In the present study, we investigated the effect of MK-4 upon the proliferation of rheumatoid synovial cells and the development of arthritis in collagen-induced arthritis.. Our results
Trang 1new potential therapeutic strategy for rheumatoid arthritis Hiroshi Okamoto, Kumi Shidara, Daisuke Hoshi and Naoyuki Kamatani
Institute of Rheumatology, Tokyo Women’s Medical University, Japan
Vitamin K is a generic term for compounds that
include phytonadione (vitamin K1), the menaquinone
series (vitamin K2) and menadione (vitamin K3) These
vitamin K compounds share a common chemical
struc-ture consisting of a naphthoquinone nucleus
Vita-min K1 has a long phytol side chain whereas
vitamin K2has an unsaturated side chain [1]
Vitamin K2 acts as a cofactor for a vitamin
K-dependent carboxylase involved in the carboxylation
of coagulation factors and is an essential substrate for
blood coagulation [2] It has been reported that
osteo-porosis and fractures frequently occurred after the
long-term use of warfarin, which inhibits the effect of
vitamin K upon coagulation [3] Vitamin K2 has been
shown to be a key inducer of bone mineralization in
human osteoblasts and has also been reported to
inhi-bit osteoclastogenesis of bone by induction of the
osteoclast apoptosis [4–6]
Human studies have demonstrated that vitamin K2
is proposed to be an effective treatment for osteoporo-sis and the prevention of fractures [7] Menaquinone-4 (MK-4), the most common form of vitamin K2 is frequently used for the treatment of osteoporosis in Japan and other Asian countries
In addition to these biological activities, vitamin K2 has been reported to exert an inhibitory effect on the growth of several cell lines and tumor cells such as hepa-toma cells [8] Several lines of evidence indicate that vitamin K2 has a potent pro-apoptotic effect on leuke-mia cell lines and primary cultured leukeleuke-mia cells [9,10]
In addition, several case studies have demonstrated the clinical benefit of vitamin K2in the treatment of patients with acute myeloid leukemia and myelodysplastic syn-drome [11–13] Thus far, there are no studies examining the effect of vitamin K2on animal models of inflamma-tory arthritis or humans with inflammainflamma-tory arthritis
Keywords
apoptosis; collagen type II-induced arthritis;
rheumatoid arthritis; vitamin K2
(menaquinone-4)
Correspondence
H Okamoto, Institute of Rheumatology,
Tokyo Women’s Medical University,
10–22 Kawada-cho, Shinjuku,
Tokyo 162–0054, Japan
Fax: +81 3 5269 1726
Tel: +81 3 5269 1725
E-mail: hokamoto@ior.twmu.ac.jp
(Received 9 December 2006, revised
26 June 2007, accepted 12 July 2007)
doi:10.1111/j.1742-4658.2007.05987.x
Vitamin K2(menaquinone-4, MK-4) has been reported to induce apoptosis
in hepatocellular carcinoma, leukemia and myelodysplastic syndrome cell lines The effects of MK-4 on the development of arthritis have never been addressed thus far In the present study, we investigated the effect of MK-4 upon the proliferation of rheumatoid synovial cells and the development of arthritis in collagen-induced arthritis We analyzed the effect of MK-4 on the proliferation of fibroblast-like synoviocytes using the 3-(4,5-de-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay The pro-apop-totic effect of MK-4 upon fibroblast-like synoviocytes was investigated with annexin V staining and DNA fragmentation and caspase 3⁄ 7 assays Moreover, we analyzed the effect of MK-4 on the development of colla-gen-induced arthritis in female dark agouti rats Our results indicated that MK-4 inhibited the proliferation of fibroblast-like synoviocytes and the development of collagen-induced arthritis in a dose-dependent manner We conclude that MK-4 may represent a new agent for the treatment of rheu-matoid arthritis in the setting of combination therapy with other disease-modifying antirheumatic drugs
Abbreviations
CIA, collagen-induced arthritis; CII, collagen type II; FLS, fibroblast-like synoviocytes; MK-4, menaquinone-4; MTT, 3-(4,5-demethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; RA, rheumatoid arthritis; TNF-a, tumor necrosis factor a.
