Europearl Journal of Pharmacology,
221 (1992) 3X1-384
3x1
8 1992 Elsevier Science Publishers B.V. All right!, reserved OOl4-2999/92/$05.00
EJP 21133
Short communication
Inhibitory effect of curcumin, an anti-inflammatory smooth muscle cell proliferation Huei-Chen Department
of Pharmacology,
Huatig, Tong-Rong
College of Medicine. National National
Yang-Ming
Jan and Sheau-Farn
agent, on vascular Yeh a
Taiwan University, Taipei, Taiwan and u I~sti~ture of Biochemistry,
Medical College, Taipei, Taiwan
Received I6 July 1992, accepted 25 August 1992
The effects of curcumin, an anti-inflammatory agent from Clfrcuma longu, on the proliferation of blood mononuclear cells and vascular smooth muscle cells were studied. Proliferative responses were determined from the uptake of tritiated thymidine. In human peripheral blood mononuclear cells, curcumin dose dependently inhibited the responses to phytohemagglutinin and mixed lymphocyte reaction at the dose ranges of lO_’ to 3 x IO-’ and 3 x lo-” to 3 x 1O-5 M, respectively. Curcumin (IO-’ to lo-” MI dose dependently inhibited the proliferation of rabbit vascular smooth muscle cells stimulated by fetal calf serum. Curcumin had a greater inhibitory cffcct on platclct-derived growth factor-stimulated proliferation than on serum-stimulated proliferation. Cinnamic acid, coumaric acid and fcrulic acid were much less effective than curcumin as inhibitors of scrum-induced smooth muscle cell proliferation, suggesting that the cinnamic acid and ferulic actd moieties alone are not sufficient for activity, and that the characteristics of the diferuloylmethane moleculc itself are necessary for activity. Curcumin may be useful as a new template for the development of better remedies for the prevention of the pathological changes of atherosclerosis and restenosis. Curcumin; Antiproliferative
effect; Mononuclear cells: Smooth muscle cells
1. Introduction The increased potential for growth of vascular smooth muscle cells is one of the key abnormalities in the development of atherosclerosis and restenosis (Ross and Glomset, 1976; Lundergan et al., 1991). Studies have shown that mononuclear cells, including monocytes and lymphocytes, are the first participants in early atherogenesis and restenosis (Jonasson et al., 1986, 1988). We have previously reported that emodin and scoparone from hypolipidemic Chinese herbs exhibit vasodilator, immunosuppressive and antiproliferative effects (H.C. Huang et al.. 1991, 1992). Recently, we found that curcumin (diferuloylmethane), an active principle from another hypolipidemic Chinese herb Curcuma longa, inhibited the proliferative responses of both rabbit vascular smooth muscle cells and human blood mononuclear cells. Curcumin has been reported to possess anti-inflammatory activity (Srimal and Dharvan, 1973; Mukhopadchyay et al., 1982). In this study,
Correspondence to: H.C. Huang, Department of Pharmacology, College of Medicine, National Taiwan University. No. 1. Jen-Ai Road, 1st Section, Taipei, Taiwan. Tel. 8X6.2.3123 456 ext. X320, fax X86.2.3513 716.
the effects of curcumin on vascular smooth mu&c cells and blood mononuclear cells were studied.
2. Materials and methods 2.1. Materials Commercially available rhizomes of Curcun:a longa were purchased from the market. Curcumin was isolated from the crude extract or purchased from Aldrich Chemical Co. (Milwaukee, WI). Cinnamic acid, coumaric acid and ferulic acid were purchased from Aldrich Co. [Methyl-“Hlthymidine (6.7 Ci/mmol) was purchased from New England Nuclear (Boston, MA). Platelet-derived growth factor (PDGF) B/B (human, recombinant sterile) was purchased from Boehringer Mannheim (Mannheim, Germany). Mitomycin C was a gift from Kyowa Hakko Kogyo Co. (Tokyo, Japan). Fetal calf serum, phytohemagglutinin (PHA) and RPM1 1640 medium were purchased from Gibco Lab. (Grand Island, NY). Ficoll-Hypacue was purchased from Pharmacia Fine Chemicals (Piscataway, NJ). New Zealand rabbits were supplied from animal center of National Taivvan University, Taipei. Taiwan.
