N-acetylcysteine (NAC), often used as an antioxidant-scavenging reactive oxygen species (ROS) in vitro, was recently shown to increase the cytotoxicity of other compounds through ROS-dependent and ROS-independent mechanisms. In this study, NAC itself was found to induce extensive ROS production in human leukemia HL-60 and U937 cells. The cytotoxicity depends on ROS-modulating enzyme expression. In HL-60 cells, NAC activated NOX2 to produce superoxide (O₂•⁻). Its subsequent conversion into H₂O₂ by superoxide dismutase 1 and 3 (SOD1, SOD3) and production of ClO⁻ from H₂O₂ by myeloperoxidase (MPO) was necessary for cell death induction. While the addition of extracellular SOD potentiated NAC-induced cell death, extracellular catalase (CAT) prevented cell death in HL-60 cells. The MPO inhibitor partially reduced the number of dying HL-60 cells. In U937 cells, the weak cytotoxicity of NAC is probably caused by lower expression of NOX2, SOD1, SOD3, and by the absence of MOP expression. However, even here, the addition of extracellular SOD induced cell death in U937 cells, and this effect could be reversed by extracellular CAT. NAC-induced cell death exhibited predominantly apoptotic features in both cell lines. Conclusions: NAC itself can induce extensive production of O₂•⁻ in HL-60 and U937 cell lines. The fate of the cells then depends on the expression of enzymes that control the formation and conversion of ROS: NOX, SOD, and MPO. The mode of cell death in response to NAC treatment bears apoptotic and apoptotic-like features in both cell lines.

In laboratory experiments, many electrophilic cytotoxic agents induce cell death accompanied by reactive oxygen species (ROS) production and/or by glutathione (GSH) depletion. Not surprisingly, millimolar concentrations of N-acetylcysteine (NAC), which is used as a universal ROS scavenger and precursor of GSH biosynthesis, inhibit ROS production, restore GSH levels, and prevent cell death. The protective effect of NAC is generally used as corroborative evidence that cell death induced by a studied cytotoxic agent is mediated by an oxidative stress-related mechanism. However, any simple interpretation of the results of the protective effects of NAC may be misleading because it is unable to interact with superoxide (O₂•⁻), the most important biologically relevant ROS, and is a very weak scavenger of H₂O₂. In addition, NAC is used in concentrations that are unnecessarily high to stimulate GSH synthesis. Unfortunately, the possibility that NAC as a nucleophile can directly interact with cytotoxic electrophiles to form non-cytotoxic NAC–electrophile adduct is rarely considered, although it is a well-known protective mechanism that is much more common than expected. Overall, apropos the possible mechanism of the cytoprotective effect of NAC in vitro, it is appropriate to investigate whether there is a direct interaction between NAC and the cytotoxic electrophile to form a non-cytotoxic NAC–electrophilic adduct(s).

The Lysosomal sequestration of weak-base anticancer drugs is one putative mechanism for resistance to chemotherapy but it has never been directly proven. We addressed the question of whether the lysosomal sequestration of tyrosine kinase inhibitors (TKIs) itself contributes to the drug resistance in vitro. Our analysis indicates that lysosomal sequestration of an anticancer drug can significantly reduce the concentration at target sites, only when it simultaneously decreases its extracellular concentration due to equilibrium, since uncharged forms of weak-base drugs freely diffuse across cellular membranes. Even though the studied TKIs, including imatinib, nilotinib, and dasatinib, were extensively accumulated in the lysosomes of cancer cells, their sequestration was insufficient to substantially reduce the extracellular drug concentration. Lysosomal accumulation of TKIs also failed to affect the Bcr-Abl signaling. Cell pre-treatment with sunitinib significantly enhanced the lysosomal accumulation of the TKIs used; however, without apparent lysosomal biogenesis. Importantly, even increased lysosomal sequestration of TKIs neither decreased their extracellular concentrations nor affected the sensitivity of Bcr-Abl to TKIs. In conclusion, our results clearly show that the lysosomal sequestration of TKIs failed to change their concentrations at target sites, and thus, can hardly contribute to drug resistance in vitro.