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Overexpression of MRP4 (ABCC4) and MRP5 (ABCC5) confer resistance to the nucleoside analogs cytarabine and troxacitabine, but not gemcitabine.

Adema AD, Floor K, Smid K, Honeywell RJ, Scheffer GL, Jansen G, Peters GJ - Springerplus (2014)

Bottom Line: Trocacitabine accumulation was similar in the 3 cell lines, but after the DFM period troxacitabine decreased 2-4-fold faster in MRP4/5 cells.Troxacitabine-nucleotides were about 25% lower in MRP4/5 cells and decreased rapidly in MRP4, but not in MRP5 cells.MRP4 and MRP5 overexpression confer resistance to troxacitabine and ara-C, but not to GEM, which was associated with a rapid decline of the ara-C and troxacitabine-nucleotides in HEK/MRP4-5 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands.

ABSTRACT

Unlabelled: We aimed to determine whether the multidrug-resistance-proteins MRP4 (ABCC4) and MRP5 (ABCC5) confer resistance to the antimetabolites cytarabine (Ara-C), gemcitabine (GEM), and the L-nucleoside analog troxacitabine. For this purpose we used HEK293 and the transfected HEK/MRP4 (59-fold increased MRP4) or HEK/MRP5i (991-fold increased MRP5) as model systems and tested the cells for drug sensitivity using a proliferation test. Drug accumulation was performed by using radioactive Ara-C, and for GEM and troxacitabine with HPLC with tandem-MS or UV detection. At 4-hr exposure HEK/MRP4 cells were 2-4-fold resistant to troxacitabine, ara-C and 9-(2-phosphonylmethoxyethyl)adenine (PMEA), and HEK/MRP5i to ara-C and PMEA, but none to GEM. The inhibitors probenecid and indomethacin reversed resistance. After 4-hr exposure ara-C-nucleotides were 2-3-fold lower in MRP4/5 cells, in which they decreased more rapidly after washing with drug-free medium (DFM). Trocacitabine accumulation was similar in the 3 cell lines, but after the DFM period troxacitabine decreased 2-4-fold faster in MRP4/5 cells. Troxacitabine-nucleotides were about 25% lower in MRP4/5 cells and decreased rapidly in MRP4, but not in MRP5 cells. Accumulation of GEM-nucleotides was higher in the MRP4/5 cells.

In conclusion: MRP4 and MRP5 overexpression confer resistance to troxacitabine and ara-C, but not to GEM, which was associated with a rapid decline of the ara-C and troxacitabine-nucleotides in HEK/MRP4-5 cells.

No MeSH data available.


Related in: MedlinePlus

Accumulation and retention of free troxacitabine (trox) (a) and troxacitabine-nucleotides (b) in the HEK, HEK/MRP4 (HEK/4) and HEK/MRP5i (HEK/5) cell lines. T‚ÄČ=‚ÄČ4: 4¬†hours exposure to 10¬†őľM troxacitabine, T‚ÄČ=‚ÄČ4‚ÄČ+‚ÄČ2: 4¬†hours exposure to troxacitabine followed by 2¬†hours drug-free medium. Values represent fmol/őľg protein‚ÄȬĪ‚ÄČSEM of 3 separate experiments. For free troxacitabine the effect of drug-free medium was significant (*,p‚ÄČ<‚ÄČ0.01) (paired t test). Accumulation was measured in the absence of indomethacin or probenecid.
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Fig5: Accumulation and retention of free troxacitabine (trox) (a) and troxacitabine-nucleotides (b) in the HEK, HEK/MRP4 (HEK/4) and HEK/MRP5i (HEK/5) cell lines. T‚ÄČ=‚ÄČ4: 4¬†hours exposure to 10¬†őľM troxacitabine, T‚ÄČ=‚ÄČ4‚ÄČ+‚ÄČ2: 4¬†hours exposure to troxacitabine followed by 2¬†hours drug-free medium. Values represent fmol/őľg protein‚ÄȬĪ‚ÄČSEM of 3 separate experiments. For free troxacitabine the effect of drug-free medium was significant (*,p‚ÄČ<‚ÄČ0.01) (paired t test). Accumulation was measured in the absence of indomethacin or probenecid.

