Abstract
Low extracellular pH (pHe), that is characteristic of many tumours, tends to reduce the uptake of weakly basic drugs, such as doxorubicin, thereby conferring a degree of physiological resistance to chemotherapy. It has been assumed, from pH-partition theory, that the effect of intracellular pH (pHi) is symmetrically opposite, although this has not been tested experimentally. Doxorubicin uptake into colon HCT116 cells was measured using the drug's intrinsic fluorescence under conditions that alter pHi and pHe or pHi alone. Acutely, doxorubicin influx across the cell-membrane correlates with the trans-membrane pH-gradient (facilitated at alkaline pHe and acidic pHi). However, the protonated molecule is not completely membrane-impermeant and, therefore, overall drug uptake is less pHe-sensitive than expected from pH-partitioning. Once inside cells, doxorubicin associates with slowly-releasing nuclear binding sites. The occupancy of these sites increases with pHi, such that steady-state drug uptake can be greater with alkaline cytoplasm, in contradiction to pH-partition theory. Measurements of cell proliferation demonstrate that doxorubicin efficacy is enhanced at alkaline pHi and that pH-partition theory is inadequate to account for this. The limitations in the predictive power of pH-partition theory arise because it only accounts for the pHi/pHe-sensitivity of drug entry into cells but not the drug's subsequent interactions that, independently, show pHi-dependence. In summary, doxorubicin uptake into cells is favoured by high pHe and high pHi. This modified formalism should be taken into account when designing manoeuvres aimed at increasing doxorubicin efficacy.
Highlights
The entry of weakly basic or weakly acidic molecules into cells can show pH-dependence arising from differences in the membrane permeability of their protonated and unprotonated forms [1,2,3]
To change pHi at constant pHe: (i) NaCl was iso-osmotically replaced with 40 mM or 80 mM NaAcetate, (ii) Na+ was replaced with N-methyl-D-glucamine, (iii) 5-(N,N-dimethyl) amiloride (DMA) was added, (iv) Cl2 was replaced with gluconate, or (v) Hepes/Mes was replaced with CO2/HCO32
Intracellular doxorubicin associates with a slowlyreleasing binding site Colon HCT116 cells were exposed transiently to solution containing 50 mM doxorubicin for 1, 2 and 5 minutes (Fig. 1A)
Summary
The entry of weakly basic or weakly acidic molecules into cells can show pH-dependence arising from differences in the membrane permeability of their protonated and unprotonated forms [1,2,3] This so-called pH-partitioning is relevant to chemotherapy because many anti-cancer drugs are protonatable and tumours typically have substantially lower extracellular pH (pHe) than normal tissue, whilst maintaining a modestly alkaline intracellular pH (pHi) [3,4,5,6]. Recent in vitro work has shown that doxorubicin forms an adduct with DNA [15] in a pH-sensitive manner [16], decreasing as pH is reduced below 7 This process may introduce physiologically-relevant pHi-sensitivity of doxorubicin uptake into cells, independently of pH-partitioning across the membrane. Further testing of pH-partition theory would require studies performed under conditions where pHi is manipulated to a greater degree than pHe or pHv
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