triacsin-c and Liver-Neoplasms

Free fatty acids (FFAs) are proposed to play a pathogenic role in both peripheral and hepatic insulin resistance. We have examined the effect of saturated FFA on insulin signalling (100 nM) in two hepatocyte cell lines. Fao hepatoma cells were treated with physiological concentrations of sodium palmitate (0.25 mM) (16:0) for 0.25-48 h. Palmitate decreased insulin receptor (IR) protein and mRNA expression in a dose- and time-dependent manner (35% decrease at 12 h). Palmitate also reduced insulin-stimulated IR and IRS-2 tyrosine phosphorylation, IRS-2-associated PI 3-kinase activity, and phosphorylation of Akt, p70 S6 kinase, GSK-3 and FOXO1A. Palmitate also inhibited insulin action in hepatocytes derived from wild-type IR (+/+) mice, but was ineffective in IR-deficient (-/-) cells. The effects of palmitate were reversed by triacsin C, an inhibitor of fatty acyl CoA synthases, indicating that palmitoyl CoA ester formation is critical. Neither the non-metabolized bromopalmitate alone nor the medium chain fatty acid octanoate (8:0) produced similar effects. However, the CPT-1 inhibitor (+/-)-etomoxir and bromopalmitate (in molar excess) reversed the effects of palmitate. Thus, the inhibition of insulin signalling by palmitate in hepatoma cells is dependent upon oxidation of fatty acyl-CoA species and requires intact insulin receptor expression.

Topics: Aminoimidazole Carboxamide; Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Enzyme Activation; Enzyme Inhibitors; Epoxy Compounds; Extracellular Signal-Regulated MAP Kinases; Fatty Acids; Fatty Acids, Nonesterified; Forkhead Transcription Factors; Glycogen Synthase Kinase 3; Hypoglycemic Agents; Insulin; Insulin Receptor Substrate Proteins; Liver; Liver Neoplasms; Mice; Mice, Knockout; Oxidation-Reduction; p38 Mitogen-Activated Protein Kinases; Palmitic Acid; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Receptor, Insulin; Ribonucleotides; Signal Transduction; Triazenes

In this study, we detected the expression of FACL4 mRNA in 40 patients with hepatic carcinoma and its adjacent normal tissues by semi-quantitative RT-PCR. The changes of proliferation and apoptosis of hepatic cancer cell line HepG2 with FACL4 protein expression were examined by MTT and flow cytometry respectively after FACL4 selective inhibitor triacsin C treatment. The activity related to apoptosis of proteinases, caspase-3, caspase-8 and caspase-9, were detected by colorimetry. The expression related to apoptosis of protein, wt-p53, Bax and Bcl-2, in HepG2 cells were evaluated by S-P immunocytochemical dyeing. The results were: (1) FACL4 mRNA was expressed in 95.0% of hepatic cancer tissue, while the positive expression of FACL4 mRNA was 82.5% in cancer adjacent normal liver tissues. Moreover, there was a statistically significant increased in quantity of FACL4 mRNA in cancer tissues compared with adjacent normal liver tissues. (2) The concentration of triacsin C (0.5-2 mg/L) could inhibit the proliferation and induce the apoptosis of HepG2 cells significantly in a dose- and time-effect. (3) During the apoptosis of HepG2 cells induced by triacsin C, flow cytometry coupled with Rhodamine 123 dyeing showed that mitochondrial transmembrane potential of HepG2 declined significantly, and the activity of caspase-9 and caspase-3 increased more remarkably than caspase-8. Besides, the increased apoptosis was accompanied by increased Bax, and decreased wtp53 and Bcl-2 protein levels. The present study suggested that FACL4 might play a role in the growth of hepatic cancer cells. FACL4 selective inhibitor triacsin C leads to a marked growth inhibition of human liver tumor cells, based on the inhibition of proliferation and induction of apoptosis. The apoptotic process was mediated by intrinsic mitochondrial apoptotic pathway due to activation of caspase-9 and caspase-3. The increased apoptosis was accompanied by upregulation of Bax, and decreased wt-p53 and Bcl-2 protein level.

Topics: Apoptosis; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Coenzyme A Ligases; Flow Cytometry; Humans; Liver Neoplasms; Membrane Potential, Mitochondrial; RNA, Messenger; Triazenes

Fatty acid-CoA ligase 4 (FACL4) is a central enzyme controlling the unesterified free arachidonic acid (AA) level in cells and the free AA is known to induce apoptosis. We have recently reported that expression of FACL4 is upregulated in about 40% of human hepatocellular carcinoma (HCC) and 50% of HCC cell lines, suggesting that FACL4 may be involved in liver carcinogenesis. In this study, we investigated whether HCC cell growth is regulated by FACL4. Immunoblot analysis showed that SNU 398 cells express very low or no detectable level of FACL4. We, therefore, transfected the SNU 398 cells with FACL4 expression vector, and clones expressing FACL4 were pooled and analyzed. We found that forced expression of FACL4 in SNU 398 promotes the growth of cells. In addition, we observed that triacsin C, a FACL4 inhibitor, inhibits the growth of Hep 3B cell line which expresses high level of endogenous FACL4. We also found that the triacsin C-mediated growth inhibition in Hep 3B cells results from the induction of apoptosis with evidence of Bcl-2 reduction. Altogether, our data show that FACL4 affects HCC cell growth and suggest that modulation of FACL4 expression/activity is an approach for treatment of HCC.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Coenzyme A Ligases; Humans; Liver Neoplasms; Proto-Oncogene Proteins c-bcl-2; Triazenes