Mohamed Abou-Shady

Enhanced glypican-3 expression differentiates the majority of hepatocellular carcinomas from benign hepatic disorders

BACKGROUND/AIMS Hepatocellular carcinoma (HCC) is a common malignant tumour worldwide, and its differential diagnosis from benign lesions of the liver is often difficult yet of great clinical importance. In the present study, we analysed whether glypican-3 is useful in differentiating between benign and malignant liver diseases and whether it influences the growth behaviour of HCC. METHODS Northern blot analysis and in situ hybridisation. RESULTS Northern blot analysis indicated that expression of glypican-3 mRNA was either low or absent in normal liver, in focal nodular hyperplasia (FNH), and in liver cirrhosis. In contrast, expression of glypican-3 mRNA was markedly increased in 20 of 30 and moderately increased in five of 30 HCC samples. The average increase in glypican-3 mRNA expression in HCC was significant compared with expression in normal liver (21.7-fold increase, p<0.01). In comparison with FNH or liver cirrhosis, glypican-3 mRNA expression in HCC was increased 7.2- (p<0.05) and 10.8-fold (p<0.01), respectively. In addition, pushing HCCs exhibited significantly higher glypican-3 mRNA expression than invading tumours (p<0.05). In situ hybridisation analysis demonstrated weak expression of glypican-3 mRNA in normal hepatocytes and bile ductular cells, and weak to occasionally moderate signals in hepatocytes forming nodules of liver cirrhosis and in regenerated hepatic nodules of FNH. In contrast, glypican-3 in situ hybridisation signals were intense in hepatic cancer cells with even higher levels in pushing HCCs than in invading HCCs. CONCLUSIONS These findings suggest that glypican-3, in many cases, has the potential to differentiate between benign and malignant liver diseases.

Transforming growth factor betas and their signaling receptors in human hepatocellular carcinoma.

BACKGROUND Transforming growth factor betas (TGF-betas) are multifunctional polypeptides that have been suggested to influence tumor growth. They mediate their functions via specific cell surface receptors (type I ALK5 and type II TGF-beta receptors). The aim of this study was to analyze the roles of the three TGF-betas and their signaling receptors in human hepatocellular carcinoma (HCC). METHODS HCC tissue samples were obtained from 18 patients undergoing partial liver resection. Normal liver tissues from 7 females and 3 males served as controls. The tissues for histological analysis were fixed in Bouins solution and paraffin embedded. For RNA analysis, freshly obtained tissue samples were snap frozen in liquid nitrogen and stored at -80 degrees C until used. Northern blot analysis was used in normal liver and HCC to examine the expression of TGF-beta1, -beta2, -beta3 and their receptors: type I ALK5 (TbetaR-I ALK5), type II (TbetaR-II), and type III (TbetaR-III). Immunohistochemistry was performed to localize the corresponding proteins. RESULTS All three TGF-betas demonstrated a marked mRNA overexpression in HCC in comparison with normal controls, whereas the levels of all three TGF-beta receptors showed no significant changes. Intense TGF-beta1, TGF-beta2, and TGF-beta3 immunostaining was found in hepatocellular carcinoma cells and in the perineoplastic stroma with immunohistochemistry, whereas no or mild immunostaining was present in the normal liver. For TbetaR-I ALK5 and TbetaR-II, the immunostaining in both HCC and normal liver was mild to moderate, with a slightly higher intensity in the normal tissues. CONCLUSION The upregulation of TGF-betas in HCC suggests an important role for these isoforms in hepatic carcinogenesis and tumor progression. Moreover, the localization of the immunoreactivity in both malignant hepatocytes and stromal cells suggests that TGF-betas act via autocrine and paracrine pathways in this neoplasm.

Group II and IV phospholipase A2 are produced in human pancreatic cancer cells and influence prognosis

BACKGROUND Phospholipase A2 (PLA2) is involved in regulating biosynthesis of arachidonic acid and its metabolites. There are three major structurally different forms of PLA2: group I, also called pancreatic PLA2 (PLA2-I); group II, referred to as secretory non-pancreatic or synovial or platelet PLA2 (PLA2-II); group IV, referred to as cytosolic PLA2 (PLA2-IV). AIMS To examine PLA2-I, PLA2-II, and PLA2-IV in normal and pancreatic cancer tissues. Patients—PLA2 was studied in 58 pancreatic adenocarcinomas, obtained from 25 women and 33 men undergoing pancreatic resection. Normal organ donor pancreas served as control. METHODS The enzymes were analysed by northern blot, in situ hybridisation, and immunohistochemistry. The molecular findings were correlated with clinical variables of the patients. RESULTS Northern blot analysis of total RNA showed enhanced PLA2 group II and IV mRNA expression in 52% and 55% of the pancreatic cancer samples respectively compared with the normal controls (p = 0.0013 and p = 0.0025). On immunohistochemical analysis, intense PLA2-I immunoreactivity was seen in acinar cells, but not in ductal cells, in the normal pancreas. In pancreatic cancer cells, PLA2-I immunostaining was absent. PLA2-II immunostaining was visible only in some acinar and ductal cells in the normal pancreas, whereas in pancreatic cancer increased PLA2-II immunoreactivity was present in 65% of the cancer samples. On in situ hybridisation, weak PLA2-IV mRNA signals were detected in acinar and ductal cells of normal samples; these signals were present to a much greater extent in pancreatic cancer cells. The presence of PLA2-II in pancreatic cancer was associated with a higher degree of fibrosis (p<0.01). Furthermore, there was a significant correlation between the enhanced expression of PLA2-II and longer survival after surgery (p<0.03), but not of PLA2-IV and longer postoperative survival. CONCLUSION These data suggest that PLA2-II and PLA2-IV are upregulated in human pancreatic cancer, and that upregulation of PLA2-II in pancreatic cancer covariates negatively with cancer cell growth.

