Robson C, Wright K A, Twentyman P R, Lambert P A, Griffin R J
Department of Chemistry, University of Newcastle, Newcastle upon Tyne, UK.
Biochem Pharmacol. 1998 Oct 1;56(7):807-16. doi: 10.1016/s0006-2952(98)00068-9.
We have synthesised a series of fluorescent analogues of methylbenzoprim, a diaminopyrimidine antifolate which we have previously shown to exhibit in vivo antitumour activity in a methotrexate (MTX) "transport-resistant" tumour cell line. The analogues bear the dansyl, nitrobenzoxodiazole or methoxycoumarin fluorophores. The cytotoxicity of the compounds was evaluated using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay against two human lung cancer cell lines, together with their multidrug resistant (MDR) sublines. H69/P is a small cell line and its multidrug resistant subline H69/LX4 overexpresses P-glycoprotein (Pgp). COR-L23/P is a large cell line and its multidrug resistant subline COR-L23/R overexpresses the multidrug resistance associated protein (MRP). IC50 values for the compounds (i.e. concentration to reduce cell growth by 50%) in the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay ranged from 0.20 to 0.81 microM in the H69 parental line and from 0.83 to 5.10 microM in the COR-L23 parent line. The MDR sublines both showed clear cross-resistance to each of the compounds, with resistance factors (ratio of IC50 value in resistant vs parental cell line) ranging from 16 to 137 in H69/LX4 and from 5 to 16 in COR-L23/R. For compounds (10) and (11) where drug accumulation was studied using flow cytometry, resistance was associated with an approximately 10-fold reduction in cellular drug accumulation over a period of 30 min. The drug resistance modifiers verapamil (used at 6.6 microM) and cyclosporin A (used at 4.2 microM) were tested for their ability to sensitise the resistant lines. Whereas verapamil showed little activity, cyclosporin A partially restored the activity of compound (10), and fully restored the activity of compound (11) in H69/LX4 cells. This sensitisation of H69/LX4 by cyclosporin A was associated with a partial restoration of the drug accumulation deficit in this line. Hence, these novel lipophilic antifolates appear to be substrates for both the P-glycoprotein and MRP resistance mechanisms. Therefore, although they have been designed to overcome one mechanism of methotrexate resistance, namely impaired drug transport, this has been achieved only at the cost of rendering them susceptible to alternative mechanisms.
我们合成了一系列甲基苯并普明的荧光类似物，甲基苯并普明是一种二氨基嘧啶抗叶酸剂，我们之前已证明其在甲氨蝶呤（MTX）“转运抗性”肿瘤细胞系中具有体内抗肿瘤活性。这些类似物带有丹磺酰基、硝基苯并恶二唑或甲氧基香豆素荧光团。使用3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四氮唑溴盐（MTT）比色法评估了这些化合物对两种人肺癌细胞系及其多药耐药（MDR）亚系的细胞毒性。H69/P是一种小细胞系，其多药耐药亚系H69/LX4过表达P-糖蛋白（Pgp）。COR-L23/P是一种大细胞系，其多药耐药亚系COR-L23/R过表达多药耐药相关蛋白（MRP）。在MTT试验中，这些化合物的IC50值（即使细胞生长减少50%的浓度）在H69亲代细胞系中为0.20至0.81微摩尔，在COR-L23亲代细胞系中为0.83至5.10微摩尔。两个MDR亚系对每种化合物均表现出明显的交叉耐药性，H69/LX4中的耐药因子（耐药细胞系与亲代细胞系IC50值之比）为16至137，COR-L23/R中的耐药因子为5至16。对于使用流式细胞术研究药物积累的化合物（10）和（11），耐药性与30分钟内细胞药物积累减少约10倍有关。测试了耐药性调节剂维拉帕米（使用浓度为6.6微摩尔）和环孢素A（使用浓度为4.2微摩尔）使耐药细胞系敏感化的能力。虽然维拉帕米几乎没有活性，但环孢素A部分恢复了化合物（10）的活性，并完全恢复了化合物（11）在H69/LX4细胞中的活性。环孢素A对H69/LX4的这种敏感化与该细胞系中药物积累缺陷的部分恢复有关。因此，这些新型亲脂性抗叶酸剂似乎是P-糖蛋白和MRP耐药机制的底物。所以，尽管它们的设计目的是克服甲氨蝶呤耐药的一种机制，即药物转运受损，但这是以使其易受其他机制影响为代价实现的。
