Butyrate increases methylglyoxal production through regulation of the JAK2/Stat3/Nrf2/Glo1 pathway in castration­resistant prostate cancer cells.

Cancer cells are characterized by increased glycolysis, known as the Warburg effect, which leads to increased production of cytotoxic methylglyoxal (MGO) and apoptotic cell death. Cancer cells often activate the protective nuclear factor erythroid 2­related factor2 (Nrf2)/glyoxalase1 (Glo1) system to detoxify MGO. The effects of sodium butyrate (NaB), a product of gut microbiota, on Nrf2/Glos/MGO pathway and the underlying mechanisms in prostate cancer (PCa) cells were investigated in the present study. Treatment with NaB induced the cell death and reduced the proliferation of PCa cells (DU145 and LNCap). Moreover, the protein kinase RNA-like endoplasmic reticulum kinase/Nrf2/Glo1 pathway was greatly inhibited by NaB, thereby accumulating MGO-derived adduct hydroimidazolone (MG-H1). In response to a high amount of MGO, the expression of Nrf2 and Glo1 was attenuated, coinciding with an increased cellular death. NaB also markedly inhibited the Janus kinase 2 (JAK2)/Signal transducer and activator of transcription 3 (Stat3) pathway. Conversely, co­treatment with Colivelin, a Stat3 activator, significantly reversed the effects of NaB on Glo1 expression, MG-H1 production, and the cell migration and viability. As expected, overexpression of Stat3 or Glo1 reduced NaB­induced cell death. The activation of calcium/calmodulin dependent protein kinase II gamma and reactive oxygen species production also contributed to the anticancer effect of NaB. The present study, for the first time, demonstrated that NaB greatly increases MGO production through suppression of the JAK2/Stat3/Nrf2/Glo1 pathway in DU145 cells, a cell line mimicking castration­resistant PCa (CRPC), suggesting that NaB may be a potential agent for PCa therapy.

Far-infrared radiation alleviates cisplatin-induced vascular damage and impaired circulation via activation of HIF-1α.

Severe vascular damage and complications are often observed in cancer patients during treatment with chemotherapeutic drugs such as cisplatin. Thus, development of potential options to ameliorate the vascular side effects is urgently needed. In this study, the effects and the underlying mechanisms of far-infrared radiation (FIR) on cisplatin-induced vascular injury and endothelial cytotoxicity/dysfunction in mice and human umbilical vein endothelial cells (HUVECs) were investigated. An important finding is that the severe vascular stenosis and poor blood flow seen in cisplatin-treated mice were greatly mitigated by FIR irradiation (30 minutes/day) for 1-3 days. Moreover, FIR markedly increased the levels of phosphorylation of PI3K and Akt, and VEGF secretion, as well as the expression and the activity of hypoxia-inducible factor 1α (HIF-1α) in cisplatin-treated HUVECs in a promyelocytic leukemia zinc finger protein (PLZF)-dependent manner. However, FIR-stimulated endothelial angiogenesis and VEGF release were significantly diminished by transfection with HIF-1α siRNA. We also confirmed that HIF-1α, PI3K, and PLZF contribute to the inhibitory effect of FIR on cisplatin-induced apoptosis in HUVECs. Notably, FIR did not affect the anticancer activity and the HIF-1α/VEGF cascade in cisplatin-treated cancer cells under normoxic or hypoxic condition, indicating that the actions of FIR may specifically target endothelial cells. It is the first study to demonstrate that FIR effectively attenuates cisplatin-induced vascular damage and impaired angiogenesis through activation of HIF-1α-dependent processes via regulation of PLZF and PI3K/Akt. Taken together, cotreatment with the noninvasive and easily performed FIR has a therapeutic potential to prevent the pathogenesis of vascular complications in cancer patients during cisplatin treatment.

Osthole Exerts Inhibitory Effects on Hypoxic Colon Cancer Cells via EIF2[Formula: see text] Phosphorylation-mediated Apoptosis and Regulation of HIF-1[Formula: see text].

