IL-32γ enhances TNF-α-induced cell death in colon cancer.

Interleukin (IL)-32 is a recently discovered cytokine that appears to play an important role in human colon cancer growth. We investigated that IL-32γ in combination with TNF-α remarkably inhibited cell growth of human colon cancer cells (HCT116 and SW620) and tumor growth in xenograft-bearing nude mice. The transient enforced overexpression of IL-32γ potentiated the inhibitory effect of TNF-α on DNA synthesis, cell number and protein content, and enhanced apoptosis in colon cancer cells. We also found that knockdown of IL-32γ by siRNA showed the abolishment of cell growth inhibitory effect of TNF-α. The IL-32γ-overexpressing colon cancer cells further increased TNF-α-mediated expression of p38 MAPK as well as that of Bax, cleaved caspase-3 and -9, but decreased that of antiapoptotic proteins such as Bcl-2, cellular inhibitor of apoptosis protein (IAP) and X chromosome IAP. In xenograft model, the lipopolysaccharide (LPS)-injected (1.25 mg/kg) mice inoculated with IL-32γ-transfected HCT116 colon cancer cells were more decrease tumor volume and weight than inoculated with vector. Tumor tissues isolated from LPS-injected mice inoculated with IL-32γ-overexpressing colon cancer cells potentiated the expression levels of pro-apoptotic proteins such as cleaved caspase-3, 9 and Bax, but decreased that of Bcl-2. Furthermore, the mice increased IL-10 production, but decreased IL-6 levels in serum. In conclusion, our results suggest that IL-32γ may potentiate TNF-α-induced cell growth inhibition through activation of p38 MAPK pathways.

Camptothecin inhibits platelet-derived growth factor-BB-induced proliferation of rat aortic vascular smooth muscle cells through inhibition of PI3K/Akt signaling pathway.

The abnormal proliferation of vascular smooth muscle cells (VSMCs) in arterial wall is a major cause of vascular disorders such as atherosclerosis and restenosis after angioplasty. In this study, we investigated not only the inhibitory effects of camptothecin (CPT) on PDGF-BB-induced VSMC proliferation, but also its molecular mechanism of this inhibition. CPT significantly inhibited proliferation with IC50 value of 0.58 µM and the DNA synthesis of PDGF-BB-stimulated VSMCs in a dose-dependent manner (0.5-2 µM ) without any cytotoxicity. CPT induced the cell cycle arrest at G0/G1 phase. Also, CPT decreased the expressions of G0/G1-specific regulatory proteins including cyclin-dependent kinase (CDK)2, cyclin D1 and PCNA in PDGF-BB-stimulated VSMCs. Pre-incubation of VSMCs with CPT significantly inhibited PDGF-BB-induced Akt activation, whereas CPT did not affect PDGF-receptor beta phosphorylation, extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and phospholipase C (PLC)-γ1 phosphorylation in PDGF-BB signaling pathway. Our data showed that CPT pre-treatment inhibited VSMC proliferation, and that the inhibitory effect of CPT was enhanced by LY294002, a PI3K inhibitor, on PDGF-BB-induced VSMC proliferation. In addition, inhibiting the PI3K/Akt pathway by LY294002 significantly enhanced the suppression of PCNA expression and Akt activation by CPT. These results suggest that the anti-proliferative activity of CPT is mediated in part by downregulating the PI3K/Akt signaling pathway.

Hinokitiol inhibits cell growth through induction of S-phase arrest and apoptosis in human colon cancer cells and suppresses tumor growth in a mouse xenograft experiment.

