Comparative Proteomics Study On Proteins Relevant To Bone Metastasis In Lung Cancer

Backgroud Metastasis is not only the malignant characteristics of lung cancer,but also the key cause of failure to cure and high mortality.Bone is one of the most common target organs for lung cancer.In order to prevent and remedy the lung cancer effectively,intensive research related to the mechanism of lung cancer metastasis to bone should be undertaken.It has been well-known that lung cancer metastasis to bone is a complex process involving multiple gene and steps.Because of the limitations in conventional techniques,researchers have been allowed to focus on a few biological target molecules at a time,and little has been known regarding to the specific alterations responsible for the development or progression of lung cancer metastasis to bone. Powerful comparative proteomic techniques with high resolution,high throughput,realtime express analysis can provide effective methods for detecting more key proteins simultaneously during complex pathological progress of multi-gene and multifactor diseases . Unlike traditional ways, more new biomarks and key molecules were found by this strategy to benefit for diagnosis and elucidation of pathological mechanism. Aims In order to detect bone metastasis biomarker,we selected two human lung cancer cell lines SBC-5 and SBC-3/DOX with high and low bone metastasis potentials.Through two-dimensional gel electrophoresis (2-DE) whole soluble proteins in each cell line was separated.The proteins expressing maps of these two cell lines were described.Through analyzing the maps,the different expressing spots were found and identified by matrix assisted laser desorption/ionization-time of flight-mass spectrometry(MALDI-TOF-MS) and searched in proteins database.Our results will provide laboratorial document to screening new molecular target for reversing or controlling lung cancer metastasis to bone.Methods Total proteins of SBC-5 and SBC-3/DOX cells were extracted and 2-DE based proteomics methods were used to analyze the two samples.The first dimension was carried out by IPG pH gradient isoelectric focusing system and the second dimension was separated by vertical SDS-PAGE;the gels were stained by Silver.The 2-DE maps were matched and analyzed by Image analysis software PDQuest 8.0 to find differrentially expressed proteins spots.Peptide Mass Fingerprint maps of differentially expressed proteins were obtained by MALDI-TOF/MS methods.Bioinformatics methods were used to identify differentially expressed protein spots and to primarily analyze the function of proteins by searching from data banks.Results Image analysis software PDQuest 8.0 demonstrated good match and reproducibility of the 2-DE maps obtained from 3 independent experiments, with 1116±64 spots detected for SBC-5 cells and 1132±72 spots for SBC-3/DOX cells with the average matching rate of 84% and 82% respectively. The spots distributed in the greatest density at the isoelectric points of 4-7 and relative molecular mass weight of 20 000-80 000.Sixteen distinctly different protein spots in-gel digested by TPCK trypsin and 14 peptide mass fingerprint maps were obtained by mass spectrometry. Eight significant differentially proteins in gel were identified by MALDI-TOF-MS, where included Annexin A1,CaN,IGFBP-1,ADAM 32,and so on.Conclusions In this study,the well-resolved reproducible two-dimensional gel electrophoresis patterns of lung cancer cell lines SBC-5和SBC-3/DOX with high and low bone metastasis potentials were established and optimized. A significantly different expression of proteins is exited among the different cell lines which have different bone metastasis abilities.Eight protein spots were indentified by MALDI TOF-MS and database search. Proteins which expressed differently in lung cancer cell lines may be associated with cell growth,proliferation,metastasis,signal transduction regulation ,and so on. These results will provide scenticfic foundation for screening the molecular biomarker used to diagnose and treat bone metastasis of lung cancer,as well as to elevate the patient's prognosis and provide new clue for the research of bone metastasis mechanism.