Hypoxic microenvironment and dysregulated endoplasmic reticulum stress/unfolded protein response (UPR) system are considered important factors that promote cancer progression. Although osthole extracted from Cnidium monnieri(Fructus Cnidii) has been confirmed to exhibit an anticancer activity in various cancers, the effects of osthole in hypoxic colon cancer cells have not been explored. Therefore, the aim of this study was to examine whether osthole has an inhibitory effect on hypoxic colon cancer HCT116 cells and further investigate the underlying molecular mechanisms. Treatment with osthole significantly attenuated the cell viability, proliferation, and migration in hypoxic HCT116 cells. Osthole also activated UPR signaling such as phospho-eukaryotic initiation factor 2 alpha (EIF2[Formula: see text]/ATF4/CHOP/DR5 cascade accompanied by upregulation of pro-apoptotic proteins. Moreover, the tubule-like formation of human umbilical vein endothelial cells, the secretion of vascular endothelial growth factor A, and the expression and activity of hypoxia-inducible factor-1[Formula: see text] (HIF-1[Formula: see text] in hypoxic HCT116 cells were markedly suppressed by osthole. However, suppressing EIF2[Formula: see text] phosphorylation with salubrinal or ISRIB markedly reversed the effects of osthole on the expressions of pro-apoptotic proteins and HIF-1[Formula: see text]. Co-treatment of hypoxic HCT116 cells with osthole greatly increased the sensitivity to cisplatin and the expressions of phospho-EIF2[Formula: see text] and cleaved caspase 3. Collectively, the inhibitory effect of osthole in hypoxic HCT116 cells may be associated with EIF2[Formula: see text] phosphorylation-mediated apoptosis and translational repression of HIF-1[Formula: see text]. Taken together, osthole may be a potential agent in the treatment of colon cancer.

Magnolol ameliorates pneumonectomy and monocrotaline-induced pulmonary arterial hypertension in rats through inhibition of angiotensin II and endothelin-1 expression.

BACKGROUND: Magnolol, a major bioactive component extracted from Magnolia officinalis, exerts several beneficial effects, such as anti-inflammatory and anti-hypertensive activities. PURPOSE: In this study, we investigated whether magnolol has a protective effect on pneumonectomy and monocrotaline-induced pulmonary arterial hypertension (PAH) in rats. DESIGN/METHODS: The alterations of right ventricular (RV) hypertrophy, pulmonary vascular remodeling, histopathological parameters, and related gene expression and signaling pathways in lungs by magnolol treatment were studied in the PAH rats. RESULTS: Administration of magnolol greatly ameliorated the characteristic features of PAH, including increased pulmonary arterial pressure, RV hypertrophy, and pulmonary vascular remodeling. Moreover, magnolol inhibited angiotensin-converting enzyme (ACE)/angiotensin II (Ang II)/Ang II type 1 receptor (AT-1R) cascade, whereas upregulates ACE2 in the lungs of PAH rats. The overexpression of endothelin-1 (ET-1) and ETA receptor occurred in the PAH rats was significantly attenuated by magnolol through inhibition of Akt/ERK1/2/GSK3ß/ß-catenin pathway. Compared with that of untreated PAH rats, higher expression of endothelial nitric oxide synthase, and lower expression of inducible nitric oxide synthase and O2- production in lungs were observed in magnolol-treated PAH rats. CONCLUSION: We demonstrated that treatment with magnolol reduces the development of PAH induced by pneumonectomy and monocrotaline in rats, and suppressing Ang II and ET-1-mediated processes may contribute to its protective effects. These findings suggest that magnolol may be a potential agent for PAH therapy.

Anti-cachectic effect of Antrodia cinnamomea extract in lung tumor-bearing mice under chemotherapy.

Skeletal muscle atrophy, the most characteristic feature of cancer cachexia, often occurs in patients with cancer undergoing chemotherapy. Antrodia cinnamomea (AC) a widely used edible medical fungus, exhibits hepatoprotective, anti-inflammatory and anticancer activities. In this study, we investigated whether combined treatment with the ethonolic extract of AC ameliorates cachexia symptoms, especially muscle wasting, in lung tumor-bearing mice treated with chemotherapy. Our results revealed that gemcitabine and cisplatin-induced severe body weight loss and skeletal muscle atrophy in the mice with cancer were greatly attenuated after AC extract administration. The protection may be attributed to the inhibition of skeletal muscle proteolysis by suppressing myostatin and activin release, muscle wasting-related FoxO3/MuRF-1/MAFbx signaling, proteasomal enzyme activity, and pro-inflammatory cytokine production. A significant decrease in insulin-like growth factor 1 (IGF-1) expression and formation was observed in the atrophying muscle of the conventional chemotherapy treatment group (CGC), and this decrease was markedly reversed by AC treatment. Additionally, the anorexia, intestinal injury and dysfunction that occurred in the CGC group were mitigated by AC extract. Taken together, these results demonstrated that the AC extract has a protective effect against chemotherapy-induced muscle atrophy mainly by attenuating muscle proteolysis, pro-inflammatory cytokine production, and anorexia, and activating IGF-1-dependent protein synthesis.

