Figure 5
CaMKII-independent P2X7-mediated killing.
(A) qPCR showing the extent of gene knockdown in 4T1.2 cells transfected with shRNA targeting the P2X7 receptor versus non-targeting (NT) control (left panel). The right panel shows that P2X7 knockdown does not compromise cancer cell growth and viability. One million wt, shRNA(NT) and shRNA(P2X7) 4T1.2 cells were plated and incubated for 48h before evaluation of cell numbers and viability. (B) P2X7 knockdown cells are more resistant to ATP and the ATP/IVM combo. 4T1.2 cells were treated for 24 h with 1–3 mM ATP, 6–10 μM Ivermectin, or combinations of ATP and Ivermectin. P2X7 knockdown suppresses the synergy between Ivermectin and high dose ATP, as shown by calculation of combination index (CI) values, see Table 2. (C) P2X7 knockdown cells are resistant to ATP/Ivermectin induced membrane permeabilization (Asterisk (*) indicates p < 0.05). (D) The combination of Ivermectin (16 μM) and exogenous ATP (3 mM) can synergistically kill even some resistant human cancer cell lines like breast (MCF7), prostate (DU-145), head and neck (A253) and ovarian (SKOV3) cancer cells. (E) Comparison of the protective effects of P2X7, CaMKII and NADPH oxidases inhibition in short-term (4 h, 32 μM IVM) and long-term (24 h, 12 μM IVM) exposure of 4T1.2 cells to Ivermectin. Asterisk (*) indicates p < 0.05 relative to Ivermectin alone controls.
