Characterization of the enhanced apoptotic response to azidothymidine by pharmacological inhibition of NF-kB.
OBJECTIVE
The present study addresses the issue of enhanced apoptotic response to AZT following co-treatment with an NF-kB inhibitor.


METHODS
To investigate this issue, different cell lines were assayed for susceptibility to AZT-mediated apoptosis without or with the addition of the NF-kB inhibitor Bay-11-7085. For further investigation, U937 cells were 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 the mitochondrial 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.


RESULTS
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.


CONCLUSIONS
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.
Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata", Rome, Italy.
Claudia
C
Matteucci
Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata", Rome, Italy.
Antonella
A
Minutolo
Department of Biological and Environmental Sciences, University of Messina, Messina, Italy.
Francesca
F
Marino-Merlo
Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata", Rome, Italy.
Sandro
S
Grelli
Department of Systems Medicine, University of Rome "Tor Vergata", Rome, Italy.
Caterina
C
Frezza
Department of Biological and Environmental Sciences, University of Messina, Messina, Italy; The Institute of Translational Pharmacology, CNR, Rome, Italy. Electronic address: antonio.mastino@unime.it.
Antonio
A
Mastino
Department of Systems Medicine, University of Rome "Tor Vergata", Rome, Italy.
Beatrice
B
Macchi
D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000096:biosynthesis / Q000235:genetics; D045304:Cytochromes c / Q000378:metabolism; D019254:Genes, bcl-2 / Q000187:drug effects; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D009570:Nitriles / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D013450:Sulfones / Q000494:pharmacology; D051996:Transcription Factor RelA / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics; D014407:Tumor Cells, Cultured; D020298:U937 Cells; D015215:Zidovudine / Q000494:pharmacology*
D051017:Apoptosis Regulatory Proteins; C416282:BAY 11-7085; D016328:NF-kappa B; D009570:Nitriles; D018894:Reverse Transcriptase Inhibitors; D013450:Sulfones; D051996:Transcription Factor RelA; D015215:Zidovudine; D045304:Cytochromes c
AZT; Apoptosis; Bcl-2; Chemotherapy; NF-kB