Baicalein Ameliorates Pulmonary Arterial Hypertension Caused by Monocrotaline through Downregulation of ET-1 and ETAR in Pneumonectomized Rats.

Baicalein (BE) extracted from Scutellaria baicalensis Georgi is able to alleviate various cardiovascular and inflammatory diseases. However, the effects of BE on pulmonary arterial hypertension (PAH) remain unknown. Therefore, the present study aimed to examine whether BE ameliorates pneumonectomy and monocrotaline-induced PAH in rats and further investigate the underlying molecular mechanisms. Administration of BE greatly attenuated the development of PAH as evidenced by an improvement of its characteristic features, including elevation of right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary vascular remodeling. Moreover, the increased protein expression of endothelin-1 (ET-1) and ETA receptor (ETAR), superoxide overproduction, and activation of Akt/ERK1/2/GSK3[Formula: see text]/[Formula: see text]-catenin pathway that occurred in the lungs of PAH rats were markedly reversed by BE treatment. Compared with the untreated PAH rats, higher expression of endothelial nitric oxide synthase (eNOS), but lower levels of inducible nitric oxide synthase and vWF were observed in BE-treated PAH rats. Collectively, treatment with BE remarkably attenuates the pathogenesis of PAH, and the protection of BE may be associated with suppressing Akt/Erk1/2/GSK3[Formula: see text]/[Formula: see text]-catenin/ET-1/ETAR signaling and preventing endothelial dysfunction. These results suggest that BE is a potential agent for treatment of PAH.

The novel compound MP407 inhibits platelet aggregation through cyclic AMP-dependent processes.

Platelet hyperactivity plays a critical role for initiating several vascular diseases such as atherothrombosis. Therefore, development of effective antiplatelet agents is necessary for ameliorating platelet-related diseases. In this study, we investigated the effects of the new synthesized compound, MP407 on platelet aggregation and further elucidated the underlying mechanisms. Our results demonstrated that MP407 dose-dependently inhibited collagen-induced platelet aggregation, thromboxane B2 (TXB2) production, intracellular Ca2+ mobilization, platelet membrane GPIIb/IIIa expression, and the phosphorylation of Akt, GSK3ß, p38MAPK, and phospho (Ser) PKC substrate (p47). Moreover, MP407 is able to increase the cyclic AMP formation both in resting and activated platelets. However, blocking cyclic AMP formation with 2'5'-ddAdo, an inhibitor of adenylate cyclase, greatly reversed the antiplatelet activity of MP407 and related platelet-activating pathways. MP407 also enhanced VASP phosphorylation at Ser157 in collagen-stimulated platelets, which was attenuated by addition of 2'5'-ddAdo. Therefore, the antiplatelet activity of MP407 may be modulated by cyclic AMP-dependent regulation of Akt, GSK3ß, p38MAPK and VASP phosphorylation. Notably, treatment with MP407 markedly reduced the pulmonary thrombosis and the numbers of paralysis and death in mice induced by ADP injection, but did not affect the bleeding time. Taken together, MP407 may be a potential candidate or lead compound for developing novel antiplatelet or antithrombotic agents for platelet hyperactivity-triggered disease therapy.

Antiangiogenic activity of phthalides-enriched Angelica Sinensis extract by suppressing WSB-1/pVHL/HIF-1α/VEGF signaling in bladder cancer.

