Tumor initiation and progression in hepatocellular carcinoma: risk factors, classification, and therapeutic targets.

Hepatocellular carcinoma (HCC) is a major health problem worldwide responsible for 500 000 deaths annually. A number of risk factors are associated with either the induction of the disease or its progression; these include infection with hepatitis B or C virus, alcohol consumption, non-alcoholic steatohepatitis and certain congenital disorders. In around 80% of the cases, HCC is associated with cirrhosis or advanced fibrosis and with inflammation and oxidative stress. In this review we focus firstly on the different risk factors for HCC and summarize the mechanisms by which each is considered to contribute to HCC. In the second part we look at the molecular processes involved in cancer progression. HCC development is recognized as a multistep process that normally develops over many years. Over this period several mutations accumulate in the cell and that stimulate malign transformation, growth, and metastatic behavior. Over the recent years it has become evident that not only the tumor cell itself but also the tumor microenviroment plays a major role in the development of a tumor. There is a direct link between the role of inflammation and cirrhosis with this microenviroment. Both in vitro and in vivo it has been shown that tumor formation and metastatic properties are linked to epithelial-mesenchymal transition (EMT), a process by which facillitates the tumor cell's attempts to migrate to a more favourable microenviroment. Several groups have analyzed the gene expression in HCC and its surrounding tissue by microarray and this has resulted in the molecular classification into a distinct number of classes. Here we also found a role for hypoxia induced gene expression leading to a clinically more aggressive gene expression in HCC. Molecular analysis also helped to identify important cellular pathways and possible therapeutic targets. The first molecule that in this way has shown clinical application for liver cancer is the multikinase inhibitor sorafenib, others are currently in different stages of clinical studies like the mTOR inhibitor everolimus.

HBx or HCV core gene expression in HepG2 human liver cells results in a survival benefit against oxidative stress with possible implications for HCC development.

Hepatitis virus replication in the liver is often accompanied by inflammation resulting in the formation of reactive oxygen species (ROS) and nitric oxide (NO) and these may induce cell death. We investigated whether the expression of HBx or HCV core protein in HepG2 cells has an influence on the sensitivity of these cells for oxidative radicals. Our previous study, using the inducible HBV model of HepAD38, revealed that oxidative-stress-related genes are upregulated by virus replication. In the present study, we examined the intracellular pro-oxidant status with dichlorofluorescein (DCF) in HepG2 cell lines transfected with HBx, HbsAg and HCV core. Baseline intracellular oxidative levels were not different in the cell lines expressing viral proteins as compared to control. However, when these cells were exposed to H(2)O(2), the viral protein expressing cells, especially those expressing HBx, showed a reduced level of ROS. This suggests that HBx and HCV core transfected cells can convert H(2)O(2) to less reactive compounds at a higher rate than the control cells. When HBx or HCV core expressing cells were exposed to peroxynitrite (a highly reactive product formed under physiological conditions through interaction of superoxide (O(2)(-)) with NO) these cells were less sensitive to induction of cell death. In addition, these cell lines were less prone to cell death when exposed to H(2)O(2) directly. In conclusion, HBx and HCV core expression in HepG2 cells leads to a survival benefit under oxidative stress which in vivo can be induced during inflammation.

Both Ca2+ -dependent and -independent pathways are involved in rat hepatic stellate cell contraction and intrahepatic hyperresponsiveness to methoxamine.

In chronic liver injury, hepatic stellate cells (HSCs) have been implicated as regulators of sinusoidal vascular tone. We studied the relative role of Ca(2+)-dependent and Ca(2+)-independent contraction pathways in rat HSCs and correlated these findings to in situ perfused cirrhotic rat livers. Contraction of primary rat HSCs was studied by a stress-relaxed collagen lattice model. Dose-response curves to the Ca(2+) ionophore A-23187 and to the calmodulin/myosin light chain kinase inhibitor W-7 served to study Ca(2+)-dependent pathways. Y-27632, staurosporin, and calyculin (inhibitors of Rho kinase, protein kinase C, and myosin light chain phosphatase, respectively) were used to investigate Ca(2+)-independent pathways. The actomyosin interaction, the common end target, was inhibited by 2,3-butanedione monoxime. Additionally, the effects of W-7, Y-27632, and staurosporin on intrahepatic vascular resistance were evaluated by in situ perfusion of normal and thioacetamide-treated cirrhotic rat livers stimulated with methoxamine (n = 25 each). In vitro, HSC contraction was shown to be actomyosin based with a regulating role for both Ca(2+)-dependent and -independent pathways. Although the former seem important, an important auxiliary role for the latter was illustrated through their involvement in the phenomenon of "Ca(2+) sensitization." In vivo, preincubation of cirrhotic livers with Y-27632 (10(-4) M) and staurosporin (25 nM), more than with W-7 (10(-4) M), significantly reduced the hyperresponsiveness to methoxamine (10(-4) M) by -66.8 +/- 1.3%, -52.4 +/- 2.7%, and -28.7 +/- 2.8%, respectively, whereas in normal livers this was significantly less: -43.1 +/- 4.2%, -40.2 +/- 4.2%, and -3.8 +/- 6.3%, respectively. Taken together, these results suggest that HSC contraction is based on both Ca(2+)-dependent and -independent pathways, which were shown to be upregulated in the perfused cirrhotic liver, with a predominance of Ca(2+)-independent pathways.

Glypican-3 expression distinguishes small hepatocellular carcinomas from cirrhosis, dysplastic nodules, and focal nodular hyperplasia-like nodules.

