The effects of antioxidants on radiation-induced apoptosis pathways in TK6 cells.
This study was designed to determine if radiation-mediated activation of the apoptotic pathways would be influenced by antioxidants and if a correlation would be found between radioprotection and changes in transduction pathways. Human lymphoblastoid TK6 cells, known to undergo apoptosis as a result of radiation, were irradiated (6 Gy) with and without antioxidants, and then whole-cell lysates were collected. Parallel studies were conducted to assess the survival (clonogenic assay) and apoptotic index. The impacts of two nitroxide antioxidants, tempol and CAT-1, differing in cell permeability, as well as the sulfhydryl antioxidant N-acetyl-L-cysteine (L-NAC), were estimated. Changes in apoptotic pathway proteins and p53 were assessed by Western blotting. Fraction of apoptotic cells was determined by flow cytometry. Tempol (10 mM), which readily enters cells, partially radioprotected TK6 cells against clonogenic killing, but had no effect on radiation-induced apoptotic parameters such as cleaved caspase 3 or cleaved PARP. Tempol alone did not induce cytotoxicity, yet did increase cleaved PARP levels. The radiation-induced increase in p53 protein was partly inhibited by tempol, but was unaffected by CAT-1 and L-NAC. Both CAT-1 (10 mM), which does not enter cells, and L-NAC (10 mM) had no radioprotective effect on cell survival. Although L-NAC did not protect against radiation-induced cytotoxicity, it completely inhibited radiation-induced increase in cleaved caspase 3 and cleaved PARP. Collectively, the results question the validity of using selected apoptosis pathway members as sole indicators of cytotoxicity.
Samuni
Ayelet M
AM
Radiation Biology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-1002, USA.
DeGraff
William
W
Cook
John A
JA
Krishna
Murali C
MC
Russo
Angelo
A
Mitchell
James B
JB
D000111:Acetylcysteine / Q000494:pharmacology*; D000975:Antioxidants / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D024161:Collagen Type XI / Q000378:metabolism; D003497:Cyclic N-Oxides / Q000494:pharmacology*; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects / Q000528:radiation effects; D011827:Radiation; D013113:Spin Labels; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D000975:Antioxidants; C499319:COL11A2 protein, human; D024161:Collagen Type XI; D003497:Cyclic N-Oxides; D013113:Spin Labels; D016159:Tumor Suppressor Protein p53; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; C001803:tempol; D000111:Acetylcysteine