Introduction
Adenosine diphosphate (ADP)-ribosylation reaction refers to the process during which mono- or poly-ADP is transferred onto amino acid residues. The process is catalyzed by ADP-ribosyltransferases (ARTS) to break the glycosidic bond between nicotinamide of nicotinamide adenine dinucleotide (NAD+) and adjacent ribose (1,2).
The mono-ADP-ARTS (mARTS) include 5 members, the only one of which, existing in human cells with a fixed sequence R-S-EXE of arginine-specific ARTS, is arginine-specific mono-ADP-ribosyltransferase-1 (ART1) (16), which can directly modify integrin α7β1. As a transmembrane protein, integrin α7β1 is highly involved in the adhesion, invasion and metastasis of cancer cells. The modified integrin α7β1 is functionally upregulated, with its affinity to laminin (LN) improved (3–5).
Meta-iodobenzylguanidine (MIBG) is a type of analogue of guanidine neurotransmitter, norepinephrine, which can inhibit the function of ARTS through competitive binding of the catalytic site. As a result, MIBG can function as an ART1-specific inhibitor (6). It has been previously reported that MIBG inhibits the proliferation and metastasis of smooth muscle cells (7).
In a previous study by our group, it was demonstrated that the gene silencing of ART1 in colorectal carcinoma inhibited the expression of ART1, downregulated the expression of matrix metalloproteinase (MMP)-2 and MMP-9, inhibited their activity and eventually affected the adhesion and invasion capacity of CT26 colorectal carcinoma cells (8). Currently, however, the effects of ART and integrin α7β1 on the invasion and metastasis of hepatocellular carcinoma (HCC) (HepG2) cells remains unknown. Consequently, this study aimed to investigate the effects of ART and integrin α7β1 on the invasion and metastasis of HCC cells, as well as to determine the effects of ART1 on the expression of integrin α7β1, focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI3K) and urokinase-type plasminogen activator (uPA) in HepG2 cells, after inhibiting the expression of ART1 by utilizing MIBG. The present study provides preliminarily relevant experimental foundation for a novel method for HCC moleculer-targeted therapy.
Materials and methods
Immunohistochemical (IHC) analysis
HCC tissue sections (n=39) were obtained from the Department of Pathology of the First Affiliated Hospital of Chongqing Medical University, Chongqing, China. Of these, 29 were from male patients and 10 were from female patients with the liver histological grade (Edmondson grade) distributed as follows: I–II (12 cases), III–IV (27 cases), HCC with metastasis (lymph node, blood vessel or other organs) (17 cases) and HCC without metastasis (22 patients). IHC was used to detect the expression of ART1 (Sigma, St. Louis, MO, USA) and integrin α7 (Beijing, Biosynthesis Biotechnology Co., Ltd., Beijing, China) in the HCC tissues according to the instructions provided with the kit. Sets of positively stained cells were divided into groups based on the antigen signal: pale yellow, weakly positive (+) 1 point; yellow, moderately positive (++) 2 points; brownish yellow, strongly positive (+++) 3 points. The slides were divided into groups based on the number of positive cells: weakly positive (+, refers to the number of positive cells up to 25%); moderately positive (++, refers to the number of positive cells between 25–49%); strongly positive (+++, >50% of cells were positive). Using integrated measurement, the following calculation formula was developed and used: (+)% × 1 + (++)% × 2 + (+++)% × 3, the total value of <1.0 by (+), 1.0 to 1.5 by (++), >1.5 by (+++). Random observations were made for at least 5–10 high-power fields (HPFs).
