Selective inhibition of human carbonic anhydrase IX in Xenopus oocytes and MDA-MB-231 breast cancer cells

Figures & data

Figure 1. Measurements of cytosolic H⁺changes in native Xenopus oocytes (B) and oocytes expressing target CA isoform (A, C–H). Cells were injected with 5 ng CA IX-cRNA (A, D, F and H) or 6 ng CA II-cRNA (C, E and G) during application of 5% CO₂/25 mM HCO₃^- (from a nominally CO₂-free, HEPES-buffered solution) in the absence of compounds and in the presence of 10 μM VD11-4-2 (C and D), 10 μM VD12-09 (E and F) and 10 μM EA2–3 (G and H). * indicates a significance level of p ≤ 0.05, ** indicate a significance level of p ≤ 0.01, *** indicate a significance level of p ≤ 0.001.

Figure 2. Dose-dependent inhibition of intracellular CA IX catalytic activity with VD11-4-2 in Xenopus oocytes. (A) Recordings of [H⁺]_i in oocytes injected with 5 ng CA IX-cRNA during application of 5% CO₂/25 mM HCO₃^- (from a nominally CO₂-free, HEPES-buffered solution) in the absence and presence of 1 nM to 10 μM of VD11-4-2. (B) The dependence of changes in [H⁺]_i on added VD11-4-2 concentrations. Black data points were obtained experimentally. The solid line was simulated according to the Hill model with variable slope.

Figure 3. Measurements of [H⁺]_s in Xenopus oocytes injected with 5 ng CA IX-cRNA (A and C) and native oocytes (B) during application of 5% CO₂/25 mM HCO₃^- (from a nominally CO₂-free, HEPES-buffered solution) in the absence and presence of 10 μM VD11-4-2 and after 15 min of washing out. ** indicate a significance level of p ≤ 0.01, *** indicate a significance level of p ≤ 0.001.

Figure 4. Dose-dependent inhibition of extracellular CA IX catalytic activity with VD11-4-2 in Xenopus oocytes. (A) Recordings of [H⁺]_s in oocytes injected with 5 ng CA IX-cRNA during application of 5% CO₂/25 mM HCO₃^- (from a nominally CO₂-free, HEPES-buffered solution) in the absence and presence of 0.1 nM to 10 μM of VD11-4-2. (B) The dependence of changes in [H⁺]_s as a function of added VD11-4-2 concentrations. The data points were obtained experimentally while the solid line was simulated according to the Hill model with varying slope.

Figure 5. Measurements of [H⁺]_s in oocytes injected with 1 ng CA IV-cRNA (A) or 5 ng CA XII-cRNA (B) during application of 5% CO₂/25 mM HCO₃^- (from a nominally CO₂-free, HEPES-buffered solution) in the absence and presence of 50 nM and 10 μM VD11-4-2 and after 15 min of washing out. ** indicate a significance level of p ≤ 0.01, *** indicate a significance level of p ≤ 0.001.

Figure 6. Recordings of the degradation of ¹⁸O-labeled CO₂ (A) and evaluations of CA IX activity (B) in the absence and presence of 1 and 10 nM VD11-4-2 in lysed oocytes as measured by mass spectrometry. The addition of oocyte lysate made from 20 cells and different concentrations of VD11-4-2 are shown by arrows. *** indicate a significance level of p ≤ 0.001.

Figure 7. Inhibition of CA activity with VD11-4-2 in MDA-MB-231 breast cancer cells. (A) Recordings of pH_i in MDA-MB-231 cells, kept under normoxic (21% O₂, A₁) or hypoxic (1% O₂, A₂) conditions, during application of 5% CO₂/15 mM HCO₃^- (pH_o 7.2) in the absence and presence of 5 nM, 50 nM and 1 μM of VD11-4-2. (B) Rate of change in pH_i in normoxic (white) and hypoxic (black) MDA-MB-231 cells, induced by application of 5% CO₂/15 mM HCO₃^-, as a function of VD11-4-2 concentrations. The asterisks refer to the values in the absence of VD11-4-2. (C) Rate of change in pH_i in normoxic (white) and hypoxic (black) MDA-MB-231 cells, induced by application of 5% CO₂/15 mM HCO₃^-, in the absence (control) and presence of 30 μM EZA.