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Background Obesity and its associated health conditions, type 2 diabetes mellitus (T2DM) and nonalcoholic fatty liver disease (NAFLD), are worldwide health problems. It has been shown that insulin resistance is associated with increased hepatic lipid and causes hepatic steatosis through a myriad of mechanisms, including inflammatory signaling. Methods Helminthostachys zeylanica (HZ) is used widely as a common herbal medicine to relieve fever symptoms and inflammatory diseases in Asia. In the present study, we evaluated whether HZ has therapeutic effects on obesity, NAFLD and insulin resistance. The protective effects of HZ extract were examined using free fatty acid-induced steatosis in human HuS-E/2 cells and a high-fat diet-induced NAFLD in mice. Results The major components of the HZ extract are ugonins J and K, confirmed by HPLC. Incubation of human hepatocytes, HuS-E/2 cells, with palmitate markedly increased lipid accumulation and treatment with the HZ extract significantly decreased lipid deposition and facilitated AMPK and ACC activation. After 12 weeks of a high-fat diet with HZ extract treatment, the HFD mice were protected from hyperlipidemia and hyperglycemia. HZ extract prevented body weight gain, adipose tissue expansion and adipocyte hypertrophy in the HFD mice. In addition, fat accumulation was reduced in mice livers. Moreover, the insulin sensitivity-associated index, which evaluates insulin function, was also significantly restored. Conclusions These results suggest that HZ has a promising pharmacological effect on high-fat diet-induced obesity, hepatic steatosis and insulin resistance, which may have the potential for clinical application.

// Jen-Ho Tseng 1, 2 , Cheng-Yi Chen 3 , Pei-Chun Chen 3 , Sheng-Huang Hsiao 1, 4 , Chi-Chen Fan 5 , Yu-Chih Liang 6 , Chie-Pein Chen 3, 7 1 Department of Neurosurgery, Taipei City Hospital, Renai Branch, Taipei 106, Taiwan 2 Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 110, Taiwan 3 Department of Medical Research, MacKay Memorial Hospital, New Taipei City 251, Taiwan 4 College of Science, National Chengchi University, Taipei 116, Taiwan 5 Department of Physiology, MacKay Memorial Hospital, Taipei 104, Taiwan 6 School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei 110, Taiwan 7 Department of Medicine, Taipei Medical University, Taipei 110, Taiwan Correspondence to: Chie-Pein Chen, email: cpchen@mmh.org.tw Keywords: cell growth, glioblastoma multiforme, histone deacetylase, paraoxonase 2, valproic acid Received: September 30, 2015 Accepted: January 10, 2017 Published: January 18, 2017 ABSTRACT We studied the potential mechanisms of valproic acid (VPA) in the treatment of glioblastoma multiforme (GBM). Using the human U87, GBM8401, and DBTRG-05MG GBM-derived cell lines, VPA at concentrations of 5 to 20 mM induced G2/M cell cycle arrest and increased the production of reactive oxygen species (ROS). Stress-related molecules such as paraoxonase 2 (PON2), cyclin B1, cdc2, and Bcl-xL were downregulated, but p27, p21 and Bim were upregulated by VPA treatment. VPA response element on the PON2 promoter was localized at position -400/-1. PON2 protein expression was increased in GBM cells compared with normal brain tissue and there was a negative correlation between the expression of PON2 and Bim. These findings were confirmed by the public Bredel GBM microarray (Gene Expression Omnibus accession: GSE2223) and the Cancer Genome Atlas GBM microarray datasets. Overexpression of PON2 in GBM cells significantly decreased intracellular ROS levels, and PON2 expression was decreased after VPA stimulation compared with controls. Bim expression was significantly induced by VPA in GBM cells with PON2 silencing. These observations were further shown in the subcutaneous GBM8401 cell xenograft of BALB/c nude mice. Our results suggest that VPA reduces PON2 expression in GBM cells, which in turn increases ROS production and induces Bim production that inhibits cancer progression via the PON2–Bim cascade.

Closely associated with visceral obesity, hepatic steatosis resulting from non-alcoholic fatty liver disease (NAFLD) exacerbates insulin resistance. Developing effective drugs to treat NAFLD is imperative. Here, we investigated the pharmacological mechanism of ugonin J (UJ) in controlling metabolic disorder and ameliorating NAFLD pathophysiology in diet-induced obese mice. The effects of UJ were assessed in 5-week-old C57BL/6 J mice fed a high-fat diet (HFD) for 12 weeks. UJ treatment averted HFD-induced body weight gain by reducing fat deposition in adipose tissues and reduced HFD-induced hyperlipidemia and hepatic inflammation. UJ also improved HFD-induced glucose tolerance and insulin resistance. Moreover, the mode of action of UJ was analyzed in palmitate (PA)-induced steatotic human HuS-E/2 hepatocytes and in hyperglycemia-simulating rat BRIN-BD11 pancreatic β cells. In PA-induced steatotic human hepatocytes, UJ treatment promoted lipid clearance via pAMPK, pACC and CPT-1 upregulation and SREBP-1c downregulation. Interestingly, UJ upregulated Akt activity in hepatocytes and increased insulin secretion from β cells in acute insulin secretion tests. Taken together, UJ improved adipocyte hypertrophy, hyperinsulinemia, hyperglycemia, hyperlipidemia and fat deposition in livers. UJ also reduced fatty acid accumulation by modulating key metabolic regulators. Our findings demonstrated the therapeutic potential of UJ for the treatment of NAFLD and diet-induced metabolic disorders.

15,16-Dihydrotanshinone I (DHTS) is extracted from Salvia miltiorrhiza Bunge which is a functional food in Asia. In this study, we investigated the apoptotic effect of DHTS on the human acute myeloid leukemia (AML) type III HL-60 cell line. We found that treatment with 1.5 μg/mL DHTS increased proapoptotic Bax and Bad protein expressions and activated caspases-3, -8, and -9, thus leading to poly ADP ribose polymerase (PARP) cleavage and resulting in cell apoptosis. DHTS induced sustained c-Jun N-terminal kinase (JNK) phosphorylation and Fas ligand (FasL) expression. The anti-Fas blocking antibody reversed the DHTS-induced cell death, and the JNK-specific inhibitor, SP600125, inhibited DHTS-induced caspase-3, -8, -9, and PARP cleavage. In a xenograft nude mice model, 25 mg/kg DHTS showed a great effect in attenuating HL-60 tumor growth. Taken together, these results suggest that DHTS can induce HL-60 cell apoptosis in vitro and inhibit HL-60 cell growth in vivo, the underlying mechanisms might be mediated through activation of the JNK and FasL signal pathways.

Objective Diseases associated with calcium-containing crystal deposition can lead to local bone erosion. We aimed to determine whether calcium-containing crystal-hydroxyapatite, β-tricalcium phosphate and CPPD enhanced osteoclastogenesis and to define underlying mechanisms of action. Methods Osteoclastogenesis was studied by culturing murine RAW 264.7 osteoclast precursor cells with RANK ligand (RANKL)/ M-CSF and/or calcium-containing crystals, and observing the tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and TRAP activity. Resorption pit formation was used to evaluate osteoclast activity. Real-time RT-PCR analysis revealed osteoclast marker genes, including TRAP, cathepsin K and calcitonin receptor (CTR). Western blotting was used to analyse the phosphorylation levels of signal transduction molecules. Results Three kinds of calcium-containing crystal significantly enhanced RANKL/M-CSF-induced osteoclastogenesis in RAW 264.7 cells, as evidenced by the increased number of TRAP-positive multinucleated cells, TRAP activity and resorption pit formation in a dose-dependent manner. Hydroxyapatite, β-tricalcium phosphate and CPPD treatments significantly enhanced RANKL/M-CSF-induced mRNA expression of TRAP, cathepsin K and CTR. Moreover, the three kinds of calcium-containing crystal enhanced the phosphorylation of extracellular-signal-regulated kinase and p38 in RANKL/M-CSF-treated cells. Conclusion We concluded that calcium-containing crystals can promote osteoclastogenesis and bone resorption through the extracellular-signal-regulated kinase and p38 pathways. Together with synovial activation, this mechanism may be important in the pathogenesis of destructive arthropathies triggered by calcium-containing crystals.

