Melatonin activates cell death programs for the suppression of uterine leiomyoma cell proliferation.

The circadian nature of melatonin has a protective effect on the progression of female reproductive cancers, including breast and ovarian cancers. However, the effect of melatonin on the growth of uterine leiomyoma is still unclear. In this study, we found that the growth of uterine leiomyoma ELT3 cells was reduced by treatment with melatonin. Treatment with melatonin increased the distribution of sub-G1 phase and increased DNA condensation in ELT3 cells. Melatonin-induced apoptosis and autophagy cell death progression were observed in ELT3 cells. Melatonin exerts a highly selective effect on primary normal human uterine smooth muscle (UtSMC) cells. The UtSMC cell cycle was arrested by melatonin treatment through up-regulation of p21, p27, and PTEN protein expression, but melatonin did not further promote apoptosis program activation. Melatonin reduced cell proliferation in ELT3 cells underlying the activation of melatonin MT1 and MT2 receptors, which in turn down-regulated the Akt-ERK1/2-NFκB signaling pathway. Melatonin reduced ELT3 tumor growth in both xenograft and orthotopic uterine tumor mice models. The extracellular matrix of the tumor was also reduced by melatonin treatment. Taken together, these results suggest that melatonin potentially plays a role in suppression of uterine leiomyoma growth.

Effects of female sex hormones on the development of atherosclerosis.

Atherosclerosis and associated pathologies, such as coronary artery disease, peripheral vascular disease, and stroke, are still the leading cause of death in Western society. The impact of female sex hormones on cardiovascular diseases has been studied intensively with conflicting findings. The controversy is mainly due to the differences in groups sampling, animal models used, hormonal treatment regimens, and the data analyzed. In the present article, the results of in vitro and in vivo studies and clinical trials are under review.

A Synergistic Anti-Cancer Effect of Troglitazone and Lovastatin in a Human Anaplastic Thyroid Cancer Cell Line and in a Mouse Xenograft Model.

Anaplastic thyroid cancer (ATC) is a malignant subtype of thyroid cancers and its mechanism of development remains inconclusive. Importantly, there is no effective strategy for treatment since ATC is not responsive to conventional therapies, including radioactive iodine therapy and thyroid-stimulating hormone suppression. Here, we report that a combinational approach consisting of drugs designed for targeting lipid metabolism, lovastatin (an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, HMGCR) and troglitazone (an agonist of peroxisome proliferator-activated receptor gamma, PPARγ), exhibits anti-proliferation in cell culture systems and leads to tumor regression in a mouse xenograft model. The composition contains a sub-lethal concentration of both drugs and exhibits low toxicity to certain types of normal cells. Our results support a hypothesis that the inhibitory effect of the combination is partly through a cell cycle arrest at G0/G1 phase, as evidenced by the induction of cyclin-dependent kinase inhibitors, p21cip and p27kip, and the reduction of hyperphosphorylated retinoblastoma protein (pp-Rb)-E2F1 signaling. Therefore, targeting two pathways involved in lipid metabolism may provide a new direction for treating ATC.

Reactivity Study of Unsymmetrical ß-Diketiminato Copper(I) Complexes: Effect of the Chelating Ring.

ß-Diketiminato copper(I) complexes play important roles in bioinspired catalytic chemistry and in applications to the materials industry. However, it has been observed that these complexes are very susceptible to disproportionation. Coordinating solvents or Lewis bases are typically used to prevent disproportionation and to block the coordination sites of the copper(I) center from further decomposition. Here, we incorporate this coordination protection directly into the molecule in order to increase the stability and reactivity of these complexes and to discover new copper(I) binding motifs. Here we describe the synthesis, structural characterization, and reactivity of a series of unsymmetrical N-aryl-N'-alkylpyridyl ß-diketiminato copper(I) complexes and discuss the structures and reactivity of these complexes with respect to the length of the pyridyl arm. All of the aforementioned unsymmetrical ß-diketiminato copper(I) complexes bind CO reversibly and are stable to disproportionation. The binding ability of CO and the rate of pyridyl ligand decoordination of these copper(I) complexes are directly related to the competition between the degree of puckering of the chelate system and the steric demands of the N-aryl substituent.

