Ação da leptina sobre o efeito terapêutico da radiação ionizante e mecanismos de degradação proteolítica no carcinoma epidermoide de boca

Abstract

Leptin, a hormone that regulates energy balance and body weight control, can be involved in the development and progression of different cancer types. In oral squamous cell carcinoma (OSCC), the leptin signaling pathway was associated with changes in the neoplastic phenotype and increased expression of genes related to proliferation, invasion and angiogenesis. Evidence indicates that patients with squamous cell carcinoma in the head and neck submitted to radiotherapy show the reduced levels of leptin after treatment. However, it is not well understood whether increased levels of leptin are able to interfere with the effect of antineoplastic treatment. In addition to the leptin signaling pathway, the ubiquitin-proteasome system, responsible for proteolytic degradation mechanisms, has been of interest target for research in the field of oral carcinogenesis. These two targets were adopted as the focus of study, since both are involved in the mechanisms of neoplastic proliferation and cell cycle deregulation. In this context, this study aimed to investigate the action of leptin on the therapeutic effect of ionizing radiation and mechanisms of proteolytic degradation in CEB. For this, two study approaches were outlined. In the first approach, through in vitro assays, we hypothesized that leptin may compromise the effect of ionizing radiation and behavior of OSCC cells. The immortalized cell lines, SCC4 and SCC9, were treated with recombinant human leptin, and exposed to 6Gy of radiation produced by cobalt-60. For phenotypic analysis, the cell proliferation, death, migration and clonogenic assays were performed. The molecular profile was investigated through the analysis of reactive oxygen species (ROS) levels, and proteomic analysis by means of mass spectrometry. Leptin promoted reduction of cell death and increased cell proliferation, migration and clonogenic formation, despite the suppressive effects induced by ionizing radiation. In addition, it was associated with a significant reduction of intracellular accumulation of ROS, and increased expression of proteins related to carcinogenesis events, such as ACTC1, KRT6A and EEF2, in irradiated cells. A second approach was proposed to investigate whether leptin can modulate the gene expression of ubiquitin-proteasome system components and neoplastic behavior in OSCC cells. Through bioinformatic simulations, it was hypothesized that leptin establishes interactions with components of the ubiquitin-proteasome system. The OSCC cell lines were treated with leptin and the levels of mRNA expression of the ubiquitin-proteasome system components were quantified through qRT-PCR, including USP2, UBA, UBC, PSMD4 and PSME3. The cell viability and death tests were performed. The In silico analysis showed a possible mechanism of activation of USP2 by leptin. The results revealed that leptin favored the neoplastic phenotype, accordling with the increase inthe expression of the genes involved in the mechanism of proteasomal degradation. The cells treated with leptin showed overexpression of the oncogene USP2 and regulatory genes of the cell cycle, UBA and UBC. The proteasomal genes PSME3 and PSMD4 were not altered by treatment. Our findings points the leptin as an important factor impairing the responsivity of OSCC cells to the ionizing radiation, reducing its suppressive effects on the neoplastic phenotype, and increasing protein expression of important genes involved in carcinogenesis pathway. We highlight yet that leptin can modulate the gene expression of ubiquitin-proteasome system components, suggesting this association as an important factor modifying the behavior of OSCC cells. However, functional analysis and in vivo assays are necessary in order to better understand the biological impact of this mechanisms connection in oral carcinogenesis. Thus, functional analyzes and in vivo assays are needed to better understand the biological impact of this mechanism binding on oral carcinogenesis, as well as the real importance of leptin in molecular events related to the radiation on oral cancer cells.