Correlation between Apoptotic and Anti-apoptotic Proteins (caspase-3 and Bcl2) in Chronic Hepatitis C: An Immunohistochemical Study

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1 KASR EL AINI MEDICAL JOURNAL VOL. 17, NO 3, October Correlation between Apoptotic and Anti-apoptotic Proteins (caspase-3 and Bcl2) in Chronic Hepatitis C: An Immunohistochemical Study Samia El-Naggar and Sahar Aly Daoud Pathology department, Faculty of medicine, Beni -Sueif University, Egypt ABSTRACT There is increasing evidence suggesting that liver cell damage in chronic HCV infection is mediated by induction of apoptosis. The morphological alterations of apoptosis are mediated by a family of intracellular proteases called caspases. Caspase- 3 is the central executioner of many if not all apoptosis pathway. The aim of the work of the present study is to analyze the apoptotic like protein caspase-3 and anti-apoptotic Bcl2 expression in tissues from patients with chronic HCV to throw more light on apoptotic process and their correlation with histopathological changes. Material and Methods: Liver biopsies from 73 patients with chronic HCV (40 patients with history of bilharziasis, 33 patients with no history of bilharziasis) selected from those referred to tropical medicine department, Beni Sueif University hospital before taking treatment, examined by H&E and immunostained for caspase-3 and Bcl2. Results: There was positive correlation between caspase-3 and the grade of activity; as well as the stage of fibrosis in cases with HCV of both groups (those associated and not associated with bilharziasis). While, Bcl2 was not expressed by liver cells. Conclusion: Finding that the anti-apoptotic protein Bcl2 was not expressed in hepatocytes in patients with chronic hepatitis C selected for our study leads to the conclusion that in these cells apoptosis is not inhibited by this protein, fact that could explain the increased apoptosis observed in liver samples from these patients. In addition caspase-3 is a noninvasive measure for detection of early liver cell injury and fibrosis. INTRODUCTION In the last years, infection with hepatitis C virus is prevalent around the world and for more than 80% of cases leads to chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. (Tran, 2008) There is a great degree of variation in the prevalence, ranging from less 1% in the United Kingdom to more than 20% in Egypt. In the developed world, the relatively low incidence is mostly due to intravenous drug abuse, while the World Health Organization (WHO) anti- Schistosomal vaccination program in Egypt 1970s and other vaccination programs in Mongolia and Bolivia have led to much higher rate in those countries.(frank, et al., 2000) These figure are alarming, since patients currently asymptomatic with relatively mild disease will eventually progress to end stage liver disease and develop hepatocellular carcinoma (HCC).Moreover, there is no proper vaccine against HCV and anti-viral treatment is not only expensive but relatively toxic, sufficiently ineffective in treating all patients. (Pearlman, 2004) This underscores the need for more effective therapies. A better understanding of the mechanism underlying the pathology of the chronic HCV infection could be helpful in identifying a novel therapeutic target against this disease. The mechanisms by which liver cell inflammation, fibrosis and sign for chronic hepatitis appear are not completely understood. Many studies suggest that apoptosis may be involved in the pathogenesis of chronic hepatitis C. (Bantel and Schultze-Osthoff, 2003), (Fisher et al, 2007). Apoptosis is a complex phenomenon that occurs through two main pathways: extrinsic (mediated by death receptors) and intrinsic (mitochondrial pathway) that converged to final common way involving activation of caspases.(hengartner, 2000 and Gupta, 2003) The intrinsic pathway of apoptosis is initiated within the cell mitochondria; playing an important role. The instability of the mitochondrial membrane induces the release of proapoptotic factors into cytosl, such as cytochrome C, leading to activation of downstream caspases; enzymes responsible for degradation of several cellular changes that define apoptotic cell death. Several intracellular proteins are involved in the regulation of apoptosis constitute the caspase family and in