Trang 2In the present study, we determined the effect of
vitamin K2 (MK-4) on the proliferation of rheumatoid
synovial cells and the development of arthritis in the
experimental model of collagen type II-induced
arthri-tis (CIA)
Results
Effect of MK-4 on the viability of fibroblast-like
synoviocytes
It has been reported that MK-4 can induce apoptosis
in several tumor cells It has been also reported that
rheumatoid arthritis (RA) synovial cells proliferate as
fierce as tumor cells [14] We hypothesized that MK-4
could also reduce the viability of synovial cells and
thus be a novel treatment for RA because the marked
proliferation of synovial cells is a key pathological
fea-ture of RA We therefore studied the biological effects
of MK-4 on the proliferation of fibroblast-like
syno-viocytes (FLS) We observed that MK-4 inhibited the
viability of FLS in a dose-dependent manner (Fig 1)
By contrast, vitamin K1, which exerts no inhibitory
effects on the proliferation of tumor cell lines [10], had
no significant effect on the viability of synovial cells
These results indicate that MK-4 exerts cytotoxic
effects on synovial cells and this may be secondary to
its side chain structure
Induction of apoptosis of fibroblast-like
synoviocytes by MK-4
Analogous to the effects of MK-4 on tumor cells, we
hypothesized that MK-4 induces apoptotic death of
synovial cells We therefore examined the level of
apoptosis in synovial cells by measuring annexin V
staining, DNA fragmentation and caspase activity As shown in Fig 2A, most of the cells treated for 30 min with MK-4 (10)6m) exhibited diffuse cytosolic annexin
V staining (mean ± SD; 45 ± 9⁄ 100 cells) compared with positive (63 ± 11⁄ 100 cells) and negative (12 ± 4⁄ 100 cells) controls To further confirm the pro-apoptotic effect of MK-4 on FLS, we conducted a DNA fragmentation assay with various concentrations
of MK-4 MK-4 exhibited dose-dependent pro-apopto-tic effects on FLS (Fig 2B) To determine whether MK-4 activates caspase 3 and 7 to induce apoptosis
on FLS, we conducted a caspase activity assay MK-4 activated caspase assay in a dose-dependent manner (Fig 2C) By contrast, vitamin K1 did not show any effects on caspase activity and DNA fragmentation (data not shown) These results indicate that the inhib-itory effect of MK-4 on the proliferation of FLS is secondary to the induction of apoptosis by MK-4 induction of caspase activation
Oral administration of MK-4 ameliorates collagen-induced arthritis
As shown in Fig 3, MK-4 suppressed it suppressed the initiation of clinical arthritis compared with con-trol rats treated with NaCl⁄ Pi, as demonstrated by paw volume (Fig 3A), arthritis score (Fig 3B) and bone destruction score (Fig 3C) MK-4 treated rats exhibited statistically significant effects in a dose-dependent manner (P < 0.01) Histological analysis of the ankle joints of MK-4-treated rats (10 mgÆkg)1Æ day)1 and 50 mgÆkg)1Æday)1 groups) at day 32 demon-strates that MK-4 inhibits synovial proliferation and pannus formation compared with control rats (NaCl⁄ Pi) (Fig 3D) Histology of a joint of a rat of similar age is also shown in Fig 3D There was no
0
25
50
75
100
125
0 25 50 75 100 125
Cell Viability (%) Cell Viability (%)
Vitamin K2 (MK-4) 0
*
**
*
*
**
*
5 2.5 1.25 0.63 0.313 0.15 Vitamin K1
Fig 1 Inhibition of synovial cell viability by vitamin K 2 (MK-4) The inhibitory effect of vitamin K 2 and vitamin K 1 was evaluated with the MTT assay The level of the absorbance in the MTT assay from untreated cells was taken as 100% The data are presented as the mean ± SD.
