Proteomic analysis of whole saliva from patients with oral cancer 132146 Proteomic analysis of whole saliva from patients with oral cancer K JESSIE, O.H. HASHIM1, R.B. Zain2 and Z.H.A. RAHIM, Department of Oral Biology, Faculty of Dentistry, 1University of Malaya Centre for Proteomics Research, 2Oral Cancer Research and Coordinating Center, University of Malaya, 50603 Kuala Lumpur, Malaysia INTRODUCTION RESULTS AND DISCUSSION Oral cancer Oral cancer is basically an abnormal growth found in the mouth region. Oral cancer is the 6th most common cancer worldwide, with estimated 350,000 new cases annually1. In Malaysia, a total of 21,464 oral cancer cases were diagnosed among Malaysian in Peninsula Malaysia in the year 2003. Other than the lips, the most common areas for oral cancer are on the tongue, gum and cheek. Main challenge in oral cancer control and prevention is to detect the cancer at early stage. Five years survival rate for oral cancer patients remains at 50% for the last 30 years2. Therefore, sensitive biomarkers are needed to improve early detection of oral cancer 7 4 100kDa pI A B D C 1 E 2 6 3 4 7 MW 8 9 Lip cancer Gum cancer Tongue cancer 5 10kDa Figure 1: 2-D gel showing five uncommon and differentially expressed proteins in the unstimulated whole saliva of patients with oral cancer Oral cancer and saliva Saliva contains large number of proteins and peptides with several important biological functions and informations for disease detection and surveillance of oral health3. Oral carcinogenesis involves a complex process of transformation of normal cells to a cancerous state. The gene products (proteins) and other organic chemicals made by the cancer cells could serve as early biomarkers. These proteins could be secreted into saliva which immersed the cancer cells. Therefore, analysis of the salivary proteome from oral cancer patients represents a potentially promising approach to finding potential biomarkers for the disease. Saliva could be an alternative to blood for diagnostic and/or prognostic use because it is easy to collect or handle for diagnostic purpose, non-invasive, cost-effective and may be collected repeatedly with minimal discomfort to the patient4. Objective: To compare the protein profiles of unstimulated whole saliva from patients with oral cancer with those of healthy control subjects using two-dimensional gel electrophoresis . Spot Protein name Fold change in disease P value 1 α1 -antitrypsin + 6.05 0.005 2 + 13.0 0.004 3 Complement C3 precursor + 10.2 0.048 4 Actin cytoplasmic 2 + 8.0 0.02 5 Transtherytin + 2.4 0.01 Spot Protein name Fold change in disease P value 6 Zinc α2- glycoprotein (one isoform) - 0.32 0.013 7 Carbonic Anhydrase VI - 0.36 0.007 8 Parotid Secretory Protein (PSP) - 0.38 0.009 9 Cystatin S - 0.3 0.03 Table 2: Proteins showed decreased secretion in whole saliva of patients Table 1: Proteins showed increased secretion in whole saliva of patients 152 protein spots were compared across all saliva samples, 5 were found to be highly abundant in whole saliva from oral cancer patients while 4 protein spots were low abundant. The predominant alteration observed in patients with oral cancer was an increase in the secretion of acute phase response proteins which were α1 antitrypsin, C3 complement and transtherytin (Table 1). Remaining proteins with altered abundance were Zinc α2 glycoprotein, carbonic anhydrase VI, parotid secertory protein, and cystatin S (Table 2). Five uncommon protein spots (A, B, C, D and E) were exclusively detected in the 2-DE profiles of the patients’ whole saliva and these proteins spots varied within patients’ samples. Cystatin S and Cystatin SA which were commonly present in all normal saliva were not detected in the 2-DE profile of one of the oral cancer patient. EXPERIMENTAL DESIGN Four patients with oral cancer and four healthy subjects of matching age and sex were recruited in this study. All were recently diagnosed and had not received any prior treatment in the form of chemotherapy, radiotherapy, surgery or alternative remedies before saliva collection. Unstimulated whole saliva samples were collected by spitting, were concentrated using TCA precipitation, dissolved in rehydration buffer and subjected to two-dimensional (2-D) gel electrophoresis over a pH range between 4-75. Identification of the proteins was performed by spot-pattern comparison with the previously established normal human unstimulated whole saliva proteome6,7. The separated proteins were analyzed using computer assisted 2D gel analysis using GE Image Master 2D Platinum version 7. Spot-volume quantification was done on all pattern-matched protein spots between the two groups of oral cancer and healthy subjects. The percentage of the volume of the spots representing a certain protein was determined in comparison with the total protein spots in the 2D gel. Data was subjected to Student t test and also one way ANOVA. Both gave similar conclusions. The significance level was defined as p value <0.05 Conclusion Proteomic analysis of the unstimulated whole saliva of patients with oral cancer showed presence of uncommon proteins that were not normally secreted in the unstimulated whole saliva of healthy controls. Eight proteins that were commonly detected in the unstimulated whole saliva of healthy controls were also found to be aberrantly secreted by the patients. These proteins can be targeted for future validation as potential saliva markers in oral cancer diagnosis Acknowledgement This study is supported by the PPP Grant no : PS105/2008A and PS071/2009A REFERENCES Fearlay J, Bray F, Pisani P, Parkin DM(2001). Cancer incidence, mortality and prevalence worldwide. Version 1.0. IARC cancer base, Vol 5. Lyon: IARC Press. Lippman SM & Hong WK (2001) Molecular markers of the risk of oral cancer. N Eng J Med, 344: 1323-1326. Lamkin MS & Oppenheim FG(1993). Structural features of saliva function. Crit Rev Oral Biol Med 4: 251-259. 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