RT-PCR detection of ART1 and integrin α7 mRNA expression
Fresh HCC samples (3 cases of HCC with metastasis; 3 cases of HCC without metastasis) were obtained from the Department of Hepatobiliary Surgery of the First Affiliated Hospital of Chongqing Medical University. The samples were stored in liquid nitrogen. Total RNA was extracted from the tissue samples using a One-Step RT-PCR kit (Takara, Shiga, Japan) according to the manufacturer’s instructions. The primers for ART1 (XM_005252934) and integrin α7 (NM_001144996) were synthesized by Shanghai Sangon Biological Engineering Technology & Services, Co., Ltd., Shanghai, China. The primer sequences were as follows: ART1 forward, 5′-CTCCATTTCC TGCTGACTGAG-3′ and reverse, 5′-AGGTCCAGATGCC GAAGAAG-3′; integrin α7 forward, 5′-GAACTCCTCCC ACCCAACTT-3′ and reverse, 5′-GACGAAACCACGAAA CCACT-3′; β-actin forward, 5′-GTCAAGAAAGGGTGTAA CGCAAC-3′ and reverse, 5′-TCCTGTGGCATCCACGA AACT-3′. The PCR reaction conditions for ART1 and integrin α7 were as follows: 30 cycles of 94°C for 3 min, 94°C for 30 sec and 60°C for 30 sec. The PCR reaction conditions for β-actin were as follows: 30 cycles of 94°C for 3 min, 94°C for 30 sec, 56°C for 60 sec and 72°C for 60 sec. After completion of the reaction, 20 μl PCR reaction solution was used for electrophoresis on an agarose gel. Band intensities were measured using Quantity One software (Bio-Rad, Hercules, CA, USA), and gene amplification was defined as the ratio of the optical density (OD) of the target gene to that of the housekeeping gene, β-actin. The experiment was performed 3 times.
Cell culture
HepG2 cells were provided by the Life Science Institute of Chongqing Medical University. The cells were grown in RPMI-1640 supplemented with 10% fetal calf serum plus ampicillin (100 μ/ml) and streptomycin (100 μg/ml), at 37°C and 5% CO2. MIBG (Sigma) was used to inhibit the ART1 signaling pathway.
Immunofluorescence (IF) detection of ART1 expression
The HepG2 cells (1×106 cells/well) were seeded into 6-well plates containing glass coverslips. When cell attachment to the glass was approximately 90% complete, the slides were removed and washed 3 times with PBS. These cells were fixed with 10% formaldehyde for 30 min, washed 3 times with PBS for 2 min each, and treated with 0.5% Triton for a further 15 min. They were then washed twice with PBS for 5 min each. The cells were then covered with 1% BSA for 30 min and incubated with 1:100 anti-ART1 antibody overnight at 4°C. Cells exposed to PBS instead of the primary antibody served as the negative controls. The following day, the slides were washed 3 times with PBS for 5 min each. Subsequently, 1:500 rabbit anti-goat IgG/FITC-labeled antibody was added at 37°C in the dark for hybridization followed by incubation for 1 h. The slides were then washed 3 times with PBS. Finally, the glass coverslips were mounted using fluorescence quenching liquid. Images were captured using a fluorescence microscope (Olympus, Tokyo, Japan) and analyzed using IPP Image software (Media Cybernetics, Inc., Rockville, MD, USA).
Cell proliferation, migration and invasion assays
Cell proliferation inhibition assay
Cell proliferation was determined by MTT assay. Briefly, the HepG2 cells, at 103 cells/well, were dispensed in 100 μl aliquots into 96-well plates. Subsequently, 24 h after inoculation, the cell culture medium was replaced with final concentrations of 50, 100, 150, 200, 250 and 300 μmol/l MIBG for 24 h. An equal volume of DMSO was used as a negative control. After 24 h, 20 μl MTT were added to each well followed by culture for a further 4 h. The supernatant was then carefully aspirated. Subsequently, 150 μl of DMSO were added to each well, and the plate was shaken until the purple crystals were completely dissolved. OD490 nm (A) was measured using a 680 microplate reader (Bio-Rad). The rate of inhibition of cell growth (IR) was calculated as follows: cell growth inhibition rate (%) = (1−A treatment/A control) ×100%. The half maximal inhibition concentration (IC50) of IMBG in the HepG2 cells was determined using IC50 calculation software (SPSS, Inc., Chicago, IL, USA).
