Profilin 1 is a potential biomarker for bladder cancer aggressiveness.
ABSTRACT Of the most important clinical needs for bladder cancer (BC) management is the identification of biomarkers for disease aggressiveness. Urine is a "gold mine" for biomarker discovery, nevertheless, with multiple proteins being in low amounts, urine proteomics becomes challenging. In the present study we applied a fractionation strategy of urinary proteins based on the use of immobilized metal affinity chromatography for the discovery of biomarkers for aggressive BC. Urine samples from patients with non invasive (two pools) and invasive (two pools) BC were subjected to immobilized metal affinity chromatography fractionation and eluted proteins analyzed by 1D-SDS-PAGE, band excision and liquid chromatography tandem MS. Among the identified proteins, multiple corresponded to proteins with affinity for metals and/or reported to be phosphorylated and included proteins with demonstrated association with BC such as MMP9, fibrinogen forms, and clusterin. In agreement to the immobilized metal affinity chromatography results, aminopeptidase N, profilin 1, and myeloblastin were further found to be differentially expressed in urine from patients with invasive compared with non invasive BC and benign controls, by Western blot or Elisa analysis, nevertheless exhibiting high interindividual variability. By tissue microarray analysis, profilin 1 was found to have a marked decrease of expression in the epithelial cells of the invasive (T2+) versus high risk non invasive (T1G3) tumors with occasional expression in stroma; importantly, this pattern strongly correlated with poor prognosis and increased mortality. The functional relevance of profilin 1 was investigated in the T24 BC cells where blockage of the protein by the use of antibodies resulted in decreased cell motility with concomitant decrease in actin polymerization. Collectively, our study involves the application of a fractionation method of urinary proteins and as one main result of this analysis reveals the association of profilin 1 with BC paving the way for its further investigation in BC stratification.
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ABSTRACT: Identification of specific protein markers for colorectal cancer (CRC) could provide a basis for its early diagnosis and detection, as well as clues to the molecular mechanisms governing cancer progression. In the present study, 2-D DIGE coupled with MS was used to screen for biomarker candidates in the serum proteome of ten human CRC samples and ten healthy control samples. After pooling identical amounts of serum proteins (based on total protein concentration), albumin/IgG was depleted under partially denaturing conditions. Subsequently, the serum samples were labeled with three different CyDyes, and separated by 2-D DIGE. After analysis with the biological variation analysis module of the DeCyder software, only three spots were found to be significantly elevated in all patient groups (with ratios from 1.52 to 9.08), whereas five spots were significantly down-regulated in patients (with ratios from -1.23 to -10.21) (t-test; p<0.05). Finally, two potential biomarkers, Transaldolase 1 and thyroid receptor interactor, were chosen for validation and analysis by ELISA with the serum of 30 CRC patients and 30 healthy controls. The serum levels of the two proteins correlated well with the 2-D DIGE results. Thus, 2-D DIGE approaches show great promise for biomarker discovery in CRC.Electrophoresis 07/2009; 30(15):2591-9. · 3.30 Impact Factor