Antioxidative function of L-FABP in L-FABP stably transfected Chang liver cells.
Liver fatty acid binding protein (L-FABP) contains amino acids that are known to possess antioxidant function. In this study, we tested the hypothesis that L-FABP may serve as an effective endogenous cytoprotectant against oxidative stress. Chang liver cells were selected as the experimental model because of their undetectable L-FABP mRNA level. Full-length L-FABP cDNA was subcloned into the mammalian expression vector pcDNA3.1 (pcDNA-FABP). Chang cells were stably transfected with pc-DNA-FABP or vector (pcDNA3.1) alone. Oxidative stress was induced by incubating cells with 400 micromol/L H2O2 or by subjecting cells to hypoxia/reoxygenation. Total cellular reactive oxygen species (ROS) was determined using the fluorescent probe DCF. Cellular damage induced by hypoxia/reoxygenation was assayed by lactate dehydrogenase (LDH) release. Expression of L-FABP was documented by regular reverse transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, and Western blot. The pcDNA-FABP-transfected cells expressed full-length L-FABP mRNA, which was absent from vector-transfected control cells. Western blot showed expression of 14-kd L-FABP protein in pcDNA-FABP-transfected cells, but not in vector-transfected cells. Transfected cells showed decreased DCF fluorescence intensity under oxidative stress (H2O2 and hypoxia/reoxygenation) conditions versus control in inverse proportion to the level of L-FABP expression. Lower LDH release was observed in the higher L-FABP-expressed cells in hypoxia/reoxygenation experiments. In conclusion, we successfully transfected and cloned a Chang liver cell line that expressed the L-FABP gene. The L-FABP-expressing cell line had a reduced intracellular ROS level versus control. This finding implies that L-FABP has a significant role in oxidative stress.
Wang
Guqi
G
Faculty of Pharmacy, University of Manitoba, Winnipeg, MB, Canada.
Gong
Yuewen
Y
Anderson
Judy
J
Sun
Dongfeng
D
Minuk
Gerald
G
Roberts
Michael S
MS
Burczynski
Frank J
FJ
D000975:Antioxidants / Q000378:metabolism*; D017209:Apoptosis; D006528:Carcinoma, Hepatocellular; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D045744:Cell Line, Tumor; D018076:DNA, Complementary; D050556:Fatty Acid-Binding Proteins; D005978:Glutathione / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D000860:Hypoxia / Q000378:metabolism; D008113:Liver Neoplasms; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D017382:Reactive Oxygen Species / Q000378:metabolism; D015427:Reperfusion Injury / Q000378:metabolism; D013482:Superoxide Dismutase / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D000975:Antioxidants; D002352:Carrier Proteins; D018076:DNA, Complementary; C493319:FABP1 protein, human; D050556:Fatty Acid-Binding Proteins; D016877:Oxidants; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase; D005978:Glutathione