Our previous studies found that osthole markedly reduced blood glucose levels in both db/db and ob/ob mice. To improve the antidiabetic activity of osthole, a series of N-hydroxycinnamide derivatives of osthole were synthesized, and their hypoglycemia activities were examined in vitro and in vivo. Both N-hydroxycinnamide derivatives of osthole, OHC-4p and OHC-2m, had the greatest potential for activating AMPK and increasing glucose uptake by L6 skeletal muscle cells. In addition, OHC-4p and OHC-2m time- and dose-dependently increased phosphorylation levels of AMPK and p38 MAPK. The AMPK inhibitor, compound C, and the p38 MAPK inhibitor, SB203580, significantly reversed activation of AMPK and p38 MAPK, respectively, in OHC-4p- and OHC-2m-treated cells. Compound C and SB203580 also inhibited glucose uptake induced by OHC-4p and OHC-2m. Next, we found that OHC-4p and OHC-2m significantly increased glucose transporter 4 (GLUT4) translocation to plasma membranes and counteracted hyperglycemia in mice with streptozotocin-induced diabetes. These results suggest that activation of AMPK and p38 MAPK by OHC-4p and OHC-2m is associated with increased glucose uptake and GLUT4 translocation and subsequently led to amelioration of hyperglycemia. Therefore, OHC-4p and OHC-2m might have potential as antidiabetic agents for treating type 2 diabetes. Our previous studies found that osthole markedly reduced blood glucose levels in both db/db and ob/ob mice. To improve the antidiabetic activity of osthole, a series of N-hydroxycinnamide derivatives of osthole were synthesized, and their hypoglycemia activities were examined in vitro and in vivo. Both N-hydroxycinnamide derivatives of osthole, OHC-4p and OHC-2m, had the greatest potential for activating AMPK and increasing glucose uptake by L6 skeletal muscle cells. In addition, OHC-4p and OHC-2m time- and dose-dependently increased phosphorylation levels of AMPK and p38 MAPK. The AMPK inhibitor, compound C, and the p38 MAPK inhibitor, SB203580, significantly reversed activation of AMPK and p38 MAPK, respectively, in OHC-4p- and OHC-2m-treated cells. Compound C and SB203580 also inhibited glucose uptake induced by OHC-4p and OHC-2m. Next, we found that OHC-4p and OHC-2m significantly increased glucose transporter 4 (GLUT4) translocation to plasma membranes and counteracted hyperglycemia in mice with streptozotocin-induced diabetes. These results suggest that activation of AMPK and p38 MAPK by OHC-4p and OHC-2m is associated with increased glucose uptake and GLUT4 translocation and subsequently led to amelioration of hyperglycemia. Therefore, OHC-4p and OHC-2m might have potential as antidiabetic agents for treating type 2 diabetes.

Free radicals play a critical role in causing hemorheologic abnormality which is highly correlated with cardiovascular disease and stroke. In this study, we established an in vitro model to evaluate the influence of free radical attacks on hemorheological parameters. A well-sealed chamber with hyperbaric oxygen was used to simulate an environment of free radical attacks. Hemorheological parameters, including whole blood viscosity, erythrocyte membrane lipid peroxidation, and erythrocyte deformability, were investigated. We then used the in vitro model to evaluate the anti-free radical effects of some well-known catechin antioxidants, such as epigallocatechin gallate (EGCG), (-)-epicatechin 3-gallate (ECG), and (-)-epigallocatechin (EGC) on abnormal hemorheological parameters induced by hyperbaric oxygen. The results show that an increase in oxygen partial pressure (1.0, 1.5, 2.0 and 2.5 atm) and exposure time (4, 8, 12 and 16 h) resulted in elevated free radical formation and viscosity of whole blood, enhanced lipid peroxidation in erythrocyte membranes, but decreased erythrocyte deformability. In addition, EGCG, ECG, and EGC (0.1, 0.5 and 1.0 μM) effectively ameliorated hemorheologic abnormality and enhanced erythrocyte deformability. Therefore, this study has provided an in vitro hyperbaric oxygen model to rapidly screen or assess the efficacy of functional foods and drugs in the prevention or improvement of hemorheologic abnormality.

it has been claimed that phycocyanin exhibits pharmaceutical functions in inhibiting histamine release and leukotriene biosynthesis. In allergic asthma, these inflammatory mediators are crucial for disease progression.the aim of this study was to evaluate the therapeutic potential of R-phycocyanin (R-PC) against allergic airway inflammation.mouse bone marrow-derived dendritic cells (BMDCs) were used to evaluate the immunomodulatory functions of R-PC. In addition, an airway inflammatory model was used to evaluate the therapeutic potential of R-PC.R-PC treatment resulted in a decrease of endocytosis, increase of costimulatory molecule expression, and enhancement of interleukin-12 production in mouse BMDCs. Moreover, R-PC-treated cultured dendritic cells were able to promote CD4(+) T-cell stimulatory capacity and increase interferon-γ expression in CD4(+) T cells. Intraperitoneal administration of R-PC suppressed ovalbumin (OVA)-induced airway hyperresponsiveness, serum levels of OVA-specific IgE and IgG1, eosinophil infiltration, Th2 cytokine levels, and eotaxin in bronchoalveolar lavage fluid of mice. Antibody against Toll-like receptor-4 was able to inhibit R-PC-induced IL-12 p70 production. Moreover, inhibition of nuclear factor-κB (NF-κB) by helenalin and inhibition of the JNK pathway by JNK inhibitor II inhibited R-PC-induced IL-12 p70 production. Western blotting and electrophoretic mobility shift assays showed that R-PC augmented phosphorylation of the inhibitors of NF-κB and inhibitors of NF-κB kinase and facilitated NF-κB activity.our data demonstrated that R-PC promoted activation and maturation of cultured dendritic cells and skewed the immunological function toward Th1 activity. Therefore, R-PC may have potential in regulating immune responses and application in reducing allergic asthma.

Many biological functions of heme oxygenase (HO) have been attributed to its enzymatic byproduct carbon monoxide (CO). CO has been demonstrated to play an important role in down-regulation of pro-inflammatory cytokines, but few studies have investigated the effects of CO on the cyclooxygenase-2 (COX-2) expression in macrophage. Here, we assessed the induction of COX-2 by CO in macrophage with or without lipopolysaccharide (LPS) stimulation. Tricarbonyldichloro ruthenium (II) dimmer (CORM-2) is a well known CO-releasing molecule, and exhibits anti-inflammatory activity in several cell types. In this study, both CORM-2 and CO gas were used to investigate the induction of COX-2 and the underlying molecular mechanisms in macrophage. Western blot and RT-PCR analysis demonstrated that CORM-2 and CO gas (500 ppm) significantly inhibited the protein and mRNA expression of iNOS in LPS-activated macrophages. In contrast, CORM-2 and CO gas up-regulated COX-2 expression and prostaglandin E2 (PGE2) production in the macrophage with or without LPS. CORM-2 time-dependently induced the phosphorylation of Akt and MAPKs, and the induction of COX-2 could be blocked by Akt, PKG, and MAPKs inhibitors. Indomethacin was used to decrease CORM-2-induced PGE2 production by inhibiting COX-2 enzyme activity. Indomethacin was unable to reverse the decrease of iNOS, but it could restore the IL-1β expression and decrease the IL-10 expression in CORM-2-treated cells. The results suggest that CO induced COX-2 expression and PGE2 production through activating the Akt, PKG, and MAPK pathways, and CO-induced PGE2 may modulate inflammation during macrophage activation by suppressing IL-1β expression and inducing IL-10 production.

An increase in mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) reportedly attenuates insulin-mediated signaling which participates in the development of brown adipose tissues (BATs). Nevertheless, the effect of MAP4K4 on brown adipogenesis remains largely uncharacterized. In this study, results of a transcriptome analysis (also referred as RNA-sequencing) showed differential expressions of MAP4K4 or SRSF3 transcripts isolated from distinct stages of embryonic BATs. The discriminative splicing profiles of MAP4K4 or SRSF3 were noted as well in brown adipocytes (BAs) with RNA-binding motif protein 4-knockout (RBM4&minus, /&minus, ) compared to the wild-type counterparts. Moreover, the relatively high expressions of authentic SRSF3 transcripts encoding the splicing factor functioned as a novel regulator toward MAP4K4 splicing during brown adipogenesis. The presence of alternatively spliced MAP4K4 variants exerted differential effects on the phosphorylation of c-Jun N-terminal protein kinase (JNK) which was correlated with the differentiation or metabolic signature of BAs. Collectively, the RBM4-SRSF3-MAP4K4 splicing cascade constitutes a novel molecular mechanism in manipulating the development of BAs through related signaling pathways.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) plays a central role in the pathogenesis of atherosclerosis. β-Sitosterol, an important phytosterol found in plant food, is known to exert antiatherosclerosis activity. However, the molecular mechanisms underlying β-sitosterol-induced antiproliferation of VSMCs were still not clear. This study demonstrated that β-sitosterol (1-20 μM) concentration-dependently inhibited proliferation of rat aortic smooth muscle cells (RASMCs) without cytotoxic effect. Flow cytometric analysis revealed that β-sitosterol arrested cell cycle progression through down-regulation of cyclin E and cyclin-dependent kinase (CDK)2 and up-regulation of p21(cip1). In the β-sitosterol-treated RASMCs, the formation of the CDK2-p21(cip1) complex was increased and the assayable CDK2 activity was decreased. Knockdown of the expression of p21(cip1) gene prevented β-sitosterol-induced cell cycle arrest in RASMCs. In conclusion, β-sitosterol inhibited VSMC proliferation by increasing the levels of p21(cip1) protein, which in turn inhibited the CDK2 activity, and finally interrupted the progress of the cell cycle.

