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. 2011 Dec 30;9:76.
doi: 10.1186/1477-5956-9-76.

S100A10 Protein Expression Is Associated With Oxaliplatin Sensitivity in Human Colorectal Cancer Cells

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Free PMC article

S100A10 Protein Expression Is Associated With Oxaliplatin Sensitivity in Human Colorectal Cancer Cells

Sayo Suzuki et al. Proteome Sci. .
Free PMC article

Abstract

Background: Individual responses to oxaliplatin (L-OHP)-based chemotherapy remain unpredictable. The objective of our study was to find candidate protein markers for tumor sensitivity to L-OHP from intracellular proteins of human colorectal cancer (CRC) cell lines. We performed expression difference mapping (EDM) analysis of whole cell lysates from 11 human CRC cell lines with different sensitivities to L-OHP by using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), and identified a candidate protein by liquid chromatography/mass spectrometry ion trap time-of-flight (LCMS-IT-TOF).

Results: Of the qualified mass peaks obtained by EDM analysis, 41 proteins were differentially expressed in 11 human colorectal cancer cell lines. Among these proteins, the peak intensity of 11.1 kDa protein was strongly correlated with the L-OHP sensitivity (50% inhibitory concentrations) (P < 0.001, R2 = 0.80). We identified this protein as Protein S100-A10 (S100A10) by MS/MS ion search using LCMS-IT-TOF. We verified its differential expression and the correlation between S100A10 protein expression levels in drug-untreated CRC cells and their L-OHP sensitivities by Western blot analyses. In addition, S100A10 protein expression levels were not correlated with sensitivity to 5-fluorouracil, suggesting that S100A10 is more specific to L-OHP than to 5-fluorouracil in CRC cells. S100A10 was detected in cell culture supernatant, suggesting secretion out of cells.

Conclusions: By proteomic approaches including SELDI technology, we have demonstrated that intracellular S100A10 protein expression levels in drug-untreated CRC cells differ according to cell lines and are significantly correlated with sensitivity of CRC cells to L-OHP exposure. Our findings provide a new clue to searching predictive markers of the response to L-OHP, suggesting that S100A10 is expected to be one of the candidate protein markers.

Figures

Figure 1
Figure 1
L-OHP sensitivity and candidate peak selection. (A) Protein expression profiles of each cell line on CM10 array at pH 4.5. The candidate peak is enclosed by the rectangle. (B) Peak intensity of the 11.1 kDa protein in 11 CRC cell lines strongly correlates with L-OHP sensitivity. The peak intensity and IC50 value of each cell line are plotted as means ± S.D. (peak intensity, n = 3; IC50, n = 3 or 4).
Figure 2
Figure 2
Characterization of the 11.1 kDa protein. (A) Estimation of pI of the 11.1 kDa protein. The affinity of the 11.1 kDa protein (indicated by the arrow) to a CM10 array changed with a pH range of 3.0-10.0. (B) Estimation of experimental molecular mass of the 11.1 kDa protein (indicated by the arrow). Bovine insulin (5733.5 Da) and equine cytochrome C (12361.0 Da) were calibrants for internal calibration.
Figure 3
Figure 3
Identification of the 11.1 kDa protein. (A) 2-DE maps of whole cell lysates from HT29 (left panel) and COLO-320 (right panel), which showed high and low expression of the 11.1 kDa protein, respectively. The 4 spots indicated by the arrows were selected on the basis of the experimental molecular mass (m/z 11,072) and experimental pI (7.0-7.5) of the candidate protein. (B) The MS/MS spectrum of tryptic-digested m/z 550.29 recorded from spot 4. The amino acid sequence of this peptide fragment is highlighted on the spectrum.
Figure 4
Figure 4
Comparison of Western blot densitometry and SELDI peak intensity. (A) A Western blot illustrating the differential expression of S100A10 derived from cell lysates of 8 CRC cell lines (a). SELDI-TOF MS gel view (b) and mass peak (c) illustrating the differential expression of the protein at m/z 11,072 in cell lysates from 8 CRC cell lines. The results are representative of three separate experiments. (B) The results of Western blot densitometry of S100A10 significantly correlated with the SELDI peak intensity at m/z 11,072. Twenty-four experimental data points (3 separate experiments with each of 8 cell lines) are plotted.
Figure 5
Figure 5
Relationship between S100A10 protein expression levels and sensitivity to L-OHP or 5-FU. The S100A10 protein expression level is associated with L-OHP sensitivity (P = 0.02, R2 = 0.27) (A), but not with 5-FU sensitivity (P = 0.40, R2 = 0.04) (B). The relationship between the sensitivity to L-OHP or 5-FU and S100A10 protein expression levels in 7 cell lines which were used in the candidate search were examined by the linear regression analysis. Twenty-one experimental data points (3 separate experiments with each of 7 cell lines) of the index data set and 3 experimental data points of HCT116 as a testing sample are plotted. Linear regression line (solid line) and 95% prediction interval (dotted line) are depicted.
Figure 6
Figure 6
Detection of S100A10 in serum-free conditioned medium (SFCM). Western blotting of SFCM from HT29 or DLD-1. DLD-1 whole cell lysate (10 μg) was used as a positive control, and conditioned medium from a 'cell-free' dish treated with the same protocol was used as a negative control.

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