Highly expressed N1-acetylpolyamine oxidase detoxifies polyamine analogue N1-cyclopropylmethyl-N11-ethylnorspermine in human lung cancer cell line A549.
BACKGROUND
The critical roles of polyamines in cell growth and differentiation have made polyamine metabolic pathway a promising target for antitumor therapy. Recent studies have demonstrated in vitro that some antitumor polyamine analogues could be used as substrates and oxidized by purified recombinant human N(1)-acetylpolyamine oxidase (APAO, an enzyme that catabolizes natural polyamines), indicating a potential role of APAO in determining the sensitivity of cancer cells to specific antitumor analogues. This study evaluated, in vivo, the effect of APAO on cytotoxicity of antitumor polyamine analogue, N(1)-cyclopropylmethyl-N(11)-ethylnorspermine (CPENS) and its mechanism when highly expressed in human lung cancer line A549.


METHODS
A clone with high expression of APAO was obtained by transfecting A549 lung cancer cell line with pcDNA3.1/APAO plasmid and selecting with quantitative realtime PCR and APAO activity assay. Cell proliferation was determined by MTT method and apoptosis related events were evaluated by DNA fragmentation, sub-G1/flow cytometric assay, western blotting (for cytochrome C and Bax) and colorimetric assay (for casapse-3 activity).


RESULTS
A clone highly expressing APAO was obtained. High expression of APAO in A549 cells inhibited accumulation of CPENS, decreased their sensitivity to the toxicity of CPENS and prevented CPENS induced apoptosis.


CONCLUSIONS
These results indicate a new drug resisting, mechanism in the tumor cells. High expression of APAO can greatly decrease the sensitivity of tumor cells to the specific polyamine analogues by detoxifying those analogues and prevent analogue induced apoptosis.
Han
Yu
Y
Three Gorges University Medical College, Three Gorges University Institute of Molecular Biology, Yichang, Hubei 443002, China.
Ren
Yu-San
YS
Cao
Chun-Yu
CY
Ren
Dong-Ming
DM
Zhou
Yong-Qin
YQ
Wang
Yan-Lin
YL
D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D005434:Flow Cytometry; D006801:Humans; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000235:genetics / Q000378:metabolism*; D011073:Polyamines / Q000378:metabolism* / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000097667:Polyamine Oxidase
D000587:Oxidoreductases Acting on CH-NH Group Donors; D011073:Polyamines; D000097667:Polyamine Oxidase; C501650:N(1)-acetylpolyamine oxidase, human