The hypoxia-inducible factor-1α (HIF-1α) plays a critical role in tumor angiogenesis. It has been reported that the acetone extract of Angelica sinensis (AE-AS) rich in phthalides is able to inhibit cancer cell proliferation. However, whether AE-AS reduces cancer angiogenesis remains unknown. In this study, we demonstrated that AE-AS significantly inhibited the angiogenesis in vitro and in vivo evidenced by attenuation of the tube formation in hypoxic human umbilical vascular endothelial cells (HUVECs), and the vasculature generation in Matrigel plug, the chicken chorioallantoic membrane, and tumors. Treatment with AE-AS markedly decreased the protein accumulation and transcriptional activity of HIF-1α, vascular endothelial growth factor (VEGF) expression/secretion, and VEGFR2 phosphorylation in hypoxic human bladder cancer (T24) cells and tumor tissues accompanied by a reduction of tumor growth. Notably, AE-AS-induced HIF-1α protein degradation may, at least partly, attribute to inhibition of WSB-1-dependent pVHL degradation. Moreover, VEGFR2-activated PI3K/AKT/mTOR signaling pathway in hypoxic T24 cells was greatly inhibited by AE-AS. Collectively, AE-AS may be a potential anticancer agent by attenuating cancer angiogenesis via suppression of WSB-1/pVHL/HIF-1α/VEGF/VEGFR2 cascade.

Far-infrared protects vascular endothelial cells from advanced glycation end products-induced injury via PLZF-mediated autophagy in diabetic mice.

The accumulation of advanced glycation end products (AGEs) in diabetic patients induces vascular endothelial injury. Promyelocytic leukemia zinc finger protein (PLZF) is a transcription factor that can be activated by low-temperature far-infrared (FIR) irradiation to exert beneficial effects on the vascular endothelium. In the present study, we investigated the influence of FIR-induced PLZF activation on AGE-induced endothelial injury both in vitro and in vivo. FIR irradiation inhibited AGE-induced apoptosis in human umbilical vein endothelial cells (HUVECs). PLZF activation increased the expression of phosphatidylinositol-3 kinases (PI3K), which are important kinases in the autophagic signaling pathway. FIR-induced PLZF activation led to autophagy in HUVEC, which was mediated through the upregulation of PI3K. Immunofluorescence staining showed that AGEs were engulfed by HUVECs and localized to lysosomes. FIR-induced autophagy promoted AGEs degradation in HUVECs. In nicotinamide/streptozotocin-induced diabetic mice, FIR therapy reduced serum AGEs and AGEs deposition at the vascular endothelium. FIR therapy also reduced diabetes-induced inflammatory markers in the vascular endothelium and improved vascular endothelial function. These protective effects of FIR therapy were not found in PLZF-knockout mice. Our data suggest that FIR-induced PLZF activation in vascular endothelial cells protects the vascular endothelium in diabetic mice from AGE-induced injury.

Combined administration of fucoidan ameliorates tumor and chemotherapy-induced skeletal muscle atrophy in bladder cancer-bearing mice.

Cancer cachexia is characterized by anorexia, skeletal muscle atrophy, and systemic inflammation. Fucoidan extracted from brown algae exhibits anti-inflammatory and anticancer activities. However, whether fucoidan ameliorates tumour and chemotherapy-induced muscle atrophy and -related cachectic symptoms remains unknown. Compared with mice with bladder cancer treated with chemotherapy alone (TGC group), those treated with a combination of low molecular weight fucoidan (LMWF) and chemotherapy drugs such as gemcitabine and cisplatin (TGCF) showed a significant reduction of body weight loss, muscle atrophy, and intestinal injury and dysfunction. Moreover, myostatin, activin A, and pro-inflammatory cytokine production, FoxO3 expression and activation, NF-κB activation, MuRF-1 and MAFbx/atrogin-1 expression, and proteasome activity in muscle were significantly decreased in the TGCF group compared with the TGC group. In addition, insulin-like growth factor 1 (IGF-1) expression and formation, and IGF-1-regulated mTOR/p70S6k/4EBP-1 protein synthesis signalling were elevated in the TGCF group compared with the TGC group. Taken together, these results suggest that LMWF is a potential agent for preventing cancer cachexia-associated muscle atrophy during chemotherapy. Furthermore, the beneficial effect of LMWF may be attributed to suppressing NF-κB-evoked inflammation, myostatin and activin A production, and subsequent muscle proteolysis, and enhancing IGF-1-dependent protein synthesis.

Supplementation of Magnolol Attenuates Skeletal Muscle Atrophy in Bladder Cancer-Bearing Mice Undergoing Chemotherapy via Suppression of FoxO3 Activation and Induction of IGF-1.

