Grape seed proanthocyanidins inhibit UV-radiation-induced oxidative
stress and activation of MAPK and NF-κB signaling in human
Sudheer K. Mantenaa, Santosh K. Katiyara,b,⁎
aDepartment of Dermatology, University of Alabama at Birmingham, Volker Hall 557, 1670 University Boulevard, P.O. Box 202, Birmingham, AL 35294, USA
bBirmingham VA Medical Center, Birmingham, AL 35294, USA
Received 20 October 2005; revised 14 December 2005; accepted 23 December 2005
Available online 26 January 2006
Solar ultraviolet (UV) radiation-induced oxidative stress has been implicated in various skin diseases. Here, we report the photoprotective
effect of grape seed proanthocyanidins (GSPs) on UV-induced oxidative stress and activation of mitogen-activated protein kinase (MAPK) and
NF-κB signaling pathways using normal human epidermal keratinocytes (NHEK). Treatment of NHEK with GSPs inhibited UVB-induced
hydrogen peroxide (H2O2), lipid peroxidation, protein oxidation, and DNA damage in NHEK and scavenged hydroxyl radicals and superoxide
anions in a cell-free system. GSPs also inhibited UVB-induced depletion of antioxidant defense components, such as glutathione peroxidase,
catalase, superoxide dismutase, and glutathione. As UV-induced oxidative stress mediates activation of MAPK and NF-κB signaling pathways, we
determined the effects of GSPs on these pathways. Treatment of NHEK with GSPs inhibited UVB-induced phosphorylation of ERK1/2, JNK, and
p38 proteins of the MAPK family at the various time points studied. As UV-induced H2O2plays a major role in activation of MAPK proteins,
NHEK were treated with H2O2with or without GSPs and other known antioxidants, viz. (−)-epigallocatechin-3-gallate, silymarin, ascorbic acid,
and N-acetylcysteine. It was observed that H2O2-induced phosphorylation of ERK1/2, JNK, and p38 was decreased by these antioxidants. Under
identical conditions, GSPs also inhibited UVB-induced activation of NF-κB/p65, which was mediated through inhibition of degradation and
activation of IκBα and IKKα, respectively. Together, these results suggest that GSPs could be useful in the attenuation of UV-radiation-induced
oxidative stress-mediated skin diseases in human skin.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Grape seed proanthocyanidins; Oxidative stress; Ultraviolet radiation; Mitogen-activated protein kinases; Hydrogen peroxide; Glutathione peroxidase;
Glutathione; NF-κB; Free radicals
Skin is constantly exposed to pro-oxidant environmental
stresses from a wide array of sources, including exposure to
solar ultraviolet (UV) radiation. Acute or chronic exposure of
the skin to UV radiation results in the development of
inflammation, oxidative stress, and DNA damage leading to
several skin disorders, including hyperpigmentation, photoa-
ging or premature aging of the skin, and melanoma and
nonmelanoma skin cancers [1–4]. The incidence of these skin
diseases is growing continuously as the population ages and
larger amounts of UV radiation reach the Earth's surface
because of depletion of the ozone layer [5,6] and the continuing
use of sun-tanning devices for cosmetic purposes [1,4].
UV-induced reactive oxygen species (ROS) or oxidative
stress is capable of oxidizing lipids, proteins, or DNA, leading
to the formation of oxidized products such as lipid hydroper-
oxides, protein carbonyls, or 8-hydroxydeoxyguanosine, which
have been implicated in the onset of skin diseases [7–9].
Endogenous enzymatic and nonenzymatic antioxidants protect
the skin from UV-induced oxidative damage . However, if
Free Radical Biology & Medicine 40 (2006) 1603–1614
Abbreviations: DNPH, dinitrophenylhydrazine; GSPs, grape seed proantho-
cyanidins; GSH, glutathione; SOD, superoxide dismutase; GPx, glutathione
peroxidase; LPO, lipid peroxidation; MAPK, mitogen-activated protein kinase;
ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; NF-
κB, nuclear factor κB; UV, ultraviolet.
⁎Corresponding author. Department of Dermatology, University of Alabama
at Birmingham, Volker Hall 557, 1670 University Boulevard, P.O. Box 202,
Birmingham, AL 35294, USA. Fax: +1 205 934 5745.
E-mail address: firstname.lastname@example.org (S.K. Katiyar).
0891-5849/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
the antioxidant defense component of the skin is overwhelmed
by the presence of ROS, it can lead to oxidative damage of
cellular constituents . Further, there is considerable
evidence that UV-induced oxidative stress mediates the
phosphorylation of protein kinases through a series of cascades,
such as mitogen-activated protein kinases (MAPK), and
activation of transcription factors, such as nuclear factor-κB
(NF-κB) [11–16]. Three structurally related but biochemically
and functionally distinct MAPK signal transduction pathways
have been identified in mammalian cells, including the
extracellular signal-regulated kinase (ERK1/2), c-Jun N-termi-
nal kinase/stress-activated protein kinase (JNK/SAPK), and the
p38 [12,13] pathways. These MAPK proteins are mediators of
signal transduction from the cell surface to the nucleus and play
a major role in triggering and coordinating gene responses .
ERKs are predominantly activated by mitogenic signals,
whereas JNK and p38 are primarily activated by environmental
stresses such as UV radiation, inflammatory cytokines, heat
shock, and DNA-damaging agents [13,15,16]. Activation of
ERKs stimulates proliferation and differentiation  and also
has a role in tumor promotion, especially stimulated by the
oxidative state . Phosphorylation of JNK and p38 has a role
in cellular differentiation and inflammatory responses [16–18].
Antioxidants have been shown to attenuate the activation of
MAPK [14,17,19] signaling, thereby indicating that the MAPK
signaling pathway is an important target of ROS. It is well
documented that NF-κB is a downstream target of the MAPK
signal transduction pathway, and activation of NF-κB has a
crucial role in inflammatory diseases, including cancers.
Therefore, the signaling pathways leading to the regulation of
NF-κB activity have become target points for chemopreventive
or chemotherapeutic agents.
