The Effects Of MIBG On The Invasive Properties Of Hepatocellular Carcinoma Cells

Objective:The aim of the present study was to investigate the effects of meta-iodobenzylguanidine (MIBG) on the invasive properties of hepatocellular carcinoma (HCC) cells and examine whether these effects are due to the ability of MIBG to inhibit arginine-specific mono-ADP-ribosylation.Methods：This experiment uses the human liver cancer tissue paraffin section and fresh human liver cancer tissue in vivo experiment;HepG2 liver cancer cell lines in vitro experiment。1.Human liver cancer tissue:HCC tissue sections (n=39) were obtained from the Department of Pathology of the First Affiliated Hospital of Chongqing Medical University, Chongqing,China.Samples from patients with HCC were divided into 2 groups, a metastatic group and a non-metastatic group;Fresh HCC samples (3 cases of HCC with metastasis; 3 cases of HCC without metastasis) were obtained from the Department of the operating room of the First Affiliated Hospital of Chongqing Medical University. Immunohistochemistry and RT-PCR were used to detect the protein and mRNA expression of arginine-specific adenosine diphosphate-ribosyltransferase 1 (ART1) and integrin α7 in the HCC tissues.2.Cultured HepG2 cells,In addition, the expression of ART1 was measured in HepG2 HCC cells by immunofluorescence;The inhibition of the metastasis of HepG2 cells by MIBG at various concentrations was measured by MTT assay;The effects of MIBG on HepG2 cell metastasis were measured using a scratch wound assay and a Transwell invasion assay;Western blot analysis was used to detect the protein expression of ART1, integrin α7, focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI3K) and urokinase-type plasminogen activator (uPA) in the HepG2 cells.Results:1.Immunohistochemical(IHC) analysis:Its expression was mostly observed on the cell surface and in the cytoplasm. The expression levels of ART1 and integrin α7 in the HCC samples with metastasis and a liver histological grade (Edmondson grade) of Ⅲ-Ⅳ were significantly higher than those in the HCC samples without metastasis and a liver histological grade (Edmondson grade) of I-II (P＜0.05). ART1 expression was significantly lower in the HCC tissues that were weakly positive for integrin α7 than in the HCC tissues that were strongly positive for integrin α7. The expression of ART1 positively correlated with the expression of integrin α7 in the HCC tissues (p=0.88410, P<0.01).2.RT-PCR assay:The RT-PCR results revealed that the mRNA expression levels of ART1 and integrin α7 were significantly higher in the HCC samples with metastasis than in the HCC samples without metastasis (P<0.05).3.Immunofluores:The HepG2 cells were incubated with anti-ART1 antibody, then bound with FITC-labeled secondary antibody. They were then observed under a fluo-rescence microscope, and the results showed green fluorescence on the cell surface and in the cytoplasm. The negative control HepG2 cells did not show green fluorescence. This indicates that ART1 is expressed in HepG2 cells.4.MTT assay:The proliferation of HepG2 cells decreased as the concentration of MIBG increased, indicating that MIBG inhibited the proliferation of the HepG2 cells. The IC50 of MIBG in the HepG2 cells was 200 μmol/1 (P＜0.05).5.Scratch wound assay:The width of the scratched area in the untreated control group of HepG2 cells at 24 h was significantly smaller than that at 0 h. This indicated that the HepG2 cells had a strong ability to migrate. In the 200 μmol/1 MIBG-treated group, the width of the scratched area of HepG2 cells at 24 h was significantly greater than that of the untreated group (P＜0.05). This indi-cated that HepG2 cell migration was inhibited by MIBG.6.Transwell Matrigel invasion assay:The number of HepG2 cells that underwent invasion through Matrigel in the MIBG-treated group was 147.2±18.23952. The number of untreated control group cells was 300.4±11.9147. These values were significantly lower in the MIBG-treated group than in the control group (P<0.01), indicating that MIBG inhibited the invasive ability of the HepG2 cells.7.Western blot:Western blot analysis revealed that after MIBG was incubated with the HepG2 cells, the protein expression of ART1, integrin α7, FAK, PI3K and uPA in the HepG2 cells was significantly lower compared with the untreated group, and this difference was dose-dependent. The difference was statistically significant (P<0.05). These results suggested that MIBG inhibited the invasion and migration of HepG2 cells, possibly through the inhibition of the ART1/integrin α7/FAK/PI3K/uPA pathway.Conclusion:Our data demonstrate that ART1 and integrin α7 may be involved in the invasive and metastatic properties of HCC cells. MIBG inhibited the migration and invasion of HepG2 cells, possibly through the inhibition of arginine-specific single-adenosine diphosphate ribosylation and the suppression of the protein expression of integrin α7β1, FAK and PI3K and the secretion of uPA, leading to reduced invasion by HepG2 cells.