Carcinogenesis vol.25 no.8 pp.

1459--1465, 2004
doi:10.1093/carcin/bgh152

Silibinin prevents ultraviolet radiation-caused skin damages in SKH-1

hairless mice via a decrease in thymine dimer positive cells and an
up-regulation of p53-p21/Cip1 in epidermis

Sivanandhan Dhanalakshmi1,*, G.U.Mallikarjuna1,*, non-melanoma skin cancer (NMSC) (1). In addition to its
Rana P.Singh1 and Rajesh Agarwal1--3 carcinogenic activity, UV also causes photoaging, immuno-
1 suppression and sunburn (2--4). There is high morbidity with
Department of Pharmaceutical Sciences, School of Pharmacy
and 2University of Colorado Cancer Center, University of Colorado NMSC, and the associated medical cost is estimated to be over
Health Sciences Center, Denver, CO 80262, USA $500 million/year. In spite of skin cancer awareness in recent
years, the prediction is a further increase in NMSC cases

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3
To whom correspondence should be addressed
Email: rajesh.agarwal@UCHSC.edu possibly because of several outdoor activities and depletion
in atmospheric ozone (5). This emphasizes the importance of
Non-melanoma skin cancer (NMSC) accounts for >1
developing additional strategies that could control and prevent
million new cases each year in the US alone suggesting
solar UV-caused NMSC. Although the usage of sunscreens
that more approaches are needed for its prevention and
has been an important protection strategy, further approaches
control. Earlier studies by us have shown that silymarin (a
are needed to more effectively protect human skin against
crude form of biologically active silibinin with some other
UV-caused damages as well as NMSC (6,7).
isomers), isolated from milk thistle, affords strong protec-
Chemoprevention of cancer is a novel and more effective
tion against ultraviolet (UV) radiation-induced NMSC in
means of cancer control where naturally occurring agents are
SKH-1 hairless mice; however, the molecular mechanisms
considered as a less toxic and more effective approach in
of its efficacy are not known. Here, we assessed the effect of
controlling various human malignancies including NMSC
silibinin on UV-induced DNA damage and p53-p21/Cip1
(8--12). For example, oral administration or topical application
accumulation, and their roles in UV-induced cell prolifera-
of green tea and black tea, with or without caffeine, has been
tion and apoptosis in SKH-1 hairless mouse epidermis.
shown to strongly prevent UV-induced skin cancer in mouse
Topical application of silibinin prior to, or immediately
skin (13), inhibit skin carcinogenesis in UV-exposed high-risk
after, UV irradiation resulted in a very strong protective
mice (12), and increase apoptotic sunburn cells by inducing
effect against UV-induced thymine dimer positive cells in
p53 and p21/Cip1 (14). Earlier studies by us have shown that
epidermis accounting for 76--85% (P 5 0.001) inhibition.
silymarin (a crude form of biologically active silibinin with
In other studies, silibinin treatment resulted in a further
some other isomers), isolated from milk thistle [Silybum
up-regulation of p53 by ~1.6-fold (P 5 0.001) together with
marianum (L.) Gaertn] plant, affords strong protection against:
an increase (~2-fold, P 5 0.001) in p21/Cip1 protein levels.
(i) UV-induced tumor initiation, promotion and complete
Proliferative cell nuclear antigen staining showed that sili-
carcinogenesis in SKH-1 hairless, and (ii) chemical carcino-
binin pre- or post-topical application significantly inhibits
genesis in SENCAR mouse skin (15--19). Completed studies
(40--52 and 20--40%, respectively, P 5 0.001) UV-induced
by us have also shown that silymarin inhibits UV- and chemi-
epidermal cell proliferation. In addition, silibinin strongly
cal tumor promoter-induced cellular and biochemical events
decreased UV-caused terminal deoxynucleotidyl transfer-
associated with skin tumor promotion (15--19); however, the
ase-mediated dUTP nick end labeling-positive apoptotic/
mechanism of its efficacy against UV-caused damages leading
sunburn cell formation (P 5 0.001). These findings suggest
to skin tumor initiation is not known, which was explored in
that silibinin affords strong protection against UV-induced
the present investigation.
damage in epidermis by a decrease in thymine dimer posi-
UV radiation is known to exert its tumor initiating effects
tive cells and an up-regulation of p53-p21/Cip1 possibly
primarily through the formation of cyclobutane pyrimidine
leading to an inhibition in both cell proliferation and apop-
dimers and 6-4 photoproducts (20) in DNA, as well as via
tosis. Comparable effects of silibinin following its pre- or
formation of reactive oxygen species that subsequently lead to
post-UV application suggest that mechanisms other than
the damage of DNA and other major cellular targets (21). The
sunscreen effect are operational in silibinin efficacy against
UV-damaged cell responds to these alterations either by
UV-caused skin damages.
enhanced DNA repair or by inducing apoptotic death, if the
damage is too severe; failure to do so, however, could lead to
the formation of an initiated cell (22). One important cellular
Introduction
response occurring after UV exposure and/or UV-caused DNA
Ultraviolet (UV) radiation, in the range of 290--320 and damage is phosphorylation of p53 at various serines that
320--400 nm for UVB and UVA wavelengths, respectively, increases its half-life causing its accumulation (23). This also
from the sunlight is the major etiologic factor for the increases the transcriptional activity of p53 leading to the
synthesis of its target genes such as p21/Cip1 that helps the
cells in: (i) arresting DNA synthesis and allowing more time
Abbreviations: H&E, hematoxylin and eosin; NMSC, non-melanoma skin for DNA repair, or (ii) modulating bax levels causing apoptosis
cancer; PCNA, proliferating cell nuclear antigen; UV, ultraviolet; TUNEL, (24,25). Consistent with above reports, in the present study, we
terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. assessed the effect of topical application of silibinin on UV-

