The effect of azacitidine on interleukin-6 signaling and nuclear factor-kappaB activation and its in vitro and in vivo activity against multiple myeloma.
BACKGROUND
Azacitidine is a DNA methyltransferase inhibitor and cytotoxic agent known to induce apoptosis of some cancer cells. This study evaluated the pre-clinical potential of azacitidine as a therapeutic agent for multiple myeloma.


METHODS
Dose responsiveness to azacitidine was determined utilizing a panel of genetically heterogenous human multiple myeloma cell lines. Azacitidine was also tested against primary multiple myeloma cells and in the 5T33MM murine model of systemic myelomatosis. Mechanistic studies included immunoblotting of key apoptosis signaling proteins, analysis of p16 gene methylation status, and characterization of both the interleukin-6 and nuclear factor-kappaB signaling pathways following azacitidine treatment.


RESULTS
Human myeloma cell lines and primary multiple myeloma cells underwent apoptosis following exposure to clinically achievable concentrations of azacitidine (1 microM-20 microM). Similarly, azacitidine prolonged survival from 24.5 days to 32 days (p=0.001, log rank) in the 5T33MM model. At a mechanistic level azacitidine down-regulated two crucial cell survival pathways in multiple myeloma. First, it inhibited the elaboration of both interleukin-6 receptor-alpha and interleukin- 6 resulting in the reduced expression of both phospho-STAT3 and Bcl-xl. Secondly, azacitidine inhibited both nuclear factor-kappaB nuclear translocation and DNA binding in a manner independent of IkappaB. The kinetics of these azacitidine-induced responses was more consistent with protein synthesis inhibition than with either hypomethylation or another DNA-mediated effect.


CONCLUSIONS
Azacitidine rapidly induces apoptosis of multiple myeloma cells, is effective in vivo against multiple myeloma and inhibits two crucial cell survival pathways in this disease. We conclude that azacitidine demonstrates novel and highly relevant anti-myeloma effects and warrants further evaluation in a clinical context.
Khong
Tiffany
T
Myeloma Research Group, Department of Clinical Haematology and Bone Marrow Transplantation, Alfred Hospital, Melbourne, Victoria, Australia.
Sharkey
Janelle
J
Spencer
Andrew
A
D021581:Active Transport, Cell Nucleus; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis; D001374:Azacitidine / Q000494:pharmacology*; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D066298:In Vitro Techniques; D015850:Interleukin-6 / Q000378:metabolism*; D009101:Multiple Myeloma / Q000188:drug therapy*; D016328:NF-kappa B / Q000378:metabolism*; D010766:Phosphorylation; D050796:STAT3 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction; D013778:Tetrazolium Salts / Q000494:pharmacology; D051020:bcl-X Protein / Q000378:metabolism
D000964:Antimetabolites, Antineoplastic; D015850:Interleukin-6; D016328:NF-kappa B; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D013778:Tetrazolium Salts; D051020:bcl-X Protein; D001374:Azacitidine