| Background: Hepatocellular carcinoma is one of common malignant tumors,accounting for the third cause of cancer death in the world.China is a big country with liver disease,for the high incidence of liver cancer.Liver cancer due to radiotherapy and chemotherapy is not sensitive,with high metastases and high recurrence rate,resulting in poor curative efficacy and prognosis.Therefore,to find a new drug that can effectively inhibit the metastasis of liver cancer and enhance the sensitivity of hepatocellular carcinoma to radiotherapy and chemotherapy may be of great significance in improving the prognosis of liver cancer.Arginase(Arg)hydrolyzes L-arginine to the products L-ornithine and urea involved in the process of ammonia detoxification.Arginase also promote tissue regeneration,cell proliferation and so on by its downstream products,involved in inflammation triggered tumor immune escape,fibrosis,immunosuppression and other pathological process.Nitric oxide synthase(NOS)competes with Arg for L-arginine to produce nitric oxide(NO),NO has played a very important role in tumor initiation,progression and metastasis,and other important tumor-related processes.The effect of NO on the tumor depends on its concentration level.A low concentration of NO can promote the progression of the tumor,while the high concentration of NO exerts cytotoxicity,induction of apoptosis,inhibition of tumor progression and metastasis.However,it is not clear whether inhibition of Arg can increase the expression of NOS,resulting in high concentrations of NO to inhibit tumor.Objective: To investigate the effect of arginase inhibitor nor-NOHA on the proliferation of HepG2 hepatocellular carcinoma cells,the induction of apoptosis in HepG2 cells,and on the invasion and migration of HepG2 cells and to explore the specific mechanisms.Methods: The inhibition of nor-NOHA(0.0,0.5,1.0,2.0,3.0)ng/?L on proliferation of HepG2 cells was detected by CCK-8 method.The effect of nor-NOHA on HepG2 cell apoptosis was detected by flow cytometry.The proteins levels of arginase 1(Arg1),p53,matrix metalloproteinase-2(MMP-2),E-cadherin(E-cad)were detected by Western blotting.Real time quantitative PCR was employed to examine the changes in the m RNA level of inducible nitric oxide synthase(i NOS).Using Griess assay to measure the concentration of nitric oxide(NO)in HepG2 cells.Transwell assay and Wound-healing assay were used to evaluate the changes of the cell invasion and migration ability,respectively.Results: 1?The survival rate of nor-NOHA-treated HepG2 cells at the concentration of 1.0,2.0 and 3.0 ng/?L was significantly lower than that of the control group(P<0.05).The apoptosis rate of nor-NOHA group was significantly higher than that of the control group(P<0.05)by flow cytometry.2?The expression of P53 and E-cad in nor-NOHA-treated HepG2 cells was significantly higher than that in the control group(P <0.05),while the expression of Arg1 and MMP-2 in the nor-NOHA group was significantly lower than that in the control group(P <0.05).3?Real-time quantitative PCR results showed that the expression of i NOS m RNA in nor-NOHA group was significantly higher than that in the control group(P <0.05).The results of Griess test showed that production of NO was significantly increased in HepG2 cells treated with nor-NOHA(P <0.05).4?Transwell assay and Wound-healing assay confirmed that the migration and invasion ability of HepG2 cells in nor-NOHA treatment group was significantly reduced than that in the control group(P <0.05).Conclusion: 1?nor-NOHA inhibits the proliferation of HepG2 cells and induces apoptosis.This process is related to the high concentration of NO up-regulated the expression of p53 protein.2?nor-NOHA reduces the invasion and migration ability of HepG2 cells,which is associated with the increased expression of E-cad and the decrease of MMP-2 expression by decreasing the expression of Arg,which resulting in increased expression of i NOS to produce high concentrations of NO. |
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