Avaliação do efeito citotóxico e polarizante de derivado sintético 1,2,4-oxadiazol em células de melanoma e MDMO murinos

Abstract

Cancer is a term generally used to refer to a set of diseases characterized by the uncontrolled proliferation of malignant cells and metastasis. The tumor microenvironment (TME) is composed of various inflammatory cells, with macrophages being the most numerous representatives. Depending on the stimulation, macrophages can play a pro-tumoral or anti-tumoral role. Skin cancer is the most frequent among them, with melanoma standing out due to its low incidence but high lethality. In the 1970s, dacarbazine was the first chemotherapy used for metastatic melanoma. Limited therapeutic success led to the use of immunotherapy to improve patient survival. Over time, immunotherapy targeting tumor-associated macrophages (TAMs) has shown promising results. Strategies include limiting TAM recruitment by blocking chemokines CCL2 and CXCL12, reducing M2 TAMs quantity, inducing their death, and repolarizing TAMs to the M1 phenotype using CD40 agonists, CD47 antagonists, PI3Kγ inhibitors, and class II HDAC inhibitors. The oxadiazole class, characterized by a heterocycle of two carbon atoms, two nitrogen atoms, one oxygen atom, and two double bonds, includes the isomer 1,2,4-oxadiazole, described as a bioisostere of amides. Our study aimed to evaluate the antitumor and immunomodulatory effects of the synthetic derivative 1,2,4- oxadiazole, N-cyclohexyl-3-(3-methylphenyl)-1,2,4-oxadiazole-5-amine, named molecule 2. In vitro assays assessed cytotoxicity in B16-F10 melanoma and bone marrow-derived macrophages (BMDMs), both murine, using the MTT method. Flow cytometry determined apoptosis/necrosis pathways in B16-F10, phenotypic changes in BMDMs after molecule 2, LPS, and murine melanoma supernatant stimulation, and cytokine levels in BMDM supernatants by ELISA. Molecule 2 inhibited proliferation in both B16-F10 (IC50 = 50.99µM) and BMDMs (EC50 = 132.7µM). Concentrations of 50.99, 76.48, and 101.98μM induced necrotic cell death in B16-F10 (7-AAD labeling between 70-75%). Stimulation with 33.17 and 66.35µM shifted BMDM phenotype to M1 (CD11b > 78%) and induced TNF-α production. Supernatants A, B, C, and D altered BMDM phenotype to M1 in varying percentages. Supernatants C and D induced NO, TNF-α, and IL-12 production. Co-stimulation with molecule 2 + LPS and molecule 2 + supernatant A shifted BMDM phenotype to M1 (CD11b > 80%) and induced NO, TNFα, and IL-12 production at different concentrations. Molecule 2 underscores the pharmacological potential of oxadiazoles, necessitating further studies to elucidate its demonstrated activities in antitumor and immunomodulation.