Cell migration assay
Cell migration was evaluated using a scratch wound assay. Briefly, 5×105 HepG2 cells/well were plated in 6-well plates and cultured overnight to yield a confluent monolayer. A 10-μl pipette tip was used to scratch the bottom of the culture plates. The remaining cells were washed twice, then cultured with fresh medium supplemented with MIBG 200 μmol/l, and fresh medium was used as a control medium. Images were captured at 0 and 24 h using an NIS-Elements Image Analysis System (Nikon Corp., Tokyo, Japan). Distance was marked at fixed intervals. Three mean values were calculated, including the width of the scratch test, according to the following formula to determine the rate at which the scratches healed. Healing rate = (initial scratch width value − corresponding points scratch width)/initial value ×100% scratch width.
Cell invasion assay
Cell invasion was evaluated using a Transwell Matrigel invasion assay. First, 105 HepG2 cells/well suspended with RPMI-1640 were seeded in the upper chamber (Millipore Corp., Billerica, MA, USA). These were coated with Matrigel (BD Biosciences, Bedford, MA, USA) and incubated with HepG2 conditioned supernatant (MIBG 200 μmol/l in serum-free RPMI-1640, and RPMI-1640 without serum served as a control). RPMI-1640 supplemented with 10% FBS was in the lower chamber. After 24 h, the invading cells on the bottom surface were fixed with 4% paraformaldehyde and quantified after staining with crystal violet. The invasion inhibition rate was caclulated as follows: invasion inhibition rate = (average untreated control group − average treated group)/average value for the dosing control group ×100%.
Western blot analysis
Protein concentrations were evaluated using BCA protein assay reagent (Beyotime, Shanghai, China). HepG2 cells from different groups (control group, MIBG 150 μmol/l-treated group and MIBG 200 μmol/l-treated group) were lysed with RIPA buffer, and 25 μg of total protein were separated by standard 6–10% SDS-PAGE and transferred onto PVDF membranes. The membranes were washed and blocked with 5% skim milk at room temperature for 1 h before being incubated with primary antibodies to ART1 (Sigma), integrin α7 (Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China), FAK, PI3K and uPA (Wuhan Mitaka Biotechnology, Wuhan, China) at 4°C overnight. The primary antibodies were diluted as follows: ART1, 1:500; integrin α7, 1:500; FAK, 1:500; PI3K, 1:500; uPA, 1:200; and β-actin, 1:1,000. An HRP-conjugated secondary anti-rabbit antibody (1:1,000) was then added for 2 h at room temperature. The reactions were detected by enhanced chemiluminescence assay (Beyotime Institute of Biotechnology). The relative intensity of each band was determined using Quantity One software (Bio-Rad). Each experiment was performed 3 times.
Statistical analysis
SPSS 18.0 statistical software was used for statistical analysis. The quantitative data are presented as the means ± standard deviation (SD). Comparisons of continuous data were performed using one-way ANOVA. The rank-sum and least significant difference (LSD) test were used to analyze the ranked data and the differences between each group. The relationship between 2 groups was analyzed by correlation analysis. A value of P<0.05 was considered to indicate a statistically significant difference.
Results
Expression of ART1 and integrin α7 in HCC tissues
IHC revealed the positive expression of ART1 and integrin α7 in the HCC tissues. 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; Table I, Fig. 1).
Correlation between ART1 and integrin α7 expression
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 (ρ=0.87629; P<0.01) (data not shown).
mRNA expression of ART1 and integrin α7 in HCC tissues
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; Fig. 2).
Immunofluorescence detection of the expression of ART1 on the surface of HepG2 cells
The HepG2 cells were incubated with anti-ART1 antibody, then bound with FITC-labeled secondary antibody. They were then observed under a fluorescence microscope (Olympus), 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 (Fig. 3).
IC<sub>50</sub> of MIBG in HepG2 cells
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/l (P<0.05; Table II).
Effect of MIBG on the migration of HepG2 cells
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 (P<0.05; Fig. 4). This indicated that the HepG2 cells had a strong ability to migrate. In the 200 μmol/l 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 indicated that HepG2 cell migration was inhibited by MIBG.
Effect of MIBG on the invasion of HepG2 cells
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; Fig. 5), indicating that MIBG inhibited the invasive ability of the HepG2 cells.
Effect of MIBG on the protein expression of ART1, integrin α7, FAK, PI3K and uPA in HepG2 cells
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; Fig. 6). 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.
