The Differential Proteomics Of Melanoma Metastasis Associated Proteins

Purpose:2D-DIGE combinding with MALDI-TOF/TOF MS were used to identify the proteins associated with melanoma metastasis. The materials were from transplanted tumors of B16F10 and corresponding lung metastases. And the function of special protein was identified. According to the results, some predictors of melanoma hematogenous metastasis should be found.Contents:1. During the passage of B16F10 transplanted tumor subcutaneously, we found some mouse bearing spontaneous lung metastases. We picked out the metastatic spots of the lung, and transplanted them to the mouse groin to be passaged stably. We termed them as B16M group and compared the differential characteristics between it with B16 group, i.e. B16F10 transplanted tumors.2.2D-DIGE (Two dimensional Differential in Gel Electrophoresis) was used to investigate the differential proteins expression between two groups which were identified by MS (Mass Spectrometry)3. The MS results of vimentin was validated by western blotting, and its clinical significance was studied in 70 samples with detailed follow-up.Methods:1. Fifty C57BL/6J mouse were divided into two groups, one was inoculated B16F10 single cell suspension (B16 group), the other was inoculated single cell suspension of transplanted tumors from lung metastases (B16M group). The mouse were sacrificed when the tumor diameter was up to 1 cm, and the tumor tissures were separated and harvested. VMD and MVD were counted. MMP-2 and MMP-9 immunohistochemical staining were performed in two group samples.2. To abstract proteins from eight pairs of B16M and B16 tissues, and mix them respectively. One group was labled by Cy3, the other was labled by Cy5, mixture of equal two groups were labled by Cy2 which was as internal control. The second gel were reversed labled. The excitated wave length of Cy2, Cy3 and Cy5 were 488/520nm,532/580nm and 633/670nm. The statistical analysis was used by DeCyder software and differential expressed proteins were identified by MALDI-TOF/TOF-MS.3. The MS results were validated by western blotting in the same samples. The clinical significance of individual protein was investigated in the patient paraffin tissues with whole follow-up.Results:1. The differential characteristics between B16M group and B16 groupThe color of B16M tumor tissues was gray-white or gray-yellow, but B16 tumor was black as usual. VM can be observed in the B16M, and VMD counts were higher significantly in it than in B16. The positively expression of MMP-2 and MMP-9 were also incresed in B16M. The H&E manifestation of B16M look like amelanotic melanoma. So we supposed that B16M has stronger metastatic and invasive property.2.2D-DIGE and MS results of B16M group and B16 groupThere were 13 metastasis-associated proteins which were identified by MS,11 of them were up-regulated in the B16M that included vimentin,PGK1,enolase 1,TPI,Bip,laminin binding protein,GAPDH,myoglobin,proteasome activator reg alpha,β-actin and y-actin,2 hypothetical proteins were down-regulated in the B16M. The function of those proteins involved in cytoskeleton,glycolytic enzymes,immunoproteasome assembly and the chaperone family of protein.3. The validation of MS result of vimentin and its clinical significanceThe result of western blotting was coincident with MS. The immunohistochemical staining of vimentin was performed in the patient paraffin tissues with whole follow-up. The positive expression of vimentin is yellow stained in the tumor cell cytoplasm. We recognized positive cells>40% as higher expression group,<=40% as lower expression group reversely. The higher expression of vimentin is correlated with melanoma hematogenous metastasis, but not associated with age,gende,location,TNM stage and lymph node metastasis.Conclusion:Vimentin is associated with melanoma hematogenous metastasis. Higher expression of vimentin is a predictor of hematogenous metastasis for primary melanomas. The significance of vimentin clinically is not only the diagnostic marker, but also one of prognostic factor and treatment target for melanoma patients.