Hinokitiol (1), a tropolone-related natural compound, induces apoptosis and has anti-inflammatory, antioxidant, and antitumor activities. In this study, the inhibitory effects of 1 were investigated on human colon cancer cell growth and tumor formation of xenograft mice. HCT-116 and SW-620 cells derived from human colon cancers were found to be similarly susceptible to 1, with IC50 values of 4.5 and 4.4 µM, respectively. Compound 1 induced S-phase arrest in the cell cycle progression and decreased the expression levels of cyclin A, cyclin E, and Cdk2. Conversely, 1 increased the expression of p21, a Cdk inhibitor. Compound 1 decreased Bcl-2 expression and increased the expression of Bax, and cleaved caspase-9 and -3. The effect of 1 on tumor formation when administered orally was evaluated in male BALB/c-nude mice implanted intradermally separately with HCT-116 and SW-620 cells. Tumor volumes and tumor weights in the mice treated with 1 (100 mg/kg) were decreased in both cases. These results suggest that the suppression of tumor formation by compound 1 in human colon cancer may occur through cell cycle arrest and apoptosis.

Ethanol extract of Magnolia officinalis prevents lipopolysaccharide-induced memory deficiency via its antineuroinflammatory and antiamyloidogenic effects.

Magnolia bark contains several compounds such as magnolol, honokiol, 4-O-methylhonokiol, obovatol, and other neolignan compounds. These compounds have been reported to have various beneficial effects in various diseases. There is sufficient possibility that ethanol extract of Magnolia officinalis is more effective in amyloidogenesis via synergism of these ingredients. Neuroinflammation has been known to play a critical role in the pathogenesis of Alzheimer's disease (AD). We investigated whether the ethanol extract of M. officinalis (10 mg/ kg in 0.05% ethanol) prevents memory dysfunction and amyloidogenesis in AD mouse model by intraperitoneal lipopolysaccharide (LPS, 250 µg/ kg/day for seven times) injection. We found that ethanol extract of M. officinalis prevented LPS-induced memory deficiency as well as inhibited the LPS-induced elevation of inflammatory proteins, such as inducible nitric oxide synthase and cyclooxygenase 2, and activation of astrocytes and microglia. In particular, administration of M. officinalis ethanol extract inhibited LPS-induced amyloidogenesis, which resulted in the inhibition of amyloid precursor protein, beta-site amyloid-precursor-protein-cleaving enzyme 1 and C99. Thus, this study shows that ethanol extract of M. officinalis prevents LPS-induced memory impairment as well as amyloidogenesis via inhibition of neuroinflammation and suggests that ethanol extract of M. officinalis might be a useful intervention for neuroinflammation-associated diseases such as AD.

Piperlongumine inhibits atherosclerotic plaque formation and vascular smooth muscle cell proliferation by suppressing PDGF receptor signaling.

Piperlongumine (piplartine, PL) is an alkaloid found in the long pepper (Piper longum L.) and has well-documented anti-platelet aggregation, anti-inflammatory, and anti-cancer properties; however, the role of PL in prevention of atherosclerosis is unknown. We evaluated the anti-atherosclerotic potential of PL in an in vivo murine model of accelerated atherosclerosis and defined its mechanism of action in aortic vascular smooth muscle cells (VSMCs) in vitro. Local treatment with PL significantly reduced atherosclerotic plaque formation as well as proliferation and nuclear factor-kappa B (NF-κB) activation in an in vivo setting. PL treatment in VSMCs in vitro showed inhibition of migration and platelet-derived growth factor BB (PDGF-BB)-induced proliferation to the in vivo findings. We further identified that PL inhibited PDGF-BB-induced PDGF receptor beta activation and suppressed downstream signaling molecules such as phospholipase Cγ1, extracellular signal-regulated kinases 1 and 2 and Akt. Lastly, PL significantly attenuated activation of NF-κB-a downstream transcriptional regulator in PDGF receptor signaling, in response to PDGF-BB stimulation. In conclusion, our findings demonstrate a novel, therapeutic mechanism by which PL suppresses atherosclerosis plaque formation in vivo.

Development and diagnostic application/evaluation of pandemic (H1N1) 2009 influenza virus-specific monoclonal antibodies.

