The important role of ADAM8 in the progression of hepatocellular carcinoma induced by diethylnitrosamine in mice

Abstract

This study focuses on investigating the concrete role of a disintegrin and metalloproteinase 8 (ADAM8) in the progression of hepatocellular carcinoma (HCC). Mice received anti-ADAM8 monoclonal antibody (mAb) of 100 μg/100 μl, 200 μg/100 μl or 300 μg/100 μl, respectively, in phosphate-buffered saline (PBS) or PBS intervention during the progression of HCC induced by diethylnitrosamine. The survival rate, body weight, and relative liver weight were determined in the mice. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and α-fetoprotein (AFP) level, hematoxylin–eosin staining, the expression level of vascular endothelial growth factor A (VEGF-A), proliferating cell nuclear antigen (PCNA), caspase 3 (Casp3), B cell leukemia 2 (Bcl2), B cell leukemia 2-associated X protein (Bax), protein p53 (P53), and ADAM8 were detected in the mice at the end of the 24th week. Our results showed that anti-ADAM8 mAb intervention effectively improved the survival rate, reduced the body weight loss and increased the relative liver weight in mice in a dose-dependent manner (p < 0.05 or p < 0.01). Anti-ADAM8 mAb intervention also significantly lowered serum AST, ALT, and AFP levels (p < 0.05 or p < 0.01), slowed the progression of HCC (p < 0.05 or p < 0.01), induced the expression of Casp3, Bax, and P53 (p < 0.05 or p < 0.01), and inhibited the expression of VEGF-A, PCNA, and Bcl2 in the liver of mice (p < 0.05 or p < 0.01) in a dose-dependent manner compared with the mice receiving PBS intervention. Our study suggested that ADAM8 might promote the progression of HCC by regulating the expression of these factors. Anti-ADAM8 mAb intervention might be suitable as a potential method for HCC therapy.

Keywords

HCC ADAM8 monoclonal antibody P53 Bcl2

Introduction

Hepatocellular carcinoma (HCC) is one of the world’s deadliest cancers, ranking third among all cancer-related mortalities.¹ Most cases occur in Asia and sub-Saharan Africa, among which half are reported from China.^1,2 Carcinogenesis is a multistep process involving multiple molecular changes, and various risk factors may accelerate this process at different steps. Identification of these risk factors may provide insight into the pathogenesis of HCC.³

A disintegrin and metalloproteinases (ADAMs) are a new gene family of proteins with sequence similar to the reprolysin family of venomous snake that share the metalloproteinase domain with matrix metalloproteinases. These molecules are involved in various biological events such as cell adhesion, cell fusion, cell migration, membrane protein shedding, and proteolysis.^4,5 Studies on the biochemical characteristics and biological functions of ADAMs are in progress, and accumulated lines of evidence have shown that some ADAMs are expressed in malignant tumors and participate in the pathology of cancers.⁶ Emerging data from model systems suggest that ADAMs, such as ADAM9, ADAM12, ADAM15, and ADAM17, are causally involved in tumor formation/progression.⁶ In human cancer, specific ADAMs are upregulated, with levels generally correlating with parameters of tumor progression and poor outcome. In preclinical models, selective ADAM inhibitors against ADAM10 and ADAM17 have been shown to synergize with existing therapies in decreasing tumor growth. The ADAMs are thus a new family of potential targets for the treatment of cancer.⁷ ADAM8 is an important member of ADAM family. ADAM8 was initially identified in macrophages⁸ and subsequently detected in neurons, osteoclasts, leukocytes, neutrophils, epithelial cells, and cancer cells.^9

–12 ADAM8 may regulate cellular functions by processing membrane receptors that are crucial for cellular signaling pathways and its catalytic activity has been demonstrated in biochemical and cell-based assays toward substrates that include human heparin-binding epidermal growth factor, epidermal growth factor, CD23, and L-selectin.¹³ The dysregulation of ADAM8 could therefore influence pathological conditions such as rheumatoid arthritis, asthma,¹³ and cancers, such as breast cancer,¹⁴ lung cancer,¹⁵ osteosarcoma,¹⁶ and HCC.²

Although some studies have shown that high expression of ADAM8 correlates with poor prognosis in HCC,² the concrete role of ADAM8 in the progression of HCC is still unknown. A well-defined liver cancer model mimicking the human situation is essential to faithfully reflect human disease for both basic studies of tumor biology and experimental therapeutic purposes.³ So the purpose of this study, therefore, is to study the function of ADAM8 utilizing anti-ADAM8 monoclonal antibody (mAb) to antagonize ADAM8 during the progression of HCC induced by diethylnitrosamine (DEN) in mice,¹⁷ which will also help to check whether anti-ADAM8 mAb intervention might be suitable as a potential method for HCC therapy.

Materials and methods

Chemicals and reagents

DEN and all other chemicals required for all biochemical assays were obtained from Sigma Chemicals Co. (St Louis, Missouri, USA).

Animals

Male BALB/c mice (approximately 6–8 weeks old and weighing 22 ± 2 g) were purchased from the experimental animals center of Henan province. Mice were weighed and maintained in an air-conditioned animal room at 25°C with free access to water and food under 12-h light/12-h dark cycles for the experiments. All animals were allowed to adapt to the environment for 1 week before the experiment and fed on laboratory chow. All protocols conformed to the guidelines from the National Animal Care and Use Committee of China. The protocol was approved by the Committee on the Ethics of Animal Experiments of Henan University of Science and Technology. All animals received care in compliance with the Principles of Laboratory Animal Care.