Trang 3Annexin V-FITC Propidium Iodide FITC + PI
FITC + PI
FITC + PI
MK-4
0
0.4
0.8
1.2
MK-4
DNA fragmentation (absorbance A
0
60
Caspase 3/7 activity (luminescence (RLU))
MK-4
B
C
**
0 IL-1β TNF-α 0.63 1.25 2.5 5 10 (x10 -7 M )
40
20
*
0 0.31 0.63 1.25 2.5 5 10 (x10 -7 M )
**
**
*
TNF-α
Fig 2 Induction of apoptosis in FLS by MK-4 (A) FLS were cultured on 2-well Labo-ratory-Tek tissue culture chamber slides Cells were double-stained with annexin V and propidium iodine using the ApoAlert Annexin V-fluorescein isothiocyanate apop-tosis kit In positive controls, cells were treated with 1 ngÆmL)1of TNF-a In negative controls, cells were treated with NaCl ⁄ P i (B) DNA fragmentation induced by MK-4 was determined with the Cellular DNA Frag-mentation ELISA kit In positive controls, cells were treated with 1 ngÆmL)1of TNF-a.
In negative controls, cells were treated with NaCl ⁄ P i Each measurement was performed
in triplicate and the results are presented as the absorbance (A 270 nm ) compared with positive (TNF-a) and negative (NaCl ⁄ P i ) con-trols (C) Caspase activity induced by MK-4 was determined using the Caspase-Glo 3 ⁄ 7 Assay kit In positive controls, cells were treated with 1 ngÆmL)1of TNF-a or
1 ngÆmL)1of interleukin-1b In negative con-trol, cells were treated with NaCl ⁄ P i Each measurement was performed in triplicate and the results are presented as the lumi-nescence (relative light units) compared with positive (interleukin-1b and TNF-a) and negative (NaCl ⁄ P i ) controls *P < 0.05,
**P < 0.01 versus control (–), by the paired t-test.
Trang 4mortality or weight loss in MK-4-treated rats These
data suggest that MK-4 has significant inhibitory
effects on arthritis in vivo because MK-4 suppressed
the initiation of arthritis in the CIA model
Discussion
Synovial hyper-proliferation has reported to be caused,
at least in part, by impaired apoptosis of FLS
Defi-cient apoptosis of FLS results from up-regulation of
anti-apoptotic molecules such as bcl-2, sumo-1 and
FLIP (Fas-associated death domain-like interleukin 1b
converting enzyme inhibitory protein) [15–18]
Defi-cient apoptosis of FLS was reported to result from
lower expression of pro-apoptotic molecules such as
PTEN (phophatase and tensin homologue deleted from
chromosome 10) [19,20] These data suggested that the
synovial hyperplasia in RA is the result of defective
apoptosis not due to antibody or complement
media-tiated cytotoxicity In support of this model, various compounds, including antirheumatic drugs, could induce apoptosis in FLS Indeed, methotrexate, hydroxychloroquine, and bucillamine have all been reported to cause apoptosis in FLS [21–24] Therefore,
a compound that induces apoptosis in FLS could potentially be efficacious in the treatment of RA Vitamin K2is significantly less toxic than other anti-proliferative agents such as methotrexate In addition, vitamin K2 has significant anti-osteoporotic effects Thus, vitamin K2 may well open up novel future strat-egies, including chemoprevention, for the management
of patients with RA Vitamin K2 is an established treatment for patients with osteoporosis in Japan and has been used for more than 10 years [7] In the pres-ent study, we have shown that vitamin K2 inhibited the proliferation of FLS through the induction of apoptosis and also inhibited the development of CIA
in a dose-dependent manner Therefore, vitamin K2
2
2.5
3
3.5
4
4.5
0 10 20 30 Days
0 0.5 1 1.5 2 2.5
0 10 20 30 Days
0
0.5
1
1.5
2
Tarsal bone Metatarsal bone Calcanus
50mg/kg 10mg/kg
Control Normal
MK-4
Paw volume (ml) Arthritis score
50mg/kg 10mg/kg Control Normal
Control (NaCl/P i ) Normal
*
*
*
Fig 3 Suppression of arthritis development by MK-4 in the collagen type-II induced arthritis model (A,B) MK-4 suppressed the progression
of clinical arthritis compared with control rats treated with NaCl⁄ P i , as demonstrated by paw volume and the clinical arthritis score The data are represented as the mean ± SD *P < 0.05 (C) Radiological examination of bone destruction at the affected joints (calcaneus, metatarsal and tarsal bone) in normal rats, MK-4-treated rats (10 mgÆkg)1Æday)1), MK-4 treated rats (50 mgÆkg)1Æday)1) and control rats (NaCl ⁄ P i ) as described in Experimental procedures (D) Histological findings of the foot joint in normal rats, MK-4-treated rats (10 mgÆkg)1Æday)1), MK-4-treated rats (50 mgÆkg)1Æday)1) and control rats (NaCl⁄ P i ).