The use of dietary botanical supplements with substantial
antioxidant activity has generated immense interest in
attenuating the UV-radiation-induced oxidative stress and
oxidative stress-mediated risk of skin diseases. Previously, we
have shown that dietary grape seed proanthocyanidins (GSPs)
inhibit UV-radiation-induced skin cancer in mice . GSPs
are a mixture of several polyphenols/flavanols and mainly
contain proanthocyanidins (89%), which constitute dimers,
trimers, tetramers, and oligomers, and monomeric flavanols
(6.6%), as has been described previously . GSPs can be
easily consumed as a dietary supplement for skin photo-
protection. It is worth mentioning that we are testing the
effects of GSPs rather than individual constituents as we
consider that at least some of the constituents present in GSPs
act synergistically and may provide better photoprotection
than a single constituent. As UV-induced skin cancer has
been associated with UV-induced oxidative stress, DNA
damage, and activation of oncogenic cellular signaling [3,21],
we determined whether GSPs have the capability to inhibit
the above-mentioned UV-induced adverse biological effects.
Therefore, we determined the photoprotective effects of GSPs
against UVB-radiation-induced: (i) oxidative stress in terms of
hydrogen peroxide production, lipid and protein oxidation,
and oxidative DNA damage in the form of 8-hydroxydeox-
yguanosine (8-OHdG), (ii) depletion of endogenous antiox-
idant defense enzymes, such as glutathione peroxidase (GPx),
glutathione (GSH), catalase (CAT), and superoxide dismutase
(SOD), and (iii) activation of MAPK and NF-κB signaling
pathways using normal human epidermal keratinocytes
(NHEK) as an in vitro model.
Materials and methods
Chemicals and antibodies
GSPs were kindly provided by the Kikkoman Corp. (Japan),
as a generous gift for our research work. Dihydrorhodamine 123
(DHR) was purchased from Molecular Probes (Eugene, OR,
USA). OxyBlot Protein Oxidation Detection Kit was purchased
from the Intergen Co. (Purchase, NY, USA). The phosphory-
lated ERK1/2 (Thr202/Tyr204), JNK (Thr183/Tyr185), and p38
(Thr180/Tyr182) and nonphosphorylated ERK1/2, JNK, and p38
antibodies and the anti-β-actin antibody were purchased from
Cell Signaling Technology, Inc. (Beverly, MA, USA). Anti-
bodies for NF-κB, IκBα, and IκB kinase α (IKKα) and the anti-
mouse IgG HRP-linked and anti-rabbit IgG HRP-linked
secondary antibodies were obtained from Santa Cruz Biotech-
nology, Inc. (Santa Cruz, CA, USA). DMEM, penicillin,
streptomycin, fetal bovine serum, and trypsin/EDTA were
purchased from CellGro (Herndon, VA, USA). Protein assay kit
was obtained from Bio-Rad (Hercules, CA, USA) and enhanced
chemiluminescence Western blotting detection reagents were
purchased from Amersham Pharmacia Biotech (Piscataway, NJ,
USA). All other chemicals employed in this study were of
analytical grade and purchased from Sigma Chemical Co. (St.
Louis, MO, USA).
The source of UVB radiation was a band of four UVB lamps
(Daavlin, UVA/UVB Research Irradiation Unit, Bryan, OH,
USA) equipped with an electronic controller to regulate UV
dosage at a fixed distance of 24 cm from the lamps to the surface
of the cell culture plates. The majority of the resulting
wavelengths were in the UVB (290–320 nm; about 80%) and
UVA (about 20%) range and the peak emission was recorded at
The NHEK cells were obtained from the Cell Culture Core
Facility of the Skin Diseases Research Center at UAB.
Primary cultures were initiated and maintained in a
keratinocyte serum-free medium (GIBCO, Invitrogen Corp.)
with L-glutamine. Complete medium was also supplemented
with EGF (2.5 μg/500 ml) and bovine pituitary extract (25
mg/500 ml). Cells were maintained in an incubator at 37°C at
95% humidity and 5% CO2environment. Cells were grown in
standard medium, without growth factors, for 24 h to obtain
quiescent cells with low levels of activated ERK1/2, JNK,
and p38 before experiments examining the effects of UVB,
H2O2, or GSPs.
1604S.K. Mantena, S.K. Katiyar / Free Radical Biology & Medicine 40 (2006) 1603–1614
Treatment of cells
GSPs were dissolved in a small amount of dimethyl
sulfoxide (DMSO) and thereafter dissolved in PBS (pH 7.4).
The concentration of DMSO was not more than 0.1% in culture
medium when cells were treated with GSPs. An identical
concentration of DMSO was added to non-GSP-treated cells. To
determine the effects of GSPs on UVB-induced markers of
oxidative stress or activation of signaling pathways, the
subconfluent (70–80%) cells were treated with GSPs (10, 20,
30, 40, or 50 μg/ml) for 3–6 h before UVB irradiation.
Thereafter, cells were washed with PBS to make them GSP free
and then cells in fresh PBS were exposed to the desired doses of
UVB. After UVB exposure, PBS was replaced with culture
medium, if required. Cells were harvested at desired time points
and whole-cell lysates and nuclear or cytosolic lysates were
prepared as described previously [14,22]. Treatments of NHEK
with other antioxidants, similar to treatment with GSPs, were
Markers of oxidative stress
Measurement of intracellular H2O2
Intracellular H2O2was measured using DHR as a specific
fluorescent dye probe as described previously [11,14]. Briefly,
confluent NHEK in a 24-well culture plate were treated with
GSPs for 3h and thereafter washed with PBS to remove residual
GSPs from the culture. The cells were then incubated with DHR
(5 mM) for 45 min, washed, and then exposed to UVB (60 mJ/
cm2). Thirty minutes after UVB exposure, the fluorescence
intensity was recorded on a Synergy HT (Bio-TEK Instruments,
Inc.) fluorescence plate reader with an excitation wavelength of
485 nm and an emission wavelength of 530 nm.