Experimental design

Our previous work showed that 200 μg/100 μl mAb in phosphate-buffered saline (PBS) containing anti-ADAM8 mAb could effectively downregulate the expression of ADAM8 in liver of mice.¹⁸ So the concentration gradient mAb around 200 μg/100 μl in PBS containing anti-ADAM8 mAb was used to study the function of ADAM8 during the progression of HCC induced by DEN in mice. Mice were divided into five groups, with 60 mice in each group. Group A only received intraperitoneal (i.p.) injection of anti-ADAM8 mAb (300 μg/100 μl in PBS) at days 1, 3, and 5 of each week for 24 weeks. The intervention groups B1, B2, B3, and B4 freely drank sterile water containing 30 µg/ml DEN for 24 weeks to induce HCC. At days 1, 3, and 5 of each week, PBS, 100 μg/100 μl, 200 μg/100 μl, or 300 μg/100 μl in PBS containing anti-ADAM8 mAbSanta Cruz Biotechnology, Santa Cruz, California, USA) were intraperitoneally administered to groups B1, B2, B3 and B4, respectively. At the end of the experiment (24 weeks), mice were weighed, then anesthetized by ether, and blood samples were collected by cardiac puncture. The survival rate within each group was calculated as: number of live animals after 24 weeks/number of animals at the start of the experiment × 100.¹⁹ Then mice were killed and their livers were dissected. Fresh liver was washed twice with ice-cold saline, dried on clean paper towels, and weighed. Relative liver weight was calculated as: liverweight (g)/final body weight(g) × 100.²⁰ Liver specimen from each liver tumor site of individual mice was divided into three sections. They were respectively used for histological examination, Western blot, and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) detection. For histological examination, samples of liver were fixed in 10% formaldehyde for 24 h and then dehydrated and embedded in paraffin. Six micrometer thick sections were cut from each paraffin-embedded tissue for histological examination. For Western blot detection, liver samples were added to PBS (0.02 M, pH 7.4) as the volume proportion (1:10). Then, liver samples were homogenized in a homogenizer, homogenates were transferred to centrifuge tubes and centrifuged at 10,000 r/min at 4°C for 10 min, and the protein sample in the middle layer of solution was collected and used for Western blot detection. For qRT-PCR, liver tissues (20 mg) for total RNA preparation were homogenized on ice in 1.0 ml of TRIzol reagent (TaKaRa, Dalian, China) according to the manufacturer’s instructions. RNA electrophoretic gels were run to determine the RNA quality. The purity and level of total RNA were estimated by the absorbance ratio at A₂₆₀ and A₂₈₀ nm. In another experiment, mice (n = 60) only freely drank sterile water containing 30 µg/ml DEN for 24 weeks to induce HCC. We detected the expression of ADAM8 in the liver of mice during the progression of HCC in the 0, 8th, 16th, and 24th weeks by Western blot.

Detection of serum AST, ALT, and AFP levels

Blood samples were collected at the end of the experiment (24 weeks). The blood is left to clot at room temperature for approximately 15–30 min. After it is completely clotted, it is rimmed using an applicator stick and then centrifuged for approximately 5–10 min at 2500 r/min. Then serum is separated. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were determined with a commercial assay kit (Nanjing Jiancheng Biological Technology, Inc., China). Enzyme activities were expressed as international units per liter. Levels of α-fetoprotein (AFP) were determined by an AFP enzyme-linked immuno sorbent assay kit (Shanghai Fengxiang Biological Technology Inc., China).

Histopathological examination

Liver specimens were obtained from the mice in the 8th, 16th, and 24th weeks after the induction of HCC. Samples of liver were fixed in 10% formaldehyde for 24 h and then dehydrated and embedded in paraffin. Six micrometer thick sections were cut from each paraffin-embedded tissue and stained with hematoxylin–eosin (H&E).

Immunohistochemistry of PCNA, Casp3 at the end of the 24th week after HCC induction

Six micrometer thick sections were cut from each paraffin-embedded tissue as prepared above. The sections of liver from the mice in different groups were immunostained with an mAb to mouse proliferating cell nuclear antigen (PCNA) or Casp3 (active form; dilution 1:300; Santa Cruz Biotechnology) as described by Li et al.¹⁸ The signal was detected using the Polink-2 plus polymer horseradish peroxidase (HRP) detection system (Zhongshan, Beijing, China) using 3,3′-diaminobenzidine (DAB; Sigma). A negative control was carried out on each slide by omitting the primary antibody. Sections were examined microscopically for specific staining, and photographs were taken using a digital image capture system (Olympus, Tokyo, Japan). Numbers of positive cells in the mice of five groups, at least 12 mm² tissue sections were measured for each mouse by ImagePro Plus software Version 6.0 (ImagePro Plus, Media Cybernetics, Maryland, Rockville, USA).

