Exposure Of Human Hepatoma Cells To Nitrite And Ammonia Promote Cell Proliferation And Invasion

Background: With the wide application of agricultural fertilizers and industrial preservatives, and the content of ammonia and nitrite nitrogen in soil nature water increased every year. Meanwhile, epidemiological studies show severe cancer with ammonia contaminated areas gradually increased morbidity and mortality, suggesting that nitrite may have carcinogenic effects. In recent years, the carcinogenic effects of nitrite has been questioned, there is no direct evidence that nitrite is a carcinogen in animal studies, but found their range of additional medicinal value, such as reducing blood pressure high cholesterol and protect the organ shortage blood reperfusion injury. Many scholars therefore proposed that nitrite could be used as drugs for clinical treatment. On this issue, the research group also requires careful care it. Because the realization of these medicinal value through nitrite converted into nitric oxide in acid environment of hypoxic in tumor tissue. The characteristic of microenvironment of the tumor cell life is lack of oxygen and acidic and the tumor microenvironment is a high ammonia environment, in the microenvironment, nitrite would back into nitric oxide, stimulates the tumor cells proliferation.To date, There is no literature reported nitrite in a high ammonia environment effect on tumor cell biology. And reveal the nitrite in a high ammonia environment influence on tumor proliferation and invasion, on a thorough knowledge of the biological effect of nitrite has very important significance.Aim: This study was to explore combined exposure to ammonia and nitrite on the effects of proliferation and invasion ability in human hepatoma cells.Methods: The experiment was divided into four groups, respectively, blank control group, sodium nitrite group, ammonium chloride group, sodium nitrite and ammonium chloride mixed group. The cell viability was analyzed by MTT assay; cell mitosis was observed by microtubules immunofluorescence; cell migration was analyzed by wound healing assay; cell mitochondria and microfilament change was observed with mitochondrial tracker and microfilament tracker detected by fluorescence microscope; cell ATP content detected by colorimetry; cell invasion was analyzed by Trans well assay; the intracellular ROS levels was detected by reactive oxygen species(ROS) test kits; the intracellular ODC levels was detected by ODC ELISA kits; the expression of MMP-2,MMP-9,ODC were analyzed by Western Blot.Results: 1. sodium nitrite and ammonium chloride combined exposure to promote proliferation of the cells to determine the optimal concentration and time MTT showed: Sodium nitrite, ammonium chloride, sodium nitrite, and ammonium chloride mixture(V: V = 1:1) for liver cancer SMMC- 7721 cells and promote proliferation activity of optimal concentration to 150?M and promote proliferation activity of suitable time for 24 h, mixed group compared with other groups to promote proliferation activity more obviously.2. Microtubules tracer observe mitosis phase showed Sodium nitrite groups,ammonium chloride groups slightly increased mitotic cells compared with the control group, mixed group compared with the other groups mitotic phase increased significantly. These results showed that the mixture has a stronger ability to promote the proliferation of hepatoma cells than sodium nitrite alone.3. Sodium nitrite and ammonium chloride combined exposure enhanced Liver cancer cell migration and invasion. Cell wound healing assay showed that compared with the control group and the individual group nitrite and ammonium chloride, a mixture of group healing scratches relatively more distance large, horizontal movement stronger; the cells form a large number actin stress fibers, a large number of lamellipodia and filopodia formation, the number of mitochondria increased compared with other groups; cell ATP content increased significantly; Tran swell invasion assay showed that the mixture group cells passes through the basement membrane significantly increased compared to the control group, a single sodium nitrite and ammonium chloride alone;Western blotting results showed that compared with the control group and simple nitrite and ammonium chloride, MMP-2 and MMP-9 expression was significantly increased in mixture group; the above of showed mixture group than alone nitrite stronger hepatocellular carcinoma cell migration and invasion capabilities.4. Sodium nitrite and ammonium chloride combined exposure via ROS / ODC pathways promote hepatocellular carcinoma cell migration and invasion DCFH-DA fluorescent probe detect intracellular ROS level, and the results show the group of sodium nitrite, ammonium chloride in the fluorescence intensity of ROS slightly stronger than the control group; a mixture of sodium nitrite and ammonium chloride group were significantly increased compared with sodium nitrite or ammonium chloride group alone. ELISA and Western blotting experiments showed that, compared with the control group and the group of nitrite and ammonium chloride group, a mixture of group ornithine decarboxylase(ODC) expression was significantly increased.Conclusion: 1. Nitrite and ammonium mixture treatment could promote human liver SMMC- 7721 cells proliferation and invasion.2. Sodium nitrite and ammonium chloride mixture may promote invasion in SMMC-7721 cells by enhancing ROS/ODC pathway.