Increased sensitivity to gemcitabine of P-glycoprotein and multidrug resistance-associated protein-overexpressing human cancer cell lines

Abstract

Gemcitabine (2',2'-difluorodeoxycytidine) is a deoxycytidine analogue that is activated by deoxycytidine kinase (dCK) to its monophosphate and subsequently to its triphosphate dFdCTP, which is incorporated into both RNA and DNA, leading to DNA damage. Multidrug resistance (MDR) is characterised by an overexpression of the membrane efflux pumps P-glycoprotein (P-gP) or multidrug resistance-associated protein (MRP). Gemcitabine was tested against human melanoma, non-small-cell lung cancer, small-cell lung cancer, epidermoid carcinoma and ovarian cancer cells with an MDR phenotype as a result of selection by drug exposure or by transfection with the mdr1 gene. These cell lines were nine- to 72-fold more sensitive to gemcitabine than their parental cell lines. The doxorubicin-resistant cells 2R120 (MRP1) and 2R160 (P-gP) were nine- and 28-fold more sensitive to gemcitabine than their parental SW1573 cells, respectively (P<0.01), which was completely reverted by 25 micro M verapamil. In 2R120 and 2R160 cells, dCK activities were seven- and four-fold higher than in SW1573, respectively, which was associated with an increased dCK mRNA and dCK protein. Inactivation by deoxycytidine deaminase was 2.9- and 2.2-fold decreased in 2R120 and 2R160, respectively. dFdCTP accumulation was similar in SW1573 and its MDR variants after 24 h exposure to 0.1 micro M gemcitabine, but dFdCTP was retained longer in 2R120 (P<0.001) and 2R160 (P<0.003) cells. 2R120 and 2R160 cells also incorporated four- and six-fold more [(3)H]gemcitabine into DNA (P<0.05), respectively. P-glycoprotein and MRP1 overexpression possibly caused a cellular stress resulting in increased gemcitabine metabolism and sensitivity, while reversal of collateral gemcitabine sensitivity by verapamil also suggests a direct relation between the presence of membrane efflux pumps and gemcitabine sensitivity.

Figure 1

Representative growth inhibition curves of the human melanoma cell line BRO (-▴-) and its transfected variant BROmdr (-▵-) (broken line) and the human ovarian carcinoma cell line A2780 (-•-) and its doxorubicin-resistant, P-gP-overexpressing variant A2780AD (-○-) (solid line). Cells were exposed to gemcitabine for 72 h.

Figure 2

Sensitivity factors to gemcitabine, without 25 μM verapamil (▪) or with 25 μM verapamil () (IC₅₀ of the MDR variants relative to IC₅₀ of SW1573 was set at 1), of the MRP1-overexpressing 2R120, the P-gP-overexpressing 2R160, the mdr1-transfected S1(1.1) and the MRP1-transfected S1(MRP). Verapamil itself did not affect cellular growth. Values are means±s.d. of at least three experiments. ^*Sensitivity with 25 μM verapamil statistically significantly different from gemcitabine alone (t-test, independent samples, P<0.05).

Figure 3

Accumulation (□), of dFdCTP after 24 h exposure to 0.1 or 1.0 μM gemcitabine and its retention after suspension in drug-free medium for 4 h (▪) or 24 h () in the human NSCLC cell line SW1573 and its doxorubicin-resistant MDR cell lines; MRP1-overexpressing 2R120 and P-gP-overexpressing 2R160. Values are means±s.d. of at least three experiments. ^*Statistically significant different from SW1573 cells (t-test, independent samples, P<0.005). n.d.=not detectable.

Figure 4

Incorporation of [³H]gemcitabine into DNA relative to incorporation of [¹⁴C]thymidine (TdR) into DNA (A) and incorporation of [³H]gemcitabine into RNA relative to incorporation of [³H]uridine (UR) into RNA (B) after 24 h exposure to 0.1 μM (▪) or 1.0 μM () gemcitabine in the human NSCLC cell line SW1573 and its doxorubicin-resistant MDR cell lines; MRP1-overexpressing 2R120 and P-gP-overexpressing 2R160. Values are means±s.d. of at least three experiments. ^*Statistically significant different from SW1573 cells (t-test, independent samples), P<0.05, ^**P<0.02. n.d.=not detectable.