Expression of Cyclooxygenase-2 and Transforming Growth Factor-Beta 1 in Patients with the Early Recurrence of Hepatocellular Carcinoma Following Hepatectomy ()
1. Introduction
Surgical resection for hepatocellular carcinoma (HCC) is a widely accepted and safe treatment with a low operative mortality as a result of advances in surgical techniques and peri-operative management [1,2]. However, the long-term survival remains unsatisfactory, mainly because of the high incidence of recurrence: a 2-year recurrence rate of up to 60% - 70% [3-5]. In HCC, there are two types at recurrence; intrahepatic metastasis and multicentric carcinogenesis derived from background liver disease. Intrahepatic metastasis occurs mainly within 2 years after hepatectomy [4].
In general, vascular invasion, number of tumors and large tumor size were thought to be conventional prognostic factors. However, some other biological molecules are also related to early intrahepatic recurrence in HCCs [5].
Cyclooxygenase-2 (COX-2) is induced by a variety of factors such as cytokines, growth factors, and carcinoma promoters [6] and has been connected to inflammation and carcinogenesis [7]. As key enzymes in the conversion of arachidonate to prostaglandin, COX-1 and COX-2 are involved in various biological processes from the production of cell specific prostaglandin, including the regulation of immune function, kidney development, reproductive biology, and gastrointestinal capability. The expression of COX-2 and the production of prostaglandins appear to provide a survival advantage to transformed cells through the inhibition of apoptosis [8], increased attachment to the extracellular matrix, increased invasiveness, and the stimulation of angiogenesis [9]. An increased COX-2 expression has been observed in several human tumor types and in selected animal and cell culture models of carcinogenesis, including hepatocellular carcinoma [6-9].
Transforming growth factor-beta (TGF-β) is a member of the multifunctional cytokine family and has been implicated in diverse cellular phenomena, including cell growth control, cell adhesion and motility, alteration of the cellular phenotype, production and degradation of the extracellular matrix protein, and apoptosis of hepatic cell lines [10]. TGF-β signaling plays an important role in the pathogenesis of fibrosis in liver cirrhosis, and hepatic stellate cells are the mediators of this protein. A large number of experimental and clinical studies have established that the TGF-β system can be activated as a tumor suppressor pathway, and that this pathway inhibits cellcycle progression during the G1 phase through the enhanced expression of cyclin-dependent kinase inhibitors such as p21.This signaling activates a tumor suppressor pathway by reversible arrest of cell proliferation [11]. However, these neoplastic epithelial cells often become resistant to TGF-β-mediated mitoinhibition, and the mechanisms for this alteration during carcinogenesis appear to be influenced by COX-2 expression [12]. COX-2 has been implicated in a number of carcinogenetic functions, but its relationship to TGF-β1-mediated HCCs is unknown. In past studies, TGF-β1 and epidermal growth factor (EGF) synergistically induced the expression of COX-2 production in mink lung epithelial cells [13]. Therefore, a major purpose of the present study was to assess the relation between TGF-β1 and COX-2 expression in the HCC and adjacent nonHCC tissues, and to assess the role of hepatic expression of COX-2 and TGF-β1 as predictors for patients with early recurrence of HCC.
2. Materials and Methods
2.1. Patients
This study was designed as a retrospective cohort study and was conducted in accordance with the Declaration of Helsinki and “ethical guidelines for clinical studies” from Ministry of Health, Labor and Welfare in Japan.
Sixty patients underwent curative liver resection and pathologically proved to be HCC between 2000 and 2003 in Wakayama Medical University Hospital were overviewed in this study. All the patients were routinely diagnosed by ultrasonography (US), contrast enhanced dynamic computed tomography (CE-CT) and tumor markers such as alpha fetoprotein (AFP). Fifty one patients underwent anatomical resection and 9 patients underwent non-anatomical resection. None of the patients received neoadjuvant and adjuvant chemotherapy or radiotherapy. Postoperative surveillance was performed every 2 to 3 months using US, CE-CT and blood examinations including tumor markers.
The presence of an intrahepatic recurrence was determined by the existence of a hypervascular nodule in early phase with a perfusion defect in the portal phase under CE-CT. If an extra-hepatic recurrence was suspected, lung CT or bone scintigraphy was performed. An extrahepatic recurrence was determined by the existence of a tumor. After detecting any recurrence, appropriate therapeutic modalities were administered, and the same surveillance was performed.
2.2. Data Collection
Clinical risk factors that may associate with early recurrence; the tumor size, preoperative blood chemical data, pathologic grading, vascular invasion, and high AFP values were reviewed.
