Abstract
Oral squamous cell carcinoma (OSCC) is often diagnosed with late stage resulting in poor prognosis. Therefore, early detection and screening methods of OSCC that has the accuracy, precision, rapidity and simplicity are urgently developed for screening of people who exposure with risk factors including smoking, alcohol consumption and betel quid chewing as well as human papillomavirus (HPV) and Epstein-Barr virus (EBV) infection and have precancerous lesion. From our preliminary result, peroxiredoxin 2 (PRDX2) and zinc-alpha-2-glycoprotein (ZAG or AZGP1) were significantly upregulated in salivary OSCC different from cancer-free control using two-dimensional gel electrophoresis (2-D) Gel which was the step of validation test. Therefore, this study aimed to examine the expression of PRDX2 and ZAG proteins in saliva from healthy control, oral inflammation, precancerous and OSCC groups which was the step of confirmation test. In addition, we also investigated the function of PRDX2 and ZAG in OSCC cell line that has not been reported yet. The saliva samples from healthy volunteers, patient with any inflammatory lesions in oral cavity, precancerous lesions with leukoplakia or lichen planus and OSCC were analyzed for the expression of PRDX2 and ZAG proteins using western blot. The function of PRDX2 and ZAG were in vitro studied by transduction to OSCC cell line (ORL-48T) with retrovirus containing pCLXSN-PRDX2 and pCLXSN-ZAG, respectively. These transduced cells were selected by the G418-containing medium and analyzed for cell proliferation, cell-cycle progression, apoptosis and cell migration using MTT, PI staining for flow cytometry, apoptosis kit for flow cytometry and wound healing assay, respectively. The results showed that the relative expression of PRDX2 protein in salivary precancerous and OSCC was significant difference from healthy volunteers and patient with any inflammatory lesions in oral cavity, whereas only the relative expression of ZAG protein in saliva of OSCC significant difference from healthy volunteers and patient with any inflammatory lesions in oral cavity. The result showed that AUC of PRDX2 in salivary precancerous and OSCC was 0.8661 and 0.9521 whereas AUC of ZAG was 0.8832 and 0.8494 respectively when compared to healthy control. In addition, AUC of PRDX2 in salivary precancerous and OSCC had higher value (0.8512 and 0.8543, respectively) than ZAG (0.7752 and 0.7848, respectively) when compared to the patient with any inflammatory lesions in oral cavity. This result demonstrated that only PRDX2 can be a biomarker for screening of precancerous and OSCC from healthy control and the patient with any inflammatory lesions in oral cavity. The AUC of the combination of PRDX2 and ZAG in precancerous and OSCC was 0.8677 and 0.9009 respectively compared to healthy control and 0.8002 and 0.8570 respectively compared to the patient with any inflammatory lesions in oral cavity. The combination of PRDX2 and ZAG can increase the sensitivity to discriminate the precancerous and OSCC from the patient with any inflammatory lesions in oral cavity. For in vitro study, the proportion of cell proliferation, cell-cycle progression and cell migration in PRDX2-overexpression ORL-48T cells were significantly higher than pCLXSN-transduced ORL-48T cells. Inversely, cell proliferation and migration in ZAG-overexpression ORL-48T cells were not different from pCLXSN-transduced ORL-48T cells.These results demonstrate that PRDX2 functions as a tumor promoter whereas ZAG plays role on induction of late apoptosis in ORL-48T cells. The expression of PRDX2 in saliva can be biomarker to screen of precancerous and OSCC and the combination of PRDX2 and ZAG can increase the sensitivity to discriminate the precancerous and OSCC from the patient with any inflammatory lesions in oral cavity. These proteins will be further used for development of OSCC screening method.
