Importance of intracellular pH in determining the uptake and efficacy of the weakly basic chemotherapeutic drug, doxorubicin

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. © 2012 Swietach et al.