Trang 5may represent a new strategy for the treatment of RA,
presumably in the setting of combination therapy with
other disease-modifying antirheumatic drugs Further
clinical studies are needed to evaluate the beneficial
effects of vitamin K2
Experimental procedures
Synovial fibroblasts
Synovial biopsy samples were obtained from six patients
with RA and synoviocytes were maintained in separate
cul-tures These patients had active RA as defined by the
clini-cal criteria of the American Rheumatism Association [25]
All RA patients were receiving treatment that included
methtrexate (8 mgÆweek)1) and nonsteroidal
anti-inflamma-tory drugs, as well as steroids (less than 5 mgÆday)1)
How-ever, patients treated with biological agents, such as tumor
necrosis factor a (TNF-a) blocking agents, were excluded
from the study
All experiments were carried out using cell cultures
dur-ing the third to seventh passage FLS were cultured at
37C in 5% CO2in DMEM (Nikken Bio Medical
Labora-tory, Kyoto, Japan) supplemented with 10% fetal bovine
serum (Bioscience International Inc., Rockville, MD,
USA)
Reagents
Vitamin K1 (2-methyl-3-phenyl-1,4-naphthoquinone) and
vitamin K2 (MK-4) were all supplied by EisaiCo., Ltd
(Tokyo, Japan)
MTT assay
FLS were seeded at a density of 1· 103
cells per well in 96-well microtiter plates in 100 lL serum-free DMEM and
were treated with various concentrations of MK-4
(1.56· 10)7m to 10)6m) for 48 h Cell proliferation was
evaluated by measuring the number of viable cells using
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide (MTT) assay [26] Experiments were performed six
times with each of the three independent cell cultures
Annexin V staining
To examine annexin V staining, FLS were cultured on
2-well Laboratory-Tek tissue culture chamber slides After
treatment with MK-4 (10)6m), cells were fixed with 4%
paraformaldehyde in NaCl⁄ Pifor 15 min at room
tempera-ture and examined by microscopy Cells were
double-stained with annexin V and propidium iodine using the
ApoAlert Annexin V-fluorescein isothiocyanate apoptosis
kit (Takara Bio Inc., Shiga, Japan)
DNA fragmentation assay Apoptosis was determined using the Cellular DNA Frag-mentation ELISA kit (Roche Diagnostics, Mannheim, Ger-many) to detect BrdU labeled DNA fragments Each measurement was performed in triplicate and the results are presented as the absorbance (A270 nm) compared with posi-tive (TNF-a) and negaposi-tive (NaCl⁄ Pi) controls
Caspase 3⁄ 7 assay Apoptosis was determined using the Caspase-Glo 3⁄ 7 assay kit (Promega Co., WI, USA) to detect caspase 3⁄ 7 activity Each measurement was performed in triplicate and the results are presented as the luminescence (relative light units) compared with positive (interleukin-1b and TNF-a) and negative (NaCl⁄ Pi) controls
CIA model in rats Seven-week-old female dark agouti rats were obtained from Japan SLC, Inc (Shizuoka, Japan) 0.3% Collagen type II (CII) solution (Koken-Cellgen, Koken, Co Tokyo, Japan) was used CII emulsion was made by mixing
1 mL of 0.9% saline, 3 mL of incomplete Freund’s adju-vant and 2 mL of 0.3% CII solution CIA was induced
