Iron chelator-induced growth arrest and cytochrome c-dependent apoptosis in immortalized and malignant oral keratinocytes.
BACKGROUND
Many studies have shown the anti-proliferative effects of iron deprivation on cancer cells, but the effects of iron-chelators on oral cancer have not been clearly elucidated.


METHODS
To investigate the effects of an iron chelator, desferrioxamine (DFO), on the growth of immortalized human oral keratinocytes (IHOK), primary oral cancer cells (HN4), metastatic oral cancer cells (HN12) and human skin keratinocytes (HaCaT) in the MTT assay, three-dimensional (3D) raft cultures, Western blotting, cell cycle analysis, nuclear staining and cytochrome c expression for apoptosis signaling pathway were used.


RESULTS
Desferrioxamine inhibited the growth of immortalized IHOK and HaCaT and malignant HN4 and HN12 keratinocytes in a time- and dose-dependent manner according to the MTT assay. The 3D organotypic culture also revealed that DFO-treated cells showed less epithelial maturation, less surface keratinization and decreased epithelial thickness. The major mechanism of growth inhibition with the micromolar DFO treatment was by the induction of apoptosis, which was supported by nuclear DAPI staining, DNA fragmentation analysis and flow cytometric analysis for sub-G(1) phase arrest and Annexin V-FITC (fluorescein isothiocyanate) staining. Furthermore, Bax expression increased together with p53 and p21(WAF1/CIP1), while the Bcl-2 expression decreased in the immortalized and malignant keratinocytes treated with DFO. Time-dependent cytochrome c from mitochondria was observed in DFO-treated IHOK and oral cancer cells and was accompanied by the activation of caspase-3 in IHOK cells.


CONCLUSIONS
These results demonstrate that DFO has growth inhibitory effects on immortalized and malignant oral keratinocytes through the induction of apoptosis and suggest that further evaluation of DFO as a potential therapeutic agent for human oral precancerous lesions is warranted.
Lee
Sun-Kyung
SK
Department of Oral and Maxillofacial Pathology, College of Dentistry, Wonkwang University, Iksan, Korea.
Lee
Jae-Jung
JJ
Lee
Hwa-Jeong
HJ
Lee
Jun
J
Jeon
Byung-Hun
BH
Jun
Chang-Duk
CD
Lee
Suk-Keun
SK
Kim
Eun-Cheol
EC
D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000502:physiology; D015153:Blotting, Western; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects*; D002461:Cell Line, Transformed / Q000187:drug effects; D045744:Cell Line, Tumor / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects*; D045304:Cytochromes c / Q000032:analysis / Q000502:physiology; D003676:Deferoxamine / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D005434:Flow Cytometry; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects*; D009062:Mouth Neoplasms / Q000473:pathology; D017262:Siderophores / Q000494:pharmacology*; D012867:Skin / Q000166:cytology / Q000187:drug effects
D051017:Apoptosis Regulatory Proteins; D061945:Caspase Inhibitors; D017262:Siderophores; D045304:Cytochromes c; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003676:Deferoxamine