ORIGINAL ARTICLE

Carbazole Is a Naturally Occurring Inhibitor of

Angiogenesis and Inflammation Isolated from
Antipsoriatic Coal Tar
Jack L. Arbiser1, Baskaran Govindarajan1, Traci E. Battle3, Rebecca Lynch3, David A. Frank3,
Masuko Ushio-Fukai2, Betsy N. Perry1, David F. Stern4, G. Tim Bowden5, Anquan Liu6, Eva Klein6,
Pawel J. Kolodziejski7, N. Tony Eissa7, Chowdhury F. Hossain8 and Dale G. Nagle8

Coal tar is one of the oldest and an effective treatment for psoriasis. Coal tar has been directly applied to the
skin, or used in combination with UV light as part of the Goeckerman treatment. The use of coal tar has caused
long-term remissions in psoriasis, but has fallen out of favor because the treatment requires hospitalization and
coal tar is poorly acceptable aesthetically to patients. Thus, determining the active antipsoriatic component of
coal tar is of considerable therapeutic interest. We fractionated coal tar into its components, and tested them
using the SVR angiogenesis inhibitor assay. Treatment of SVR endothelial cells with coal tar fractions resulted in
the isolation of a single fraction with antiangiogenic activity. The active antiangiogenic compound in coal tar is
carbazole. In addition to antiangiogenic activity, carbazole inhibited the production of inflammatory IL-15 by
human mononuclear cells. IL-15 is elevated in psoriasis and is thought to contribute to psoriatic inflammation.
Carbazole treatment also reduced activity of inducible nitric oxide synthase (iNOS), which is proinflammatory
and elevated in psoriasis. The effect of carbazole on upstream pathways in human psoriasis was determined,
and carbazole was shown to inhibit signal transducer and activator of transcription (stat)3-mediated
transcription, which has been shown to be relevant in human psoriasis. IL-15, iNOS, and stat3 activation
require the activation of the small GTPase rac for optimal activity. Carbazole was found to inhibit rac activation
as a mechanism for its inhibition of downstream inflammatory and angiogenic pathways. Given its
antiangiogenic and anti-inflammatory activities, carbazole is likely a major component of the antipsoriatic
activity of coal tar. Carbazole and derivatives may be useful in the therapy of human psoriasis.
Journal of Investigative Dermatology (2006) 126, 1396–1402. doi:10.1038/sj.jid.5700276; published online 13 April 2006

INTRODUCTION agents such as vitamin D and retinoids have a smaller role

Psoriasis is a common inflammatory condition found in
between 1 and 2% of the population. Patients can range from
mildly to severely affected, and the degree of involvement
dictates the treatment of psoriasis. Mild psoriasis is treated
primarily with topical glucocorticoids, and other topical
1
Department of Dermatology, Emory University School of Medicine, Atlanta,
Georgia, USA; 2Department of Cardiology, Emory University School of
Medicine, Atlanta, Georgia, USA; 3Dana Farber Cancer Institute, Harvard
Medical School, Boston, Massachusetts, USA; 4Department of Pathology,
Yale University, New Haven, Connecticut, USA; 5Arizona Cancer Center,
University of Arizona School of Medicine, Tuscon, Arizona, USA;
6
Microbiology and Tumor Center, Karolinska Institute, Stockholm, Sweden;
7
Baylor College of Medicine, Houston, Texas, USA and 8Department of
Pharmacognosy, School of Pharmacy, University of Mississippi University,
Mississippi, USA
Correspondence: Dr Jack L. Arbiser, Department of Dermatology, Emory
University School of Medicine, WMB 5309, Atlanta, Georgia 30322, USA.
E-mail: jarbise@emory.edu
Abbreviations: AP-1, activator protein-1; EGF, epidermal growth factor;
iNOS, inducible nitric oxide synthase; NK, natural killer; NMR, nuclear
magnetic resonance; stat, signal transducer and activator of transcription;
TNFa, tumor necrosis factor-alpha; VEGF, vascular endothelial growth factor
Received 9 August 2005; revised 19 January 2006; accepted 27 January 2006;
published online 13 April 2006
because of decreased efficacy. Severe psoriasis is often
treated with systemic medications, including methotrexate,
cyclosporine, and oral retinoids, and more recently with
biological agents, such as soluble receptors to tumor necrosis
factor-alpha (TNFa), or antibodies to TNFa receptors (Nickol-
off and Wrone-Smith, 1999; Saurat, 1999; Oh et al., 2000;
Gordon et al., 2003; Nickoloff and Nestle, 2004). Although
all of these therapies are effective in various settings, they are
all associated with side effects. Hepatotoxicity is a major
issue for long-term use of methotrexate and retinoids,
whereas hypertension, nephropathy, and systemic immuno-
suppression complicate the use of cyclosporine. Topical
glucocorticoids are effective in the control of mild psoriasis,
but even mild disease may become refractory to topical
glucocorticoids, or disease may flare once topical glucocor-
ticoids are withdrawn. Skin atrophy and breakdown are
common side effects of long-term topical glucocorticoid use,
due to its suppression of collagen synthesis. UV light is
effective in a significant number of patients with severe
psoriasis, but is complicated by the well-described develop-
ment of skin cancer after long-term use (Stern et al., 1984).