Discussion
Metastatic disease is the most common cause of liver cancer-related deaths, thus research on effective anti-metastatic drugs has become a hotspot in drug development. MIBG is currently being used for the diagnosis and treatment of neuroendocrine tumors (9,10).
The invasion and metastasis of tumor cells is a complex process. Adherence to the extracellular matrix (ECM) is the first step in cancer invasion. It is mediated by a specific cell surface receptor. Adhesion molecules are one of the most important molecules in the integrin family.
Integrin adhesion molecules include the non-covalently bound α-subunit and β-subunit. These are connected by heterodimeric transmembrane glycoproteins. There are 18 α-subunits and 8 β-subunits in the 24 different integrins (11). Integrins α3β1, α6β1, α6β4 and α7β1 mediate cell interaction with the ECM, whereas integrin α7β1 binds to laminin and promotes tumor cell invasion and migration (12–14).
ART1 is an ADP-ribosyltransferase expressed at a low level in eukaryotes. It is specifically involved in the arginine ADP-ribosylation process. It transfers the ADP-ribose of NAD+ to histidine residues of a targeted protein. This reaction can regulate a variety of important cellular functions, including immune responses, cell adhesion, cell signaling and metabolism (15). However, only α7 subunits can be modified in integrin α7β1. The β1 subunit cannot undergo ribosylation. The ribosylation of integrin α7β1 causes a conformational change in integrin dimers, leading to greater affinity to a ligand and the enhancement of the function of integrin α7β1 (16). The maximum rate of migration of tumor cells depends on the concentration of ligands, the levels of integrin expression and affinity between ligands and integrins (17). For this reason, as shown by our results, when integrin α7β1 was modified with ART1, the ability of the tumor cells to migrate improved.
The integrin signaling pathway involves a variety of protein kinases, such as FAK, proline rich tyrosine kinase 2 (Pyk2) and Src kinase homologs. FAK plays a pivotal role in signal transduction mediated by integrins (18,19). FAK is a non-receptor tyrosine kinase with a molecular weight of 125 kD. It binds to the integrin β-subunit to activate the latter (20,21). FAK binds to and phosphorylates the proline-rich region of the p85 subunit of PI3K. This takes place directly or indirectly through Src, and then the invasion signal is mediated by integrins (22–24). This prompts uPA secretion. It has been reported that inhibitors of PI3K and AKT may reduce the expression of uPA (25–28). uPA is a serine protease, and activated uPA can activate collagenase, which decomposes the collagen matrix. Activated uPA can degrade the basement membrane and extracellular matrix components directly, which promotes tumor metastasis (29,30). Increased levels of uPA and invasive properties of tumor cells have been found to be closely related to the overexpression of uPA in breast cancer. This is a strong indicator of a poor prognosis (31). In HCC, uPA upregulation is closely related to vascular invasion and intrahepatic metastasis (32).
The results of the present study demonstrated that there was a higher expression of ART1 and integrin α7 in HCC with metastasis than in carcinoma without metastasis. These elevated expression levels were found to positively correlate with each other. As the expression of integrin α7 increased, the expression of ART1 also increased, indicating that the expression of integrin α7 correlated with the expression of ART1. This suggests that these two proteins are synergistically involved in HCC invasion and metastasis. MIBG inhibitedthe expression of ART1, downregulated the expression of integrin α7β1, continually inhibited its downstream pathway, FAK/PI3K, and further inhibited the expression of uPA. The migratory and invasive capabilities of the HepG2 cells were reduced, suggesting that MIBG inhibited ART1, decreased the secretion of uPA and affected the ability of the HepG2 cells to migrate and invade.
The expression levels of ART1 and integrin α7 are closely related to the degree of differentiation and metastasis of HCC; thus, these two proteins can be used as indicators for the degree of malignancy and invasiveness of HCC. The role of ART1 and integrin α7 in HCC invasion has important clinical significance for the treatment of liver cancer. MIBG can suppress HCC invasion and metastasis through the inhibition of the expression of ART1 and integrin α7. Based on our observations, MIBG shows promise as a novel anticancer drug for use in the treatment of HCC.