Meccanismi estrinseci ed intrinseci del processo apoptotico nell'epatocarcinogenesi

The mechanisms underlying the strict correlation between viral infection, cirrhosis and HCC development have been investigated in deep, but, despite this several aspects are still largely unknown or understood only in part. Hepatitis B and C viruses seem to be involved in neoplastic transformation through both direct and indirect mechanisms, due to inflammation process and the capacity of viral proteins in modulating the proliferation and the death of infected hepatocytes. Moreover it has been hypothesized that dysregulation of apoptotic process in infected cells could be a crucial step for the cronicity of infection, in the failure to antiviral treatment and in the neoplastic transformation. Although apoptosis can be triggered by several different stimuli, apoptotic signalling within the cell is transduced mainly via two defined molecular pathways: the death receptor pathways, also called extrinsic pathway (Fas/FasL, TNF-?/TNF-R1-R2) and the mitochondrial pathway also called the intrinsic pathway (Bcl-2, Bax, Bad). Other plasma membrane receptors such as IL-1b and TGF-b1 and intrinsic mediators as BI-1 and NF-kB are involved in the apoptotic pathways. In this study mRNA expression of mediators of the extrinsic and intrinsic apoptotic process was evaluated. The liver biopsies and the surgical hepatic tissues obtained from patients with different degree of liver disease were used. mRNA transcripts were detected by gene amplifications techniques. on the basis of our results human hepatocytes cultures were performed. Primary hepatocytes were treated with TNF-a, Staurosporine and LY294002, protein-kinase inhibitors, in order to evaluate the activation and/or the inhibition of these mediators in the apoptotic process. Aims In the first part the study was performed in order to: 1) evaluate the mRNA expression of Fas/FasL, TNF-?, IL-1?, TGFß-1, Bcl-2, Bax, Bad, BI-1 and NF-kB in liver tissues from patients with different degrees of liver damage, from chronic hepatitis, to cirrhosis and hepatocellular carcinoma. 2) determine whether HCV and HBV infection modulates their expression 3) analyze the protein expression of the mediators identified by Western Blot, in liver tissues from patients with HCC the second part included an "in vitro" study in order to: 4) culture primary human hepatocytes 5) evaluate the different apoptotic pathways in human hepatocytes after stimulation with: a) TNF-?, mainly proinflammatory cytokine found in abundance in the inflammatory process relating to liver injury b) Staurosporine (STS), a potent protein tyrosine/serine kinase inhibitor with an antiproliferative and anti-apoptotic activity in many tumoral cells c) LY294002 synthetic inhibitor of PI3K (phosphoinositide 3-kinase) Material and methods We examined 62 patients: 39 with chronic hepatitis (CH), 7 with cirrhosis (CIRR), 13 with hepatocellular carcinoma HCC) and 3 controls (Contr). The Fas/FasL, IL-ß and TNF-? mRNAs were quantified by Semiquantitative RT-PCR, while the Bcl-2, Bax, Bad, BI-1, NF-kB and TGFß-1 mRNAs were quantified by quantitative Real Time PCR, using ß-actin as the housekeeping gene. The protein expression of NF-kB in tissues with HCC was analyzed by Western Blot. Results Tissues: Fas/FasL mRNA expression: Fas mRNA levels were significantly increased in CIRR when compared with HCC (p=0.026) while FasL mRNAs showed a statistically significant differences between CIRR and cirrhotic tissues surrounding tumours (PHCC) (p=0.018) or HCC (p=0.005). IL-1ß mRNA expression: PHCC tissues had higher IL-1b levels than either CH tissues (p<0,0001) or CIRR tissues (p=0,005). TNF-? mRNA expression: TNF-ß mRNAs were increased in CIRR tissues compared with PHCC (p=0,038) and HCC tissues (p=0,05). TGFß-1 mRNA expression: In CH tissues, TGFß-1 mRNA expression was significantly lower than in HCC (p=0,014) or PHCC tissues (p=0,028). Bcl-2 mRNA expression: Bcl-2 mRNA levels were statistically higher in PHCC when compared with normal liver tissues (p=0,026), with CH (p=0,004), and with CIRR (p=0,05). BI-1 mRNA expression: CH tissues expressed the highest BI-1 mRNA amount that was significantly higher when compared to cirrhotic tissues (p=0,033), cirrhotic tissues surrounding tumours (p<0,001) and HCC (p<0,001). Bad mRNA expression: Contr tissues showed the highest Bad transcript levels, which were statistically higher than in CH (p=0,006), PHCC (p=0,005), or HCC (p=0,002). CH coincided with higher Bad levels than in PHCC or HCC tissues (p=0,00006 and p=0,0007, respectively) Bax mRNA expression: No differences in Bax expression were observed. NF-kB mRNA expression: CONTR expressed the lowest NF-kB mRNA amount, significantly lower when compared with CH (p=0.05) and PHCC (p=0.05). HBV HCV infection: In CH, only TNF-?, IL-1ß and Bad mRNA expression were significantly different in HBV-related infection versus HCV-related infection. Human hepatocytes: TNF-? treatment: Human hepatocytes incubated with TNF-? showed the highest levels of FasL (solo a 12h), IL-1ß(until 24h), TGFß-1 (24 e 48h), Bcl-2 (4h, 24h), Bad (24 and 48h) and NF-kB (8h) respect