Effect Of Nitrite On Mitochondrial Biogenesis And The Potentials Of Migration And Invasion Of Human Hepatocellular Carcinoma SMMC-7721 Cells

Background The concept that nitrite is a carcinogen has been deep-rooted, but it is now lack of direct experimental evidences. In recent years, plenty of studies have found that low doses of nitrite have many pharmacological effects, such as decreasing blood pressure, lowering blood cholesterol, preventing atherosclerosis, reducing ischemia/reperfusion injury in organs and so on. We must understand the effect of nitrite on patients who also suffered from cancer before developing nitrite as a clinical drug. The occurrence and progression of carcinoma not only needs initiation, but also relys on promotion. Cancer promotion includes Tumor Cells Proliferation,invasion and metastasis. These behaviours of carcinoma cells need energy,and almost all of the energy generates in mitochondria. Mitochondria are highly dynamic organelles which regulate mitochondrial quality through mitochondrial biogenesis and mitophagy. Mitochondial mass changes and carcinoma promotion have close relationship. To further realize the influence of nitrite on carcinoma promotion, it has important significance that finding out the role of nitrite on mitochondrial biogenesis of cancer.Aim To study the influence of nitrite on the mitochondrial biogenesis of human hepatocellular carcinoma SMMC-7721 cells and provide theoretical basis for the view that nitrite is a tumor promoter.Methods After exposure of human hepatocellular carcinoma SMMC-7721 cells to a serial concentrations of sodium nitrite for 24 h under normal oxygen,showed the cell vitality by MTT assay. DCFH-DA staining was used to test the intracellular level of ROS concentration. the impact of nitrite on the potentials of migration and invasion for SMMC-7721 cells was tested by transwell assay.the effect of nitrite on stress fibers and mitochondrial distribution in SMMC-7721 cells were detected by cell staining with Actin-Tracker Green and Mito-Tracker Red. RT-PCR was used to detect the expression levels of COX? and COX? m RNA. laser scanning confocal microscopy showed mitochondrial mass detected by Mito-Tracker Green staining. the expression levels of HIF-1?, PGC-1? and cytochrome c were tested by Western blot.Results After exposure of human hepatocellular carcinoma SMMC-7721 cells to a serial concentrations of sodium nitrite for 24 h under normal oxygen, the cell vitality by MTT assay showed maximal increase after insulting with 16 mg·L-1 sodium nitrite. DCFH-DA staining with fluorescent microscopy showed that the intracellular level of ROS concentration increased after sodium nitrite treatment. In addition, the potentials of migration and invasion for SMMC-7721 cells increased significantly after exposure of cells to sodium nitrite for 24 h. Furthermore, sodium nitrite exposure displayed an increase of stress fibers, lamellipodum and perinuclear mitochondrial distribution by cell staining with Actin-Tracker Green and Mito-Tracker Red, which could be reversed by N-acetylcysteine(NAC, an reactive oxygen scavenger). RT-PCR results indicated that the expression levels of COX? and COX? m RNA decreased significantly after sodium nitrite treatment. Meanwhile, laser scanning confocal microscopy showed that sodium nitrite significantly reduced mitochondrial mass detected by Mito-Tracker Green staining. The expression levels of HIF-1? increased remarkably and the expression levels of PGC-1? and cytochrome c(mitochondrial biogenesis marker molecular) decreased significantly after sodium nitrite treatment, which were reversed by NAC.Conclusions low doses of sodium nitrite decreased mitochondrial biogenesis and then enhanced the potentials of invasion and migration in hepatic cancer SMMC-7721 cells via ROS/HIF-1? pathways.