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Multidrug Resistance-Associated Protein 2 (MRP2) Mediated Transport of Oxaliplatin-Derived Platinum in Membrane Vesicles.

Myint K, Li Y, Paxton J, McKeage M - PLoS ONE (2015)

Bottom Line: However, the role of multidrug resistance-associated protein 2 (MRP2) in controlling oxaliplatin membrane transport, in vivo handling, toxicity and therapeutic responses is unclear.In conclusion, MRP2 mediates the ATP-dependent active membrane transport of oxaliplatin-derived platinum.Intact oxaliplatin and its anionic monochloro oxalate ring-opened intermediate appear likely candidates as substrates for MRP2-mediated transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Clinical Pharmacology, University of Auckland, Auckland, New Zealand.

ABSTRACT
The platinum-based anticancer drug oxaliplatin is important clinically in cancer treatment. However, the role of multidrug resistance-associated protein 2 (MRP2) in controlling oxaliplatin membrane transport, in vivo handling, toxicity and therapeutic responses is unclear. In the current study, preparations of MRP2-expressing and control membrane vesicles, containing inside-out orientated vesicles, were used to directly characterise the membrane transport of oxaliplatin-derived platinum measured by inductively coupled plasma mass spectrometry. Oxaliplatin inhibited the ATP-dependent accumulation of the model MRP2 fluorescent probe, 5(6)-carboxy-2,'7'-dichlorofluorescein, in MRP2-expressing membrane vesicles. MRP2-expressing membrane vesicles accumulated up to 19-fold more platinum during their incubation with oxaliplatin and ATP as compared to control membrane vesicles and in the absence of ATP. The rate of ATP-dependent MRP2-mediated active transport of oxaliplatin-derived platinum increased non-linearly with increasing oxaliplatin exposure concentration, approaching a plateau value (Vmax) of 2680 pmol Pt/mg protein/10 minutes (95%CI, 2010 to 3360 pmol Pt/mg protein/10 minutes), with the half-maximal platinum accumulation rate (Km) at an oxaliplatin exposure concentration of 301 μM (95% CI, 163 to 438 μM), in accordance with Michaelis-Menten kinetics (r2 = 0.954). MRP2 inhibitors (myricetin and MK571) reduced the ATP-dependent accumulation of oxaliplatin-derived platinum in MRP2-expressing membrane vesicles in a concentration-dependent manner. To identify whether oxaliplatin, or perhaps a degradation product, was the likely substrate for this active transport, HPLC studies were undertaken showing that oxaliplatin degraded slowly in membrane vesicle incubation buffer containing chloride ions and glutathione, with approximately 95% remaining intact after a 10 minute incubation time and a degradation half-life of 2.24 hours (95%CI, 2.08 to 2.43 hours). In conclusion, MRP2 mediates the ATP-dependent active membrane transport of oxaliplatin-derived platinum. Intact oxaliplatin and its anionic monochloro oxalate ring-opened intermediate appear likely candidates as substrates for MRP2-mediated transport.

No MeSH data available.


Related in: MedlinePlus

ATP-dependent accumulation of CDCF in MRP2-expressing membrane vesicles: inhibition by oxaliplatin.MRP2-expressing and control membrane vesicles were incubated with CDCF (5 μM) for 5 min with or without ATP (4 mM) and oxaliplatin (400 μM), before measurement of CDCF accumulation by fluorescence. The P values shown as numbers are from two-way ANOVA and those shown as *** (P< 0.001) and N.S. (P> 0.05) are from Tukey’s multiple comparison post-tests following two-way ANOVA for comparisons with the respective membrane vesicles incubated with ATP but no oxaliplatin. Bars represent the means and standard deviations of individual values (open symbols) pooled from two independent experiments. Grey bars, MRP2-expressing membrane vesicles. Black bars, control membrane vesicles.
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pone.0130727.g001: ATP-dependent accumulation of CDCF in MRP2-expressing membrane vesicles: inhibition by oxaliplatin.MRP2-expressing and control membrane vesicles were incubated with CDCF (5 μM) for 5 min with or without ATP (4 mM) and oxaliplatin (400 μM), before measurement of CDCF accumulation by fluorescence. The P values shown as numbers are from two-way ANOVA and those shown as *** (P< 0.001) and N.S. (P> 0.05) are from Tukey’s multiple comparison post-tests following two-way ANOVA for comparisons with the respective membrane vesicles incubated with ATP but no oxaliplatin. Bars represent the means and standard deviations of individual values (open symbols) pooled from two independent experiments. Grey bars, MRP2-expressing membrane vesicles. Black bars, control membrane vesicles.

Mentions: To validate the MRP2-mediated membrane transport assay, MRP2 expressing and control membrane vesicles were exposed to a model fluorescence probe (CDCF; 5 μM) for 5 min in the presence or absence of ATP (4 mM) with or without oxaliplatin (400 μM), before measurement of CDCF accumulation by fluorescence. Membrane vesicle accumulation of CDCF was significantly increased by 9.4-fold by MRP2 (P<0.0001 Two-way ANOVA) and by 7.7-fold by ATP (P<0.0001 Two-way ANOVA) as compared to control membrane vesicles and the absence of ATP, respectively (Fig 1). Oxaliplatin significantly inhibited the ATP-dependent accumulation of CDCF in MRP2-expressing membrane vesicles by 25 ± 15% (P<0.0001 Tukey’s multiple comparison post-test following two-way ANOVA) but not in control membrane vesicles.