These authors contributed equally to this work and share first authorship. induced DNA damage in terms of thymine dimer positive
Carcinogenesis vol.25 no.8 # Oxford University Press 2004; all rights reserved. 1459
S.Dhanalakshmi et al.
cells, cell proliferation and apoptosis as well as the role of p53
and p21/Cip1 molecules in these UV-caused early adaptive
changes, in female SKH-1 hairless mouse epidermis.
Materials and methods
Animals and UV light source
Female SKH-1 hairless mice (5 weeks old) were obtained from Charles River
Laboratories (Wilmington, MA) and maintained in animal house facility at the
University of Colorado Health Sciences Center. Animals were fed with Purina
Chow and water ad libitum for a week before the start of the experiments. The
UV light source was a bank of four FS-40-T-12-UVB sunlamps equipped with
a UVB Spectra 305 Dosimeter (Daavlin, Bryan, OH), which emitted ~80%
radiation in the range of 280--340 nm with a peak emission at 314 nm as
monitored with a SEL 240 photodetector, 103 filter and 1008 diffuser attached
to an IL1400A Research Radiometer (International Light, Newburyport, MA).
The UVB irradiation doses were also calibrated using IL1400A radiometer.
Mice were exposed to UV irradiation as reported earlier (15) with a distance of
23 cm between the light source and the target skin. Silibinin used in the study
was obtained commercially from Sigma Chemical Company (St Louis, MO),
and was analyzed by HPLC as a pure agent as reported earlier (17).
Experimental design
Animals were divided into five groups containing five animals in each group
for each time-point of the study as follows: (i) unexposed and untreated control
mice, (ii) animals topically applied with 9 mg silibinin in 200 ml acetone/
mouse, (iii) animals irradiated once with 180 mJ/cm2 UV dose, (iv) animals
topically applied with 9 mg silibinin in 200 ml acetone/mouse 30 min prior to
identical UV exposure as in group 3, and (v) animals topically applied with
9 mg silibinin in 200 ml acetone/mouse immediately after identical UV
exposure as in group 3. Animals were killed after UV exposure at various
time points, and dorsal skin was collected, fixed in 10% formalin for 8--10 h at
4 C and processed for immunohistochemical analyses.
Immunohistochemical and hematoxylin and eosin (H&E) staining analyses
Skin samples were processed conventionally before paraffin embedding.
Briefly, skin samples were dehydrated in 70, 95 and 100% ethanol, cleared
in xylene and embedded in paraffin. Serial, 4 mm sections were cut and used
for all immunohistochemical and H&E analyses. The sections were deparaffi-
nized, re-hydrated with water and used for measurement of thymine dimer,
p53, p21/Cip1, proliferating cell nuclear antigen (PCNA), terminal deoxy-
nucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and apopto-
tic sunburn positive cells. All samples were coded and evaluated by two
investigators in a blinded manner and the mean  SE values were obtained
from the evaluation of multiple fields in each group. Five to ten representative
fields were counted for each mouse at 400 magnification, and the data
represent the results from at least five mice in each group.
Measurement of thymine dimer positive cells
Thymine dimer positive cells in epidermal layer were measured as described
earlier (26). Briefly, endogenous peroxidase activity was blocked by 10 min
incubation with 3% hydrogen peroxide, and slides were then incubated with
0.125% trypsin for 10 min at 37 C and then with 1 N HCl for 30 min at room
temperature. The sections were then blocked with 5% goat serum for 10 min
and incubated with peroxidase-conjugated monoclonal anti-thymine dimer
antibody (Kamiya Biomedical Company, Seattle, WA) for 90 min at room
temperature. Color was developed by incubating the sections in 3,30 -diamino-
benzidine (DAB) for 10 min at room temperature. The sections were counter-
stained with Harris Hematoxylin, dehydrated and mounted.
Immunostaining for p53, p21/Cip1 and PCNA
Skin sections were processed conventionally for immunostaining. Briefly,
tissue sections after deparaffinization and re-hydration were treated with
0.01 M sodium citrate buffer (pH 6.0) in a microwave for 5 min at full power
for antigen-retrieval. Sections were then quenched of endogenous peroxidase
activity by immersing in 3% hydrogen peroxide for 5 min at room temperature.
The sections were then blocked using 5% rabbit serum (only for p53 staining)
for 30 min at room temperature in a humid chamber, and then incubated at 4 C
overnight or at room temperature for 2 h (in case of PCNA) with anti-p53 (cat.
