| Abstract |
Tumor necrosis factor (TNF), a pleiotropic cytokine involved in a broad spectrum of inflammatory and immune responses, exerts its biological effects by engaging two distinct cell surface receptors, the 55-kD TNFR1 and the 75-kD TNFR2. Both TNF Receptors (TNFRs) have the ability to mediate cell death via various mechanisms. TNFR1 stimulation may also result in inhibition of apoptosis through induction of the transcription factor NF-kappaB, specifically the heterodimer p50/p65. In response to TNF, p65 undergoes several post-translational modifications, including phosphorylation on serines 536 and 468, and translocates to the nucleus, where it typically mediates expression of many pro-survival genes. Depending on the cell type and the stimulus involved, NF-kappaB may also have a proapoptotic function.
TNF in liver is mainly produced by Kupffer cells (KC), the resident liver macrophages, and has a pivotal role in hepatic homeostasis and pathophysiology, since it holds the capacity to induce both hepatocyte cell death and proliferation. KC have been demonstrated to be implicated in the defence against the development of hepatic metastases from various extrahepatic tumors, but on the other hand, they may also be detrimental in the development of liver cancer.
Hepatocellular carcinoma (HCC), the main histological type of liver cancer, is a major cause of morbidity and mortality worldwide. Because of its innate resistance to the conventional treatment regiments, the investigation of new therapeutic agents is required. A large number of clinical and experimental data indicate that Octreotide, a potent synthetic somatostatin analogue with antiproliferative, antisecretory and immunomodulatory properties, may be a promising anti-cancer therapeutic agent. Octreotide has been reported to prolong survival in approximately 40% of
patients with unresectable hepatocellular carcinoma. Although the favourable findings have been disputed, these negative studies have been criticized for the selection of the participating patients.
Previous work in our laboratory has demonstrated that Octreotide has a direct antiproliferative effect on human hepatoma cells HepG2 and modulates the expression of pro- or antifibrotic agents, inflammatory mediators, chemokines and apoptosis related proteins in rat KC.
To further explore the mechanisms by which Octreotide exerts its antineoplastic actions, we evaluated its influence, at the clinically feasible concentration of 10-8 Μ, on TNFRs expression, NF-kappaB activation and apoptotic response of KC and HepG2/Hep3B human hepatoma cells to TNF.
Rat KC were isolated by centrifugal elutriation. TNFR1 and TNFR2 expression was studied by RT-PCR, quantitative PCR, Western Blot and immunofluorescence. TNF mRNA expression in KC was also assessed by semiquantitative PCR. Intracellular localization of NF-kappaB in HepG2/Hep3B was detected by immunofluorescent analysis and a cell-based ELISA assay was used to quantitate total and active (phosphorylated at serine 536 and serine 468) p65. Apoptosis was examined by a DNA nucleosomal fragmentation ELISA assay and TUNEL assay.
Our experiments show that TNFR1, TNFR2 and TNF mRNA are constitutively expressed in KC. Octreotide does not influence the expression of TNF but increases the expression of both receptors with a stronger effect on TNFR2.
TNF alone had no effect on KC apoptosis. Octreotide significantly increased their apoptosis and this effect might be a further mechanism of its antineoplastic activity in HCC. We hypothesize that this is possibly due to TNFR2 overexpression, while the fact that TNF co-stimulation slightly ameliorates this effect is possibly due to TNFR1 stimulation.
We also show that TNFR1 but no TNFR2 receptor was constitutively expressed in both HCC cell lines. Octreotide caused an early reduction of both mRNA and TNFR1 protein in HepG2 and a late reduction in Hep3B cells.
TNF induced NF-kappaB nuclear translocation, stronger in Hep3B, which was not influenced by Octreotide. TNF significantly increased p65 Ser536 and p65 Ser468 phosphorylation, more pronounced in Hep3B cells. Octreotide significantly decreased TNF-induced Ser536 phosphorylation only in HepG2 cells, whereas it caused a significant increase in p65 Ser468 phosphorylation in Hep3B only.
Octreotide or TNF alone had no effect on apoptosis in HepG2 cells, but TNF greatly increased apoptosis in Hep3B cells. Co-incubation with Octreotide and TNF increased apoptosis in HepG2 cells and decreased TNF-induced apoptosis in Hep3B cells. This differential effect of Octreotide on apoptosis may be due to its different effect on TNFR1 expression in the two cell lines and could 4
explain why not all patients with HCC do not respond to somatostatin treatment. The increase in p65 Ser468 phosphorylation that Octreotide caused in Hep3B could account for a slight increase in their apoptosis. Although TNF-induced NF-kappaB nuclear translocation in both HCC cell lines seems to be proapoptotic, phosphorylation seems to play a protective role, since in HepG2 only it was decreased by Octreotide and this was related to a substantial increase in TNF-induced apoptosis.
Our results indicate that Octreotide may indirectly act in HCC through TNF-induced apoptosis in Kupffer and HCC cells. Clearly further investigation is required to clarify this hypothesis and to completely elucidate the mechanisms by which Octreotide exerts its antineoplasmatic actions.
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