NF-kB protects RAT ARL-6 hepatocellular carcinoma cells against hydrogen peroxide-induced apoptosis

Abstract

Background and Aims: Refractoriness of some turnouts to apoptosis has been related to over-expression of NF-kB, but NF-kB inhibition call also promote apoptosis in several cell types. We compared NF-kB activation profiles between normal rat hepatocytes and ARL-6 tat hepatocellular carcinoma (HCC) cells exposed to hydrogen peroxide (H202). We then examined whether NF-kB activation could explain tile observed resistance of ARL-6 cells to HAOA-induced apoptosis by blocking NF-kB activation with adenovirus-mediated mutant (non-degradable) IkBa (mlkB a). We also studied tile effects of NF-kI3 on cell proliferation. Methods: Hepatocytes is isolated from male Wistar rats by collagenase perfusion were established in primary culture in DMEM. ARL-6 cells were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum. Inhibition of NF-kB was achieved by infecting cultured ARL-6 cells with mlkBa (or b-sat for controls) for 24h. Infection efficiency was determined by X-gut staining. Cytotoxicity was determined by WST-1 assay. NF-!d3 translocation was determined by Western blot and iraraunofluorescence microscopy, and NF-kB DNA binding by electrophoretic mobility shift, assay (EMSA); cell proliferation was by [H3]- thymidine incorporation. Apoptosis was detected by H-33342 fluorescent microscopy, scanning electron microscopy, and TUNEL assay. Protein expression was measured by Western blotting. Results: At low concentrations, H;O; induced more apoptosis in primary hepatocytes than in ARL-6 cells, and was associated with greater NF-kB activation. In ARL-6 cells, nuclear translocation of NF-kB took place within 2h of administering HAO 2 and remained prominent at 36h. Inhibition of NF-kB by rnIkBa sensitized ARL-6 cells to HAOA-induced apoptosis, but cell proliferation was minimally suppressed. Conelusious: Hepatocytes are exquisitely sensitive to HAOA-induced apoptosis and relatively insensitive to NF-kB activation. In contrast, ARL-6 cells are refractory to apoptosis after exposure to low doses of H202, and this is associated with NF-kB activation. Sustained NF-kB activation in these HCC cells may protect them against apoptosis produced by physiological levels of oxidative stress. Conversely, NF-kB inhibition sensitises ARL-6 cells to oxidative stress-induced apoptosis.