Assay for protein oxidation
Oxidized proteins were detected using the OxyBlot Protein
Oxidation Detection kit (Intergen) following the manufacturer's
protocol as described by us previously . Briefly, 10-μg
samples of proteins were subjected to dinitrophenylhydrazine
(DNPH) derivatization. Incubation of equal aliquots with a
control solution lacking DNPH served as negative controls. The
dinitrophenylhydrazine-derivatized protein samples were sepa-
rated by 10% SDS–PAGE gel electrophoresis and blotted onto
nitrocellulose membranes. After nonspecific binding was
blocked, the membranes were incubated with a rabbit anti-
dinitrophenylhydrazine antibody for 1 h and then with a
peroxidase-coupled goat anti-rabbit IgG antibody at room
temperature. The membranes were then treated with an ECL
(Amersham Life Sciences, Arlington, IL, USA) detection
system to visualize protein bands.
Assay for 8-OHdG
Cells were UVB irradiated with or without pretreatment with
GSPs, harvested after 24 h, washed with cold PBS, and
cytospun. The cells were fixed in methacarn (methanol/
chloroform/acetic acid, 6/3/1, v/v) for 1 h at room temperature.
Endogenous peroxidase was blocked by incubation with 3%
H2O2in methanol for 30 min, and nonspecific binding sites
were blocked by incubation with 10% normal goat serum in
Tris-buffered saline (150 mM Tris/HCl and 150 mM NaCl, pH
7.6) for 15 min. The cells were then treated with proteinase K
(20 mg/ml in PBS) for 15 min at room temperature. The anti-8-
OHdG monoclonal antibody was used to detect oxidized
nucleosides (OxisResearch, Portland, OR, USA). As a negative
control, cells were incubated in PBS without the primary
Assay for lipid peroxidation
Lipid peroxidation (LPO) was determined in microsomal
fractions from the various treatment groups using the
thiobarbituric acid (TBA) reaction method, as described
previously . Briefly, 0.2 ml of the microsomal fraction
was treated with 0.2 ml of 8.1% SDS and 3 ml of TBA reagent.
Total volume was made up to 4 ml with distilled water and kept
at 95°C in a water bath for 1 h. Color was extracted with n-
butanol and pyridine (15:1, v/v). The absorbance was measured
at 530 nm, and the resultant data are expressed in terms of
percentage of control.
Assay for hydroxyl radical scavenging
Hydroxyl radical scavenging activity of GSPs was measured
using the deoxyribose method in a cell-free system . To the
reaction mixture containing deoxyribose (3 mM), ferric chloride
(0.1 mM), EDTA (0.1 mM), ascorbic acid (0.1 mM), and
hydrogen peroxide (2 mM) in phosphate buffer (pH 7.4, 20
mM), various concentrations of GSPs (20–100 μg/ml) were
thiobarbituric acid (0.5 ml, 1% w/v) were added. The reaction
mixture was kept in a boiling water bath for 30 min and cooled
and absorbance was measured at 532 nm.
Direct scavenging of superoxide radical was determined
using the competition kinetic method. For these studies,
superoxide ion was prepared in a cell-free system by dissolving
potassium superoxide (KO2) in DMSO in crown ether complex
. The solubility and the stability of the superoxide were
increased by such complexation with crown ether. In this
method superoxide radical is made to react with nitroblue
tetrazolium (NBT2+, 9 μg/ml) at pH 7.0, in the absence and
presenceof GSPs. Inthe absence ofGSPs thesuperoxide radical
completely reacts with NBT2+to produce NBT+. The formation
of NBT+was observed by monitoring its absorbance at 560 nm.
However, the presence of GSPs results in a decrease in the
absorbance of NBT+.From the extent of the decrease, the rate of
scavenging of superoxide radical by GSPs was determined.
Assays for antioxidant defense enzymes
Preparation of cytosols and microsomal fractions
After treatment with GSPs and UVB irradiation, the cells
were harvested and washed in PBS, and cytosolic and
microsomal fractions were prepared as described earlier .
1605S.K. Mantena, S.K. Katiyar / Free Radical Biology & Medicine 40 (2006) 1603–1614
Briefly, cells were homogenized with a Polytron homogenizer
in PBS buffer containing potassium chloride (1.19%, w/v) and
centrifuged at 18,000g for 15 min at 4°C to prepare cytosolic
and microsomal fractions . Cytosols were used to determine
the endogenous antioxidant defense components like GSH,
GPx, CAT, and SOD, whereas the microsomal fraction was used
to determine LPO.
Assays for endogenous antioxidant enzymes
The levels of GPx, GSH, CAT, and SOD were measured in
cytosolic fractions following the standard analytical methods of
Flohe and Gunzler , Akerboom and Sies , Nelson and
Kiesow , and Misra and Fridovich , respectively. The
data are expressed in terms of percentage of control.
The experiments for all the markers of oxidative stress and
antioxidant enzymes were repeated at least three times.
Western blot analysis
For Western blot analysis, the proteins (25–50 μg) were re-
solved over 8–12% SDS–PAGE gels and transferred onto a
nitrocellulose membrane, as detailed previously . After the
nonspecific binding sites in were blocked using blocking buffer
(5% nonfat dry milk, 1% Tween 20 in 20 mM TBS, pH 7.6), the
blots were then incubated overnight with primary antibodies
specific for the protein to be assessed. The blot was washed and
a wash, the protein expression was detected by chemilumi-
nescence using an ECL detection system (Amersham Life
Science, Inc.). The intensities of the bands were measured using
the digitized scientific software program UN-SCAN-IT (Silk
Scientific Corp., Orem, UT, USA) to determine the chemopre-
ventive effect of GSPs. To ensure equal protein loading, the
membranes were stripped and reprobed with anti-β-actin
antibodies using the protocol detailed above. The experiments
were repeated three times.
Statistical analysis was done using ANOVA followed by post
hoc multiple comparison tests. The chemopreventive effect of
GSPs was considered significant if p b 0.05.