Transforming growth factor betas and their receptors in human liver cirrhosis

Background Transforming growth factor betas (TGF-βs) are a group of homologous polypeptides that exert pleiotropic effects on various cell types and stimulate the formation of extracellular matrix and fibrosis. To evaluate whether TGF-β isoforms (TGF-β1, TGF-β2 and TGF-β3) and their receptors (types l-lll) are also of importance in the pathophysiology of liver cirrhosis, we analysed their concomitant expression and localization in human liver cirrhosis. Patients Cirrhotic liver tissue samples were obtained from 17 patients (four women, 13 men) with a median age of 41 years (range 22–67). Normal liver tissues from ten patients (seven women, three men) with a median age of 55 years (range 45–75) served as controls. Methods The tissues were fixed in Bouins solution and paraffin-embedded for histological analysis. For RNA analysis, freshly obtained tissue samples were snapfrozen in liquid nitrogen and stored at −80°C until analysed. Northern blot analysis was used to examine the expression of TGF-β1, β2 and β3 and their receptors, type I (TβR-I), type II (TβR-ll) and type III (TβR-lll). Immunohistochemistry was performed to determine the localization of the corresponding proteins in the normal and the cirrhotic liver. Results Northern blot analysis revealed enhanced expression (P<0.05) of TGF-β1 (twofold increase), TGF-β2 (threefold increase) and TGF-β3 (8.5-fold increase) and of TβR-ll (threefold increase) mRNA in liver cirrhosis in comparison with normal controls. In contrast, TβR-l (ALK-5) and TβR-lll mRNA expression showed no significant changes. No TGF-β isoform immunoreactivity was present in hepatocytes in either normal livers or in liver cirrhosis. However, in liver cirrhosis, intense TGF-β1 immunoreactivity was present in bile duct and ductular epithelial cells (including ductular proliferations) and In inflammatory cells. In a few sinusoidal lining cells, faint TGF-β1 and moderate TGF-β2 immunoreactivity was present TGF-β3 immunostaining was present in bile duet and ductular epithelial cells, in inflammatory cells and in fibroblast-like spindle cells in liver cirrhosis. For TβR-l and TβR-ll, the immunoreactivity was localized in hepatocytes and biliary cells in normal and cirrhotic liver tissues, with higher intensity for TβR-ll in the cirrhotic liver. Conclusion Enhanced expression of all three TGF-β isoforms and of TβR-ll in liver cirrhosis suggests, their involvement in this fibrotic disorder. The higher immunoreactivity of the three TGF-β isoforms in the bile duct epithelial cells in cirrhotic tissues suggests a possible role of these cells in the pathogenesis of liver cirrhosis.

Laparoscopic Partial Hepatectomy in the Rat: A New Resectional Technique

Background: Rats are widely used for basic research in laparoscopic surgery. We have developed a new technique of laparoscopic partial hepatectomy in the rat. Methods: 40 American Cancer Institute rats were randomized into 3 groups. Group A (n = 14) underwent laparoscopic liver resection using a CO2 pneumoperitoneum. Group B (n = 14) was operated on with a gasless laparoscopic technique using a lifting device. A control group C (n = 12) underwent conventional open liver resection. In each group half of the animals underwent single lobectomy and the other half bilobectomy. Results: The liver resection was performed successfully in all 40 rats. No conversion to open surgery was necessary. No mortality or morbidity was observed. Conclusions: This new technique of laparoscopic partial hepatectomy proved to be feasible and safe. It is the first description of a laparoscopic hepatic resection in the rat that could prove valuable in further investigations of liver physiology and pathology.

Connective tissue growth factor in human liver cirrhosis

BACKGROUND Connective tissue growth factor (CTGF) belongs to a family of factors that regulate fibrogenesis and wound healing. While the significance of transforming growth factor beta (TGF-beta) in liver fibrosis is well established, the role of CTGF in fibrosing hepatopathy is still unknown. METHODS CTGF was analyzed in 10 normal and in 16 cirrhotic liver tissue samples. Northern blot analysis was used to examine the concomitant expression of CTGF and TGF-beta1 mRNAs, and the cellular localization of CTGF mRNA was studied by in situ hybridization. For identification of myofibroblasts and activated hepatic stellate cells, alpha-smooth muscle actin (alpha-SMA) immunohistochemistry was used. RESULTS Northern blot analysis showed 6.5-fold enhanced expression of CTGF mRNA and 7.8-fold enhanced expression of TGF-beta1 mRNA in liver cirrhosis in comparison with normal controls (p<0.01). By in situ hybridization, CTGF mRNA was detectable in only a few spindle cells in the portal tracts in normal liver samples. In contrast, there was strong expression of CTGF mRNA in fibroblasts and myofibroblast-like cells present in fibrous septa surrounding the cirrhotic nodules, in stellate cells, in endothelial cells and in mesenchymal cells around ductular proliferations, and in ductular epithelial cells. There was a strong correlation between CTGF mRNA and TGF-beta1 mRNA as well as the degree of fibrosis (p<0.01). CONCLUSIONS Overexpression of CTGF in liver cirrhosis, especially in fibroblasts/myofibroblasts and stellate cells, suggests that this novel factor may play an important role in hepatic fibrosis.