Changes of integrin expression in rat hepatocarcinogenesis induced by 3’-Me-DAB

Abstract

AIM: To investigate the expression of integrins in rats liver during 3'-Me-DAB induced hepatocarcinogenesis and to find out the relationship between integrins and liver cancer metastasis.

METHODS: The expressions of integrins α₁, α₂, α₃ and α₅ and epidermal keratin (EK) were observed by immunohistochemical PAP method.

RESULTS: In the normal liver tissues, hepatocytes express integrins α₁ and α₅ and in the bile duct epithlium, EK. In liver cirrhosis, hepatocytes highly express integrins α₁, α₂, α₃ and α₅ and in hyperplastic bile duct epithelium, integrins α₁, α₅ and EK. Expression of integrins α₁, α₂, α₃ and α₅ were obviously decreased in the preneoplastic nodules and primary carcinoma but expressions of integrins α₁ and α₅ in metastasis in the lung and diaphragma were higher than those in primary carcinoma.

CONCLUSION: Integrins α₁ and α₅ may play a major role in chemically induced hepatocarcinogenesis and metastasis in rats.

Key Words: integrins, 3’Me-DAB, liver neoplasm/chemically induced, neoplasm metastasis, rats

INTRODUCTION

The integrin superfamily of heterodimeric cell-surface receptors composed of distinct α and β subunits mediates the adhesion of cells to the extracellular matrix and in some instance, the intercellular adhesion. The integrins are thought to play important roles in differentiation and development, cell migration, and the complex process of tumor cell invasion and metastasis, as well as adhesion[1]. Alterations in integrin expressions upon malignant transformation and in naturally occurring human malignances are now well established. However, the mechanisms that give rise to altered integrin protein expression following malignant transformation are poorly understood. We investigate the expression of integrins α₁, α₂, α₃ and α₅ in rat liver during 3’-Me’-DAB induced hepatocarcinogenesis and expect to find out the relationship between integrins and liver cancer metastasis.

MATERIALS AND METHODS

Animal model[3]

Male Wistar rats (n = 100, provided by Experimental Animal Center, Shanghai Medical University), weighing 100 g-150 g, were divided into four groups: group A (n = 44), fed with low choline maize powder and 0.03% 3’-Me-DAB for 12 wk; group B (n = 28), fed with low choline maize powder only for 12 wk and group C (n = 28), fed with standard diet only. After 12 wk, the rats of group A and B were fed with standard diet. Four to rats were killed in each group at week 3, 6, 9, 12 and 16 and 8-12 at week 20.

Tissue preparation

Small pieces of liver samples (including tumor and metastasis) were divided in two parts, one embedded in O.C.T (Miles, USA), rapidly frozen and stored at -70 °C, and the other was fixed with 10% neutral buffered formalin, embedded in paraffin and used for routine histological examination.

Immunohistochemistry

The antibodies against integrins α₁, α₂, α₃ and α₅ were purchased from Chemicon International INC and those against epidermal keratin (EK) from Nichi Lei Co. Tokyo, Japan. The rabbit PAP kits were prepared by our department[4]. Integrins α₁, α₂, α₃ and α₅ and EK in normal liver, liver cirrhosis and liver tumor were detected by PAP method. Briefly, 5 μm cryostat sections of the frozen tissues were fixed with cold acetone and 10% neutral buffered formalin (9∶1) for 10 min, and washed in 0.01 mol/L phosphate- buffered saline (PBS, pH7.4) for 5 times, each for 3 min. The sections were treated with methanol (containing 0.2 mL/L H₂O₂) at 37 °C for 30 min and washed in PBS, and then incubated in PBS with 100 mL/L normal goat serum at 37 °C for 30 min. The sections were incubated with rabbit anti-integrins α₁ (1∶1200 dilution), α₂ (1∶1200 dilution), α₃ (1∶1200 dilution), α₅ (1∶1500 dilution) and EK (1∶1000 dilution) respectively at 37 °C for 30 min and then at 4 °C overnight. The sections were washed in PBS and incubated with goat anti-rabbit IgG (1∶200 dilution) at 37 °C for 40 min. After washing in PBS, the sections were incubated with rabbit PAP complex (1∶200 dilution). The color was developed with 0.5 g/L 3,3’diaminobenzidine/0.5 mL/L H ₂O₂/0.05 mol/L PBS (pH7.6) for 10 min. Normal rabbit serum instead of the specific primary antibodies was used as negative control.

Semiqantitation of the results

Only cells with membranous staining were considered; cells with intense dotlike cytoplasm immunoreactivity caused by endogenous peroxidase were ignored. The intensity of staining was divided into +++ (strong), ++ (moderate), + (weak), +/- (equivocal), - (negative). Variable patterns were indicated by combining the two extremes of staining intensity, e.g. +++/+/-.

RESULTS

Morphologic changes

Liver cancer model of the rats in group A was successfully induced by fed 3’-Me-DAB with low choline maize powder. In the early experiment (week 1-9), the main morphologic changes were steatosis and necrosis of hepatocytes, proliferation of oval cells and cholangiofibrosis. In the middle of the experiment (week 10-16), massively proliferated oval cells, and extensive areas of cholangio fibrosis were visible. In some livers, few hepatocytes remained; they were either entrapped in cholangio fibrotic structures or located at the peripheries of these lesions. Then, liver cirrhosis and preneoplastic nodules were obvious. At the end of the experiment, most of the rats in group A had liver tumor, mainly mixed hepatocarcinoma (HCC) and cholangiocarcinoma (CCC). Tumor metastasized to the lung, spleen and diaphragm in some rats (Figure 1). There were no obvious morphologic changes in the livers of rats in group B and C.

