The effects of antioxidants on radiation-induced apoptosis pathways in TK6 cells.
The effects of antioxidants on radiation-induced apoptosis pathways in TK6 cells.
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DOI：
10.1016/j.freeradbiomed.2004.08.006
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发表时间：
2004-11
影响因子：
7.4
通讯作者：
A. Samuni;W. Degraff;J. Cook;M. Krishna;A. Russo;James B. Mitchell
中科院分区：
文献类型：
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作者：
A. Samuni;W. Degraff;J. Cook;M. Krishna;A. Russo;James B. Mitchell
本研究旨在确定辐射介导的凋亡途径的激活是否会受到抗氧化剂的影响，以及是否会发现辐射防护和转导途径变化之间的相关性。已知由于辐射而经历细胞凋亡的人淋巴母细胞TK 6细胞在有和没有抗氧化剂的情况下被辐射（6戈伊），然后收集全细胞裂解物。进行平行研究以评估存活（克隆形成测定）和凋亡指数。两个氮氧抗氧化剂，tempol和CAT-1，不同的细胞渗透性，以及巯基抗氧化剂N-乙酰基-L-半胱氨酸（L-NAC）的影响，估计。通过Western印迹法评估凋亡途径蛋白和p53的变化。流式细胞仪检测凋亡细胞比例。Tempol（10 mM），它很容易进入细胞，部分辐射保护TK 6细胞对克隆杀伤，但没有影响辐射诱导的凋亡参数，如裂解半胱天冬酶3或裂解PARP。Tempol单独不诱导细胞毒性，但确实增加了裂解的PARP水平。Tempol可部分抑制辐射诱导的p53蛋白的增加，但CAT-1和L-NAC不影响辐射诱导的p53蛋白的增加。不进入细胞的CAT-1（10 mM）和L-NAC（10 mM）对细胞存活没有辐射保护作用。虽然L-NAC不能防止辐射诱导的细胞毒性，但它完全抑制了辐射诱导的裂解半胱天冬酶3和裂解PARP的增加。总的来说，结果质疑使用选定的细胞凋亡途径成员作为细胞毒性的唯一指标的有效性。
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.