Apoptosis analysis by TUNEL at the end of the 24th week after HCC induction

Apoptosis in the hepatocytes of mice in different groups were further analyzed using a commercial kit (GENMED, Shanghai, China) based on the terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate -digoxigenin nick end-labeling (TUNEL) of apoptotic cells. Sections were examined microscopically for specific staining, and photographs were taken using a digital image capture system (Olympus).

Western blot of VEGF-A, PCNA, Casp3, Bcl2, Bax, P53, and ADAM8

Vascular endothelial growth factor A (VEGF-A) is the main subtype of VEGF. So the expression level of VEGF-A, PCNA, Casp3 (active form), B cell leukemia 2 (Bcl2), Bcl2-associated X protein (Bax), and protein p53 (P53) were detected by Western blot to compare liver blood vessel genesis, cell proliferation, and cell apoptosis in the liver of mice at the end of 24th week after the induction of HCC. In addition, we also detected the expression of ADAM8 in the liver of mice during the progression of HCC in the 0, 8th, 16th, and 24th weeks by Western blot. Protein samples of 70 μg from the mice in different groups were adjusted to the composition of the electrophoresis sample buffer (50 mM Tris, pH 6.8, 10% glycerol, 5% β-mercaptoethanol, 2% sodium dodecyl sulfate (SDS), 0.1% bromphenol blue) and boiled for 5 min prior to analysis. SDS-polyacrylamide gel electrophoresis (10% polyacrylamide gels) in 1 mm slab gel was performed as described by Li et al.²¹ The proteins were transferred from the gel to the nitrocellulose membranes. Then, the membrane was probed with an mAb to mouse VEGF-A, PCNA, Casp3, Bcl2, Bax, and P53 (Santa Cruz Biotechnology), respectively. The signal was detected by HRP detection system using DAB. Protein bands were quantified with Gel Pro Analyzer software 4.0 (Media Cybernetics Inc., Bethesda, Maryland, USA), and the intensities of the bands were normalized against β-actin (Actb). Each experiment was repeated three times.

Quantitative real-time RT-PCR for mRNA expression detection of VEGF-A, PCNA, Casp3, Bcl2, Bax, and P53

The expression level of VEGF-A, PCNA, Casp3, Bcl2, Bax, and P53 were also detected by qRT-PCR in the liver of mice at the end of 24th week after the induction of HCC. The specific primers for these genes were designed using Primer3²² and are listed in Table 1. Samples of RNA extracted from individual mice were used in the qRT-PCR analysis. Briefly, complementary DNA (cDNA) was synthesized from 1 µg of RNA in the presence of ribonuclease inhibitor (BBI, Toronto, Canada), deoxynucleotide triphosphates (Sangon, Shanghai, China), Oligo(dT)18 primers, and RevertAid™ M-Mulv reverse transcriptase (Fermentas, Glen Burnie, Maryland, USA) in a total of 25 µL reaction mix. Real-time RT-PCR was conducted by Applied Biosystems PRISM 7900HT system (Applied Biosystems, Foster City, California, USA) and SYBR Green I chemistry (TaKaRa) according to the manufacturer’s instructions. The final concentration of each primer was 200 nM. The signal of the target gene was normalized to Actb and compared with the control by a $2^{- Δ Δ C_{T}}$ method.²³ A plot of the log cDNA dilution versus ΔC_T was made to calculate the amplification efficiencies of the target and reference.²³

Table 1.

Oligonucleotide primers used in quantitative real-time RT-PCR.

Gene name	GenBank accessionno.	Forward primer	Reverse primer
Actb	NM_007393	5′-CTGTCCCTGTATGCCTCTG	5′-ATGTCACGCACGATTTCC
VEGF	NM_001025250.3	5′-GGCAGACTATTCAGCGGACTC	5′-CTCAAACCGTTGGCACGA
PCNA	AK034723	5′-CTGAAGAAGGTGCTGGAGGCT	5′-TTTGGACATGCTGGTGAGGTT
Bcl2	NM_009741	5′-GTGTTCCATGCACCAAGTCCA	5′-AGGTACAGGCATTGCCGCATA
Bax	NM_007527	5′-CAGGATGCGTCCACCAAGAA	5′-AGTTGCCATCAGCAAACATGTCA
Casp3	NM_009810	5′-CTGCCGGAGTCTGACTGGAA	5′-ATCAGTCCCACTGTCTGTCTCAATG
P53	NM_030989	5′-GCTGAGTATCTGGACGACA-3′	5′-CAGGCACAAACACGAACC-3′

RT-PCR: reverse transcription polymerase chain reaction; VEGF: vascular endothelial growth factor; PCNA: proliferating cell nuclear antigen; Bcl2: B-cell leukemia/lymphoma 2; Bax: B-cell leukemia/lymphoma 2-associated X protein; Casp3: caspase 3; P53: protein p53.

Statistical analysis

All data were presented as the mean ± standard deviation. Statistical comparisons were made using one-way analysis of variance with Tukey’s post hoc test for multiple comparisons. All statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) 13.0 (SPSS Inc., Chicago, Illinois, USA).