1396 Journal of Investigative Dermatology (2006), Volume 126 & 2006 The Society for Investigative Dermatology

Inhibitor of Angiogenesis and Inflammation Isolated from Antipsoriatic Coal Tar
Thus, there is a pressing need for effective topical therapies
for psoriasis, especially those working through mechanisms
that differ from those currently available.
Coal tar has been widely used in the treatment of psoriasis,
but little is known of its mode of action (Joshi and Pathak,
1984). We fractionated coal tar using chromatography and
tested the fractions using the SVR angiogenesis assay.
Potential mechanisms of activity of carbazole included
inhibition of EGFR activation, inhibition of activator pro-
tein-1 (AP-1) activity, and inhibition of proinflammatory
cytokine synthesis. We found that carbazole inhibits the
production of IL-15 by mononuclear cells, a critical cytokine
in psoriasis. We also found that carbazole inhibits activity of
nitric oxide synthase (NOS) and reduces inducible NOS
(iNOS) protein levels by a mechanism that involves co-
translational downregulation (Kolodziejski et al., 2004).
Signal transducer and activator of transcription (stat)3 has
recently been implicated as a major signaling pathway in
psoriasis, and is thought to be involved in the signaling of
both IL-15 and nitric oxide (Guren et al., 1999; Bromberg and
Chen, 2001; Niu et al., 2002). Stat3-mediated transcription is
inhibited by carbazole, although levels of phosphorylated
stat3 are not decreased by it, implying that a modifier of stat3
may underlie the activity of carbazole. Given that rac
activation has been implicated in stat3 activation, IL-15
production and NOS activity, we examined the effect of
carbazole on rac activation (Faruqi et al., 2001; Strengell
et al., 2003) and determined that carbazole inhibits rac
activation in endothelial cells. Carbazole and its derivatives
should be tested as antipsoriatic agents in humans.
RESULTS
Carbazole is the active ingredient of coal tar
9H-Carbazole 1. Compound 1 was obtained as white
amorphous solid; 1H nuclear magnetic resonance (NMR)
(CDCl3, 600 MHz) d 7.23 (2H, ddd, J ¼ 8.3, 7.2, 1.2 Hz, H-3,
H-6), 7.42 (2H, ddd, J ¼ 8.3, 7.8, 1.2 Hz, H-2, H-7), 7.43 (2H,
d, J ¼ 7.8 Hz, H-1, H-8), and 8.08 (2H, d, J ¼ 7.2 Hz, H-4, H-
5); 13C NMR (CDCl3, 150 MHz) d 110.8 (CH  2, C-1, C-8),
119.7 (CH  2, C-3, C-6), 120.5 (CH  2, C-4, C-5), 123.6
(C  2, C-4a, C-4b), 126.0 (CH  2, C-2, C-7), 139.7 (C  2,
C-8a, C-9a); EIMS m/z M þ (%) 167 (100), 139 (15), 113 (4),
and 83 (8).
Carbazole and derivatives inhibit endothelial proliferation
Coal tar fractions were tested for their ability to inhibit the
proliferation of SVR cells, an established angiogenic bioas-
say. SVR cells are murine endothelial cells, which have been
transformed with oncogenic ras. One fraction was found to
have activity, and induce a characteristic morphologic
change in SVR cells. Chemical analysis of the active fraction
demonstrated that the active principle is carbazole. As
carbazole is known to be metabolized by mammals through
hydroxylation, we also tested hydroxylated and oxidized
derivatives of carbazole, including 2-, 3-, and 4-hydroxylated
carbazoles (Johns and Wright, 1964; Resnick et al., 1993).
Carbazole itself had mild antiproliferative activity, whereas
hydroxylated carbazoles had more potent antiproliferative
JL Arbiser et al.
Carbazole and its derivatives inhibit proliferation
of SVR cells in vitro
No. of viable cells
250,000
EtOH control
200,000
* 0.5
g/ml
150,000 *
* *
* 1
g/ml
100,000 *
2
g/ml
50,000
0 4
g/ml
8
g/ml
e
l
l
zo
zo
zo
ba
ba
ba
ar
ar
ar
yc
yc
ox
ox
r
yd
yd
H
H
2-
4-
Treatment compound
Figure 1. Dose-dependent effect of carbazole and carbazole derivatives on
the proliferation of SVR endothelial cells. SVR cells were treated with doses
of carbazole and carbazole derivatives (see legend). There is a significant
difference (Po0.05) between ethanol control and the following treatment
groups: 8 mg/ml carbazole, 1 mg/ml, 4 mg/ml, and 8 mg/ml 2-hydroxycarbazole,
and 4 mg/ml and 8 mg/ml 4-hydroxycarbazole. The experiment was performed
in triplicate twice and the colored bars represent the average of these
experiments. Error bars represent the standard error of the mean.
activity (Figure 1). These findings suggest that carbazole and
its derivatives have antiangiogenic activity.
Carbazole does not inhibit AP-1 activation
Maximum clinical efficacy of coal tar is observed when
combined with UV light (Goeckerman treatment). UV light is
thought to inhibit keratinocyte proliferation by direct DNA
damage, and also has proinflammatory effects through the
induction of AP-1 and NF-kB transcription factors (Li and
Karin, 1998; Wang et al., 1998; Shaulian et al., 2000;
Bhoumik et al., 2001). One possibility is that coal tar inhibits
the UV induction of AP-1 and NF-kB while allowing the
antiproliferative ability. In order to test this possibility, we
studied the effect of carbazole and its derivatives to inhibit
UV-induced AP-1 and NF-kB activation. Neither carbazole
nor carbazole derivatives has significant effect on UV-
induced AP-1/NF-kB activation (data not shown).
Carbazole does not inhibit EGFR activity
Epidermal growth factor and epidermal growth factor
receptor (EGF-EGFR) signaling has been shown to be elevated
in human psoriasis. COS cells expressing EGFR were
stimulated with exogenous EGF and treated with carbazole.
No inhibition of EGFR phosphorylation was noted (data not
shown).
Carbazole prevents mononuclear cell synthesis of IL-15
Cytokine production by mononuclear cells plays an impor-
tant role in psoriasis. One of the cardinal cytokines involved
in the pathogenesis of psoriasis is IL-15 (Campbell et al.,
2000; Villadsen et al., 2003). IL-15 plays several roles that
may be important in psoriasis. First, it causes activation of
lymphocyte subsets demonstrated to be pathogenic in
psoriasis, including natural killer (NK)-like T cells (McInnes
and Gracie, 2004). In addition, IL-15 is angiogenic in vivo or
not (Angiolillo et al., 1997). In order to determine whether
www.jidonline.org 1397
 JL Arbiser et al.
Inhibitor of Angiogenesis and Inflammation Isolated from Antipsoriatic Coal Tar

a The effect of carbazole on the IL-15 b The effect of carbazole on the PSK-induced T a HEK293-iNOS b RAW264.7
production induced by PSK and NK cell activation tested by SAP expression kDa
2.0 kDa 131 iNOS
Exp 1 Actin 131 iNOS
1.6
L15 103 pg/ml

