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Introduction
Increasing evidence over the past decade has indicated that UV irradiation provokes premature aging, cystitis, and skin
cancer, which reinforces the need for novel chemopre-vention strategies involving the use of anti-oxidants or other
biological events occurring following UV exposure to the skin. UV that reaches the earth's surface consists of 90%_99% UVA
(wavelength 320_400 nm), 1%_10% UVB (290_320 nm), and a very small portion of
UVC[1]. UVB penetrates the epidermis, and
to a lesser extent, the upper part of the dermis, while UVA penetrates more deeply into human skin. Although UVC can
damage skin to a larger extent than UVA and UVB, most of it is absorbed by ozone. In the present study, we irradiated cells
by UVA plus UVB to mimic the UV light in the sunlight.
Relative molecular mass of polypeptide from Chlamys
farreri (PCF) is 912. PCF is a novel, marine-active product isolated
from the Chinese scallop Chlamys farreri, which has been served as a seafood for thousands of years. The extraction yield
of PCF was 1.14 g/kg Chlamys farreri by our techniques. As an octapeptide, PCF consists of Pro, Asn, Ser, Thr, Arg, Lys, and
Cys. However, the primary structure of PCF is kept secret. Our previous studies demonstrated that PCF was found to have
an anti-apoptotic effect against UVB irradiation in HeLa
cells[2] and hairless
mice[3], which implied a potential clinical
usage of preventing skin damage induced by UV light or solar
light. However, relatively little is known about the effect of
PCF on UVA plus UVB-mediated damage on normal human
skin cells in vitro. HaCaT cell, a spontaneously
immortalized human keratinocyte cell line, has similar characteristic
to the normal keratinocyte line and thus may be considered
as a good experimental model for testing the effects of PCF
on UV irradiation-damaged skin cells.
UV irradiation can induce reactive oxygen species (ROS)
accumulation and further cause cell
apoptosis[4]. More and more studies have provided evidence that the activation of
c-Jun amino-terminal kinase (JNK) is required for UV
irradiation-induced apoptosis[5_7]. In addition, the JNK signaling
pathway is known to play a fundamental role in UV-triggered,
caspase-dependent apoptosis[8,9]. In view of this information,
we hypothesized there may be a relation between ROS, JNK,
and caspase-3 during the process of apoptosis induced by
UV irradiation, and PCF may protect cells from apoptosis via
blocking one of the signal pathways.
The purpose of this study was, by mimicking the action
of environmental UV light on human skin, to investigate the
anti-apoptotic function and protective mechanism of PCF
on HaCaT cells damaged by UV irradiation.
Materials and methods
Reagents PCF (purity >96%) was purified and analyzed
by high-performance liquid chromatography, dissolved in
sterile deionized water, and stored at 4 °C. Antibodies against
phospho-JNK, cleaved caspase-3 (17/19 kDa), β-actin, and
Ac-DEVD-CHO (an inhibitor of caspase-3), were acquired
from Cell Signaling (Beverly, MA, USA).
2',7'-Dichlorofluo-rescein diacetate (DCFH-DA), SP600125 (an inhibitor of JNK),
and N-acetyl-L-cysteine (NAC, the scavenger of ROS) were
obtained from Beyotime Biotechnology (Haimen, China).
Cell culture and treatment The HaCaT cells were
cultured in the complete culture medium composed of
Dulbecco's modified Eagle's medium (Gibco, Grand Island,
NY, USA), 10% fetal bovine serum, 100 U/mL penicillin, and
100 U/mL streptomycin. The cells were maintained in a
humidified atmosphere of 95% air and 5%
CO2 at 37 °C. UV irradiation was carried out using a UVA plus UVB light source
(Beijing Normal University, Beijing, China). After
standardization of conditions, the distance between the plate and the
lamp was maintained at 7.0 cm, and the dose of UV
irradiation was controlled by changing the time for radiation. When
the confluent cells were radiating, the lid of the culture dish
was removed and the culture medium was replaced by
D-Hanks. The cells were randomly divided into several groups,
including the control group (normal group), model group
(UV-irradiated group), PCF group (PCF-pretreated and then
UV-irradiated group), and the inhibitor groups (the
corresponding inhibitor pretreated and then UV-irradiated group).