Skeletal muscle atrophy, the most prominent phenotypic feature of cancer cachexia, is often observed in cancer patients undergoing chemotherapy. Magnolol (M) extracted from Magnolia officinalis exhibits several pharmacological effects including anti-inflammatory and anticancer activities. In this study, we investigated whether magnolol supplementation protects against the development of cachexia symptoms in bladder cancer-bearing mice undergoing chemotherapy. Combined treatment of magnolol with chemotherapeutic drugs, such as gemcitabine and cisplatin (TGCM) or gemcitabine (TGM), markedly attenuates the body weight loss and skeletal muscle atrophy compared with conventional chemotherapy (TGC). The antiatrophic effect of magnolol may be associated with inhibition of myostatin and activin A formation, as well as FoxO3 transcriptional activity resulting from Akt activation, thereby suppressing ubiquitin ligases MuRF-1 and MAFbx/atrogin-1 expression, as well as proteasomal enzyme activity. Notably, magnolol-induced insulin-like growth factor 1 (IGF-1) production and related protein synthesis may also contribute to its protective effects. The decreased food intake, and intestinal injury and dysfunction observed in the mice of TGC group were significantly improved in the TGCM and TGM groups. Moreover, the increased inflammatory responses evidenced by elevation of proinflammatory cytokine formation and NF-κB activation occurred in the atrophying muscle of TGC group were markedly inhibited in mice of combined treatment with magnolol. In summary, these findings support that magnolol is a promising chemopreventive supplement for preventing chemotherapy-induced skeletal muscle atrophy associated with cancer cachexia by suppressing muscle protein degradation, and inflammatory responses, as well as increasing IGF-1-mediated protein synthesis.

Low Molecular Weight Fucoidan Inhibits Tumor Angiogenesis through Downregulation of HIF-1/VEGF Signaling under Hypoxia.

Activation of hypoxia-induced hypoxia-inducible factors-1 (HIF-1) plays a critical role in promoting tumor angiogenesis, growth and metastasis. Low molecular weight fucoidan (LMWF) is prepared from brown algae, and exhibits anticancer activity. However, whether LMWF attenuates hypoxia-induced angiogenesis in bladder cancer cells and the molecular mechanisms involved remain unclear. This is the first study to demonstrate that LMWF can inhibit hypoxia-stimulated H2O2 formation, HIF-1 accumulation and transcriptional activity vascular endothelial growth factor (VEGF) secretion, and the migration and invasion in hypoxic human bladder cancer cells (T24) cells. LMWF also downregulated hypoxia-activated phosphorylation of PI3K/AKT/mTOR/p70S6K/4EBP-1 signaling in T24 cells. Blocking PI3K/AKT or mTOR activity strongly diminished hypoxia-induced HIF-1α expression and VEGF secretion in T24 cells, supporting the involvement of PI3K/AKT/mTOR in the induction of HIF-1α and VEGF. Additionally, LMWF significantly attenuated angiogenesis in vitro and in vivo evidenced by reduction of tube formation of hypoxic human umbilical vascular endothelial cells and blood capillary generation in the tumor. Similarly, administration of LMWF also inhibited the HIF-1α and VEGF expression in vivo, accompanied by a reduction of tumor growth. In summary, under hypoxia conditions, the antiangiogenic activity of LMWF in bladder cancer may be associated with suppressing HIF-1/VEGF-regulated signaling pathway.

Mechanisms of Nifedipine-Downregulated CD40L/sCD40L Signaling in Collagen Stimulated Human Platelets.