GSPs inhibit UVB-induced intracellular release of H2O2in
were exposed to a UVB dose of 30 mJ/cm2, which resulted in
1A), which was determined as a marker of UVB-induced
oxidative stress. Pretreatment of NHEK cells with GSPs (10–50
μg/ml) before UVB (30 mJ/cm2) exposure resulted in a dose-
dependent inhibition of UVB-induced intracellular release of
H2O2(30–89%; *p b 0.01, ¶p b 0.001) when measured in terms
of relative fluorescence intensity of oxidized DHR. In another
set of experiments, NHEK were exposed to 10, 30, and 60 mJ/
cm2of UVB, which resulted in a significant dose-dependent
increase in H2O2production compared to non-UVB-exposed
NHEK. Pretreatment of NHEK with GSPs (30 μg/ml)
significantly inhibited (80–92%, ¶p b 0.001) UVB-induced
intracellular production of H2O2(Fig. 1B) at each dose level
of UVB tested. These data indicated that GSPs possess
antioxidant activity and confirmed that GSPs have the
capability to inhibit UVB radiation-induced oxidative stress
in target cells.
These preliminary studies indicate that exposure of NHEK to
30 mJ/cm2of UVB significantly induced intracellular produc-
tion of H2O2, and treatment with a 30 μg/ml dose of GSPs to
NHEK induced significant inhibition of UVB-induced H2O2
production in target cells. Therefore in all further experiments,
we used the optimum doses of UVB (30 mJ/cm2) and GSPs (30
μg/ml) to define the photoprotective effects of GSPs on UVB-
Fig. 1. In vitro treatment of NHEK with GSPs inhibits UVB-induced
intracellular release of H2O2. (A) Pretreatment of NHEK with GSPs dose-
dependently inhibits UVB (60 mJ/cm2)-induced intracellular release of H2O2.
(B) Pretreatment of NHEK with GSPs (30 μg/ml) inhibits UVB dose-dependent
induction of intracellular release of H2O2. UVB-induced H2O2production in
different treatment groups is expressed in terms of relative fluorescence intensity
experiments. Significant difference from UVB alone, *p b 0.01; ¶p b 0.001.
1606 S.K. Mantena, S.K. Katiyar / Free Radical Biology & Medicine 40 (2006) 1603–1614
induced oxidative stress and activation of MAPK and NF-κB
signaling pathways in NHEK cells in vitro.
GSPs inhibit UVB-induced photo-oxidative damage to lipids
The hallmarks of UVB-induced oxidative damage are the
oxidation of biomacromolecules like lipids and proteins. The
compared to non-UVB-exposed control cells. Treatment of
NHEK with GSPs (30 μg/ml) decreased UVB-induced LPO by
77% (p b 0.001) in NHEK. Similar to LPO, the oxidation of
protein molecules also adversely affects the functionality of the
derivatives that affect the nature and biological activity of
proteins . The presence of carbonyl groups has become a
widely accepted measure of oxidative damage of proteins under
conditions of oxidative stress, because the groups react with
DNPH to form stable hydrazone derivatives . Therefore, we
oxidative damage and found that exposure of NHEK to UVB
resulted in an increase in protein oxidation as evident from the
darker and/or new bands of proteins (Fig. 2B, lane 3) compared
to non-UVB-irradiated NHEK cells (lanes 1 and 2 from left).
Treatment of GSPs markedly inhibited UVB-induced oxidation
treated UVB-irradiated cells (Fig. 2B, lane 3). Treatment of
NHEK with GSPs alone did not induce carbonyl formation or
oxidation of protein molecules (Fig. 2B, lane 2).
GSPs scavenge hydroxyl radicals and superoxide anions in a
radicals from the system, a cell-free in vitro system was utilized.
Fig. 2. In vitro treatment of NHEK with GSPs inhibits UVB-induced markers of
oxidative stress. NHEK were exposed to UVB (30 mJ/cm2) with or without
treatment of GSPs (30 μg/ml) for 6 h before UVB irradiation. At the time of
UVB irradiation, GSPs were washed from the culture medium with PBS buffer.
Cells were harvested 3 h after UVB irradiation, and the microsomal fraction was
prepared for the analysis of LPO and cell lysates were prepared for the
determination of protein oxidation using the OxyBlot Protein Oxidation
Detection Kit following the manufacturer's protocol. (A) Treatment of NHEK
with GSPs inhibits UVB-induced LPO. The inhibition of LPO by GSPs is
expressed in terms of percentage of control cells not exposed to UVB radiation
and the data are presented as the means ± SEM from three independent
experiments, *p b 0.001. (B) Treatment of NHEK with GSPs inhibits UVB-
induced oxidation of proteins. Relative intensity of various oxidized proteins in
different treatment groups is visible by the thickness or darkness of the bands. A
representative blot is shown from three separate experiments with similar
results. Treatment of NHEK with GSPs alone did not affect the LPO or protein
oxidation. (C) Treatment of GSPs in an in vitro cell-free system scavenges
hydroxyl radicals dose dependently. The data are expressed in terms of
percentage of control in which the cell-free system was not treated with GSPs.
(D) GSPs in an in vitro cell-free system scavenge superoxide anion dose
dependently. The scavenging capacity of superoxide anion by GSPs was
expressed in terms of the ratio between GSPs and NBT. Data are presented as
means ± SEM from three independent experiments.
1607 S.K. Mantena, S.K. Katiyar / Free Radical Biology & Medicine 40 (2006) 1603–1614
As shown in Fig. 2C, the hydroxyl radical scavenging ability of
GSPs was increased dose dependently and thus confirmed their
antioxidant capability. In order to quantitatively evaluate the
scavenging ability of GSPs for superoxide radicals, we studied
the kinetics of its reaction using a UV spectrometer. For this
purpose, at constant superoxide concentration, conditions were
selected such that NBT2+and GSPs compete for superoxide
ion. Absorbance (A) due to NBT+formed at 560 nm was
monitored under various GSP concentrations, both in the
absence (A0) and in the presence of GSPs (A), and was found to
decrease with increasing GSP content. The slope of the linear
plot for A0/A − 1 vs [GSPs]/[NBT2+], both expressed in μg/ml,
will give the comparative ability of GSPs in reacting with
superoxide radical with respect to NBT2+. Thus from the linear
plot (Fig. 2D), it seems that GSPs have almost half (0.43) the
reactivity toward superoxide radicals as NBT2+. The inset in
Fig. 2D shows the percentage scavenging of superoxide radical
with increasing concentration of GSPs. From this experiment, it
can be concluded that GSPs are potential superoxide radical
GSPs inhibit UVB-induced oxidative DNA damage
The formation of 8-OHdG after UVB exposure is another
biomarker of UVB-induced photo-oxidative damage. We found
that UVB irradiation of NHEK resulted in DNA oxidation,
which was detected in the form of 8-OHdG using immunocy-
tochemistry (Fig. 3). Irradiation of NHEK with UVB resulted in
8-OHdG formation in about 15 ± 3% cells, whereas in GSPs +
UVB-treated cells only 4 ± 1% cells were 8-OHdG+. Thus
pretreatment of NHEK with GSPs decreased the number of
UVB-induced 8-OHdG+cells by 73% (p b 0.001) compared to
non-GSP-treated but UVB-irradiated cells (Fig. 3), thus
indicating the photoprotection against DNA damage from
UVB radiation by GSPs.