We prepared mAb specific to the H1N1 2009 virus (H1N1 2009) to facilitate development of an RDT with enhanced sensitivity and specificity. Among these antibodies, we identified two clones--hybridomas 1H7E1 and 3A3H7-that specifically bound to H1N1 2009 (non-seasonal) and were very suitable for application to a diagnostic kit. The affinity constants (K(a)) of 1H7E1 and 3A3H7 were 1.10 × 10(10) and 2.35 × 10(10), respectively. To identify the antibodies, we performed ELISA and immunoblot analyses and found that 1H7E1 recognized a conformational epitope of HA while 3A3H7 recognized a linear epitope. In clinical evaluations using specimens from 215 patients, a lateral flow rapid testing kit comprising these mAb showed a sensitivity of 81.5% (75/92) and a specificity of 96.7% (119/123). Results using the RDT kit were well correlated with conventional RT-PCR methods as commonly and commercially used. Based on our findings, we believe that use of these mAb with a rapid evaluation kit could serve as a good diagnostic tool for H1N1 2009.

Clitocybin A, a novel isoindolinone, from the mushroom Clitocybe aurantiaca, inhibits cell proliferation through G1 phase arrest by regulating the PI3K/Akt cascade in vascular smooth muscle cells.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) plays an essential role in the pathogenesis of vascular diseases, such as atherosclerosis, hypertension, and restenosis. Clitocybin A, a novel isoindolinone, isolated from the culture broth of mushroom Clitocybe aurantiaca has been reported to possess free radical scavenging activity. However, the antiproliferative effects of clitocybin A on VSMCs are unknown. In the present study, we investigated the effect of clitocybin A on platelet-derived growth factor (PDGF)-BB-induced proliferation of VSMCs and examined the molecular basis of the underlying mechanism. Clitocybin A inhibited DNA synthesis and cell proliferation. In accordance with these findings, clitocybin A blocked the PDGF-BB-inducible progression through G0/G1 to S phase of the cell cycle in synchronized cells and decreased the expression of cyclin-dependent kinase (CDK) 2, CDK4, cyclin D1, cyclin E, and proliferative cell nuclear antigen. In addition, clitocybin A inhibited the PDGF-BB-induced phosphorylation of phosphatidylinositol 3 kinase (PI3K) / Akt kinase. However, clitocybin A did not change the expression levels of extracellular signal-related kinase (ERK) 1/2, phospholipase C-γ1, and PDGF-Rß phosphorylation. These results indicate that clitocybin A may inhibit VSMCs proliferation through G1 phase arrest by regulating the PI3K/Akt pathway.

Ceramide 1-phosphate induces neointimal formation via cell proliferation and cell cycle progression upstream of ERK1/2 in vascular smooth muscle cells.

Ceramide 1-phosphate (C1P) is a novel bioactive sphingolipid formed by ceramide kinase (CERK)-catalyzed phosphorylation of ceramide. It has been implicated in the regulation of such vital pathophysiological functions as phagocytosis and inflammation, but there have been no reports ascribing a biological function to CERK in vascular disorders. Here the potential role of CERK/C1P in neointimal formation was investigated using rat aortic vascular smooth muscle cells (VSMCs) in primary culture and a rat carotid injury model. Exogenous C8-C1P stimulated cell proliferation, DNA synthesis, and cell cycle progression of rat aortic VSMCs in primary culture. In addition, wild-type CERK-transfected rat aortic VSMCs induced a marked increase in rat aortic VSMC proliferation and [(3)H]-thymidine incorporation when compared to empty vector transfectant. C8-C1P markedly activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) within 5min, and the activation could be prevented by U0126, a MEK inhibitor. Also, K1, a CERK inhibitor, decreased the ERK1/2 phosphorylation and cell proliferation on platelet-derived growth factor (PDGF)-stimulated rat aortic VSMCs. CERK expression and C1P levels were found to be potently increased during neointimal formation using a rat carotid injury model. However, ceramide levels decreased during the neointimal formation process. These findings suggest that C1P can induce neointimal formation via cell proliferation through the regulation of the ERK1/2 protein in rat aortic VSMCs and that CERK/C1P may regulate VSMC proliferation as an important pathogenic marker in the development of cardiovascular disorders.