Mentions: Exposure of the MRP 4 and 5 overexpressing cell lines to Ara-C, dFdC, troxacitabine and PMEA showed a differential resistance compared to the parental cell line both at 4 compared to 72¬†hours exposure (Table¬† 1). PMEA was used as a positive control since this compound is known to be a substrate for MRP4 and MRP5 (Reid et al 2003). The modest 4-fold resistance was within the expected range. However, the highest resistance, although still modest, was observed for troxacitabine in the HEK/MRP4 cell line, while no real resistance was observed in the HEK/MRP5 cell line. Resistance to Ara-C was observed in both the overexpressing cell lines. The effects at 72¬†hours exposure were either absent (dFdC) or lower. No resistance was seen for dFdC; for troxacitabine just a low1.4-fold in MRP4 cells and for Ara-C an equally low 2- and 1.6-fold in MRP4 and MRP5 cells, respectively.For both Ara-C and troxacitabine we investigated whether inhibition of MRP4 or MRP5 would increase sensitivity. The inhibitors indomethacin and probenecid increased sensitivity to both Ara-C and troxacitabine in HEK/MRP4 cells (Figure¬† 4a); inhibition of MRP5 with indomethacin only increased the sensitivity to Ara-C (Figure¬† 4b). Although these transporters are not specific, they would only inhibit MRP4 or MRP5, since that is the only detectable transporter in the transfected cells. Moreover, in wild-type cells neither indomethacin or probenecid affected the sensitivity of either drugs, excluding the possibility that inhibition of another transporter might be responsible for the observed effect in the transfected cells.To further elucidate the mechanisms explaining the resistance we investigated the accumulation and retention of troxacitabine, Ara-C, dFdC and their respective phosphorylated forms. Accumulation of phosphorylated troxacitabine was lower in both the HEK/MRP4 and HEK/MRP5i cell lines compared to the wild-type HEK cells (Figure¬† 5a), this decrease was most pronounced in the MRP4 cell line after 4¬†hours exposure to troxacitabine and after 2¬†hours incubation in drug-free medium. Next to the phosphorylated troxacitabine, the free troxacitabine also showed a markedly decreased retention in both the HEK/MRP4 and the HEK/MRP5i cell lines (Figure¬† 5b), which was most pronounced in the HEK/MRP4 cell line. None of the nucleotides was detectable outside the cells; apparently in case they are effluxed their concentration would be so far diluted that it would be even below the detection limit of our sensitive LC-MS-MS assay (troxacitabine) or radioactivity (ara-C).Accumulation of both Ara-C and phosphorylated Ara-C were lower in both the HEK/MRP4 and HEK/MRP5i cell lines compared to the wild-type HEK cells (Figure¬† 6), however the decrease was most pronounced in the HEK/MRP4. The differences in both Ara-C and phosphorylated Ara-C were larger after incubation in drug-free medium. In all three cell lines the concentration of Ara-C decreased about 10-fold during incubation in drug-free medium, but that of Ara-C nucleotide only 5-fold in the wild-type. However, in both MRP4 and MRP5 cells remaining nucleotides were much lower than in the wild-type cells.The accumulation and retention of dFdCTP showed a completely different pattern compared to Ara-C and troxacitabine. First, after 4¬†hours exposure dFdCTP accumulation was higher in the MRP4 and MRP5 cells compared to the wild-type HEK (Figure¬† 7). Furthermore dFdCTP accumulation continued to increase upon withdrawal of gemcitabine both after exposure to 25 or 50¬†őľM gemcitabine (data not shown).Figure 4


Overexpression of MRP4 (ABCC4) and MRP5 (ABCC5) confer resistance to the nucleoside analogs cytarabine and troxacitabine, but not gemcitabine.