PCF and the inhibitors of ROS, JNK, and caspase-3 were
added into the medium for 2 h before radiation. After
radiation, the cells were cultured in compete culture medium
again for the appropriate time.
Cell viability assay Cell viability was evaluated via
conventional
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction assays. The HaCaT cells
were planted into 96-well culture plates at an optimal density
of 5×103 cells/mL with 100 µL culture medium per well. After
1_2 d culture to 90% confluence, the cells were radiated with
different doses of UV irradiation, or incubated in medium
containing different concentrations of PCF, or were
pre-incubated with different concentrations of PCF for 2 h
followed by UV irradiation. At 24 h after treatment, MTT
solution was added to each well and incubated at 37 °C for 4 h.
The medium was gently aspirated, and then 150 mL DMSO
was added to each well to solubilize the formazan crystals.
The absorbance of each sample was immediately measured
using an ELISA reader at 490 nm.
Apoptosis assay Apoptosis in the HaCaT cells was
analyzed by 2 methods: (i) Hoechst 33258 staining, where the
cells were seeded on sterile cover glasses placed in the
6-well plates. At 18 h after treatment, the cells were fixed,
washed twice with D-Hanks, and stained with Hoechst 33258
staining solution according to the manufacturer's
instructions (Beyotime Biotechnology, Jiangsu, China). Stained
nuclei were observed under a fluorescence microscope; and
(ii) DNA fragmentation, where the HaCaT cells were washed
twice with D-Hanks solution and harvested. The cells were
incubated in lysis buffer [150 mmol/L NaCl, 10 mmol/L
Tris-HCl (pH 7.5), 10 mmol/L EDTA, 0.5% SDS, and 500 mg/L
proteinase K] overnight at 50 °C. DNA was extracted with an
equal volume of phenol and then extracted with an equal
volume of phenol:chloroform:isoamyl alcohol (25:24:1) and
precipitated with 0.1 volume of 3 mol/L NaAc and 2.0 volume
of absolute ethanol, and incubated overnight at -20 °C. The
DNA pellets were dissolved in Tris-EDTA buffer and
analyzed on a 1.5 g/L agarose gel containing 0.5 g/L ethidium
bromide, and visulized by UV transillumination.
Measurement of intracellular ROS levels Changes in
the intracellular ROS levels were determined by measuring
the oxidative conversion of cell permeable DCFH-DA to
fluorescent dichlorofluorescein (DCF) by flow cytometry. After
pretreatment with different concentrations of PCF or 5.68
mmol/L vitamin C for 2 h, the cells in the 6-well culture dishes
were radiated by 4 J/cm2 UVA plus 10
mJ/cm2 UVB and cultured for another 30 min. Then the cells were collected and
adjusted to 1×107/mL and incubated with DCFH-DA at 37 °C
for 20 min. Subsequently, the cells were washed 3 times with
D-Hanks, and a cover was placed on top of the dish. The
fluorescent signal intensity of DCF was detected by flow
cytometry at an excitation wavelength of 488 nm and at an
emission wavelength of 535 nm.
