Carbonic anhydrase IX promotes tumor growth and necrosis in vivo and inhibition enhances anti-VEGF therapy.

PURPOSE: Bevacizumab, an anti-VEGFA antibody, inhibits the developing vasculature of tumors, but resistance is common. Antiangiogenic therapy induces hypoxia and we observed increased expression of hypoxia-regulated genes, including carbonic anhydrase IX (CAIX), in response to bevacizumab treatment in xenografts. CAIX expression correlates with poor prognosis in most tumor types and with worse outcome in bevacizumab-treated patients with metastatic colorectal cancer, malignant astrocytoma, and recurrent malignant glioma. EXPERIMENTAL DESIGN: We knocked down CAIX expression by short hairpin RNA in a colon cancer (HT29) and a glioblastoma (U87) cell line which have high hypoxic induction of CAIX and overexpressed CAIX in HCT116 cells which has low CAIX. We investigated the effect on growth rate in three-dimensional (3D) culture and in vivo, and examined the effect of CAIX knockdown in combination with bevacizumab. RESULTS: CAIX expression was associated with increased growth rate in spheroids and in vivo. Surprisingly, CAIX expression was associated with increased necrosis and apoptosis in vivo and in vitro. We found that acidity inhibits CAIX activity over the pH range found in tumors (pK = 6.84), and this may be the mechanism whereby excess acid self-limits the build-up of extracellular acid. Expression of another hypoxia inducible CA isoform, CAXII, was upregulated in 3D but not two-dimensional culture in response to CAIX knockdown. CAIX knockdown enhanced the effect of bevacizumab treatment, reducing tumor growth rate in vivo. CONCLUSION: This work provides evidence that inhibition of the hypoxic adaptation to antiangiogenic therapy enhances bevacizumab treatment and highlights the value of developing small molecules or antibodies which inhibit CAIX for combination therapy

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

The role of carbonic anhydrase IX in tumour biology

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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 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

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