1.2 1.11
SAP 50 -Tubulin
Carbazole
0 10 100 0 10 100
0.8 0.61 (
M) Carbazole

MoCar 10
M+PSK
MoCar 20
M+PSK
0.6 (
M)

Ex vito
Jurkat
0.4

LCL
0.2
0.0
0 Figure 3. Translational effects of iNOS. (a) Effect of carbazole on iNOS

MoPSK
expression in cultured cells human embryonic kidney 293 (HEK293). HEK293

Mo
cells, stably expressing iNOS, were incubated for 18 h in the presence of
CB-Mo+EBV 0–100 mm carbazole. Western blotting of cell lysates (50 mg per lane) was
Figure 2. Carbazole inhibits T- and NK cell activation in peripheral blood performed by using an anti-iNOS antibody. Shown data represent two
mononuclear cells. (a) Production of IL-15 is stimulated by treatment with independent experiments carried out in duplicates. (b) Effect of carbazole on
polysaccharide K (PSK), and is downregulated by carbazole treatment. (b) PSK iNOS expression in cultured cells RAW264.7. RAW264.7 cells were
induces signaling lymphocyte activation molecule (SLAM)-associated protein stimulated with lipopolysaccharide (LPS) 10 ng/ml and mouse IFN-g (mIFN-g)
(SAP) expression in lymphocytes, and SAP expression is inhibited by 10 U/ml for 18 hours in either the absence or the presence of carbazole in
carbazole. Data shown represent two independent experiments performed in concentrations from 10 to 100 mM. Cells were harvested and Western analysis
duplicate. of cell lysates (50 mg per lane) was performed using anti-iNOS (upper panel)
antibody. Anti-g-tubulin antibody (lower panel) was used to ensure equivalent
amounts of whole-cell lysates. Shown data represent three independent
experiments carried out in duplicate.
carbazole affected IL-15 production and activation of lympho-
cytes, we activated cord lymphocytes with polysaccharide
K (PSK), a stimulus resulting in IL-15 synthesis (Liu et al., 90

2005). Carbazole had no significant effect on lympho- 80

cyte viability, but significantly inhibited IL-15 production 70

(Figure 2). Nitrites (
M) 60

50

Co-translational downregulation of iNOS by carbazole 40

We found a previously unrecognized co-translational down- 30

regulation by which some dimerization inhibitors of iNOS 20

lead to reduced iNOS expression. We also found that newly 10

discovered pyrimidineimidazole-based allosteric dimeriza- 0

0 (
M) 10 (
M) 100 (
M)
tion inhibitors and imidazole-based clotriomazole and Carbazole
miconazole are able to reduce iNOS expression and activity.
Figure 4. iNOS activity in RAW264.7 cells treated with carbazole.
Since it was found that a synthetic carbazole compound, 9-(2- RAW264.7 cells were stimulated with lipopolysaccharide (LPS) 100 ng/ml
chlorobenzyl)-9H-carbazole-3-carbaldehyde (LCY-2-CHO), and mouse IFN-g (mIFN-g) 10 U/ml for 18 hours. During stimulation,
had an inhibitory effect on lipopolysaccharide-stimulated carbazole was added in concentrations from 0 to 100 mM. iNOS activity was
NO generation in RAW264.7 macrophages (Bromberg, 2000; evaluated by measuring nitrite accumulation in culture media (means7SD,
Tsao et al., 2002), we hypothesized that carbazole might n ¼ 4). Shown data represent two independent experiments carried out
in duplicates; 0 mM carbazole–76.195 mM nitrites average (SD ¼ 7.31);
harbor a similar mechanism of action caused by co-trans-
10 mM carbazole–75.32 mM nitrites average (SD ¼ 4.97); and 100 mM
lational downregulation of iNOS. carbazole–54.28 mM nitrites average (SD ¼ 4.96).
To investigate this hypothesis, we examined the effect of
carbazole on iNOS expression in human embryonic kidney
293 cells stably expressing iNOS and treated with carbazole
in concentrations of 10 or 100 mM or vehicle only (data not stably expressing iNOS human embryonic kidney 293 cells.
shown). Cells were incubated in the presence of reagents for The effect was observed at 10 and 100 mM concentrations.
18 hours. As Western blot analysis showed, iNOS expression Importantly, there was a reduction of iNOS activity measured
was slightly diminished in cells treated with 100 mM carbazole by nitrite accumulation observed only at the higher dose of
(Figure 3a). There were no significant differences in iNOS carbazole (Figure 4). The above results suggest that carbazole
activity measured by assay of nitrites released into the cell is able to reduce iNOS (both human and murine counterpart)
growth medium (data not shown). expression in living cells. The underlying mechanism could
To further confirm this finding, we extended our experi- include transcriptional and/or post-transcriptional effects.
ment to murine macrophages cell line RAW264.7, which Given that stat3 is a major mediator of immune and
express iNOS upon stimulation with lipopolysaccharide and angiogenic characteristics of psoriasis, we examined the
IFN-g (Figure 3b). In this cell line, we took advantage of the effect of carbazole and 2-hydroxycarbazole, a carbazole
inducible nature of iNOS by adding carbazole immediately metabolic product, on stat3-mediated transcription. Both
before iNOS induction. Thus, carbazole could potentially carbazole and 2-hydroxycarbazole significantly downregu-
interact with almost all newly synthesized iNOS. This lated stat3-mediated transcription. Inhibition of stat3 was
resulted in a much more marked decrease in iNOS protein specific, as treatment with carbazoles had no effect on a
expression analyzed by Western blotting in comparison with constitutive promoter (Figure 5).

