title: Selective downregulation of EGF receptor and downstream MAPK pathway in human cancer cell lines by active components partially purified from the seeds of Livistona chinensis R. Brown.
abstract: Deregulation of protein kinase-mediated signaling events is one of the major causes to malignant transformation. In this work, we tried to purify protein kinase inhibitory activity and antitumor activity from ethanol extracts of the seeds of Livistona chinensis R. Brown (LC), a traditional herb used for the treatment of nasopharyngeal carcinoma (NPC). Both activities were found to be co-purified in various chromatography steps, and a highly purified fraction, LC-X, was obtained and its biological effects were characterized further. LC-X inhibited the activities of various protein kinases in vitro, including PAK2, PKA, PKC, GSK-3alpha, CK2, mitogen-activated protein kinase (MAPK), and JNK1, with IC(50) between approximately 1 and 40microg/ml. The proliferation of two NPC (NPC-TW02 and -TW04) and one breast cancer (MCF-7) cell lines, but not the epidermoid (A431) and cervical (HeLa) carcinoma cell lines, were significantly blocked by LC-X at the dose of >50microg/ml. Cell cycle arrested at G(2)/M phase and apoptosis were detected in NPC-TW02 cells treated with LC-X for 24h. Further studies revealed that epidermal growth factor (EGF)-induced activation of epidermal growth factor receptor (EGFR) and MAPK could be potently inhibited by LC-X in both NPC-TW02 and A431cells in a dose-dependent manner. More interestingly, the level of EGFR protein detected by Western blot decreased drastically in LC-X-treated A431 and NPC-TW02 cells in a dose- and time-dependent fashion. Further analysis of the plasma membrane and cytosolic fractions from LC-X-treated and untreated A431 cells showed that a 170kDa protein selectively disappeared from the plasma membrane of LC-X-treated cells. The protein was identified as EGFR by MALDI-TOF mass spectrometry, indicating EGFR as a selective target for LC-X. Moreover, the electrophoretic mobility of purified EGFR in SDS-PAGE was altered dramatically post LC-X treatment, suggesting that LC-X may chemically modify EGFR. In conclusion, the active components with both antitumor and protein kinases inhibitor activities were highly purified from LC, which can inhibit the EGF signaling events mainly through EGFR modification. Blockage of the functions of EGFR may account for the antitumor activity of these active components.
authors.affiliation: Graduate Institute of Basic Medical Sciences, Medical College of Chang Gung University, Tao-Yuan, Taiwan, Republic of China.
authors.forename: Wen-Chuang
authors.initials: WC
authors.lastname: Huang
authors.forename: Rae-Mann
authors.initials: RM
authors.lastname: Hsu
authors.forename: Lang-Ming
authors.initials: LM
authors.lastname: Chi
authors.forename: Yann-Lii
authors.initials: YL
authors.lastname: Leu
authors.forename: Yu-Sun
authors.initials: YS
authors.lastname: Chang
authors.forename: Jau-Song
authors.initials: JS
authors.lastname: Yu
mesh_terms: D000970:Antineoplastic Agents / Q000302:isolation & purification / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D028023:Arecaceae / Q000737:chemistry*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects; D066246:ErbB Receptors / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008565:Membrane Proteins / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009303:Nasopharyngeal Neoplasms / Q000378:metabolism / Q000473:pathology; D010936:Plant Extracts / Q000302:isolation & purification / Q000494:pharmacology*; D047428:Protein Kinase Inhibitors / Q000302:isolation & purification / Q000494:pharmacology; D012639:Seeds / Q000737:chemistry*
chemical_list: D000970:Antineoplastic Agents; D008565:Membrane Proteins; D010936:Plant Extracts; D047428:Protein Kinase Inhibitors; D066246:ErbB Receptors; D020928:Mitogen-Activated Protein Kinases