Mushroom product induces a Th1 response and may possess a potential effect in regulating immune responses. Ganoderma lucidum, an oriental medicinal mushroom, has been widely used in Asia to promote health and longevity. LZ-8 is a protein derived from the fungus G. lucidum and has immunomodulatory capacities. In this study, we investigated the immune modulatory effects of rLZ-8 on human monocyte-derived DCs. Treatment of DC with rLZ-8 resulted in the enhanced cell-surface expression of CD80, CD86, CD83, and HLA-DR, as well as the enhanced production of IL-12 p40, IL-10, and IL-23, and the capacity for endocytosis was suppressed in DCs. In addition, treatment of DCs with rLZ-8 resulted in an enhanced, naïve T cell-stimulatory capacity and increased, naïve T cell secretion of IFN-γ and IL-10. Neutralization with antibodies against TLR4 inhibited the rLZ-8-induced production of IL-12 p40 and IL-10 in DCs. rLZ-8 can stimulate TLR4 or TLR4/MD2-transfected HEK293 cells to produce IL-8. These results suggested an important role for TLR4 in signaling DCs upon incubation with rLZ-8. Further study showed that rLZ-8 was able to augment IKK, NF-κB activity, and also IκBα and MAPK phosphorylation. Further, inhibition of NF-κB by helenalin prevented the effects of rLZ-8 in the expression of CD80, CD86, CD83, and HLA-DR and production of IL-12 p40 and IL-10 in various degrees. To confirm the in vitro data, we investigated the effect of rLZ-8 further on antigen-specific antibody and cytokine production in BALB/c mice. Immunization with OVA/rLZ-8 showed that the anti-OVA IgG2a, IFN-γ, and IL-2 were increased significantly compared with OVA alone in BALB/c mice. In conclusion, our experiments demonstrated that rLZ-8 can effectively promote the activation and maturation of immature DCs, preferring a Th1 response, suggesting that rLZ-8 may possess a potential effect in regulating immune responses.

Previously, we demonstrated that 5,5-diphenyl-2-thiohydantoin (DPTH) exerts an anti-proliferation effect on subcultured human umbilical vein endothelial cells (HUVEC). In the present study, we show that 2(naphthalen-2-ylmethylsulfanyl)-5,5-diphenyl-1,5-dihydro-imidazol-4-one (DPTH-N10), a derivative compound of DPTH, exerts a 5 times stronger inhibition of [3H]thymidine incorporation into HUVEC as compared with DPTH and at very low concentrations (0–20 μM) inhibited DNA synthesis and decreased cell number in cultured HUVEC in a concentration- and time-dependent manner, but not in human fibroblasts. [3H]thymidine incorporation analysis demonstrated that treatment of HUVEC with DPTH-N10 arrested the cell at the G0/G1 phase of the cell cycle. Western blot analysis revealed that the protein level of p21 in HUVEC increased after DPTH-N10 treatment. In contrast, the protein levels of p27, p53, cyclins A, D1, D3 and E, cyclin-dependent kinase (CDK)2, and CDK4 in HUVEC were not changed significantly after DPTH-N10 treatment. Immunoprecipitation showed that the formation of the CDK2–p21 complex, but not the CDK2–p27, CDK4–p21, and CDK4–p27 complex, was increased in the DPTH-N10-treated HUVEC. Kinase assay further demonstrated that CDK2, but not CDK4, kinase activity was decreased in the DPTH-N10-treated HUVEC. Pretreatment of HUVEC with a p21, but not p27, antisense oligonucleotide reversed the DPTH-N10-induced inhibition of [3H]thymidine incorporation into HUVEC. Taken together, these data suggest that DPTH-N10 inhibits HUVEC proliferation by increasing the level of p21 protein, which in turn inhibits CDK2 kinase activity, and finally interrupts the cell cycle. Capillary-like tube formation, aortic ring culture, and chick embryo chorioallantoic membrane (CAM) assays further demonstrated the anti-angiogenic effect of DPTH-N10.

Several reports have demonstrated that cantharidin is a strong anticancer compound in vitro; however, its in vivo usefulness is often limited due to its high systemic toxicity. In this study, we encapsulated cantharidin into pegylated liposomes and studied its activity against human breast cancer MCF-7 cells in vitro and its systemic toxicity in mice. Another two methods were also used to reduce the dosage of cantharidin, including labeling liposomal cantharidin with octreotide and exposing cells to hyperbaric oxygen. The cytotoxic activity of pegylated liposomal cantharidin was drastically reduced compared with free cantharidin in vitro. Octreotide-labeled pegylated liposomal cantharidin induced cell death by specifically targeting somatostatin receptors in MCF-7 cells. Cell death was augmented with a low dose of cantharidin under hyperbaric oxygen. Liposomal cantharidin had significantly less systemic toxicity than free cantharidin in vivo and also exhibited a high efficacy against antitumor growth in nude mice. These results suggest that the systemic toxicity of cantharidin can be mitigated by liposome encapsulation; however, that did not decrease its antitumor activity.

Taiwanofungus camphoratus (T. camphoratus), a fungus and a Taiwan-specific, well-known traditional Chinese medicine, has long been used to treat diarrhea, hypertension, itchy skin, and liver cancer. To gain a large amount of T. camphoratus, several culture techniques have been developed, including solid-state culture and liquid-state fermentation. Peroxisome proliferator-activated receptor gamma (PPARgamma) has been described as a hypoglycemic agent that increases insulin sensitivity in peripheral tissues and results in reduced blood glucose, insulin, and triglyceride levels in insulin-resistant animals and in type-2 (non-insulin-dependent) diabetic patients. In this study, we investigate the possibility that T. camphoratus might activate PPARgamma in vitro and hypolipidemic activity in vivo. The results show that an aqueous extract of the wild fruiting bodies of T. camphoratus was able to increase the PPARgamma activity in cells transfected with the PPARgamma expression plasmid and the AOx-TK reporter plasmid. Based on the cell experiment, we examined the hypolipidemic effect of wild fruiting bodies (WFT) and a solid-state culture (SST) of T. camphoratus on SD rats fed on a high-cholesterol (HC) diet. The results show that WFT significantly decreased the serum triglyceride level, but could not affect the cholesterol level. SST only slightly decreased the serum triglyceride level. In addition, both WFT and SST significantly decreased the serum alanine transaminase (ALT) level and protected against the liver damage induced by the HC diet from the results of a histological examination. These results suggest that T. camphoratus might contain PPARgamma ligands and result in a hypotriglyceridemic effect, and that it also exhibits a liver protective activity.

Chemotherapeutic drugs are usually designed to induce cancer cell death via cell cycle arrest and/or apoptosis pathways. In this study, we used the chemical drug 15,16-dihydrotanshinone I (DHTS) to inhibit breast cancer cell proliferation and tumor growth, and investigate the underlying molecular mechanisms. Human breast cancer cell lines MCF-7 and MDA-MB-231 were both used in this study, and DHTS was found to significantly decrease cell proliferation by a dose-dependent manner in both cells. Flow cytometry indicated that DHTS induced G1 phase arrest in synchronous MCF-7 and MDA-MB-231 cells. When analyzing the expression of cell cycle-related proteins, we found that DHTS reduced cyclin D1, cyclin D3, cyclin E, and CDK4 expression, and increased CDK inhibitor p27 expression in a dose-dependent manner. In addition, DHTS inhibited the kinase activities of CDK2 and CDK4 by an immunocomplex kinase assay. In addition, DHTS also induced apoptosis in both cells through mainly mitochondrial apoptosis pathways. We found that DHTS decreased the anti-apoptotic protein Bcl-xL level and increased the loss of mitochondria membrane potential and the amount of cytochrome c released. Moreover, DHTS activated caspase-9, caspase-3, and caspase-7 and caused cell apoptosis. The fact that DHTS-induced apoptosis could be blocked by pretreating cells with pan-caspase inhibitor confirmed that it is mediated through activation of the caspase-3-dependent pathway. In a nude mice xenograft experiment, DHTS significantly inhibited the tumor growth of MDA-MB-231 cells. Taken together, these results suggest that DHTS can inhibit human breast cancer cell proliferation and tumor growth, and might have potential chemotherapeutic applications.

Taiwanofungus camphoratus (syn. Antrodia camphorata), a medicinal mushroom in Taiwan, is reputed to provide several therapeutic benefits, but the wild fruiting body is very rare. In this study, we used Taiwanofungus camphoratus extracts from wild fruiting bodies and two types of artificial cultivation (solid-state culture and liquid-state fermentation) to examine their anti-inflammatory effects in microglia cells and their possible roles in protection against neurodegenerative diseases. First, EOC13.31 microglia was treated with various kinds of Taiwanofungus camphoratus extracts and lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) to evaluate the iNOS expression. Western blot and RT-PCR analysis showed that among the various kinds of extracts from wild fruiting bodies, methanol extracts were the most potent inhibitors of iNOS expression. Secondly, the potency of methanol extracts could be ranked as follows: extracts of wild fruiting body>solid-state culture>liquid-state fermentation. To clarify the mechanisms involved, methanol extracts from fruiting body were found to inhibit the phosphorylation of extracellular signal-regulated protein kinases (ERK), c-Jun NH2-terminal protein kinases (JNK) and signal transducer and activator of transcription-1 (STAT-1) induced by LPS/IFN-gamma. Methanol extracts from fruiting body also inhibited NF-kappaB activation through the prevention of inhibitor kappaB (IkappaB) degradation. Moreover, methanol extracts from wild fruiting body inhibited both the iNOS and cyclooxygenase-2 (COX-2) expression induced by beta-amyloid in microglia in a dose-dependent manner. In an animal model, we confirmed that methanol extracts from fruiting bodies were able to suppress ear edema, indicating that they have anti-inflammatory activity in vivo. These results suggest that Taiwanofungus camphoratus exhibits an anti-inflammatory activity that might contribute to the prevention of neurodegenerative diseases.

Phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P(2)) has been known to serve as a substrate for phosphatidylinositol 3-kinase (PI(3)K) and phosphoinositide-specific phospholipase C (PI-PLC), which can produce PtdIns(3,4,5)P(3) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) and diacylglycerol (DAG), respectively. In this study, we elucidated the role of PI-PLC during the LPS-activated mouse macrophages RAW264.7 treated with PI(3)K inhibitor wortmannin. First, wortmannin treatment enhanced Ins(1,4,5)P(3) production and iNOS expression in LPS-activated macrophages. Inhibition of PI(3)K by p85 siRNA also showed an enhancement of iNOS expression. On the other hand, overexpression of PI(3)K by ras-p110 expression plasmid significantly decreased iNOS expression in LPS-activated macrophages. In addition, overexpression of wild-type or dominant-negative Akt expression plasmid did not affect the iNOS expression in LPS-activated macrophages. Second, treatment of PI-PLC inhibitor U73122 reversed the enhancement of iNOS expression, the increase of phosphorylation level of ERK, JNK and p38, and the increase of AP-1-dependent gene expression in wortmannin-treated and LPS-activated macrophages. However, NF-kappaB activity determined by EMSA assay and reporter plasmid assay did not change during LPS-activated macrophages with or without wortmannin. We propose that the inhibition of PI(3)K by wortmannin in mouse macrophages enhances the PI-PLC downstream signals, and subsequently increases the LPS induction of iNOS expression independently of Akt pathway.

Combined effects of hyaluronate and indomethacin in the treatment of rabbits with antigen-induced arthritis (AIA) were evaluated by assessing joint swelling, C-reactive protein (CRP) and prostaglandin E(2) (PGE(2)) levels with periodic intra-articular (ia) injections of hyaluronate alone (HA group) and with either a low or high concentration of indomethacin (LI-HA or HI-HA group). End-point analyses included matrix metalloproteinases-3 (MMP-3) activity and macroscopic and histological joint examinations. Results demonstrated that treatment in LI-HA and HI-HA groups resulted in statistically significant suppression of CRP, PGE(2, )and MMP-3 in comparison with those of HA group. Inhibition of serum CRP was only observed in LI-HA group. The order of serum MMP-3 inhibition was LI-HAHI-HAHA. Based on macroscopic and histological analyses of pannus formation, hyperplasia, inflammation, joint leakage and erosion, and loss of proteoglycan, the only statistically significant improvement was shown in LI-HA group compared to HA group and HI-HA group compared to control group.

Wild and solid-state cultures (SSC) of Taiwanofungus camphoratus (aka Antrodia camphorata and Chang-chih [CC]) were sequentially extracted with cold water, methanol, and hot water to get cold-water-soluble (CWS), methanol-soluble (MS), and hot-water-soluble (HWS) extracts, respectively. Only the MS extract exhibited angiotensin-converting enzyme (ACE) inhibitory activities. The antihypertensive effects of the MS extract (10 mg/kg BW) were measured in spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats. MS extract of the SSC type was able to effectively lower the systolic blood pressure (SBP) and diastolic blood pressure (DBP) of SHR, but not of WKY rats, the results being significantly different from those for distilled water only (the blank). However, wild CC and its MS extract were not as effective as the SSC type in reducing SHR blood pressure and had no effect on WKY rats. SSC-type CC might be developed into a health food with the ability to regulate blood pressure.

Pectin is composed of complex polysaccharides rich in galactoside residues, and it is most abundant in citrus fruits. Pectin and modified pectin have been found to exhibit anti-mutagenic activity and inhibit cancer metastasis and proliferation, with no evidence of toxicity or other serious side effects. In this study, we investigated the inhibitory effect of pectin at different degrees of esterification (DEs) on the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-activated macrophages. Western blot and RT-PCR analyses demonstrated that 30% esterified pectin (DE30), DE60 pectin, and DE90 pectin significantly inhibited the protein and mRNA expressions of iNOS and COX-2 in LPS-activated macrophages, and DE90 pectin was the most-potent inhibitor. To clarify the mechanisms involved, DE90 pectin was found to inhibit the phosphorylation of MAPKs and IKK kinase activity. In addition, DE90 pectin inhibited the activation of NF-kappaB and AP-1 by electrophoretic mobility shift assay and transient transfection experiments. Finally, we found that DE90 pectin could bind with LPS, and might result in decreased binding of LPS to its receptor. These results suggest that modulation of iNOS and COX-2 expressions by dietary pectin may be important in cancer chemoprevention and anti-inflammation.

Quercetin is one of the most ubiquitous bioflavonoids in foods of plant origin. Although quercetin is generally considered to provide protection against oxidative injury and inflammation, recent studies have demonstrated that its cytoprotective effects occur within a narrow concentration range. We attempted to examine the concentration-dependent effect on proliferation and inflammation in the primary culture of rat aortic smooth muscle cells. We demonstrate that quercetin inhibited [3H]thymidine incorporation into rat aortic smooth muscle cells only at concentrationsor =50 microM in a concentration-dependent manner. Nevertheless, quercetin, at concentrationsor =100 microM, reduced cell viability; this was further characterized as being due to apoptosis, which occurred through the proteolytic activation of pro-caspase-3. Additionally, the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK) substantially increased in rat aortic smooth muscle cells exposed to 100 microM quercetin, results which differ from observations by others and ourselves of cells exposed toor =50 microM quercetin. Unlike P-JNK and P-p38, the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/ERK2) was not significantly affected by the concentration-dependent effects of quercetin. Surprisingly, the adverse effects of higher concentrations of quercetin could be ameliorated by adding the antioxidants, catalase, and N-acetylcysteine (NAC). Furthermore, the electrophoretic mobility shift assay (EMSA) showed that rat aortic smooth muscle cells exposed to quercetin at concentrations ofor =50 microM caused concentration-dependent inhibition of nuclear factor kappa B (NF-kappaB) activity, whereas concentrations ofor =100 microM resulted in increased NF-kappaB binding activity. We demonstrate for the first time that quercetin at low concentrations has antiproliferative and antiinflammatory effects, but at concentrations ofor =100 microM, is likely to induce the opposite effects on rat aortic smooth muscle cells.

Heme oxygenase-1 (HO-1) is induced as a beneficial and adaptive response in cells and tissues exposed to oxidative stress. Herein we examined how various eicosanoids affect the induction of HO-1, and the possible mechanism underlying 15-deoxy-Delta(12,14)- prostaglandin J(2) (15d-PGJ(2))-induced HO-1 expression. PGH(2), PGD(2) and its metabolites of the PGJ(2) series, and PGA(1) markedly induced the protein expression of HO-1. Arachidonic acid (AA), docosahexaenoic acid (DHA), PGE(2), PGF(2 alpha), and thromboxane B(2) (TXB(2)) were shown to have no effect on the induction of HO-1. 15d-PGJ(2) was the most potent activator achieving significance at 5 microM. Although 15d-PGJ(2) significantly activated the MAPKs of JNK and ERK, the activation of JNK and ERK did not contribute to the induction of HO-1 as determined using transfection of dominant-negative plasmids and MAPKs inhibitors. Additional experiment indicated that 15d-PGJ(2) induced HO-1 expression through peroxisome proliferator-activated receptor (PPAR)-independent pathway. 15d-PGJ(2) significantly decreased the intracellular level of reduced glutathione; and the thiol antioxidant, N-acetyl-L-cysteine (NAC), and the thiol-reducing agent, dithiothreitol (DTT), inhibited the induction of HO-1 by 15d-PGJ(2). Finally, NAC and DTT exhibited significant inhibition of HO-1 mRNA and HO-1 promoter reporter activity induced by 15d-PGJ(2). These results suggest that thiol antioxidant and reducing agents attenuate the expression of HO-1 induced by 15d-PGJ(2), and that the cellular thiol-disulfide redox status may be linked to HO-1 activation.

Garcinol is a polyisoprenylated benzophenone derivative of Garcinia indica fruit rind and other species. Recent studies have demonstrated that garcinol exhibited antioxidative effects in vitro. In this study, we found that garcinol inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-activated macrophages. Western blot analyzes and gel-shift assays revealed that garcinol strongly blocks the activation of eukaryotic transcription factor nuclear factor-kappa B (NF-kappa B)-induced by LPS. Moreover, transient transfection experiments showed that garcinol inhibited the NF-kappa B-dependent transcriptional activity. Based on these data, we demonstrated that inhibition of LPS-induced NF-kappa B activation occurred through suppressing the phosphorylation of I kappa B alpha and p38 mitogen-activated kinase (MAPK). Garcinol also lowers the LPS-induced increase of intracellular reactive oxygen species (ROS), which contributes to the activation of NF-kappa B. The NF-kappa B signaling pathway leads to inflammatory reaction and our results suggest that garcinol suppresses the expression of iNOS in this pathway.

