Radiotherapy Changes Proteins of Saliva in Patients with Head-and-neck Cancer

Objectives: This study evaluated the changes in the proteomic profile in saliva of head and neck cancer (HNC) patients, both before and during radiotherapy.
Methods: Stimulated saliva was collected from nine HNC patients before radiotherapy and during treatment (2 to 5 weeks). For the proteomic analysis, the salivary proteins were extracted with a solution containing 6 M urea, 2 M thiourea in 50 mM ammonium bicarbonate. The samples were then concentrated, reduced, alkylated and digested with trypsin (37°C, 14 hours). Samples were purified using Spin C18 columns and total protein quantification was performed by the Bradford method. The samples were then processed for label-free quantitative LC-ESI-MS/MS.
Results: A total of 153 proteins were identified in the saliva in the two groups. Among them, 77 and 38 were exclusively found before and during radiotherapy, respectively. Thirty-eight proteins were identified in both periods among which are Cystatins -S, -SA and –SN, Lysozyme C, Proline-rich protein 4, Serum albumin and Statherin. Regarding the quantitative analysis, 10 and 13 proteins were up- and down-regulated, respectively, during radiotherapy. The following proteins were increased more than 2-fold during radiotherapy, such as Histatin-1, Lactotransferrin, Proline-rich protein 4, Salivary acidic proline-rich phosphoprotein ½ and Statherin. On the other hand, radiotherapy decreased more than 2-fold the following proteins: Basic salivary proline-rich protein 2, BPI fold-containing family B member 2, Immunoglobulin heavy constant alpha 1 and 2, Immunoglobulin J chain and Polymeric immunoglobulin receptor.
Conclusions: During radiotherapy, profound alterations are found in the salivary proteome of HNC patients. Several proteins with important functions in the homeostasis of the oral cavity are changed, which might modify the protective role of saliva in the oral homeostasis and should be evaluated in further studies.