JJK694, a synthesized obovatol derivative, inhibits platelet activation by suppressing cyclooxygenase and lipoxygenase activities.

Obovatol has various biological activities, including anti-proliferative, neurotrophic, anti-fibrillogenic, anti-platelet, anti-fungal and anti-inflammatory activities. In this study, we investigated the effects of JJK694, a synthesized obovatol derivative, on rabbit platelet activation and its molecular mechanisms. JJK694 significantly inhibited washed rabbit platelet aggregation and serotonin secretion induced by collagen and arachidonic acid, but had little effect on thrombin- or U46619-induced aggregation. These results suggest that JJK694 selectively inhibits collagen- and arachidonic acid-mediated signaling. JJK694 also showed a concentration-dependent decrease in cytosolic Ca(2+) mobilization, but it had no effect on arachidonic acid liberation. On the other hand, it significantly inhibited the formation of arachidonic acid metabolites, including thromboxane A(2) (TXA(2)), prostaglandin D(2), and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), by suppression of cyclooxygenase (COX)-1 and lipoxygenase (LOX) activities. These results indicate that JJK694 hasanti-platelet activities through inhibition of arachidonic acid metabolite production by suppression of COX-1 and LOX activities.

Quantitative analysis of sphingomyelin by high-performance liquid chromatography after enzymatic hydrolysis.

Sphingomyelin is the most abundant sphingolipid in mammalian cells and is mostly present in the plasma membrane. A new analytical method using high-performance liquid chromatography (HPLC) was developed to quantify sphingomyelin in mouse plasma and tissues, 3T3-L1 cells, rat aortic smooth muscle cells, and HT-29 cells. Sphingomyelin and dihydrosphingomyelin, an internal standard, were separated by high-performance thin-layer chromatography and simultaneously hydrolyzed with sphingolipid ceramide N-deacylase and sphingomyelinase to release sphingosine and dihydrosphingosine, respectively. Sphingomyelin content was measured by HPLC following o-phthalaldehyde derivatization. Sphingomyelin concentrations in 3T3-L1 cells, rat aortic smooth muscle cells, and HT-29 cells were 60.10 ± 0.24, 62.69 ± 0.08, and 58.38 ± 0.37 pmol/µg protein, respectively, whereas those in brain, kidney, and liver of ICR mice were 55.60 ± 0.43, 43.75 ± 0.21, and 22.26 ± 0.14 pmol/µg protein. The sphingomyelin concentration in mouse plasma was 407.40 ± 0.31 µM. The limits of detection and quantification for sphingomyelin were 5 and 20 pmol, respectively, in the HPLC analysis with fluorescence detection. This sensitivity was sufficient for analyzing sphingomyelin in biological samples. In conclusion, this analytical method is a sensitive and specific technique for quantifying sphingomyelin and was successfully applied to diverse biological samples with excellent reproducibility.

Increases in blood pressure and heart rate induced by caffeine are inhibited by (-)-epigallocatechin-3-O-gallate: involvement of catecholamines.

In a previous experiment, (-)-epigallocatechin-3-O-gallate (EGCG) reduced caffeine-induced locomotor activity and stereotyped behaviors and inhibited caffeine-induced neuronal stimulant activity. This research was performed to give additional evidence that EGCG counteracts caffeine-induced stimulant effects in animals. EGCG inhibited caffeine-induced cardiovascular activation measures, such as arterial pressure and heart rate. In addition, the increases in the levels of adrenaline and noradrenaline in the blood induced by caffeine was reduced by EGCG. We suggest that EGCG may reduce caffeine-induced increases in blood pressure and heart rate and may decrease the levels of catecholamines in the blood. Therefore, EGCG counteracts caffeine-induced cardiovascular activity. The stimulant effects of caffeine should be reduced by the amount of EGCG in green tea.

