N‑acetylcysteine induces apoptosis via the mitochondria‑dependent pathway but not via endoplasmic reticulum stress in H9c2 cells

Abstract

N‑acetylcysteine (NAC), a precursor of glutathione, is a widely used thiol‑containing antioxidant and modulator of the intracellular redox state. Our previous study demonstrated that excess reduced glutathione (GSH) from NAC treatment paradoxically led to a reduction in glutathione redox potential, increased mitochondrial oxidation and caused cytotoxicity at lower reactive oxygen species levels in H9c2 cells. However, no detailed data are available on the molecular mechanisms of NAC‑induced cytotoxicity on H9c2 cells. In the present study, it was demonstrated that NAC‑induced cytotoxicity towards H9c2 cells was associated with apoptosis. The activation of caspase‑9 and ‑3, and cleavage of procaspase‑9 and ‑3, but not of caspase‑8, were involved in NAC‑induced apoptosis. The dissipation of mitochondrial transmembrane potential, release of cytochrome c, translocation of B cell lymphoma‑2 (Bcl‑2)‑associated X protein (Bax) to the mitochondria, and the increased ratio of Bax/Bcl‑2 mRNA indicated that NAC treatment‑induced apoptosis occurred mainly through the mitochondria‑dependent pathway. Redox western blot analysis demonstrated that NAC did not disrupt the highly oxidized environment of the endoplasmic reticulum, which was indicated by maintenance of the oxidized form of protein disulfide isomerase, an essential chaperone in the formation of disulfide bond formation in the endoplasmic reticulum. In addition, no significant changes in the expression of binding immunoglobulin protein or C/EBP homologous protein were apparent in the process of NAC‑induced apoptosis. Taken together, the present study demonstrated for the first time, to the best of our knowledge, that NAC induced apoptosis via the mitochondria‑dependent pathway but not via endoplasmic reticulum stress in H9c2 cells, and the exogenous GSH from NAC did not alter the oxidized milieu of the endoplasmic reticulum.

Figure 1.

NAC induces concentration- and time-dependent apoptosis in H9c2 cells. (A) Cell viabilities were determined using an alamarBlue^® assay. The values were calculated relative to the control group. (B) LDH activity was measured in the supernatant of NAC-treated H9c2 cells. The results are presented as the mean ± standard deviation and are representative of three independent experiments. (C) Cells were treated with NAC and H₂O₂ for 24 h. The nuclear morphology of H9c2 cells was observed under a fluorescent microscope following Hoechst 33342 and propidium iodide staining (magnification, ×400). (D) NAC-treated cells were stained with Annexin V-FITC and propidium iodide followed by flow cytometric analysis. The Annexin V-positive cells were regarded as apoptotic. Values are presented as the mean ± standard deviation (n=3). *P<0.05 and **P<0.01 vs. Ctrl group. NAC, N-acetylcysteine; Ctrl, control; LDH, lactate dehydrogenase.

Figure 2.

NAC-induced apoptosis is mediated by the activation of caspase-9 and −3 in H9c2 cells. H9c2 cells were incubated with 4 µM NAC for indicated durations. The enzymatic activities of (A) caspase-3, (B) caspase-9 and (C) caspase-8 were measured using fluorescence assay kits. The results are presented as the mean ± standard deviation and are representative of three independent experiments. **P<0.01 vs. Ctrl group. (D) Whole cell lysates were subjected to western blot analysis to detect cleavage of procaspase-3 and −9 using cleaved procaspase-9 and −3 antibodies. The data shown are representative of three independent experiments. NAC, N-acetylcysteine; Ctrl, control.

Figure 3.

NAC induces apoptosis through activation of the intrinsic mitochondrial signaling pathway in H9c2 cells. (A) Whole cell lysates were subjected to western blot analysis to detect Fas and FasL. Equal loading was confirmed by reprobing the blots with an antibody against GAPDH. The data shown are representative of three independent experiments. (B) Mitochondrial membrane potential was detected using JC-1 on an Flx800 plate reader. The results are presented as the mean ± standard deviation and are representative of three independent experiments. *P<0.05 and **P<0.01 vs. Ctrl group. (C) Levels of cytochrome c and Bax were measured in the cytosolic and mitochondrial fractions using western blot analysis. VDAC and GAPDH were used as internal controls for the mitochondrial and the cytosolic fractions, respectively. All data shown are representative of three independent experiments. (D) mRNA expression levels of BAX and Bcl-2 were detected using reverse transcription-quantitative polymerase chain reaction analysis. Values are presented as the mean ± standard deviation (n=3). **P<0.01 vs. Ctrl group. NAC, N-acetylcysteine; Ctrl, control; FasL, Fas ligand; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.

Figure 4.

NAC treatment does not induce ER stress. (A) Redox western blot analysis was performed to investigate the redox state of PDI following NAC or DTT treatment. For reducing SDS-PAGE, 5% DTT was added to the samples. (B) Whole cell lysates were subjected to western blot analysis to detect BiP and CHOP following NAC or tunicamycin treatment. Equal loading was confirmed by reprobing of the blots with an antibody against GAPDH. All data shown are representative of three independent experiments. (C) Densitometry values of BiP and CHOP after NAC or tunicamycin treatment are represented as relative intensities to GAPDH in mean arbitrary units using ImageJ software version 1.50i. The levels were averaged over three experiments and plotted. *P<0.05 or ^#P<0.001 vs. Ctrl. (D) H9c2 cells were incubated with 4 µM NAC for the indicated durations. The enzymatic activities of caspase-12 were measured using fluorescence assay kits. The results are presented as the mean ± standard deviation and are representative of three independent experiments. NAC, N-acetylcysteine; Ctrl, control; DTT, dithiothreitol; PDI, protein disulfide isomerase; BiP, binding immunoglobulin protein; CHOP, C/EBP homologous protein.