The immunosuppressive effect wxs assayed as the ability to suppress the responses of human peripheral baa ~nonu~~ear cells to PHA and mixed lymphocyte reaction (MLR) (H.C. Huang et al., 1991). Human peripheral blood mononuclear cells were isolated on Ficoll-Hypaque dansity gradients from heparinized No& from healthy adult donors. The cell population, termed mon~~nuctear cek, contained macrophages. T cells and 6 cells. The cell culture medium consisted of RPM1 16-N)supplemented with 10% fetal calf serum. 5 mM HEPES. 2 mM L-glutamine, 100 U/mI penicillin and 100 yg/m! streptomycin. Cells were incubated at 37°C in a homidified atmosphere of 5% CO? in air. Ceil viab~~i~ was determined by using the trypan blue dye exclusion method, In preliminary experiments, optimal concentrations ( < WJ M) of curcumin were found 13 have a minimal effect on cell viability in a 72-h ehpsur; period.
For assaying the response to PI-IA, PHA (25 @g/ml, 1),25% v/v) and curcumin were added directly to mononuclear cells (2 x 10’ cells/well) in RPMI 1640 medium upon the initiation of culture at 37°C in a humidi~ed atmosphere of 5% CO, in air for 72 h. Tritiated thymidine (0.1 @Yi) was added to cultures during the last 6 h of culture. At the end of culture, the cells were harvested on filter paper and radioactivity was counted in a liquid scintillation counter. For assaying the response to mixed lyrnph~~cyte reaction, human mononuclear responder cells f10” ceils/ml) were cocultured with an equal number of mitomycin C-treated stimulator ceils (25 @g/ml mitomycin C at 37°C for 20 min) in RPM1 1640 medium. Curcumin was added when the culture was started. The cells were incubated at 37°C in a humidified atmos~herc of 5% CO, in air for 5 days. The cultures were pulsed with 2 ,uCi of [>H]thymidine for 18 h and harvested on filkr paper; cellular radioactivity was countrd in a liquid scintillation counter.
I o-o
o .’ o Cinnamic acid Coumaric acid o-e Fe$ulic acid A--I Curcumin
6
5
4
Curcumin,-Log[M]
Concentration,-Log@] ‘L,, Fk. I. (A) Effect of curcuminon the proliferation of blood mononuclear cells and vascular smooth muscle cells. (B) Effects of ciircumin and r&ted compounds on vascular smooth muscle cell proliferation. The proliferative response was delerminrd from the uptake ‘lflf tritiated tbmidine. {AI Human mononuclear cell:, (2x IO” ceils/well) were stimulated with 0.25% pbytob~m~~~iutinin (WA) fof and mixed &mphocyte reaction fML.Rt@I. The suppressive activity of the compound is expressed as a percentage of the untreated cctntrol stimu~tcd with PHA or MLR !% nf control). The untreaied control values for [ ~Hlthymidine incorpsration were 8525.0 & 332.6 cpm for PIIA and X41 I.3 + 3311.: cpm fc>r MI-R. Rabbit vascular smmth muscle cells (2.5 x IO” cells/well) were stimulated with 54 fetal calf serum (FCS) ( A ).The inhibitory ;tctivity of the compound is expressed as a percentage of the untreated contrnl stimulated with FCS (% of control). The untreated control vrjue was 2 lhj.tj+ ltt3.l cpnl.(Bl Rabbit vascular smooth muscle cells were stimulated with 5% FCS. The inhibitory activities of cinnamic acid (c?- --01. coumaric acid (q-o 1. ferulic acid (of and curcumio ( A 1 are expressed as percentages of untreated controls stimulated with FCS IQ of c”ntroll. Points with vertical lipes represent the means and S.E.M. nf a triplicate experiment which was repeated more than three times with similar results,
TABLE I Chemicalstructuresof curctiminand other cinnamicacirlde-k~rjves. -