Simvastatin Inhibits Cell Proliferation and Migration in Human Anaplastic Thyroid Cancer.

Malignant human anaplastic thyroid cancer (ATC) is pertinacious to conventional therapies. The present study investigated the anti-cancer activity of simvastatin and its underlying regulatory mechanism in cultured ATC cells. Simvastatin (0-20 µM) concentration-dependently reduced cell viability and relative colony formation. Depletions of mevalonate (MEV) and geranylgeranyl pyrophosphate (GGpp) by simvastatin induced G1 arrest and increased apoptotic cell populations at the sub-G1 phase. Adding MEV and GGpp prevented the simvastatin-inhibited cell proliferation. Immunoblotting analysis illustrated that simvastatin diminished the activation of RhoA and Rac1 protein, and this effect was prevented by pre-treatment with MEV and GGpp. Simvastatin increased the levels of p21cip and p27kip proteins and reduced the levels of hyperphosphorylated-Rb, E2F1 and CCND1 proteins. Adding GGpp abolished the simvastatin-increased levels of p27kip protein, and the GGpp-caused effect was abolished by Skp2 inhibition. Introduction of Cyr61 siRNA into ATC cells prevented the epidermal growth factor (EGF)-enhanced cell migration. The EGF-induced increases of Cyr61 protein expression and cell migration were prevented by simvastatin. Taken together, these results suggest that simvastatin induced ATC proliferation inhibition through the deactivation of RhoA/Rac1 protein and overexpression of p21cip and p27kip, and migration inhibition through the abrogation of Cyr61 protein expression.

Involvement of cysteine-rich protein 61 in the epidermal growth factor-induced migration of human anaplastic thyroid cancer cells.

Anaplastic thyroid cancer (ATC) is among the most aggressive types of malignant cancer. Epidermal growth factor (EGF) plays a crucial role in the pathogenesis of ATC, and patients with thyroid carcinoma typically exhibit increased cysteine-rich protein 61 (Cyr61). In this study, we found that EGF treatment induced cell migration, stress fiber formation, Cyr61 mRNA and protein expressions, and Cyr61 protein secretion in ATC cells. The recombinant Cyr61 protein significantly induced cell migration; however, inhibition of Cyr61 activity by a Cyr61-specific antibody abrogated EGF-induced cell migration. EGF treatment also affected epithelial-to-mesenchymal transition (EMT)-related marker protein expression, as evidenced by an increase in vimentin and a decrease in E-cadherin expression. Inhibition of Cyr61 expression by Cyr61 siRNA decreased cell migration and reversed the EMT-related marker protein expression. EGF treatment increased the phosphorylation of the extracellular signal-regulated kinase (ERK) and cAMP response element-binding protein (CREB), and finally activated Cyr61 promoter plasmid activity. Our results suggest that Cyr61 is induced by EGF through the ERK/CREB signal pathway and that it plays a crucial role in the migration and invasion of ATC cells; moreover, Cyr61 might be a therapeutic target for metastatic ATC.

