Nitrite Promotes The Proliferation And Invasion Of SMMC-7721 Cells By Activating Nrf2

Background: Nitrite levels in the blood and tissue of cancer patients are significantly increased.Moreover,nitrite promotes cancer growth at a certain concentration,but the specific mechanism of nitrite promoting cancer is not fully understood.nitrite exists in the form of NO2-in the body,and NO2-can directly inhibit mitochondrial respiratory chain complexes and lead to the increase of superoxide anion radical(O2·-).Under the condition of hypoxia,nitrite is reduced to nitric oxide radical(·NO),·NO can quickly interaction with O2·-to yield peroxynitrite anion(ONOO-).Nitrite may increase reactive oxygen species(ROS)levels in cancer cells.Additionally,Nuclear factor erythroid 2-related factor-2(Nrf2)is the master regulator of the cellular antioxidant response.Excessive ROS can promote the oxidation of cysteine residues on Kelch-like ECH-associated protein1(Keap1),inducing the activation of Nrf2,which translocates into the nucleus.And accumulating evidence has established Nrf2 activation promotes progression of pre-existing cancer.Aim: This study investigated the effect of nitrite on intracellular ROS level and the relationship between growth of human hepatocellular carcinoma SMMC-7721 cells induced by nitrite and Nrf2 activation.Methods: The SMMC-7721 cells were transfected with Nrf2 si RNA,RT-PCR and Western Blot were used to detect the changes of m RNA and protein levels of Nrf2 in SMMC-7721 cells.And then DCFH-DA staining was used to detect the changes of intracellular ROS.Additionally,the content of NO was detected by NO Nitrogen detection kit.Total antioxidant capacity of cells was examined using total antioxidant capacity detection kit.Moreover,cloneformation test was used to detect cells proliferation ability.And MTT method was used to detect the viability of SMMC-7721 cells.DAPI staining was used to observe nuclear morphology of SMMC-7721 cells.Furthermore,apoptosis was examined using Annexin V-FITC/PI double staining method.The migration and invasion of SMMC-7721 cells were detected by Transwell migration and invasion experiment.Results:(1)After SMMC-7721 cells were transfected using Nrf2 si RNA,Nrf2 m RNA and Nrf2 protein expression in cytoplasm and nucleus were significantly reduced.(2)DCFH-DA staining results showed that nitrite(SN:16mg/L)increased the ROS in SMMC-7721 cells.And Nrf2-depleted SMMC-7721 cells were treated by nitrite,the intracellular ROS were significantly increased.(3)Nitrite increased the accumulation of Nrf2 protein in the nucleus.However,the Nrf2-depleted SMMC-7721 cells were treated with nitrite,the expression of Nrf2 protein in the nucleus was significantly reduced.Additionally,Nitrite enhanced the total antioxidant capacity of SMMC-7721 cells significantly.However,the Nrf2-depleted SMMC-7721 cells were treated with nitrite,the total antioxidant capacity of cells decreased significantly.(4)The results of cells cloning experiments showed that nitrite significantly increased the number of cells clones.However,when SMMC-7721 cells were transfected with Nrf2 si RNA,and then the Nrf2-depleted SMMC-7721 cells were treated with nitrite,the cells clones was significantly reduced.Moreover,the cells viability results of MTT assay showed that nitrite enhanced cells viability.However,when SMMC-7721 cells were transfected using Nrf2 si RNA,and then nitrite treated Nrf2-depleted SMMC-7721 cells,the cells viability decreased significantly.(5)DAPI staining results showed that when SMMC-7721 cells were transfected with Nrf2 si RNA,and then the Nrf2-depleted SMMC-7721 cells were treated with nitrite,the cells showed characteristic changesin apoptosis.Furthermore,the apoptosis rate was increased significantly in the(SN+)+(Nrf2si RNA+)group.(6)The distribution of microfilaments showed that SMMC-7721 cells were treated with nitrite,the spindle cells were increased,what's more,stress fibers and Pseudopods were increased.However,the Nrf2-depleted SMMC-7721 cells were treated with nitrite,the cortex of cells became thicker,and stress fibers decreased or even disappeared.Transwell migration and invasion experiment results showed that nitrite enhanced the cell migration and invasion ability significantly,but when the Nrf2-depleted SMMC-7721 cells were treated with nitrite,the migration and invasion ability of cells were significantly weakened.Conclusion: Nitrite(16mg/L)increases the ROS level in human hepatocellular carcinoma SMMC-7721 cells,and further promotes the proliferation,migration and invasion ability of human hepatocellular carcinoma SMMC-7721 cells by activating Nrf2.