A cautionary note on using N-acetylcysteine as an antagonist to assess isothiocyanate-induced reactive oxygen species-mediated apoptosis

Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057, USA.
Analytical Biochemistry (Impact Factor: 2.22). 10/2010; 405(2):269-71. DOI: 10.1016/j.ab.2010.06.015
Source: PubMed


N-Acetylcysteine (NAC) has been widely used in cell culture-based studies for the role of reactive oxygen species (ROS) generation in apoptosis induction by isothiocyanates (ITCs). Here we have demonstrated, using [(14)C]phenethyl ITC and [(14)C]sulforaphane, that NAC pretreatment significantly reduces ITC cellular uptake by conjugating with ITCs in the medium, suggesting that reduced uptake of ITCs, rather than the antioxidant activity of NAC itself, is responsible for the diminished downstream apoptotic effects. The study provides a cautionary note on the assay in studying mechanisms of apoptosis by ITCs and other electrophilic and thiol-reactive compounds.

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    • "However, an alternative interpretation of our results is that XB05 can react directly with thiol groups, which are present in NAC or GSH but not AA. Reaction with NAC or GSH could potentially inhibit XB05 activity by sequestering the compound in the medium and prevent it from entering cells, as previously described for another compound [33]. To assess this possibility, we chose to examine the in vitro reactivity of XB05 with relevant thiols using the 5,5′-dithiobis-2-nitrobenzoic acid assay. "
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    ABSTRACT: We have synthesized a novel molecule named XB05 (1-bromo-1,1-difluoro-non-2-yn-4-ol) and evaluated its effects in a variety of human cell lines. XB05 displayed potent antiproliferative activity against cell lines derived from leukemia or solid tumors, but had less effect on non-malignant cells. To identify factors that contribute to the cancer-selectivity of XB05, we chose three cell lines that had high sensitivity to XB05 (U937 leukemia), moderate sensitivity (A549 lung cancer), or low sensitivity (Hs27 non-malignant skin fibroblasts), and proceeded to assess cell death and oxidative stress in these cells. XB05 was found to induce cell death via both apoptotic and nonapoptotic mechanisms in U937 and A549 cells, whereas it had no cytotoxicity against Hs27 cells at comparable concentrations. Treatment with XB05 caused an increase in reactive oxygen species in all cell lines tested, but levels were higher in malignant compared to non-malignant cells. XB05 treatment also induced DNA damage exclusively in the malignant cells. Differences in antioxidant responses were observed between cell lines. For example, XB05 caused a decrease in levels of glutathione and nuclear Nrf2 in the most sensitive cells (U937), whereas the least sensitive cells (Hs27) displayed increased glutathione levels and no change in nuclear Nrf2. XB05 could react in vitro with cysteine and glutathione, but had much lower reactivity compared to typical thiol-reactive electrophiles, diethyl maleate and maleimide. In summary, XB05 is a novel compound that selectively kills malignant cells, most likely by disrupting cellular redox homeostasis, making it a promising candidate for development as a chemotherapeutic agent.
    Free Radical Biology and Medicine 12/2013; 68. DOI:10.1016/j.freeradbiomed.2013.12.002 · 5.74 Impact Factor
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    • "In addition, NAC and GSH rescued the decrease of the mitochondria membrane potential and restored apoptosis-related proteins, such as capsase-9, caspase-3, PARP, p53 and p21, after physalin F treatment in A498 cells. Because NAC is a precursor of GSH, it has been reported to increase intracellular levels of glutathione, which is important to the prevention of oxidative stress in cells [30]. In addition, NAC and GSH might protect cells from damage caused by conjugation with electrophiles [30], [31]. "
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    ABSTRACT: The aim of this study was to determine the molecular mechanisms of physalin F, an effective purified extract of Physalis angulata L. (Solanacae), in renal carcinoma A498 cells. Physalin F was observed to significantly induce cytotoxicity of three human renal carcinoma A498, ACHN, and UO-31 cells in a concentration-dependent manner; this was especially potent in A498 cells. The physalin F-induced cell apoptosis of A498 cells was characterized by MTT assay, nuclear DNA fragmentation and chromatin condensation. Using flow cytometry analysis, physalin F induced A498 cell apoptosis as demonstrated by the accumulation of the sub-G1 phase in a concentration- and time-dependent manner. Moreover, physalin F-mediated accumulation of reactive oxygen species (ROS) caused Bcl-2 family proteins, Bcl-2, and Bcl-xL degradation, which led to disruption of mitochondrial membrane potential and release of cytochrome c from the mitochondria into the cytosol. These effects were associated with induction of caspase-3 and caspase-9 activity, which led to poly(ADP-ribose) polymerase cleavage. However, the antioxidant N-acetyl-(L)-cysteine (NAC) and glutathione (GSH) resulted in the inhibition of these events and reversed physalin F-induced cell apoptosis. In addition, physalin F suppressed NF-κB activity and nuclear translocation of p65 and p50, which was reversed by NAC and GSH. Physalin F induced cell apoptosis through the ROS-mediated mitochondrial pathway and suppressed NF-κB activation in human renal cancer A498 cells. Thus, physalin F appears to be a promising anti-cancer agent worthy of further clinical development.
    PLoS ONE 07/2012; 7(7):e40727. DOI:10.1371/journal.pone.0040727 · 3.23 Impact Factor
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