Huei-Chen Huang

Effect of curcumin on cell cycle progression and apoptosis in vascular smooth muscle cells

1 The possible mechanisms of the antiproliferative and apoptotic effects of curcumin (diferuloylmethane), a polyphenol in the spice turmeric, on vascular smooth muscle cells were studied in rat aortic smooth muscle cell line (A7r5). 2 The proliferative response was determined from the uptake of [3H]‐thymidine. Curcumin (10−6–10−4 m) inhibited serum‐stimulated [3H]‐thymidine incorporation of both A7r5 cells and rabbit cultured vascular smooth muscle cells in a concentration‐dependent manner. Cell viability, as determined by the trypan blue dye exclusion method, was unaffected by curcumin at the concentration range 10−6 to 10−5 m in A7r5 cells. However, the number of viable cells after 10−4 m curcumin treatment was less than the basal value (2×105 cells). 3 To analyse the various stages of the cell cycle, [3H]‐thymidine incorporation into DNA was determined every 3 h. After stimulation with foetal calf serum, quiescent A7r5 cells started DNA synthesis in 9 to 12 h (G1/S phase), then reached a maximum at 15 to 18 h (S phase). Curcumin (10−6–10−4 m) added during either the G1/S phase or S phase significantly inhibited [3H]‐thymidine incorporation. 4 Following curcumin (10−6–10−4 m) treatment, cell cycle analysis utilizing flow cytometry of propidium iodide stained cells revealed a G0/G1 arrest and a reduction in the percentage of cells in S phase. Curcumin at 10−4 m also induced cell apoptosis. It is suggested that curcumin arrested cell proliferation and induced cell apoptosis, and hence reduced the [3H]‐thymidine incorporation. 5 The apoptotic effect of 10−4 m curcumin was also demonstrated by haematoxylin‐eosin staining, TdT‐mediated dUTP nick end labelling (TUNEL), and DNA laddering. Curcumin (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 curcumin at the concentration range 10−5 to 10−4 m. On the other hand, the cytosolic protein kinase C activity stimulated by phorbol ester 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 analysed using a reverse transcription‐polymerase chain reaction (RT‐PCR) technique. The level of c‐myc mRNA was significantly reduced by curcumin (10−5–10−4 m) treatment. And, the level of bcl‐2 mRNA was significantly reduced by 10−4 m curcumin. However, the alteration of the p53 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 demonstrate that curcumin inhibited cell proliferation, arrested the cell cycle progression and induced cell apoptosis in vascular smooth muscle cells. Curcumin may be useful as a template for the development of drugs to prevent the pathological changes of atherosclerosis and post‐angioplasty restenosis. Our results suggest that the antiproliferative effect of curcumin may partly be mediated through inhibition of protein tyrosine kinase activity and c‐myc mRNA expression. And, the apoptotic effect may partly be mediated through inhibition of protein tyrosine kinase activity, protein kinase C activity, c‐myc mRNA expression and bcl‐2 mRNA expression.

VASORELAXANTS FROM CHINESE HERBS, EMODIN AND SCOPARONE, POSSESS IMMUNOSUPPRESSIVE PROPERTIES

Emodin and scoparone, the active principles isolated from Polygonum multiflorum and Artemisia scoparia, respectively, both exhibit vasorelaxant and immunosuppressive effects. Emodin (10(-6)-3 x 10(-5) M) and scoparone (10(-6)-3 x 10(-5) M) dose dependently relaxed rat thoracic aortic rings precontracted with phenylephrine. Emodin (3 x 10(-7)-10(-4) M) and scoparone (10(-6)-3 x 10(-4) M) also dose dependently suppressed the responses of human mononuclear cells to phytohemagglutinin and mixed lymphocyte reaction. These compounds may be useful as new templates for the development of better immunosuppressive agents with vasorelaxant actions for use against transplantation rejection and autoimmune disease.

