Department of Nuclear Medicine, Samsung Medical Center, Seoul, Korea.
Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University School of Medicine, Seoul, Korea.
Sci Rep. 2019 Apr 23;9(1):6462. doi: 10.1038/s41598-019-42954-9.
Aldehyde dehydrogenase (ALDH) assays measure the accumulated fluorescence of enzyme products. However, cancer cells frequently co-express ALDH and ATP-binding cassette (ABC) transporters, which might mediate efflux of ALDH assay reagents. We demonstrate expression of active multidrug resistance protein1 (MDR1), multidrug resistance-associated protein (MRP), and breast cancer resistance protein (BCRP) in CT26 cancer cells as well as expression of MRP and BCRP in HT29 cancer cells. Without transporter inhibition, only small portions of both cell types were estimated to be ALDH-positive based on Aldefluor and AldeRed588 assays. However, MK-571 (MRP inhibitor) and novobiocin (BCRP inhibitor) substantially increased the rate of ALDH-positive CT26 cells based on either Aldefluor or AldeRed588 assays. Verapamil (MDR inhibitor) did not influence assay results. MK-571 also substantially increased the rate of ALDH-positive HT29 cells. Limiting dilution assays demonstrated greater numbers of tumor-spheres formed by Aldefluor-positive compared to -negative CT26 cells selected in the presence of MK-571 or novobiocin but not in their absence. These results reveal that Aldefluor and AldeRed588 products are efficient substrates for MRP- and BCRP-mediated efflux and substantially reduce estimated ALDH positivity rates in cancer cells. These findings demonstrate that complete blockade of these transporters is important to ensure accurate ALDH assay results and to develop newer assay techniques.
醛脱氢酶 (ALDH) 测定法测量酶产物的累积荧光。然而，癌细胞经常共表达 ALDH 和三磷酸腺苷结合盒 (ABC) 转运蛋白，这可能介导 ALDH 测定试剂的外排。我们证明 CT26 癌细胞中表达有活性的多药耐药蛋白 1 (MDR1)、多药耐药相关蛋白 (MRP) 和乳腺癌耐药蛋白 (BCRP)，HT29 癌细胞中表达有 MRP 和 BCRP。没有转运蛋白抑制时，根据 Aldefluor 和 AldeRed588 测定法，只有很小一部分这两种细胞类型被估计为 ALDH 阳性。然而，MK-571（MRP 抑制剂）和诺维本（BCRP 抑制剂）显著增加了基于 Aldefluor 或 AldeRed588 测定法的 CT26 细胞中 ALDH 阳性细胞的比率。维拉帕米（MDR 抑制剂）不影响测定结果。MK-571 也显著增加了基于 Aldefluor 的 HT29 细胞中 ALDH 阳性细胞的比率。有限稀释测定法表明，与未用 MK-571 或诺维本选择时相比，在用 MK-571 或诺维本选择时形成的 Aldefluor 阳性 CT26 细胞形成的肿瘤球数量更多。这些结果表明，AldeRed588 产物是 MRP 和 BCRP 介导的外排的有效底物，大大降低了癌细胞中估计的 ALDH 阳性率。这些发现表明，完全阻断这些转运蛋白对于确保准确的 ALDH 测定结果和开发新的测定技术非常重要。