The platelet-derived soluble CD40L (sCD40L) release plays a critical role in the development of atherosclerosis. Nifedipine, a dihydropyridine-based L-type calcium channel blocker (CCB), has been reported to have an anti-atherosclerotic effect beyond its blood pressure-lowering effect, but the molecular mechanisms remain unclear. The present study was designed to investigate whether nifedipine affects sCD40L release from collagen-stimulated human platelets and to determine the potential role of peroxisome proliferator-activated receptor-ß/-γ (PPAR-ß/-γ). We found that treatment with nifedipine significantly inhibited the platelet surface CD40L expression and sCD40L release in response to collagen, while the inhibition was markedly reversed by blocking PPAR-ß/-γ activity with specific antagonist such as GSK0660 and GW9662. Meanwhile, nifedipine also enhanced nitric oxide (NO) and cyclic GMP formation in a PPAR-ß/-γ-dependent manner. When the NO/cyclic GMP pathway was suppressed, nifedipine-mediated inhibition of sCD40L release was abolished significantly. Collagen-induced phosphorylation of p38MAPK, ERK1/2 and HSP27, matrix metalloproteinase-2 (MMP-2) expression/activity and reactive oxygen species (ROS) formation were significantly inhibited by nifedipine, whereas these alterations were all attenuated by co-treatment with PPAR-ß/-γ antagonists. Collectively, these results demonstrate that PPAR-ß/-γ-dependent pathways contribute to nifedipine-mediated downregulation of CD40L/sCD40L signaling in activated platelets through regulation of NO/ p38MAPK/ERK1/2/HSP27/MMP-2 signalings and provide a novel mechanism regarding the anti-atherosclerotic effect of nifedipine.

Immunomodulatory activity of Melaleuca alternifolia concentrate (MAC): inhibition of LPS-induced NF-κB activation and cytokine production in myeloid cell lines.

Melaleuca alternifolia concentrate (MAC) is a mixture predominantly composed of monoterpenoids and sesquiterpenes, refined from the essential oil of the tea tree by removing up to 99% of the more toxic, hydrophobic monoterpenes. MAC was examined here for its immunomodulatory effects on the human THP1 and murine RAW264.7 myeloid leukemic cell lines as models for macrophage-like cells. Firstly, MAC levels were determined that did not affect either the survival or proliferation of these cell lines in vitro. Next, the levels of lipopolysaccharide (LPS)-induced production of cytokines (IL-6, TNFα, IL-10, GM-CSF, IFNγ and IL-3) were examined from the myeloid cell lines using multiplex assays. Many of the LPS-inducible cytokines produced by either cell lines could be significantly inhibited by MAC. Closer examination of the mechanism of action of MAC showed that it inhibited the LPS-induced activation of IκB phosphorylation and nuclear factor (NF)-κB signalling and translocation, inhibiting iNOS protein expression and NO production. These results demonstrate that MAC exerts its immunomodulatory effects by inhibiting NF-κB signalling activation and levels of cytokine production by macrophage-like cell lines.

Magnolol Inhibits RANKL-induced osteoclast differentiation of raw 264.7 macrophages through heme oxygenase-1-dependent inhibition of NFATc1 expression.

Magnolol (1) isolated from Magnolia officinalis exhibits many beneficial effects such as anti-inflammatory and antioxidant activity. The aim of this study was to evaluate the effects of magnolol (1) on RANKL-induced osteoclast differentiation and investigate the underlying molecular mechanisms. Treatment with magnolol (1) significantly inhibited osteoclast differentiation of RAW 264.7 macrophages and bone-resorbing activity of osteoclasts in the RANKL-induced system. Moreover, RANKL-activated JNK/ERK/AP-1 and NF-κB signaling, ROS formation, and NFATc1 activation were attenuated by magnolol (1). A novel finding of this study is that magnolol (1) can increase heme oxygenase-1 (HO-1) expression and Nrf2 activation in RANKL-stimulated cells. Blocking HO-1 activity with tin protoporphyrin IX markedly reversed magnolol (1)-mediated inhibition of osteoclast differentiation, NFATc1 nuclear translocation, and MMP-9 activity, suggesting that HO-1 contributes to the attenuation of NFATc1-mediated osteoclastogenesis by magnolol (1). Therefore, the inhibitory effect of magnolol (1) on osteoclast differentiation is due to inhibition of MAPK/c-fos/AP-1 and NF-κB signaling as well as ROS production and up-regulation of HO-1 expression, which ultimately suppresses NFATc1 induction. These findings indicate that magnolol (1) may have potential to treat bone diseases associated with excessive osteoclastogenesis.

Mechanisms of antiplatelet activity of nifedipine: role of peroxisome proliferator-activated receptor-ß-γ-dependent processes.