Inhibitory effects of docetaxel on platelet-derived growth factor (PDGF)-BB-induced proliferation of vascular smooth muscle cells through blocking PDGF-receptor ß phosphorylation.

The abnormal proliferation of vascular smooth muscle cells (VSMCs) in arterial wall is an important pathogenic factor for vascular disorders such as atherosclerosis and restenosis after angioplasty. The present study was designed to investigate the inhibitory effects of docetaxel on VSMC proliferation, as well as the molecular mechanism of this inhibition. Docetaxel at 10, 20 and 40 µM significantly inhibited both the proliferation and the DNA synthesis of fetal bovine serum (FBS)- and platelet-derived growth factor (PDGF)-BB-stimulated VSMCs in a concentration-dependent manner. In accordance with these findings, docetaxel blocked the FBS- and PDGF-BB-induced progression of synchronized cells through the G0/G1 phase of the cell cycle. Docetaxel also decreased the expressions of cell cycle-related proteins, including cyclin-dependent kinase (CDK) 2, cyclin E, CDK4, cyclin D1, retinoblastoma protein, and proliferative cell nuclear antigen in PDGF-BB-stimulated VSMCs. Docetaxel significantly inhibited the phosphorylation of extracellular signal-regulated kinase 1/2, Akt, and phospholipase C-γ1, downstream molecule in the PDGF-BB signaling pathway. Docetaxel suppressed the phosphorylation of PDGF receptor (PDGF-R) ß, the upstream molecule in PDGF-BB signaling cascade, suggesting that the inhibitory effect of docetaxel on the proliferation of VSMCs may occur by blocking PDGF-Rß phosphorylation. Thus, docetaxel may be a potential antiproliferative agent for the treatment of atherosclerosis and angioplasty restenosis.[Supplementary Figures: available only at http://dx.doi.org/10.1254/jphs.10276FP].

Sauchinone attenuates oxidative stress-induced skeletal muscle myoblast damage through the down-regulation of ceramide.

We investigated the effects of sauchinone, isolated from the root of Saururus chinensis, on muscle disorders and the underlying mechanism of oxidative stress-induced C(2)C(12) skeletal muscle myoblast damage. To assess the protective effects of sauchinone on oxidative stress-induced C(2)C(12) skeletal muscle myoblasts, we measured the viability of the cells, showing that sauchinone pre-treatment significantly reduced the decreased cell viability after H(2)O(2) treatment. We also investigated the mechanism of this protective effect of sauchinone. In Western blot analysis, the heat shock protein (HSP)-70 level increased significantly in the sauchinone-pretreated myoblasts. We used high performance liquid chromatography (HPLC) to examine the level of endogenous ceramide after pre-treatment with sauchinone followed by exposure to H(2)O(2). While hydrogen peroxide increased the ceramide content to approximately 166.60±38.93% of the control level, pre-treatment with sauchinone inhibited this increase, maintaining the ceramide content at the control level. We demonstrated that sauchinone regulates intracellular HSP70 expression as well as ceramide levels to protect against oxidative stress-induced C(2)C(12) muscle myoblast damage. We suggest the potential benefits of herbal medicines in the treatment of oxidative stress-related muscle disorders.

Inverse relationship between adipocyte differentiation and ceramide level in 3T3-L1 cells.