Figure 1 Tumor metastasis in diaphragm. HE × 200

Expression of integrins and EK

The overall staining patterns are shown in Table 1.

Table 1 Pattern of integrins and EK in normal liver, liver cirrhosis and liver tumor.

	α1	α2	α3	α5	EK
Normal liver tissue
Hepatocytes	+	-	-	+	-
Sinusoidal endothelial cells	+	-	-	+	-
Bile duct epithelium	-	-	-	-	+++
Liver cirrhosis
Hepatocytes	+++	++	++	+++	-
Sinusoidal endothelial cells	++	+	+	++	-
Oval cell	+/-	+/-	+/-	+/-	+++/-
Hyperplastic bile duct epithelium	++	-	-	++	+++
Preneoplastic nodules	+	+/-	+/-	+	-
Tumor
Primary	-/+	-	-	-/+	+++/-
Metastasis	+++/-	-	-	+++/-	+++/-

In the normal liver tissues, integrins α₁ and α₅ were weakly expressed in hepatocytes and sinusoidal endothelial cells but not in bile duct epithelium. The predominant staining was located on the sinusoidal side plasma membranes of hepatocytes. EK was highly expressed in the bile duct epithelium but not in hepatocytes.

In liver cirrhosis, the expression of integrins α₁, α₂, α₃ and α₅ were increased in hepatocytes and sinusoidal endothelial cells but the distribution did not significantly change (Figure 2). However, hyperplastic bile duct epithelium only moderately expressed α₁ and α₅. Oval cells were equivocal on expression of integrins but most of them highly expressed EK.

Figure 2 In liver cirrhosis, the expression of integr in α₁ was increased in hepatocytes and sinusoidal endothelial cells. PAP × 200

Expression of integrins α₁, α₂, α₃ and α₅ were obviously decreased in the preneoplastic nodules and primary carcinoma (Figure 3) but expression of integrins α₁ and α₅ in metastasis in the lung and diaphragm were higher than in primary carcinoma (Figure 4).

Figure 3 Expression of α₁ was obviously decreased in the primary carcinoma (right) and in para-tumor liver cirrhosis (left), the expression of integrin α₁ was increased in hepatocytes and sinusoidal endothelial cells. PAP × 100

Figure 4 Expression of α₁ in metastasis in the lung was higher than in primary carcinoma. PAP × 200

DISCUSSION

The β₁ subgroup of the integrin superfamily of cell-surface receptors (also known as VLA integrins) contains six heterodimers (VLA-1 to VLA-6), in which different α-subunits share a common β₁ subunit. They serve as receptor for extracellular matrix components as laminin (VLA-1, VLA-2, VLA-3, and VLA-6), collagen (VLA-1, VLA-2, and VLA-3), and fibronectin (VLA-3, VLA-4, and VLA-5) and are involved as counter-receptors for other cell-surface molecules (VLA-4 recognizes the vascular cell adhesio nmolecule-1 [VCAM-1]) in a cell-to-cell type of interaction[2].

The integrins are thought to play important roles in differentiation and development, cell migration, and the complex process of tumor cell invasion and metastasis, as well as adhesion. The integrin β₁ and fibronectin were mainly distributed along sinusoids in normal liver, and the expression was weak in adult normal liver[5]. The distribution did not significantly change during the regeneration and proliferation, but the expression was increased. They acquire some characteristics of fetal and neonatal hepatocytes, including alternations in cell surface properties, such as the composition of glycoconjugates, and the level of receptors for hormones and growth factors and of pro teins involved in cell-cell contacts. In the early phase of liver carcinogenesis, proliferation of oval cells, cholangiofibrosis, proliferation of hepatocytes and the extracellular matrix (cirrhosis) were obvious. The high expression of integrins plays an important role in cirrhosis[6].

Alterations in integrins receptor expression upon malignant transformation and naturally occurring human malignances are now well established[7,8]. It was one of the properties of tumor cells that the expressions of integrins and fibronectin were decreased[9]. Integrins such as fibronection receptor transduce important growth inhibitory stimuli from the extracellular matrix. Integrins are involved not only in the adhesive, motile and invasive behavior of tumor cells but also in their growth regulation.

Expression of intergrins α₁, α₂, α₃ and α₅ was obviously decreased in the preneoplastic nodules and primary carcinoma but expression of integrins α₁ and α₅ in metastasis in the lung and diaphragma was higher than in primary carcinoma. Integrins α₁ and α₅ may play a major role in chemically induced hepatocarcinogenesis and metastasis in rats, and it was necessary for tumor cells to adher to the extracellular matrix in metastatic focus[10,11].

EK is a mark of epidermal cells. EK was highly expressed by the bile duct epithelium but not by hepatocytes and might be a helpful tool in the differential diagnosis between HCC and CCC. Some oval cells expressed EK but some not. It suggested that oval cells are the progeny of hepatic stem cells, which might differentiate into preneoplastic parenchyma cells and might give rise to HCC.