Molecular and Cellular Pharmacology
Withaferin A inhibits iNOS expression and nitric oxide production by Akt inactivation
and down-regulating LPS-induced activity of NF-κB in RAW 264.7 cells
Jung Hwa Oh, Tae-Jin Lee
, Jong-Wook Park, Taeg Kyu Kwon
Department of Immunology, School of Medicine, Keimyung University, 194 DongSan Dong Jung-Gu, Taegu, 700-712, South Korea
Received 7 April 2008
Received in revised form 26 August 2008
Accepted 4 September 2008
Available online 24 September 2008
Induction of inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production is thought to
have beneﬁcial immunomodulatory effects in acute and chronic inﬂammatory disorders. In Raw 264.7 cells
stimulated with lipopolysaccharide (LPS) to mimic inﬂammation, withaferin A inhibited LPS-induced
expression of both iNOS protein and mRNA in a dose-dependent manner. To investigate the mechanism by
which withaferin A inhibits iNOS gene expression, we examined activation of mitogen-activated protein
kinases (MAPKs) and Akt in Raw 264.7 cells. We did not observe any signiﬁcant changes in the
phosphorylation of p38 MAPK in cells treated with LPS alone or LPS plus withaferin A. However, LPS-
induced Akt phosphorylation was markedly inhibited by withaferin A, while the phosphorylation of p42/p44
extracellular signal-regulated kinases (ERKs) was slightly inhibited by withaferin A treatment. Withaferin A
prevented IκB phosphorylation, blocking the subsequent nuclear translocation of nuclear factor-κB (NF-κB)
and inhibiting its DNA binding activity. LPS-induced p65 phosphorylation, which is mediated by extracellular
signal-regulated kinase (ERK) and Akt pathways, was attenuated by withaferin A treatment. Moreover, LPS-
induced NO production and NF-κB activation were inhibited by SH-6, a speciﬁc inhibitor of Akt. Taken
together, these results suggest that withaferin A inhibits inﬂammation through inhibition of NO production
and iNOS expression, at least in part, by blocking Akt and subsequently down-regulating NF-κB activity.
© 2008 Elsevier B.V. All rights reserved.
The intercellular messenger nitric oxide (NO) is a short-lived free
radical that plays an important role in the physiology and pathophysiol-
ogy of the central nervous, cardiovascular, and immune systems
(Bogdan et al., 2001; Moncada et al., 1991). NO is synthesized by nitric
oxide synthase (NOS) from
-arginine using NADPH and molecular
oxygen (MacMicking et al., 1997). Three isoforms of NOS have been
identiﬁed and are classiﬁed into two major categories: constitutive and
inducible. The overproduction of NO by inducible NOS (iNOS) is
implicated in the pathogenesis of various inﬂammatory diseases
(Moncada et al., 1991; MacMicking et al., 1997; Liu et al., 1995; Alderton
et al., 2001). The various inducers of iNOS expression have been shown
to activate different signaling pathways (Kleinert et al., 2003). Expres-
sion of the iNOS gene is regulated at different levels, including
transcriptional, posttranscriptional, translational and posttranslational
(Vodovotz et al., 1996; Rao et al., 2000). The transcription factor, nuclear
factor-κB(NF-κB), has been implicated as a central target for stimuli that
activate or inhibit iNOS expression (Ghosh et al., 1998).
Withaferin A is a steroidal lactone puriﬁed from Withania somnifera.
It exhibits a wide variety of activities, including antitumor, anti-
inﬂammatory, and immunomodulatory properties (Devi et al., 1992;
Agarwal et al., 1999). Recent reports have helped to clarify certain
aspects of withaferin A's bioactive properties, demonstrating that it
alters cytoskeletal architecture by covalently binding annexin II (Falsey
et al., 2006), exerts antitumor activity by inhibiting proteasomal
chymotrypsin-like activity (Yang et al., 2007), and induces apoptosis
through theinhibition of protein kinase C (Sen et al., 2007). However, the
cellular and molecular mechanisms underlying withaferin A-induced
inhibition of NO production in macrophages are not known.
In this study, we found that withaferin A inhibited LPS-induced NO
production and iNOS expression in Raw 264.7 cells and showed that
these effects are mediated, at least in part, by inhibiting Akt activation
and subsequently down-regulating NF-κB activity.
2. Materials and methods
2.1. Cells and materials
LPS (Escherichia coli serotype O26:B6), withaferin A, and all
reagents were purchased from Sigma-Aldrich unless otherwise stated.
SH-6 was purchased from Alexis Biochemicals (San Diego CA). The
macrophage cell line, Raw 264.7, was obtained from the American
Type Culture Collection (Rockville, MD) and cultured in RPMI 1640
European Journal of Pharmacology 599 (2008) 11–17
⁎ Corresponding author. Department of Immunology, School of Medicine, Keimyung
University, 194 DongSan-Dong Jung-Gu, Taegu, 700-712, South Korea. Tel.: +82 53 250
7846; fax: +82 53 250 7074.
E-mail address: email@example.com (T.K. Kwon).
Present address: Department of Anatomy, College of Medicine, Yeungnam
University, 317-1 Daemyoung-Dong Nam-Gu, Taegu 705-717, Korea.
0014-2999/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
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