An integrated analysis of dysregulated SCD1 in human cancers and functional verification of miR-181a-5p/SCD1 axis in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC), one of the most lethal cancers, has become a global health issue. Stearoyl-coA desaturase 1 (SCD1) has been demonstrated to play a crucial role in human cancers. However, pan-cancer analysis has revealed little evidence to date. In the current study, we systematically inspected the expression patterns and potential clinical outcomes of SCD1 in multiple human cancers. SCD1 was dysregulated in several types of cancers, and its aberrant expression acted as a diagnostic biomarker, indicating that SCD1 may play a role in tumorigenesis. We used ESCC as an example to demonstrate that SCD1 was dramatically upregulated in tumor tissues of ESCC and was associated with clinicopathological characteristics in ESCC patients. Furthermore, Kaplan-Meier analysis showed that high SCD1 expression was correlated with poor progression-free survival (PFS) and disease-free survival (DFS) in ESCC patients. The protein-protein interaction (PPI) network and module analysis by PINA database and Gephi were performed to identify the hub targets. Meanwhile, the functional annotation analysis of these hubs was constructed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Functionally, the gain-of-function of SCD1 in ESCC cells promoted cell proliferation, migration, and invasion; in contrast, loss-of-function of SCD1 in ESCC cells had opposite effects. Bioinformatic, QPCR, Western blotting and luciferase assays indicated that SCD1 was a direct target of miR-181a-5p in ESCC cells. In addition, gain-of-function of miR-181a-5p in ESCC cells reduced the cell growth, migratory, and invasive abilities. Conversely, inhibition of miR-181a-5p expression by its inhibitor in ESCC cells had opposite biological effects. Importantly, reinforced SCD1 in miR-181a-5p mimic ESCC transfectants reversed miR-181a-5p mimic-prevented malignant phenotypes of ESCC cells. Taken together, these results indicate that SCD1 expression influences tumor progression in a variety of cancers, and the miR-181a-5p/SCD1 axis may be a potential therapeutic target for ESCC treatment.

Folic acid inhibits endothelial cell proliferation through activating the cSrc/ERK 2/NF-κB/p53 pathway mediated by folic acid receptor.

Folate is important for normal cell division. Folate deficiency has been implicated in various diseases, including atherosclerosis, neural tube defects, and cancer. However, the effect of folate on angiogenesis was unclear. The aim of this study was to investigate the anti-angiogenic action of folic acid (FA). FA (0-10 µmol/L) concentration-dependently decreased DNA synthesis and proliferation in cultured human umbilical venous endothelial cells (HUVEC). Western blot analyses demonstrated that the levels of p21, p27 and p53 protein in HUVEC were increased by FA. The FA-inhibited [3H]thymidine incorporation was completely blocked when the expressions of p21 and p27 were knocked-down together. Knock-down of p53 prevented the FA-induced increases in p21 and p27 protein level. The levels of phosphorylated Src (p-Src) and p-Src-FA receptor (FR) complex in HUVEC were increased by FA. Knock-down of FR reduced the FA-induced increases of p-Src and p53. The FA-induced increases of p21, p27 and p53 protein levels were abolished when cSrc was knocked-down. FA also increased NF-κB nuclear translocation and binding onto the p53 promoter. The FA-induced up-regulation of the p53 promoter activity was prevented by knocked-down of ERK. Matrigel angiogenesis assay in mice demonstrate the anti-angiogenic effect of FA in vivo. In conclusion, our data indicate that FA bound to FR in HUVEC, subsequently activated the cSrc/ERK 2/NF-κB/p53 signaling pathway, which in turn up-regulated the expression of p21 and p27, and finally resulted in cell cycle arrest at the G0/G1 phase. In the present study, we uncover a completely novel role of FA for anti-angiogenesis.

PEP-sNASP Peptide Alleviates LPS-Induced Acute Lung Injury Through the TLR4/TRAF6 Axis.

Acute lung injury (ALI) is a severe inflammatory lung disease associated with macrophages. Somatic nuclear autoantigenic sperm protein (sNASP) is a negative regulator of Toll-like receptor (TLR) signaling that targets tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) in macrophages, which is required to maintain homeostasis of the innate immune response. In the present study, we generated a cell permeable PEP-sNASP peptide using the sNASP protein N-terminal domain, and examined its potential therapeutic effect in a mouse model of ALI induced by the intranasal administration of lipopolysaccharide (LPS) and elucidated the underlying molecular mechanisms in RAW 264.7 cells. In vivo, PEP-sNASP peptide treatment markedly ameliorated pathological injury, reduced the wet/dry (W/D) weight ratio of the lungs and the production of proinflammatory cytokines (interleukin (IL)-1ß, IL-6, and TNF-α). In vitro, we demonstrated that when the PEP-sNASP peptide was transduced into RAW 264.7 cells, it bound to TRAF6, which markedly decreased LPS-induced proinflammatory cytokines by inhibiting TRAF6 autoubiquitination, nuclear factor (NF)-κB activation, reactive oxygen species (ROS) and cellular nitric oxide (NO) levels. Furthermore, the PEP-sNASP peptide also inhibited NLR family pyrin domain containing 3 (NLRP3) inflammasome activation. Our results therefore suggest that the PEP-sNASP may provide a potential protein therapy against oxidative stress and pulmonary inflammation via selective TRAF6 signaling.