Adema AD, Floor K, Smid K, Honeywell RJ, Scheffer GL, Jansen G, Peters GJ - Springerplus (2014)

Accumulation and retention of free troxacitabine (trox) (a) and troxacitabine-nucleotides (b) in the HEK, HEK/MRP4 (HEK/4) and HEK/MRP5i (HEK/5) cell lines. T‚ÄČ=‚ÄČ4: 4¬†hours exposure to 10¬†őľM troxacitabine, T‚ÄČ=‚ÄČ4‚ÄČ+‚ÄČ2: 4¬†hours exposure to troxacitabine followed by 2¬†hours drug-free medium. Values represent fmol/őľg protein‚ÄȬĪ‚ÄČSEM of 3 separate experiments. For free troxacitabine the effect of drug-free medium was significant (*,p‚ÄČ<‚ÄČ0.01) (paired t test). Accumulation was measured in the absence of indomethacin or probenecid.
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Related In: Results  -  Collection

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Fig5: Accumulation and retention of free troxacitabine (trox) (a) and troxacitabine-nucleotides (b) in the HEK, HEK/MRP4 (HEK/4) and HEK/MRP5i (HEK/5) cell lines. T‚ÄČ=‚ÄČ4: 4¬†hours exposure to 10¬†őľM troxacitabine, T‚ÄČ=‚ÄČ4‚ÄČ+‚ÄČ2: 4¬†hours exposure to troxacitabine followed by 2¬†hours drug-free medium. Values represent fmol/őľg protein‚ÄȬĪ‚ÄČSEM of 3 separate experiments. For free troxacitabine the effect of drug-free medium was significant (*,p‚ÄČ<‚ÄČ0.01) (paired t test). Accumulation was measured in the absence of indomethacin or probenecid.
Mentions: Exposure of the MRP 4 and 5 overexpressing cell lines to Ara-C, dFdC, troxacitabine and PMEA showed a differential resistance compared to the parental cell line both at 4 compared to 72¬†hours exposure (Table¬† 1). PMEA was used as a positive control since this compound is known to be a substrate for MRP4 and MRP5 (Reid et al 2003). The modest 4-fold resistance was within the expected range. However, the highest resistance, although still modest, was observed for troxacitabine in the HEK/MRP4 cell line, while no real resistance was observed in the HEK/MRP5 cell line. Resistance to Ara-C was observed in both the overexpressing cell lines. The effects at 72¬†hours exposure were either absent (dFdC) or lower. No resistance was seen for dFdC; for troxacitabine just a low1.4-fold in MRP4 cells and for Ara-C an equally low 2- and 1.6-fold in MRP4 and MRP5 cells, respectively.For both Ara-C and troxacitabine we investigated whether inhibition of MRP4 or MRP5 would increase sensitivity. The inhibitors indomethacin and probenecid increased sensitivity to both Ara-C and troxacitabine in HEK/MRP4 cells (Figure¬† 4a); inhibition of MRP5 with indomethacin only increased the sensitivity to Ara-C (Figure¬† 4b). Although these transporters are not specific, they would only inhibit MRP4 or MRP5, since that is the only detectable transporter in the transfected cells. Moreover, in wild-type cells neither indomethacin or probenecid affected the sensitivity of either drugs, excluding the possibility that inhibition of another transporter might be responsible for the observed effect in the transfected cells.To further elucidate the mechanisms explaining the resistance we investigated the accumulation and retention of troxacitabine, Ara-C, dFdC and their respective phosphorylated forms. Accumulation of phosphorylated troxacitabine was lower in both the HEK/MRP4 and HEK/MRP5i cell lines compared to the wild-type HEK cells (Figure¬† 5a), this decrease was most pronounced in the MRP4 cell line after 4¬†hours exposure to troxacitabine and after 2¬†hours incubation in drug-free medium. Next to the phosphorylated troxacitabine, the free troxacitabine also showed a markedly decreased retention in both the HEK/MRP4 and the HEK/MRP5i cell lines (Figure¬† 5b), which was most pronounced in the HEK/MRP4 cell line. None of the nucleotides was detectable outside the cells; apparently in case they are effluxed their concentration would be so far diluted that it would be even below the detection limit of our sensitive LC-MS-MS assay (troxacitabine) or radioactivity (ara-C).Accumulation of both Ara-C and phosphorylated Ara-C were lower in both the HEK/MRP4 and HEK/MRP5i cell lines compared to the wild-type HEK cells (Figure¬† 6), however the decrease was most pronounced in the HEK/MRP4. The differences in both Ara-C and phosphorylated Ara-C were larger after incubation in drug-free medium. In all three cell lines the concentration of Ara-C decreased about 10-fold during incubation in drug-free medium, but that of Ara-C nucleotide only 5-fold in the wild-type. However, in both MRP4 and MRP5 cells remaining nucleotides were much lower than in the wild-type cells.The accumulation and retention of dFdCTP showed a completely different pattern compared to Ara-C and troxacitabine. First, after 4¬†hours exposure dFdCTP accumulation was higher in the MRP4 and MRP5 cells compared to the wild-type HEK (Figure¬† 7). Furthermore dFdCTP accumulation continued to increase upon withdrawal of gemcitabine both after exposure to 25 or 50¬†őľM gemcitabine (data not shown).Figure 4

