Abstract
Leflunomide (LFN) is a well-known immunomodulatory and anti-inflammatory prodrug of teriflunomide (TFN). Due to pyrimidine synthesis inhibition TFN also exhibits potent anticancer effect. Because, there is the strict coupling between the pyrimidine synthesis and the mitochondrial respiratory chain, the oxygen level could modify the cytostatic TNF effect.The aim of the study was to evaluate the cytostatic effect of pharmacologically achievable teriflunomide (TFN) concentrations at physiological oxygen levels, i.e. 1% hypoxia and 10% tissue normoxia compared to 21%oxygen level occurred in routine cell culture environment.The TFN effect was evaluated using TB, MTT and FITC Annexin tests for human primary (SW480) and metastatic (SW620) colon cancer cell lines at various oxygen levels.We demonstrated significant differences between proliferation, survival and apoptosis at 1, 10 and 21% oxygen in primary and metastatic colon cancer cell lines (SW480, SW620) under TFN treatment. The cytostatic TFN effect was more pronounced at hypoxia compared to tissue and atmospheric normoxia in both cancer cell lines, however metastatic cells were more resistant to antiproliferative and proapoptotic TFN action. The early apoptosis was predominant in physiological oxygen tension while in atmospheric normoxia the late apoptosis was induced.Our findings showed that anticancer TFN effect is more strong in physiological oxygen compared to atmospheric normoxia. It suggests that results obtained from in vitro studies could be underestimated. Thus, it gives assumption for future comprehensive studies at real oxygen environment involving TNF use in combination with other antitumor agents affecting oxygen-dependent pyrimidine synthesis.
Highlights
Specific changes in cellular metabolism lead to key processes important for cancer development – uncontrolled growth and prolifer ation
dihydroorotate dehydrogenase (DHODH) is located in the mito chondrial inner membrane with the active site facing in and is being functionally connected to the Electron Transport Chain (ETC) by a flavin prosthetic group that couples dihydroorotate oxidation with reduction of ubiquinone to ubiquinol followed by electron flow through the ETC
Cell culture conditions and treatments SW480, SW620 and HaCaT cells were purchased from the American Type Culture Collection (ATCC) and cultured according to protocol
Summary
Specific changes in cellular metabolism lead to key processes important for cancer development – uncontrolled growth and prolifer ation. Pyrimidine biosynthesis dependent on respiration-linked dihydroorotate dehydrogenase (DHODH) is crucial for cell growth, while mitochondrial ATP generation is not essential for proliferation [2,3]. It seems that pharmacological inhibi tion of mitochondrial metabolism can be used as a potential therapeutic strategy in some cancers. Among the enzymes of de novo pyrimidine biosynthesis pathway only DHODH is localized in mitochondria. The integral step of de novo pyrimidines synthesis is catalyzed by the flavoenzyme dihydroorotate dehydrogenase (DHODH) which provides oxidative reaction relies on the conversion of dihydroorotate to orotate. DHODH is located in the mito chondrial inner membrane with the active site facing in and is being functionally connected to the Electron Transport Chain (ETC) by a flavin prosthetic group that couples dihydroorotate oxidation with reduction of ubiquinone to ubiquinol followed by electron flow through the ETC
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