Efeito da terapia fotodinâmica no fenótipo tronco de linhagens celulares de carcinoma epidermóide de boca

Abstract

Among the various types of cancer that affect the oral cavity, squamous cell carcinoma of the mouth (OSCC) is the most prevalent malignant neoplasm. Despite advances in treatment, patients with OSCC often present metastasis at diagnosis and recurrence, both associated with a poor prognosis. Recent studies have shown that the presence of cancer stem cells (CTC) are responsible for tumor growth and resistance to conventional therapy. An alternative for the treatment of CEB is Photodynamic Therapy (PDT). However, the effect of PDT on CTC in the CEB is unknown. Thus, this study aimed to evaluate in vitro the effect of PDT mediated by the photosensitizer 5-aminoulevulinic acid (5-ALA) on cell viability, maintenance of the stem cells phenotype, cell migration and expression of the genes Bmi1, Clagranulin, IVL and E- cadherin in two cell lines deriveted of OSCC (Ca1 and Luc4). All in vitro assays were conducted with both cell lines, which were divided into the following groups: Control, 5-ALA, LED 6J/cm² and PDT (5-ALA+6J/cm2). Cell viability was assessed by MTT and Neutral Red assays and the stem phenotype was assessed by flow cytometry to analyze the expression of CD44 and ESA to identify the CD44high/ESAhigh, CD44high/ESAlow and CD44low populations, as well as by colony and tumor sphere formation assays. Additionally, the PpIX production in each CTC population was evaluated. Cell migration was analyzed by wound healing assay and gene expression was analyzed by RT-qPCR. Both cell lines showed a significant reduction in cell viability after PDT. A percentage reduction of CD44high/ESAhigh cells was observed after PDT when compared to the other groups, which was followed by an increase in the percentage of cells with the CD44low phenotype, indicating cell differentiation induction. Additionally, reduced colony formation capacity and tumoral sphere formation after PDT. A CD44high / ESAhigh population of both cell lines had greater production of PpIX, indicating greater sensitivity of them to PDT. Cells from the Ca1 and Luc4 cell lines of the 5-ALA and PDT groups had less capacity to migrate compared to the other groups. An analysis of gene expression after PDT, showed a decrease in Bmi1 expression and increased Calgranulin expression in the Ca1 cell line, confirming the loss of the stem potential and induction of cell differentiation. Thus, the results shown here, indicate that a 5-ALA-PDT is capable of decreasing the cell viability and cell lines migration derived from OSCC, as well as reducing the percentage of cells with stem phenotype, inducing their differentiation.