Shrieve D C, Harris J W
Int J Radiat Oncol Biol Phys. 1986 Jul;12(7):1171-4. doi: 10.1016/0360-3016(86)90251-8.
The effects of depletion of cellular glutathione (GSH) on the sensitivity of cultured EMT6/SF cells to chemotherapy agents or x rays under hypoxic and aerated conditions were investigated. Buthionine sulfoximine (BSO), a potent inhibitor of the enzyme gamma-glutamyl-cysteine synthetase, was used to deplete cellular GSH. Addition of BSO (50 microM) to EMT6/SF cultures depleted cellular GSH with a half-time of approximately 2 hr. Cellular GSH reached very low levels within hours of addition of BSO. After removal of BSO, cellular GSH recovered with approximately the same kinetics as was seen for depletion. Incubation of EMT6/SF cells with BSO concentrations of up to 1 mM did not reduce the viability or inhibit growth when exposure was limited to times less than 24 hr. However, for longer exposure times, toxicity and growth inhibition were demonstrated in a dose dependent fashion. EMT6/SF cells were treated with chemotherapy agents under either aerated or extremely hypoxic conditions. Cells were more sensitive to cis-dichlorodiammino Pt(II) (DDP), mitomycin C (MitC), L-phenylalanine mustard (L-PAM), and nitrogen mustard (HN2) when treatment was under hypoxic conditions. The magnitude of this sensitization under hypoxic conditions ranged from a dose modifying factor (DMF) of 1.4 (HN2) to 4.1 (MitC), measured at the 0.1 level of cell survival. Hypoxic EMT6/SF cells were more resistant to the cytotoxic effects of actinomycin D (ActD) under hypoxic conditions (DMF = 10 at SF = 0.3). When cellular GSH was depleted to less than 5% of control by treatment with 50 microM BSO for 12-14 hr, cells were sensitized to DDP, L-PAM and HN2 under both aerated and hypoxic conditions. DMF's ranged from 1.4-6.5, depending on the agent. Hypoxic cell sensitization was never significantly greater than that seen in aerated cells, as was the case for X radiation (DMF = 1.3 for hypoxic cells only). GSH depletion also sensitized to MitC, but only under aerated conditions (DMF = 2.1). Hypoxic EMT6/SF cells were not sensitized to MitC by depletion of GSH. GSH depletion afforded slight protection against ActD toxicity under both aerated and hypoxic conditions. These studies suggest that cellular GSH plays an important role in modifying cellular response to cytotoxic drugs. GSH depletion may sensitize tumor cells to some chemotherapy agents, but differential sensitization of tumors compared to normal tissues, based on hypoxic tumor cells as targets, would not be expected based on these in vitro experiments.
研究了细胞内谷胱甘肽（GSH）耗竭对培养的EMT6/SF细胞在缺氧和通气条件下对化疗药物或X射线敏感性的影响。丁硫氨酸亚砜胺（BSO）是γ-谷氨酰半胱氨酸合成酶的有效抑制剂，用于耗竭细胞内GSH。向EMT6/SF培养物中添加BSO（50μM）可使细胞内GSH耗竭，半衰期约为2小时。添加BSO后数小时内，细胞内GSH水平降至非常低的水平。去除BSO后，细胞内GSH以与耗竭时大致相同的动力学恢复。当暴露时间限制在24小时以内时，用浓度高达1mM的BSO孵育EMT6/SF细胞不会降低细胞活力或抑制生长。然而，对于更长的暴露时间，毒性和生长抑制呈剂量依赖性。在通气或极度缺氧条件下，用化疗药物处理EMT6/SF细胞。当在缺氧条件下处理时，细胞对顺二氯二氨合铂（II）（DDP）、丝裂霉素C（MitC）、L-苯丙氨酸氮芥（L-PAM）和氮芥（HN2）更敏感。在缺氧条件下这种增敏作用的幅度范围为剂量修正因子（DMF）从1.4（HN2）到4.1（MitC），在细胞存活0.1水平下测量。缺氧的EMT6/SF细胞在缺氧条件下对放线菌素D（ActD）的细胞毒性作用更具抗性（在SF = 0.3时DMF = 10）。当用50μM BSO处理12 - 14小时使细胞内GSH耗竭至对照的5%以下时，细胞在通气和缺氧条件下对DDP、L-PAM和HN2均敏感。DMF范围为1.4 - 6.5，取决于药物。缺氧细胞的增敏作用从未显著大于通气细胞，X射线照射情况也是如此（仅缺氧细胞的DMF = 1.3）。GSH耗竭也使细胞对MitC敏感，但仅在通气条件下（DMF = 2.1）。缺氧的EMT6/SF细胞不会因GSH耗竭而对MitC敏感。在通气和缺氧条件下，GSH耗竭对ActD毒性均有轻微保护作用。这些研究表明，细胞内GSH在改变细胞对细胞毒性药物的反应中起重要作用。GSH耗竭可能使肿瘤细胞对某些化疗药物敏感，但基于这些体外实验，以缺氧肿瘤细胞为靶点，预计肿瘤与正常组织之间不会有差异增敏作用。
