Upregulation of cellular glutathione levels in human ABCB5- and murine Abcb5-transfected cells.
Upregulation of cellular glutathione levels in human ABCB5- and murine Abcb5-transfected cells.
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DOI：
10.1186/s40360-015-0038-5
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发表时间：
2015-12-15
影响因子：
2.9
通讯作者：
Sugimoto Y
中科院分区：
文献类型：
--
作者：
Kondo S;Hongama K;Hanaya K;Yoshida R;Kawanobe T;Katayama K;Noguchi K;Sugimoto Y
以前，我们已经证明，人ABCB 5是一个完整的大小ATP结合盒转运，与ABCB 1/P-糖蛋白有很强的同源性。ABCB 5转染细胞表现出对紫杉烷类和蒽环类抗生素的抗性。在此，我们进一步筛选ABCB 5的底物，并探讨其耐药机制。使用细胞生长抑制测定评价细胞对测试化合物的敏感性。使用HPLC和基于酶的测定来测量丁硫氨酸亚砜亚胺（BSO）、谷胱甘肽和氨基酸的细胞水平。谷胱甘肽的细胞和囊泡运输进行了评价放射性标记的底物。通过RT-PCR评估谷胱甘肽代谢酶的表达水平。人ABCB 5转染的293/B5-11细胞和鼠Abcb 5转染的293/mb 5 -8细胞对BSO的抗性分别比模拟转染的293/mock细胞高6.5倍和14倍。BSO是γ-谷氨酰半胱氨酸连接酶（GCL）的抑制剂，GCL是谷胱甘肽合成的关键酶。293/B5-11和293/mb 5 -8细胞对另一种GCL抑制剂甲硫氨酸亚砜亚胺也表现出耐药性。细胞摄取实验显示，BSO在293/B5-11和293/mb 5 -8细胞中的蓄积与293/mock细胞中的蓄积相似，表明BSO不是ABCB 5底物。293/B5-11和293/mb 5 -8细胞的谷胱甘肽含量显著高于293/mock细胞。BSO对细胞谷胱甘肽含量影响的评价显示，与293/模拟细胞相比，293/B5-11和293/mb 5 -8细胞中谷胱甘肽含量降低50%所需的BSO浓度约高2- 3倍。该结果表明，ABCB 5和Abcb 5转染细胞的BSO抗性可归因于BSO对转染子的作用降低。细胞和囊泡转运试验表明，放射性标记的谷胱甘肽在293/B5-11细胞中的转运与在293/mock细胞中的转运相似。293/B5-11细胞中谷胱甘肽代谢酶编码基因的mRNA表达与293/mock细胞相似。293/B5-11和293/mb 5 -8细胞中谷胱甘肽前体Glu的含量高于293/mock细胞。ABCB 5/Abcb 5转染的细胞显示出对BSO的抗性，BSO不是ABCB 5的底物。我们的研究结果表明，ABCB 5/Abcb 5上调细胞谷胱甘肽水平，以保护细胞免受各种毒物的侵害。
Previously, we have demonstrated that human ABCB5 is a full-sized ATP-binding cassette transporter that shares strong homology with ABCB1/P-glycoprotein. ABCB5-transfected cells showed resistance to taxanes and anthracyclines. Herein, we further screened ABCB5 substrates, and explored the mechanism of resistance. Sensitivity of the cells to test compounds was evaluated using cell growth inhibition assay. Cellular levels of buthionine sulfoximine (BSO), glutathione and amino acids were measured using HPLC and an enzyme-based assay. Cellular and vesicular transport of glutathione was evaluated by a radiolabeled substrate. Expression levels of glutathione-metabolizing enzymes were assessed by RT-PCR. Human ABCB5-transfected 293/B5-11 cells and murine Abcb5-transfected 293/mb5-8 cells showed 6.5- and 14-fold higher resistance to BSO than the mock-transfected 293/mock cells, respectively. BSO is an inhibitor of gamma-glutamylcysteine ligase (GCL), which is a key enzyme of glutathione synthesis. 293/B5-11 and 293/mb5-8 cells also showed resistance to methionine sulfoximine, another GCL inhibitor. A cellular uptake experiment revealed that BSO accumulation in 293/B5-11 and 293/mb5-8 cells was similar to that in 293/mock cells, suggesting that BSO is not an ABCB5 substrate. The cellular glutathione content in 293/B5-11 and 293/mb5-8 cells was significantly higher than that in 293/mock cells. Evaluation of the BSO effect on the cellular glutathione content showed that compared with 293/mock cells the BSO concentration required for a 50 % reduction in glutathione content in 293/B5-11 and 293/mb5-8 cells was approximately 2- to 3-fold higher. This result suggests that the BSO resistance of the ABCB5- and Abcb5-transfected cells can be attributed to the reduced effect of BSO on the transfectants. Cellular and vesicular transport assays showed that the transport of radiolabeled glutathione in 293/B5-11 cells was similar to that in 293/mock cells. The mRNA expression of genes encoding glutathione-metabolizing enzymes in 293/B5-11 cells was similar to that in 293/mock cells. The cellular content of Glu, a precursor of glutathione, in 293/B5-11 and 293/mb5-8 cells was higher than that in 293/mock cells. ABCB5/Abcb5-transfected cells showed resistance to BSO, which is not a substrate of ABCB5. Our results suggest that ABCB5/Abcb5 upregulates cellular glutathione levels to protect cells from various poisons.