by an intradermal injection of 200 lL of CII emulsion (collagen II 1 mgÆmL)1) at the base of the tail Treatment with MK-4 was commenced at the onset of the disease MK-4 and control NaCl⁄ Piwere orally administered once per day at the specified dose for 32 days Each group was comprised with ten female dark agouti rats MK-4 was freshly suspended in 0.5% methyl cellulose diluted in NaCl⁄ Pi In each experiment, a group of control rats were administered 1% methyl cellulose orally Rats were examined for signs of CIA at days 1, 7 (onset of arthri-tis), 14, 18, 21, 25, 28 and 32 after immunization using the clinical parameters of paw swelling and clinical score The footpad volume was measured with a plethysmome-ter TK-101 (Unicom Japan, Tokyo, Japan) A scoring system from 0–4 was used for the clinical evaluation of CIA as follows: 0, normal; 1, mild swelling; 2, moderate swelling; 3, severe swelling; 4, severe and non-weight-bearing arthritis Each limb was graded giving a maximal clinical score of 4 per animal [27] For histological evalu-ation, we performed hematoxylin and eosin staining of tissue specimens of the ankle in five rats in both groups Radiological examination of bone destruction at the affected joints (calcaneus, metatarsal and tarsal bone) of two rats in both group was graded from 0–3 as follows:
0, normal; 1, minor signs of destruction; 2, up to 30% destruction; 3, more than 30% destruction [27] There was no mortality and no body weight loss in MK-4-trea-ted rats
Trang 6Statistical analysis
The Mann–Whitney U-test was used to compare
nonpara-metric data for statistical significance This test was used to
evaluate the histological examination of ankle joints, paw
volume and the clinical arthritis score
Acknowledgements
The expert technical help of Yukiko Katagiri is
grate-fully acknowledged
References
1 Suttie JW (1993) Synthesis of vitamin K-dependent
pro-teins FASEB J 7, 445–452
2 Liu Y, Nelson AN & Lipsky JJ (1996) Vitamin
K-dependent carboxylase: mRNA distribution and effects
of vitamin K-deficiency and warfarin treatment
Biochem Biophys Res Commun 224, 549–554
3 Simon RR, Beaudin SM, Johnston M, Walton KJ &
Shaughnessy SG (2002) Long-term treatment with
sodium warfarin results in decreased femoral bone
strength and cancellous bone volume in rats Thromb
Res 105, 353–358
4 Yamaguchi M, Taguchi H, Gao YH, Igarashi A &
Tsu-kamoto Y (1999) Effect of vitamin K2 (menaquinone-7)
in fermented soybean (natto) on bone loss in
ovariecto-mized rats J Bone Miner Metab 17, 23–29
5 Akiyama Y, Hara K, Tajima T, Murota S & Morita I
(1994) Effect of vitamin K2 (menatetrenone) on
osteo-clast-like cell formation in mouse bone marrow cultures
Eur J Pharmacol 263, 181–185
6 Kameda T, Miyazawa K, Mori Y, Yuasa T, Shiokawa
M, Nakamaru Y, Mano H, Hakeda Y, Kameda A &
Kumegawa M (1996) Vitamin K2 inhibits osteoclastic
bone resorption by inducing osteoclast apoptosis
Biochem Biophys Res Commun 220, 515–519
7 Shiraki M, Shiraki Y, Aoki C & Miura M (2000)
Vita-min K2 (menatetrenone) effectively prevents fractures
and sustains lumbar bone mineral density in
osteoporo-sis J Bone Miner Res 15, 515–521
8 Wang Z, Wang M, Finn F & Carr BI (1995) The
growth inhibitory effect of vitamins K and their actions
on gene expression Hepatology 2, 875–882
9 Yaguchi M, Miyazawa K, Katagiri T, Nishimaki J, Kizaki
M, Tohyama K & Toyama K (1997) Vitamin K2 and its
derivatives induce apoptosis in leukemia cells and enhance
the effect of all-trans retinoic acid Leukemia 11, 779–787
10 Yaguchi M, Miyazawa M, Otawa M, Katagiri T,
Nishi-maki J, Uchida Y, Iwase O, Gotoh A, Kawanishi Y &
Toyama K (1998) Vitamin K2 selectively induces
apop-tosis of blastic cells in myelodysplastic syndrome: flow
cytometric detection of apoptotic cells using APO2.7
monoclonal antibody Leukemia 12, 1392–1397
11 Miyazawa K, Nishimaki J, Ohyashiki K, Enomoto S, Kuriya S, Fukuda R, Hotta T, Teramura M, Mizoguchi
