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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

Kinetic analysis of MRP2-mediated active transport of oxaliplatin-derived platinum.Rates of ATP-dependent platinum accumulation in MRP2-expressing membrane vesicles were derived, plotted against oxaliplatin exposure concentration and fitted to a non-linear model. Symbols represent individual values pooled from two independent experiments. The line represents a nonlinear Michaelis-Menten regression fit (r2 = 0.954) with a Vmax of 2680 pmol Pt/mg protein/10 min (95%CI, 2010 to 3360 pmol Pt/mg protein/10 min) and a Km of 301 μM (95% CI, 163 to 438 μM).
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pone.0130727.g004: Kinetic analysis of MRP2-mediated active transport of oxaliplatin-derived platinum.Rates of ATP-dependent platinum accumulation in MRP2-expressing membrane vesicles were derived, plotted against oxaliplatin exposure concentration and fitted to a non-linear model. Symbols represent individual values pooled from two independent experiments. The line represents a nonlinear Michaelis-Menten regression fit (r2 = 0.954) with a Vmax of 2680 pmol Pt/mg protein/10 min (95%CI, 2010 to 3360 pmol Pt/mg protein/10 min) and a Km of 301 μM (95% CI, 163 to 438 μM).

Mentions: The concentration dependence of MRP2-mediated active transport of oxaliplatin-derived platinum was investigated by incubating MRP2-expressing membrane vesicles with oxaliplatin at 6.25 to 400 μM, with or without ATP (4 mM) for 10 min followed by measurement of platinum accumulation by ICPMS. Membrane vesicle platinum accumulation increased with increasing oxaliplatin exposure concentration (P<0.0001 Two-way ANOVA) (Fig 3). Membrane vesicle platinum accumulation was increased by ATP by up to 5.9-fold compared to the absence of ATP (P<0.0001 Two-way ANOVA). The kinetics of MRP2-mediated active transport of oxaliplatin was investigated by plotting rates of ATP-dependent platinum accumulation in MRP2-expressing membrane vesicles against oxaliplatin exposure concentration and fitting these data to a nonlinear model (Fig 4). The rate of ATP-dependent MRP2-mediated active transport of oxaliplatin-derived platinum increased nonlinearly with oxaliplatin exposure concentration, approaching a plateau value (Vmax) of 2680 pmol Pt/mg protein/10 min (95%CI, 2010 to 3360 pmol Pt/mg protein/10 min), with the half-maximal platinum accumulation rate occurring at an oxaliplatin exposure concentration of 301 μM (95% CI, 163 to 438 μM), in accordance with Michaelis-Menten kinetics (r2 = 0.954).


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)

Kinetic analysis of MRP2-mediated active transport of oxaliplatin-derived platinum.Rates of ATP-dependent platinum accumulation in MRP2-expressing membrane vesicles were derived, plotted against oxaliplatin exposure concentration and fitted to a non-linear model. Symbols represent individual values pooled from two independent experiments. The line represents a nonlinear Michaelis-Menten regression fit (r2 = 0.954) with a Vmax of 2680 pmol Pt/mg protein/10 min (95%CI, 2010 to 3360 pmol Pt/mg protein/10 min) and a Km of 301 μM (95% CI, 163 to 438 μM).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130727.g004: Kinetic analysis of MRP2-mediated active transport of oxaliplatin-derived platinum.Rates of ATP-dependent platinum accumulation in MRP2-expressing membrane vesicles were derived, plotted against oxaliplatin exposure concentration and fitted to a non-linear model. Symbols represent individual values pooled from two independent experiments. The line represents a nonlinear Michaelis-Menten regression fit (r2 = 0.954) with a Vmax of 2680 pmol Pt/mg protein/10 min (95%CI, 2010 to 3360 pmol Pt/mg protein/10 min) and a Km of 301 μM (95% CI, 163 to 438 μM).
Mentions: The concentration dependence of MRP2-mediated active transport of oxaliplatin-derived platinum was investigated by incubating MRP2-expressing membrane vesicles with oxaliplatin at 6.25 to 400 μM, with or without ATP (4 mM) for 10 min followed by measurement of platinum accumulation by ICPMS. Membrane vesicle platinum accumulation increased with increasing oxaliplatin exposure concentration (P<0.0001 Two-way ANOVA) (Fig 3). Membrane vesicle platinum accumulation was increased by ATP by up to 5.9-fold compared to the absence of ATP (P<0.0001 Two-way ANOVA). The kinetics of MRP2-mediated active transport of oxaliplatin was investigated by plotting rates of ATP-dependent platinum accumulation in MRP2-expressing membrane vesicles against oxaliplatin exposure concentration and fitting these data to a nonlinear model (Fig 4). The rate of ATP-dependent MRP2-mediated active transport of oxaliplatin-derived platinum increased nonlinearly with oxaliplatin exposure concentration, approaching a plateau value (Vmax) of 2680 pmol Pt/mg protein/10 min (95%CI, 2010 to 3360 pmol Pt/mg protein/10 min), with the half-maximal platinum accumulation rate occurring at an oxaliplatin exposure concentration of 301 μM (95% CI, 163 to 438 μM), in accordance with Michaelis-Menten kinetics (r2 = 0.954).

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