1398 Journal of Investigative Dermatology (2006), Volume 126

 JL Arbiser et al.
Inhibitor of Angiogenesis and Inflammation Isolated from Antipsoriatic Coal Tar

Effect of carbazole on Rac1 activity

a 8.0 of HUVEC after VEGF stimulation
pSTAT3-luc
Relative luciferase activity

7.0 Carbazole
6.0 0 v5 0 v5 VEGF stimulation (min)
5.0
4.0
3.0 Figure 6. Carbazole blocks rac activation in response to VEGF. Cells were
2.0 treated for 5 minutes with VEGF (20 ng/ml) in the presence or absence of
1.0 carbazole. Lysates bound to PAK-1 agarose were immunoblotted with anti-
0.0 Rac antibody to measure Rac1 activity.
CB − − − + −
2HCB − − − − +
Vehicle − − + − −
IL-6 − + + + +

b 1.2 and decreased maturation are evident, as well as a

pGL3-luc
Relative luciferase activity

1 lymphocytic infiltrate and angiogenesis. Psoriasis is main-

0.8
0.6
0.4
0.2
0.0
CB − −
2HCB − −
Vehicle − +
Figure 5. Carbazole and 2-hydroxycarbazole inhibit stat-3-mediated tran-
scription. Cells were transiently transfected with a (a) stat-3-responsive or
(b) constitutive promoter-driving luciferase, and stimulated with IL-6, a
known stimulator of stat3. Cells were treated with carbazole (CB) or
2-hydroxycarbazole (2HCB), or vehicle.
We then examined whether carbazole treatment inhibited
phosphorylation of stat3. No effect was seen on levels of stat3
phosphorylation, suggesting that carbazoles inhibit stat3-
mediated transcription by an alternative mechanism. Given
that rac activation is required for optimal stat3 activation, we
examined the effect on rac activation by vascular endothelial
growth factor (VEGF). Carbazole treatment significantly
inhibited rac activation by VEGF (Figure 6).
DISCUSSION
Natural products are a potent source of antiangiogenic, anti-
inflammatory, and antitumor agents. Previously isolated
compounds from plants include curcumin, epicatechin
gallate, indole carbinol, and nordihydroguiaretic acid (Allen
et al., 1984; Arbiser et al., 1998; Bradlow et al., 1999; Katiyar
et al., 2001; Li et al., 2004). We have previously used the SVR
endothelial bioassay as simple methods for the isolation and
characterization of small molecules from complex mixtures
of natural products (Arbiser et al., 1999; Bai et al., 2003).
Recently, we isolated honokiol as the active ingredient of
Magnolia grandiflora extracts, and found it to have potent
antiangiogenic and antitumor activity in vivo (Arbiser et al.,
1999). Similarly, we have found polycinnamate esters of
quinic acid isolated from Ilex paraguayensis to be inhibitors
of the 26S proteasome (Arbiser et al., 2005). We used the SVR
bioassay to fractionate clinical grade coal tar used for the
treatment of psoriasis, and found carbazole to be the active
principle.
Psoriasis is a common human disease characterized by a
complex interaction of keratinocytes, lymphocytes, and endo-
thelial cells. Histologically, increased keratinocyte proliferation
tained by chronic production of proinflammatory cytokines
including TNFa, IL-15, IFN-g, and VEGF (Grossman et al.,
1989; Detmar et al., 1994; Austin et al., 1999; McInnes and
Gracie, 2004; Nickoloff and Nestle, 2004). These cytokines
stimulate multiple events, such as increased endothelial
+ −
expression of adhesion molecules, increased production of
− + iNOS, increased endothelial proliferation, and increased
− −
numbers of IFN-g-secreting NK-like T lymphocytes (Uyemura
et al., 1993; Lebwohl, 2004; Sano et al., 2005). Production of
these inflammatory cytokines is mediated through several
signaling pathways, including stat3, AP-1, and NF-kB.
Current therapies target either the transcription factors
and/or aberrant cytokine production. Novel biological
therapies include soluble receptors to TNFa or antibodies
that cause apoptosis of TNFa-containing cells. Topical
therapies indirectly target AP-1 and NF-kB signaling, and
include glucocorticoids, vitamin D derivatives, and retinoids.
None of these agents is known to target stat3, which has
recently been implicated in human psoriasis (Turksen et al.,
1992; Sano et al., 2004). Coal tar was initially used as the
mainstay of psoriasis treatment before the discovery of newer
agents, and in combination with UV light, caused long-term
remissions. Coal tar has fallen out of favor in more recent
years, for several reasons. First, coal tar is not aesthetic from a
visual or olfactory point of view. Second, day treatments with
coal tar and UV light are expensive. Thus, there is a strong
impetus to discover novel therapies for psoriasis.
We initially examined AP-1 and NF-kB signaling as initial
targets of carbazole. We looked that these targets initially
because UV light, which works in combination with
carbazole, is known to activate these pathways. In addition,
EGFR, which is activated in psoriasis, is known to activate
AP-1 and NF-kB (Elder et al., 1990). Carbazole had no effect
on basal or stimulated AP-1 or NF-kB activation, nor did it
modulate autophosphorylation of EGFR. Psoriasis is known to
have elevated levels of NK-like T lymphocytes, and has high
levels of IL-15, a cytokine required for survival and activation
of NK-like T lymphocytes (Liu et al., 2005). Carbazole
treatment caused a reduction in IL-15 synthesis by mono-
nuclear lymphocytes, as well as decreased activation of NK-
like T cells, yet had little effect on lymphocyte viability. We
then examined production of iNOS, which has been found to
be activated in psoriasis, and serves as a potential target of
antipsoriatic therapy (Qureshi et al., 1996; Ormerod et al.,
1998). Carbazole treatment led to modest decreases in iNOS
activity and protein levels.

