The role of glutathione in resistance to cisplatin in a human small cell lung cancer cell line

C Meijer,Ege De Vries,Dra Uges,Gap Hospers,Nh Mulder

doi:10.1038/bjc.1990.232

Abstract

The role of glutathione (GSH) in resistance to cisplatin (CDDP) was studied in a human small cell lung carcinoma cell line (GLC4) and a CDDP-resistant subline (GLC4-CDDP). In addition to studying the steady state of GSH, the kinetics of this defence system were also studied via the monitoring of the GSH status of the cells under continuous pressure of CDDP. GLC4-CDDP maintained its elevated GSH level whereas GLC4 (under pressure of CDDP) quickly synthesised GSH to about twice its initial level, corresponding with 80% of the GSH level of GLC4-CDDP. D,L-buthionine-S,R-sulphoximine (BSO) was used to analyse the role of GSH in resistance to CDDP. Pretreatment with BSO (48 h, 50 microM, GSH not detectable) increased the CDDP-induced cytotoxicity 2.8-fold in GLC4-CDDP and 1.7-fold in GLC4. In GLC4 no changes in the amount of platinum (Pt) bound to DNA could be observed after GSH depletion. Changes in formation of interstrand cross-links or the main Pt-containing intrastrand cross-link in digested DNA, the Pt-GG adduct, were also not observed. In GSH depleted GLC4-CDDP cells, an increase in the amount of Pt bound to DNA and in the Pt-GG adduct was observed. Pretreatment with BSO substantially reduced the repair of Pt bound to DNA in both cell lines. We conclude that an increased GSH level and GSH synthesis capacity were demonstrated in CDDP resistant cells. The observations after BSO treatment suggest two roles for GSH in CDDP resistance, namely that of a cytosolic elimination resulting in less DNA platination and a nuclear effect on the formation and repair of DNA platinum adducts.

Highlights

Dose modifying factors (DMF) were calculated at the ICIO, IC20, ..., IC90 for both cell lines to confirm a significantly higher BSO modulating effect on the CDDP induced cytotoxicity in GLC4-CDDP compared to GLC4
Given the sulphydryl reactive properties of CDDP, alterations in cellular thiol content may be a defence mechanism generated in CDDP resistant cells
When BSO is used to deplete GSH levels, the effect on CDDP induced cytotoxicity varied in the different studies

Summary

Methods

RPMI 1640 medium was obtained from Gibco, Paisley, UK. Fetal calf serum was obtained from Flow Lab, Irvine, UK. GLC4, a human small cell lung cancer cell line and a CDDP resistant subline, GLC4-CDDP, characterised before by Hospers et al (1988), were cultured in RPMI 1640 medium with 10% heat-inactivated fetal calf serum at 37°C, 5% CO2. GLC4-CDDP was cultured under constant challenge of a monthly dose of 75 lg ml' CDDP. During the period in which the experiments for this study were performed, doubling times were 16 h and 28 h for GLC4 and GLC4-CDDP, respectively. Total GSH levels were 4.6 ± 0.5 and 11.5 ± 2.4 ytg mg-' protein, respectively in GLC4 and GLC4CDDP (mean ± s.d.)

Results

Discussion

Conclusion