Results

Effect of anti-ADAM8 mAb intervention on the survival rate, body weight, and relative liver weight of mice that were induced HCC

Table 2 shows that anti-ADAM8 mAb intervention significantly improved the survival rate (Figure 1), reduced the body weight loss and the increasing of relative liver weight in the mice that were induced HCC in a dose-dependent manner (p < 0.05 or p < 0.01). The survival rate and body weight were significantly higher and relative liver weight were remarkably lower in the mice with only 300 μg/100 μl anti-ADAM8 mAb injection than those with 300 μg/100 μl anti-ADAM8 mAb intervention and DEN induction for HCC (p < 0.05).

Table 2.

Effect of anti-ADAM8 mAb intervention on the survival rate, body weight, and relative liver weight of mice that were induced HCC.^a

Group	Survival rate (%)	Body weight difference (g)	Relative liver weight (g)
B1 (PBS + DEN)	40	(−) 4.05 ± 0.25	(+) 5.94 ± 0.27
B2 (100ADAM8 + DEN)	55^b,c	(−) 3.25 ± 0.36^b,c	(+) 5.12 ± 0.35^b,c
B3 (200ADAM8 + DEN)	70^b,c	(−) 2.35 ± 0.23^b,c	(+) 4.77 ± 0.29^b,c
B4 (300ADAM8 + DEN)	80^d	(−) 1.45 ± 0.19^d	(+) 4.35 ± 0.38^d
A (300ADAM8)	100^d,e	(+) 2.76 ± 0.32^d,e	(+) 3.85 ± 0.41^d,e

ADAM8: a disintegrin and metalloprotease 8; HCC: hepatocellular carcinoma; PBS: phosphate buffered saline; DEN: diethylnitrosamine; i.p.: intraparetoneal; mAb: monoclonal antibody; (+): increasing; (−): decreasing.

^aPBS + DEN, 100ADAM8 + DEN, 200ADAM8 + DEN and 300ADAM8 + DEN: mice freely drank sterile water containing 30 µg ml⁻¹ DEN for 24 weeks to induce HCC and respectively received i.p. injection of PBS or anti-ADAM8 mAb (100 μg/100 μl, 200 μg/100 μl or 300 μg/100 μl in PBS) at days 1, 3, and 5 of each week. 300ADAM8: mice only received i.p. injection of anti-ADAM8 mAb (300 μg/100 μl in PBS) at days 1, 3, and 5 of each week for 24 weeks. Each group contains 60 mice. Body weight difference = final bodyweight − initial body weight: Survival rate = number of live animals after 24 weeks/number of animals at the start of the experiment × 100. Relative liver weight = liver weight(g)/final body weight(g) × 100.

^bp < 0.05: significant difference in the each ADAM8 mAb + DEN group or 300ADAM8 group as compared to the PBS + DEN group.

^cp < 0.05: significant difference in the 300ADAM8 + DEN group as compared to the 100ADAM8 + DEN group and 200ADAM8 + DEN group.

^dp < 0.01: significant difference in the each ADAM8 mAb + DEN group or 300ADAM8 group as compared to the PBS + DEN group.

^ep < 0.05: significant difference in the 300ADAM8 + DEN group as compared to the 300ADAM8 group.

Figure 1.

The survival rate of mice in different groups in the 0, 8th, 16th, and 24th weeks after the induction of HCC. (a), (b), (c), (d), and (e) represent the survival rate of mice in group B1 (PBS + DEN), group B2 (100ADAM8 + DEN), group B3 (200ADAM8 + DEN), group B4 (300ADAM8 + DEN), and group A (300ADAM8), respectively. Groups B1, B2, B3, and B4: mice freely drank sterile water containing 30 µg ml⁻¹ DEN for 24 weeks to induce HCC and respectively received i.p. injection of PBS or anti-ADAM8 mAb (100 μg/100 μl, 200 μg/100 μl, or 300 μg/100 μl in PBS) at days 1, 3, and 5 of each week. Group A: mice only received i.p. injection of anti-ADAM8 mAb (300 μg/100 μl in PBS) at days 1, 3, and 5 of each week for 24 weeks. The results were analyzed using the log-rank test and expressed as the Kaplan–Meier survival curves. HCC: hepatocellular carcinoma; PBS: phosphate-buffered saline; DEN: diethylnitrosamine; ADAM8: a disintegrin and metalloproteinase 8; i.p.: intraperitoneal.

Alterations of serum AST, ALT, and AFP levels in the mice at the end of 24th week after HCC induction

Figure 2 indicates that anti-ADAM8 mAb intervention significantly lowered serum AST, ALT, and AFP levels in the mice that were induced HCC in a dose-dependent manner (p < 0.05 or p < 0.01). The levels of serum AST, ALT, and AFP in the mice with only 300 μg/100 μl anti-ADAM8 mAb injection were significantly lower than those with 300 μg/100 μl anti-ADAM8 mAb intervention in the HCC progression (p < 0.05).

Figure 2.

Serum AST (a), ALT (b), and AFP (c) levels in the mice with PBS or anti-ADAM8 mAb of different concentrations intervention at the end of 24th week after the induction of HCC. **p < 0.01 or *p < 0.05: significant difference in the each ADAM8 mAb + DEN group or 300ADAM8 group as compared to the PBS + DEN group. ^#p < 0.05: significant difference in the 300ADAM8 + DEN group as compared to the 100ADAM8 + DEN group and 200ADAM8 + DEN group. ^&p < 0.05: significant difference in the 300ADAM8 + DEN group as compared with the 300ADAM8 group. AST: aspartate aminotransferase; ALT: alanine aminotransferase; AFP: α-fetoprotein; PBS: phosphate-buffered saline; ADAM8: a disintegrin and metalloproteinase 8; mAB: monoclonal antibody; HCC: hepatocellular carcinoma; DEN: diethylnitrosamine.