Immunosuppressive effect of emodin, a free radical generator

The possible mechanism of immunosuppressive effect of emodin (1,3,8-trihydroxy-6-methylanthraquinone) was investigated in this study. Human mononuclear cells (10(6) cells/ml) were stimulated with 0.25% phytohemagglutinin for 24, 48 and 72 h, and the proliferative response was determined by the uptake of tritiated thymidine. In the presence of emodin (10(-6) to 3 x 10(-5) M), the proliferative response was reduced in a dose-dependent manner. Emodin (3 x 10(-7) to 3 x 10(-5) M) also dose dependently reduced the proliferative response to mixed lymphocyte reaction. After 72 h exposure to emodin (10 microM), interleukin-1 (IL-1), interleukin-2 (IL-2) production and IL-2 receptor expression were all reduced. The structure-activity relationship of emodin and 10 other anthraquione derivatives indicates that the free hydroxyl group at the beta-position of the anthraquinone nucleus plays an important role in the immunosuppressive effect. The suppressive activity of emodin was significantly inhibited by catalase (a scavenger of hydrogen peroxide), but little affected by superoxide dismutase (a scavenger of superoxide radical) and mannitol (a scavenger of hydroxyl radical). Methylene blue and hemoglobin, guanylate cyclase inhibitors, did not significantly affect the suppressive activity of emodin. Nordihydroguaiaretic acid (a lipoxygenase inhibitor) significantly potentiated the suppressive activity whereas quinacrine (a phospholipase A2 inhibitor) and indomethacin (a cyclooxygenase inhibitor) did not significantly affect it. The results suggest that the immunosuppressive effect of emodin may be partly mediated through hydrogen peroxide generated from semiquinone and regulated by arachidonic acid metabolites or byproducts.

Antiproliferative effect of baicalein, a flavonoid from a Chinese herb, on vascular smooth muscle cell.

The effects of baicalein, baicalin and wogonin, the flavonoids from Scutellaria baicalensis, on the proliferative responses of cultured rabbit vascular smooth muscle cells were studied. The proliferative response was determined from the uptake of tritiated thymidine. In rabbit vascular smooth muscle cells, all three flavonoids dose dependently inhibited the proliferative response induced by 5% fetal calf serum at the dose range of 10(-6) to 10(-4) M. Baicalin and wogonin were less effective than baicalein as inhibitors of the serum-induced smooth muscle cell proliferation, indicating that the three hydroxyl groups on positions 5, 6 and 7 seem to be necessary and sufficient for full inhibitory activity against the proliferative response of smooth muscle cells. Baicalein had a greater inhibitory effect on the proliferative reponse stimulated by platelet-derived growth factor than on serum-stimulated proliferation. Baicalein, a flavonoid with antiproliferative and lipoxygenase-inhibitory activities, may be useful as another template for the development of better drugs to prevent the pathological changes of atherosclerosis and restenosis.

The possible mechanisms of the antiproliferative effect of fullerenol, polyhydroxylated C60, on vascular smooth muscle cells

1 The possible mechanisms of the antiproliferative effect of polyhydroxylated fullerene (fullerenol), a novel free radical trapper, were studied in rat vascular smooth muscle cells (A7r5 cells) and compared with the effect of ascorbic acid. 2 Fullerenol‐1 and ascorbic acid inhibited the proliferative responses in a number of cells, including rat aortic smooth muscle cells (A7r5 cells), human coronary artery smooth muscle cells, and human CEM lymphocytes (CEM cells) in a concentration dependent manner. 3 At the concentration range of 10−6 to 10−2 M, fullerenol‐1 and ascorbic acid concentration‐dependently inhibited the proliferative responses stimulated by serum in A7r5 cells. Fullerenol‐1 was more potent than ascorbic acid. 4 The production of O2− induced by alloxan, a diabetogenic compound, was reduced by fullerenol‐1 (10−4 M) in the presence of A7r5 cells. 5 The cytosolic protein kinase C activity of A7r5 cells stimulated by phorbol ester was reduced by 10−3 M fullerenol‐1, but not ascorbic acid (10−4–10−2 M) and fullerenol‐1 at lower concentrations (10−6–10−4 M). 6 In contrast, the membraneous protein tyrosine kinase activity of A7r5 cells stimulated by foetal calf serum was significantly reduced by fullerenol‐1 (10−6–10−3 M) and ascorbic acid (10−4–10−2 M). Again, the inhibitory activity of fullerenol‐1 was greater than that of ascorbic acid. 7 Our results demonstrate that fullerenol‐1 and ascorbic acid exhibit inhibitory effects on transduction signals in addition to their antioxidative property. It is suggested that the antiproliferative effect of fullerenol‐1 on vascular smooth muscle cells may partly be mediated through the inhibition of protein tyrosine kinase.

Effects of baicalein and esculetin on transduction signals and growth factors expression in T-lymphoid leukemia cells.

