Figure 2.

Flow chart of major steps in CAP leading to selective apoptosis of tumor cells. Step 1: CAP generates NO₂⁻ and H₂O₂ in cell containing medium for 1 minute. Alternatively, CAP is used to treat medium, creating PAM (step 1′). Defined concentrations of NO₂⁻ and H₂O₂ containing medium are used in reconstitution experiments (step 1”). Step 2: NO₂⁻- and H₂O₂ create primary ¹O₂ near cells following O₂NOOH pathway, as described in Figure 1. Step 3: Few catalase molecules on a few cells are inactivated due to primary ¹O₂ near cells. Step 4: At the site of inactivated catalase, H₂O₂ and ONOO⁻ (generated through NOX1 and NOS) are no longer decomposed. Step 5: The reaction between H₂O₂ and ONOO⁻ is leading ultimately to secondary ¹O₂. Step 6: This additional ¹O₂ leads to further catalase inactivation and the process cycles back to step 4. Step 7: Increased H₂O₂ resulting from catalase loss from secondary ¹O₂ leads to H₂O₂ entering cells via aquaporins, leading to antioxidant glutathione depletion. Step 8: In parallel with step 7, increased H₂O₂ resulting from catalase loss from secondary ¹O₂ also leads to HOCl generation by peroxidase, in the presence of Cl⁻. The interaction between NOX1-derived O₂^●− leads to ^●OH formation near the cell membrane and lipid oxidation. Step 8′: If HOCl signaling is suppressed, an alternative ^●NO/ONOO⁻ signaling can also lead to lipid peroxdiation. Step 9: If both lipid peroxidation and glutathione depletion occurs, then caspase-associated apoptosis can take place, finally leading to cell death. Steps 1–3 correspond to CAP triggering or activation of a few cells, thereby initiating propagating bystander signaling in steps 4–6. Steps 7–9 are the steps that lead to the final cell apoptosis. These steps are activated only if the repeated performance of steps 4–6 has caused a sufficiently high degree of catalase inactivation for reactivation of HOCl or ^●NO/ONOO⁻ - mediated apoptosis-inducing signaling.