Our previous studies demonstrated that the oral antifungal agent ketoconazole (KT) induces apoptosis and G0/G1 phase cell cycle arrest in human cancer cell lines. In this study, we first demonstrated that KT (1 microM) potentiated the apoptotic effects of nocodazole (ND, 1 nM) in COLO 205 cancer cells. We further demonstrated the therapeutic efficacy of a combined treatment of KT (50 mg/kg/three times per week) and ND (5 mg/kg/three times per week) in vivo by treating athymic mice bearing COLO 205 tumor xenografts. The antitumor effects of ND were significantly potentiated by KT in mice after 6 wk of treatment. No gross signs of toxicity were observed in mice receiving these treatment regimens. The apoptotic cells were detected in a microscopic view of the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining and by observation of DNA fragmentation in KT + ND-treated tumor tissues. The levels of cell cycle regulatory proteins were determined by Western blot analysis. Treatment with KT inhibits tumor growth through elevation of p53, p21/CIP1, and p27/KIP1 as well as inhibition of cyclin D3 and cyclin-dependent kinase 4 protein expression. Immunohistochemical staining analysis showed that p53, p21/CIP1, and p27/KIP1 immunoreactivity were induced in the tumor tissues. To clarify the roles of the p21/CIP1 and p27/KIP1 protein expression involved in G(0)/G(1) arrest and/or apoptosis induced by a combined treatment with KT and ND, antisense oligodeoxynucleotides (ODNs) specific to p21/CIP1 and p27/KIP1 were used. Our results demonstrated that apoptotic phenomena, including BAX induction and cytochrome C released from mitochondria induced by KT + ND, were significantly attenuated by pretreatment the cells with the p27/KIP1-specific antisense ODNs. These results indicate that p27/KIP1 protein does indeed play a critical role in the KT + ND-induced apoptosis. Our study revealed the molecular mechanism of KT + ND in regression of the tumor growth. The apoptotic effects of KT in a great variety of cancer cells make it a very attractive agent for cancer chemotherapy.

Carnosol is a naturally occurring phytopolyphenol found in rosemary. Carnosol functions as antioxidant and anticarcinogen. In the present study, we compared the antioxidant activity of carnosol and other compounds extracted from rosemary. Carnosol showed potent antioxidative activity in alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH) free radicals scavenge and DNA protection from Fenton reaction. High concentrations of nitric oxide (NO) are produced by inducible NO synthase (iNOS) in inflammation and multiple stages of carcinogenesis. Treatment of mouse macrophage RAW 264.7 cell line with carnosol markly reduced lipopolysaccharide (LPS)-stimulated NO production in a concentration-related manner with an IC50 of 9.4 microM; but other tested compounds had slight effects. Western blot, reverse transcription-polymerase chain reaction, and northern blot analyses demonstrated that carnosol decreased LPS-induced iNOS mRNA and protein expression. Carnosol treatment showed reduction of nuclear factor-kappaB (NF-kappaB) subunits translocation and NF-kappaB DNA binding activity in activated macrophages. Carnosol also showed inhibition of iNOS and NF-kappaB promoter activity in transient transfection assay. These activities were referred to down-regulation of inhibitor kappaB (IkappaB) kinase (IKK) activity by carnosol (5 microM), thus inhibited LPS-induced phosphorylation as well as degradation of IkappaBalpha. Carnosol also inhibited LPS-induced p38 and p44/42 mitogen-activated protein kinase (MAPK) activation at a higher concentration (20 microM). These results suggest that carnosol suppresses the NO production and iNOS gene expression by inhibiting NF-kappaB activation, and provide possible mechanisms for its anti-inflammatory and chemopreventive action.

Miconazole (MIC), a promising oral antifungal agent, has been used worldwide in the treatment of superficial mycosis. In this study, we demonstrated that MIC dose dependently arrested various human cancer cells at the G0/G1 phase of the cell cycle. The protein levels of p53, p21/Cip1, and p27/Kip1 were significantly elevated by MIC treatment in COLO 205 cells. Electrophoretic mobility gel shift assays showed that the nuclear extracts of the MIC-treated COLO 205 cells exerted a significant binding between wild-type p53 and its consensus-binding site present in the p21/Cip1 promoter. These results suggested that the p53-associated signaling pathway is involved in the regulation of MIC-induced cancer cell growth arrest. By immunoblot analysis, we demonstrated that cyclin D3 and cyclin-dependent kinase-4 (CDK4) protein levels were inhibited by MIC treatment in the cancer cells. Significant therapeutic effect was further demonstrated in vivo by treating nude mice bearing COLO 205 tumor xenografts with MIC (50 mg/kg ip). The protein expression of p53 was significantly increased in MIC-treated tumor tissues by immunohistochemical staining and Western blotting analysis. DNA fragmentation and TUNEL assay were performed and demonstrated that apoptosis occurred in tumor tissues treated with MIC. Our study provides the novel mechanisms of antitumor effects of MIC and such results may have significant applications for cancer chemotherapy.

Among black tea polyphenols, theaflavins were generally considered to be the most effective in cancer chemoprevention. In this study, we examined the inhibitory effects of black tea polyphenols, including theaflavin (TF-1), a mixture (TF-2) of theaflavin-3-gallate and theaflavin-3'-gallate, theaflavin-3,3'-digallate (TF-3), and the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced edema and ornithine decarboxylase (ODC) activity. Topical application of these polyphenols onto the mouse resulted in inhibition of TPA-induced ear edema and skin epidermal ODC activity. The inhibitory order was as follows: TF-3 > TF-2 approximately equal to EGCG > TF-1. Western and Northern blots indicated that TF-3 significantly reduced the protein and mRNA levels of ODC in TPA-treated mouse skin and NIH 3T3 cells, whereas EGCG showed less activity. EGCG and TF-3 were able to inhibit the ODC enzyme activity in vitro. Furthermore, TF-3 also significantly reduced the basal promoter activity of the ODC gene in NIH 3T3 cells that were transiently transfected with ODC reporter plasmid. These results suggested that TF-3 was a potential inhibitor of ODC activity and TPA-induced edema and might be effective in cancer chemoprevention.

Peroxisome proliferator-activated receptor (PPAR)gamma transcription factor has been implicated in anti-inflammatory response. Of the compounds tested, apigenin, chrysin, and kaempferol significantly stimulated PPAR gamma transcriptional activity in a transient reporter assay. In addition, these three flavonoids strongly enhanced the inhibition of inducible cyclooxygenase and inducible nitric oxide synthase promoter activities in lipopolysaccharide-activated macrophages which contain the PPAR gamma expression plasmids. However, these three flavonoids exhibited weak PPAR gamma agonist activities in an in vitro competitive binding assay. Limited protease digestion of PPAR gamma suggested these three flavonoids produced a conformational change in PPAR gamma and the conformation differs in the receptor bound to BRL49653 versus these three flavonoids. These results suggested that these three flavonoids might act as allosteric effectors and were able to bind to PPAR gamma and activate it, but its binding site might be different from the natural ligand BRL49653.

Disulfiram is frequently used in the treatment of alcoholism. In this study, we found that CuCl(2) (1-10 microM), but not other metal ions (Fe(2+), Zn(2+), Pb(2+)), markedly potentiated disulfiram-induced cytotoxicity by 440-fold in primary astrocytes. Thus, the molecular mechanisms of the cytotoxic effects induced by the disulfiram-Cu(2+) complex were explored. The changes in morphology (nuclear condensation and apoptotic body formation) and hypodiploidy of DNA suggested that the disulfiram-Cu(2+) complex induced an apoptotic process. Our studies of the death-signaling pathway reveal that decreased mitochondrial membrane potential, increased free radical production, and depletion of non-protein-thiols (glutathione) were involved. The disulfiram-Cu(2+) complex activated c-Jun-amino-terminal kinase (JNK) and caspase-3 followed by poly (ADP-ribose) polymerase degradation in a time-dependent manner. Moreover, the cellular Cu content was markedly increased and the copper chelator bathocuproine disulfonate abolished all of these cellular events, suggesting that Cu(2+) is essential for death signaling. The antioxidants N-acetylcysteine and vitamin C also inhibited the cytotoxic effect. Thus, we conclude that the disulfiram-Cu(2+) complex induces apoptosis and perhaps necrosis at a late stage mediated by oxidative stress followed by sequential activation of JNK, caspase-3 and poly (ADP-ribose) polymerase degradation. These findings imply that the axonal degeneration and neurotoxicity observed after the chronic administration of disulfiram are perhaps, at least in part, due to the cytotoxic effect of the disulfiram-Cu(2+) complex formed endogenously.