#NCL-p53-CM5p rabbit polyclonal antibody against recombinant mouse p53
protein, Novocastra Laboratories, Newcastle upon Tyne, UK), anti-p21/Cip1
(cat. #556430 purified mouse anti-p21 monoclonal antibody, BD Biosciences,
San Diego, CA) or anti-PCNA (cat. #M0879 mouse monoclonal anti-PCNA
antibody, Dako, Carpinteria, CA) antibody. In each case, negative controls
were treated only with PBS under identical conditions. The sections were then
incubated with biotinylated anti-rabbit secondary antibody (in case of p53) or
1460
anti-mouse secondary antibody (in case of p21 and PCNA) for 1 h at room
temperature followed by 30 min incubation with HRP-conjugated streptavidin.
Color development was achieved by incubation with DAB for 10 min at room
temperature. The sections were counterstained with Harris Hematoxylin, dehy-
drated and mounted.
TUNEL staining for apoptotic cells
Apoptotic cells were detected using the DeadEnd Colorimetric TUNEL system
(Promega, Madison, WI) following manufacturer's protocol with some mod-
ifications. In brief, tissue sections after deparaffinization and re-hydration,
were permeabilized with Proteinase K (30 mg/ml) for 1 h at 37 C. Thereafter,
the sections were quenched of endogenous peroxidase activity using 3%
hydrogen peroxide for 10 min. After thorough washing with 1 PBS, sections
were incubated with equilibration buffer for 10 min, and then TdT reaction
mixture was added to the sections, except for the negative control, and incu-
bated at 37 C for 1 h. The reaction was stopped by immersing the sections in
2 saline-sodium citrate buffer for 15 min. Sections were then added with
streptavidin-HRP (1:500) for 30 min at room temperature, and after repeated
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washings, sections were incubated with substrate DAB until color develop-
ment (~5--10 min). Sections were then mounted after re-hydration and
observed under 400 for TUNEL-positive cells.
Measurement of apoptotic sunburn cells
Skin sections were stained conventionally with H&E as mentioned earlier (15)
for the identification of sunburn cells. Apoptotic cells are morphologically
distinct due to cell shrinkage and nuclear condensation that stain darker by
H&E. Dark stained cells were scored in 5 random fields/sample and the
percentage/field was calculated.
Immunohistochemical and statistical analyses
All the microscopic immunohistochemical analyses were done using Zeiss
Axioscop 2 microscope (Carl Zeiss, Jena, Germany). Pictures were taken by
Kodak DC290 camera under 400 magnification and processed by Kodak
Microscopy Documentation System 290 (Eastman Kodak Company, Roche-
ster, NY). For statistical analysis, data were analyzed using the Jandel Scien-
tific SigmaStat 2.03 software (San Rafael, CA). The statistical significance of
difference between control and UV-exposed, and UV-exposed versus silibinin
plus UV or UV plus silibinin-treated groups was determined by one way
analysis of variance (one-way ANOVA) followed by Bonferroni t-test for
multiple comparisons. A P 5 0.05 was considered statistically significant.
Results
Silibinin inhibits UV-induced DNA damage in SKH-1 hairless
mouse epidermis
Formation of thymine dimers is one of the characteristics of
UV-induced DNA damage, and occurs three times more often
as compared with other photoproducts (27). Based on an ear-
lier study showing that thymine dimer formation occurs as
early as 5 min and peaks at 1 h after UV exposure (26), we
assessed the effect of silibinin on thymine dimer positive cells
in mouse epidermis 1 h after a single UV exposure at 180 mJ/
cm2 dose. As shown in Figure 1, compared with unexposed
control mice, UV irradiation resulted in a very strong immu-
nostaining for thymine dimer in the mouse epidermis; how-
ever, topical application of silibinin prior to or immediately
after (immunostaining data not shown) UV exposure showed a
remarkably reduced immunostaining for UV-caused thymine
dimer positive cells in epidermis (Figure 1A--C). In quantita-
tive analysis, UV exposure resulted in 78  2% thymine dimer
positive cells in the epidermis while no such cells were
observed in unexposed controls (Figure 1D). Pre- or post-
topical application of silibinin, to UV exposure, resulted in
only 12  1 and 19  1% thymine dimer positive cells,
accounting for 85 and 76% inhibition (P 5 0.001), respec-
tively (Figure 1D). These data clearly suggest that preventive
efficacy of silibinin against UV-caused skin tumor initiation
could be, in part, via an inhibition in UV-induced DNA
damage.

