Cross-resistance to antifolates in multidrug resistant cell lines with P-glycoprotein or multidrug resistance protein expression

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.