Adipocyte differentiation has been a target in anti-obesity strategies and is known to be closely related to lipid metabolism. Ceramide, a major sphingolipid metabolite, has been implicated in differentiation. In this study, we investigated whether ceramide biosynthesis is related to adipogenesis in 3T3-L1 cells. Preadipocytes can be differentiated synchronously by a mixture of adipogenic inducers including 3-isobutyl-1-methylxanthine, dexamethasone and insulin. The number of lipid droplets and the triglyceride content, which are differentiation biomarkers, gradually increased during adipogenesis. Interestingly, ceramide and sphingosine contents in the differentiated cells were decreased compared to those in preadipocytes. When the preadipocytes were treated with an 3-isobutyl-1-methylxanthine- or dexamethasone- or insulin-deficient mixture of inducers, the cellular ceramide levels were significantly increased compared with those in cells treated with the complete set of inducers. When preadipocytes were treated with 0, 0.1 or 1 µg/ml insulin along with 3-isobutyl-1-methylxanthine and dexamethasone, the ceramide levels were decreased and the triglyceride content was increased in a concentration-dependent manner. When the cells were treated with epigallocatechin gallate, an adipocyte differentiation inhibitor, during adipogenesis, the ceramide levels of adipocytes were increased and the fat content was decreased. In conclusion, our findings demonstrate that cellular ceramide levels are inversely correlated with adipocyte differentiation.

Inhibitory effects of BST406, a newly synthesized benzylideneacetophenone derivative, on abnormal vascular smooth muscle cell proliferation.

Benzylideneacetophenone analogues are known to have several significant biological activities, including antiinflammatory, antitumor, antibacterial, antiviral, and gastric-protective activities. However, the antiproliferative effects of benzylideneacetophenone analogues on vascular smooth muscle cells (VSMCs) are unknown. The aim of this study was to elucidate the antiproliferative effects and molecular mechanism of BST406, a newly synthesized benzylideneacetophenone derivative, on platelet-derived growth factor (PDGF)-BB-stimulated rat aortic VSMCs. BST406 inhibited [(3)H]-thymidine incorporation into DNA in VSMCs following treatment with PDGFBB 25 ng/ml. PDGF-BB-stimulated DNA synthesis was significantly reduced. Moreover, pretreatment with BST406 (0-10microM) suppressed the proliferation of PDGF-BB-stimulated cells in a concentration-dependent manner. We also investigated the mechanism of the antiproliferative effects of BST406 in PDGF-BB-stimulated VSMCs. In Western blot analysis, PDGF-BB-stimulated (25 ng/ml) phospholipase-C (PLC)gamma1 and Akt phosphorylation was inhibited by BST406 (0-10microM). However, BST406 did not inhibit the PDGF-receptor beta-chain (PDGF-Rbeta) and extracellular-regulated kinase 1 and 2 (ERK1/2) phosphorylation induced by PDGF-BB. To confirm that the inhibitory effects of BST406 are mediated through the inhibition of PLCgamma1 or Akt, the effects of inhibitors on cell viability were examined. U73122 completely inhibited PDGF-BB-induced proliferation of VSMCs. However, LY294002 10microM had no significant effects on PDGF-BB-induced proliferation. These findings suggest that the inhibitory effects of BST406 on the proliferation of PDGF-BB-stimulated VSMCs are mediated by suppression of the PLCgamma1 signaling pathways. Our observations may explain, in part, the mechanistic basis for the prevention of cardiovascular disease (such as atherosclerosis and restenosis after coronary angioplasty) by BST406.

Green tea (-)-epigallocatechin-3-gallate inhibits beta-amyloid-induced cognitive dysfunction through modification of secretase activity via inhibition of ERK and NF-kappaB pathways in mice.