Fig. 1. Silibinin decreases UV-caused thymine dimer positive cells in
epidermis. Mice were (A) unexposed, (B) exposed with UV (180 mJ/cm2 ) or
(C) topically applied with 9 mg silibinin/200 ml acetone/mouse 30 min prior
to UV exposure, and killed 1 h thereafter. (A--C) Skin was removed, fixed,
paraffin-embedded and processed for immunohistochemical analysis of
thymine dimer positive cells as described in the Materials and methods.
(D) Percent thymine dimer positive cells were calculated as mean of five
randomly selected fields (400) from each skin sample. Data shown are
mean  SE of 25 fields/5 mice/group. Sb, Silibinin;  P 5 0.001.
Silibinin up-regulates UV-induced activation of p53-p21/Cip1
cascade
p53 is known to play a crucial role in DNA damage response
where it arrests cell proliferation when the damage is mild or
Silibinin protects mouse skin against UV damage
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Fig. 2. Silibinin up-regulates UV-induced p53 and p21/Cip1 levels. Mice
were (A) unexposed, (B) exposed with UV (180 mJ/cm2 ) or (C) topically
applied with 9 mg silibinin/200 ml acetone/mouse 30 min prior to UV
exposure, and killed 8 h thereafter. Skin was processed conventionally for
immunohistochemical analysis (A--C for p53) as described in the Materials
and methods. Percent p53 (D) and p21/Cip1 (E) positive cells were calculated
as mean of five randomly selected fields (400) from each skin sample. Data
shown are mean  SE of 25 fields/5 mice/group. Sb, Silibinin;  P 5 0.001.
induces apoptosis when the damage is severe (28--30). Based
on our findings showing that silibinin strongly reduces UV-
caused thymine dimer positive cells, we next focused our
efforts on assessing the effect of silibinin on UV-caused
p53 accumulation. As shown in Figure 2, compared with a
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S.Dhanalakshmi et al.

