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Combined effects of GSTP1 and MRP1 in melanoma drug resistance.

Depeille P, Cuq P, Passagne I, Evrard A, Vian L - Br. J. Cancer (2005)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Toxicologie du Médicament - EA 2994, Faculté de Pharmacie, BP 14491, 15 avenue Charles Flahault, 34093 Montpellier cedex 5, France.

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.

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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.
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fig2: 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.

Mentions: Next, the clones were analysed for inhibition level of GSTP1 by AS RNA expressed under tetracycline control. The clone A375-ASPi1 displayed, in the presence of doxycycline (0.2 μg ml− 1, 24 h), a specific 40% inhibition of GSTP1 mRNA and protein expression (Figure 1 and Table 1), and a significant decrease (about 40%) of total GST activity (Figure 2). Neither the expression of the other GSTs (A1 and M1) nor that of the MRPs was affected by doxycycline treatment in these cells (Figure 1).


Combined effects of GSTP1 and MRP1 in melanoma drug resistance.

Depeille P, Cuq P, Passagne I, Evrard A, Vian L - Br. J. Cancer (2005)

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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2361556&req=5

fig2: 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.
Mentions: Next, the clones were analysed for inhibition level of GSTP1 by AS RNA expressed under tetracycline control. The clone A375-ASPi1 displayed, in the presence of doxycycline (0.2 μg ml− 1, 24 h), a specific 40% inhibition of GSTP1 mRNA and protein expression (Figure 1 and Table 1), and a significant decrease (about 40%) of total GST activity (Figure 2). Neither the expression of the other GSTs (A1 and M1) nor that of the MRPs was affected by doxycycline treatment in these cells (Figure 1).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Toxicologie du Médicament - EA 2994, Faculté de Pharmacie, BP 14491, 15 avenue Charles Flahault, 34093 Montpellier cedex 5, France.

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.

Show MeSH
Related in: MedlinePlus