title: Ephedrae herba stimulates hepatocyte growth factor-induced MET endocytosis and downregulation via early/late endocytic pathways in gefitinib-resistant human lung cancer cells.
abstract: The MET tyrosine kinase receptor and its ligand, hepatocyte growth factor (HGF), are known to be overexpressed in a variety of malignant tumor cells, and are implicated in the development of gefitinib-resistance in human non-small cell lung cancer (NSCLC) cells. Ephedrae herba was previously reported to prevent HGF-induced cancer cell motility by directly suppressing HGF/MET signaling through the inhibition of MET tyrosine kinase, and treatment with its extract also considerably reduced MET protein levels. To further investigate the mechanism underlying the Ephedrae herba-induced inhibition of MET phosphorylation as well as its degradation and subsequent disappearance, we examined the effect of Ephedrae herba on HGF-stimulated MET endocytosis and downregulation via early/late endocytic pathways in an NSCLC cell line. Using immunofluorescence microscopy, we found that pretreatment of cells with Ephedrae herba extract dramatically changed the intracellular distribution of plasma membrane-associated MET, and that the resultant MET staining was distributed throughout the cytoplasm. Pretreatment of the cells with Ephedrae herba extract also led to the rapid loss of MET and phosphorylated (p)-MET in HGF-stimulated cells. In contrast, inefficient endocytic delivery of MET and p-MET from early to late endosomes was observed in the absence of Ephedrae herba extract, since considerable amounts of the internalized MET accumulated in the early endosomes and were not delivered to lysosomes up to 1 h after HGF-stimulation. Furthermore, large amounts of MET and p-MET that had accumulated in late endosomes of Ephedrae herba-pretreated cells after HGF stimulation were observed along with bafilomycin A1. Therefore, we inferred that degradation of MET occurred in the late endosome/lysosome pathway. Moreover, western blot analysis revealed the accelerated degradation of MET and p-MET proceeds in cells pretreated with Ephedrae herba extract. Collectively, our results suggest that some components of Ephedrae herba have a novel role in promoting HGF-stimulated MET and p-MET endocytosis followed by its downregulation, likely mediated by the early/late endocytic pathways.
authors.affiliation: Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
authors.forename: Yukio
authors.initials: Y
authors.lastname: Nishimura
authors.affiliation: Department of Clinical Research, Oriental Medicine Research Center of Kitasato University, Tokyo 108-8642, Japan.
authors.forename: Sumiko
authors.initials: S
authors.lastname: Hyuga
authors.affiliation: Institute for Clinical Research, National Kyushu Cancer Center, Fukuoka 811-1395, Japan.
authors.forename: Soichi
authors.initials: S
authors.lastname: Takiguchi
authors.affiliation: Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Tokyo 158-8501, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Hyuga
authors.affiliation: Department of Biology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka 537-8511, Japan.
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Itoh
authors.affiliation: Department of Clinical Research, Oriental Medicine Research Center of Kitasato University, Tokyo 108-8642, Japan.
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Hanawa
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D002289:Carcinoma, Non-Small-Cell Lung / Q000378:metabolism*; D045744:Cell Line, Tumor; D015536:Down-Regulation / Q000187:drug effects; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D004705:Endocytosis / Q000187:drug effects; D029789:Ephedra / Q000737:chemistry*; D000077156:Gefitinib; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D017228:Hepatocyte Growth Factor / Q000378:metabolism*; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D010936:Plant Extracts / Q000494:pharmacology*; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism*; D011799:Quinazolines / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D000972:Antineoplastic Agents, Phytogenic; C066855:HGF protein, human; D010936:Plant Extracts; D011799:Quinazolines; D017228:Hepatocyte Growth Factor; C491743:MET protein, human; D019859:Proto-Oncogene Proteins c-met; D000077156:Gefitinib