The possible mechanisms of antiproliferative effect of baicalein were studied in human T-lymphoid leukemia cells (CEM cells) and compared with those of esculetin. Baicalein, esculetin and related compounds, baicalein, wogonin, esculin and scoparone, inhibited CEM cell proliferation. Baicalein exhibited the greatest antiproliferative activity with an IC50 of 4.7 +/- 0.5 microM and the maximal suppression of 91.5 +/- 1.4% in CEM cells. The protein tyrosine kinase activity in the CEM cells was significantly reduced by baicalein (10(-6)-10(-4) M) and esculetin (10(-4) M). Baicalein exhibited a greater inhibitory activity on the protein tyrosine kinase than did esculetin (74.1 +/- 3.3% vs. 64.6 +/- 2.8% inhibition at 10(-4) M). On the other hand, the protein kinase C activity stimulated by phorbol-12-myristate 13-acetate was reduced by directly incubating with baicalein (10(-6)-10(-4) M) and esculetin (10(-4) M). However, the inhibitory activities on protein kinase C did not show a dose-dependency. The reverse transcription-polymerase chain reaction analysis of platelet-derived growth factor-A (PDGF-A) and transforming growth factor-beta 1 (TGF-beta 1) messenger RNA levels demonstrates that baicalein and esculetin reduced the PDGF-A mRNA level, but less affected the TGF-beta 1 mRNA. Baicalein exhibited the greater reduction on the expression of PDGF-A mRNA than did esculetin. It is suggested that baicalein and esculetin may affect cell proliferation by direct inhibition of growth-related signal, protein tyrosine kinase, as well as reduction of mRNA expression of growth factor, platelet-derived growth factor.

Effect of saikosaponin, a triterpene saponin, on apoptosis in lymphocytes: association with c-myc, p53, and bcl-2 mRNA

The mechanisms involved in the apoptotic effect of saikosaponin‐d, a triterpene saponin from Bupleurum falcatum L., were studied in human CEM lymphocytes and compared with those of dexamethasone (3×10−7 M). Saikosaponin‐d (10−8 to 10−5 M) inhibited the serum‐stimulated [3H]‐thymidine incorporation in a concentration‐dependent manner. Dexamethasone also inhibited serum‐stimulated [3H]‐thymidine incorporation. Cell viability was unaffected by saikosaponin‐d until 10−5–10−4 M. Dexamethasone significantly reduced the number of viable cells. Following saikosaponin‐d (10−5–10−4 M) treatment, flow cytometry analysis of propidium iodide‐stained cells showed a significant increase in the percentage of cells in the apoptotic region. Dexamethasone also significantly increased the percentage of apoptotic cells. The supravital exposure to propidium iodide and annexin V labelling demonstrated that saikosaponin‐d (10−5–10−4 M) induced apoptosis as well as necrosis. The apoptotic effect of saikosaponin‐d (3×10−6–10−4 M) was also demonstrated by TUNEL analysis and DNA laddering. The percentage of apoptotic cells induced by saikosaponin‐d (3×10−6–10−5 M) was unaffected by the presence of Z‐VAD‐FMK, indicating that saikosaponin‐d‐induced apoptosis may not be mediated by caspase activity. However, the percentage of apoptotic cells induced by dexamethasone was significantly reduced by the presence of Z‐VAD‐FMK. Levels of c‐myc, p53, and bcl‐2 mRNA were analysed by the reverse transcription‐polymerase chain reaction. Levels of c‐myc and p53 mRNA were significantly increased, while the level of bcl‐2 mRNA was decreased, by saikosaponin‐d (10−5 M) treatment. Dexamethasone did not significantly change the expression of these genes. It is suggested that the apoptotic effect of saikosaponin‐d may be partly mediated by increases in c‐myc and p53 mRNA levels accompanied by a decrease in bcl‐2 mRNA level.

Antiproliferative effect of esculetin on vascular smooth muscle cells: possible roles of signal transduction pathways

