Cisplatin efflux, binding and intracellular pH in the HTB56 human lung adenocarcinoma cell line and the E-8/0.7 cisplatin-resistant variant.
OBJECTIVE
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 microM 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 (pHi) 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 microM DDP, and 111 vs 666 microM DDP, respectively. Platinum was assayed by flameless atomic absorption spectrophotometry.


RESULTS
At time 0 (end 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 (KR1) was 0.17 s(-1), whereas that for sensitive cells (KS1) was 0.14 s(-1). From 10 s to 50 s, however, KR2 and KS2 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 microM 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 microM 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.
Chau
Q
Q
Cancer Care Ontario, Ottawa Regional Cancer Center, Ontario, Canada.
Stewart
D J
DJ
D000230:Adenocarcinoma / Q000378:metabolism* / Q000473:pathology; D000970:Antineoplastic Agents / Q000378:metabolism / Q000493:pharmacokinetics*; D001692:Biological Transport; D002945:Cisplatin / Q000378:metabolism / Q000493:pharmacokinetics*; D019008:Drug Resistance, Neoplasm; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008175:Lung Neoplasms / Q000378:metabolism* / Q000473:pathology; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D002945:Cisplatin