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

Carbonic anhydrase IX is a pH-stat that sets an acidic tumour extracellular pH in vivo

Background Tumour Carbonic Anhydrase IX (CAIX), a hypoxia-inducible tumour-associated cell surface enzyme, is thought to acidify the tumour microenvironment by hydrating CO2 to form protons and bicarbonate, but there is no definitive evidence for this in solid tumours in vivo. Methods We used 1H magnetic resonance spectroscopic imaging (MRSI) of the extracellular pH probe imidazolyl succinic acid (ISUCA) to measure and spatially map extracellular pH in HCT116 tumours transfected to express CAIX and empty vector controls in SCID mice. We also measured intracellular pH in situ with 31P MRS and measured lactate in freeze-clamped tumours. Results CAIX expressing tumours had 0.15 pH-unit lower median extracellular pH than control tumours (pH 6.71 tumour vs pH 6.86 control, P = 0.01). Importantly, CAIX expression imposed an upper limit for tumour extracellular pH at 6.93. Despite the increased lactate concentration in CAIX-expressing tumours, 31P MRS showed no difference in intracellular pH, suggesting that CAIX acidifies only the tumour extracellular space. Conclusions CAIX acidifies the tumour microenvironment, and also provides an extracellular pH control mechanism. We propose that CAIX thus acts as an extracellular pH-stat, maintaining an acidic tumour extracellular pH that is tolerated by cancer cells and favours invasion and metastasis.We are grateful for the support of CRUK [grant number C14303/A17197], the Breast Cancer Research Foundation, the Royal Society, Worldwide Cancer Research and the European Research Council [SURVIVE: 723397]. JP-T and SC received support from the Spanish Ministry of Economy and Competitiveness SAF2014-23622

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

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

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

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

Carbonic anhydrase IX is a pH-stat that sets an acidic tumour extracellular pH in vivo

[Background]: Tumour carbonic anhydrase IX (CAIX), a hypoxia-inducible tumour-associated cell surface enzyme, is thought to acidify the tumour microenvironment by hydrating CO2 to form protons and bicarbonate, but there is no definitive evidence for this in solid tumours in vivo.[Methods]: We used 1H magnetic resonance spectroscopic imaging (MRSI) of the extracellular pH probe imidazolyl succinic acid (ISUCA) to measure and spatially map extracellular pH in HCT116 tumours transfected to express CAIX and empty vector controls in SCID mice. We also measured intracellular pH in situ with 31P MRS and measured lactate in freeze-clamped tumours.[Results]: CAIX-expressing tumours had 0.15 pH-unit lower median extracellular pH than control tumours (pH 6.71 tumour vs pH 6.86 control, P = 0.01). Importantly, CAIX expression imposed an upper limit for tumour extracellular pH at 6.93. Despite the increased lactate concentration in CAIX-expressing tumours, 31P MRS showed no difference in intracellular pH, suggesting that CAIX acidifies only the tumour extracellular space.[Conclusions]: CAIX acidifies the tumour microenvironment, and also provides an extracellular pH control mechanism. We propose that CAIX thus acts as an extracellular pH-stat, maintaining an acidic tumour extracellular pH that is tolerated by cancer cells and favours invasion and metastasis.We are grateful for the support of CRUK (grant number C14303/A17197), the Breast Cancer Research Foundation, the Royal Society, Worldwide Cancer Research and the European Research Council (SURVIVE: 723397). J.P.-T. and S.C. received support from the Spanish Ministry of Economy and Competitiveness SAF2014-23622.Peer reviewe