Download Resveratrol induces apoptosis human rheumatoid arthritis synovial

Resveratrol induces apoptosis human rheumatoid arthritis synovial cells
in a sirtuin 1-dependent manner
1
+1Nakayama, H; 1Yaguchi, T; 1Nishizaki, T; 1 Yoshiya, S; 2 Miyazawa, K
Hyogo College of Medicine, Nishinomiya, Japan, 2Central Research Laboratories, Kissei Pharmaceutical Co., Ltd. Nagano
[email protected]
INTRODUCTION:
In the treatment of rheumatoid arthritis (RA), administration of TNF-
inhibitors, anti-TNF antibodies, a soluble TNF receptor-fusion protein,
and an IL-1 receptor antagonist has been attempted, but a big problem
for those materials is side effects such as serious infections and inducible
malignant tumors1). Resveratrol is a phytoalexin that is present in grape
skin and red wine and exerts a variety of actions, that reduce superoxides,
suppress cartinogenesis and angiogenesis, or prolong life span. The
present study aimed at understanding the mechanism of resveratrolinduced apoptosis in MH7A cells (Riken cell bank, Ibaraki, Japan) a
human RA synovial cell line2). This supports the possibility for
resveratrol as a beneficial drug for treatment of RA.
MATERIALS AND METHODS:
Evaluation of Cell viability:
Cell viability was evaluated by the method using 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) as previously
described 3).
Assessment of apoptosis:
Apoptosis of the MH7A cells were examined with TUNEL assay.
Assay of mitochondrial membrane potentials:
Mitochondrial membrane potentials were measured using a DePsipher TM
kit as previously described (3).
Enzymatic assay of caspase activity:
Caspase-3,8,9 activation was measured using a caspase fluorometric
assay kit as previously described (3).
Reverse transcription-polymerase chain reaction (RT-PCR):
RT-PCR was carried out with a Takara Thermal cycler Dice (Takara Co.
Ltd., Shiga, Japan) using primers.
Western blotting:
Each fraction was loaded on SDS-PAGE. Blotting membranes reacted
with an anti-cytochrome c antibody (1:400) (Chemicon, Billerica, MA,
USA).
RESULTS:
Fig. 1. (A) MTT assay
(B) TUNEL assay
Resveratrol reduced MH7A cell viability in a concentration and
treatment time dependent manner (Fig.1A), and increased TUNELpositive cells in a concentration dependent manner (Fig.1B).
Fig.2. MTT assay
Fig. 3. Mitochondrial
membrane potentials
The resveratrol-induced MH7A cell death was inhibited by the SIRT1
inhibitors sirtinol (10 M) and tricostatin A (30 nM) (Fig. 2).
To see the mechanism of resveratrol induced cell apoptosis,
mitochondrial membrane potentials were monitored. For untreated cells,
orange-red fluorescent signals alone were found (Fig.3 A,B). Resveratrol
produced green fluorescent signal accumulation without orange-red
fluorescent signal (Fig.3 C,D). The resveratrol effect on mitochondrial
membrane potentials were abolished by sirtinol (10 M) (Fig.3 E,F).
Fig. 4. RT-PCR analysis
Resveratrol (100 M) increased expression of the sirtuin 1 mRNA in
MH7A cells in a treatment time dependent manner (Fig.4A) and
downregulated expression of the Bcl-XL mRNA in MH7A cells from 1-h
through 3-h treatment (Fig.4B), while it had no effect on expression of
mRNAs for Bcl-2, Bad, Bax, and Bak.
Fig. 5. Western blot analysis
Fig. 6. Caspase activity
Resveratrol (100M) increased presence of cytosolic cytochrome c in
parallel with a treatment time dependent decrease in the presence of
mitochondrial cytochrome c (Fig.5) and significantly activated caspase-3
and -9, but no activation of caspase-8 was obtained. Activation of
caspase-3 and -9 was also inhibited by sirtinol (10 M) (Fig.6).
DISCUSSION:
In the present study, resveratrol reduced viability of MH7A cells, a
human RA synovial cell line. Resveratrol increased TUNEL-positive
cells, which confirms resveratrol-induced MH7A cell apoptosis.
Resveratrol disrupted mitochondrial membrane potentials in MH7A cells,
and promoted cytochrome c release from the mitochondria into the
cytosol. Resveratrol appears to induce MH7A cell apoptosis by
activating caspase-9 and the effector caspase-3 in concert with
mitochondrial disruption allowing cytochrome c release from the
mitochondria into the cytosol. Of particular interest are the findings that
resveratrol upregulates expression of the sirtuin 1 mRNA in MH7A cells
and that resveratrol-induced MH7A cell death, mitochondrial damage,
and caspase-3/-9 activation were prevented by sirtuin 1 inhibitors. This
accounts for the implication of sirtuin 1 in resveratrol-induced MH7A
cell apoptosis. Resveratrol downregulated expression of the Bcl-XL
mRNA in MH7A cells, although expression of mRNAs for Bcl-2, Bax,
Bad, and Bak was not affected. This suggests that resveratrol disrupts
mitochondrial membrane potentials by upregulating sirtuin 1 expression
to downregulate Bcl-XL expression. This raises the possibility that
resveratrol is capable of preventing hyperplasia of synovial cells.
Resveratrol, therefore, could be developed as a promising drug for
treatment of RA.
REFERENCES:
1.den Broeder A et al, Journal of Rheumatology 9 (11), 2288-2298, 2002.
2. Miyazawa K et al, J Biochem. Dec 1;124(6):1153-62,1998.
3. Yasuda Y et al, Journal of Gastroenterology 44 (1), 56-65,2009.
Poster No. 2035 • ORS 2011 Annual Meeting