Plasma proteomic analysis of patients with squamous cell carcinoma of the uterine cervix

Abstract

Objective: To compare plasma protein expression between patients with squamous cell carcinoma (SCC) of the cervix and normal controls.

Methods: Plasma samples from patients with benign gynecological disease (normal cervix, n=6) and cervical cancer (SCC, n=6) were subjected to plasma proteomic analysis using two dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization mass spectroscopy (MALDI-MS). Western blotting and immunoturbidimetric assay were performed to validate the results of 2-DE.

Results: Eight proteins showed differential expression between controls and SCC patients; six (ceruloplasmin, complement C3, afamin precursor, alpha-1-B-glycoprotein, transferrin, alpha-fibrinogen precursor) were up-regulated, while two (chain A, crystal structure of antithrombin and apolipoprotein A-IV precursor) were down-regulated in the plasma of SCC patients. Western blotting analysis revealed significant elevation of ceruloplasmin, complement C3, afamin, and alpha-1-B-glycoprotein in the plasma of SCC patients in comparison to controls. Immunoturbidimetric assay of a larger group confirmed the results of 2-DE and Western blotting, and showed that ceruloplasmin and complement C3 were significantly elevated in the plasma of SCC patients in comparison with controls and patients with carcinoma in situ (CIS) of the uterine cervix.

Conclusion: Plasma protein expression determined using 2-DE and MALDI-MS will give a chance to identify tumor-specific biomarkers for SCC of the cervix.

Keywords: MALDI-MS; Plasma proteins; Squamous cell carcinoma; Two dimensional gel electrophoresis; Uterine cervical cancer.

Fig. 1

2-DE analysis of plasma proteins in normal controls (A) and patients with squamous cell carcinoma of the cervix (B). Fifteen spots that showed significant changes were selected for MALDI-MS analysis.

Fig. 2

(A) Enlarged 2-DE images showing up- and down-regulation of expression of plasma proteins in squamous cell carcinoma of the cervix compared to normal controls. (B) Histogram of different plasma protein levels in normal and cervical cancer.

Fig. 3

MALDI-MS multipotent spectra of afamin isolated from a 2-DE plasma protein gel. Representative spectra for 17 peaks are shown. The 13 matched peptides are shown in bold on the peaks.

Fig. 4

Plasma proteins obtained from normal controls and patients with squamous cell carcinoma of the cervix were subjected to Western blotting analysis with antibodies to ceruloplasmin, afamin, complement C3, and alpha-1-B glycoprotein (A1BG). (A) Equal amounts of protein (20 µg) were analyzed by SDS-PAGE. (B) Histogram of the four different proteins in normal controls and patients with squamous cell carcinoma of the cervix (p<0.05).

Fig. 5

Plasma proteins obtained from 24 normal controls, 26 patients with CIS of the cervix, and 27 patients with squamous cell carcinoma of the cervix were subjected to immunoturbidimetric assays with ceruloplasmin and complement C3 antibodies. These proteins were significantly elevated in the plasma of patients with squamous cell carcinoma of the cervix compared with normal controls and patients with CIS of the cervix (p<0.05).