The antiproliferative effect was.assayed as the abiiity to inhibit the proliferation of cultured rabbit aortic SMC induced by 5% fetal calf serum or PDGF (Huang et al,, 1992). The abdominal aorta of a rabbit was dissected and placed in cold Hanks balanced salt soiution. The intima and inner two-thirds of the media were as I-mm strips, cut into squares and put in a dry Petri dish. The ceils were cultured in Duibecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. When ceils approached confluence, the aol;ta pieces were removed. Ceils between passages 3 dnd 8 were used. Ceil viability was determined with the trypan blue dye exclusion method. In pre~imina~ experiments, optimal concentrations i < IO-” MI of curcumin were found to have a minimal effect on cell viability. The proliferative response of vascular SMC was determined from the uptake of tritiated thymidine. Prior to ail experiments, confluent SMC (2-3 x 104 cells/well) were rendered quiescent by culturing for 48 h (with one medium change after 24 h) in 0.5% fetal caif serum instead of 10%. Stimulator, 5% fetal calf serum or PDGF, and test drug were then added to medium for 24 h before [3H]tymidine (0.05 pCi/weiiJ was added to the medium. Twenty-four hours after the addition of ~~H]thym~dine, ceffs were harvested and [“Hlthymidine incorporated to DNA was counted in a liquid ycintiilation counter. Each experiment was performed in triplicate. The inhibitory activities of curcumin are expressed as percentages of untreated control values in response to stimulators or mito~ens_ The concentration evoking 50% of maximal inhibition (ICs,,I was calculated for each experiment. The data are expressed as means + S.E.M. Significance was tested using Student’s t-test. Values of P < O,U5 were considered significant.
3. Results
Human peripheral blood mononuclear cells wcrc stimulated with PHA or MLR. The suppressive activity of curcumin is expressed as a percentage of the untreated control value. Its shuwn in fig. IA, eurcumin dose dependently suppressed the response to PHA in the dose range lo-” to 3 x lo-’ M and that to MLR in the dose range 3 X lQ-” to 3 x lO_” M. The ECso values (n = 5) were 2.5 & 1.0 FM for the response to PHA and 5.0 + 1.O PM for the MLR.
Gnamic acid Cnumaricacid Feruii~acid
R,
R2
H
U
Curcumin
The control value for [ ‘Hlthymidine incorporation rabbit vascular SMC in 5% fetal calf serum was 2 165.0 & 103.1 cpm/weii. Exposure of SMC to curcumin (lOMh to IO-’ M) significantly inhibited [“H] thymidine incorporation in a dose-dependent manner (fig. 1A). The E&, value was 23.3 i 0.7 PM (n = 6). PDGF, a potent growth factor, is thought to piay a prominent role in disorders of SMC. Curcumin is more potent in inhibiting PDGF (1 nM)-stimulated SMC pruiifierat~on than fetal calf serum-stimulated prohferation (82.5 f 2.5 vs. 36.3 _t 9.0% at 10 PM). The effects of some phenoiic acids that are structurally related to curcumin (table I) were a!so examined and the results are shown in fig. 1E. Exposure of SMC to cinnamic acid, coumaric acid and ferulic acid ~nhibjted serum-induced proliferation, and higher doses of the phenolic acids resulted in greater inhibition. The inhibitory activities of cinnamic acid, coumaric acid and ferulic acid at 10-j M were 56.3, SO.0 and 41.4r/r, respectively. In contrast, curcumin (IO-” MI completely abolished the proliferative response ( lOll%~inhibition). irs
4. Discussion The present results demonstrated for the first time that curcumin has an ar~ti~ro~~ferat~veeffect on both blood mononuclear cells and vascular SMC. The processes of atherogenesis, immune defense and inflammation consist of sevcrai proliferative responses at the tissue and ccii icvci. Curcumin has been reparted to bc an effective anti~in~amrnato~ agent in acute and