Alzheimer's disease (AD) is characterized by the extracellular deposition of beta-amyloid peptide (Abeta) in cerebral plaques. Abeta is derived from the beta-amyloid precursor protein (APP) by the enzymes alpha-, beta- and gamma-secretase. Compounds that enhance alpha-secretase, but inhibit beta- or gamma-secretase activity, have therapeutic potential in the treatment of AD. Green tea, or its major polyphenolic compound, has been shown to have neuroprotective effects. In this study, we investigated the possible effects of (-)-epigallocatechin-3-gallate (EGCG) on memory dysfunction caused by Abeta through the change of Abeta-induced secretase activities. Mice were pretreated with EGCG (1.5 or 3 mg/kg body weight in drinking water) for 3 wk before intracerebroventricular administration of 0.5 microg Abeta(1-42). EGCG dose-dependently reduced the Abeta(1-42)-induced memory dysfunction, which was evaluated using passive avoidance and water maze tests. Abeta(1-42) induced a decrease in brain alpha-secretase and increases in both brain beta- and gamma-secretase activities, which were reduced by EGCG. In the cortex and the hippocampus, expression of the metabolic products of the beta- and gamma-secretases from APP, C99, and Abeta also were dose-dependently suppressed by EGCG. Paralleled with the suppression of beta- and gamma-secretases by EGCG, we found that EGCG inhibited the activation of extracellular signal-regulated kinase and nuclear transcription factor-kappaB in the Abeta(1-42)-injected mouse brains. In addition, EGCG inhibited Abeta(1-42)-induced apoptotic neuronal cell death in the brain. To further test the ability of EGCG to affect memory, EGCG (3 mg/kg body weight) was administered in drinking water for 1 wk to genetically developed preseniline 2 (PS2) mutant AD mice. Compared with untreated mutant PS2 AD mice, treatment with EGCG enhanced memory function and brain alpha-secretase activity but reduced brain beta- and gamma-secretase activities as well as Abeta levels. Moreover, EGCG inhibited the fibrillization of Abeta in vitro with a half maximal inhibitory concentration of 7.5 mg/L. These studies suggest that EGCG may be a beneficial agent in the prevention of development or progression of AD.

Antiproliferative action of cudraflavone B, isolated from Cudrania tricuspidata, through the downregulation of pRb phosphorylation in aortic smooth muscle cell proliferation signaling.

Cudrania tricuspidata has been proposed to possess anti-inflammatory, antioxidant, hepatoprotective, and antitumor activities. Although cudraflavone B, isolated from the root bark of C. tricuspidata, has a variety of pharmacological effects, its effects on rat aortic smooth muscle cells (RASMCs) are unclear. In the present study, cudraflavone B was found to inhibit cell proliferation and DNA synthesis in cultured RASMCs. Pretreatment with cudraflavone B (0.1-4 microM) suppressed platelet-derived growth factor-BB (PDGF-BB)-stimulated cell number in a concentration-dependent manner. The inhibition percentages were 19.7%, 36.4%, 52.3%, and 99.1% at concentrations of 0.1, 1, 2, and 4 microM, respectively. Moreover, cudraflavone B inhibited [H]-thymidine incorporation into DNA in RASMCs in response to 25 ng/mL PDGF-BB. PDGF-BB-stimulated DNA synthesis was significantly reduced by 15.9%, 31.7%, 43.1%, and 78.2% at concentrations of 0.1, 1, 2, and 4 muM, respectively. Thus, cudraflavone B blocked the PDGF-BB-inducible progression through G0/G1 to S phase of the cell cycle in synchronized cells. Furthermore, PDGF-BB-induced phosphorylation of retinoblastoma protein (pRb), the hyperphosphorylation of which is a hallmark of the G1-S transition in the cell cycle, was significantly inhibited by cudraflavone B. Because pRb phosphorylation is regulated by cyclin-dependent kinases (CDKs), we investigated the expression of CDK2, CDK4, cyclin E, and cyclin D1 and the CDK inhibitors p21 and p27. Treatment with cudraflavone B downregulated the cyclins and CDKs and upregulated the expression of p21 and p27, a CDK inhibitor. These findings suggest that cudraflavone B inhibits RASMC proliferation via the induction of p21 and p27 expression and subsequent cell cycle arrest with reduction of pRb phosphorylation at the G1-S phase.

Hesperetin, a bioflavonoid, inhibits rat aortic vascular smooth muscle cells proliferation by arresting cell cycle.