Allopregnanolone suppresses glioblastoma survival through decreasing DPYSL3 and S100A11 expression.

Allopregnanolone (allo) is a physiological regulator of neuronal activity that treats multiple neurological disorders. Allo penetrates the blood-brain barrier with very high efficiency, implying that allo can treat CNS-related diseases, including glioblastoma (GBM), which always recurs after standard therapy. Hence, this study aimed to determine whether allo has a therapeutic effect on GBM. We found that allo enhanced temozolomide (TMZ)-suppressed cell survival and proliferation of TMZ-resistant cells. In particular, allo enhanced TMZ-inhibited cell migration and TMZ-induced apoptosis. Additionally, allo strongly induced DNA damage characterized by γH2Ax. Furthermore, quantitative proteomic analysis, iTRAQ, showed that allo significantly decreased the levels of DPYSL3, S100A11, and S100A4, reflecting the poor prognosis of patients with GBM confirmed by differential gene expression and survival analysis. Moreover, single-cell RNA-Seq revealed that S100A11, expressed in malignant cells, oligodendrocytes, and macrophages, was significantly associated with immune cell infiltration. Furthermore, overexpression of DPYSL3 or S100A11 prevented allo-induced cell death. In conclusion, allo suppresses GBM cell survival by decreasing DPYSL3/S100A11 expression and inducing DNA damage.

17ß-estradiol induces temozolomide resistance through NRF2-mediated redox homeostasis in glioblastoma.

Glioblastoma multiforme (GBM) is the most fatal cancer among brain tumors, and the standard treatment of GBM patients is surgical tumor resection followed by radiotherapy and temozolomide (TMZ) chemotherapy. However, tumors always recur due to the developing drug resistance. It has been shown that neurosteroids, including dehydroepiandrosterone and 17ß-estradiol, are synthesized in TMZ-resistant GBM tumors. Therefore, we sought to explore the possible role of 17ß-estradiol in the development of drug resistance in GBM. Bioinformatics analysis revealed that aromatase/cytochrome P450 19A1 expression was gradually increased in the development from normal, astrocytoma to GBM. The level of 17ß-estradiol was significantly increased in TMZ-resistant cells characterized by ultra performance liquid chromatography-tandem mass spectrometry. Furthermore, 17ß-estradiol attenuated TMZ-induced cell death and reduced reactive oxygen species production by mitochondria. In addition, 17ß-estradiol attenuated oxidative stress by increasing the expression of superoxide dismutase 1/2, catalase, and nuclear factor erythroid 2-related factor (NRF) 2. We found that NRF2 expression was essential for the induction of drug resistance by 17ß-estradiol through the reduction of oxidative stress in GBM.

Investigation of Anti-Tumor Effects of an MLK1 Inhibitor in Prostate and Pancreatic Cancers.

It was shown that mixed lineage kinase 1 (MLK1) regulates pancreatic cancer growth; however, its role in prostate cancer remains unclear. We showed that MLK1 is a tumor marker in prostate cancer by analyzing clinical gene expression data and identified a novel MLK1 inhibitor (NSC14465) from the compound library of the National Cancer Institute (NCI) using a MLK1 protein structure. The inhibitory effects of MLK1 were validated by an in vitro kinase assay and by monitoring phosphorylation signaling, and the anti-proliferation function was shown in several prostate and pancreatic cancer cell lines. We also demonstrated anti-tumor ability and prevention of cancer-related weight loss in a syngeneic orthotopic mouse model of pancreatic cancer that mimicked the tumor growth environment in the pancreas. Our results demonstrate that the MLK1 inhibitor is an anti-tumor agent for malignant prostate and pancreatic cancers.