&WEIC‘~~$~~~R~m~~ti~~n teats (Srimal and Dharvan, 1973; ~~~~~~~n~.~dhy~~y t’t al.. 1982). In blood mononuclear c&s. ~~~~rn~~ irnp~~ir~dnot only the response of C&S to mitugen. PHA. but also the response to alloantigcn. MER. In vascular SMC, curcumin was an effective ~~h~bjt~~rof both strum-induced and PD~F-dependent mitogenesis. Thus. curcumin may be clinically useful in transpiant atherosclerosis. In mouse fibroblast ceils, cureumin has been reported to suppress c-fun/AP-1 activity. which is known to be stimulated by different mitogens (T.S. Huang et al.. 19911. Further studies are in progress to elucidate the mechanisms of action of curcumin in the proliferation of mononuclear ceils and SMC. Cinnamic acid. coumaric acid and ferulic acid,wre less potent in inhibiting vascular SMC proliferation than curcumin. This itidicates thai the cinnamic acid or ferulic acid moieties alone are not sufficient for the antiproliferative activity of curcumin. The antiproliferative activity of ~urcumin is not mediated by the release of ferulic atid in the system. but may be caused by the characteristics of the diferuloyimethane molecule itself. Alteration of the control of vascular SMC proliferation is a major factor in the development of atherosclerosis and restenosis (Ross and Glomset, !976; Lundergan et a!.. 199!). Mononuclear cells, including both monocytes and Iymphocytes, have been shown to be among the first participants in early atherogenesis and restenosis (Jonasson et al., 1986, 1988). Moreover, several important immunovascular interactions have been reported (Hansson et al., 1989). it has been suggested that vascular disease could be treated by interfering with mechanisms for immunological activation. Thus, curcumin, which posseses both immunosuppressive and antiproliferative activities, may be useful as a new template for the development of better remedies for the prevention of the vascutar pathoIogica1 changes of
atherosclerosis atherosclerosis.
and
restenosis,
especially
transplant
Acknowledgement This investigation was supported by grants from the National Science Council and Department of Health, Taiwan.
References Hansson, G.K.. J. Helm, L. Jonasson, PS Seifert and S. Stemme, IYXY. Immunological control of vascular cell growth and differentiation, J. Cardiovasc. Pharmacol. 14 (Suppl. 61, SX2. Huang. H.C.. S.H. Chu and P.D. Lee Chao, 1991. Vasorelaxants from Chinese herbs, emodin and scoparone. possess immunosuppressive properties. Eur. J. Pharmacol. 198, 211. Huang. H.C.. W.W. Lin and Y.T. Lee, 1992, Antiproliferative effect of vasodilators. emodin and scoparone, on vascular smooth muscle cells from normal and hyperlipidemic diabetic rabbits, The Seventh Joint Annual Conference of Biomedical Science, Taipei, Taiwan. p. 57. Huanp, T.S.. S.C. Lee and 3.K. Lin, 1991. Suppression of c-Jun/AP-1 activation by the inhibitor of tumor promotion in mouse fibmblast cells. Pmt. Nat]. Acad. Sci. U.S.A. 88. 5292. Jonasson, L., J. Holm, 0. Skalli, G. Bondjers and C.K. Hansson, 198h, Regional accumulations of T cells, macrophages. and smooth muscle cells in the human atherosclerotic plaque, Arteriosclerosis 6, 131. Jonasson. Lx. J. Holm and G.K. Hansson, 1988. Cyclosporin A inhibits smooth mucle proliferaiiun in the vascular response to injury, Proc. Natl. Acad. Sci. U1S.A. 8.5, 2X03. Mukhopadhyay. A., N. Basu. N. Ghatak and P.K. Gujzal. 19X2, Anti-inflammatory and irritant activities of curcumin analogues in rats, Agents Actions 12. 508. Lundergan. C.F.. M.L. Foegh and P.W. Ramwell, 1991. Peptide inhibition of myoi&mal proliferation by angiopeptin. a somatostatin analogue, J. Am. Coil. Cardioi. 17. l32B. Ross, R. and J.A. Glomset. 1976. The pathr~~enesisof atherosclerosis. N. Engl. J. Med. 29S, 369. Srimal, R.C. and B.N. Dharvan. 1973, Pharmacology of curcumin. A non-st~ro~dal and anti-in~ammato~ agents, J. Pharm. Pharmacol. 25, 447.