OBJECTIVE: Nifedipine, an L-type calcium channel blocker, is widely used in the treatment of hypertension and coronary heart diseases, and also exhibits an antiplatelet activity. Activation of peroxisome proliferator-activated receptors (PPARs; α, ß/δ, and γ) inhibits the platelet aggregation. Therefore, the purpose of this study was to evaluate the contribution of PPAR-mediated processes to the antiplatelet activity of nifedipine. METHODS AND RESULTS: We assessed human platelet aggregation by using an aggregometer and measured several platelet activating markers and related signaling pathways in platelets treated with nifedipine in the presence or absence of PPAR agonists. Nifedipine treatment (1, 5  µmol/l) dose-dependently increased the activity and intracellular expression of PPAR-ß/-γ by inhibiting the release of PPAR-ß/-γ from activated platelets. Nifedipine treatment also upregulated cyclic 3',5'-cyclic monophosphate (GMP)/protein kinase G (PKG) expression, and increased PI(3)K/Akt pathway, endothelial nitric oxide synthase, and soluble guanylyl cyclase activities. In the presence of a selective PPAR-ß antagonist (GSK0660) or PPAR-γ antagonist (GW9662), the inhibitory effects of nifedipine on collagen-induced platelet aggregation, intracellular Ca mobilization, and protein kinase C (PKC-α) activation were abrogated. Similarly, PPAR-ß-γ antagonists markedly attenuated nifedipine-mediated upregulation of nitric oxide/cyclic GMP/PKG cascade. In a mouse model of thrombosis, the administration of nifedipine substantially inhibited fluorescein sodium-induced vessel thrombus formation; however, the antithrombotic effect was considerably reduced in the presence of PPAR-ß/-γ antagonists. CONCLUSION: This study is the first to show that the PPAR-ß/-γ-dependent upregulation of PI(3)K/Akt/nitric oxide/cyclic GMP/PKG pathway and the inhibition of PKC-α activity and intracellular Ca(+) mobilization in platelets may be the mechanisms underlying the antiplatelet and antithrombotic activities of nifedipine.

Magnolol suppresses hypoxia-induced angiogenesis via inhibition of HIF-1α/VEGF signaling pathway in human bladder cancer cells.

The hypoxic environment in tumors is an important factor causing tumor angiogenesis by activating the key transcription factor, hypoxia-inducible factors-1α (HIF-1α). Magnolol isolated from Magnolia officinalis has been reported to exhibit an anticancer activity via elevation of apoptosis. However, whether magnolol inhibits tumor angiogenesis remains unknown. In the present study, we demonstrated that magnolol significantly inhibited angiogenesis in vitro and in vivo evidenced by the attenuation of hypoxia and vascular endothelial growth factor (VEGF)-induced tube formation of human umbilical vascular endothelial cells, vasculature generation in chicken chorioallantoic membrane and Matrigel plug. In hypoxic human bladder cancer cells (T24), treatment with magnolol inhibited hypoxia-stimulated H2O2 formation, HIF-1α induction including mRNA, protein expression, and transcriptional activity as well as VEGF secretion. Additionally, the enhanced degradation of HIF-1α protein via enhancing prolyl hydroxylase activity and the decreased newly-synthesized HIF-1α protein in hypoxic T24 cells may involve the reduction of HIF-1α protein accumulation by magnolol. Interestingly, magnolol also acts as a VEGFR2 antagonist, and subsequently attenuates the down-stream AKT/mTOR/p70S6K/4E-BP-1 kinase activation both in hypoxic T24 cells and tumor tissues. As expected, administration of magnolol greatly attenuated tumor growth, angiogenesis and the protein expression of HIF-1α, VEGF, CD31, a marker of endothelial cells, and carbonic anhydrase IX, an endogenous marker for hypoxia, in the T24 xenograft mouse model. Collectively, these findings strongly indicate that the anti-agngiogenic activity of magnolol is, at least in part, mediated by suppressing HIF-1α/VEGF-dependent pathways, and suggest that magnolol may be a potential drug for human bladder cancer therapy.

The antiplatelet activity of magnolol is mediated by PPAR-ß/γ.

