Cisplatin efflux, binding and intracellular pH in the HTB56 human lung adenocarcinoma cell line and the E-8/0.7 cisplatin-resistant variant
Cisplatin efflux, binding and intracellular pH in the HTB56 human lung adenocarcinoma cell line and the E-8/0.7 cisplatin-resistant variant
复制标题
DOI：
10.1007/s002800050967
复制
发表时间：
1999-09-01
影响因子：
3
通讯作者：
Stewart, DJ
中科院分区：
文献类型：
--
作者：
Chau, Q;Stewart, DJ
目的：许多对顺铂（DDP）耐药的细胞系减少了DDP的积累。我们假设耐药细胞中DDP积累减少可能是由于细胞内DDP结合减少，导致被动外排增加。方法：比较HTB56人肺腺癌细胞株及其获得DDP耐药的E-8/0.7变异株的总细胞（[T-DDP]）、细胞内超滤（[F-DDP]）和可沉淀细胞结合（[B-DDP]） DDP浓度。细胞暴露于509 μ M DDP中20 min。超滤与500分子量截止分离细胞游离顺铂结合。超声破碎和微离心纺丝沉淀细胞结合顺铂。采用流式细胞术测定HTB56细胞系、E-8/0.7细胞系、OV2008细胞系及其C13耐药变异株的细胞内pH值（pH(i)）。我们还比较了dna结合的DDP和蛋白质结合的DDP ([P-DDP])，当敏感细胞和抗性细胞分别暴露于509 / 911 μ M DDP和111 / 666 μ M DDP两对DDP浓度下1小时获得相同的[T-DDP]时。采用无焰原子吸收分光光度法测定铂。结果：在顺铂暴露时间0时，敏感的HTB56亲本细胞系[T-DDP]和[B-DDP]显著升高（P < 0.02和P < 0.001），而[F-DDP]无显著差异（P = 0.62）。观察到两个不同阶段的T-DDP外排。在DDP暴露后的前10 s，抗性细胞（K-R1）的速率常数为0.17 s(-1)，敏感细胞（K-S1）的速率常数为0.14 s（-1）。从10 s到50 s， K-R2和K-S2分别变为0.005 s（-1）和0.004 s。在10、30和50 s时，耐药细胞的T-DDP仍低于敏感细胞（均P < 0.0001）。509 μ M顺铂浓度与911 μ M顺铂浓度在敏感和耐药细胞系中可提供相当的[T-DDP]，暴露1小时后，只有[dna结合的DDP]在敏感细胞中显著升高（P = 0.002），而[F-DDP]和[P-DDP]没有显著差异（P = 0.18, P = 0.75）。另一方面，当使用111 μ M DDP与666 μ M DDP时，两种细胞系之间的[F-DDP]， [P-DDP]和[dna结合DDP]无显著差异。流式细胞术数据显示，与敏感亲本细胞系相比，E-8/0.7 （P < 0.0186）和C13 （P < 0.0169）耐药变异体的pHi显著升高。结论：尽管游离药物量相当，但DDP在耐药肺癌细胞中的结合速度比敏感肺癌细胞慢。耐药型的早期外排较高。细胞系之间DNA结合的差异可能与DDP浓度有关。我们推测耐药系早期结合减少和早期外排增加可能与耐药系较高的pH有关。较高的pH值被认为有利于中性羟基代谢物的产生，而不是带电的水代谢物，这些中性代谢物预计不太容易与细胞内分子反应，更容易穿过细胞膜流出。由于我们之前已经记录了人类HTB56肺癌细胞系耐ddp变体中葡萄糖利用率和乳酸产量增加了三倍，并且这种增加的乳酸产量预计会降低而不是提高细胞内pH值，因此我们的耐碱性细胞可能具有更高的Na+/H+交换活性，从而保护它们免受细胞内酸化。
Purpose: Many cell lines resistant to cisplatin (DDP) have reduced DDP accumulation. We postulated that reduced accumulation of DDP in resistant cells might be due to decreased intracellular DDP binding, leading to increased passive efflux. Methods: The total cellular ([T-DDP]), intracellular ultrafiltrable ([F-DDP]) and precipitable cellular bound ([B-DDP]) DDP concentrations were all compared in the HTB56 human lung adenocarcinoma cell line and its E-8/0.7 variant that has acquired DDP resistance. Cells were exposed to 509 mu M DDP for 20 min. Ultrafiltration with a 500 molecular weight cut-off separated cellular free from bound cisplatin. Fragmentation by sonication and microcentrifugal spinning precipitated cellular bound cisplatin. Flow cytometry was used to measure the intracellular pH (pH(i)) of the HTB56 cell line, the E-8/0.7 cell line, as well as of the OV2008 cell line and its C13 resistant variant. The DNA-bound DDP and protein-bound DDP ([P-DDP]) were also compared when equal [T-DDP] was achieved for both sensitive and resistant cells by exposing them for 1 h to two pairs of DDP concentrations, i.e. 509 vs 911 mu M DDP, and 111 vs 666 mu M DDP, respectively. Platinum was assayed by flameless atomic absorption spectrophotometry. Results: At time 0 tend of cisplatin exposure), [T-DDP] and [B-DDP] were significantly higher in the sensitive HTB56 parent cell line (P < 0.02 and P < 0.001, respectively), whereas [F-DDP] did not differ significantly (P = 0.62). Two distinct phases of T-DDP efflux were observed. In the first 10 s after DDP exposure, the rate constant for resistant cells (K-R1) was 0.17 s(-1), whereas that for sensitive cells (K-S1) was 0.14 s(-1). From 10 s to 50 s, however, K-R2 and K-S2 became 0.005 s(-1) and 0.004 s, respectively. [T-DDP] remained lower in resistant cells than in sensitive cells at 10, 30 and 50 s (all P < 0.0001). For 1 h drug exposure to 509 vs 911 mu M cisplatin concentrations designed to give comparable [T-DDP] in the sensitive and resistant cell lines, only [DNA-bound DDP] was found to be significantly higher in sensitive cells (P = 0.002), whereas both [F-DDP] and [P-DDP] did not differ significantly (P = 0.18, P = 0.75, respectively). On the other hand, there were no significant differences found in [F-DDP], [P-DDP] and [DNA-bound DDP] between the two cell lines when 111 vs 666 mu M DDP was used. Flow cytometry data indicated that the pHi was significantly higher in the E-8/0.7 (P < 0.0186) and C13 (P < 0.0169) resistant variants than in the sensitive parent cell lines. Conclusions: DDP binds more slowly in resistant than in sensitive lung cancer cells, despite comparable amounts of free drug. Early efflux is higher in the resistant variant. Differences between the lines with respect to DNA binding may be DDP concentration-dependent. We speculate that the reduced early binding and increased early efflux in the resistant line may be related to the higher pH in this line. A higher pH is supposed to favor production of neutral hydroxyl metabolites rather than charged aquated metabolites, and these neutral metabolites would be expected to react less readily with intracellular molecules and to efflux more readily across cell membranes.Since we have previously documented a threefold increase in glucose utilization and lactate production in the DDP-resistant variants of the human HTB56 lung cancer cell lines, and this increased lactate production would have been expected to reduce the intracellular pH instead of raising it, it is possible that our alkaline-resistant cells have a higher Na+/H+ exchanger activity which would protect them from intracellular acidification.