Fig 1.
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.
Fig 2.
Membrane vesicle accumulation of oxaliplatin-derived platinum: dependence upon MRP2, ATP and oxaliplatin exposure time.
MRP2-expressing and control membrane vesicles were incubated with oxaliplatin (100 μM), with or without ATP (4 mM) for 5, 10 and 20 min, followed by measurement of platinum accumulation by ICPMS. The P values shown as numbers are from two-way ANOVA and those shown as *** (P< 0.001) and **** (P < 0.0001) are from Tukey’s multiple comparisons post-tests following two-way ANOVA for comparisons with MRP2-expressing membrane vesicles incubated with ATP at each respective time point. Bars represent means and standard deviations of individual values (open symbols) pooled from two independent experiments. Light grey bars, MRP2-expressing membrane vesicles with ATP. Dark grey bars, MRP2-expressing vesicles without ATP. Black bars, control membrane vesicles.
Fig 3.
MRP2-mediated membrane vesicle accumulation of oxaliplatin-derived platinum: dependence upon oxaliplatin exposure concentration and ATP.
MRP2-expressing membrane vesicles were incubated with oxaliplatin (6.25 to 400 μM) with or without ATP (4 mM) for 10 min before measurement of platinum accumulation by ICPMS. The P values shown as numbers are from two-way ANOVA and those shown as **** (P< 0.0001) are from Tukey’s multiple comparison post-tests following two-way ANOVA. The bars represent means and standard deviations from individual values (open symbols) pooled from two independent experiments. Light grey bars, with ATP. Dark grey bars, without ATP.
Fig 4.
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).
Fig 5.
Effects of MRP2 inhibitors on membrane vesicle accumulation of oxaliplatin-derived platinum.
MRP2-expressing and control membrane vesicles were incubated with oxaliplatin (100 μM), ATP (4 mM) and MRP2 inhibitors for 10 minutes before measurement of platinum accumulation by ICPMS. The P values shown as numbers are from two-way ANOVA and those shown as * (P < 0.05), ** (P < 0.01) and *** (P < 0.001) are from Tukey’s multiple comparison post-tests following two-way ANOVA for comparisons to MRP2-expressing membrane vesicles incubated with oxaliplatin and ATP but without MRP2 inhibitors. Bars represent means and standard deviations of individual values (open symbols) pooled from at least two independent experiments. Light grey bars, MRP2-expressing membrane vesicles. Black bars, control membrane vesicles.
Fig 6.
HPLC-UV detection of oxaliplatin, glutathione and degradation products in membrane vesicle incubation buffer.
Injection of blank membrane vesicle incubation buffer (A) and authentic standards showed chromatographic separation of glutathione (B, peak 1, retention time 6.5 min), oxaliplatin (C, peak 2, retention time 12.5 min) and Pt(DACH)Cl2 (D, peak 3, retention time 10.5 min) with no interference from components of the blank membrane vesicle incubation buffer. Oxaliplatin was incubated (100 μM, pH 7.4, 37˚C) in membrane vesicle incubation buffer with (I-L) or without glutathione (2 mM) (E-H) before analysis of samples by HPLC-UV after 0 hours (E,I), 0.3 hours (F,J), 2 hours (G,K) or 7 hours (H,L) incubation time. After 0.3 hours incubation time (F,J), HPLC-UV chromatograms appeared more-or-less unchanged from the start of the incubation (E,I) and similar with (J) and without glutathione (F). With an increasing incubation time, the oxaliplatin peak areas progressively reduced, and new peaks appeared, corresponding to Pt(DACH)Cl2 (G,H: peak 3) in solutions containing no glutathione, and an unknown peak (K,L: peak 4) in solutions containing glutathione. Chromatograms are representative of those from two independent experiments.
Fig 7.
Kinetic analysis of oxaliplatin degradation in membrane vesicle incubation buffer containing glutathione.
Open symbols represent individual values of oxaliplatin chromatographic peak areas pooled from two independent experiments. The line represents a nonlinear one phase exponential decay regression fit (r2 = 0.964) to the data giving an oxaliplatin degradation half-life of 2.24 hours (95%CI, 2.08 to 2.43 hours).