221 (1992) 3X1-384
3x1
8 1992 Elsevier Science Publishers B.V. All right!, reserved OOl4-2999/92/$05.00
EJP 21133
Short communication
Inhibitory effect of curcumin, an anti-inflammatory smooth muscle cell proliferation Huei-Chen Department
of Pharmacology,
Huatig, Tong-Rong
College of Medicine. National National
Yang-Ming
Jan and Sheau-Farn
agent, on vascular Yeh a
Taiwan University, Taipei, Taiwan and u I~sti~ture of Biochemistry,
Medical College, Taipei, Taiwan
Received I6 July 1992, accepted 25 August 1992
The effects of curcumin, an anti-inflammatory agent from Clfrcuma longu, on the proliferation of blood mononuclear cells and vascular smooth muscle cells were studied. Proliferative responses were determined from the uptake of tritiated thymidine. In human peripheral blood mononuclear cells, curcumin dose dependently inhibited the responses to phytohemagglutinin and mixed lymphocyte reaction at the dose ranges of lO_’ to 3 x IO-’ and 3 x lo-” to 3 x 1O-5 M, respectively. Curcumin (IO-’ to lo-” MI dose dependently inhibited the proliferation of rabbit vascular smooth muscle cells stimulated by fetal calf serum. Curcumin had a greater inhibitory cffcct on platclct-derived growth factor-stimulated proliferation than on serum-stimulated proliferation. Cinnamic acid, coumaric acid and fcrulic acid were much less effective than curcumin as inhibitors of scrum-induced smooth muscle cell proliferation, suggesting that the cinnamic acid and ferulic actd moieties alone are not sufficient for activity, and that the characteristics of the diferuloylmethane moleculc itself are necessary for activity. Curcumin may be useful as a new template for the development of better remedies for the prevention of the pathological changes of atherosclerosis and restenosis. Curcumin; Antiproliferative
effect; Mononuclear cells: Smooth muscle cells
1. Introduction The increased potential for growth of vascular smooth muscle cells is one of the key abnormalities in the development of atherosclerosis and restenosis (Ross and Glomset, 1976; Lundergan et al., 1991). Studies have shown that mononuclear cells, including monocytes and lymphocytes, are the first participants in early atherogenesis and restenosis (Jonasson et al., 1986, 1988). We have previously reported that emodin and scoparone from hypolipidemic Chinese herbs exhibit vasodilator, immunosuppressive and antiproliferative effects (H.C. Huang et al.. 1991, 1992). Recently, we found that curcumin (diferuloylmethane), an active principle from another hypolipidemic Chinese herb Curcuma longa, inhibited the proliferative responses of both rabbit vascular smooth muscle cells and human blood mononuclear cells. Curcumin has been reported to possess anti-inflammatory activity (Srimal and Dharvan, 1973; Mukhopadchyay et al., 1982). In this study,
Correspondence to: H.C. Huang, Department of Pharmacology, College of Medicine, National Taiwan University. No. 1. Jen-Ai Road, 1st Section, Taipei, Taiwan. Tel. 8X6.2.3123 456 ext. X320, fax X86.2.3513 716.
the effects of curcumin on vascular smooth mu&c cells and blood mononuclear cells were studied.
2. Materials and methods 2.1. Materials Commercially available rhizomes of Curcun:a longa were purchased from the market. Curcumin was isolated from the crude extract or purchased from Aldrich Chemical Co. (Milwaukee, WI). Cinnamic acid, coumaric acid and ferulic acid were purchased from Aldrich Co. [Methyl-“Hlthymidine (6.7 Ci/mmol) was purchased from New England Nuclear (Boston, MA). Platelet-derived growth factor (PDGF) B/B (human, recombinant sterile) was purchased from Boehringer Mannheim (Mannheim, Germany). Mitomycin C was a gift from Kyowa Hakko Kogyo Co. (Tokyo, Japan). Fetal calf serum, phytohemagglutinin (PHA) and RPM1 1640 medium were purchased from Gibco Lab. (Grand Island, NY). Ficoll-Hypacue was purchased from Pharmacia Fine Chemicals (Piscataway, NJ). New Zealand rabbits were supplied from animal center of National Taivvan University, Taipei. Taiwan.