The effect of esculetin, a coumarin derivative with lipoxygenase inhibitor activity, on the proliferation response of cultured rabbit vascular smooth muscle cells was studied. Proliferation response was determined by the uptake of tritiated thymidine. Esculetin (10(-5)-10(-4) M) dose dependently inhibited the enhanced proliferation stimulated by 5% fetal calf serum. The structure-activity relationship of esculetin and eight other coumarin derivatives indicates that two adjacent phenolic hydroxyl groups at the C-6 and C-7 positions in the coumarin skeleton are necessary for the potent antiproliferative effect. The antiproliferative effects of other lipoxygenase inhibitors, 5,8,11,14-eicosatetraynoic acid (ETYA) and ketoconazole, were comparable to the effect of esculetin. However, esculetin exhibited the greatest maximal suppression. The enhanced releases of 12-hydroxyeicosatetraenoic acid (12-HETE), prostaglandin E2 and 6-keto-prostaglandin F1 alpha in the culture medium of smooth muscle cells stimulated by 5% fetal calf serum were significantly reduced by esculetin. Furthermore, the fetal calf serum-stimulated protein tyrosine kinase activity was reduced by esculetin (10(-5)-10(-4) M) in a dose-dependent manner. In contrast, the protein kinase C activity stimulated by phorbol-12-myristate-13-acetate was not affected by esculetin (10(-6)-10(-4) M). These results suggest that the antiproliferative effect of esculetin on vascular smooth muscle cells may be partly mediated through inhibition of protein tyrosine kinase and modulated by inhibition of lipoxygenase.

Possible mechanism of immunosuppressive effect of scoparone (6,7-dimethoxycoumarin).

The possible mechanism of the immunosuppressive effect of scoparone (6,7-dimethoxycoumarin) was investigated. Human peripheral blood mononuclear cells (10(6) cells/ml) were stimulated with 0.25% phytohemagglutinin (PHA) and the proliferative response was determined from the uptake of tritiated thymidine. Scoparone (10(-6) to 3 x 10(-4) M) reduced the proliferative response in a dose-dependent manner. The proliferative response of mononuclear cells to mixed lymphocyte reaction was also reduced by scoparone (10(-5) to 10(-4) M). Interleukin-1, interleukin-2 production and interleukin-2 receptor expression were all reduced in the presence of scoparone. Scoparone (10 and 30 microM) significantly reduced the suppression elicited by the diabetogenic drug, alloxan (10 mM). The suppressive activity of scoparone was significantly reduced by quinacrine (a phospholipase A2 inhibitor), indomethacin (a cyclooxygenase inhibitor) and nordihydroguaiaretic acid (a lipoxygenase inhibitor). The levels of prostaglandin E2, prostaglandin F2 alpha, leukotriene B4 and 2,3-dinor-thromboxane B2 in culture medium of PHA-stimulated mononuclear cells, measured with an enzyme immunoassay, were elevated by scoparone treatment. We compared the effect of scoparone on the mononuclear cell response to genistein, a specific inhibitor of protein tyrosine kinase and demonstrated the non-additivity and cross-desensitization of the two compounds. Our results suggest that the immunosuppressive effect of scoparone may be exerted in part through inhibition of protein tyrosine kinase and release of arachidonic acid metabolites.

Relaxant effect of phospholipase A2 from Vipera russelli snake venom on rat aorta

The effect of the acidic phospholipase A2 (PLA2) from Vipera russelli venom on the rat aortic ring was studied and compared with that of acetylcholine (ACh). PLA2 induced relaxation of the aortic ring precontracted with noradrenaline (NA) in a dose-dependent manner. Removal of the endothelium did not reduce the relaxant effect of PLA2. Replacement of Ca2+ by Sr2+ in the medium to inhibit the PLA2 enzyme activity reduced the relaxant effect. Atropine, a muscarinic receptor antagonist, did not affect the relaxant response. The cyclooxygenase inhibitor indomethacin, when equilibrated for 50 min, potentiated the relaxation. The lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) partially reduced the relaxation. This relaxation was also partially reduced by the guanylate cyclase inhibitor methylene blue. In contrast, the relaxation elicited by ACh was abolished by de-endothelialization, atropine, NDGA or methylene blue. 6-keto-PGF1 alpha (degradation product of prostacyclin) and PGE2 produced by aortic rings were measured by radioimmunoassay. PLA2 (3 X 10(-6) g/ml) increased the output of 6-keto-PGF1 alpha about 10-fold. The production of PGE2 was also increased but to a lesser extent. ACh also increased the output of 6-keto-PGF1 alpha and PGE2. However, prostacyclin released by PLA2 and ACh appears not to contribute to the relaxant effect, since prostacyclin does not relax the rat aorta. It is concluded that the relaxation elicited by PLA2 in the rat aorta is endothelium-independent and partially mediated by lipoxygenase product(s) and cyclic GMP whereas the relaxation induced by ACh was endothelium-dependent, mediated by lipoxygenase product(s) and cyclic GMP, and blocked by atropine.