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The physiological role of autophagy in the progression of liver diseases is still debated. To understand the clinical relevance of autophagy in primary e secondary hepatic tumors, we analyzed the expression of mTOR (mammalian target of rapamycin), a key regulator of autophagy; Raptor (regulatory-associated protein of mTOR); ULK1 (Unc-51 like kinase 1) determinant in the autophagy initiation; LC3 (microtubule-associated protein 1A/1B-light chain 3), a specific marker of autophagosomes; and p62, a selective autophagy receptor. Samples from subjects with chronic hepatitis (n.58), cirrhosis (n.12), hepatocellular carcinoma (HCC, n.56), metastases (n.48) from colorectal cancer and hyperplasia or gallbladder stones (n.7), the latter considered as controls, were examined. Gene expression analysis was carried out in n.213 tissues by absolute q-PCR, while protein expression by Western Blot in n.191 lysates, including tumoral, surrounding tumoral and normal tissues. Nonparametric statistical tests were used for comparing expression levels in the above-mentioned groups. Subgroup analysis was performed considering viral infection and chemotherapy treatment. The mTOR transcriptional level was significantly lower in metastases compared to HCC (P = 0.0001). p-mTOR(Ser2448) and LC3II/LC3I protein levels were significantly higher in metastases compared to HCC (P = 0.008 and P<0.0001, respectively). ULK(Ser757) levels were significantly higher in HCC compared to metastases (P = 0.0002) while the HCV- and HBV- related HCC showed the highest p62 levels. Chemotherapy induced a down-regulation of the p-mTOR(Ser2448) in metastases and in non-tumor surrounding tissues in treated patients compared to untreated (P = 0.001 and P = 0.005, respectively). Conclusions: the different expression of proteins considered, owning their interaction and diverse tissue microenvironment, indicate an impairment of the autophagy flux in primary liver tumors that is critical for the promotion of tumorigenesis process and a coexistence of autophagy inhibition and activation mechanisms in secondary liver tumors. Differences in mTOR and LC3 transcripts emerged in tumor-free tissues, therefore particular attention should be considered in selecting the control group., Competing Interests: The authors have declared that no competing interests exist.

The histo-pathologic and molecular mechanisms leading to initiation and progression of hepatocellular carcinoma (HCC) are still ill-defined; however, there is increasing evidence that the gradual accumulation of mutations, genetic and epigenetic changes which occur in preneoplastic hepatocytes results in the development of dysplastic foci, nodules, and finally, overt HCC. As well as many other neoplasias, liver cancer is considered an "inflammatory cancer", arising from a context of inflammation, and characterized by inflammation-related mechanisms that favor tumor cell survival, proliferation, and invasion. Molecular mechanisms that link inflammation and neoplasia have been widely investigated, and it has been well established that inflammatory cells recruited at these sites with ongoing inflammatory activity release chemokines that enhance the production of reactive oxygen species. The latter, in turn, probably have a major pathogenic role in the continuum starting from hepatitis followed by chronic inflammation, and ultimately leading to cancer. The relationship amongst chronic liver injury, free radical production, and development of HCC is explored in the present review, particularly in the light of the complex network that involves oxidative DNA damage, cytokine synthesis, telomere dysfunction, and microRNA regulation.

Background: Dysregulation of autophagy is important in the pathogenesis of many diseases, including cancer. Several aspects of the biological role of autophagy are however still unclear and the relationship between apoptosis and autophagy, particularly in the liver has yet to be thoroughly explored. In this study we evaluated the expression of Beclin 1 (one of the main autophagocytic agents, which bridges autophagy, apoptosis and both differentiation), and both pro- (Bad, Bax) and anti-apoptotic (Bcl-2, Bcl-xL) factors in liver samples from patients with different stages of liver disease., Methods: The study concerned 93 patients from 49 cases of chronic hepatitis (CH) (30 HCV and 19 HBV-related), 13 of cirrhosis (CIRR) (10 HCV and 3 HBV-related), 21 of hepatocellular carcinoma (both HCC and peritumoral tissues [PHCC]), and 10 controls (CONTR). Real-time PCR and Western blotting were used to measure mRNA and protein expression levels., Results: Beclin 1 mRNA levels were lower in HCC than in CH (P = 0.010) or CIRR (P = 0.011), and so were the Bcl-xL transcripts (P < 0.0001). Bad mRNA levels were higher in CH and CIRR than in CONTR, while Bax transcripts were increased in all tissues (P = 0.036). PHCC expressed the highest Bcl-2 mRNA levels. HBV-related CH tissues showed significantly higher Bcl-xL and Bad mRNA levels than HCV-related CH (P = 0.003 and P = 0.016, respectively)., Conclusions: High Beclin 1, Bcl-xL and Bad levels in CH and CIRR tissues suggest an interaction between autophagy and apoptosis in the early and intermediate stages of viral hepatitis. In HCC these processes seem to be downregulated, probably enabling the survival and growth of neoplastic hepatocytes.