GSPs prevent UVB-induced depletion of endogenous
antioxidant defense enzymes
It is well recognized that acute or chronic UV exposure
depletes the antioxidant defense capability in an in vivo
system . By using NHEK as an in vitro model we further
confirmed that UVB irradiation depletes the levels of
endogenous antioxidant defense enzymes (Fig. 4). For
instance, our data demonstrated that exposure of NHEK to
UVB radiation significantly decreased GPx (23%, p b 0.05)
and endogenous antioxidant GSH (36%, p b 0.01) compared
to non-UVB-exposed control cells (Fig. 4). Pretreatment of
NHEK with GSPs before UVB exposure not only prevented
the depletion of GPx but also enhanced the level of GPx by
16% compared to non-UVB-exposed normal cells. Similarly,
the treatment with GSPs also prevented UVB-induced
depletion of endogenous antioxidant GSH in NHEK. The
nucleophilic and reducing properties of GSH play a central
role in metabolic pathways, as well as in the antioxidant
system of aerobic cells. Catalase is another endogenous
antioxidant enzyme involved in the catalytic conversion of
H2O2to oxygen and water and thus decreases the level of
oxidative stress. The irradiation of NHEK with UVB resulted
in reduction of catalase (30%, p b 0.05) compared to non-
UVB-exposed NHEK (Fig. 4), whereas pretreatment of
NHEK with GSPs restored the activities of catalase. Similar
to other enzyme levels, UVB irradiation of NHEK cells
depleted the level of SOD by 32% compared to non-UVB-
exposed cells, and pretreatment of NHEK with GSPs restored
Fig. 3. Treatment of NHEK with GSPs inhibits UVB-induced DNA damage in
the form of 8-OHdG. (A) NHEK cells were exposed to UVB (30 mJ/cm2) with
or without prior treatment of GSPs (30 μg/ml) for 3 h. Cells were harvested
24 h after UVB exposure and immunocytostaining was performed to detect
8-OHdG+cells as described under Materials and methods. 8-OHdG+cells were
not visible in the control group as well as in the GSPs-alone-treated group (non-
UVB exposed), whereas they were observed in the UVB-exposed NHEK
groups. 8-OHdG+cells are shown by dark brown staining and arrows indicate
their presence. (B) Percentage of 8-OHdG+cells in different treatment groups is
summarized from three independent experiments and reported as means ± SD.
*p b 0.001 vs UVB-alone-exposed cells.
1608 S.K. Mantena, S.K. Katiyar / Free Radical Biology & Medicine 40 (2006) 1603–1614
the activity of SOD enzyme, thus indicating photoprotective
effects of GSPs against UVB-induced depletion of antioxidant
defense in an in vitro model. Moreover, treatment of NHEK
with GSPs alone did not significantly affect the original levels
of antioxidant enzymes (Fig. 4).
Kinetics of UVB radiation-induced phosphorylation of
ERK1/2, JNK, and p38 MAPK proteins in NHEK and
prevention by GSP treatment
Previous studies, including the studies from our laboratory,
have shown that UVB radiation induces the phosphorylation of
MAPKs, such as ERK1/2, JNK, and p38, which have been
implicated in skin carcinogenesis. The phosphorylation of these
proteins has been shown to be mediated through UVB-induced
oxidative stress [11,34,35]. In order to investigate the photo-
protective effect of GSPs, we determined their effects on UVB-
induced phosphorylation of the above-mentioned three MAPK
proteins using Western blot analysis. The data on the kinetics of
MAPK activation in UVB-irradiated NHEK showed that
maximum phosphorylation of ERK1/2 (p42/p44) occurred at
15–30 min post-UVB irradiation and thereafter gradually
decreased (Fig. 5, left). Western blotting and subsequent
analysis of the intensity of bands relative to β-actin indicated
that GSP treatment inhibited UVB-induced phosphorylation of
ERK1/2 in NHEK at each time point studied (80–90%,
p b 0.001, right).
Similar to UVB-induced phosphorylation of ERK1/2, a
marked induction in JNK (p46/p54) phosphorylation was found
within 15 min and remained elevated until 60 min and thereafter
declined (Fig. 5, left). Western blotting and subsequent
measurement of the intensity of bands relative to β-actin
indicated that treatment with GSPs markedly inhibited UVB-
induced phosphorylation of JNK1 and JNK2 at each time point
studied. Exposure of NHEK to UVB induced phosphorylation
of p38 as early as 5 min (data not shown), and elevated levels of
phosphorylation of p38 were observed until 2 h after UVB
irradiation (Fig. 5, left) and thereafter declined. Measurement of
intensities of bands relative to β-actin indicated that treatment
of GSPs significantly inhibited (80–85%, p b 0.001) UVB-
induced phosphorylation of p38 compared to non-GSP-treated
(UVB alone) NHEK. Importantly, treatment of NHEK with
GSPs alone did not induce the phosphorylation of ERK1/2,
JNK, or p38 proteins of the MAPK family (data not shown).
Further, the total amount of ERK1/2, JNK, and p38 remain
unchanged at each time point studied.
