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.
Highlights
The platinum-based anticancer drug oxaliplatin, and its combination therapies, are clinically important for treating colorectal cancer and other gastrointestinal malignancies [1]
To validate the multidrug resistance-associated protein 2 (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
To investigate the role of MRP2 in the membrane transport of oxaliplatin, MRP2-expressing and control membrane vesicles were incubated with oxaliplatin (100 μM) with or without ATP (4 mM) for 5, 10 or 20 min followed by measurement of platinum accumulation by Inductively coupled plasma mass spectrometry (ICPMS)
Summary
The platinum-based anticancer drug oxaliplatin, and its combination therapies, are clinically important for treating colorectal cancer and other gastrointestinal malignancies [1]. Oxaliplatin-based chemotherapy is limited by poor efficacy and high toxicity in a proportion of treated patients, who exhibit disease progression or severe adverse drug reactions early after the commencement of therapy [2,3,4,5]. Recent evidence has pointed to alternative membrane transport mechanisms involving transporter proteins whereby oxaliplatin moves into and out of cells [10,11,12,13,14,15,16,17,18,19,20,21]. As this field of research is relatively new, it seems likely that many interactions involving oxaliplatin and membrane transporter proteins remain to be characterised
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