(-)-Epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, was shown to have cancer chemopreventive activity. In this study, we examined the antimetastatic effects of EGCG or the combination of EGCG and dacarbazine on B16-F3m melanoma cells in vitro and in vivo. First, the antimetastatic potentials of five green tea catechins were examined by soft agar colony formation assay, and the results show that EGCG was more effective than the other catechins in inhibiting soft agar colony formation. Second, EGCG dose-dependently inhibited B16-F3m cell migration and invasion by in vitro Transwell assay. Third, EGCG significantly inhibited the spread of B16-F3m cells on fibronectin, laminin, collagen, and Matrigel in a dose-dependent manner. In addition, EGCG significantly inhibited the tyrosine phosphorylation of focal adhesion kinase (FAK) and the activity of matrix metalloproteinase-9 (MMP-9). In animal experiments, EGCG alone reduced lung metastases in mice bearing B16-F3m melanomas. However, a combination of EGCG and dacarbazine was more effective than EGCG alone in reducing the number of pulmonary metastases and primary tumor growths, and increased the survival rate of melanoma-bearing mice. These results demonstrate that combination treatment with EGCG and dacarbazine strongly inhibits melanoma growth and metastasis, and the action mechanisms of EGCG are associated with the inhibition of cell spreading, cell-extracellular matrix and cell-cell interactions, MMP-9 and FAK activities.

Ketoconazole is an oral-antifungal agent that has been used worldwide in the treatment of some hormone-dependent human cancer. In this study, we demonstrated that ketoconazole (20 microM) induced various types of human cancer cell growth arrest in the G0/G1 phase. Our results revealed that ketoconazole-induced growth arrest was more profound in COLO 205 and Hep G2 (with wild-type p53) than in HT 29 (p53 His(273) mutant) and Hep 3B (with deleted p53) cells. The protein levels of p53, p21/Cip1, and p27/Kip1 were significantly elevated by ketoconazole (10 microM) treatment in COLO 205 but not in HT 29 cells. The ketoconazole-induced G0/G1 phase arrest in COLO 205 cells was attenuated by p53-specific antisense oligodeoxynucleotides (20 microM) treatment. These results suggested that the p53-associated signaling pathway is involved in the regulation of ketoconazole-induced cancer cell growth arrest. By Western blot analysis, we demonstrated that cyclin D3 and CDK4 protein but not other G0/G1 phase regulatory protein levels were decreased by ketoconazole-treatment in both COLO 205 and HT 29 cells. Our study provides the basis of molecular mechanisms for ketoconazole in growth inhibition of human cancer cells and such results may have significant applications for cancer chemotherapy.

Prostaglandins biosynthesis and nitric oxide production have been implicated in the process of carcinogenesis and inflammation. In this study, we investigated the effect of various flavonoids and (-)-epigallocatechin-3-gallate on the activities of inducible cyclooxygenase (COX-2) and inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. Apigenin, genistein and kaempferol were markedly active inhibitors of transcriptional activation of COX-2, with IC(50) < 15 microM. In addition, apigenin and kaempferol were also markedly active inhibitors of transcriptional activation of iNOS, with IC(50) < 15 microM. Of those compounds tested, apigenin was the most potent inhibitor of transcriptional activation of both COX-2 and iNOS. Western and northern blot analyses demonstrated that apigenin significantly blocked protein and mRNA expression of COX-2 and iNOS in LPS-activated macrophages. Transient transfection experiments showed that LPS caused an approximately 4-fold increase in both COX-2 and iNOS promoter activities, these increments were suppressed by apigenin. Moreover, electrophoretic mobility shift assay (EMSA) experiments indicated that apigenin blocked the LPS-induced activation of nuclear factor-kB (NF-kB). The inhibition of NF-kB activation occurs through the prevention of inhibitor kB (IkB) degradation. Transient transfection experiments also showed that apigenin inhibited NF-kB-dependent transcriptional activity. Finally, we showed that apigenin could inhibit the IkB kinase activity induced by LPS or interferon-gamma. The results of further studies suggest that suppression of transcriptional activation of COX-2 and iNOS by apigenin might mainly be mediated through inhibition of IkB kinase activity. This study suggests that modulation of COX-2 and iNOS by apigenin and related flavonoids may be important in the prevention of carcinogenesis and inflammation.

Osthole is extracted from the Chinese herbs Cnidium monnieri and Angelica pubescens, and it was found to have antitumor activity in vitro and in vivo. A series of osthole derivatives have been synthesized, and the N-hydroxycinnamide derivatives of osthole, WJ1376-1 and WJ1398-1 were found to have the greatest potential against human colon adenocarcinoma cells. In contrast to the parental osthole, both WJ1376-1 and WJ1398-1 were found to induce multinucleation and polyploidy by microscopic observation and flow cytometry. WJ1376-1 and WJ1398-1 significantly activated ataxia telangiectasia and rad3 related (ATR) kinase, which triggered activation of the checkpoint kinase 2 (Chk2) signaling pathway and then down regulated Cdc25 phosphatase and Cdc2/cyclin B kinase activities. WJ1376-1 and WJ1398-1 also inhibited the phosphorylation of Aurora A kinase, which is associated with important processes during mitosis. The presence of a "comet" DNA fragment and phosphorylation of p53 at Ser 15 clearly indicated that DNA damage occurred with WJ1376-1 and WJ1398-1 treatment. WJ1376-1 and WJ1398-1 ultimately induced apoptosis as evidenced by the upregulation of Bad and activation of caspases-3, -7, and -9. Furthermore, WJ1376-1 and WJ1398-1 also showed a great effect in attenuating tumor growth without affecting the body weight of xenograft nude mice. Taken together, these results suggest that the toxic activities of WJ1376-1 and WJ1398-1 were dissimilar to that of the parental osthole, which can induce cell polyploidy and G2/M cell cycle arrest in colon adenocarcinoma cells and may provide a potential therapeutic target for colon cancer treatment in the future.

Autooxidation of polyphenolic metabolites of benzene, such as hydroquionone (HQ), catechol (CT), 1,2,4-benzenetriol (BT) and pyrogallol (PG), produced several kinds of active oxygen species (AOS). BT and PG induced DNA breaks in the absence of metal ions, especially when producing AOS such as H 2 O 2 , O 2 − , HO • or 1 ΔgO 2 . HQ and CT did not result in double-strand DNA breaks, except when ferrous ion was added, indicating the participation of the Fenton reaction. Polyphenolic fractions isolated from green tea (GTP) exerted inhibitory effects on the autooxidation of BT and suppressive effects on H2O2 or HO• generated from phenolic metabolites of benzene in the presence of S9 or an in vivo system. Additionally, although the activities of antioxidant and phase II enzymes were elevated by both GTP and phenolic metabolites of benzene, GTP counteracted the lowering GSH caused by phenolic metabolites of benzene in rat liver. The above results suggest that GTP and phenolic metabolites of benzene are antagonistic in their response to AOS, especially hydroxyl radical.

In chronic liver diseases, regardless of their etiology, the development of fibrosis is the first step toward the progression to cirrhosis, portal hypertension, and hepatocellular carcinoma. Hepatic stellate cells (HSCs) are the main profibrogenic cells that promote the pathogenesis of liver fibrosis, and so it is important to identify the molecules that regulate HSCs activation and liver fibrosis. Niemann-Pick type C2 (NPC2) protein plays an important role in the regulation of intracellular cholesterol homeostasis by directly binding with free cholesterol. However, the roles of NPC2 in HSCs activation and liver fibrosis have not been explored in detail. Since a high-cholesterol diet exacerbates liver fibrosis progression in both rodents and humans, we propose that the expression of NPC2 affects free cholesterol metabolism and regulates HSCs activation. In this study, we found that NPC2 is decreased in both thioacetamide- and carbon tetrachloride-induced liver fibrosis tissues. In addition, NPC2 is expressed in quiescent HSCs, but its activation status is down-regulated. Knockdown of NPC2 in HSC-T6 cells resulted in marked increases in transforming growth factor-β1 (TGF-β1)-induced collagen type 1 α1 (Col1a1), α-smooth muscle actin (α-SMA) expression, and Smad2 phosphorylation. In contrast, NPC2 overexpression decreased TGF-β1-induced HSCs activation. We further demonstrated that NPC2 deficiency significantly increased the accumulation of free cholesterol in HSCs, increasing Col1a1 and α-SMA expression and activating Smad2, and leading to sensitization of HSCs to TGF-β1 activation. In contrast, overexpression of NPC2 decreased U18666A-induced free cholesterol accumulation and inhibited the subsequent HSCs activation. In conclusion, our study has demonstrated that NPC2 plays an important role in HSCs activation by regulating the accumulation of free cholesterol. NPC2 overexpression may thus represent a new treatment strategy for liver fibrosis.

AMP-activated protein kinase (AMPK) is an energy sensor that regulates cellular metabolism. Activation of AMPK in skeletal muscles, the liver, and adipose tissues results in a favorable metabolic milieu for preventing and treating type 2 diabetes, i.e., decreased levels of circulating glucose, plasma lipids, and ectopic fat accumulation and enhanced insulin sensitivity. Osthole was extracted from a Chinese herbal medicine, and we found that it had glucose lowering activity in our previous study. However, the detailed glucose lowering mechanisms of osthole are still unclear. In this study, we used skeletal muscle cells to examine the underlying molecular mechanisms of osthole's glucose lowering activity. A Western blot analysis revealed that osthole significantly induced phosphorylation of AMPK and acetyl-CoA carboxylase (ACC). Next, we found that osthole significantly increased the level of translocation of glucose transporter 4 (GLUT4) to plasma membranes and glucose uptake in a dose-dependent manner. Osthole-induced glucose uptake was reversed by treatment with Compound C, an AMPK inhibitor, suggesting that osthole-induced glucose uptake was mediated in an AMPK-dependent manner. The increase in the AMP:ATP ratio was involved in osthole's activation of AMPK. Finally, we found that osthole counteracted hyperglycemia in mice with streptozotocin-induced diabetes. These results suggest that the increase in the AMP:ATP ratio by osthole triggered activation of the AMPK signaling pathway and led to increases in plasma membrane GLUT4 content and glucose uptake level. Therefore, osthole might have potential as an antidiabetic agent for treating diabetes.