Treatment of NHEK with various antioxidants inhibits
H2O2-induced phosphorylation of ERK1/2, JNK, and p38 proteins
To confirm whether UVB-induced H2O2 mediates phos-
phorylation of MAPK proteins, we treated the NHEK with a
Fig. 4. Treatment of NHEK with GSPs inhibits UVB-induced depletion of
antioxidant enzymes. Cells were irradiated with 30 mJ/cm2of UVB radiation
with or without pretreatment with GSPs (30 μg/ml). Cells were treated with
GSPs for 3 h before UVB exposure, and GSPs were removed after washing with
PBS buffer at the time of irradiation. Non-UVB-exposed cells were used as
controls. Three hours after UVB exposure, NHEK were harvested and cytosols
were prepared for the analysis of antioxidant enzymes, as described under
Materials and methods. Data are presented in terms of percentage of control
(non-UVB-exposed) and reported as the means ± SD from three independent
experiments. In vitro treatment of NHEK with GSPs inhibits UVB-induced
depletion of (A) GPx, (B) GSH, (C) catalase, and (D) SOD. Significant
difference vs UVB alone, *p b 0.01; ¶p b 0.05.
1609S.K. Mantena, S.K. Katiyar / Free Radical Biology & Medicine 40 (2006) 1603–1614
predetermined/prestandardized dose of H2O2 (100 μM) as
described previously [11,14]. To examine whether inhibition
of H2O2-mediated phosphorylation of MAPK proteins in
NHEK by GSPs is because of their antioxidant property, we
treated the NHEK with known antioxidants, such as (−)-
epigallocatechin-3-gallate (EGCG), ascorbic acid, silymarin,
and N-acetylcysteine, as positive controls and compared the
results with those from the GSPs. Moreover, it has been
shown that H2O2treatment of NHEK induces phosphoryla-
tion of MAPK proteins and that treatment with EGCG and
ascorbic acid inhibits either UVB- or H2O2-induced phos-
phorylation of MAPK signaling pathways [11,14]. Using
Western blot analysis, we found that pretreatment with GSPs
as well as other known antioxidants inhibited H2O2-induced
phosphorylation of ERK1/2, JNK, and p38 proteins of
MAPK family but at different levels (Fig. 6). This
experiment further confirmed that inhibition of H2O2-induced
activation of MAPK signaling is due to the antioxidant
property of GSPs.
GSPs inhibit UVB-induced activation and translocation of
NF-κB in NHEK
Our results indicated that in vitro treatment of NHEK with
GSPs inhibits UVB-induced phosphorylation of MAPK
proteins. Therefore, we determined whether GSPs could inhibit
activation of the transcription factor NF-κB/p65, which is a
downstream target of the MAPK signal transduction pathways.
As shown in Fig. 7, exposure of NHEK to UVB resulted in
enhanced activation and translocation of NF-κB/p65 to the
nucleus (Fig. 7A). In a time-dependent study we found that
UVB-induced activation of NF-κB in NHEK started as early as
30 min (data not shown) after UVB irradiation, with the
maximum activation at 3 h post-UVB exposure, and thereafter
declined at and after 6 h. Western blotting and subsequent
measurement of the intensity of bands relative to β-actin
indicated that treatment of NHEK with GSPs before UVB
irradiation markedly abrogated the UVB-induced activation and
subsequently translocation of NF-κB to the nucleus in a time-
dependent manner, which was studied at 3, 6, and 12 h post-
GSPs inhibit UVB-induced degradation of IκBα and activation
Exposure of NHEK to UVB radiation resulted in the
degradation of IκBα protein and subsequent activation and
translocation of NF-κB/p65 to the nucleus . We determined
whether UVB-induced degradation of IκBα is inhibited by GSP
treatment, which will in turn inhibit the activation and
translocation of NF-κB/p65. Western blot analysis and
subsequent measurement of the intensity of bands relative to
β-actin indicated that exposure of NHEK to UVB radiation
resulted in degradation of IκBα protein at each time point
studied compared to non-UVB-exposed cells (Fig. 7B). The
UVB-induced degradation of IκBα was inhibited at all the time
points studied and almost completely inhibited at 6 and 12
h after UVB exposure in those NHEK cells which were
pretreated with GSPs. The induction of IKKα activity has been
shown to be essential for UVB-induced phosphorylation/
Fig. 5. In vitro treatment of NHEK with GSPs inhibits UVB-induced phosphorylation of (A) ERK1/2, (B) JNK, and (C) p38 proteins of the MAPK family. Inhibition
was determined in a time-dependent (15–180 min) manner after UVB irradiation of the cells. Cells were exposed to UVB (30 mJ/cm2) with or without pretreatment
with GSPs (30 μg/ml) for 3 h. Cells were harvested at different time points as indicated and cell lysates were prepared to determinethe phosphorylated and total protein
levelsof ERK1/2,JNK,andp38 usingWesternblot analysis,as describedunderMaterialsandmethods.Arepresentative blot fromthreeindependentexperimentswith
identical observations is shown, and equivalent protein loading was confirmed by probing stripped blots for β-actin as shown.
1610S.K. Mantena, S.K. Katiyar / Free Radical Biology & Medicine 40 (2006) 1603–1614
degradation of IκBα. To determine the possible inhibitory
mechanism of GSPs on IκBα protein degradation, we
determined the level of IKKα in our system. Our Western
blot analysis indicated that irradiation of NHEK with UVB
resulted in the activation of IKKα protein in NHEK within 3
h and thereafter gradually declined (Fig. 7C). Pretreatment of
NHEK with GSPs markedly inhibited UVB-induced activation
of IKKα at all the time points studied, thus confirming that
inhibition of UVB-induced activation of NF-κB by GSPs is
mediated through inhibition of activation of IKKα.
