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

ABSTRACT: 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.

SUBMITTER: Mi L

PROVIDER: S-EPMC2992988 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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

Mi Lixin L Sirajuddin Paul P Gan Nanqin N Wang Xiantao X

Analytical biochemistry 20100610 2

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 ...[more]

PMID: 20541518

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Project description:Microglia activation and proliferation are hallmarks of many neurodegenerative disorders and may contribute to disease pathogenesis. Neurons actively regulate microglia survival and function, in part by secreting the microglia mitogen interleukin (IL)-34. Both IL-34 and colony stimulating factor (CSF)-1 bind colony stimulating factor receptor (CSFR)1 expressed on microglia. Systemic treatment with central nervous system (CNS) penetrant, CSFR1 antagonists, results in microglia death in a dose dependent matter, while others, such as GW2580, suppress activation during disease states without altering viability. However, it is not known how treatment with non-penetrant CSF1R antagonists, such as GW2580, affect the normal physiology of microglia. To determine how GW2580 affects microglia function, C57BL/6J mice were orally gavaged with vehicle or GW2580 (80mg/kg/d) for 8 days. Body weights and burrowing behavior were measured throughout the experiment. The effects of GW2580 on circulating leukocyte populations, brain microglia morphology, and the transcriptome of magnetically isolated adult brain microglia were determined. Body weights, burrowing behavior, and circulating leukocytes were not affected by treatment. Analysis of Iba-1 stained brain microglia indicated that GW2580 treatment altered morphology, but not cell number. Analysis of RNA-sequencing data indicated that genes related to reactive oxygen species (ROS) regulation and survival were suppressed by treatment. Treatment of primary microglia cultures with GW2580 resulted in a dose-dependent reduction in viability only when the cells were concurrently treated with LPS, an inducer of ROS. Pre-treatment with the ROS inhibitor, YCG063, blocked treatment induced reductions in viability. Finally, GW2580 sensitized microglia to hydrogen peroxide induced cell death. Together, these data suggest that partial CSF1R antagonism may render microglia more susceptible to reactive oxygen and nitrogen species.

Project description:In our previous studies, we have shown that benzyl isothiocyanate (BITC) inhibits the growth of human pancreatic cancer cells by inducing apoptosis. In the present study, we demonstrate the activation of all the three (MAPK) family members [extracellular signal-regulated protein kinase (ERK), c-jun N-terminal kinase (JNK) and P38] in response to BITC treatment. Exposure of Capan-2 cells with varying concentrations of BITC for 24 h resulted in the phosphorylation (activation) of ERK at Thr202/Tyr204, JNK at Thr183/Tyr185 and P38 at Thr180/Tyr182, leading to the induction of apoptosis. Similar MAPK activation was also observed in MiaPaCa-2 cells in response to BITC treatment. However, normal human pancreatic ductal epithelial cells did not show the activation of MAPK's and remained unaffected by BITC treatment. To confirm the role of ERK, JNK and P38 in BITC-induced G(2)/M arrest and apoptosis, Capan-2 cells were pre-treated with MAPK-specific inhibitors or MAPK8-short hairpin RNA (shRNA) prior to BITC treatment. Significant protection from BITC-induced G(2)/M arrest was observed in the cells pre-treated with MAPK kinase (MEK-1) but not JNK or P38 inhibitors. On the other hand, BITC-induced apoptosis was almost completely abrogated in the cells pre-treated with MEK-1, JNK or P38 inhibitors. Similarly, MAPK8-shRNA also offered almost complete protection against BITC-induced G(2)/M arrest and apoptosis. Furthermore, we observed that BITC treatment leads to the generation of reactive oxygen species (ROS) in Capan-2 and MiaPaCa-2 cells, which in part was orchestrated by depletion of reduced glutathione (GSH) level. Blocking ROS generation with N-acetyl-L-cysteine (NAC) significantly prevented GSH depletion and activation of ERK and JNK but not P38. Further, NAC or tiron prevented G(2)/M arrest by blocking G(2)/M regulatory proteins and completely protected the cells from BITC-induced apoptosis. Taken together, our results suggest that BITC-mediated G(2)/M arrest is mediated through ERK activation, whereas apoptosis is via ERK, JNK and P38.

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

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

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