Analysis of pathological change in the mice after HCC induction by H&E staining

Our results showed that the HCC starting point was at the end of the 8th week after DEN induction in the mice that received PBS intervention only (Figure 3). At the end of the 24th week after HCC induction, our results showed that anti-ADAM8 mAb intervention significantly slowed the progression of HCC induced by DEN in the mice compared with those that received PBS injection only (p < 0.05 or p < 0.01). Anti-ADAM8 mAb of 300 μg/100 μl intervention significantly slowed the progression of HCC compared with anti-ADAM8 mAb of 200 μg/100 μl or 100 μg/100 μl intervention in the mice (p < 0.05). Only receiving anti-ADAM8 mAb of 300 μg/100 μl injection did not induce liver injury in the mice (Figure 4).

Figure 3.

Histologic examination of HCC starting point in mice by H&E staining at the end of the 8th week. (a), (b), (c), (d) and (e) represent the liver pathological condition of mice in the group B1 (PBS + DEN) group, group B2 (100ADAM8 + DEN), group B3 (200ADAM8 + DEN), group B4 (300ADAM8 + DEN), and group A (300ADAM8), respectively. HCC: hepatocellular carcinoma; H&E: hematoxylin–eosin; PBS: phosphate-buffered saline; ADAM8: a disintegrin and metalloprotease 8; DEN: diethylnitrosamine.

Figure 4.

Histologic examination of liver injury in mice by H&E staining at the end of the 24th week after the induction of HCC. (a), (b), (c), (d), and (e) represent the degree of liver injury of mice in the group B1 (PBS + DEN), group B2 (100ADAM8 + DEN), group B3 (200ADAM8 + DEN), group B4 (300ADAM8 + DEN), and group A (300ADAM8), respectively. (f) Necrotic areas. Pane refers to the representative necrotic area. Representative findings from measure tissue sections that were at least 10 mm² for each mouse by ImagePro Plus software Version 6.0 (Media Cybernetics, Maryland, Rockville, USA). **P < 0.01 or *P < 0.05: significant difference in the each ADAM8 mAb+DEN group or 300ADAM8 group as compared with the PBS+DEN group. ^#P < 0.05: significant difference in the 300ADAM8+DEN group as compared with the 100ADAM8+DEN group and 200ADAM8+DEN group. ^&P < 0.05: significant difference in the 300ADAM8+DEN group as compared with the 300ADAM8 group. (Scale bar: 50 µm.) H&E: hematoxylin–eosin; HCC: hepatocellular carcinoma; PBS: phosphate buffered saline; ADAM8: a disintegrin and metalloprotease 8; DEN: diethylnitrosamine.

Detection of the effect of anti-ADAM8 mAb intervention on the hepatocytes proliferation by immunohistochemistry of PCNA

Figure 5 shows that PCNA was mainly expressed in the cytoplasm of hepatocytes of mice. The results show that anti-ADAM8 mAb intervention significantly inhibited the expression of PCNA in the liver of mice with induction of HCC by DEN in a dose-dependent manner (p < 0.05 or p < 0.01), which indicated that anti-ADAM8 mAb intervention significantly inhibited the proliferation of hepatocytes to slow the progression of HCC in the mice.

Figure 5.

Photomicrographs of immunohistochemical staining of PCNA in the liver of mice at the end of the 24th week after the induction of HCC. The arrowheads indicate the PCNA-positive cells in the liver of mice in the group B1 (PBS + DEN) (a), group B2 (100ADAM8 + DEN) (b), group B3 (200ADAM8 + DEN) (c), group B4 (300ADAM8 + DEN) (d), and group A (300ADAM8) (e). (f) Numbers of PCNA + cells in the liver of mice in the different groups; tissue sections that were at least 12 mm² were measured for each mouse by ImagePro Plus software Version 6.0 (Media Cybernetics, Maryland, Rockville, USA). The results show that anti-ADAM8 mAb intervention significantly inhibited the expression of PCNA in the liver of mice in a dose-dependent manner after HCC induction. **P < 0.01 or *P < 0.05: significant difference in the each ADAM8 mAb+DEN group or 300ADAM8 group as compared with the PBS+DEN group. ^#P < 0.05: significant difference in the 300ADAM8+DEN group as compared with the 100ADAM8+DEN group and 200ADAM8+DEN group. ^&P < 0.05: significant difference in the 300ADAM8+DEN group as compared with the 300ADAM8 group. (Scale bar: 50 µm.). PCNA: proliferating cell nuclear antigen; HCC: hepatocellular carcinoma; PBS: phosphate-buffered saline; DEN: diethylnitrosamine; ADAM8: a disintegrin and metalloprotease 8; mAb: monoclonal antibody.