Bottom Line: Trocacitabine accumulation was similar in the 3 cell lines, but after the DFM period troxacitabine decreased 2-4-fold faster in MRP4/5 cells.Troxacitabine-nucleotides were about 25% lower in MRP4/5 cells and decreased rapidly in MRP4, but not in MRP5 cells.MRP4 and MRP5 overexpression confer resistance to troxacitabine and ara-C, but not to GEM, which was associated with a rapid decline of the ara-C and troxacitabine-nucleotides in HEK/MRP4-5 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands.

ABSTRACT

Unlabelled: We aimed to determine whether the multidrug-resistance-proteins MRP4 (ABCC4) and MRP5 (ABCC5) confer resistance to the antimetabolites cytarabine (Ara-C), gemcitabine (GEM), and the L-nucleoside analog troxacitabine. For this purpose we used HEK293 and the transfected HEK/MRP4 (59-fold increased MRP4) or HEK/MRP5i (991-fold increased MRP5) as model systems and tested the cells for drug sensitivity using a proliferation test. Drug accumulation was performed by using radioactive Ara-C, and for GEM and troxacitabine with HPLC with tandem-MS or UV detection. At 4-hr exposure HEK/MRP4 cells were 2-4-fold resistant to troxacitabine, ara-C and 9-(2-phosphonylmethoxyethyl)adenine (PMEA), and HEK/MRP5i to ara-C and PMEA, but none to GEM. The inhibitors probenecid and indomethacin reversed resistance. After 4-hr exposure ara-C-nucleotides were 2-3-fold lower in MRP4/5 cells, in which they decreased more rapidly after washing with drug-free medium (DFM). Trocacitabine accumulation was similar in the 3 cell lines, but after the DFM period troxacitabine decreased 2-4-fold faster in MRP4/5 cells. Troxacitabine-nucleotides were about 25% lower in MRP4/5 cells and decreased rapidly in MRP4, but not in MRP5 cells. Accumulation of GEM-nucleotides was higher in the MRP4/5 cells.

In conclusion: MRP4 and MRP5 overexpression confer resistance to troxacitabine and ara-C, but not to GEM, which was associated with a rapid decline of the ara-C and troxacitabine-nucleotides in HEK/MRP4-5 cells.

No MeSH data available.


Related in: MedlinePlus