Activation of peroxisome proliferator-activated receptor (PPAR) isoforms (α, ß/δ, and γ) is known to inhibit platelet aggregation. In the present study, we examined whether PPARs-mediated pathways contribute to the antiplatelet activity of magnolol, a compound purified from Magnolia officinalis. Magnolol (20-60 µM) dose-dependently enhanced the activity and intracellular level of PPAR-ß/γ in platelets. In the presence of selective PPAR-ß antagonist (GSK0660) or PPAR-γ antagonist (GW9662), the inhibition of magnolol on collagen-induced platelet aggregation and intracellular Ca(2+) mobilization was significantly reversed. Moreover, magnolol-mediated up-regulation of NO/cyclic GMP/PKG pathway and Akt phosphorylation leading to increase of eNOS activity were markedly abolished by blocking PPAR-ß/γ activity. Additionally, magnolol significantly inhibited collagen-induced PKCα activation through a PPAR-ß/γ and PKCα interaction manner. The arachidonic acid (AA) or collagen-induced thromboxane B(2) formation and elevation of COX-1 activity caused by AA were also markedly attenuated by magnolol. However, these above effects of magnolol on platelet responses were strongly reduced by simultaneous addition of GSK0660 or GW9662, suggesting that PPAR-ß/γ-mediated processes may account for magnolol-regulated antiplatelet mechanisms. Similarly, administration of PPAR-ß/γ antagonists remarkably abolished the actions of magnolol in preventing platelet plug formation and prolonging bleeding time in mice. Taken together, we demonstrate for the first time that the antiplatelet and anti-thrombotic activities of magnolol are modulated by up-regulation of PPAR-ß/γ-dependent pathways.

Simvastatin inhibits pro-inflammatory mediators through induction of heme oxygenase-1 expression in lipopolysaccharide-stimulated RAW264.7 macrophages.

It has been reported that the anti-inflammatory activity of 3-hydroxy-3-methyl-glutary coenzyme A (HMG-CoA) reductase inhibitors (statins) is independent of their hypocholesterolemic effect. Previous studies indicated that induction of heme oxygenase-1 (HO-1) exerts a cytoprotective activity in several inflammatory diseases. Here, the possibility that HO-1 is involved in the anti-inflammatory action of simvastatin, using lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages as a model system has been specifically addressed. Our results demonstrated that in the presence of LPS, simvastatin significantly increased HO-1 expression and activity in a dose-dependent manner compared to that of LPS-stimulated alone macrophages. Moreover, simvastatin significantly inhibited LPS-induced inducible nitric oxide synthase (NOS) expression, and formation of pro-inflammatory mediators, including tumor necrosis factor-α (TNF-α), nitrite and free radicals, but enhanced interleukin-10 (IL-10) production. Similarly, the IκB-α degradation and nuclear transcription factor-κB translocation and activation caused by LPS were significantly suppressed by simvastatin. However, these anti-inflammatory activities of simvastatin were markedly reversed by addition of a HO-1 inhibitor zinc protoporphyrin (ZnPP). Accordingly, the present results indicate that the anti-inflammatory activity of simvastatin could, at least in part, be regulated by induction of HO-1-mediated processes.

Inhibitory effect of α-lipoic acid on platelet aggregation is mediated by PPARs.

Peroxisome proliferator-activated receptors (PPARs) isoforms (α, ß/δ, and γ are present in human platelets, and activation of PPARs inhibits platelet aggregation. α-Lipoic acid (ALA), occurring naturally in human food, has been reported to exhibit an antiplatelet activity. However, the mechanisms underlying ALA-mediated inhibition of platelet aggregation remain unknown. The aim of this study was to investigate whether the antiplatelet activity of ALA is mediated by PPARs. ALA itself significantly induced PPARα/γ activation in platelets and increased intracellular amounts of PPARα/γ by blocking PPARα/γ secretion from arachidonic acid (AA)-activated platelets. Moreover, ALA significantly inhibited AA-induced platelet aggregation, Ca(2+) mobilization, and cyclooxygenase-1 (COX-1) activity, but increased cyclic AMP production in rabbit washed platelets. Importantly, ALA also enhanced interaction of PPARα/γ with protein kinase Cα (PKCα) and COX-1 accompanied by an inhibition of PKCα activity in resting and AA-activated platelets. However, the above effects of ALA on platelets were markedly reversed by simultaneous addition of selective PPARα antagonist (GW6471) or PPARγ antagonist (GW9662). Taken together, the present study provides a novel mechanism by which ALA inhibition of platelet aggregation is mediated by PPARα/γ-dependent processes, which involve interaction with PKCα and COX-1, increase of cyclic AMP formation, and inhibition of intracellular Ca(2+) mobilization.