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 JL Arbiser et al.
Inhibitor of Angiogenesis and Inflammation Isolated from Antipsoriatic Coal Tar
Recently, stat3 has been implicated as a signaling
molecule that is activated in human psoriasis. Stat3-mediated
transcription is inhibited by carbazole, and its metabolite,
2-hydroxycarbazole, but phosphorylation of stat3 is unaffected.
Prior studies have shown that stat3 transcription, IL-15
production, and iNOS activity can be mediated by the small
GTPase rac (Kone and Kuncewicz, 1999; Campbell et al.,
2000; Faruqi et al., 2001). We thus looked at rac activation
and found that rac activation in human endothelial cells is
inhibited by carbazole.
Interestingly, carbamazepine, an anticonvulsant, was
found to have great therapeutic benefit in a patient with
generalized psoriasis (Smith and Skelton, 1996). Carbamaze-
pine is structurally similar to carbazole. Our data suggest that
carbazole is the active property of coal tar, and that carbazole
and its derivatives can be used topically for the treatment of
psoriasis and other inflammatory disorders. The use of
carbazole adds a new agent with a novel mechanism of
action for the treatment of human psoriasis.
MATERIALS AND METHODS
Isolation, purification, and identification of active principle
from coal tar
Liquid extract of coal tar was concentrated under vacuum to get
crude extract (20.0 mg). This extract was applied on a Sephadex LH
20 (3.0  100 cm) column and eluted with methanol. The elution
was monitored by thin-layer chromatography and similar fractions
are combined to get 11 fractions. This fraction was assayed for
inhibition of SVR cell viability (Arbiser et al., 1999; Bai et al., 2003).
Fraction 5 (0.5 mg) and fraction 6 (1.2 mg) tested positive in the
bioassay. Thin-layer chromatography comparison of these two
fractions revealed the presence of one common UV-active compo-
nent in both fractions that produced a blue char upon spraying with
H2SO4. The fraction 6 (1.2 mg) was subjected to normal phase-HPLC
(1:1 hexanes in CH2Cl2 (v/v), 1.0 ml/min, photodiode-array detection
monitored at 254 nm) to obtain 1 (0.7 mg). Compound 1 was
identified as carbazole from its NMR and mass spectra spectra, and
confirmed by direct comparison of authentic sample of carbazole
(Sigma-Aldrich Chemical Company, St Louis, MO). Carbazole,
2-hydroxycabazole, 4-hydroxycarbazole and 3,6-dichloro-9-cyclo-
propylmethylcarbazole were obtained from Sigma-Aldrich Chemical
Company (St Louis, MO), and 3-hydroxycarbazole was obtained
from David Gibson (University of Iowa, Iowa City, IA) (Resnick et al.,
1993).
General experimental procedures of carbazole identification
The 1H NMR, 1H-1H COSY, 13C NMR, DEPT, HMQC, and HMBC
spectra were recorded on a Varian Unity Inova spectrometer. The 1H
NMR and 13C NMR of carbazole (isolated and authentic sample
from Aldrich) spectra were recorded on a Bruker DRX 400
spectrometer. Varian NMR spectrometer was operating at
600 MHz for 1H and 150 MHz for 13C and Bruker NMR spectrometer
was operating at 400 MHz for 1H and 100 MHz for 13C, respectively.
The NMR spectra were recorded running gradients and using
residual solvent peaks (d 7.25 s for 1H and d 77.2 middle of the
triplet for 13C) as internal references. The electron impact mass
spectra and gas chromatography data were acquired on a Hewlett
Packard Series II 5890 gas chromatography-mass spectroscopy
1400 Journal of Investigative Dermatology (2006), Volume 126
spectrometer. Thin-layer chromatography analysis were run on
Merck thin-layer chromatography plates precoated with Si60 F254
observed under UV ray at 254 nm and then visualized by spraying
with 1:1 H2SO4 in ethyl alcohol and heating. HPLC separation was
carried out on a Waters Millennium system with a 996 photodiode-
array detector (Prodigys Si-gel, 5 mm, 4.6  250 mm photodiode-
array detection monitored at 254 nm).
Cell culture
Human embryonic kidney 293 and murine macrophage RAW264.7
cell lines were purchased from American Type Culture Collection
(Manassas, VA). Cells were cultured in the manufacturer’s recom-
mended media. Each medium was supplemented with 2 mM
glutamine, and 10% heat-inactivated fetal bovine serum.
Transfection and stable cell line production of human embryo-
nic kidney 293 cells
Cationic lipid-mediated transient transfection was carried out by
using ‘‘LipofectAMINE 2000’’ (Invitrogen Corporation, Carlsbad, CA)
following the manufacturer’s instructions. Stable cell line expressing
iNOS were produced by using transfection of iNOS cDNA followed
by positive colony selection using G418 (Invitrogen) at a concentra-
tion of 600 mg/ml.
Cell lysis
After gently rinsing twice with phosphate-buffered saline, the cell
layer was lysed on ice for 30 minutes in 40 mM Bis–Tris propane
buffer, pH 7.7, 150 mM NaCl, 10% glycerol, 1% Triton X-100 in the
presence of protease inhibitors (phenylmethylsulfonyl fluoride
(1 mM), pepstatin-A (10 mg/ml), leupeptin (10 mg/ml), aprotinin
(10 mg/ml), phenanthroline (10 mg/ml), benzamidine HCl (16 mg/ml))
(BD-Pharmingen, BD-Biosciences, San Jose, CA). Lysates were
centrifuged (16,000  g, 5 minutes, 41C), and supernatants were
stored at 801C. Total protein concentrations were determined by
using a bicinchoninic acid reagent, following the manufacturer’s
instructions (Pierce Biotechnology Inc., Rockford, IL).
iNOS induction
Mouse macrophages RAW264.7 were incubated with a mixture of
mouse IFN-g (10 m/ml) and lipopolysaccharide (100 ng/ml).
Western analysis
Aliquots of cell lysates (50 mg) were mixed with one-third volume of
4  Laemmli sample buffer (200 mM Tris-HCl, pH 6.8/8% SDS/
0.004% bromophenol blue/40% glycerol/400 mM dithiothreitol), and
heated at 951C for 5 minutes. Proteins were resolved on SDS/PAGE
(NOVEX, San Diego, CA) at 125 V and transferred to nitrocellulose
membranes for 1 h at 20 V by using semidry transfer cell (Bio-Rad
Lifesciences, Hercules, CA). Immunoreactive bands were visualized
with the use of enhanced chemiluminescence system (SuperSignal,
Pierce Biotechnology Inc., Rockford, IL). Images were acquired
using a cooled charge-coupled device camera (Eagle Eye II Still
Video System; Stratagene, La Jolla, CA, USA).
iNOS activity assay
iNOS activity was assayed by assay of nitrite released into the cell
growth medium. To measure the amount nitrite in the cell medium
100 ml of culture medium was mixed with 100 ml of Griess reagent

