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Li LH, Wu LJ, Tashiro SI, Onodera S, Uchiumi F, Ikejima T.
Activation of the SIRT1 pathway and modulation of the cell cycle were
involved in silymarin's protection against UV-induced A375-S2 cell
J Asian Nat Prod Res. 2007 Apr-May;9(3):245-52.
Silymarin, derived from the milk thistle plant, Silybum marianum, has been
traditionally used in the treatment of liver disease. Our previous study
demonstrated that silymarin has an anti-apoptotic effect against UV
In this study, SIRT1, a human deacetylase that was reported to promote cell
survival, was activated by silymarin (5 × 10^-4 mol/L) in UV-irradiated
human malignant melanoma, A375-S2 cells, followed by down-regulated
expression of Bax and decreased release of cytochrome c. Cleavage of
procaspase-3 and digestion of its substrates, the inhibitor of
caspase-activated DNase (ICAD) and poly(ADP-ribose) polymerase (PARP), were
also reduced. Consistent with its protective effect on UV-induced apoptosis,
silymarin (5 × 10^-4 mol/L) also increased G2/M phase arrest, possibly
providing a prolonged time for efficient DNA repair.
Consequently, that silymarin protected A375-S2 cell against UV-induced
apoptosis was partially through SIRT1 pathway and modulation of the cell
Keywords: Silymarin; A375-S2 cell; UV irradiation; Anti-apoptosis; SIRT1;
Cell cycle arrest
... Here, we found that silymarin's inhibitory mechanism on UV-induced
A375-S2 cell apoptosis has a relationship with SIRT1, a member of the
conserved sirtuin family of nicotinamide adenine dinucleotide
(NAD+)-dependent deacetylases, which is a key regulator of cell defences and
survival in response to a variety of stresses 4-6 ...
2. Results and discussion
2.1 Silymarin protected A375-S2 cells against UV-induced cell death
Table 1 shows the viability ratio of A375-S2 cells, which were treated with
various concentrations of silymarin for 1 h and then further incubated for
12 h after UV irradiation. It was found that silymarin protected
UV-irradiated A375-S2 cells from death in a dose-dependent manner (cell
viability ratio was increased to 92.5% at the concentration of 5 × 10^-4
mol/L) and that it had no cytotoxic effect on the cells.
Table 1. Protective effect of silymarin on cell viability in
UV-irradiated A375-S2 cells (%).
Silymarin (mol/L)---Cell viability (%)
----UV irradiation Without UV irradiation
0 18.2±3.4 100.0±2.8
1 × 10^-5 17.8±2.6 100.3±2.4
5 × 10^-5 18.9±4.3 98.5±3.7
1 × 10^-4 28.3±2.5 103.1±1.6
2 × 10^-4 34.7±4.2 105.7±4.9
3 × 10^-4 45.6±1.6 105.4±4.1
4 × 10^-4 69.7±3.8 107.3±6.2
5 × 10^-4 92.5±5.6** 103.5±3.1
Mean±SD, n = 3.
**P < 0.01 vs 0 mol/L.
2.2 Silymarin reversed UV irradiation-induced morphologic changes in A375-S2
In response to cellular insults, cells attempt to repair and defend
themselves, but if unsuccessful, they often undergo programmed cell death,
or apoptosis. Therefore, in order to determine whether silymarin protected
A375-S2 cells against UV-induced cell death through anti-apoptotic pathway,
the morphologic changes were observed. When A375-S2 cells were cultured for
12 h after UV irradiation, marked morphologic changes were observed as
compared with the untreated control (figure 2a,c). The majority of cells
became round, and some of these cells showed membrane blebbing (figure 2c),
which were hallmarks of apoptosis, while silymarin pre-treatment (5 × 10^-4
mol/L) reversed these morphologic changes (figure 2d).
2.3 The expression of SIRT1 was up-regulated in UV-irradiated A375-S2 cells
after silymarin pre-treatment
SIRT1 play an important role in cell defences and survival in response to
stress 4-6. To investigate whether SIRT1 might be responsible for the
ability of silymarin to protect A375-S2 cells from UV-induced apoptosis, the
expression of SIRT1 was examined by Western blot analysis, which was found
to be markedly up-regulated by silymarin (5 × 10^-4 mol/L) in UV-irradiated
A375-S2 cells as compared to that of silymarin-untreated cells (figure 3),
suggesting that silymarin's protection against UV irradiation might be
through SIRT1 pathway.
2.4 The protein expressions involved in SIRT1 pathway
Since up-regulated SIRT1 activity  could deacetylate the DNA repair
factor Ku70, causing it to sequester the proapoptotic factor Bax away from
the outer mitochondrial membrane to the cytoplasm, forming a complex with
Ku70, the subsequent release of cytochrome c was inhibited as the result of
Bax protein relocalisation. Downstream events including caspase activation
and cleavage of ICAD and PARP were attenuated, thereby inhibiting
stress-induced apoptotic cell death. In our study, it was found that the
expression of Bax and release of cytochrome c from mitochondria were
attenuated in UV-irradiated A375-S2 cells after silymarin pre-treatment
(figure 3). Cleavage of procaspase-3 to caspase-3 (figure 4) and digestion
of its substrates, ICAD and PARP, were also inhibited subsequently (figure
2.5 The effect of silymarin on UV-induced cell cycle modulation
Cell cycle progression is important for maintaining homeostasis, especially
when there is an insult to DNA [12,13]. Physiological stress or an insult to
DNA could cause arrest in different stages of the cell cycle. Since UV
irradiation is known to damage DNA directly, the effect of UV irradiation
and silymarin pre-treatment on cell cycle progression was assessed. It was
found that UV exposure caused a S arrest (29.62 versus 16.00% in control) at
the expense of a decrease in G2/M phase cells (0 versus 7.86% in control)
(figure 6a,c, table 2). Pre-treatment with silymarin (5 × 10^-4 mol/L),
however, reversed the UV-induced S arrest, resulting in an increase in G2/M
phase cells (7.45% in silymarin+UV versus 0 in UV alone) (figure 6c,d, table
2). In general, an arrest in G2/M phase of the cell cycle allows cells more
time to repair damaged DNA before mitosis (M phase), until the damage of the
genome is repaired. Since silymarin treatment resulted in an accumulation of
UV-irradiated cells at G2/M phase, part of the protective effect of
silymarin against UV-induced apoptosis might be due to its effect on cell
cycle distribution. However, detailed studies remain to be conducted to
delineate the molecular mechanism involved in this action of silymarin. In
addition, apoptotic sub-G0/G1 phase peak, caused by UV irradiation, was
reduced obviously by silymarin pre-treatment (figure 6c,d).
Table 2. Effects of silymarin on cell cycle distribution (%).
Group---Cell phage (%)
---G0/G1 S G2/M
Medium control 76.14 16.00 7.86
Silymarin (5 × 10^-4 mol/L) 75.60 16.45 7.95
UV irradiation (52.1 J/m2) 70.38 29.62 0
Silymarin (5 × 10^-4 mol/L)+UV irradiation (52.1 J/m2) 76.39 16.16 7.45
In conclusion, silymarin promoted UV-irradiated A375-S2 cell survival partly
through SIRT1 pathway. It also modulated the distribution of the cell cycle
to allow more time for the damaged cells to repair. Present results may
broaden silymarin's potential therapy use for many diseases in the future.
Edited by fredrik, 19 June 2007 - 01:49 PM.