The immunosuppressive effect wxs assayed as the ability to suppress the responses of human peripheral baa ~nonu~~ear cells to PHA and mixed lymphocyte reaction (MLR) (H.C. Huang et al., 1991). Human peripheral blood mononuclear cells were isolated on Ficoll-Hypaque dansity gradients from heparinized No& from healthy adult donors. The cell population, termed mon~~nuctear cek, contained macrophages. T cells and 6 cells. The cell culture medium consisted of RPM1 16-N)supplemented with 10% fetal calf serum. 5 mM HEPES. 2 mM L-glutamine, 100 U/mI penicillin and 100 yg/m! streptomycin. Cells were incubated at 37°C in a homidified atmosphere of 5% CO? in air. Ceil viab~~i~ was determined by using the trypan blue dye exclusion method, In preliminary experiments, optimal concentrations ( < WJ M) of curcumin were found 13 have a minimal effect on cell viability in a 72-h ehpsur; period.
For assaying the response to PI-IA, PHA (25 @g/ml, 1),25% v/v) and curcumin were added directly to mononuclear cells (2 x 10’ cells/well) in RPMI 1640 medium upon the initiation of culture at 37°C in a humidi~ed atmosphere of 5% CO, in air for 72 h. Tritiated thymidine (0.1 @Yi) was added to cultures during the last 6 h of culture. At the end of culture, the cells were harvested on filter paper and radioactivity was counted in a liquid scintillation counter. For assaying the response to mixed lyrnph~~cyte reaction, human mononuclear responder cells f10” ceils/ml) were cocultured with an equal number of mitomycin C-treated stimulator ceils (25 @g/ml mitomycin C at 37°C for 20 min) in RPM1 1640 medium. Curcumin was added when the culture was started. The cells were incubated at 37°C in a humidified atmos~herc of 5% CO, in air for 5 days. The cultures were pulsed with 2 ,uCi of [>H]thymidine for 18 h and harvested on filkr paper; cellular radioactivity was countrd in a liquid scintillation counter.
I o-o
o .’ o Cinnamic acid Coumaric acid o-e Fe$ulic acid A--I Curcumin
6
5
4
Curcumin,-Log[M]
Concentration,-Log@] ‘L,, Fk. I. (A) Effect of curcuminon the proliferation of blood mononuclear cells and vascular smooth muscle cells. (B) Effects of ciircumin and r&ted compounds on vascular smooth muscle cell proliferation. The proliferative response was delerminrd from the uptake ‘lflf tritiated tbmidine. {AI Human mononuclear cell:, (2x IO” ceils/well) were stimulated with 0.25% pbytob~m~~~iutinin (WA) fof and mixed &mphocyte reaction fML.Rt@I. The suppressive activity of the compound is expressed as a percentage of the untreated cctntrol stimu~tcd with PHA or MLR !% nf control). The untreaied control values for [ ~Hlthymidine incorpsration were 8525.0 & 332.6 cpm for PIIA and X41 I.3 + 3311.: cpm fc>r MI-R. Rabbit vascular smmth muscle cells (2.5 x IO” cells/well) were stimulated with 54 fetal calf serum (FCS) ( A ).The inhibitory ;tctivity of the compound is expressed as a percentage of the untreated contrnl stimulated with FCS (% of control). The untreated control vrjue was 2 lhj.tj+ ltt3.l cpnl.(Bl Rabbit vascular smooth muscle cells were stimulated with 5% FCS. The inhibitory activities of cinnamic acid (c?- --01. coumaric acid (q-o 1. ferulic acid (of and curcumio ( A 1 are expressed as percentages of untreated controls stimulated with FCS IQ of c”ntroll. Points with vertical lipes represent the means and S.E.M. nf a triplicate experiment which was repeated more than three times with similar results,
TABLE I Chemicalstructuresof curctiminand other cinnamicacirlde-k~rjves. -