Detection of the effect of anti-ADAM8 mAb intervention on the hepatocytes apoptosis by immunohistochemistry of TUNEL and Casp3

Our results showed that anti-ADAM8 mAb intervention significantly induced hepatocytes apoptosis compared with the PBS intervention in the mice at the end of the 24th week after the induction of HCC (p < 0.05 or p < 0.01; Figure 6). In addition, Casp3 was mainly expressed in the cytoplasm of hepatocytes of mice. Our results show that anti-ADAM8 mAb intervention significantly induced the expression of Casp3 in the liver of mice that were induced HCC in a dose-dependent manner (p < 0.05 or p < 0.01; Figure 7).

Figure 6.

Photomicrographs of results of TUNEL assay of liver sections prepared from the mice at the end of the 24th week after the induction of HCC. The arrowheads indicate the apoptotic cells in the liver of mice in the group B1 (PBS + DEN) (a), group B2 (100ADAM8 + DEN) (b), group B3 (200ADAM8 + DEN) (c), group B4 (300ADAM8 + DEN) (d), and group A (300ADAM8) (e). (f) Percentages of TUNEL-positive cells among total hepatocytes, at least 12 mm² tissue sections were counted for each mouse. **P < 0.01 or *P < 0.05: significant difference in the each ADAM8 mAb+DEN group or 300ADAM8 group as compared with the PBS+DEN group. ^#P < 0.05: significant difference in the 300ADAM8+DEN group as compared with the 100ADAM8+DEN group and 200ADAM8+DEN group. ^&P < 0.05: significant difference in the 300ADAM8+DEN group as compared with the 300ADAM8 group. (Scale bar: 50 µm.) TUNEL: terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate -digoxigenin nick end labeling; HCC: hepatocellular carcinoma; PBS: phosphate-buffered saline; DEN: diethylnitrosamine; ADAM8: a disintegrin and metalloproteinase 8.

Figure 7.

Photomicrographs of immunohistochemical staining of Casp3 in the liver of mice at the end of the 24th week after the induction of HCC. The arrowheads indicate the Casp3-positive cells in the liver of mice in the group B1 (PBS + DEN) (a), group B2 (100ADAM8 + DEN) (b), group B3 (200ADAM8 + DEN) (c), group B4 (300ADAM8 + DEN) (d), and group A (300ADAM8) (e). (f) Numbers of Casp3 + cells in the liver of mice in the different groups; tissue sections that were at least 12 mm² were measured for each mouse by ImagePro Plus software Version 6.0 (Media Cybernetics, Maryland, Rockville, USA). The results show that anti-ADAM8 mAb intervention significantly induced the expression of Casp3 in the liver of mice that were induced HCC in a dose-dependent manner P < 0.05: significant difference in the each ADAM8 mAb+DEN group or 300ADAM8 group as compared with the PBS+DEN group. ^#P < 0.05: significant difference in the 300ADAM8+DEN group as compared with the 100ADAM8+DEN group and 200ADAM8+DEN group. ^&P < 0.05: significant difference in the 300ADAM8+DEN group as compared with the 300ADAM8 group. (Scale bar: 50 µm.) Casp3: caspase 3; HCC: hepatocellular carcinoma; PBS: phosphate-buffered saline; DEN: diethylnitrosamine; ADAM8: a disintegrin and metalloproteinase 8; mAb: monoclonal antibody.

Analysis of the expression of ADAM8 in the liver of mice during HCC progression with only DEN induction by Western blot

Figure 8 shows that the expression of ADAM8 in the liver of mice was significantly increased in the 8th, 16th, and 24th weeks when compared with 0th week after HCC induction with only DEN by Western blot (p < 0.05 or p < 0.01).

Figure 8.

The change of ADAM8 expression in the liver of mice during HCC progression with only DEN induction by Western blot. The expression of ADAM8 was detected by Western blot (a). The protein bands were quantified for ADAM8 (b) with the Gel-Pro Analyzer software Version 4.0 (Media Cybernetics Inc.) and the intensities of the bands were normalized against Actb. AU represents arbitrary unit. Each experiment was repeated three times. All data were presented as the mean ± SD. **p < 0.01 or *p < 0.05: significant difference when compared with the expression of ADAM8 in the liver of mice in the 0th week. W: week; ADAM8: a disintegrin and metalloprotease 8; HCC: hepatocellular carcinoma; DEN: diethylnitrosamine; SD: standard deviation; Actb: beta-actin.

Determination of anti-ADAM8 mAb intervention effect by Western blot of VEGF-A, PCNA, Casp3, Bcl2, Bax, and P53 in the liver of mice

Our results showed that anti-ADAM8 mAb intervention significantly induced the expression of Casp3, Bax, and P53 and inhibited the expression of VEGF-A, PCNA, and Bcl2 in the liver of mice in a dose-dependent manner compared with the mice that only received PBS injection at the end of 24th week after HCC induction (p < 0.05 or p < 0.01; Figure 9). The expression of Casp3, Bax, and P53 in the liver of mice with HCC induction and anti-ADAM8 mAb of 300 μg/100 μl intervention was significantly lower than the mice with only anti-ADAM8 mAb of 300 μg/100 μl injection (p < 0.05; Figure 9(d), (f) and (g)). But the expression of VEGF-A, PCNA, and Bcl2 in the liver of mice with HCC induction and anti-ADAM8 mAb of 300 μg/100 μl intervention was significantly higher than the mice with only anti-ADAM8 mAb of 300 μg/100 μl injection (p < 0.05; Figure 9(b), (c) and (e)).

