title: [Establishment of drug resistant cell line of MGC-803 and analysis of differential secretome].
abstract: OBJECTIVE
  To identify chemoresistance-associated secretory proteins by proteomic approaches, and to provide the basis for selecting suitable chemotherapy in gastric cancer treatment.
  
  
  METHODS
  Drug resistant cell lines were established by gradient drug treatment with MGC-803 gastric cancer cells. The secreted proteins of MGC-803 parental and resistant cells were collected from the conditional medium without serum and separated by two-dimensional gel electrophoresis (2-DE).The proteins were analyzed by PD Quest 7.1.0 software and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Real-time RT-PCR was performed to confirm the difference of expression on the mRNA level.
  
  
  RESULTS
  The 5-fluorouracil (5FU), paclitaxel (TA) and cisplatin (DDP)-resistant gastric cancer cell lines with the resistance indexes of 110.6, 70.0 and 13.3 respectively, were established successfully. DDP-resistant cells had strong cross-resistance to 5FU and TA, and the resistance indexes were 23.5 and 114.0. 5FU-resistant cells also had strong cross-resistance to TA with the resistance index 70.0. The 2-DE patterns of protein component spectra from the conditional medium were obtained with 18 proteins whose abundances were increased in all chemoresistance cells for more than 2-fold, 13 of which were identified by mass spectrometry, including protease and proteins involved in signal transduction. Compared with the parental cell MGC-803, SLMAP, TOP3A, DYNC1H1, RHPN1, PUF60 and SIAH1 were significantly up-regulated in three drug resistant cells, IFT172 and FILIP were up-regulated in 5FU-resistant and TA-resistant cells, PLVAP and LMNA were up-regulated in TA- and DDP-resistant cells. Further validation revealed that SIAH1 protein was enriched in cell lysates and the conditional medium from all the drug resistant cells.
  
  
  CONCLUSIONS
  By establishing the 5FU-,TA- and DDP-resistant gastric cancer cell lines and assisted by 2-DE and mass spectrometry, we demonstrated the different secretory protein profiling and found that SIAH1 had significantly increased in both cell lysates and the conditional medium of the drug-resistant cells, which are potential candidates for developing chemoresistance markers in sera from gastric cancer patients.
authors.affiliation: Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education; Department of Biochemistry and Molecular Biology, Peking University Cancer Hospital & Institute, Beijing 100142, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Huang
authors.affiliation: Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education; Department of Biochemistry and Molecular Biology, Peking University Cancer Hospital & Institute, Beijing 100142, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Han
authors.affiliation: Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education; Department of Biochemistry and Molecular Biology, Peking University Cancer Hospital & Institute, Beijing 100142, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Wu
authors.affiliation: Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education; Department of Biochemistry and Molecular Biology, Peking University Cancer Hospital & Institute, Beijing 100142, China.
authors.forename: Zhi-hua
authors.initials: ZH
authors.lastname: Tian
authors.affiliation: Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education; Department of Biochemistry and Molecular Biology, Peking University Cancer Hospital & Institute, Beijing 100142, China.
authors.forename: Li-ke
authors.initials: LK
authors.lastname: Qu
authors.affiliation: Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education; Department of Biochemistry and Molecular Biology, Peking University Cancer Hospital & Institute, Beijing 100142, China.
authors.forename: Cheng-chao
authors.initials: CC
authors.lastname: Shou
mesh_terms: D045744:Cell Line, Tumor / Q000187:drug effects / Q000378:metabolism; D002945:Cisplatin / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D015180:Electrophoresis, Gel, Two-Dimensional; D005472:Fluorouracil / Q000494:pharmacology; D006801:Humans; D020543:Proteome / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013274:Stomach Neoplasms / Q000378:metabolism
chemical_list: D020543:Proteome; D002945:Cisplatin; D005472:Fluorouracil