Distinguishing small hepatocellular carcinoma (HCC) from other types of small focal lesions that occur in a cirrhotic liver can be difficult on the basis of morphologic features alone. We investigated whether the expression of glypican-3 (GPC3) could be an ancillary tool in the histopathologic diagnostic process. We performed immunohistochemistry for GPC3 on 16 low-grade dysplastic nodules, 33 high-grade dysplastic nodules, 13 focal nodular hyperplasia-like nodules, and 59 HCCs with a diameter less or equal to 3 cm present in the cirrhotic liver of 66 patients. Both resected lesions and lesions biopsied by needle were included and nonlesional cirrhotic parenchyma was also stained. In a subset of cases (23 samples of cirrhosis, 4 low-grade dysplastic nodules, 5 high-grade dysplastic nodules, 2 focal nodular hyperplasia-like nodules, and 18 HCCs), real time reverse transcriptase-polymerase chain reaction for GPC3 was performed. GPC3 expression was, both on immunohistochemistry and by real time reverse transcriptase-polymerase chain reaction, much higher in small HCCs than in cirrhosis and other types of small focal lesions, indicating that the transition from premalignant lesions to small HCC is associated with a sharp increase of GPC3 expression in a majority of cases. The sensitivity and specificity of a positive GPC3-staining for the diagnosis of HCC in small focal lesions was 0.77 and 0.96, respectively, in resected cases, and 0.83 and 1, respectively, for needle biopsies. Because the result of the staining was easily interpretable, immunohistochemistry for GPC3 is valuable ancillary tool in the histopathologic diagnosis of small focal lesions in cirrhosis.

Hepatitis B virus replication causes oxidative stress in HepAD38 liver cells.

UNLABELLED: We used human hepatoma HepAD38 cells, in which HBV production is under the control of a tetracycline-regulated promotor, to investigate changes induced in the host cell by HBV replication that could contribute to malignant transformation. Parameters of oxidative stress (malondialdehyde, glutathione) and cell proliferation were determined at different times after induction (0-96 h). In HBV-producing cells, the redox status peaked at 72 h. cDNA micro array analysis at 72 h post induction revealed 3 groups of genes that were up-regulated by HBV: (i) heat shock proteins, (ii) oxidative and metabolic stress and (iii) growth and apoptosis related genes. Continuous HBV production did not accelerate karyotypic changes in cells cultured for 4 months (18 passages). IN CONCLUSION: HBV replication modulates host gene expression and induces oxidative stress. In this HepAD38 model early events (0-4 days) in the host cell after induction of HBV replication can be studied under strictly defined conditions.

A role for asymmetric dimethylarginine in the pathophysiology of portal hypertension in rats with biliary cirrhosis.

Reduced intrahepatic endothelial nitric oxide synthase (eNOS) activity contributes to the pathogenesis of portal hypertension (PHT) associated with cirrhosis. We evaluated whether asymmetric dimethylarginine (ADMA), a putative endogenous NOS inhibitor, may be involved in PHT associated with cirrhosis. Two rat models of cirrhosis (thioacetamide [TAA]-induced and bile duct excision [BDE]-induced, n = 10 each), one rat model of PHT without cirrhosis (partial portal vein-ligated [PPVL], n = 10), and sham-operated control rats (n = 10) were studied. We assessed hepatic NOS activity, eNOS protein expression, plasma ADMA levels, and intrahepatic endothelial function. To evaluate intrahepatic endothelial function, concentration-effect curves of acetylcholine were determined in situ in perfused normal rat livers and livers of rats with TAA- or BDE-induced cirrhosis (n = 10) that had been preincubated with either vehicle or ADMA; in addition, measurements of nitrite/nitrate (NOx) and ADMA were made in perfusates. Both models of cirrhosis exhibited decreased hepatic NOS activity. In rats with TAA-induced cirrhosis, this decrease was associated with reduced hepatic eNOS protein levels and immunoreactivity. Rats with BDE-induced cirrhosis had eNOS protein levels comparable to those in control rats but exhibited significantly higher plasma ADMA levels than those in all other groups. In normal perfused liver, ADMA induced impaired endothelium-dependent vasorelaxation and reduced NOx perfusate levels, phenomena that were mimicked by N(G)-nitro-L-arginine-methyl ester. In contrast to perfused livers with cirrhosis induced by TAA, impaired endothelial cell-mediated relaxation in perfused livers with cirrhosis induced by BDE was exacerbated by ADMA and was associated with a decreased rate of removal of ADMA (34.3% +/- 6.0% vs. 70.9% +/- 3.2%). In conclusion, in rats with TAA-induced cirrhosis, decreased eNOS enzyme levels seem to be responsible for impaired NOS activity; in rats with biliary cirrhosis, an endogenous NOS inhibitor, ADMA, may mediate decreased NOS activity.

Expression of hepatitis C virus core protein impairs DNA repair in human hepatoma cells.

Several studies have documented the important association between hepatitis C virus (HCV) infection and hepatocellular carcinoma. The mechanisms involved are still unknown and could involve viral proteins. We investigated the effect of HCV-core protein on DNA repair after UV-induced DNA damage. Therefore, we developed and characterized stably transfected HepG2 cell lines that express HCV-core protein as demonstrated by immunohistochemistry. These cells were significantly less capable to repair the DNA damage than control cells. This suppression of DNA repair by HCV-core protein renders the cells more sensitive to acquire mutations that in combination with enhanced in vivo cell turnover in the infected liver might increase the likelihood of malignant transformation of HCV-infected cells by other viral factors or upon exposure to environmental factors (food, drugs, smoking, alcohol, etc.). Interestingly, expression of the full-length HCV core did increase the cell doubling time in one of the cell lines we had developed that could not be attributed to an increase in apoptosis or change in telomerase activity in these cells.