Effect of intracellular and extracellular pH on drug uptake and accumulation.

<p>(A) Initial rate of doroxubicin uptake, measured at constant intracellular pH, over a range of extracellular pH values (data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035949#pone-0035949-g002" target="_blank">Fig. 2A</a>) with best-fit. (B) Intracellular doxorubicin at steady-state, normalized to extracellular concentration, over a range of extracellular pH values. Secondary axis plots steady-state pH_i attained at given pH_e. (C) Initial rate of doxorubicin uptake, measured at constant extracellular pH, over a range of intracellular pH values (data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035949#pone-0035949-g002" target="_blank">Fig. 2B</a>) with best-fit. Model predictions for the steady-state relationship between intracellular pH, extracellular pH and either (D) free, (E) bound or (F) total doxorubicin. Contour labels denote total intracellular doxorubicin concentration, normalized to its extracellular concentration (50 µM).</p

Effect of changing extracellular and intracellular pH on doxorubicin uptake.

<p>Time-courses show the average (±SEM) of at least 25 cells. (A) <i>(i)</i> Extracellular pH was changed by switching to superfusates titrated to pH 6.8 or 6.4, simultaneously with the application of 50 µM doxorubicin. <i>(ii)</i> Intracellular pH measured in separate experiments using carboxy-SNARF-1. <i>(iii)</i> Intracellular doxorubicin, normalized to its extracellular signal. Inset shows intracellular fluorescence at steady state, attained after 2.8 hours of drug-exposure. <i>(iv)</i> Simulated doxorubicin time-courses. (B) <i>(i)</i> Intracellular pH was reduced to 6.7 at constant extracellular pH by superfusing cells with 80 mM acetate in the presence of the Na⁺/H⁺ exchange inhibitor, dimethyl amiloride (DMA; 30 µM). Doxorubicin was applied once pH_i attained a steady-state. <i>(ii)</i> Intracellular pH measured with carboxy-SNARF-1. <i>(iii)</i> Intracellular doxorubicin fluorescence, showing the cross-over of time-courses for pH_i = 7.2 and 6.7. <i>(iv)</i> Simulation of doxorubicin-time-courses.</p

Importance of intracellular pH in determining doxorobucin accumulation.

<p>(A) <i>(i)</i> Specimen histogram of intracellular doxorubicin fluorescence (from >5000 cells) at different extracellular pH. <i>(ii)</i> Plot of intracellular doxorubicin (±coefficient of variation) versus intracellular pH. <i>Black circles:</i> intracellular pH manipulated by varying extracellular pH. <i>Grey symbols:</i> intracellular pH manipulated at constant extracellular pH. (B) Data from HCT116 monolayers treated with doxorobicin and Hoechst 33342. Ratio of doxorubicin fluorescence in nuclear (Hoechst 33342 positive) and non-nuclear regions quantifies the degree of drug accumulation in the nucleus.</p

Doxorubicin efficacy as a function of intracellular pH.

<p>Cell proliferation was measured using the CellTiter blue assay kit (quantified as a ratio of absorbance at 562 nm and 595 nm). Doxorubicin efficacy was determined from dose-response curves as the concentration which results in a 50% decrease in proliferation (EC₅₀). EC₅₀ was measured under six different conditions that change pH_i, with or without an associated change in pH_e. (A) Determining EC₅₀ under incubation with normal Tyrode solutions titrated to pH 7.4 (the control), 6.4 or 7.8. Incubation under these conditions also changes pH_i (n = 8 each). (B) Determining EC₅₀ under incubation with solutions at pH_e = 7.4 containing 50 µM DMA or lacking Na⁺ salts or Cl⁻ salts (n = 8 each). These manoeuvres change pH_i by altering the balance of acid/base fluxes across membranes (but do not alter pH_e significantly because of the dilution effect into the large extracellular volume). (C) EC₅₀ plotted against pH_i (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035949#pone-0035949-g004" target="_blank">Fig. 4Aii</a>). Alkaline pH_i increases doxorubicin efficacy (decreases EC₅₀).</p

Intracellular doxorubicin associates with a slowly-releasing intracellular binding site.

<p>(A) HCT116 cells superfused with Hepes/Mes buffer at pH = 7.4, 37°C. Doxorubicin (DOX; 50 µM) was applied transiently by switching rapidly between drug-free and drug-containing solution (average of 25 cells, ±SEM). (B) Proposed model with equilibria involving free and bound doxorubicin. (C) Mathematical simulation showing the fast rise of intracellular doxorubicin upon exposure, and its slow release upon reversal of the trans-membrane concentration gradient.</p