2 KASR EL AINI MEDICAL JOURNAL VOL. 17, NO 3, October particular, the Bcl-2 family of proteins, which includes both pro- and antiapoptotic members; are perhaps the most important regulators of the intrinsic pathway. These proteins act upstream and at the level of the mitochondria to integrate death and survival signals, and the balance between pro- and antiapoptotic members of the family, as well as their reciprocal interactions, determines whether or not the intrinsic pathway of apoptosis is initiated.(garrido et al, 2006 and Rasola, 2007) Aim of the work The aim of the present work is to analyze the apoptotic like protein caspase-3 and antiapoptotic Bcl2 expression in tissues from patients with chronic HCV to throw more light on apoptotic process and their correlation with histopathological changes. MATERIAL & METHODS The current study was carried on 73 patients with chronic HCV selected from those referred to tropical medicine department, Beni Sueif University hospital before taking treatment. Patients with co-infection of other viruses such as HBV, HDV, HIV infection, patients using hepatotoxic drugs, herbal medication within 6 months prior to liver biopsy, patients having diabetes mellitus or hyperlipidemia were excluded. Demographic data (age, gender, and clinical symptoms), biochemical data (aspartate aminotransferase AST, ALT) and serological findings as viral load were recorded. Two pathologists reviewed liver biopsies. Cases were divided into two groups: Group I: Included 40 HCV with history of schistosomiasis. Group II: included 33 HCV with no history of schistosomiasis. Liver biopsies were included in paraffin, processed and cut at 3 µm sections were used for Hematoxylin and Eosin (H&E) stains for histopathological diagnosis, Masson trichrome stain for detection of fibrosis and other sections were fixed on adhesive slide for immunohistochemical staining. Examination of sections stained with Hematoxylin and Eosin (H&E): Several histological features were evaluated as follows: Histological grading of necroinflammatory score based on modified HAI grading by Ishak et al.,(1995) including interface hepatitis (0-4), focal spotty necrosis (0-4), confluent necrosis (0-6) and portal inflammation (0-4). Grades of activity 1-3 were considered minimal, grades 4-8 were considered mild, grades 9-12 were considered moderate and grades were considered severe. Degree of steatosis 0: absent 1 : < 33% 2: 33-66% 3: > 66 Presence of portal lymphoid follicles, presence of bile duct damage (lymphocytic intraepithelial or periductuler infiltration), the involvement pattern in each portal tract ( total involvement > 80%, partial involvement < 80% or no involvement) the knodell histology activity index and fibrosis score of Ishak modified HAI.(Ishak 1995) were also evaluated. Histological staging of fibrosis of chronic hepatitis was done according to fibrosis score (0-6). (Ishak et al.,1995) Immunohistochemical staining for caspase-3 and Bcl2 was performed using the following primary antibodies: polyclonal rabbit caspase-3 diluted (1:50) and monoclonal mouse anti-bcl2, diluted 1:75 (Dako) followed by incubation with appropriate secondary antibody. We use avidinbiotin-peroxidase complex (Vector laboratories) to amplify the reactions and 3, 3'- diaminobenzidine hydrochloride/h2o2 (Sigma Aldrich Co.) to develop them. Nuclear counterstaining was done with Meyer's hematoxylin. The expression of caspase-3 was measured in 10 successive high power fields (x400). It was expressed as brown coloration in hepatocyte cytoplasm. The degree of caspase-3 expression within the hepatic lobules was evaluated semiquantitatively according to the percentage of positively stained cells and classified into four groups. (-)= no immunostaining, (+) = (<25%of cells), (++) = (25%-50cells), (+++) = (>50%- 75% of cells), (++++) =>75%of cells. (Bantel et al 2001). Positive immunostaining expression of Bcl2 was considered if brownish granules in the cytoplasm or in the nucleus of different cells were expressed. A semi-quantitative analysis was

3 KASR EL AINI MEDICAL JOURNAL VOL. 17, NO 3, October performed after examination of five microscopic fields (X400) on each slide, assignment of scores, based on the number of positive cells identified. The score was +: up to 5 positive hepatocyte per field, ++: 5-10 positive hepatocytes per field.(borner 2003) Statistical analysis was performed using windows soft ware. The relation was obtained by Pearson Chi- square test. P value less than 0.05 was considered significant. RESULTS According to age of the present studied cases; mean age in group I was 43y and group II was 48y. As regards sex distribution: male to female ratio in group I was 2:1 and 3:2 in group II, the difference between groups of patients in age and sex distribution was statistically insignificant (P= and P= 0.086) respectively. Histological examination of liver biopsies done on group I revealed minimal activity in 10 cases, mild activity in 7 cases, moderate activity in 11 cases, severe activity in 12 cases. As regard group II: we found minimal activity in 3 cases, mild activity in 10 cases, moderate activity in 5 cases, severe activity in 15 cases. <5% steatosis was found in 18 cases(45%) in group I and 13 cases(39%) in group II. Immunohistochemical stains for detection of caspase-3 and Bcl2: Expression of the two apoptotic protein caspase- 3 and Bcl2, which are involved in apoptosis execution and anti-apoptotic function, respectively, were assessed. There was no significant correlation between Caspase-3 with any of the histological features. Immunohistochemical assessment of caspase-3 and activity in liver biopsy specimens was shown in (table1): The grading of caspase-3 activity was expressed as percentage of positively stained cells. Minimal activity was found in (25%). Mild activity was found in (17.5%), moderate activity was found in 27.5%, marked activity was found in (30%) of liver biopsy in group I. In group II, minimal activity was found in 9.1% of cases. Mild activity was found in 30.3% of cases, moderate activity was found in 15% of cases. Severe activity was found in 45.6% of cases (fig: 1 a,b,c,d), P value < 0.05 for each, however there was no significant correlation between both groups (P value = 0.099). Table (1): correlation between caspase-3 immunostaining and grade of hepatitis activity in liver biopsy of studied cases Caspase-3 immunostaining Grade of Hepatic activity Group I(n=40) n (%) Group II ( n33) n (%) Minimal 10 (25%) 3 (9.1 %) Mild 7 (17.5%) 10 (30.3%) Moderate 11 (27.5%) 5 (15 %) Severe 12(30%) 15 (45.6%) X2 P Chi-Square: P- value 0.099

