Generation of oxygen deficiency in cell culture using a two-enzyme system to evaluate agents targeting hypoxic tumor cells.
Generation of oxygen deficiency in cell culture using a two-enzyme system to evaluate agents targeting hypoxic tumor cells.
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DOI：
10.1667/rr1431.1
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发表时间：
2008-11
影响因子：
3.4
通讯作者：
Sartorelli AC
中科院分区：
文献类型：
--
作者：
Baumann RP;Penketh PG;Seow HA;Shyam K;Sartorelli AC
实体瘤的不良且异常的血管化使它们容易受到局部缺氧区域的影响，这些区域可以被认为是肿瘤对前药敏感的部位，这些前药在低氧合条件下优先被激活为细胞毒性物质。为了方便地选择针对实体瘤中缺氧细胞的药物，我们开发了一种简单方便的双酶系统来在细胞培养物中产生缺氧。葡萄糖氧化酶通过选择性氧化葡萄糖并将分子氧还原为过氧化氢来消耗培养基中的氧气；过量的过氧化氢酶也用于清除过氧化物分子。即使在液体/空气界面存在氧气的情况下，培养基或缓冲液中也会发生快速且持续的氧气消耗。使用 CHO/AA8 中国仓鼠细胞、EMT6 鼠乳腺癌细胞和 U251 人神经胶质瘤细胞的研究表明，该系统产生缺氧，从而激活缺氧靶向前药 KS119。这种在细胞培养物中产生缺氧的方法价格低廉，不需要繁琐的设备，允许使用更长的孵育时间而不损失样品体积，并且应该适用于 96 孔板中的高通量筛选。
The poor and aberrant vascularization of solid tumors makes them susceptible to localized areas of oxygen deficiency that can be considered sites of tumor vulnerability to pro-drugs that are preferentially activated to cytotoxic species under conditions of low oxygenation. To readily facilitate the selection of agents targeted to oxygen-deficient cells in solid tumors, we have developed a simple and convenient two-enzyme system to generate oxygen deficiency in cell cultures. Glucose oxidase is employed to deplete oxygen from the medium by selectively oxidizing glucose and reducing molecular oxygen to hydrogen peroxide; an excess of catalase is also used to scavenge the peroxide molecules. Rapid and sustained depletion of oxygen occurs in medium or buffer, even in the presence of oxygen at the liquid/air interface. Studies using CHO/AA8 Chinese hamster cells, EMT6 murine mammary carcinoma cells, and U251 human glioma cells indicate that this system generates an oxygen deficiency that produces activation of the hypoxia-targeted prodrug KS119. This method of generating oxygen deficiency in cell culture is inexpensive, does not require cumbersome equipment, permits longer incubation times to be used without the loss of sample volume, and should be adaptable for high-throughput screening in 96-well plates.