negligible immunostaining for p53 in unexposed mouse epi-

dermis, a large number of p53-positive cells were observed in
the epidermis at 8 h after a single UV exposure of mice. These
results are consistent with an earlier report showing p53 accu-
mulation in mouse epidermis following 8--12 h of UV exposure
at an identical dose used in the present study (26). Silibinin
topical application prior to or immediately after UV exposure,
however, resulted in a further increase in p53-positive cells
(Figure 2A--C). Overall, in quantitative analysis, compared
with 0.81  0.32% p53-positive cells in unexposed control
epidermis, 8 h after UV exposure resulted in 23.8  1.8% p53-
positive cells, which were further increased by pre- or post-
topical application of silibinin to 36.4  2.1 and 36.9  1.2%
(P 5 0.001) p53-positive cells, respectively, accounting for
~1.6-fold increase (Figure 2D).

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UV-induced increase in p53 protein accumulation is known to
enhance the synthesis of p21/Cip1 protein, which plays a crucial
role in the adaptive responses after UV exposure of the skin by
inhibiting cell proliferation (29). Based on our finding that
silibinin further enhances UV-induced p53-positive cells in epi-
dermis, same samples were also analyzed for p21/Cip1-positive
cells. Compared with almost no immunostaining in unexposed
controls, a strong immunoreactivity for p21/Cip1-positive cells
was observed in the epidermis at 8 h after a single UV irradiation
at 180 mJ/cm2 dose, which, similar to p53 results, was further
enhanced by pre- or post-application of silibinin (data not
shown). In quantitative estimates, UV exposure showed 9.9 
0.9% p21/Cip1-positive cells in epidermis compared with 0.34
 0.09% in unexposed controls; however, pre- or post-UV
silibinin treatment resulted in 21.0  1.8 and 18.2  1.8% p21/
Cip1-positive cells, respectively, accounting for ~2-fold increase
(P 5 0.001) over UV-alone group (Figure 2E). Together, the
data shown in Figures 1 and 2 clearly suggest that silibinin
reduces UV-caused thymine dimer positive cells and further
increases UV-activated p53-p21/Cip1 cascade in mouse epider-
mis, which might be involved in an overall protection against
UV-caused biological responses such as cell proliferation and
apoptosis induction. Accordingly, we next assessed the effect of
silibinin on these two biological endpoints.

Silibinin inhibits UV-induced cell proliferation in SKH-1

hairless mouse skin
As shown in Figure 3, compared with unexposed controls, a
single UV irradiation at the dose of 180 mJ/cm2 resulted in a
strong PCNA staining within 1 h of the irradiation; however,
topical application of silibinin prior to or immediately after UV
irradiation resulted in a strong inhibition in UV-induced PCNA-
positive cells in the epidermis (Figure 3A--C). A quantitative
analysis of the PCNA immunostaining showed that UV irradia-
tion caused 22.4, 18.4, 19.6 and 16.1% PCNA-positive cells at 1,
4, 8 and 12 h post-UV irradiation as compared with 3.8%
PCNA-positive cells in un-irradiated control epidermis, respect-
ively (Figure 3D). However, topical application of silibinin at
9 mg dose in 200 ml acetone/mouse, 30 min prior to or immedi-
ately after UV exposure resulted in a strong and statistically
significant (P 5 0.005 to P 5 0.001) inhibition in UV-induced
cell proliferation at all the time points studied, and accounted for
40--52 and 20--40% inhibition, respectively (Figure 3D).
Silibinin inhibits UV-induced apoptotic and sunburn cell
formation
Consistent with its effect on a reduction in UV-caused thymine
dimer positive cells, and a further increase in UV-activated
1462
Fig. 3. Silibinin inhibits UV-induced cell proliferation as observed by PCNA
immunostaining. Mice were (A) unexposed, (B) exposed with UV (180 mJ/
cm2 ) or (C) topically applied with 9 mg silibinin/200 ml acetone/mouse 30 min
prior to UV exposure and killed 1 h thereafter. Skin was removed, fixed,
paraffin-embedded and processed for immunohistochemical analysis of
PCNA as described in the Materials and methods. (D) Under identical
experimental conditions and treatments, mice were killed at 1, 4, 8 and 12 h
after UV exposure, and skin tissue samples were subjected to PCNA staining
as described in the Materials and methods. Percent PCNA-positive cells were
calculated as mean of five randomly selected fields (400) from each skin
sample. Data shown are mean  SE of 25 fields/5 mice/group. Sb, Silibinin.
p53-p21 accumulation, silibinin treatment prior to or immedi-
ately after UV exposure also significantly inhibited UV-
induced apoptosis (Figure 4A--D). As observed by TUNEL
staining, compared with 2.9  0.73% (Figure 4A) apoptotic

