van Triest B, Pinedo H M, Telleman F, van der Wilt C L, Jansen G, Peters G J
Department of Medical Oncology, University Hospital Vrije Universiteit, Amsterdam, The Netherlands.
Biochem Pharmacol. 1997 Jun 15;53(12):1855-66. doi: 10.1016/s0006-2952(97)82448-3.
Resistance to some (lipophilic) antifolates has been associated with P-glycoprotein (P-gp)-mediated multidrug resistance (MDR). A possible relationship with non-P-gp MDR has not been established. We studied resistance to antifolates in SW-1573 human lung carcinoma cells, a P-gp overexpressing variant SW-1573/2R160 and a multidrug resistance protein (MRP) overexpressing variant SW-1573/2R120. In this study, thymidylate synthase (TS) inhibitors with different properties concerning the efficiency of membrane transport and the efficiency of polyglutamylation were tested for cross-resistance in SW-1573/2R120 and SW-1573/2R160 cells. Growth inhibition patterns in this cell line panel were measured by the Sulforhodamine B (SRB) assay. Resistance factors for TS inhibitors were: 2.4 and 0.4 for 5-fluorouracil (5FU), 18.8 and 8.8 for ZD1694, 17 and 0.7 for AG337, and 40 and 8.3 for BW1843U89 in SW-1573/2R160 and SW-1573/2R120, respectively. This study showed changes in the TS enzyme kinetics during the induction of doxorubicin resistance in both SW-1573 variants, resulting in 2-fold lower Km values for 2'-deoxyuridine-5'-monophosphate (dUMP) in both resistant variants compared to the parental cell line. TS activity, TS protein induction and TS mRNA expression all had 2-fold increased in the SW-1573/2R120 compared to the SW-1573/2R160. 3H-MTX influx was 2-fold lower in SW-1573/2R160 cells compared to SW-1573/2R120 and SW-1573 cells. In the SW-1573/2R160 cell line, an aberrant intracellular trafficking towards the target TS was observed, compared to SW-1573/2R120 and SW-1573 cells as measured by the TS in situ assay. The rate of TS inhibition by the TS inhibitors used in this study was similar in all cell lines. In conclusion, collateral sensitivity to 5FU and the lipophilic AG337 and cross-resistance to other antifolates were observed in non-P-gp MDR SW-1573/2R120 cells, as well as resistance to all antifolates in P-gp SW-1573/2R160 cells. The mechanism of resistance in SW-1573/2R160 cells possibly involves reduced influx and changes in intracellular trafficking routes. For the SW-1573/2R120 cell line, several changes related to the TS enzyme possibly play a role in the observed cross-resistance and collateral sensitivity pattern.
对某些（亲脂性）抗叶酸药物的耐药性与P-糖蛋白（P-gp）介导的多药耐药性（MDR）有关。与非P-gp介导的MDR之间的可能关系尚未明确。我们研究了人肺癌SW-1573细胞、P-gp过表达变体SW-1573/2R160以及多药耐药相关蛋白（MRP）过表达变体SW-1573/2R120对抗叶酸药物的耐药性。在本研究中，针对膜转运效率和多聚谷氨酰化效率具有不同特性的胸苷酸合成酶（TS）抑制剂，在SW-1573/2R120和SW-1573/2R160细胞中进行了交叉耐药性测试。通过磺酰罗丹明B（SRB）测定法测量该细胞系组中的生长抑制模式。TS抑制剂的耐药因子分别为：在SW-1573/2R160和SW-1573/2R120细胞中，5-氟尿嘧啶（5FU）的耐药因子分别为2.4和0.4，ZD1694的耐药因子分别为18.8和8.8，AG337的耐药因子分别为17和0.7，BW1843U89的耐药因子分别为40和8.3。本研究表明，在两种SW-1573变体中，阿霉素耐药诱导过程中TS酶动力学发生了变化，与亲代细胞系相比，两种耐药变体中2'-脱氧尿苷-5'-单磷酸（dUMP）的米氏常数（Km）值降低了2倍。与SW-1573/2R160相比，SW-1573/2R120中的TS活性、TS蛋白诱导和TS mRNA表达均增加了2倍。与SW-1573/2R120和SW-1573细胞相比，SW-1573/2R160细胞中3H-甲氨蝶呤（MTX）内流降低了2倍。通过TS原位测定法测量发现，与SW-1573/2R120和SW-1573细胞相比，在SW-1573/2R160细胞系中观察到向靶标TS的异常细胞内转运。本研究中使用的TS抑制剂对TS的抑制率在所有细胞系中相似。总之，在非P-gp MDR的SW-1573/2R120细胞中观察到对5FU和亲脂性AG337的间接敏感性以及对其他抗叶酸药物的交叉耐药性，以及在P-gp的SW-1573/2R160细胞中对所有抗叶酸药物的耐药性。SW-1573/2R160细胞中的耐药机制可能涉及内流减少和细胞内转运途径的改变。对于SW-1573/2R120细胞系，与TS酶相关的几种变化可能在观察到的交叉耐药性和间接敏感性模式中起作用。