The antiproliferative effect was.assayed as the abiiity to inhibit the proliferation of cultured rabbit aortic SMC induced by 5% fetal calf serum or PDGF (Huang et al,, 1992). The abdominal aorta of a rabbit was dissected and placed in cold Hanks balanced salt soiution. The intima and inner two-thirds of the media were as I-mm strips, cut into squares and put in a dry Petri dish. The ceils were cultured in Duibecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. When ceils approached confluence, the aol;ta pieces were removed. Ceils between passages 3 dnd 8 were used. Ceil viability was determined with the trypan blue dye exclusion method. In pre~imina~ experiments, optimal concentrations i < IO-” MI of curcumin were found to have a minimal effect on cell viability. The proliferative response of vascular SMC was determined from the uptake of tritiated thymidine. Prior to ail experiments, confluent SMC (2-3 x 104 cells/well) were rendered quiescent by culturing for 48 h (with one medium change after 24 h) in 0.5% fetal caif serum instead of 10%. Stimulator, 5% fetal calf serum or PDGF, and test drug were then added to medium for 24 h before [3H]tymidine (0.05 pCi/weiiJ was added to the medium. Twenty-four hours after the addition of ~~H]thym~dine, ceffs were harvested and [“Hlthymidine incorporated to DNA was counted in a liquid ycintiilation counter. Each experiment was performed in triplicate. The inhibitory activities of curcumin are expressed as percentages of untreated control values in response to stimulators or mito~ens_ The concentration evoking 50% of maximal inhibition (ICs,,I was calculated for each experiment. The data are expressed as means + S.E.M. Significance was tested using Student’s t-test. Values of P < O,U5 were considered significant.
3. Results
Human peripheral blood mononuclear cells wcrc stimulated with PHA or MLR. The suppressive activity of curcumin is expressed as a percentage of the untreated control value. Its shuwn in fig. IA, eurcumin dose dependently suppressed the response to PHA in the dose range lo-” to 3 x lo-’ M and that to MLR in the dose range 3 X lQ-” to 3 x lO_” M. The ECso values (n = 5) were 2.5 & 1.0 FM for the response to PHA and 5.0 + 1.O PM for the MLR.
Gnamic acid Cnumaricacid Feruii~acid
R,
R2
H
U
Curcumin
The control value for [ ‘Hlthymidine incorporation rabbit vascular SMC in 5% fetal calf serum was 2 165.0 & 103.1 cpm/weii. Exposure of SMC to curcumin (lOMh to IO-’ M) significantly inhibited [“H] thymidine incorporation in a dose-dependent manner (fig. 1A). The E&, value was 23.3 i 0.7 PM (n = 6). PDGF, a potent growth factor, is thought to piay a prominent role in disorders of SMC. Curcumin is more potent in inhibiting PDGF (1 nM)-stimulated SMC pruiifierat~on than fetal calf serum-stimulated prohferation (82.5 f 2.5 vs. 36.3 _t 9.0% at 10 PM). The effects of some phenoiic acids that are structurally related to curcumin (table I) were a!so examined and the results are shown in fig. 1E. Exposure of SMC to cinnamic acid, coumaric acid and ferulic acid ~nhibjted serum-induced proliferation, and higher doses of the phenolic acids resulted in greater inhibition. The inhibitory activities of cinnamic acid, coumaric acid and ferulic acid at 10-j M were 56.3, SO.0 and 41.4r/r, respectively. In contrast, curcumin (IO-” MI completely abolished the proliferative response ( lOll%~inhibition). irs
4. Discussion The present results demonstrated for the first time that curcumin has an ar~ti~ro~~ferat~veeffect on both blood mononuclear cells and vascular SMC. The processes of atherogenesis, immune defense and inflammation consist of sevcrai proliferative responses at the tissue and ccii icvci. Curcumin has been reparted to bc an effective anti~in~amrnato~ agent in acute and
