Identification of host-immune response protein candidates in the sera of human oral squamous cell carcinoma patients

Abstract

One of the most common cancers worldwide is oral squamous cell carcinoma (OSCC), which is associated with a significant death rate and has been linked to several risk factors. Notably, failure to detect these neoplasms at an early stage represents a fundamental barrier to improving the survival and quality of life of OSCC patients. In the present study, serum samples from OSCC patients (n = 25) and healthy controls (n = 25) were subjected to two-dimensional gel electrophoresis (2-DE) and silver staining in order to identify biomarkers that might allow early diagnosis. In this regard, 2-DE spots corresponding to various up- and down-regulated proteins were sequenced via high-resolution MALDI-TOF mass spectrometry and analyzed using the MASCOT database. We identified the following differentially expressed host-specific proteins within sera from OSCC patients: leucine-rich α2-glycoprotein (LRG), alpha-1-B-glycoprotein (ABG), clusterin (CLU), PRO2044, haptoglobin (HAP), complement C3c (C3), proapolipoprotein A1 (proapo-A1), and retinol-binding protein 4 precursor (RBP4). Moreover, five non-host factors were detected, including bacterial antigens from Acinetobacter lwoffii, Burkholderia multivorans, Myxococcus xanthus, Laribacter hongkongensis, and Streptococcus salivarius. Subsequently, we analyzed the immunogenicity of these proteins using pooled sera from OSCC patients. In this regard, five of these candidate biomarkers were found to be immunoreactive: CLU, HAP, C3, proapo-A1 and RBP4. Taken together, our immunoproteomics approach has identified various serum biomarkers that could facilitate the development of early diagnostic tools for OSCC.

Figure 1. Representative 2-DE serum protein profiles of normal controls and OSCC patients.

Unfractionated serum samples of (a) normal controls and (b) OSCC patients were subjected to 2-DE and silver staining. The labeled spot clusters are proteins which are consistently identified in profiles of normal controls and OSCC patients. α2-HS-glycoprotein (AHS) and α1-antitrysin (AAT) are high abundance proteins that typically appeared in protein profiles.

Figure 2. Results from 2-DE immunoblots for (a) normal pooled sera probed with normal pooled sera, (b) normal pooled sera probed with OSCC pooled sera, (c) OSCC pooled sera probed with normal pooled sera, (d) OSCC pooled sera probed with OSCC pooled sera.

Unfractionated, pooled serum samples from control and OSCC patients were subjected to 2-DE and blotted onto nitrocellulose membranes, which were then probed with pooled sera and monoclonal anti-human IgM-HRP.

Figure 3. Interaction networks of identified host specific proteins using STRING v9.1.

STRING database is a curated knowledge database of known and predicted protein-protein interactions. Most of the identified host-specific proteins have an established link with each other in the interaction network.