Figure 9.

The expression of VEGF-A, PCNA, Casp3, Bcl2, Bax, and P53 in the liver of mice at the end of the 24th week after HCC induction. The expression of VEGF-A, PCNA, Casp3, Bcl2, Bax, and P53 were detected by Western blot (a). 1: group B1 (PBS + DEN); 2: group B2 (100ADAM8 + DEN); 3: group B3 (200ADAM8 + DEN); 4: group B4 (300ADAM8 + DEN); 5: group A (300ADAM8). The protein bands were quantified for VEGF-A (b), PCNA (c), Casp3 (d), Bcl2 (e), Bax (f), and P53 (g) with the Gel-Pro Analyzer software Version 4.0 (Media Cybernetics Inc.) and the intensities of the bands were normalized against Actb. AU represents arbitrary unit. Every experiment was repeated three times. All data were presented as the mean ± SD. **P < 0.01 or *P < 0.05: significant difference in the each ADAM8 mAb+DEN group or 300ADAM8 group as compared with the PBS+DEN group. ^#P < 0.05: significant difference in the 300ADAM8+DEN group as compared with the 100ADAM8+DEN group and 200ADAM8+DEN group. ^&P < 0.05: significant difference in the 300ADAM8+DEN group as compared with the 300ADAM8 group. VEGF-A: vascular endothelial growth factor A; PCNA: proliferating cell nuclear antigen; Casp3: caspase 3; Bcl2: B cell leukemia 2; Bax: B cell leukemia 2 associated X protein; P53: protein p53; PBS: phosphate buffered saline; DEN: diethylnitrosamine; SD: standard deviation; Actb: beta-actin.

Analysis of the mRNA expression of VEGF-A, PCNA, Casp3, Bcl2, Bax, and P53 in the liver of mice by qRT-PCR

Figure 10 shows that anti-ADAM8 mAb intervention remarkably induced the messenger RNA (mRNA) expression of Casp3, Bax, and P53 and inhibited the mRNA expression of VEGF-A, PCNA, and Bcl2 in the liver of mice in a dose-dependent manner compared with the mice that only received PBS injection at the end of 24th week after HCC induction (p < 0.05 or p < 0.01). The mRNA expression of Casp3, Bax, and P53 in the liver of mice with HCC induction and anti-ADAM8 mAb of 300 μg/100 μl intervention was significantly lower than the mice with only anti-ADAM8 mAb of 300 μg/100 μl injection (p < 0.05). But the mRNA expression of VEGF-A, PCNA, and Bcl2 in the liver of mice with HCC induction and anti-ADAM8 mAb of 300 μg/100 μl intervention was significantly higher than the mice with only anti-ADAM8 mAb of 300 μg/100 μl injection (p < 0.05). These are consistent with the results of protein level.

Figure 10.

Fold changes in mRNA expression of VEGF-A, PCNA, Casp3, Bcl2, Bax, and P53 in the liver of mice at the end of the 24th week after HCC induction. The expression of VEGF-A (a), PCNA (b), Casp3 (c), Bcl2 (d), Bax (e), and P53 (f) were detected by quantitative real-time RT-PCR and normalized against Actb. Fold changes in mRNA expression was relative to that in group B1 (PBS + DEN). Each experiment was repeated three times. All data were presented as the mean ± SD. **P < 0.01 or *P < 0.05: significant difference in the each ADAM8 mAb+DEN group or 300ADAM8 group as compared with the PBS+DEN group. ^#P<0.05: significant difference in the 300ADAM8+DEN group as compared with the 100ADAM8+DEN group and 200ADAM8+DEN group. ^&P <0.05: significant difference in the 300ADAM8+DEN group as compared with the 300ADAM8 group. mRNA: messenger mRNA; VEGF-A: vascular endothelial growth factor A; PCNA: proliferating cell nuclear antigen; Casp3: caspase 3; Bcl2: B cell leukemia 2; Bax: B cell leukemia 2-associated X protein; P53: protein p53; RT-PCR: reverse transcription polymerase chain reaction; PBS: phosphate-buffered saline; DEN: diethylnitrosamine; Actb: beta-actin.

Discussion

Our study first analyzed the important roles of ADAM8 in HCC progression of mice. The results indicated that anti-ADAM8 mAb intervention could effectively slow the progression of HCC.