&WEIC‘~~$~~~R~m~~ti~~n teats (Srimal and Dharvan, 1973; ~~~~~~~n~.~dhy~~y t’t al.. 1982). In blood mononuclear c&s. ~~~~rn~~ irnp~~ir~dnot only the response of C&S to mitugen. PHA. but also the response to alloantigcn. MER. In vascular SMC, curcumin was an effective ~~h~bjt~~rof both strum-induced and PD~F-dependent mitogenesis. Thus. curcumin may be clinically useful in transpiant atherosclerosis. In mouse fibroblast ceils, cureumin has been reported to suppress c-fun/AP-1 activity. which is known to be stimulated by different mitogens (T.S. Huang et al.. 19911. Further studies are in progress to elucidate the mechanisms of action of curcumin in the proliferation of mononuclear ceils and SMC. Cinnamic acid. coumaric acid and ferulic acid,wre less potent in inhibiting vascular SMC proliferation than curcumin. This itidicates thai the cinnamic acid or ferulic acid moieties alone are not sufficient for the antiproliferative activity of curcumin. The antiproliferative activity of ~urcumin is not mediated by the release of ferulic atid in the system. but may be caused by the characteristics of the diferuloyimethane molecule itself. Alteration of the control of vascular SMC proliferation is a major factor in the development of atherosclerosis and restenosis (Ross and Glomset, !976; Lundergan et a!.. 199!). Mononuclear cells, including both monocytes and Iymphocytes, have been shown to be among the first participants in early atherogenesis and restenosis (Jonasson et al., 1986, 1988). Moreover, several important immunovascular interactions have been reported (Hansson et al., 1989). it has been suggested that vascular disease could be treated by interfering with mechanisms for immunological activation. Thus, curcumin, which posseses both immunosuppressive and antiproliferative activities, may be useful as a new template for the development of better remedies for the prevention of the vascutar pathoIogica1 changes of
atherosclerosis atherosclerosis.
and
restenosis,
especially
transplant
Acknowledgement This investigation was supported by grants from the National Science Council and Department of Health, Taiwan.
References Hansson, G.K.. J. Helm, L. Jonasson, PS Seifert and S. Stemme, IYXY. Immunological control of vascular cell growth and differentiation, J. Cardiovasc. Pharmacol. 14 (Suppl. 61, SX2. Huang. H.C.. S.H. Chu and P.D. Lee Chao, 1991. Vasorelaxants from Chinese herbs, emodin and scoparone. possess immunosuppressive properties. Eur. J. Pharmacol. 198, 211. Huang. H.C.. W.W. Lin and Y.T. Lee, 1992, Antiproliferative effect of vasodilators. emodin and scoparone, on vascular smooth muscle cells from normal and hyperlipidemic diabetic rabbits, The Seventh Joint Annual Conference of Biomedical Science, Taipei, Taiwan. p. 57. Huanp, T.S.. S.C. Lee and 3.K. Lin, 1991. Suppression of c-Jun/AP-1 activation by the inhibitor of tumor promotion in mouse fibmblast cells. Pmt. Nat]. Acad. Sci. U.S.A. 88. 5292. Jonasson, L., J. Holm, 0. Skalli, G. Bondjers and C.K. Hansson, 198h, Regional accumulations of T cells, macrophages. and smooth muscle cells in the human atherosclerotic plaque, Arteriosclerosis 6, 131. Jonasson. Lx. J. Holm and G.K. Hansson, 1988. Cyclosporin A inhibits smooth mucle proliferaiiun in the vascular response to injury, Proc. Natl. Acad. Sci. U1S.A. 8.5, 2X03. Mukhopadhyay. A., N. Basu. N. Ghatak and P.K. Gujzal. 19X2, Anti-inflammatory and irritant activities of curcumin analogues in rats, Agents Actions 12. 508. Lundergan. C.F.. M.L. Foegh and P.W. Ramwell, 1991. Peptide inhibition of myoi&mal proliferation by angiopeptin. a somatostatin analogue, J. Am. Coil. Cardioi. 17. l32B. Ross, R. and J.A. Glomset. 1976. The pathr~~enesisof atherosclerosis. N. Engl. J. Med. 29S, 369. Srimal, R.C. and B.N. Dharvan. 1973, Pharmacology of curcumin. A non-st~ro~dal and anti-in~ammato~ agents, J. Pharm. Pharmacol. 25, 447.
