Immunomodulatory aspects in the progression and treatment of oral leukoplakia: in vitro and in vivo studies

Abstract

Squamous cell carcinoma (SCC) in the head and neck is one of the most common malignancies in the world. One way of preventing it is by early detection and treatment of oral potentially malignant lesions (OPML). One of the most common OPML is oral leukoplakia (OLP); its malignant transformation rate is related to its clinical form and the presence or absence of epithelial dysplasia. To date, there is no evidence that any treatment for OLP reduces its progression to oral cancer. The objectives of this study will be: to evaluate whether treatments with artemisinin and/or with Rubus occidentalis extract (BRB) are capable of inducing immunogenic cell death (ICD) in human cell lines of OLP and in a preclinical model; to characterize the population of inflammatory cells; to verify the gene expression of inflammatory cytokines and immunogenic cell death. Material and methods: in the in vitro study, commercial cell lines of OLP will be treated by artemisinin and BRB in a combined or isolated way in order to analyze the expression of intracellular high mobility Box protein (HGMB), caspase 3, 8, and 9 and, Calreticulin (CALR) by immunofluorescence. Extracellular HGMB will be checked using the ELISA test. In the in vivo trial, 72 male mice will receive water with 4-Nitroquinoline N-oxide for 16 weeks. In the 8th week, after the detection of OLPs, the animals will be treated with a subepithelial injection of artemisinin weekly and/or by BRB in the daily feed. In the 12th and 16th weeks, the animals will be euthanized, and the collected tongues will be analyzed clinically and photographed. The qRT-PCR reaction will be performed for inflammatory cytokines and ICDs transcripts evaluation. Afterward, the other tongue's part will be employed to evaluate the protein expression of inflammatory cytokines using the ELISA test. The classification of the degree of epithelial dysplasia will be performed under conventional light microscopy, and the characterization and quantification of the population of immune cells will be carried out using the immunohistochemistry technique. The appropriate statistical tests will be used with ± = 0.05. (AU)