Inhibitor of Angiogenesis and Inflammation Isolated from Antipsoriatic Coal Tar
for 10 minutes at room temperature, and absorbance at 543 nm was
recorded in a microplate reader. Serial dilutions of sodium nitrite
were used as standards.
In vitro transcription/translation of iNOS
Human iNOS was transcribed and translated from plasmid DNA
templates using rabbit reticulocyte system (TnT Quick Coupled
Transcription/Translation Systems, Promega, Madison, WI). Each
reaction contained 40 ml of TnT Quick Master Mix consisting of
reticulocyte lysate, amino acids, T7 RNA polymerase, and reaction
buffer, 2 ml of DNA (0.5 mg/ml), 2 ml Redivue L-[35S]methionine
(10 mCi/ml) (Amersham Pharmacia Biotech, Piscataway, NJ), and
nuclease-free water was added to a final volume of 50 ml. Reactions
were performed in the presence of 10 mM or 100 mM iNOS
dimerization inhibitors (BBS-1, miconazole) and carbazole or
vehicle was used to dissolve them, at 301C for 90 minutes, and
terminated by placing the samples at 201C. Products of reaction
were analyzed by SDS denaturing gel visualized by Phosphoimager.
Reporter assays
U3A human fibrosarcoma (kindly provided by Dr George Stark,
Cleveland Clinic, Cleveland, OH) and NIH3T3 murine fibroblast
cells were cultured in DMEM containing 10% fetal bovine serum in
a 5% CO2 incubator at 371C. To determine the effects of organic
molecules on signaling pathways, stable cell lines were generated in
which expression of a luciferase reporter gene was under the control
of specific promoter sequences. The pSTAT3-luc plasmid, which has
a stat3-responsive element upstream of a luciferase reporter gene, as
well as a neomycin phosphotransferase gene, was transfected into
U3A cells using Lipofectamine 2000 reagent (Invitrogen) and stable
clones were selected in G418 (500 mg/ml). The pNF-kB-luc plasmid
contains an NF-kB-responsive element upstream of a luciferase
reporter gene. The pGL3-luc plasmid contains an element that
constitutively expresses the luciferase gene from an SV40 promoter.
pNF-kB-luc and pGL3-luc were transfected into 3T3 cells using
Lipofectamine 2000 reagent and stable clones were selected in
G418 (500 mg/ml).
Luciferase quantitation. Reporter cell lines were seeded at
3  105 cells/ml in a 96-well plate (Costar, Corning, NY) and
allowed to adhere overnight. Cells were then pre-treated for 1 h with
1% ethanol (vehicle control) or 30 mM each of carbazole or
2-hydroxycarbazole before stimulation with 30 ng/ml of human
IL-6 (R&D Systems Inc., Minneapolis, MN) or 10 ng/ml of murine
TNFa (R&D Systems). After 5 hours of incubation with the compound
and cytokine, luciferase activity was measured on a luminometer
(Labsystems) using the Bright-Glo Luciferase Assay System (Promega
Corporation, Madison, WI).
Rac activation assay
Human umbilical vein endothelial cells were grown to confluence
and made quiescent in 0.5% fetal bovine serum for overnight. Cells
were pretreated with carbazole (5 mg/ml) for 1 hour and then
stimulated with VEGF (20 ng/ml) for 5 minutes. Cells were lysed
with ice-cold lysis buffer, pH 7.5, containing 25 mmol/l HEPES,
150 mmol/l NaCl, 1% IGEPAL CA-630, 0.25% sodium deoxycho-
late, 10 mmol/l MgCl2, 10% glycerol, 25 mmol/l NaF, 1 mmol/l
JL Arbiser et al.
EDTA, 1 mmol/l sodium orthovanadate, 10 mg/ml leupeptin, 10 mg/
ml aprotinin, and 1 mmol/l phenylmethylsulfonyl fluoride. Activated
(GTP-bound) rac was affinity-precipitated with p21-activated kinase-
1 protein binding domain peptide, which binds only to Rac-GTP and
not Rac-GDP (Ushio-Fukai et al., 2002; Bai et al., 2003). p21-
activated kinase-1 protein binding domain-agarose (7.5 mg/mg cell
lysate; Upstate Cell Signaling Solutions, Charlottesville, VA) was
added, and the reaction mixture was gently rocked at 41C for
60 minutes. The agarose beads were collected by pulsing for
5 seconds in a microcentrifuge at 14,000  g, and the beads were
washed three times with 0.5 ml of lysis buffer. The agarose beads
were resuspended in 40 ml of 1  SDS sample buffer and boiled for
5 minutes. The supernatant was separated by SDS-PAGE on a 12%
gel, and the proteins were transferred to nitrocellulose membrane.
After blocking for 1 h in phosphate-buffered saline containing 5%
nonfat dry milk and 0.1% Tween 20, the membrane was incubated
with anti-Rac antibody (1:1000 dilution; Upstate Biotechnology)
overnight. After incubation with the secondary antibody, Rac was
detected by enhanced chemiluminescence.
CONFLICT OF INTEREST
The authors state no conflict of interest.
ACKNOWLEDGMENTS
This work was supported by NIAMS Grants RO1AR 47901 and RO1 AR
050727 to J.L.A., T32 NIHNRSA Training Grant (B.N.P.), R01CA45708 to
D.F.S., Emory Skin Disease Research Core Center P30 AR 42687,
