Combined effects of GSTP1 and MRP1 in melanoma drug resistance

Abstract

Glutathione-S-transferase Pi1 (GSTP1) and multidrug resistance protein 1 (MRP1) are overexpressed in melanoma, a skin cancer notoriously resistant to all current modalities of cancer therapy. To investigate the involvement of these detoxifying enzymes in the drug resistance of melanoma, an inducible (Tet-On system) antisense (AS) RNA strategy was used to specifically inhibit GSTP1 expression in A375 cells, a human melanoma cell line expressing high levels of GSTP1 and MRP1. Stable transfectant clones were established and analysed for GSTP1 inhibition by AS RNA. The clone A375-ASPi1, presenting a specific 40% inhibition of GSTP1 expression in the presence of doxycycline, was selected. Lowering the GSTP1 level significantly increased (about 3.3-fold) the sensitivity of A375-ASPi1 cells to etoposide. Inhibitors of glutathione synthesis (BSO), GSTs (curcumin, ethacrynic acid), and also of MRPs (MK571, sulphinpyrazone) improved the sensitising effect of GSTP1 AS RNA. All these inhibitors had stronger sensitising effects in control cells expressing high GSTP1 level (A375-ASPi1 cells in the absence of doxycycline). In conclusion, GSTP1 can act in a combined fashion with MRP1 to protect melanoma cells from toxic effects of etoposide.

Figure 1

Expression of GSTs and MRPs in A375 cell lines. Levels of GSTs and MRPs mRNA (A) and (B) were determined by RT–PCR and Western blot, respectively, as described under ‘Materials and Methods’. Examined are cellular mRNA (4 ng each lane) and proteins (20 μg and 100 μg each lane for GSTs and MRPs, respectively) from parental cells (A375-wt) and cells transfected with pTet-On and ppTRE2-ASPi (A375-ASPi1). Experiments were made in the presence (+) or absence (−) of doxycycline (0.2 μg ml^{− 1}, 24 h). Positive controls were plasmids encoding GSTs (A1, M1, P1) and MRPs (1 and 2) or cDNA from Caco-2 cells for MRP3 for PCR experiments. β-Actin was used as a standardisation control in two detection experiments.

Figure 2

GST activities in A375-ASPi1 cells. Cytosolic proteins were extracted from A375-ASPi1 cells incubated in presence (+) or absence (−) of doxycycline (0.2 μg ml^{− 1}, 24 h) and assessed for their ability to conjugate CDNB to GSH as described in ‘Material and Methods’. Glutathione-S-transferase activities, expressed as nmol min^{− 1} CDNB conjugated with GSH per mg of cytosolic protein, are means±s.e.m. of at least three separate experiments. ^**P<0.01 according to Student's t-test comparing values obtained in studied cells with those obtained in A375-ASPi1 cells in the absence of doxycycline.

Figure 3

Duration of GSTP1 inhibition after doxycycline removal. Level of GSTP1 protein inhibition was determined by Western blot, as described under ‘Materials and Methods’. Examined are cytosolic proteins (20 μg each lane) from A375-ASPi1 cultured in presence (+) or absence (−) of doxycycline (0.2 μg ml^{− 1}, 24 h). Lane 1, A375-ASPi1 were cultured in absence of doxycycline, corresponding to control cell line. Lanes 2, 3 and 4, A375-ASPi1 were cultured in presence of doxycycline during 24 h and lysed immediately, 7 and 20 h after doxycycline removal. β-Actin was used as a standardisation control in detection experiment.

Figure 4

Effects of GSTP1 and MRP inhibition on the etoposide sensitivity of A375 cell lines. A375-ASPi1 cells were preincubated in presence (open symbols) or absence (closed symbols) of doxycycline (0.2 μg ml^{− 1}, 24 h) and then exposed for 4 h to etoposide alone (triangles) or with 30 μM MK571 (square), 3.3 μM EA (circle) or 30 μM MK571 + 3.3 μM EA (diamond) as described under ‘Materials and Methods’. Data are means±s.e.m. of three independent determinations.

Figure 5

Functional activity of MRP1 in A375-ASPi1 cell lines. Cells were preincubated in presence (+) or in absence (−) of doxycycline (0.2 μg ml^{− 1} 24 h). Then cells were incubated for 1 h at 37°C with 5 μM [³H]-etoposide in culture medium. After washing with PBS, cells were incubated at 37°C for 3 h in the absence (control) or presence of inhibitors (2 mM sulphinpyrazone (S), 30 μM MK571 (MK), 30 μM curcumin (C), 3.3 μM ethacrynic acid (EA)). For BSO 50 μM (B), cells were preincubated 48 h before etoposide treatement. The intracellular [³H]-etoposide concentration was evaluated by β-scintillation counting as described in ‘Materials and Methods’. Data expressed as percentage of [³H]-etoposide concentration of control cells corresponding to A375-ASPi1 unexposed to inhibitors and to doxycycline are means±s.e.m. of three separate experiments. ^**P<0.01; ^***P<0.001 according to Student's t-test comparing values obtained with inhibitor in cells with those obtained without inhibitor.