The goal of this study was to assess the incorporation of exudates of human platelet-rich fibrin (hPRF) that is abundant in platelet cytokines and growth factors into biodegradable fibrin (FB) scaffolds as a regeneration matrix for promoting chondrocyte proliferation and re-differentiation. hPRF was obtained from human blood by centrifugation without an anticoagulant, and the exudate of hPRF was collected and mixed with bovine fibrinogen, and then thrombin was added to form the FB scaffold. Proliferation and differentiation of human primary chondrocytes and a human chondrosarcoma cell line, the SW-1353, embedded in the three-dimensional (3D) scaffolds and on the two-dimensional (2D) surface of the FB scaffolds so produced were evaluated in comparison with an agarose (AG) scaffold serving as the control. Results demonstrated that the amounts of these cytokines and growth factors in hPRF exudates were higher than those in the blood-derived products except for TGF-β1. Chondrocytes and SW1353 cells on the 2D and 3D FB scaffolds with the addition of the exudates of PRF exhibited more-available proliferation and differentiation than cells on 2D and 3D FB and AG scaffolds. It was concluded that FB scaffolds can provide an appropriate environment for chondrocyte proliferation and re-differentiation, and it could be improved by adding exudates of hPRF. These 3D scaffolds have great promise for cartilage tissue engineering.

Vitis thunbergii var. taiwaniana (VTT) is a wild grape native to Taiwan, where it has been used as a folk medicine. In this study, we found that the branch and leaf ethanol extracts of VTT significantly inhibited the inducible nitric oxide (NO) synthase protein expression and NO production in BV2 microglia. Using primary neuronal cells, kainic acid (KA) significantly increased hydrogen peroxide production in a dose-dependent manner. All four ethanol extracts of VTT significantly decreased hydrogen peroxide production. However, only root and branch ethanol extracts were able to prevent the neuronal cell death induced by KA in vitro. In the animal study, administration of all four plant part extracts of VTT delayed the onset of seizure and decreased the hippocampus neuronal cell loss, and the neuroprotective activity could be ranked as follows: branch approximately leafroottrunk. The results suggest that VTT extracts have a potential to prevent neurodegeneration through the antioxidative activity by their ability to inhibit NO and hydrogen peroxide production.

BackgroundPropolis, an ancient herbal medicine, has been reported the beneficial effect both in asthma patients and murine model of asthma, but the mechanism was not clearly understood. In this study, the effect of caffeic acid phenethyl ester (CAPE), the most extensively studied components in propolis, on the functions of human monocyte-derived dendritic cells (MoDCs) was investigated.ResultsCAPE significantly inhibited IL-12 p40, IL-12 p70, IL-10 protein expression in mature healthy human MoDCs stimulated by lipopolysaccharides (LPS) and IL-12 p40, IL-10, IP-10 stimulated by crude mite extract. CAPE significantly inhibited IL-10 and IP-10 but not IL-12 expression in allergic patients' MoDCs stimulated by crude mite extract. In contrast, the upregulation of costimulatory molecules in mature MoDCs was not suppressed by CAPE. Further, the antigen presenting ability of DCs was not inhibited by CAPE. CAPE inhibited IκBα phosphorylation and NF-κB activation but not mitogen-activated protein kinase (MAPK) family phosphorylation in human MoDCs.ConclusionThese results indicated that CAPE inhibited cytokine and chemokine production by MoDCs which might be related to the NF-κB signaling pathway. This study provided a new insight into the mechanism of CAPE in immune response and the rationale for propolis in the treatment of asthma and other allergic disorders.

Osthole is an agent isolated from Cnidium monnieri (L.) Cusson and Angelica pubescens and has been used to treat several diseases, including metabolic syndromes. To investigate the hypoglycemic effects of osthole in diabetic db/db mice and the underlying mechanisms of these effects by in vitro assay, diabetic db/db mice and cell experiments were utilized to understand its possible effects. Osthole significantly activated both PPARalpha and PPARgamma in a dose-dependent manner based on the results of the transition transfection assay. The activation of PPARalpha and PPARgamma by osthole also resulted in an increase in the expression of PPAR target genes such as PPAR itself, adipose fatty acid-binding protein 2, acyl-CoA synthetases, and carnitine palmitoyltransferase-1A. In vitro results suggested that osthole might be a dual PPARalpha/gamma activator, but its chemical structure differed from that of the thiazolidinedione class of antidiabetic drugs. In addition, osthole markedly activated the AMP-activated protein kinase and its downstream acetyl CoA carboxylase molecules by increasing their phosphorylation levels. Finally, obese diabetic db/db mice were treated with osthole by different administered routes, and osthole was found to markedly reduce blood glucose level. Interestingly, osthole did not reduce the blood insulin or lipid levels, two phenomena that did occur in animals treated with insulin sensitizers like PPAR agonists. These results suggest that osthole can alleviate hyperglycemia and could be potentially developed into a novel drug for treatment of diabetes mellitus.

Astrocytes play a critical neurotrophic and neuroprotective role in the brain, and improper function of these cells may contribute to the onset of neurodegenerative diseases. Because astrocytes are known to be enriched with Cu chaperone proteins, it is important to understand the factors that may lead to cytotoxic effects of Cu on astrocytes. In this report, we demonstrated a dramatic potentiating effect of neocuproine (NCP), a membrane permeable metal chelator, on Cu, but not Fe or Pb, in inducing apoptosis of cultured astrocytes. It was estimated that individually, CuCl2 and NCP only weakly exhibited cytotoxic effects on astrocytes, with EC50 of 180 and 600 microM, respectively. However, NCP at a nontoxic concentration of 10 microM markedly reduced EC50 of Cu to 0.35 microM (physiological concentration) and Cu (10 microM) reduced EC50 of NCP down to 0.06 microM. The mechanisms underlying these dramatic potentiation effects are elucidated. NCP increased the intracellular concentration of Cu in astrocytes and a nonpermeable Cu chelator, bathocuproine disulfonate was able to abolish all of the apoptotic signaling. Cell death was determined to be via apoptosis due to increased reactive oxygen species production, mitochondrial dysfunction, depletion of glutathione and adenosine triphosphate, cytochrome c release, c-Jun N-terminal kinase, and caspase-3 activation, and poly-ADP-ribose polymerase degradation. This finding, coupled with our previous reports, suggests that metal chelators (NCP, dithiocarbamate and disulfiram) should be cautiously used as they may potentiate a cytotoxic effect of endogenous Cu on astrocytes. Their clinical implications in the etiology of neurodegenerative diseases deserve further investigation.

BACKGROUND Epigallocatechin-3-gallate (EGCG) is the most potent antioxidant of all the green tea catechins. The objective of the present study was to find out whether it improved the age-induced hemorheological abnormalities or not. METHODS AND RESULTS Twenty-four-month-old aging guinea pigs were used to test the effects of EGCG on hemorheological properties. Orally feeding EGCG at 30 mg x kg(-1) x day (-1) for 28 days resulted in a decrease in erythrocyte membrane malondialdehyde, and further improved erythrocyte deformability and blood viscosity at high and middle shear rates. In addition, it also significantly reduced erythrocyte aggregation, and improved blood viscosity at low shear rates and viscoelasticity at oscillatory flow. Consequently, efficiency of blood oxygen transport in aged guinea pigs increased after administration with EGCG. CONCLUSIONS Orally feeding EGCG 30 mg x kg(-1) x day(-1) for 28 days significantly improves the abnormal hemorheological parameters. These results suggest that EGCG has considerable potential as a substantial component for the development of new drugs or functional foods in improving the age-induced hemorheological abnormalities.

AIM: To evaluate the preventive effect of Ginkgo biloba extract (GbE) on ethanol-induced gastric mucosal injuries in rats. METHODS: Female Wistar albino rats were used for the studies. We randomly divided the rats for each study into five subgroups: normal control, experimental control, and three experimental groups. The gastric ulcers were induced by instilling 1 mL 50% ethanol into the stomach. We gave GbE 8.75, 17.5, 26.25 mg/kg intravenously to the experimental groups respectively 30 min prior to the ulcerative challenge. We removed the stomachs 45 min later. The gastric ulcers, gastric mucus and the content of non-protein sulfhydryl groups (NP-SH), malondialdehyde (MDA), c-Jun kinase (JNK) activity in gastric mucosa were evaluated. The amount of gastric juice and its acidity were also measured. RESULTS: The findings of our study are as follows: (1) GbE pretreatment was found to provide a dose-dependent protection against the ethanol-induced gastric ulcers in rats; (2) the GbE pretreatment afforded a dose-dependent inhibition of ethanol-induced depletion of stomach wall mucus, NP-SH contents and increase in the lipid peroxidation (increase MDA) in gastric tissue; (3) gastric ulcer induced by ethanol produced an increase in JNK activity in gastric mucosa which also significantly inhibited by pretreatment with GbE; and (4) GbE alone had no inhibitory effect on gastric secretion in pylorus-ligated rats. CONCLUSION: The finding of this study showed that GbE significantly inhibited the ethanol-induced gastric lesions in rats. We suggest that the preventive effect of GbE may be mediated through: (1) inhibition of lipid peroxidation; (2) preservation of gastric mucus and NP-SH; and (3) blockade of cell apoptosis.