Our results showed that anti-ADAM8 mAb intervention inhibited the expression of VEGF-A in a dose-dependent manner compared with the mice that only received PBS injection during the progression of HCC (p < 0.05 or p < 0.01) (Figures 9(a), (b) and 10(a)). The formation of new blood vessels is critical for normal development and tissue repair as well as for pathological events such as retinal neovascularization, rheumatoid arthritis, and tumor growth.^24

–29 During physiological angiogenesis, VEGF-A expression in response to local hypoxia drives the development of new blood vessels.^30
–32 In addition, angiogenesis is known to play a critical role in pathological settings like chronic inflammatory and tumor growth.^33
–35 VEGF-A is considered to be the central angiogenic factor during chronic liver injury. It has been demonstrated that expression of VEGF-A is upregulated during liver fibrosis, and its expression is increased in activated hepatic stellate cells (HSCs).^33,34,36,37 Activated HSCs can express VEGF-A and VEGF-A receptors in the liver after carbon tetrachloride (CCl₄) intoxication.^38,39 Inflammatory mediators cause the HSCs to differentiate into myofibroblasts. They play a role in angiogenesis and act by releasing the proangiogenic mediators VEGF-A and angiopoietin-1 during the development of liver fibrosis.^40,41 Our results indicated that anti-ADAM8 mAb intervention could slow the progression of HCC by inhibiting the expression of VEGF-A in a dose-dependent manner (p < 0.05 or p < 0.01; Figures 9(a), (b) and 10(a)). ADAM8 is characterized by a metalloproteinase and a disintegrin domain, which are membrane-anchored glycoproteins involved in proteolysis and cell adhesion.⁴² VEGF-A shedding was mediated by the ADAM8 metalloproteinase. Thus, inhibiting expression of ADAM8 in hepatic cells in vivo could potentially downregulate the shedding of VEGF-A and inhibit angiogenesis, which slowed the progression of HCC in mice. At the same time, our results suggested that ADAM8 may promote the progression of HCC by inducing the expression of VEGF-A and promoting angiogenesis in the liver of mice.

Our results also indicated that anti-ADAM8 mAb intervention inhibited the expression of PCNA in a dose-dependent manner compared with the mice that only received PBS injection during the progression of HCC (p < 0.05 or p < 0.01) (Figures 5, 9(a), (c) and 10(b)). PCNA is a subunit of the mammalian DNA polymerase delta and is synthesized primarily during the S phase of the cell cycle.⁴³ PCNA is a relay or an anchoring molecule that functions as a molecular integrator for proteins involved in the control of the cell cycle, DNA replication, DNA repair, and cell death.^44,45 PCNA has been shown to be a good marker to distinguish proliferating cells.^46,47 Our results further proved that anti-ADAM8 mAb intervention could slow the progression of HCC by inhibiting the expression of PCNA in a dose-dependent manner (p < 0.05 or p < 0.01), which indicated that ADAM8 may promote the progression of HCC by inducing the expression of PCNA and promoting cell proliferation in the liver of mice.

Our results showed that anti-ADAM8 mAb intervention significantly induced hepatocytes apoptosis during the progression of HCC (Figure 6) Casp3, Bax, and Bcl2 are important factors related to apoptosis. Our study showed that anti-ADAM8 mAb intervention significantly induced the expression of Casp3 (p < 0.05 or p < 0.01; Figures 7, 9(a), (d), and 10(c)) and Bax (p < 0.05 or p < 0.01; Figures 9(a), (f), and 10(e)) but inhibited the expression of Bcl2 (p < 0.05 or p < 0.01; Figures 9(a), (e) and 10(d)) compared with the mice that only received PBS injection during the progression of HCC. Casp3 is a member of the interleukin-1 beta-converting enzyme or cell death effector-3 family, which is involved in the induction of apoptosis and has been considered to be correlated with apoptosis because of the most downstream enzyme activities in their apoptosis-inducing pathway.^48,49 Bax is an important pro-apoptotic marker and Bcl2 is an important anti-apoptotic marker, which play important roles in regulating apoptosis.⁵⁰ We think that anti-ADAM8 mAb intervention could slow the progression of HCC by upregulating the pro-apoptotic factor and apoptosis execution factor and downregulating anti-apoptotic factors to promote apoptosis, which also suggested that ADAM8 may promote the progression of HCC by downregulating the pro-apoptotic factor and apoptosis execution factor and upregulating anti-apoptotic factors to inhibit apoptosis.

Our study also showed that anti-ADAM8 mAb intervention significantly induced the expression of P53 in a dose-dependent manner compared with the mice that only received PBS injection during the progression of HCC (p < 0.05 or p < 0.01) (Figures 9(a), (g) and 10(f)). Majority of studies provided evidence that the tumor suppressor gene p53 plays a major role in hepatocarcinogenesis and the prognosis of HCC.^51

–55 Our results further indicated that anti-ADAM8 mAb intervention was likely to slow the progression of HCC through the P53-Bax-Casp3 apoptotic pathway.⁵⁶

In summary, our study suggested that ADAM8 might promote the progression of HCC by increasing the level of serum AST, ALT, and AFP and regulating the expression of VEGF-A, PCNA, Casp3, Bcl2, Bax, P53, and so on. We need to investigate more factors to find out the mechanism with which ADAM8 promotes the progression of HCC. Anti-ADAM8 mAb intervention might be a suitable potential method for HCC therapy.

Footnotes

Acknowledgment

The authors thank all the members in the laboratory who helped in carrying out the experimental work. The experiments complied with the current laws of China.

Conflict of interest

The authors declared no conflicts of interest.

Funding

This work was supported by grants from the National Natural Science Foundation of China (#81201558) to SQL, Program for Science & Technology Innovation Talents in Universities of Henan Province (#13HASTIT025) to SQL, Foundation for Henan Province’s Key Project to Tackle Key Problems of Science and Technology (#122102310030) to SQL, and the Key Projects for Science and Technology Research of the Education Department of Henan province (12B310003) to SQL.

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