İnsan Makrofajlarında Nitrik Oksit Sentaz 2 (Nos2) İfade ve Aktivitesinin Bazal Benzeri Meme Kanseri Hücrelerinden Üretilen Faktörler Varlığında İncelenmesi

Abstract

Macrophages are the leading cells of innate immunity in the immune response against pathogens and are involved in the regulation of adaptive immune responses. Macrophages with microbicidal properties promote the proinflammatory response by stimulating bacterial products such as LPS, cytokines such as IFN-γ or TNF-α. Production of nitric oxide (NO) and reactive oxygen species (ROS) is essential in the biochemical mechanism of antimicrobial responses. NO production is provided by nitric oxide synthase (NOS) enzymes. While the existence of the inducible NOS isoform (NOS2) in mouse macrophages has been proven by many studies, there are many conflicting publications on NOS2 expression and NO production level in humans. This thesis is based on the hypothesis that factors produced from basal-like breast cancer cells during monocyte-macrophage transformation can change NOS2 expression and NO during the activation and differentiation processes of myeloid cells. To this end, the THP-1 monocytic cell line was differentiated into macrophage in the presence of basal-like breast cancer cell line supernatants and stimulated with lipopolysaccharide (LPS) and interferon (IFN)-γ. The phenotypic (CD14, TLR4, MD2, CD40, CD11c and IFNγR), functional (ROT and NO production) and morphological (May-Grünwald Giemsa, actin cytoskeleton staining) differentiation parameters in cells were examined. In addition, experiments were performed with fibronectin and Matrigel coated surfaces during differentiation of THP-1 cells under these conditions. Under these conditions, NOS2 expression level was investigated at the mRNA and protein levels. Confirmation experiments were performed in the presence of mouse macrophage and breast cancer cells and in the presence of supplemental human cell lines supernatants. It was determined that ROS and NO production were modulated in THP-1 cells differentiated in the presence of factors secreted from MDA-MB-231 cells used as a human basal-like breast cancer cell line prototype. It was observed that the effect of LPS could be limited in the presence of cancer supernatants, but IFN-γ had a more pronounced effect. It was determined that human NOS2 levels were negatively affected in the presence of breast cancer supernatants and this was also reflected in NO production. The findings indicate that human NOS2 expression and thus NO production can be regulated at transcriptional-translational levels depending on the degree of monocyte- macrophage differentiation, especially in the presence of factors secreted from cancer cells.