2.3. Immunohistochemical Staining of COX-2
Tissue Samples were routinely fixed in 10% neutralbuffered formalin and were subsequently embedded in paraffin. Serial sections 4 μm thick were prepared from paraffin blocks and mounted on silanized slides (DAKO Japan, Kyoto, Japan). Sections were deparaffinized and hydrated by sequential immersion in xylenes and a graded alcohol series. Tissue sections were heated with a water bath in 0.01 M/L citric buffer (pH 6.0) at 95˚C for 40 minutes, and were washed in Tris-buffered saline containing 0.05% Tween 20 (TBS-T). Slides were incubated in 10% (vol/vol) H2O2 in methanol for 20 minutes to block the endogenous peroxidase activity and were treated with distilled water obtained from the same species in which the secondary antibody was developed for 25 minutes to block nonspecific staining. Subsequently, the slides were incubated with primary antibody [anti COX-2 (IBL, Gunma, Japan) at 1:50 for overnight at 4˚C]. After washing in TBS-T, immunostaining was performed using the avidin-biotin-peroxidase complex method. The slides were treated with a biotin-conjugated secondary antibody (Life Science, Tokyo, Japan) at 1:100 for 30 minutes followed by incubation with peroxidase-conjugated streptavidin (Life Science, Tokyo, Japan) for 30 minutes at room temperature.
The reaction products were visualized using 0.1% 3-3’-diamino-benzidine-tetrahydrochloride and 0.0005% H2O2 in 0.05 mol/L Tris-buffer (pH 7.6). Finally, the sections were counterstained with hematoxylin. All samples were stained twice to confirm the replication.
2.4. Immunohistochemical Staining of TGF-β
Samples sections were routinely treated as mentioned in the procedures for COX-2. Tissue sections were heated in a water bath in 0.05 M/L Trypsin buffered (pH 7.6) at 37˚C for 45 minutes, and were washed in TBS-T. The slides were incubated in 10% (vol/vol) H2O2 in methanol for 20 minutes to block the endogenous peroxidase activity. The slides were treated with distilled water obtained from the same species in which the secondary antibody was developed for 25 minutes to block nonspecific staining. Subsequently, slides were incubated with the primary antibody [anti TGF-β1 (Abcam, UK) at 1:100 for 18 h at room temperature].
After washing in TBS-T, all slides were treated as the same avidin-biotin-peroxidase complex method for COX- 2 staining and stained twice to confirm the replication.
2.5. Evaluation of COX-2 Expression
The immunoreactivity and distribution pattern of COX-2 were examined in both the HCC and adjacent nonHCC tissues of the liver. All immunostained sections were evaluated by two of us (NM, MU) who were blinded to clinical and pathologic information. Cases with discrepant results were re-evaluated jointly until agreement was reached. For each section, the intensity of staining was scored on a scale from 0 to 2, where 0 represented negative staining; 1, moderate; and 2, strong staining (Figures 1-2). Tissue samples with a score of 2 were defined as the high COX-2 group, and scores of 1 and 0 were defined as the low COX-2 group. COX-2 expression was very faint or undetectable in the vascular epithelium, whereas epithelial cells of the bile ducts generally expressed strong levels of COX-2. Accordingly, the latter level of staining was used as an internal control, which was designated arbitrarily as an intensity level of 2. COX-2 expression was generally homogeneous in each sample.
2.6. Evaluation of TGF-β Expression
The immunoreactivity and distribution pattern of TGF-β1 were also examined in both the HCC and adjacent nonHCC tissues of the liver by the same way as evaluation of COX-2 expression. For each section, the intensity of staining was scored on a scale from 0 to 2, where 0 represented negative staining; 1, moderate; and 2, strong staining (Figures 3-4). Tissue samples with scores of 2 and 1 were defined as the positive TGF-β1 group and, and a score of 0 was the negative TGF-β group. The positive signals of TGF-β expression stained yellow or brown, primarily in the cytoplasm. TGF-β1 expression was generally homogeneous in each sample.
2.7. Statistical Analysis
Data were analyzed using the Stat View J-4.5 software program (Abacus Concepts, Inc., Berkeley, CA). The relations between categorical variables were tested using a chi-squared analysis and Fisher’s exact probability test. Patient survival was calculated using the Kaplan-Meier method, and comparisons of survival curves were made using the log-rank test. Risk factors for early recurrence were evaluated by multivariate Cox regression analysis. p-values of less than 0.05 were considered to be statistically significant.
3. Results
3.1. COX-2 and TGF-β1 Expression by Immunohistochemical Staining
In 60 HCC specimens, COX-2 staining was high in 16 (27%) samples and low in 44 (73%) samples. In the nonHCC specimens, COX-2 staining was high in 20 (33%) samples and low in 40 (67%) samples.
TGF-β1 expression of HCC sections was positive in 51 (85%) and negative in 9 (15%) samples. In the nonHCC sections, TGF-β1 expression was positive in 55 (92%) and negative in 5 (8%) samples.
Table 1 shows the relationship between COX-2 expression and clinicopathological factors among the 60 samples of HCC and nonHCC sections. There was no relationship between COX-2 expression and underlying liver disease (HBs antigen, HCV antibody) and blood examinations (total bilirubin, serum albumin, prothrombin time, AST and ALT). On the other hand, pathologically negative vascular invasion and a tumor size of less than 5