H, Uchiyama T et al (2000) Vitamin K2 therapy for myelodysplastic syndrome (MDS) and post-MDS acute myeloid leukemia: information through a questionnaire survey of multi-center pilot studies in Japan Leukemia
14, 1156–1157
12 Takami A, Nakao S, Ontachi Y, Yamauchi H & Mat-suda T (1998) Successful therapy of myelodysplastic syndrome with menatetrenone, a vitamin K2 analog Int J Hematol 69, 24–26
13 Fujita H, Tomiyama J & Tanaka T (1998) Vitamin K2 combined with all-trans retinoic acid induced complete remission of relapsing acute promyelocytic leukemia
Br J Haematol 103, 584–585
14 Firestein GS (2003) Evolving concepts of rheumatoid arthritis Nature 423, 356–361
15 Franz JK, Pap T, Hummel KM, Nawrath M, Aicher
WK, Shigeyama Y, Muller-Ladner U, Gay RE & Gay S (2000) Expression of sentrin, a novel anti-apoptotic mole-cule, at sites of synovial invasion in rheumatoid arthritis Arthritis Rheum 43, 599–607
16 Perlman H, Liu H, Georganas C, Koch AE, Shamiyeh E, Haines GK & 3rd & Pope RM (2001) Differential expres-sion pattern of the anti-apoptotic proteins, Bcl-2 and FLIP, in experimental arthritis Arthritis Rheum 44, 2899–2908
17 Schedel J, Gay RE, Kuenzler P, Seemayer C, Simmen B, Michel BA & Gay S (2002) FLICE-inhibitory protein expression in synovial fibroblasts and at sites of cartilage and bone erosion in rheumatoid arthritis Arthritis Rheum 46, 1512–1518
18 Catrina AI, Ulfgren AK, Lindblad S, Grondal L & Klareskog L (2002) Low levels of apoptosis and high FLIP expression in early rheumatoid arthritis synovium Ann Rheum Dis 61, 934–936
19 Goberdhan DC & Wilson C (2003) PTEN: tumour sup-pressor, multifunctional growth regulator and more Hum Mol Genet 12, R239–R248
20 Pap T, Franz JK, Hummel KM, Jeisy E, Gay R & Gay S (2000) Activation of synovial fibroblasts in rheumatoid arthritis: lack of expression of the tumour suppressor PTEN at sites of invasive growth and destruction Arthritis Res 2, 59–64
21 Kim WU, Yoo SA, Min SY, Park SH, Koh HS, Song
SW & Cho CS (2006) Hydroxychloroquine potentiates Fas-mediated apoptosis of rheumatoid synoviocytes Clin Exp Immunol 144, 503–511
22 Wunder A, Schellenberger E, Mahmood U, Bogdanov
A Jr, Muller-Ladner U, Weissleder R & Josephson L (2005) Methotrexate-induced accumulation of fluores-cent annexin V in collagen-induced arthritis Mol Imaging 4, 1–6
23 Nakazawa F, Matsuno H, Yudoh K, Katayama R, Sawai T, Uzuki M & Kimura T (2001) Methotrexate
Trang 7inhibits rheumatoid synovitis by inducing apoptosis.
J Rheumatol 28, 1800–1808
24 Sawada T, Hashimoto S, Furukawa H, Tohma S, Inoue T
& Ito K (1997) Generation of reactive oxygen species is
required for bucillamine, a novel anti-rheumatic drug, to
induce apoptosis in concert with copper
Immunopharma-cology 35, 195–202
25 Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries
JF, Cooper NS, Healey LA, Kaplan SR, Liang MH,
Luthra HS et al (1987) The American Rheumatism
Association 1987 revised criteria for the classification of
rheumatoid arthritis Arthritis Rheum 31, 315–324
26 Okamoto H, Cujec TP, Okamoto M, Peterlin BM, Baba
M & Okamoto T (2000) Inhibition of the RNA-depen-dent transactivation and replication of human immuno-deficiency virus type 1 by a fluoroquinoline derivative K-37 Virology 272, 402–408
27 Larsson E, Harris HE, Palmblad K, Mansson B, Saxne
T & Klareskog L (2005) CNI-1493, an inhibitor of pro-inflammatory cytokines, retards cartilage destruction in rats with collagen induced arthritis Ann Rheum Dis 64, 494–496