The transcription factor Nrf2 regulates the basal and inducible expression of a battery of cytoprotective genes. Whereas numerous Nrf2-inducing small molecules have been reported, very few chemical inhibitors of Nrf2 have been identified to date. The quassinoid brusatol has recently been shown to inhibit Nrf2 and ameliorate chemoresistance in vitro and in vivo. Here, we show that brusatol provokes a rapid and transient depletion of Nrf2 protein, through a posttranscriptional mechanism, in mouse Hepa-1c1c7 hepatoma cells. Importantly, brusatol also inhibits Nrf2 in freshly isolated primary human hepatocytes. In keeping with its ability to inhibit Nrf2 signaling, brusatol sensitizes Hepa-1c1c7 cells to chemical stress provoked by 2,4-dinitrochlorobenzene, iodoacetamide, and N-acetyl-p-benzoquinone imine, the hepatotoxic metabolite of acetaminophen. The inhibitory effect of brusatol toward Nrf2 is shown to be independent of its repressor Keap1, the proteasomal and autophagic protein degradation systems, and protein kinase signaling pathways that are known to modulate Nrf2 activity, implying the involvement of a novel means of Nrf2 regulation. These findings substantiate brusatol as a useful experimental tool for the inhibition of Nrf2 signaling and highlight the potential for therapeutic inhibition of Nrf2 to alter the risk of adverse events by reducing the capacity of nontarget cells to buffer against chemical and oxidative insults. These data will inform a rational assessment of the risk:benefit ratio of inhibiting Nrf2 in relevant therapeutic contexts, which is essential if compounds such as brusatol are to be developed into efficacious and safe drugs.
PEGylation of therapeutic proteins is commonly used to
extend half-lives
and to reduce immunogenicity. However, reports of antibodies toward
PEGylated proteins and of poly(ethylene glycol) (PEG) accumulation
suggest that efficacy and safety concerns may arise. To understand
the relationship among the pharmacology, immunogenicity, and toxicology
of PEGylated proteins, we require knowledge of the disposition and
metabolic fate of both the drug and the polymer moieties. The analysis
of PEG by standard spectrophotometric or mass spectrometric techniques
is problematic. Consequently, we have examined and compared two independent
analytical approaches, based on gel electrophoresis and nuclear magnetic
resonance (NMR) spectroscopy, to determine the biological fate of
a model PEGylated protein, 40KPEG-insulin, within a rat
model. Both immunoblotting with an antibody to PEG and NMR analyses
(LOD 0.5 μg/mL for both assays) indicated that the PEG moiety
remained detectable for several weeks in both serum and urine following
intravenous administration of 40KPEG-insulin (4 mg/kg).
In contrast, Western blotting with anti-insulin IgG indicated that
the terminal half-life of the insulin moiety was far shorter than
that of the PEG, providing clear evidence of conjugate cleavage. The
application of combined analytical techniques in this way thus allows
simultaneous independent monitoring of both protein and polymer elements
of a PEGylated molecule. These methodologies also provide direct evidence
for cleavage and definition of the chemical species present in biological
fluids which may have toxicological consequences due to unconjugated
PEG accumulation or immunogenic recognition of the uncoupled protein.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.