Fig. 4. Inhibitory effect of silibinin on UV-induced apoptosis. Mice were
(A) unexposed, (B) exposed with UV (180 mJ/cm2 ) or (C) topically applied
with 9 mg silibinin/200 ml acetone/mouse 30 min prior to UV exposure, and
killed 12 h thereafter. Skin was removed, fixed, paraffin-embedded and
processed for the analysis of apoptotic cells by TUNEL (A--C) as well as for
the analysis of sunburn cells by H&E staining as described in the Materials
and methods. (D) Percent TUNEL-positive cells and (E) sunburn cells were
calculated as mean of five randomly selected fields (400) from each skin
sample. Data shown are mean  SE of 25 fields/5 mice/group. Sb, Silibinin;

P 5 0.001.
cells in unexposed controls, a single UV irradiation resulted in
30.5  1.34% (Figure 4B) TUNEL-positive apoptotic cell
population. However, pre- or post-application of silibinin
showed only 9.0  0.81 (Figure 4C) and 20.6  1.08% apop-
totic cells accounting for 70 and 32% decrease (P 5 0.001),
respectively (Figure 4A--D). UV-caused apoptotic/sunburn
cell formation was further confirmed by H&E staining, in
Silibinin protects mouse skin against UV damage
which exposure of mouse skin to UV resulted in 10.2 
0.94% (P 5 0.001) sunburn cells as compared with 0.88 
0.16% in un-irradiated control (Figure 4E). Consistent to
TUNEL staining results, the UV-caused sunburn cell forma-
tion was inhibited significantly to 5.0  0.37 and 6.8  0.62%
(P 5 0.001) sunburn cells, respectively, by pre- or post-topical
application of silibinin (Figure 4E).
Discussion
The central finding in the present study is that silibinin protects
SKH-1 hairless mouse skin from UV-induced DNA damage,
and that UV-caused cell proliferation and apoptotic sunburn
cell formation are prevented by silibinin possibly via further
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induction in p53-p21/Cip1 cascade. It is also important to
emphasize here that we employed two different silibinin topi-
cal application protocols in which silibinin was applied 30 min
prior to UV exposure or immediately thereafter. This approach
was important to dissect out the silibinin efficacy against UV-
caused skin damage independent of its sunscreen effects. The
findings reported here clearly support our conclusion that
silibinin efficacy against UV-caused skin damage and photo-
carcinogenesis is not only due to its sunscreen effect.
One of the most important characteristics of UV-caused
carcinogenesis is DNA damage and mutagenesis, and thymine
dimers are known as `hot spots' of UV mutagenesis (20). In
our study, silibinin treatment resulted in a remarkable decrease
in UV-caused thymine dimer positive cells. Analysis of the
mismatch repair enzyme MSH2 showed that silibinin treat-
ment does not result in an increase in MSH2 levels (data not
shown). This suggests the possible involvement of other mis-
match repair enzymes. Further, there are some reports showing
that DNA mismatch repair system is inactivated by oxidative
stress (31). Strong anti-oxidant properties of silibinin reported
earlier by us in mouse skin studies (15,16,18) suggest that
suppression of oxidative stress by silibinin could be one of
the possible mechanisms that resulted in the activation of
repair enzymes much earlier as compared with UV alone;
thereby it is possible that thymine dimers were removed
much earlier in silibinin-treated animals than 1 h, the time
point used in our study. More studies are needed in future as
a function of time employing time points earlier than 1 h,
specifically between 5 and 60 min, to assess whether silibinin
causes a faster repair of UV-caused DNA damage ultimately
leading to a strong reduction in thymine dimer positive cells in
epidermis as observed in the present study at 1 h after UV
exposure of SKH-1 mouse skin. The other possibilities could
be that silibinin protects epidermal cells from UV-caused
thymine dimer positive cells by modulating DNA repair
enzymes other than MSH2 and/or by an alteration in ATM/
ATR pathways. In this regard, recent studies have shown the
role and involvement of ATM/ATR in UV-induced DNA
damage (32), and accordingly future studies are needed to
assess silibinin effect on these pathways as an upstream
response for its efficacy against UV-caused thymine dimer
positive cells in epidermis.
p53 up-regulation following UV irradiation is known to
protect the cells from UV-caused cellular damage/s via several
mechanisms including an induction in cell cycle arrest via
transcriptional activation of p21/Cip1, or by inducing apopto-
sis through an activation of apoptotic proteins bax, Fas/Apo-1,
DR5 or by down-regulation of anti-apoptotic proteins such as
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S.Dhanalakshmi et al.
Fig. 5. Proposed mechanisms for the protective effect of silibinin on UV-induced photodamage in epidermal cells. Silibinin protects SKH-1 mouse epidermis
