Ο ρόλος της σωματοστατίνης και άλλων νευροπεπτιδίων στο ηπατοκυτταρικό καρκίνωμα
Hepatocellular carcinoma (HCC) represents more than 5% of all cancers and the estimated annual number of cases exceeds 500,000. It is also estimated to be the seventh most common cancer in men and the ninth in women. Patients with HCC tumours that are inoperable have an unfavourable natural course despite various therapeutic modalities. Such patients should be included in prospective investigations aiming to identify therapies that ultimately may lead to a survival improvement. Recently, a randomized controlled trial of octreotide in the treatment of advanced HCC has shown a significant survival benefit in the treated patients. The results of the present thesis provide data in regard to the role of the somatostatin and opioid systems in hepatocellular carcinoma. HepG2 cells were found to express mRNA for the subtypes sst2, sst3 and sst5 of the somatostatin receptors (ssts). Immunocytochemistry verified the presence of these receptors on HepG2 cells and sst2 was found to be of the sst2A variant. Ssts were found to be located mainly intracellularly in HepG2 cells and the pattern of distribution of the three subtypes was unique and characteristic for each one of them, implying different mechanisms of regulation of each subtype. Binding assays led to the characterization of functional receptors in the membranes of HepG2 cells that belong mainly in the sst3 and sst5 subtypes while minimal sst2 binding sites were detected. Octreotide was found to bind specifically to the ssts present on HepG2 cells membranes. Furthermore octreotide was found to inhibit the proliferation of HepG2 cells in a dose dependent manner and in concentrations that can be achieved in the blood of patients receiving octeotide for the treatment of HCC. Inhibition of the Protein Tyrosine Phosphatases pathway (PTPs) blocked the antiproliferative effect of octreotide in a dosedependent manner. The mainly intracellular localization of ssts in HepG2 cells led to the suspicion that this cell line produces a somatostatinergic peptide which regulates ssts in an autocrine manner. HepG2 cells were found to express and produce cortistatin (CST) but not somatostatin (SRIF). Incubation of HepG2 cells with additional CST did not have an effect in their proliferation while extracellular binding of CST with the use of a specific antibody led to a significant inhibition of cellular proliferation. The opioids EKC, aS1-casomorphin, DADLE and DAGO were also found to inhibit the proliferation of HepG2 cells. EKC and aS1-casomorphin were the most effective opioid agonists. The antiproliferative effect of EKC could not be blocked by the general opioid inhibitor diproenorphine. HepG2 cells were found to express mRNA for the cloned receptor KOR (_1) while no DOR (!2) or MOR (μ1 and μ2) mRNA transcripts were detected. However no _1 functional binding sites were detected on HepG2 cells. Furthermore probable !1 binding sites were detected but the presence _2 and _3 binding sites cannot be excluded. The inability of diproenorphine to block the antiproliferative effect of EKC in combination with presence of only low affinity opioid receptors in HepG2 cells, led us to the study of possible interaction of EKC with ssts. EKC was found to bind specifically on the ssts present on HepG2 cells. It is therefore possible that EKC exerts its antiproliferative effect via these receptors. Inhibition of PTPs, like in the case of octreotide, blocked EKCs effect on cellular proliferation. Octreotide and opioids were found to induce the production of nitric oxide metabolites from HepG2 cells, but this induction was not related to the antiproliferative effect of these substances. In conclusion the results of the present study are in accordance with those of the in vivo trial of octreotide for the treatment of HCC. They also characterize PTPs as a key intracellular pathway for the regulation of octreotide's antiproliferative effect. CST production and its possible role in the autocrine regulation of ssts in HepG2 cells, is an interesting result that gives further insight on the role of this natural peptide on the biology of ssts. Moreover in this study we report for the first time a method for the quantification of CST production. Finally opioids were found to have antiproliferative effects on HepG2 cells and in particular EKC which was also found to act via binding to ssts and activating the PTPs pathway.