EFFECT OF CURCUMIN ON CELL-CYCLE PROGRESSION AND APOPTOSIS IN VASCULAR SMOOTH-MUSCLE CELLS

1 The possible mechanisms of the antiproliferative and apoptotic effec ts of curcumin (diferuloylmethane), a polyphenol in the spice turmeric , on vascular smooth muscle cells were studied in rat aortic smooth mu scle cell line (A7r5). 2 The proliferative response was determined fro m the uptake of [H-3]-thymidine. Curcumin (10(-6)-10(-4) M) inhibited serum-stimulated [H-3]-thymidine incorporation of both A7r5 cells and rabbit cultured vascular smooth muscle cells in a concentration-depend ent manner. Cell viability, as determined by the trypan blue dye exclu sion method, was unaffected by curcumin at the concentration range 10( -6) to 10(-5) M in A7r5 cells. However, the number of viable cells aft er 10(-4) M curcumin treatment was less than the basal value (2 x 10(5 ) cells).3 To analyse the various stages of the cell cycle, [H-3]-thym idine incorporation into DNA was determined every 3 h. After stimulati on with foetal calf serum, quiescent A7r5 cells started DNA synthesis in 9 to 12 h (G(1)/S phase), then reached a maximum at 15 to 18 h (S p hase). Curcumin (10(-6)-10(-4)) added during either the G(1)/S phase o r S phase significantly inhibited [H-3]-thymidine incorporation. 4 Fol lowing curcumin (10(-6)-10(-4) M) treatment, cell cycle analysis utili zing flow cytometry of propidium iodide stained cells revealed a G(0)/ G(1) arrest and a reduction in the percentage of cells in S phase. Cur cumin at 10(-4) M also induced cell apoptosis. It is suggested that cu rcumin arrested cell proliferation and induced cell apoptosis, and hen ce reduced the [H-3]-thymidine incorporation. 5 The apoptotic effect o f 10(-4) M curcumin was also demonstrated by haematoxylin-eosin staini ng, TdT-mediated dUTP nick end labelling (TUNEL), and DNA laddering. C urcumin (10(-4) M) induced cell shrinkage, chromatin condensation, and DNA fragmentation. 6 The membranous protein tyrosine kinase activity stimulated by serum in A7r5 cells was significantly reduced by curcumi n at the concentration range 10(-5) to 10(-4) M. On the other hand, th e cytosolic protein kinase C activity stimulated by phorbol eater was reduced by 10(-4) M curcumin, but unaffected by lower concentrations ( 10(-6)-10(-5) M). 7 The levels of c-myc, p53 and bcl-2 mRNA were analy sed using a reverse transcription-polymerase chain reaction (RT-PCR) t echnique. The level of c-myc mRNA was significantly reduced by curcumi n (10(-5)-10(-4) M) treatment. And, the level of bcl-2 mRNA was signif icantly reduced by 10(-4) M curcumin. However, the alteration of the p 53 mRNA level by curcumin (10(-5)-10(-4) M) treatment did not achieve significance. The effects of curcumin on the levels of c-myc and bcl-2 mRNA were then confirmed by Northern blotting. 8 Our results demonstr ate that curcumin inhibited cell proliferation, arrested the cell cycl e progression and induced cell apoptosis in vascular smooth muscle cel ls. Curcumin may be useful as a template for the development of drugs to prevent the pathological changes of atherosclerosis and post-angiop lasty restenosis. Our results suggest that the antiproliferative effec t of curcumin may partly be mediated through inhibition of protein tyr osine kinase activity and c-myc mRNA expression And, the apoptotic eff ect may partly be mediated through inhibition of protein tyrosine kina se activity, protein kinase C activity, c-myc mRNA expression and bcl- 2 mRNA expression.