from DNA damaging effect of UV such as thymine dimer positive cells, thereby decreasing UV-caused apoptotic/sunburn cells. Further, silibinin inhibits
UV-induced epidermal cell proliferation via decreasing PCNA possibly through activation of p53-p21/Cip1.
bcl-2 and cellular inhibitor of apoptosis protein 2 (c-IAP2)
(23,29). Some chemopreventive agents have been shown to
selectively induce apoptosis of UV-damaged cells by up-
regulation of p53 (14,33). Earlier reports have shown that the
level of p53 accumulation depends on the extent of DNA
damage and that lower levels of p53 correspond to cell cycle
arrest (34). In our study, silibinin treatment resulted in a further
increase in UV-induced p53 accumulation with a concomitant
increase in p21/Cip1 protein levels, which is in accord with the
inhibition of UV-induced cell proliferation and apoptosis by
silibinin, suggesting their possible role in cell growth inhibi-
tion rather than apoptosis induction (Figure 5).
UV-induced increase in cell proliferation is an early neces-
sary event associated with UV-caused carcinogenesis that helps
the initiated cells to proceed further into cell cycle (24); this
response, however, could be prevented by arresting the cells at
G1 or S phase of the cell cycle (35). We observed that UV-
induced PCNA-positive cells were strongly inhibited by silibi-
nin treatment. It has been reported that UV-induced cell
proliferation reaches the maximum level as early as 1 h after
exposure, and we observed that silibinin treatment showed a
strong inhibitory effect even at that early time point. In addi-
tion, the inhibitory effect of silibinin on UV-caused cell pro-
liferation was sustained as observed up to 12 h after exposure.
These results suggest that inhibiting cell proliferation could be
one of the mechanisms by which silibinin protects damaged
cells from entering the cell cycle, thereby providing damaged
cells additional time for repair and preventing their entry into
apoptotic pathway in case the damage is severe. This sugges-
tion is in accord with the observation that silibinin treatment
further up-regulates p21/Cip1 levels, which is known to inhibit
cell proliferation by a direct binding with PCNA (36).
An earlier study by us has shown that topical application of
silymarin strongly protects against UV-caused sunburn cell
formation in SKH-1 hairless mice (15). The results of the
present study showing the inhibitory effect of silibinin on
UV-caused sunburn cell formation further support the previous
finding. In addition, the TUNEL staining results in the present
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study are consistent with an early report regarding apoptotic
nature of the sunburn cells (37).
Use of conventional chemical sunscreens has been an effec-
tive measure in protecting skin against UV-caused damages and
NMSC; several cancer chemopreventive agents, however, have
also been shown to protect cells from UV by acting as a
sunscreen (10,38,39). However, sunscreen usage as an only
measure is not adequate because of infrequent use, incomplete
protection and associated toxicity (7). In this regard, topical
application of antioxidants has been shown to be beneficial
against skin cancer as well as associated hazards such as
photoaging. It is important to emphasize here that silibinin is
a flavonoid antioxidant with no known toxicity in both animal
and human studies. Further, since the chemopreventive efficacy
of silibinin has been well studied and established in several
epithelial cancer models (16--18,40,41), it could be a potent
chemopreventive agent against NMSCs as well as UV-caused
skin damages and photoaging. In the present study, as pre-
treatment of silibinin showed a moderately better protective
effect (than its post-treatment) against some of the UV-caused
alterations in SKH-1 mouse epidermis, a sunscreen effect may
be an additional factor in silibinin-afforded protection.
In conclusion, the results of the present study suggest that
silibinin protects SKH-1 hairless mouse epidermis from UV-
induced cell proliferation and apoptosis through a reduction in
DNA damage and via an activation of p53-p21/Cip1 cascade.
These results warrant the development of silibinin as a phar-
macological non-toxic chemopreventive agent against human
NMSC and photoaging. In addition, more mechanistic studies
are needed in future to define the protective effect of silibinin
on UV-induced DNA damage and apoptosis, and their biolo-
gical significance in an overall efficacy of silibinin against
UV-caused skin damages and photocarcinogenesis.
Acknowledgement
This work was supported by USPHS grant CA64514.

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Received January 22, 2004; revised February 25, 2004;
accepted March 9, 2004
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