The UVB (290–320 nm) component of solar ultraviolet
radiation acts as a tumor initiator, a tumor promoter, and a
complete carcinogen . It has been recognized that UVB-
induced oxidative stress in an in vitro or in vivo system
contributes to several adverse biological effects on the skin
[1,2,21]. There is considerable evidence that the tumor-
promoting effects of UVB radiation are mediated through
UVB-induced oxidative stress-mediated activation of signal
transduction pathways that control gene expression . In
previous studies we have shown that dietary GSPs prevent
UVB-induced skin carcinogenesis in terms of tumor incidence,
tumor multiplicity, and tumor growth in mice . Because
GSPs, as a dietary supplement, prevent UV-induced skin
carcinogenesis in mice, we further attempted to define their
photoprotective mechanism of action using NHEK as an in vitro
model. As the tumor-promoting effect of UV radiation is
mediated through the induction of oxidative stress, we
determined the effects of GSPs on UVB-induced oxidative
stress and their effects on the activation of MAPK and NF-κB
signaling pathways. We observed that in vitro treatment of
NHEK with GSPs resulted in the prevention of UVB-induced
depletion of antioxidant defense enzymes like GPx, catalase,
SOD, and GSH; inhibition of H2O2production; and scavenging
of superoxide anion and hydroxyl radicals (Figs. 1–4), thus
Fig.6. Treatment of NHEK with GSPs and known antioxidants, such as EGCG,
silymarin, ascorbic acid (AA), and N-acetylcysteine (NAC), inhibits H2O2-
induced phosphorylation of (A) ERK1/2, (B) JNK, and (C) p38 proteins of the
MAPK family. NHEK were treated with H2O2(100 μM) for 30 min. NHEK
were treated with equal concentrations (30 μg/ml) of GSPs, EGCG, silymarin,
ascorbic acid, or NAC for 3 h before H2O2treatment of NHEK. Thirty minutes
after H2O2treatment, cells were washed with PBS and harvested and cell
lysates were prepared to determine H2O2-induced phosphorylation and total
protein levels of ERK1/2, JNK, and p38 using Western blot analysis, as
described under Materials and methods. A representative blot is shown from
three independent experiments with similar observations, and equivalent
protein loading was confirmed by probing stripped blots for β-actin as
Fig. 7. Treatment of NHEK with GSPs inhibits UVB-induced activation of NF-
κB and IKKα and degradation of IκBα. NHEK were exposed to UVB (30 mJ/
cm2) with or without pretreatment withGSPs (30 μg/ml). Cells weretreated with
GSPs for at least 3 h before UVB exposure. Cells were harvested at 3-, 6-, and
12-h time points after UVB exposure, and cell lysates were prepared to
determine the activation of NF-κB or IKKα or degradation of IκBα using
Western blot analysis, as described under Materials and methods. A
representative blot from three independent experiments with almost identical
observations is shown. The relative intensities of each band after normalization
for the levels of β-actin are shown under each blot. The equivalent protein
loading was confirmed by probing stripped blots for β-actin as shown.
1611 S.K. Mantena, S.K. Katiyar / Free Radical Biology & Medicine 40 (2006) 1603–1614
providing a possible mechanism for the photoprotection of
GSPs. Antioxidant enzymes function cooperatively, and any
change in one of them may affect the equilibrium state of
oxidative stress or ROS. If ROS remain without being
scavenged in the biological system they may induce biochem-
ical alterations such as inflammation, lipid and protein
oxidation, DNA damage, and certain enzyme activation or
inactivation [10,21,37–40]. Thus, prevention of UVB-induced
depletion of antioxidant defense enzymes, and scavenging
superoxide anion and hydroxyl radicals by GSPs, would result
in inhibition of the tumor-promoting activity of UV radiation.
UVB-induced LPO and protein oxidation were used as
markers of oxidative damage in our system, and these were
significantly inhibited by the treatment with GSPs (Fig. 2). LPO
in biological membranes is a free radical-mediated event and is
regulated by the availability of substrates in the form of
polyunsaturated fatty acids, pro-oxidants which promote
peroxidation, and antioxidant defenses such as α-tocopherol,
GSH, β-carotene, and superoxide dismutase [39–41]. Elevated
levels of LPO have been linked to injurious effects such as loss
of fluidity, inactivation of membrane enzymes, increases in
permeability to ions, and eventually disruption of cell
membrane leading to release of cell organelles [39–41].
Peroxidation products can also damage DNA [21,39,40].
Thus, inhibition of UV-induced LPO by GSPs should reduce
the risk factors associated with the UV-induced tumor-
promoting effects. The UV-induced oxidative stress-mediated
protein oxidation may also be a detrimental factor for skin
disorders. We observed that UV exposure to NHEK increases
protein oxidation compared to non-UV-exposed cells (Fig. 2).
The inhibition of UVB-induced protein oxidation by GSPs
would result in the reduction of photodamage. The exact
mechanism of inhibition of protein oxidation by GSPs is not
clear; however, it can be suggested that GSPs may be
preventing protein oxidation by scavenging free radicals,
activating or enhancing the antioxidant defenses of the target
cells, or activating the repair or proteolytic enzymes that repair
or degrade damaged proteins . Similarly, in vitro treatment
of GSPs prevents UV-induced DNA damage in the form of 8-
OHdG (Fig. 3). UVB-induced DNA damage is considered an
induction of the tumor initiation stage, and the prevention of
UVB-induced DNA damage by GSPs may also be considered
as one of the possible mechanisms of chemoprevention of
UVB radiation promotes tumor development by activating
various intracellular signaling cascades that play major roles in
cell growth, differentiation, and proliferation, leading to clonal
expansion of UVB-initiated cells into skin tumors . MAPKs
are important upstream regulators of transcription factor
activities and their signaling is critical to the transduction of a
wide variety of extracellular stimuli into intracellular events and
thus they control the activities of various downstream
transcription factors implicated in tumor promotion . Our
data demonstrate that UVB-induced phosphorylation of
proteins of the MAPK family, such as ERK1/2, JNK, and
p38, in in vitro NHEK was significantly inhibited by GSP
treatment at different time points studied (Fig. 5). However,
their kinetics of activation and inhibition are a little different.
