cells (HUVECs). ACEE suppresses phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) and expression of pro-angiogenic kinases in vascular endothelial growth factor (VEGF)-treated HUVECs, in addition to reducing expression of Janus kinase 2 (JAK2) and phosphorylation of signal transducer and activator of transcription 3 (STAT3). ACEE treatment inhibits VEGF-induced microvessel formation in Matrigel plugs in vivo. In addition, ACEE significantly reduces VEGFR2 expression in Lewis lung carcinoma cells and downregulates the expression of cluster of differentiation 31 (CD31) and VEGFR2 in murinelung metastases. CONCLUSION: These results indicate that A. cinnamomea produces anti-angiogenic effects by inhibiting the VEGFR2 signaling pathway.