RO1HL069033, and R01HL075421 (N.T.E.) and AHA postdoctoral fellowship
(P.J.K.). We thank Rajani Ramabhadran for EGFR assays.
REFERENCES
Allen LM, Wara W, Gonzaga A, Parkhurst R (1984) Evaluation of antitumor-
activity of nordihydroguiaretic acid (Na) with human mammary
adenocarcinoma xenografts(Mx-1) in athymic nude-mice. Proceedings
of the American Association for Cancer Research 25:329
Angiolillo AL, Kanegane H, Sgadari C, Reaman GH, Tosato G (1997)
Interleukin-15 promotes angiogenesis in vivo. Biochem Biophys Res
Commun 233:231–7
Arbiser JL, Klauber N, Rohan R, van Leeuwen R, Huang MT, Fisher C et al.
(1998) Curcumin is an in vivo inhibitor of angiogenesis [in process
citation]. Mol Med 4:376–83
Arbiser JL, Li XC, Hossain CF, Nagle DG, Smith DM, Miller P et al. (2005)
Naturally occurring proteasome inhibitors from mate tea (Ilex para-
guayensis) serve as models for topical proteasome inhibitors. J Invest
Dermatol 125:207–12
Arbiser JL, Panigrathy D, Klauber N, Rupnick M, Flynn E, Udagawa T et al.
(1999) The antiangiogenic agents TNP-470 and 2-methoxyestradiol
inhibit the growth of angiosarcoma in mice. J Am Acad Dermatol
40:925–9
Austin LM, Ozawa M, Kikuchi T, Walters IB, Krueger JG (1999) The majority
of epidermal T cells in Psoriasis vulgaris lesions can produce type 1
cytokines, interferon-gamma, interleukin-2, and tumor necrosis factor-
alpha, defining TC1 (cytotoxic T lymphocyte) and TH1 effector
populations: a type 1 differentiation bias is also measured in circulating
blood T cells in psoriatic patients. J Invest Dermatol 113:752–9
Bai X, Cerimele F, Ushio-Fukai M, Waqas M, Campbell PM, Govindarajan B
et al. (2003) Honokiol, a small molecular weight natural product,
inhibits angiogenesis in vitro and tumor growth in vivo. J Biol Chem
278:35501–7
Bhoumik A, Ivanov V, Ronai Z (2001) Activating transcription factor 2-derived
peptides alter resistance of human tumor cell lines to ultraviolet
irradiation and chemical treatment. Clin Cancer Res 7:331–42
www.jidonline.org 1401
 JL Arbiser et al.
Inhibitor of Angiogenesis and Inflammation Isolated from Antipsoriatic Coal Tar
Bradlow HL, Sepkovic DW, Telang NT, Osborne MP (1999) Multifunctional
aspects of the action of indole-3-carbinol as an antitumor agent. Cancer
Prev Novel Nutr Pharmaceut Dev 889:204–13
Bromberg J (2000) Signal transducers and activators of transcription as
regulators of growth, apoptosis and breast development. Breast Cancer
Res 2:86–90
Bromberg J, Chen XM (2001) STAT proteins: signal tranducers and activators
of transcription. Methods Enzymol 333:138–51
Campbell JDM, Cook G, Robertson SE, Gracie JA, Boyd KS, Franklin IM
(2000) Interleukin-15 maintains IL-2-induced STAT3 signaling and
protects T cells from multiple myeloma induced immunosuppression.
Exp Hematol 28:82
Detmar M, Brown LF, Claffey KP, Yeo KT, Kocher O, Jackman RW et al.
(1994) Overexpression of vascular permeability factor/vascular endothe-
lial growth factor and its receptors in psoriasis. J Exp Med 180:1141–6
Elder JT, Klein SB, Tavakkol A, Fisher GJ, Nickoloff BJ, Voorhees JJ (1990)
Growth factor and proto-oncogene expression in psoriasis. J Invest
Dermatol 95:7S–9S
Faruqi TR, Gomez D, Bustelo XR, Bar-Sagi D, Reich NC (2001) Rac1 mediates
STAT3 activation by autocrine IL-6. Proc Natl Acad Sci USA 98:9014–9
Gordon KB, Papp KA, Hamilton TK, Walicke PA, Dummer W, Li N et al.
(2003) Efalizumab for patients with moderate to severe plaque psoriasis:
a randomized controlled trial. JAMA 290:3073–80
Grossman RM, Krueger J, Yourish D, Granelli-Piperno A, Murphy DP, May LT
et al. (1989) Interleukin 6 is expressed in high levels in psoriatic skin and
stimulates proliferation of cultured human keratinocytes. Proc Natl Acad
Sci USA 86:6367–71
Guren TK, Abrahamsen H, Thoresen GH, Babaie E, Berg T, Christoffersen T
(1999) EGF-induced activation of Stat1, Stat3, and Stat5b is unrelated to
the stimulation of DNA synthesis in cultured hepatocytes. Biochem
Biophys Res Commun 258:565–71
Johns SR, Wright SE (1964) Metabolism carbazole in rats+rabbits. J Med Chem
7:158–61
Joshi PC, Pathak MA (1984) The role of active oxygen (1O2 and O(2)) induced
by crude coal tar and its ingredients used in photochemotherapy of skin
diseases. J Invest Dermatol 82:67–73
Katiyar SK, Afaq F, Perez A, Mukhtar H (2001) Green tea polyphenol ()-
epigallocatechin-3-gallate treatment of human skin inhibits ultraviolet
radiation-induced oxidative stress. Carcinogenesis 22:287–94
Kolodziejski PJ, Koo J-S, Eissa NT (2004) Regulation of inducible nitric oxygen
synthetase by rapid cellular turnover and co-translational downregula-
tion by dimerization inhibitors. Proc Natl Acad Sci USA 101:18141–6