Author Correction: Simvastatin reduces the carcinogenic effect of 3-methylcholanthrene in renal epithelial cells through histone deacetylase 1 inhibition and RhoA reactivation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Involvement of the JNK activation in terbinafine-induced p21 up-regulation and DNA synthesis inhibition in human vascular endothelial cells.

Previously, we demonstrated that the extracellular signal-regulated kinase (ERK)-mediated pathway contributes to the terbinafine (TB)-induced increases of p21 and p53 protein level as well as decrease of DNA synthesis in human umbilical venous endothelial cells (HUVEC). The aim of this study is to examine the involvement of c-Jun NH2-terminal kinase (JNK) in the TB-induced increase of p21 protein level and DNA synthesis inhibition. Western blot analysis and kinase assay demonstrated that TB treatment increased both the protein level and the kinase activity of JNK1/2 in HUVEC. Transfection of HUVEC with JNK1 dominant negative (DN-JNK1) prevented the TB-induced increases of p21 and p53 protein level and decrease of DNA synthesis, suggesting that JNK1/2 activation is involved in the TB-induced cell cycle arrest in HUVEC. Moreover, over-expression of mitogen-activated protein kinase (MEK)-1 prevented the TB-induced increase of JNK1/2 protein levels, suggesting that MEK-1 is an upstream inhibitor of JNK. Transfection of HUVEC with DN-JNK1 prevented the TB-induced inhibition of ERK phosphorylation, suggesting that JNK1/2 might serve as a negative regulator of ERK. Taken together, our results suggest that JNK activation is involved in the TB-induced inhibition of ERK phosphorylation, p53 and p21 up-regulation and DNA synthesis inhibition in HUVEC.

Progesterone up-regulates p27 through an increased binding of the progesterone receptor-A-p53 protein complex onto the non-canonical p53 binding motif in HUVEC.

We previously demonstrated that progesterone (P4) up-regulated p53 expression, which in turn increased p21 and p27 expression, and finally resulted in proliferation inhibition in human umbilical vein endothelial cells (HUVEC). While a direct transcriptional activation of p21 by p53 protein has been clearly elucidated, the mechanism by which p53 induces p27 expression has not been documented. In this study, we identified three putative p53 protein binding domains at the p27 promoter. Luciferase assay showed that the activity of ectopically introduced p27 promoter constructs containing the potential p53 protein binding region was significantly increased by P4. Immunoblotting analysis indicated that P4 increased the level of p53 protein. Treatment with pifithrin-α-HBr (PFTα), a specific blocker of p53-responsive gene transactivation, reduced the P4-increased p27 promoter activity and p27 protein expression. Transfection with dominant-negative mutants of p53 (C135Y, R175H and R248 W) abolished the P4-increased p27 promoter activity. Moreover, deletion or TCCT nucleotide sequence fill-in at the core site of any of p53 protein binding domains led to the irresponsiveness of the p27 promoter to P4 treatment. Interestingly, immunoprecipitation and chromatin-immunoprecipitation analyses demonstrated that P4 increased the complex of p53-P4 receptor (PR) protein in the nucleus and the assembly of PR protein to the p53 protein binding region of the p27 promoter. Ectopic co-overexpression of p53 and PR-A constructs further augmented the P4-increased p27 promoter activity. Taken together, the results from the present study suggest that P4-increased p53 expression might directly up-regulate p27 transactivation, and PR-A protein might promote this effect by forming complex with p53 protein.

Simvastatin reduces the carcinogenic effect of 3-methylcholanthrene in renal epithelial cells through histone deacetylase 1 inhibition and RhoA reactivation.