Detection of superoxide dismutase (SOD) and
glutathione peroxidase (GSH-px) activities and
total anti-oxidative capacity (T-AOC) The cells in the 6-well culture dishes
were washed 3 times with ice-cold D-Hanks and lysed in the
extraction buffer [50 mmol/L Tris-HCl (pH7.4), 1 mmol/L
ethyleneglycol-bis(2-aminoethylether)tetraacetic acid, 150
mmol/L NaCl, 1% (v/v) Triton X-100, 1 mmol/L
phenyl-methylsulfonyl, 10 µg/mL aprotinin, 10 mmol/L EDTA, 1
mmol/L NaF, and 1 mmol/L
Na3VO4] on ice for 30 min. Then the cells
were scraped from the plates and the lysates were subjected
to 20 000×g centrifugation at 4 °C for 10 min. The amount of
proteins in the cleared lysates was quantified with a
bicincho-ninic acid assay (Beyotime Biotechnology, China). After
determining the amount of total proteins in the supernatants,
we detected T-AOC and the enzymes that include SOD and
GSH-px using biochemical methods following the
instructions for the reagent kits (Nanjing Institute of Jiancheng
Biological Engineering, Nanjing, China).
Western blotting The phosphorylated JNK (P-JNK), JNK,
and cleaved caspase-3 were analyzed by Western blotting.
At 6 h (for P-JNK and JNK) or 9 h (for cleaved caspase-3)
after treatment, the protein was extracted and quantitated as
previously described in this study. An equal amount of
protein was separated on a 10% SDS-PAGE and transferred
electrophoretically to the nitrocellulose membranes. The
membrane was blocked with 5% bovine serum
albumin and
0.1% Tween-20 in Tris-buffered saline for 2 h at room
tempera-ture. The bolts were incubated with antibodies against
P-JNK, JNK, cleaved caspase-3, or β-actin (dilution 1:1000)
overnight at 4 °C, and then with peroxidase-conjugated
secondary antibodies (dilution 1:2000) for 2 h at room
tempera-ture. The bands were detected using the diamino-benzidine
detection kit (Boster Biotechnology, Wuhan, China), and
the result was analyzed by Quantity One software (Bio-Rad
Laboratories, Hercules, CA, USA).
Statistical analysis Data are expressed as mean±SD.
Statistical analysis was performed with one-way ANONA,
followed by the Bonferroni test using Origin7.5 (OriginLab
Corporation, Northampton, MA, USA). The difference were
considered significant if P<0.05.
Results
Effects of PCF and UV irradiation on the viability of
HaCaT cells UV irradiation decreased cell viability in a
dose-dependent manner. When the doses of UV irradiation were
4 J/cm2 UVA plus 10 mJ/cm2 UVB and 6
J/cm2 UVA plus 15 mJ/cm2 UVB, cell viability decreased to about 69% and 41%
of the control group, respectively (Figure 1A). In this study,
4 J/cm2 UVA plus 10 mJ/cm2 UVB was applied to establish the
apoptosis model. PCF treatment stimulated cell proliferation
at low concentrations, but not at high concentration (Figure
1B). PCF pretreatment before radiation stimulated cell
proliferation in a concentration-dependent manner in the range of
1.42_5.68 mmol/L (Figure 1C).
Effects of PCF on apoptosis of HaCaT cells induced by
UV irradiation Hoechst 33258 staining of cells damaged by
UV irradiation in the model group showed condensed, bright
nuclei typical of apoptotic dead cells (Figure 2B), but the
number of apoptotic nuclei in the PCF groups decreased in a
concentration-dependent manner (Figure 2C, 2D, 2E), while
almost no apoptotic nuclei were observed in the cells of the
control and Ac-DEVD-CHO groups (Figure 2A, 2F). Since
the cleavage of chromosomal DNA into fragments is a
biochemical hallmark of apoptosis, the DNA fragmentation in
HaCaT cells radiated by UV irradiation was examined by DNA
laddering assay. With the cells in the control and
Ac-DEVD-CHO groups, no obvious DNA fragmentation was observed,
while clear DNA laddering was detected at 18 h after cell
damage, and previous treatment by PCF prevented UV
irradiation-induced DNA fragmentation in the HaCaT cells in a
concentration-dependent manner (Figure 3).
Effects of PCF on the intracellular ROS level in UV
irradiation-treated HaCaT cells UV irradiation significantly
increased the intracellular level of ROS. Pretreatment with
PCF significantly inhibited the elevated intracellular
concentration of ROS by UVA plus UVB in a
concentration-dependent manner (Figure 4).