4 KASR EL AINI MEDICAL JOURNAL VOL. 17, NO 3, October A B C D Figure 1: Liver biopsy of HCV showed mild activity in group I (H&E X100) (a), the hepatocytes were positive for caspase-3 immunostaining; 75% of cells were scored +++ (b). Liver biopsy of HCV showing mild activity in group II (H&E X200) (c), 50 % of hepatocytes were positive for caspase 3 scored as ++ (x400) (d). Fibrosis of different stages 0-6 was diagnosed by Masson trichrome stain. The relation between caspase-3 and the stage of fibrosis was tabulated in table 2. It was notable that caspase-3 was expressed more in cases with fibrosis stages 4, 5 and 6 in both groups (statistically insignificant p value > 0.05) (fig:2 a,b). Table (2): correlation between caspase-3 immunostaining and stage of fibrosis in liver biopsy of studied cases Caspase-3 immunostaining Group I(n=40) n (%) Group II ( n33) n (%) Stage of fibrosis 0 1 ( 2.5 % ) 3 (9.1 %) 1 6 ( 15 % ) 5 ( 15.1%) 2 3 ( 7.5 %) 4 ( 12.2%) 3 8 ( 20%) 4 ( 12.2%) 4 5 (12.5%) 5 (15.1%) 5 9 ( 22.5%) 7 ( 21.2%) 6 8 ( 20%) 5 ( 15.1%) X2 P Chi-Square: P- value Hepatocytes didn't reveal positivity for Bcl2.We noted Bcl2 positive cells in the areas of inflammatory infiltrate, these cells being most likely lymphocytes (fig 2, c)

5 KASR EL AINI MEDICAL JOURNAL VOL. 17, NO 3, October A B C Figure 2: Liver biopsy of HCV, showing cirrhosis (stage 6) (masson trichrome X200), the hepatocytes showed positive caspase-3 immunostaining score++++ (X400) (b), negative immunoreaction for bcl2, the inflammatory cells were positive (X400) (c). DISCUSSION Hepatitis C viral infection is a major cause of liver disease, characterized by inflammation, cell damage and fibrotic reaction of hepatocytes.there is increasing evidence suggesting that liver cell damage in chronic HCV infection is mediated by induction of apoptosis. (Bantel and Schulze-Osthoff., (2003) The morphological alterations of apoptosis are mediated by a family of intracellular proteases called caspases.(los et al., 1999) Caspase- 3 is the central executioner of many if not all apoptosis pathway. (Van Molle et al. (1999) In the present study apoptosis could not be seen by H&E Stains. By immunohistochemistry caspase- 3 activity in liver tissue was detected as brown stains in cell cytoplasm of chronic HCV liver tissue with positive correlation to the grade of activity; regardless the intensity of the stain. These finding were in agreement with previous studies by Ibrahim (2004) who obtained a direct evidence for variable degree of liver cell apoptosis in the liver of patients with chronic HCV and a significant correlation was found between the amount of apoptosis, expressed as apoptotic index and inflammatory activity in the liver. Also the results of the present study are in agreement with Bantel et al, (2001) who found that liver cell damage in chronic HCV infection is mediated by induction of apoptosis. Caspases are activated in human biopsy specimens of chronic hepatitis patients, activation of Caspase- 3 correlated significantly with inflammatory activity. Similar results were also found by Bantel et al., (2004) who found that chronic HCV infection is characterized by massive caspase activation, hepatocytes exhibiting immunoreactivity for active caspases and apoptotic features. So, they suggested that measurement of caspase-3 activation may be a suitable marker for detecting very early signs of liver damage. Although the pattern of HCV/schistosomal infection frequent in many geographic areas previous reports showed more severe liver disease in co-infected patients, the inter-actions between HCV and schistosomal infection are not fully understood.(kamal et al.,2000) In the present study, no significant difference was found between co-infected patients in group I and mono- infected patients in group II as regard liver disease severity. This was in agreement with Blanton et al., (2002) study in Egypt, who found that there was no interaction between schistosomal infection or disease and the prevalence and severity of hepatitis C. For both infections, the intensity or prevalence of infections was a poor predictor of morbidity. The prevalence of chronic liver disease in the Egyptian population from different pathogens suggests a generalized susceptibility to inflammatory liver disease. In the present study caspase-3 was expressed with higher stages of fibrosis (4, 5, and 6) with slight higher incidence in cases associated of schistosomiasis statistically insignificant. Anglico et al., (1997) and Kamal et al., (2000) stated that patients with concomitant schistosoma and HCV infection have higher incidence of cirrhosis and hepato-cellular carcinoma than patients with chronic HCV mono-infection, matched for age, disease duration, and HCV genotype. Kamal et al., (2000) found that, fibrosis was significantly accelerated in co-infected patients than mono-

