Reduced 293T cell susceptibility to acrolein due to aldose reductase-like-1 protein expression.
Acrolein is a highly reactive alpha,beta-unsaturated aldehyde produced endogenously during lipid peroxidation and naturally distributed pervasively in living environments, posing serious threats to human health if not properly metabolized. In this study, we report aldose reductase-like-1 (ARL-1) as a novel enzyme that catalyzes the reduction of acrolein and protects cells from their toxicity. Using purified ARL-1 protein, we determined its enzymatic activity in response to acrolein and defined its steady-state kinetics with K(m) and V(max) at 0.110 +/- 0.012 mM and 3122.0 +/- 64.7 nmol/mg protein/min, respectively. By introducing a functional Enhanced Green Fluorescent Protein (EGFP)/ARL-1 fusion protein into 293T cells, we demonstrated that plating efficiency in liquid culture and focus formation in soft agar increased by more than 60% (p < 0.05), compared to the vector control cells. More significantly, at a low dose of 5 microM acrolein, EGFP/ARL-1 expression enhanced both plating efficiency and focus formation by more than threefold, and the foci (in soft agar) of 293T cells expressing EGFP/ARL-1 were significantly larger than those of the vector control cells. At high concentrations of acrolein (25 and 50 microM), EGFP/ARL-1 protein prevented oncotic death of 293T cells induced by acrolein. In summary, our data demonstrated for the first time that the ARL-1 protein protects 293T cells from acrolein toxicity. Due to the high toxicity and wide distribution of acrolein, this finding is important to the understanding of its detoxification mechanisms.
Zu
Xuyu
X
Department of Medical Microbiology, Immunology, and Cell Biology, SimmonsCooper Cancer Institute, Southern Illinois University School of Medicine, Springfield, IL 62702, USA.
Yan
Ruilan
R
Robbins
Sarah
S
Krishack
Paulette A
PA
Liao
Duan-Fang
DF
Cao
Deliang
D
D000171:Acrolein / Q000633:toxicity*; D000449:Aldehyde Reductase / Q000096:biosynthesis* / Q000235:genetics; D000074408:Aldo-Keto Reductases; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D003470:Culture Media; D053938:DNA Fragmentation / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D003470:Culture Media; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D000171:Acrolein; C120471:AKR1B10 protein, human; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase; D007770:L-Lactate Dehydrogenase