Multidrug Resistance-Associated Protein 2 (MRP2) Mediated Transport of Oxaliplatin-Derived Platinum in Membrane Vesicles.

Myint K, Li Y, Paxton J, McKeage M - PLoS ONE (2015)

ATP-dependent accumulation of CDCF in MRP2-expressing membrane vesicles: inhibition by oxaliplatin.MRP2-expressing and control membrane vesicles were incubated with CDCF (5 μM) for 5 min with or without ATP (4 mM) and oxaliplatin (400 μM), before measurement of CDCF accumulation by fluorescence. The P values shown as numbers are from two-way ANOVA and those shown as *** (P< 0.001) and N.S. (P> 0.05) are from Tukey’s multiple comparison post-tests following two-way ANOVA for comparisons with the respective membrane vesicles incubated with ATP but no oxaliplatin. Bars represent the means and standard deviations of individual values (open symbols) pooled from two independent experiments. Grey bars, MRP2-expressing membrane vesicles. Black bars, control membrane vesicles.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4488857&req=5

pone.0130727.g001: ATP-dependent accumulation of CDCF in MRP2-expressing membrane vesicles: inhibition by oxaliplatin.MRP2-expressing and control membrane vesicles were incubated with CDCF (5 μM) for 5 min with or without ATP (4 mM) and oxaliplatin (400 μM), before measurement of CDCF accumulation by fluorescence. The P values shown as numbers are from two-way ANOVA and those shown as *** (P< 0.001) and N.S. (P> 0.05) are from Tukey’s multiple comparison post-tests following two-way ANOVA for comparisons with the respective membrane vesicles incubated with ATP but no oxaliplatin. Bars represent the means and standard deviations of individual values (open symbols) pooled from two independent experiments. Grey bars, MRP2-expressing membrane vesicles. Black bars, control membrane vesicles.
Mentions: To validate the MRP2-mediated membrane transport assay, MRP2 expressing and control membrane vesicles were exposed to a model fluorescence probe (CDCF; 5 μM) for 5 min in the presence or absence of ATP (4 mM) with or without oxaliplatin (400 μM), before measurement of CDCF accumulation by fluorescence. Membrane vesicle accumulation of CDCF was significantly increased by 9.4-fold by MRP2 (P<0.0001 Two-way ANOVA) and by 7.7-fold by ATP (P<0.0001 Two-way ANOVA) as compared to control membrane vesicles and the absence of ATP, respectively (Fig 1). Oxaliplatin significantly inhibited the ATP-dependent accumulation of CDCF in MRP2-expressing membrane vesicles by 25 ± 15% (P<0.0001 Tukey’s multiple comparison post-test following two-way ANOVA) but not in control membrane vesicles.

Bottom Line: However, the role of multidrug resistance-associated protein 2 (MRP2) in controlling oxaliplatin membrane transport, in vivo handling, toxicity and therapeutic responses is unclear.In conclusion, MRP2 mediates the ATP-dependent active membrane transport of oxaliplatin-derived platinum.Intact oxaliplatin and its anionic monochloro oxalate ring-opened intermediate appear likely candidates as substrates for MRP2-mediated transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Clinical Pharmacology, University of Auckland, Auckland, New Zealand.

ABSTRACT
The platinum-based anticancer drug oxaliplatin is important clinically in cancer treatment. However, the role of multidrug resistance-associated protein 2 (MRP2) in controlling oxaliplatin membrane transport, in vivo handling, toxicity and therapeutic responses is unclear. In the current study, preparations of MRP2-expressing and control membrane vesicles, containing inside-out orientated vesicles, were used to directly characterise the membrane transport of oxaliplatin-derived platinum measured by inductively coupled plasma mass spectrometry. Oxaliplatin inhibited the ATP-dependent accumulation of the model MRP2 fluorescent probe, 5(6)-carboxy-2,'7'-dichlorofluorescein, in MRP2-expressing membrane vesicles. MRP2-expressing membrane vesicles accumulated up to 19-fold more platinum during their incubation with oxaliplatin and ATP as compared to control membrane vesicles and in the absence of ATP. The rate of ATP-dependent MRP2-mediated active transport of oxaliplatin-derived platinum increased non-linearly with increasing oxaliplatin exposure concentration, approaching a plateau value (Vmax) of 2680 pmol Pt/mg protein/10 minutes (95%CI, 2010 to 3360 pmol Pt/mg protein/10 minutes), with the half-maximal platinum accumulation rate (Km) at an oxaliplatin exposure concentration of 301 μM (95% CI, 163 to 438 μM), in accordance with Michaelis-Menten kinetics (r2 = 0.954). MRP2 inhibitors (myricetin and MK571) reduced the ATP-dependent accumulation of oxaliplatin-derived platinum in MRP2-expressing membrane vesicles in a concentration-dependent manner. To identify whether oxaliplatin, or perhaps a degradation product, was the likely substrate for this active transport, HPLC studies were undertaken showing that oxaliplatin degraded slowly in membrane vesicle incubation buffer containing chloride ions and glutathione, with approximately 95% remaining intact after a 10 minute incubation time and a degradation half-life of 2.24 hours (95%CI, 2.08 to 2.43 hours). In conclusion, MRP2 mediates the ATP-dependent active membrane transport of oxaliplatin-derived platinum. Intact oxaliplatin and its anionic monochloro oxalate ring-opened intermediate appear likely candidates as substrates for MRP2-mediated transport.

No MeSH data available.


Related in: MedlinePlus