Characterization of the enhanced apoptotic response to azidothymidine by pharmacological inhibition of NF-kB.

The Aims: The present study addresses the issue of enhanced apoptotic response to AZT following co-treatmentwith an NF-kB inhibitor. Main methods: To investigate this issue, different cell lines were assayed for susceptibility to AZT-mediated apoptosiswithout orwith the addition of theNF-kB inhibitor Bay-11-7085. For further investigation,U937 cellswere selected as good-responder cells to the combination treatment with 32 or 128 μM AZT, and 1 μM Bay-11-7085. Inhibition of NF-kB activation by Bay-11-7085 in cells treated with AZT was assayed through Western blot analysis of p65 expression and by EMSA. Involvement of themitochondrial pathway of apoptosis in mechanisms underlying the improved effect of AZT following Bay-11-7085 co-treatment, was evaluated by assaying the cytochrome c release and the mitochondrial membrane potential (MMP) status using the JC-1 dye. Moreover, the transcriptional activity of both anti- and pro-apoptotic genes in U937 cells after combination treatment was quantitatively evaluated through real-time PCR. Key findings: We found that the combined treatment induced high levels of cytochrome c release and of MMP collapse in association with evident changes in the expression of both anti- and pro-apoptotic genes of the Bcl-2 family. Overexpression of Bcl-2 significantly suppressed the sensitization of U937 cells to an enhanced apoptotic response to AZT following co-treatment with the NF-kB inhibitor. Significance: The new findings suggest that a combination regimen based on AZT plus an NF-kB inhibitor could represent a new chemotherapeutic tool for retrovirus-related pathologies.present study addresses the issue of enhanced apoptotic response to AZT following co-treatment with an NF-kB inhibitor.