NF-kB is activated in response to temozolomide and confers protection against drug-induced cell growth inhibition

We previously demonstrated that AKT is activated by the methylating agent temozolomide (TMZ) in mismatch repair (MMR)-proficient but not in MMR-deficient cells, and protects against drug-induced cytotoxicity. In this study we investigated whether activation of NF-kB occurs downstream of AKT in TMZ-treated cells. Moreover, we explored the role of NF-kB activation in cell response to the drug. The MMR-proficient melanoma cell line M10, the MMR-proficient colon carcinoma cell line HCT116/3-6 and its MMR-deficient counterpart HCT116 were incubated with 50 microM TMZ + 10 microM O6-benzylguanine (BG), to inhibit the repair of methyl adducts at O6-guanine. After 48 h of culture, phosphorylation of AKT on Ser473 and IkB-alfa degradation were evaluated by Western blotting, while NF-kB activation was determined using a dual-luciferase reporter assay. NF-kB activation was also assessed in MCF-7/KD12 and MCF-7/pUSE cell lines, generated by stable transfection of MMR-proficient MCF-7 cells with a vector encoding a dominant negative mutant form of AKT1, or with the empty vector, respectively. The levels of two transcriptional targets of NF-kB, i.e. IL-8 and MCP-1, were determined in control and TMZ-treated M10 cells. To assess the effect of NF-kB inhibition on cell sensitivity to TMZ, M10 and HCT116/3-6 cells were transfected with siRNAs directed against the p65 subunit of NF-kB and after 24 h incubated with graded concentrations of TMZ + BG. Cell proliferation was evaluated after 5 days of culture by the MTT assay. M10 cells were also exposed to 50 microM NEMO peptide (an inhibitor of NF-kB function) for 24 h and then tested for sensitivity to TMZ + BG as described above. TMZ treatment induced phosphorylation of AKT on Ser473, IkB-alfa degradation and NF-kB activation in M10 and HCT116/3-6 cells but not in HCT116 cells. NF-kB activation was impaired in MCF-7/KD12 cells. Increased secretion of IL-8 and MCP-1 was observed in TMZ-treated M10 cells as compared with control cells. Transfection of M10 and HCT116/3-6 cells with siRNAs against NF-kB significantly increased the growth suppressive effects of TMZ. Inhibition of NF-kB by NEMO peptide in M10 cells also potentiated the antiproliferative activity of TMZ. Our results demonstrate that NF-kB is activated in response to TMZ in a MMR- and AKT-dependent manner, and that pharmacological inhibition of NF-kB might represent an efficient strategy to increase melanoma cell sensitivity to TMZ.