6 KASR EL AINI MEDICAL JOURNAL VOL. 17, NO 3, October infected patients and explained the cause of fibrosis progression by additive effects of 2 injurious agents or the difference in immune response and alteration of host signaling pathway inflicted by schistosomal infection, resulting in fibrogenesis. The results of the current study may be explained on the basis of the small number of the studied cases. The effect of co-infection of HCV and Schistosomiasis need long term follow up, which is not included in the current study. Repeated courses of antischistosomal therapy which is a very common attitude in Egyptians may greatly diminish the effect of schistosomal infection. As regards immunoreactivity for Bcl2 in liver biopsies taken from patients with chronic hepatitis C from both groups I and II, hepatocytes didn't reveal positivity for Bcl2. We noted Bcl2 positive cells in the areas of inflammatory infiltrate, these cells being most likely lymphocytes. Several studies have shown that proteins of the Bcl2 family are important regulators of apoptosis. They integrate various signals for survival or cell death generated within or outside the cells, the reaction between pro-and anti-apoptotic members of this family setting limits that cause cell death or survival. (Burlacu, 2003 and Borner, 2003) Our results regarding the expression of Bcl2 protein are consistent with those obtained in other studies which have revealed that this protein is not expressed or have negligible expression in hepatocytes in patients with hepatitis (Tsamandas et al., 2003 and Sarfraz et al., 2008), but in contradiction with those studies saying that Bcl2 is expressed in the cytoplasm of hepatocytes in chronic liver disease. (Chen et al., 2004) Conclusion: Caspase-3 is a noninvasive measure for detection of early liver cell injury and fibrosis.finding that the anti-apoptotic protein Bcl2 was not expressed in hepatocytes in patients with chronic hepatitis C leads to the conclusion that apoptosis is not inhibited by this protein, fact that could explain the increased apoptosis observed in liver samples from these patients. REFERENCES 1. Angelico ME, Rengansthan CG, Gandin, et al., (1997): Chronic liver disease in the Alexandria governorate, Egypt contribution of schistosomiasis and hepatitis virus infection. J Hepatology; 26: Bantel H., Lugering A., Porembo C. (2001): Caspase activation correlated with the degree of inflammatory liver injury in chronic hepatitis C virus infection. Hepatology; 34 (4): Bantel H., Schultze-Osthoff K. (2003): Apoptosis in hepatitis C virus infection. Cell death diff; 10: Bantel H., Lugering A., Heidemann I., et al., (2004): Detection of apoptosis caspases activation in sera from patients with chronic HCV infection is associated with fibrotic liver injury. Hepatology; 40: Blanton R.E., Salam E.A., Katiuki H.C., et al., (2002): Population- based difference in schistosomsa- mansoni and hepatitis C induced disease. J Infect Dis ;185 (11) : Borner C. (2003): The Bcl-2 protein family: sensors and checkpoints for lifeor-death decision. Mol.Immunol; 39: Burlacu A. (2003): Regulation of apoptosis by Bcl-2family proteins. J.Cell.Mol. Med; 7(3): Chen N.L.,Bai L., Chen P.l.,Zhang, Ch., et al., (2004): Apoptosis pathways of liver cells in chronic hepatitis.world J.Gastroentrol; 10 (21): Fisher R., Baumert Th., Blum H. (2007): Hepatitis C viral infection and apoptosis. World J.Gastroentrol; 13 (36): Frank C., Mohamed M.K., Strickland K.T., et al., (2000): The role of parenteral anti-schitosomal therapy in the spread of HCV in Egypt. Lancet;355: Garrido C., Galluzzi L., Brunet M., Puig P.E., et al.,(2006): Mechanism of cytochrome c release from mitochondria. Cell death diff ;13: Gupta S. (2003): Molecular signaling in death receptor and mitochondrial pathways of apoptosis.int. J. Oncol;

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