Resveratrol-mediated heme oxgenase-1 (HO-1) induction has been shown to occur in primary neuronal cultures and has been implicated as having potential neuroprotective action. Further, antioxidant properties of resveratrol have been reported to protect against coronary heart disease. We attempted to examine the HO-1 inducing potency of resveratrol and the regulatory mechanism of its induction in rat aortic smooth muscle cells (RASMC). We showed that resveratrol-induced HO-1 expression was concentration- and time-dependent. The level of HO-1 expression and its promoter activity mediated by resveratrol was attenuated by nuclear factor-kappa B (NF-kappaB) inhibitors, but not by mitogen-activated protein kinase (MAPK) inhibitors. Deletion of NF-kappaB binding sites in the promoter region strongly reduced luciferase activity. Collectively, we suggest that resveratrol-mediated HO-1 expression occurs, at least in part, through the NF-kappaB pathway.

AIM: To explore both the in vitro and in vivo effects of denbinobin against colon cancer cells and clarify its underlying signal pathways. METHODS: We used COLO 205 cancer cell lines and nude mice xenograft model to study the in vitro and in vivo anti-cancer effects of denbinobin. RESULTS: Denbinobin at concentration of 10-20 μmol/L dose-dependently suppressed COLO 205 cell proliferation by MTT test. Flow cytometry analysis and DNA fragmentation assay revealed that 10-20 μmol/L denbinobin treatment induced COLO 205 cells apoptosis. Western blot analysis showed that caspases 3, 8, 9 and Bid protein were activated by denbinobin treatment to COLO 205 cells accompanied with cytochrome c and apoptosis-inducing factor (AIF) translocation. Pretreatment of MEK 1 inhibitor ({"type":"entrez-nucleotide","attrs":{"text":"U10126","term_id":"794227","term_text":"U10126"}}U10126), but not p38 inhibitor (SB203580) and JNK inhibitor (SP600125), reversed denbinobin-induced caspase 8, 9 and Bid activation in COLO 205 cells suggesting that extracellular signal-regulated kinase were involved in the denbinobin-induced apoptosis in COLO 205 cells. Significant regression of tumor up to 68% was further demonstrated in vivo by treating nude mice bearing COLO 205 tumor xenografts with denbinobin 50 mg/kg intraperitoneally. CONCLUSION: Our findings suggest that denbinobin could inhibit colon cancer growth both in vitro and in vivo. Activation of extrinsic and intrinsic apoptotic pathways and AIF were involved in the denbinobin-induced COLO 205 cell apoptosis.

We have demonstrated previously that terbinafine (TB), an oral antifungal agent used in the treatment of superficial mycosis, suppresses proliferation of various cultured human cancer cells in vitro and in vivo by inhibiting DNA synthesis and activating apoptosis. In our study, we further demonstrated that TB at a range of concentrations (0-120 microM) dose-dependently decreased cell number in cultured human umbilical vascular endothelial cells (HUVEC). Terbinafine was not cytotoxic at a concentration of 120 microM, indicating that it may have an inhibitory effect on the cell proliferation in HUVEC. The TB-induced inhibition of cell growth rate is reversible. [(3)H]thymidine incorporation revealed that TB reduced the [(3)H]thymidine incorporation into HUVEC during the S-phase of the cell-cycle. Western blot analysis demonstrated that the protein levels of cyclin A, but not cyclins B, D1, D3, E, CDK2 and CDK4, decreased after TB treatment. The TB-induced cell-cycle arrest in HUVEC occurred when the cyclin-dependent kinase 2 (CDK2) activity was inhibited just as the protein level of p21 was increased and cyclin A was decreased. Pretreatment of HUVEC with a p21 specific antisense oligonucleotide reversed the TB-induced inhibition of [(3)H]thymidine incorporation. Taken together, these results suggest an involvement of the p21-associated signaling pathway in the TB-induced antiproliferation in HUVEC. Capillary-like tube formation and chick embryo chorioallantoic membrane (CAM) assays further demonstrated the anti-angiogenic effect of TB. These findings demonstrate for the first time that TB can inhibit the angiogenesis.

Previously we demonstrated that progesterone at physiologic levels dose dependently inhibited cell proliferation in cultured rat aortic smooth muscle cells (RASMCs). However, the molecular mechanism underlying of progesterone-induced antiproliferation was not clear. Here we demonstrated that progesterone induced a reduction of the [(3)H]thymidine incorporation into RASMCs during the S-phase of the cell cycle. Western blotting analysis revealed that the protein levels of cyclin A, cyclin E, and cyclin-dependent-kinase (CDK) 2 but not cyclin D1 and CDK4 decreased after progesterone treatment, but those of CDK-inhibitory proteins, p21 and p27, increased. Immunoprecipitation showed that the formations of the CDK2-p21 and CDK2-p27 complex were increased and the assayable CDK2 kinase activity was decreased in the progesterone-treated RASMCs. In contrast, the formations of the CDK4-p21 and CDK4-p27 complex and the assayable CDK4 kinase activity were not changed significantly by progesterone treatment. Pretreatment of RASMCs with a p21 or p27 antisense oligonucleotide reduced the progesterone-induced inhibition of [(3)H]thymidine incorporation into RASMCs. In conclusion, these data suggest that progesterone inhibits RASMCs proliferation by increasing the levels of p21 and p27 protein, which in turn inhibit CDK2 kinase activity, and finally interrupt the cell cycle.

Terfenadine (TF) is a highly potent histamine H1 receptor antagonist that in clinically effective doses is free of significant central nervous system side effects. Ketoconazole (KT) is a worldwide used oral antifungal agent with a broad spectrum of activity against both superficial and systemic mycosis. Simultaneously administration of KT and TF has been reported to induce several potent symptoms including cardiotoxicity, excitotoxicity, inhibition of blood mononuclear cells proliferation, and cardiovascular toxicity. However, the intracellular molecular mechanisms of TF-KT interactions in cells were still uncertain. In this study, we first demonstrated that TF (5-30 microM) induced apoptosis in several types of human cancer cell lines including human hepatoma (Hep G2), colorectal cancer (COLO 205), and fibroblast (CCD 922SK) cells for 24 h. The cellular responses to TF-induced apoptosis were demonstrated to be associated with the p53-signaling pathway, including induction of p53, p21/Cip1, p27/Kip1, bax protein expression and inhibition of bcl-2 protein expression. To realized the role of H1 receptor involved in TF-induced apoptosis, different H1 receptor antagonists including promethazine, mequitazine, and chlorpheniramin (50-100 microM) were administered and demonstrated that these chemicals cannot induced apoptosis through the H1 receptor signaling pathway. Interestingly, we found that the apoptotic effect of TF (2.5 microM) was significantly potentiated by KT (1 microM) treatment in Hep G2 cells through inhibition of the cytochrome p450 3A4 (CYP 3A4) activity. Such results were demonstrated by decreased of the TF activity with recombinant CYP 3A4, which prepared from baculovirus-infected insect cells. Our results provide the molecular basis of TF-KT interaction and this information should allow more rational forecasting of the risk for TF therapy during co-administration of KT.

Tea is one of the most popular beverages in the world. Several reports have shown that both green tea and black tea were able to inhibit tumor cell proliferation in animal models. In this study, we investigated the inhibitory effects of black tea polyphenols including theaflavin (TF-1), the mixture (TF-2) of theaflavin-3-gallate (TF-2a), and theaflavin-3'-gallate (TF-2b), theaflavin-3,3'-digallate (TF-3), thearubigin (TR), and a major green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) on 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced protein kinase C (PKC) and transcription activator protein-1 (AP-1) binding activities in NIH3T3 cells. On analysis of PKC activity with partial purified preparation, TPA (100 ng/mL) treatment was able to elevate membrane-associated PKC activity approximately 3-fold, and treatment with TF-3 (20 microM) and EGCG (20 microM) showed 94.5% and 9.4% suppression on TPA-induced PKC activity, respectively. Translocation of PKCalpha protein from cytosol to membrane was detected in TPA-treated NIH3T3 cells, and TF-3 was able to block its translocation. By in vitro kinase assay using myelin basic protein (MBP) as a PKC-specific substrate, we found that TPA treatment was able to increase PKC kinase activity by detection of phosphorylated MBP protein and TF-3 showed strongest inhibitory effect on its phosphorylation while EGCG was shown to be less effective. We also analyzed the AP-1 binding activity by electrophoretic mobility shift assay and c-Jun gene expression by northern blot and western blot, the results showed that TF-3 is the most potent inhibitor on TPA-induced AP-1 binding activity and c-Jun gene expression among these five tea polyphenols. Our results might provide new molecular basis for understanding the inhibitory effects of tea polyphenols on TPA-mediated tumor promotion.