The therapeutic effects of simvastatin for renal cell carcinoma (RCC) are controversial. In this study, the effects of simvastatin on the carcinogenic properties of 3-methylcholanthrene (3MC; an aryl-hydrocarbon receptor [AhR] agonist) in human renal epithelial cells (hRECs) were investigated. We exposed in vitro and in vivo models to 3MC to induce RCC onset. 3MC upregulated the epithelial-mesenchymal transition (EMT) and tumor biomarkers; the models exhibited the reciprocal expression of histone deacetylase 1 (HDAC1) and RhoA, namely increased HDAC1 and decreased RhoA expression, through hypoxia-inducible-factor (HIF)- and AhR-dependent mechanisms. In addition to inducing EMT biomarkers, 3MC decreased von Hippel-Lindau protein levels (a risk factor for RCC) and increased CD44 expression in hRECs, which were reversed by digoxin (a HIF inhibitor) and HDAC inhibitors (suberoylanilide hydroxamic acid and trichostatin A [TSA]). Simvastatin abolished the detrimental effects of 3MC by reducing HDAC1 expression, with resulting RhoA upregulation, and reactivating RhoA in vitro and in vivo. Notably, the protective effects of simvastatin were negated by an HDAC activator (ITSA) through TSA suppression. The crucial role of RhoA in RCC carcinogenesis was verified by the overexpression of constitutively active RhoA. Collectively, these results demonstrate that simvastatin restores RhoA function through HDAC1 inhibition; therefore, simvastatin might serve as adjunct therapy for RCC induced by 3MC.

Folic acid inhibits colorectal cancer cell migration.

We recently showed that folic acid (FA) could decrease the proliferation rate of colorectal cancer cells in vitro and reduce the volume of COLO-205 tumor in vivo. Since cancer cell proliferation and migration are two major events during cancer development, we further examined whether FA could also affect the migration of colorectal cancer cells. Transwell invasion assays demonstrated that FA reduced the invasion ability of colorectal cancer cell lines, COLO-205, LoVo and HT-29. Using COLO-205 as a cell model, we further delineated the molecular mechanism underlying FA-inhibited colorectal cancer cell invasion. Western blot analyses showed that FA (10 µM) activated cSrc, ERK1/2, NFκB, and p27 at serine 10 (Ser10), and up-regulated p53, p27, and KIS protein. Subcellular fractionation illustrated that FA treatment increased cytosolic translocation of p27, formation of the p27-RhoA complex, and RhoA degradation. The FA-induced migration inhibition in COLO-205 was abolished by blockade of the cSrc or ERK1/2 activity, knockdown of p27 or KIS using the siRNA technique, or over-expression of a constitutive active RhoA cDNA. Our results suggest that FA up-regulated p27 through increasing the cSrc/ERK1/2/NFκB/p53-mediated pathway. In the nucleus, FA up-regulated KIS, which in turn increased p27 phosphorylation at serine 10 (Ser10), subsequently resulting in cytosolic translocation of p27 and forming the p27-RhoA complex, thereby causing RhoA degradation, and eventually inhibited COLO-205 cell migration. Together with our previous findings suggest that FA reduced colorectal cancer development through inhibiting colorectal cancer cell proliferation and migration.

ETF-QO Mutants Uncoupled Fatty Acid ß-Oxidation and Mitochondrial Bioenergetics Leading to Lipid Pathology.

The electron-transfer flavoprotein dehydrogenase gene (ETFDH) that encodes the ETF-ubiquinone oxidoreductase (ETF-QO) has been reported to be the major cause of multiple acyl-CoA dehydrogenase deficiency (MADD). ETF-QO is an electron carrier that mainly functions in mitochondrial fatty acid ß-oxidation and the delivery of electrons to the ubiquinone pool in the mitochondrial respiratory chain. A high frequency of c.250G>A has been found in Taiwanese patients with late-onset MADD. We postulated that the ETFDH c.250G>A mutation may concomitantly impair fatty acid ß-oxidation and mitochondrial function. Using MADD patient-derived lymphoblastoid cells and specifically overexpressed ETFDH c.92C>T, c.250G>A, or coexisted c.92C>T and c.250G>A (c.92C>T + c.250G>A) mutated lymphoblastoid cells, we addressed the genotype-phenotype relationship of ETFDH variation in the pathogenesis of MADD. The decreased adenosine triphosphate synthesis, dissipated mitochondrial membrane potentials, reduced mitochondrial bioenergetics, and increased neutral lipid droplets and lipid peroxides were found in the MADD patient-derived lymphoblastoid cells. Riboflavin and/or coenzyme Q10 supplementation rescued cells from lipid droplet accumulation. All three mutant types, c.92C>T, c.250G>A, or c.92C>T + c.250G>A, had increased lipid droplet accumulation after treatment with palmitic acid. These results help to clarify the molecular pathogenesis of MADD as a result of the high frequency of the ETFDH c.250G>A and c.92C>T mutations.