ERK1/2 has been shown to be strongly activated by tumor
promoters, growth factors, and UV radiation and has a critical
role in transmitting signals initiated by them . In the
present study we observed that in vitro treatment of NHEK
with GSPs inhibits UV-induced activation of ERK1/2. It has
been shown that a potent inhibitor of the ERK pathway, BAY
43-9006, exhibited a broad spectrum of anti-tumor activities in
breast, colon, and non-small-cell lung cancer xenograft models
. Activation of JNK regulates activator protein-1 (AP-1)
transcription in response to environmental stress such as UV
radiation . Increased AP-1 activity has been implicated in
inflammation, invasion, metastasis, and angiogenesis and also
in the promotion and progression of various types of cancers
[13,47,48]. Therefore, inhibition of JNK activation may be a
relevant molecular target for potential chemopreventive agents.
Our data indicate that in vitro treatment of NHEK with GSPs
inhibits UVB-induced activation of JNK and p38 and that
inhibition may lead to the inhibition of photocarcinogenesis. It
is well known that UVB radiation induces H2O2production in
the target cells [11,14], also as shown in Fig. 1, which in turn
initiates phosphorylation of MAPK and activation of down-
stream signals and expression of genes having direct relevance
in carcinogenesis [11,14]. Therefore, it can be suggested that
treatment with GSPs prevents UVB-induced phosphorylation
of MAPK by inhibiting UVB-induced oxidative stress, such as
depletion of antioxidant enzymes and production of H2O2.
Inhibition of UVB-induced phosphorylation of MAPK pro-
teins may thus prevent the downstream events such as
activation of NF-κB, which would lead to the prevention of
photocarcinogenic events in an in vivo system. ERK and p38
proteins of the MAPK family have been shown to modulate
NF-κB activation [49,50]. We suggest that significant
inhibition of UVB-induced phosphorylation of MAPKs by
GSPs might be responsible for their inhibitory effects on the
activation of transcription factor NF-κB. Therefore, the
inhibition of the MAPK and NF-κB signaling pathways
could potentially be utilized by GSPs to activate antioxidant-
responsive element-dependent genes.
ERK1/2, JNK, and p38 proteins are all phosphorylated by
exposure to exogenous H2O2and this phenomenon can be
inhibited by pretreatment with classical antioxidants [17,35,51].
Thus the chemopreventive effect of GSPs may be associated
with the inhibition of UVB-induced oxidative stress-mediated
activation of these MAPK pathways. We know that H2O2
treatment or UVB-induced intracellular release of H2O2
mediates phosphorylation of MAPK proteins in an NHEK in
vitro system [11,14]. The inhibition of phosphorylation of these
pathways initiated with the treatment of H2O2(an oxidant) by
antioxidants such as GSPs, ascorbic acid, EGCG, silymarin, and
NAC suggests the antioxidant potential of GSPs (Fig. 6).
Although the exact mechanism of inhibition of UVB-induced
phosphorylation of MAPK proteins by GSPs may not be clearly
explained on the basis of the present set of data, it seems that the
antioxidant property of GSPs contributed to the inhibition of
UVB-induced phosphorylation of MAPK through: (i) inhibition
of UVB-induced H2O2production and (ii) inhibition of UVB-
1612S.K. Mantena, S.K. Katiyar / Free Radical Biology & Medicine 40 (2006) 1603–1614
induced depletion of antioxidant defense enzymes. Additional-
ly, the inhibition of the H2O2-mediated phosphorylation of
MAPK by GSPs in in vitro NHEK indicates that GSPs have the
ability to neutralize the effect of H2O2.
The activation of NF-κB has an important regulatory role in
inflammation, cell proliferation, and oncogenesis [52–54].
Therefore, the signaling pathways leading to the regulation of
NF-κB activity have become a focal point for drug discovery
efforts. The activation of NF-κB by the extracellular inducers
depends on the phosphorylation and subsequent degradation of
IκB proteins. Activation of NF-κB is achieved through the
action of a family of serine/threonine kinases known as IKK.
The IKKs (IKKα and/or IKKβ) phosphorylate IκB proteins and
the members of the NF-κB family. These phosphorylation
events lead to the immediate polyubiquitination of IκB proteins
and rapid degradation by the proteasomal pathway. Therefore,
the inhibitors of IKK have long been sought as specific
regulators of NF-κB. NF-κB is commonly activated by
oxidants, including H2O2, and by agents that generate
ROS, such as UV radiation [56,57]. Agents that scavenge ROS
inhibit NF-κB activation . In our study, we demonstrated
that NF-κB is activated in NHEK after UVB exposure and
subsequently translocated to the nucleus (Fig. 7); however, its
activation and translocation to the nucleus were effectively
inhibited by GSPs. UVB exposure also resulted in an increased
degradation of IκBα protein (Fig. 7). As GSPs block IκBα
degradation in UVB-exposed NHEK, our study suggests that
the inhibitory effect of GSPs on NF-κB/p65 activation may be
mediated through the inhibition of proteolysis of IκBα protein.
It is well documented that through a protein–protein interac-
tion, IκBα is bound to NF-κB/p65 and thus prevents migration
of NF-κB/p65 into the nucleus . Additionally, the IKK
complex is believed to be an important site for integrative
signals that regulate the NF-κB pathway. In the present study,
we observed that exposure of NHEK to UVB resulted in an
increase in IKKα protein expression which was markedly
inhibited by GSP treatment. These data suggest that in vitro
treatment of NHEK with GSPs inhibits UVB-induced activa-
tion and nuclear translocation of NF-κB/p65 through the
inhibition of activation of IKKα and degradation of IκBα
In summary, it seems that the photoprotective effects of
GSPs are mediated, at least, through: (i) protection of the
endogenous antioxidant defense system, (ii) prevention of
photodamage of macromolecules, lipids, proteins, and DNA,
which leads to (iii) inhibition of activation of the MAPK and
NF-κB pathways. As the early activation of cellular signaling
pathways in response to UV irradiation is involved in the
inflammatory reactions photoaging, photodermatoses, and
photocarcinogenesis, the use of GSPs as a dietary supplement
may have beneficial effects in protecting against these skin
disorders in humans. Further in vivo mechanism-based studies
are required to examine whether GSPs can be used as a safe
pharmacologic agent in skin care products, such as moistur-
izing creams, skin care lotions, and sunscreens, for the
chemoprevention of solar UV light-induced human skin
This work was supported by funds from USPHS Grant
CA104428, the Merit Review Award from the Veterans
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