Effects of PCF on cellular SOD, GSH-px, and T-AOC
UV irradiation can decrease the activities of SOD, GSH-px
and T-AOC. Pretreatment with PCF for 2 h increased SOD,
GSH-px, and T-AOC activities in a concentration-dependent
manner (Table 1).
Effects of PCF on the UV irradiation-activated, JNK
signaling pathway Western blotting studies showed that
the total JNK protein level in cells at 6 h after UV irradiation
was not significantly different from the cells of the control
group. However, P-JNK protein expression was strongly
increased. The treatment of cells with PCF inhibited UV
irradiation-induced phosphorylation of JNK in a
concentration-dependent manner. NAC, a scavenger of ROS, also
decreased the P-JNK protein level in cells damaged by UV
irradiation (Figure 5).
Effects of PCF on the expression of cleaved caspase-3
induced by UV irradiation During apoptosis, an inactive
zymogen of precaspase-3 is cleaved into catalytically-active
caspase-3 fragments, including a large fragment (17/19 kDa)
and a small fragment (12 kDa). The expression of cleaved
caspase-3 at 17/19 kDa in the HaCaT cells increased after
exposure to UV irradiation; PCF pretreatment obviously
decreased the expression of cleaved caspase-3 at 17/19 kDa in
a concentration-dependent manner. Meanwhile, SP600125,
an inhibitor of JNK, blocked the cleavage of precaspase-3 in
the cells to a large extent. In addition, the decrease of cleaved
caspase-3 at 17/19 kDa expression was found in the vitamin
C group (Figure 6).
Discussion
Our results indicated that UVA plus UVB could inhibit
the proliferation of HaCaT cells and induce cell apoptosis.
So we successfully established the UV irradiation-induced
apoptosis model of HaCaT cells by mimicking the action of
environmental UV light on human skin in this study.
Apoptosis is a tightly regulated form of cell death and a
multifactor-related process, including gene expression and
mutation. The execution of the apoptosis program is
characterized by morphological and biochemical changes. In our
experiment, Hoechst 33258 staining and DNA laddering
assay demonstrated that UV irradiation obviously induced the
formulation of DNA laddering and karyopyknosis in cells,
and PCF protected the cells from apoptosis. In the following
studies, we tried to determine the mechanism of PCF
preventing HaCaT cells from apoptosis induced by UVA plus
UVB.
A role for oxidative stress in the induction of apoptosis
is provided by studies where the addition of low levels of
ROS can induce apoptosis, and the observation that various
anti-oxidants such as N-acetylcysteine can inhibit cell
death[10,11]. Additionally, ROS generation has been reported
to occur following the treatment of cells with various agents,
including UV irradiation and chemotherapeutic
drugs[12]. Our results showed that the ROS level was low in the control
group. Upon UV exposure, the ROS level increased, and
PCF pretreatment reduced the ROS accumulation. Lee
et
al[13] suggested that UV irradiation could suppress the
activities of anti-oxidative enzymes in cells. Several
anti-oxidative enzymes, including GSH-px and SOD, scavenged
free radicals produced by UV irradiation. In our experiments,
PCF enhanced T-AOC and the activities of GSH-px and SOD.
Generally speaking, the anti-oxidant system in healthy cells
can automatically ameliorate insults induced by a slight
increase of ROS. Our study showed that exposure to UVA
plus UVB increased the intracellular ROS levels in cells by
about 50% and decreased almost half of GSH-Px and T-AOC
compared with normal cells. According to Rezvani
et al's[14] suggestion that UVB induced an increase in the ROS levels
at 2 distinct stages: immediately following irradiation and
around 3 h after irradiation, we speculated that the
significant decrease of GSH-Px and T-AOC contributed to the
amount of ROS in all stages. In the present study, we
detected the ROS 30 min after UV radiation. Furthermore, with
the exception of the formation of ROS, UV irradiation can
induce apoptosis also via the triggering of death receptors
or via DNA damage. Further study is needed to confirm our
thoughts, but at least we can confirm that PCF works as a
strong anti-oxidant.