Background: MicroRNAs expression has been extensively studied in hepatocellular carcinoma but little is known regarding the relationship, if any, with inflammation, production of reactive oxygen species (ROS), host's repair mechanisms and cell immortalization. This study aimed at assessing the extent of oxidative DNA damage (8-hydroxydeoxyguanosine - 8-OHdG) in different phases of the carcinogenetic process, in relation to DNA repair gene polymorphism, telomeric dysfunction and to the expression of several microRNAs, non-coding genes involved in post-transcriptional regulation, cell proliferation, differentiation and death., Methods: Tissue samples obtained either at surgery, [neoplastic (HCC) and adjacent non-cancerous cirrhotic tissues (NCCT)] at percutaneous or laparoscopic biopsy (patients with HCV or HBV-related hepatitis or patients undergoing cholecystectomy) were analysed for 8-OHdG (HPLC-ED), OGG1 (a DNA repair gene) polymorphism (PCR-RFLP), telomerase activity, telomere length (T/S, by RT-PCR), Taqman microRNA assay and Bad/Bax mRNA (RT-PCR). Fifty-eight samples from 29 HCC patients (obtained in both neoplastic and peritumoral tissues), 22 from chronic hepatitis (CH) and 10 controls (cholecystectomy patients - CON) were examined., Results: Eight-OHdG levels were significantly higher in HCC and NCCT than in CH and CON (p=0.001). Telomerase activity was significantly higher in HCC than in the remaining subgroups (p=0.002); conversely T/S was significantly lower in HCC (p=0.05). MiR-199a-b, -195, -122, -92a and -145 were down-regulated in the majority of HCCs while miR-222 was up-regulated. A positive correlation was observed among 8-OHdG levels, disease stage, telomerase activity, OGG1 polymorphisms and ALT/GGT levels. In HCC, miR-92 expression correlated positively with telomerase activity, 8-OHdG levels and Bad/Bax mRNA., Conclusions: The above findings confirm the accumulation, in the progression of chronic liver damage to HCC, of a ROS-mediated oxidative DNA damage, and suggest that this correlates with induction of telomerase activity and, as a novel finding, with over-expression of miR-92, a microRNA that plays a role in both the apoptotic process and in cellular proliferation pathways.

The Fas / Fas-ligand (FasL) system is an important death signal pathway in the liver. An enhanced local inflammatory response prompted by FasL expression, which contributes to neutrophil recruitment and interleukin-1 beta (IL-1beta) release, seems to be crucial to chronic liver damage, persistence of viral infections, and probably initiation and / or promotion of HCC. In order to evaluate the expression of Fas, FasL, and IL-1beta in different stages of human liver disease and to determine whether hepatitis B virus (HBV) and hepatitis C virus (HCV) infections modulate their expression, also in relation to apoptosis, we examined 87 liver samples obtained from patients with: chronic hepatitis (CH) (n.42), cirrhosis (n.9) and hepatocellular carcinoma (HCC) (n.16) and corresponding peritumoural tissues (n.16); histologically-normal liver (n.4) as controls. Fas, FasL and IL-1beta mRNA were quantified using reverse transcriptase-polymerase chain reaction. The apoptotic index was evaluated by TUNEL analysis. Our data showed a progressive Fas / FasL increase from CH to cirrhosis followed by a decline from the latter to HCC. In histological sections apoptosis was detected in HCC. A significant difference emerged between HCV and HBV-related disease for IL-1beta expression only in CH. A significant positive correlation between IL-1beta and FasL in HCV-related disease (P = 0.014) and an inverse correlation between IL-1beta and Fas in HBV-related disease (P = 0.021) were observed. The different pattern of IL-1beta, Fas and FasL expression found in HCV- and HBV-mediated liver disease, points to a different modulation of immune response B and C virus induced, while the decline in Fas / FasL expression in HCC may be related to defence mechanisms adopted by HCC cells against the immune system.

Epidemiological evidence clearly identifies chronic infection with hepatitis C virus (HCV) as a major risk factor for the development of hepatocellular carcinoma (HCC). Among the mechanisms that have been implicated in the pro-carcinogenic effect of HCV infection, an increased production of reactive oxygen species in the liver seems to have a major pathogenetic role in leading from chronic inflammation to cancer. Recent data have also demonstrated that HCV is capable of inducing this active production of free radicals per se, not just through inflammation, a feature peculiar to this virus and the specific activity of its core protein. This paper provides an overview of the inter-relationships between HCV, liver damage, free radical production and HCC, describing at least in part the complex network involving DNA oxidative damage, cytokine synthesis, proto-oncogene activation and oestrogen receptor expression, that may all be deeply involved in liver carcinogenesis.

Background: Kupffer cells, monocytes and infiltrating T cells have been considered the major source of interleukin-1beta and tumour necrosis factor-alpha in the liver. AIMS; To explore the expression of interleukin-1beta and tumour necrosis factor-alpha and to evaluate the density and the distribution of T lymphocytes and monocytes/macrophages in the liver of patients with primary and secondary tumours., Methods: Tumoural and peritumoural liver samples were examined from 21 patients with hepatocellular carcinoma, 10 with hepatic metastases, 5 with benign focal liver lesions and 4 healthy adult livers. Interleukin-1beta and tumour necrosis factor-alpha mRNAs were detected by a semiquantitative comparative reverse transcriptase polymerase chain reaction. T lymphocytes and monocytes/macrophages were detected by immunohistochemistry., Results: Higher levels of interleukin-1beta, tumour necrosis factor-alpha, CD3+ and CD68+ cells were found in the tissue surrounding hepatocellular carcinoma and metastases than in the tumour itself. A strong expression of CD68+ and CD3+ cells was found mainly along the tumour-host interface but the highest expression of CD3+ cells was found at the metastasis interfaces. Interleukin-1beta expression, CD3+ and CD68+ cell densities were higher in peritumoural samples than in so-called "normal" liver tissue., Conclusions: An increased production of interleukin-1beta and, to a lesser extent, of tumour necrosis factor-alpha mRNA coincides with the presence of cancer be it primary or secondary, both in healthy and cirrhotic livers. The presence of cancer, irrespective of the presence of underlying liver damage, appears to play the most important role.