Pro-oxidant and antioxidant effects of N-acetylcysteine regulate doxorubicin-induced NF-kappa B activity in leukemic cells

Abstract

Clinical debate has arisen over the consequences of antioxidant supplementation during cancer chemotherapy. While antioxidants may impede the efficacy of chemotherapy by scavenging reactive oxygen species and free radicals, it is also possible that antioxidants alleviate unwanted chemotherapy-induced toxicity, thus allowing for increased chemotherapy doses. These contradictory assertions suggest that antioxidant supplementation during chemotherapy treatment can have varied outcomes depending on the cellular context. To gain a more robust understanding of the role that antioxidants play in chemotherapy, we investigated the dose-dependent effects of the antioxidant, N-acetylcysteine (NAC), on the redox-mediated regulation of intracellular signaling. In this study, we systematically evaluated the effect of Dox-induced ROS on the NF-κB pathway in a pediatric acute lymphoblastic leukemia (ALL) cell line by measuring the thiol-based oxidative modifications of redox-sensitive proteins within the pathway. We report a functional consequence of NAC supplementation during doxorubicin (Dox) chemotherapy administration via the NF-kappa B (NF-κB) signal transduction pathway. The ability of NAC to alter Dox-induced NF-κB activity is contingent on the ROS-mediated S-glutathionylation of IKK-β. Moreover, the NAC-dependent alteration of intracellular glutathione redox balance, through pro-oxidant and antioxidant mechanisms, can be exploited to either promote or inhibit Dox-induced NF-κB activity in an NAC-concentration-dependent manner. We developed an electron-transfer-based computational model that predicts the effect of NAC pretreatment on Dox-induced NF-κB signaling for a range of NAC and Dox treatment combinations.

Fig. 1. Mechanistic model of glutathione-dependent IKK-β S-glutathionyl-ation

Schematic representation of the proposed reactions involved in glutathione-dependent IKK-β S-glutathionylation. Doxorubicin treatment promotes the formation of hydrogen peroxide (H₂O₂) and protein thiyl radicals (RS^•). Once formed, H₂O₂ mediates the S-glutathionylation of reduced IKK-β via the peroxide-dependent oxidation of IKK-β. Concurrently, doxorubicin-induced RS^• formation induces IKK-β S-gluta-thionylation via the radical-dependent oxidation of IKK-β. However, increased thiyl radical levels simultaneously promote the radical oxidation of GSH into the glutathione thiyl radical (GS^•) which serves to effectively diminish reduced GSH levels from the intracellular environment. NAC, the GSH precursor, regulates the peroxide-dependent and the radical-dependent mechanisms of IKK-β S-glutathionylation via its ability to promote glutathione synthesis as well as its ability to contribute to free-radical formation in the presence of H₂O₂.

Fig. 2. Doxorubicin treatment induces ROS in EU1 cells that can be attenuated by N-acetylcysteine (NAC) pre-treatment

Time-dependent doxorubicin-induced H₂O₂ in EU1 cells, with and without NAC pre-treatment, quantified by plate reader measurement of DCF fluores-cence (MFI = mean fluorescent intensity). ([NAC] = 1 mM, 30 min pre-treatment; [Dox] = 5 μM, 1 h treatment; *p o 0.05)

Fig. 3. Differential sensitivities of NF-κB-related proteins result in selective ROS-induced IKK-β S-glutathionylation

(A) Schematic of a subset of the proteins in the NF-κB activation pathway, their redox sensitive cysteine residues, and the oxidative modifications of the cysteine residues that have been reported to alter NF-κB activity. (B) NF-κB activity, quantified by relative luciferase induction (RLU = relative light units), in untreated and doxorubicin-treated EU1 cells with and without pre-treatment with NAC or IKK-β inhibitor, SC-514. Representative immunoblot analysis, with accompanying densitometry quantification normalized to lane 1, of (C) doxorubicin-induced NEMO dimerization, (D) doxorubicin-induced IKK-β S-glutathionylation, and (E) doxorubicin-induced IκB-α S-glutathionylation in EU1 cells, with and without NAC pretreatment. ([NAC] = 1 mM, 30 min pre-treatment; [SC-514] = 100 μM, 1 h pre-treatment; [Dox] = 5 μM, 4 h treatment; *p < 0.05).

Fig. 4. NAC can modulate glutathione redox status and control Dox-induced IKK-β S-glutathionylation and NF-κB activity in a dose-dependent manner

(A) and (B) EU1 cells were treated with various concentrations of NAC prior to doxorubicin administration. (A) the intracellular GSH/GSSG ratio as quantified by an enzymatic recycling assay, and (B) intracellular ROS measurements of DCF fluorescence. (C) and (D) Luciferase-transfected and non-transfected EU1 cells, respectively, were pretreated with various concentrations of NAC prior to doxorubicin administration and then lysed. (C) intracellular levels of S-glutathionylated IKK-β quantified by IP and western blot, and (D) NF-κB reporter activity as detected by luciferase luminescence.

Fig. 5. An electron-transfer-based computational model predicts pro-oxidant and anti-oxidant mechanisms by which NAC modulates Dox-induced IKK-β S-glutathionylation.<

br>(A) Model-fitted and experimentally-derived values for doxorubicin-induced IKK-β S-glutathionylation after various NAC pretreatment regimens. (B) Model-predicted and experimentally-derived values for IKK-β S-glutathionylation after various NAC pretreatment regimens without doxorubicin treatment.

Fig. 6. The combinatorial effect of NAC and Dox on NF-κB signaling is complex and adaptable

In silico predictions of the degree of IKK-β S-glutathionylation (% of total) that would occur after 1 h treatments with various dose combinations of Dox and NAC under two distinctly different GSH : GSSG redox balances (30 : 1 and 100 : 1). Color scale represents the percentage of IKK-β-SSG.