The JNK signaling pathway is activated by a wide range
of cellular stimulus such as UV
light[15], radiation[16],
cera-mide[17], DNA-damaging
drugs[18], TNF-α[19], and interleukin
1[20]. In addition, mitogenic signals, including growth
factors[21] and CD40
ligation[22] can induce the activation of JNK,
which were originally identified through their involvement
in c-Jun NH2-terminal phosphorylation following exposure
to mammalian cells to short wavelength UV
radiation[23]. The activation of JNK requires the phosphorylation of both Thr
and Tyr residues located in its Thr-Pro-Tyr motif. In our
experiments, the upregulation of P-JNK was observed at 6 h
after the cells were exposed to UVA plus UVB, whereas, the
increment of the ROS level was detected at 30 min after
radiation. In addition, NAC, the scavenger of ROS, can block
the activation of JNK. These results suggested that the
ROS accumulation is upstream of the JNK activation induced
by UV irradiation, which is consistent with the suggestion
that inhibiting UVC light-induced ROS production inhibits
JNK activation induced by UVC[24].
Our results also showed that HaCaT cell apoptosis by
UVA plus UVB was dependent on the activation of caspase-3.
Further studies showed that when SP600125, an inhibitor of
JNK, was used, the activation of caspase-3 in the cells
damaged by UV irradiation was blocked to a large extent. In
addition, the upregulation of cleaved caspase-3 was found
at 12 h after radiation, and was 6 h later than the activation of
JNK. Although the activation of caspase-3 was not
inhibited completely by SP600125, we can confirm that the
activation of the JNK pathway led to the upregulation of the
expres-sion of cleaved caspase-3 in HaCaT cells damaged by UVA
plus UVB. Two possible mechanisms can explain how JNK
activates caspase-3, one is the death receptor pathway. JNK
activation may upregulate the expression of death receptor
ligands[25]; the subsequent binding of FasL or TNF to its
receptor induces the trimerization of the receptor and
formation of a death-inducing complex. This complex recruits, via
the adaptor molecule Fas-associated with death domain
protein or TNF-R1-associated death domain, multiple
procaspase-8 molecules, resulting in caspase-8 activation.
The activated caspase-8 cleaves various proteins, including
procaspase-3, which results in the activation of caspase
cascades and the completion of apoptosis. Although it has
been demonstrated that FasL can induce JNK activation, the
activation is a delayed event[26] that probably requires the
prior activation of caspases[27], which differs from this
situation we describe. This places JNK activation in a primary
role from where it may induce the expression of FasL or TNF
to commit cells to apoptosis. The other mechanism that can
explain how JNK activates caspase-3 is the mitochondrial
death pathway. Bcl-2 family proteins localize or translocate
to the mitochondrial membrane and modulate apoptosis by
permeabilization of the inner and/or outer membrane.
In vitro, JNK activation phosphorylates Bcl-2 and
Bcl-XL[28_30], which significantly alters the susceptibility of cells to UV
irradiation stimuli and causes cytochrome c
release[31]. Then cytosolic cytochrome c forms an apoptosome complex with
Apaf-1, dATP, and the initiator procaspase-9 to cause the
activation of caspase-9 and trigger the subsequent effector
caspase-3 activation[32_35], which results in the cleavage of
cellular substrates and apoptosis. As to which pathway was
involved in the activation of caspase-3 by JNK needs
additional exploration.
In summary, our data demonstrated that the
ROS-JNK-caspase-3-apoptosis cascade pathway is the main signal
pathway of HaCaT cell apoptosis induced by UVA plus UVB.
We found that PCF could protect HaCaT cells from damage
by UV irradiation via scavenging ROS and increasing the
activities of anti-oxidative enzymes as an anti-oxidant to block
the signal pathway.
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