to unstimulated cells. No differences in Fas, Bax, Bad (until 12h) , NF-kB (until 4h) e TGFß-1 (until 12h) mRNA levels were observed. Staurosporine (STS) treatment: In cells treated with STS, Bcl-2 mRNA expression until 48h of culture, was significantly lower than in unstimulated cells. No difference in TGFß-1, Bax, and NF-kB mRNAs levels were observed at the different time of incubation. While an increase in Bad mRNA at 12h and in BI-1 mRNA at 8 and 24h were observed. LY294002 treatment: LY294002 induce a strong inhibition of Bcl-2 mRNA (until 24h) in hepatocyte culture when compared with hepatocyte culture with medium alone. No difference in TGFß-1, Bax, and NF-kB mRNAs levels were observed while high levels of Bad mRNA at 12, 24 and 48h and of BI-1 from 8 to 24h of incubation were observed. Pre-treatment with STS and LY294002 : The pre-treatment of human hepatocytes for 1h with LY294002 and the following treatment with TNF-? inhibits the Bcl-2 mRNA expression until 48h when compared with cell treated with TNF-? without the pre-treatment. This inhibition was completely resolved after 48h of incubation. In cells pretreated with LY294002 respect to the cells treated with TNF-? alone, were observed the highest levels of Bax after 4h, Bad after 24h e BI-1 after 24h of incubation. No difference in TGFß-1 and NF-kB mRNAs expression were observed In the cells pre-treated with STS only the BI-1 transcript levels were increased until 24h. TISSUES Fas/FasL system expression is upregulated in chronically-damaged livers while the onset of cancer is associated with a shut-down of this system in HCC, as a defense mechanisms adopted by HCC against the immune system. High levels of IL-1ß mRNA transcripts observed in cirrhotic tissues surrounding tumours, may be the result of an increased activation of lymphocytes o monocytes in the liver. Contrary, the low expression of TNF-? mRNA transcripts in cirrhotic tissues surrounding tumours and HCC may be the result of loss function of Kupffer cells, one of the main sources of production of TNF-? in the liver. The Bcl-2 hyperexpression observed in cirrhotic peritumoral tissues seems to play an important part in regulating cell survival in damaged liver cells, which may contribute to the persistence of HBV or HCV and thus activate cell proliferation and cell transformation. The upregulation of the Bad-induced pro-apoptotic pathway in CH seems to be more relevant in controlling apoptosis in liver tissues with HBV infection suggesting an important role of the proteins of HBV particularly in early liver diseases. Moreover the BI-1 expression decrease correlates closely with the progression of liver damage from CH to cirrhosis and HCC and indicate a role of BI-1 as regulator of apoptosis in early diseases. The high BI-1 mRNA levels observed in CH may provide protection against apoptosis of liver cells virus infected, on the other hand the down regulation of BI-1 observed in HCC tissues seems to favour the hapatocellular carcinogenesis. The persistently elevated levels of NF-kB in all stage of liver diseases, seems to depends on a chronic inflammatory with an consequently increased risk of hepatic fibrosis and of an HCC milieu. The different pattern of IL-1ß, TNF-? and Bad expression in HCV- and HBV-related liver disease points to a different modulation of the immune response on the one hand, and to a different apoptotic activation pathways on the other. HUMAN HEPATOCYTES IL-1ß and TNF-? are proinflammatory cytokines always secreted during inflammation. The high IL-1ß mRNA transcripts induced by TNF-? stimulation in primary human hepatocytes suggest that the presence of TNF-? in the hepatic microenvironment contributes to the increase in IL-1ß with a consequent intensification of inflammatory, proliferative and regenerative processes. No difference in Fas/FasL mRNAs expression was observed after TNF-? treatment, suggesting that the expression of these mediators was not modulated by the extracellular TNF-?  factor in our experimental conditions. Bcl-2 is an antiapoptotic member of the intrinsic Bcl-2 family and its activation by TNF-? might explain the resistance to apoptotic process induced by TNF-? also observed in mouse and rat hepatocyte cultures. Pre-treatment and treatment with STS, LY924002 and TNF-? reduce the Bcl-2 transcript levels by comparison with the levels obtained from cells treated with TNF-? alone. These effects were particularly evident using LY924002. STS is considered a scarcely-selective kinase inhibitor known for its toxicity, while LY924002 represents a highly specific PI3K inhibitor. Bax and Bad are pro-apoptotic members of the BCl-2 family. TNF-?, STS and LY294002 had no effect on Bax expression in human hepatocytes compared with hepatocytes incubated with medium alone, whereas Bad mRNA levels increased after stimulation with TNF-? STS and LY294002, particularly after 12 to 48 hours of incubation. Costimulation with LY294002 and TNF-? increases the Bad mRNAs after 24h and 48h of incubation. So, when the antiapoptotic pathway (Bcl-2) was inhibited in primary human hepatocytes, members of the apoptotic pathway (Bad and Bax) were activated.