The copper chelator ATN-224 induces peroxynitrite-dependent cell death in hematological malignancies

Abstract

Chemoresistance due to oxidative stress resistance or upregulation of Bcl-2 contributes to poor outcome in the treatment of hematological malignancies. In this study, we utilize the copper-chelator drug ATN-224 (choline tetrathiomolybdate) to induce cell death in oxidative stress-resistant cells and cells overexpressing Bcl-2 by modulating the cellular redox environment and causing mitochondrial dysfunction. ATN-224 treatment decreases superoxide dismutase 1 (SOD1) activity, increases intracellular oxidants, and induces peroxynitrite-dependent cell death. ATN-224 also targets the mitochondria, decreasing both cytochrome c oxidase (CcOX) activity and mitochondrial membrane potential. The concentration of ATN-224 required to induce cell death is proportional to SOD1 levels, but independent of Bcl-2 status. In combination with doxorubicin, ATN-224 enhances cell death. In primary B-cell acute lymphoblastic leukemia patient samples, ATN-224 decreases the viable cell number. Our findings suggest that ATN-224's dual targeting of SOD1 and CcOX is a promising approach for treatment of hematological malignancies either as an adjuvant or as a single agent.

Figure 1. ATN-224 induced cell death in WEHI7.2 and WEHI7.2 variant cells

A. Immunoblot showing Bcl-2 (26 kDa) protein levels in WEHI7.2 (W), Hb12 (H) and 200R (R) cells. Immunoblot showing actin (42 kDa) protein levels to demonstrate similar loading. B. Cell viability in WEHI7.2 and WEHI7.2 variant cells treated with nanomolar concentrations of ATN-224. C. Caspase 3 activity measured in WEHI7.2 (24 h), Hb12 (18 h) and 200R (30 h) cells treated with vehicle (C) or ATN-224 (A). D. Propidium iodide uptake in WEHI7.2 (30 h), Hb12 (30 h) and 200R (36 h) cells treated with vehicle (C) or ATN-224 (A). All values are mean + SEM (n ≥). *, significantly different from vehicle treated control cells (p ≤ 0.05).

Figure 2. ATN-224 targets SOD1 and increases ROS

A. SOD activity in WEHI7.2 (W), Hb12 (H) and 200R (R) cells. B. SOD1 activity in WEHI7.2 and WEHI7.2 variant cells treated with ATN-224 at 3, 6, 12 and 24 h time points, relative to vehicle (C). C. Immunoblots showing SOD1 (18 kDa) protein levels in the WEHI7.2 and WEHI7.2 variant cells treated with vehicle (C) or ATN-224 at 6, 12 and 24 h time points. Immunoblots showing actin (42 kDa) protein levels to demonstrate similar loading. D. TEMPO-9-AC fluorescence in WEHI7.2 (18 h), Hb12 (12 h) and 200R (24 h) cells treated with vehicle (C) or ATN-224 (A). All values are mean + SEM (n ≥). *, significantly different from vehicle-treated control cells (p ≤ 0.05).

Figure 3. ATN-224 induced cell death is peroxynitrite-dependent

A. Cell viability in WEHI7.2 and WEHI7.2 variant cells treated with vehicle, ATN-224, 50 nM MnTE-2-PyP⁵⁺, 1 μM (OH)FeTM-4-PyP⁴⁺, or a combination of ATN-224 and either 50 nM MnTE-2-PyP⁵⁺ or 1 μM (OH)FeTM-4-PyP⁴⁺ for 48 h. B. Nitrotyrosine levels in WEHI7.2 and WEHI7.2 variant cells treated with vehicle (C) or ATN-224 (A) for 24 h. C. Cell viability in WEHI7.2 and WEHI7.2 variant cells treated with vehicle, ATN-224, 100 μM MnTBAP³⁻ or a combination of ATN-224 and 100 μM MnTBAP³⁻ for 48 h. D. Cell viability in U937 and Molt-4 cells treated with vehicle, ATN-224, 50 nM MnTE-2-PyP⁵⁺, 1 μM (OH)FeTM-4-PyP⁴⁺, or a combination of ATN-224 and either 50 nM MnTE-2-PyP⁵⁺ or 1 μM (OH)FeTM-4-PyP⁴⁺ for 72 h. E. Cell viability in U937 and Molt-4 cells treated with vehicle, ATN-224, 50 μM MnTBAP³⁻ or a combination of ATN-224 and 50 μM MnTBAP³⁻ for 72 h. All values are mean + SEM (n ≥). *, significantly different from ATN-224 treated control cells (p ≤ 0.05).

Figure 4. ATN-224 effects on the mitochondria

A. Immunoblot showing Bcl-2 (26 kDa) protein levels in WEHI7.2 and WEHI7.2 variant cells treated with vehicle or ATN-224 before and after caspase 3 activation. B. CcOX activity in WEHI7.2 and WEHI7.2 variant cells treated with vehicle (C) or ATN-224 (A) for 12 h. KCN (K) included as a positive control. C. Immunoblots showing CcOX subunits V_a (16.8 kDa) and V_b (13.7 kDa) protein levels in WEHI7.2 and WEHI7.2 variant cells treated with vehicle control or ATN-224. Immunoblots showing actin (42 kDa) protein levels to demonstrate similar loading. D. JC-1 aggregates in WEHI7.2 and WEHI7.2 variant cells treated with vehicle (C), 50 nM MnTE-2-PyP⁵⁺ (Mn), ATN-224 (A) or a combination of 50 nM MnTE-2-PyP⁵⁺ and ATN-224 (MnA) for 6 h. All values are mean + SEM (n ≥). *, significantly different from vehicle treated control cells (p ≤ 0.05).

Figure 5. Inhibition of SOD1 is important for ATN-224 induced cell death

A. Propidium iodide uptake in WEHI7.2 and WEHI7.2 variant cells treated with vehicle (C) or 2 mM KCN for 48 h. B. Tyrpan blue staining in Molt-4 ρ⁰ cells treated with ATN-224 for 24 and 48 h. All values are mean + SEM (n ≥). *, significantly different from vehicle treated control cells and ** significantly different from 24 and 48 h ATN-224 treated cells (p ≤ 0.05).

Figure 6. ATN-224 enhances paraquat and chemotherapeutic drug-induced cell death

A. Caspase 3 activity measured in WEHI7.2 (24 h), Hb12 (18 h) and 200R (30 h) cells treated with vehicle, ATN-224, 50 μM paraquat (PQ) or a combination of ATN-224 and paraquat. B. Cell viability in WEHI7.2 and WEHI7.2 variant cells treated with vehicle, ATN-224 (3 nM WEHI7.2; 4.5 nM Hb12 and 200R), doxorubicin (5 nM and 10 nM WEHI7.2 and 200R; 10 nM and 20 nM Hb12) or a combination of ATN-224 and doxorubicin. All values are mean + SEM (n ≥). *, significantly different from vehicle treated control cells (p ≤ 0.05). **, significantly different from ATN-224 treated cells (p ≤ 0.05).

Figure 7. ATN-224 induces cell death in primary B-ALL cells

7-AAD uptake in primary B-ALL cells with vehicle control or ATN-224 (12.5 nM and 25 nM). Each bar represents a single measurement.