Kone BC, Kuncewicz T (1999) Physical and functional interactions of Rac2
with nitric oxide synthase-2. FASEB J 13:A133
Lebwohl M (2004) Innovations in the treatment of psoriasis. J Am Acad
Dermatol 51:S40–1
Li L, Aggarwal BB, Shishodia S, Abbruzzese J, Kurzrock R (2004) Nuclear
factor-kappa B and I kappa B kinase are constitutively active in human
pancreatic cells, and their down-regulation by curcumin (diferuloyl-
methane) is associated with the suppression of proliferation and the
induction of apoptosis. Cancer 101:2351–62
Li NX, Karin M (1998) Ionizing radiation and short wavelength UV activate
NF-kappa B through two distinct mechanisms. Proc Natl Acad Sci USA
95:13012–7
Liu A, Arbiser JL, Holmgren A, Klein G, Klein E (2005) PSK and Trx80 inhibit
B-cell growth in EBV-infected cord blood mononuclear cells through T
cells activated by the monocyte products IL-15 and IL-12. Blood
105:1606–13
McInnes IB, Gracie JA (2004) Interleukin-15: a new cytokine target for the
treatment of inflammatory diseases. Curr Opin Pharmacol 4:392–7
1402 Journal of Investigative Dermatology (2006), Volume 126
Nickoloff BJ, Nestle FO (2004) Recent insights into the immunopathogenesis
of psoriasis provide new therapeutic opportunities. J Clin Invest
113:1664–75
Nickoloff BJ, Wrone-Smith T (1999) Injection of pre-psoriatic skin with CD4+
T cells induces psoriasis. Am J Pathol 155:145–58
Niu G, Wright KL, Huang M, Song L, Haura E, Turkson J et al. (2002)
Constitutive Stat3 activity up-regulates VEGF expression and tumor
angiogenesis. Oncogene 21:2000–8
Oh CJ, Das KM, Gottlieb AB (2000) Treatment with anti-tumor necrosis factor
alpha (TNF-alpha) monoclonal antibody dramatically decreases the
clinical activity of psoriasis lesions. J Am Acad Dermatol 42:829–30
Ormerod AD, Weller R, Copeland P, Benjamin N, Ralston SH, Grabowksi P
et al. (1998) Detection of nitric oxide and nitric oxide synthases in
psoriasis. Arch Dermatol Res 290:3–8
Qureshi AA, Lerner LH, Lerner EA (1996) From bedside to the bench and back
– nitric oxide and the cutis. Arch Dermatol 132:889–93
Resnick SM, Torok DS, Gibson DT (1993) Oxidation of carbazole to
3-hydroxycarbazole by naphthalene 1, 2-dioxygenase and biphenyl 2,
3-dioxygenase. FEMS Microbiol Lett 113:297–302
Sano S, Chan KS, Carbajal S, Clifford J, Peavey M, Kiguchi K et al. (2005) Stat3
links activated keratinocytes and immunocytes required for development
of psoriasis in a novel transgenic mouse model. Nat Med 11:43–9
Sano S, Chan KS, Peavy M, Carbajal S, Clifford J, Kiguchi K et al. (2004)
Constitutive activation of Stat3 in keratinocytes is essential for develop-
ment of psoriasis. J Invest Dermatol 122:A94
Saurat JH (1999) Retinoids and psoriasis: novel issues in retinoid pharmacol-
ogy and implications for psoriasis treatment. J Am Acad Dermatol
41:S2–6
Shaulian E, Schreiber M, Piu F, Beeche M, Wagner EF, Karin M (2000) The
mammalian UV response: c-jun induction is required for exit from p53-
imposed growth arrest. Cell 103:897–907
Smith KJ, Skelton HG (1996) Accidental success with carbamazepine for
psoriatic erythroderma. N Engl J Med 335:1999–2000
Stern RS, Laird N, Melski J, Parrish JA, Fitzpatrick TB, Bleich HL (1984)
Cutaneous squamous-cell carcinoma in patients treated with PUVA.
N Engl J Med 310:1156–61
Strengell M, Matikainen S, Siren J, Lehtonen A, Foster D, Julkunen I et al.
(2003) IL-21 in synergy with IL-15 or IL-18 enhances IFN-gamma
production in human NK and T cells. J Immunol 170:5464–9
Tsao LT, Lee CY, Huang LJ, Kuo SC, Wang JP (2002) Inhibition of
liposaccharide-stimulated nitric oxygen production in RAW 264.7
macrophages by a synthetic carbazole, LCY-2-CHO. Biochem Pharma-
col 63:1961–8
Turksen K, Kupper T, Degenstein L, Williams I, Fuchs E (1992) Interleukin 6:
insights to its function in skin by overexpression in transgenic mice. Proc
Natl Acad Sci USA 89:5068–72
Ushio-Fukai M, Tang Y, Fukai T, Dikalov SI, Ma YX, Fujimoto M et al. (2002)
Novel role of gp91(phox)-containing NAD(P)H oxidase in vascular
endothelial growth factor-induced signaling and angiogenesis. Circ Res
91:1160–7
Uyemura K, Yamamura M, Fivenson DF, Modlin RL, Nickoloff BJ (1993) The
cytokine network in lesional and lesion-free psoriatic skin is character-
ized by a T-helper type 1 cell-mediated response. J Invest Dermatol
101:701–5
Villadsen LS, Schuurman J, Beurskens F, Dam TN, Dagnaes-Hansen F, Skov L
et al. (2003) Resolution of psoriasis upon blockade of IL-15 biological
activity in a xenograft mouse model. J Clin Invest 112:1571–80
Wang Y E Y, Zhang X, Lebwohl M, DeLeo V, Wei H (1998) Inhibition of
ultraviolet B (UVB)-induced c-fos and c-jun expression in vivo by a
tyrosine kinase inhibitor genistein. Carcinogenesis 19:649–54