Diet can be one of the most important factors that influence risks for cardiovascular diseases. Hesperetin, a flavonoid present in grapefruits and oranges, is one candidate that may benefit the cardiovascular system. In this study, we have investigated the effect of hesperetin on the platelet-derived growth factor (PDGF)-BB-induced proliferation of primary cultured rat aortic vascular smooth muscle cells (VSMCs). Hesperetin significantly inhibited 50 ng/ml PDGF-BB-induced rat aortic VSMCs proliferation and [(3)H]-thymidine incorporation into DNA at concentrations of 5, 25, 50, and 100 microM. In accordance with these findings, hesperetin revealed blocking of the PDGF-BB-inducible progression through G(0)/G(1) to S phase of the cell cycle in synchronized cells. Western blot showed that hesperetin inhibited not only phosphorylation of retinoblastoma protein (pRb) and expressions of cyclin A, cyclin D, cyclin E, cyclin-dependent kinase 2 (CDK2) as well as proliferating cell nuclear antigen (PCNA) protein, but also downregulation of cyclin-dependent kinase inhibitor (CKI) p27(kip1), while did not affect CKI p21(cip1), p16(INK4), p53, and CDK4 expressions as well as early signaling transductions such as PDGF beta-receptor, extracellular signal-regulated kinase (ERK) 1/2, Akt, p38, and JNK phosphorylation. These results suggest that hesperetin inhibits PDGF-BB-induced rat aortic VSMCs proliferation via G(0)/G(1) arrest in association with modulation of the expression or activation of cell-cycle regulatory proteins, which may contribute to the beneficial effect of grapefruits and oranges on cardiovascular system.

Enhanced release of sphingosine-1-phosphate from hypercholesterolemic platelets: role in development of hypercholesterolemic atherosclerosis.

Although it is well known that sphingosine-1-phosphate (S1P), which induces many biological responses, is present in plasma and is mainly released from activated platelets, little is known whether the release of S1P is increased when platelets are activated in the hypercholesterolemic condition, and what are the roles of increased S1P generation in the development or progression of the atherosclerosis. Results show that 0.5% cholesterol diet for 16 weeks induces platelet hyperaggregability to low doses of agonists as well as development of hypercholesterolemic atherosclerosis in the rabbits. The generation and released level of S1P were significantly increased in the hypersensitized platelets and blood plasma in hypercholesterolemic rabbits. We also demonstrated that S1P increased VSMC proliferation via endothelial differentiation gene (EDG)-1 receptor dependent pathway. Our results indicate that release of S1P from activated platelets was increased by enhanced platelet sensitivity in hypercholesterolemia, which potentiated the ox-LDL-induced VSMC proliferation via EDG-1 receptor pathway.

YSK2821, a newly synthesized indoledione derivative, inhibits cell proliferation and cell cycle progression via the cell cycle-related proteins by regulating phosphatidylinositol-3 kinase cascade in vascular smooth muscle cells.

Indoledione derivatives have pronounced biological effects, i.e., cytotoxic activities against cancer cell lines and antifungal and antibacterial activities. The present study was designed to investigate the effects of YSK2821, a newly synthesized indoledione derivative, on platelet-derived growth factor (PDGF-BB)-induced vascular smooth muscle cell (VSMC) proliferation, as well as the molecular mechanisms of the anti-proliferative effects of YSK2821 in VSMCs. We found that YSK2821 caused the accumulation of cells in the G1 phase of the cell cycle and inhibited [3H]-thymidine incorporation. We demonstrated that YSK2821 remarkably decreased Akt kinase phosphorylation as the mechanism by which YSK2821 suppressed cell signal transduction events in VSMC proliferation. Furthermore, in terms of the effects of YSK2821 on cell cycle-related proteins, YSK2821 enhanced the expression of the cyclin-dependent protein kinase (CDK) inhibitor p27 and down-regulated CDK2 and cyclin E expression, but did not affect CDK4 and cyclin D1 expression. YSK2821 also inhibited the phosphorylation of Rb, a key regulator in the cell cycle. These results indicate that YSK2821, a newly synthesized indoledione derivative, may inhibit VSMC proliferation via a phosphatidylinositol (PI)-3 kinase-dependent pathway, and thus shed light on a novel role for YSK2821 as a potential preventive regulator of cardiovascular disease.