Molecular mechanisms of p21 and p27 induction by 3-methylcholanthrene, an aryl-hydrocarbon receptor agonist, involved in antiproliferation of human umbilical vascular endothelial cells.

We previously reported that 3-methylcholanthrene (3MC), an aryl-hydrocarbon receptor (AhR) agonist, inhibits the proliferation of human umbilical vascular endothelial cells (HUVECs; Juan et al., 2006, Eur J Pharmacol 530: 1-8). Herein, pretreatment of HUVECs with p21 or p27 small interfering (si)RNA reduced 3MC-induced elimination of [(3)H]thymidine incorporation, demonstrating their essential roles in the antiproliferation of HUVECs. The molecular mechanisms of p21 and p27 involved in the antiproliferative effects of 3MC were elucidated in this study. 3MC time- and concentration-dependently increased p21 and p27 levels, and decreased the protein level of CDK2 with no apparent alteration of p53. Interestingly, 3MC-mediated p21 and p27 inductions were eliminated by resveratrol, an AhR antagonist, suggesting their AhR dependency, further confirmed by AhR siRNA. Among the relevant pathways, p38MAPK activation sustained the levels of p21 and p27 induced by 3MC, which was eliminated by AhR antagonists and N-acetylcysteine (NAC), an antioxidant. 3MC concentration-dependently enhanced not only the consensus dioxin-responsive element (DRE)-driven luciferase activity, but also the binding activity of the AhR to the putative DRE derived from the p21 and p27 promoters. A deletion of the DRE (-285/-270) in p21 (-2,300/+8) only partially alleviated the 3MC-induced luciferase activity unless NAC was added, suggesting that there may be a DRE-independent mechanism associated with oxidative stress. However, a deletion of the DRE (-660/-645) in p27 (-1,358/-100) almost completely abrogated the activation. Our study demonstrated that both the functional DRE and the phosphorylation of p38MAPK are essential for the induction of p21 and p27, resulting in the antiproliferative action of 3MC in HUVECs.

Molecular mechanisms of the antiproliferative effect of beraprost, a prostacyclin agonist, in murine vascular smooth muscle cells.

Prostacyclin (PGI2) has been shown to inhibit proliferation in vascular smooth muscle cells. To clarify the underlying molecular mechanism, we investigated the vasoprotection of beraprost (a PGI2 agonist) both in vivo and in vitro. Beraprost eliminated increases in proliferation of rat aortic smooth muscle cells (RASMCs) by 12-O-tetradecanoylphorbol 13-acetate, and enhanced the peroxisome proliferator-activated receptor-delta (PPARdelta) and inducible nitric oxide synthetase (iNOS) expressions, which were associated with the antiproliferative action of beraprost according to inhibition experiments by [(3)H]thymidine incorporation. Additionally, elimination of iNOS activity by PPARdelta antagonists suggested that iNOS is the downstream target of PPARdelta. Furthermore, beraprost increased both consensus PPARdelta-responsive element (PPRE)-driven luciferase activity and the binding activity of the PPARdelta to the putative PPRE in the iNOS promoter; nevertheless, it was abolished by PPARdelta antagonists. Deletion of PPRE (-1,349/-1,330) in the iNOS promoter region (-1,359/+2) strongly reduced promoter-driven activity, representing a novel mechanism of iNOS induction by beraprost. Consistent with this, PPARdelta and the concomitant iNOS induction by beraprost were also evident in vivo. Beraprost-mediated protection in a murine model of balloon angioplasty was significantly attenuated by 13S-HODE, a PPARdelta antagonist. Taken together, the results suggest that the causal relationship between PPARdelta and iNOS contributes to the vasoprotective action of beraprost in RASMCs.