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PURPOSE H1-antihistamines (AHs) may exert protective effects against cancer. This study investigated the association of AH use with the risk of hepatocellular carcinoma (HCC) in patients with hepatitis B virus (HBV), hepatitis C virus (HCV), or dual HBV-HCV virus infection. MATERIALS AND METHODS Patients with HBV, HCV, or dual HBV-HCV infection were enrolled from Taiwan's National Health Insurance Research Database and examined for the period from January 1, 2006, to December 31, 2015. We used the Kaplan-Meier method and Cox proportional hazards regression to evaluate the association between AH use and HCC risk. RESULTS We included patients with HBV infection (n = 521,071), HCV (n = 169,159), and dual HBV-HCV (n = 39,016). Patients with HBV, HCV, or dual virus infection who used AHs exhibited significantly lower risk of HCC relative to patients who did not use AH, with their adjusted hazard ratio being 0.489 (95% CI, 0.455 to 0.524), 0.484 (95% CI, 0.450 to 0.522), and 0.469 (95% CI, 0.416 to 0.529), respectively. Furthermore, there was a dose-response relationship between AH use and the risk of HCC in the HBV cohort. The adjusted hazard ratios were 0.597 (95% CI, 0.530 to 0.674), 0.528 (0.465 to 0.600), 0.470 (0.416 to 0.531), and 0.407 (0.362 to 0.457) for AH use of 28-42, 43-63, 64-119, and ≥ 120 cumulative defined daily doses, respectively, relative to no AH use. Additionally, there was also a dose-response relationship between AH use and the risk of HCC in the HCV and dual HBV-HCV cohorts. CONCLUSION AH use may reduce the risk for HCC among patients with HBV, HCV, or dual infection in a dose-dependent manner. Further mechanistic research is needed.

Background Honokiol has been reported to possess anti-inflammatory and neuroprotective activities. However, the poor aqueous solubility of honokiol limits its clinical application for systemic administration. Purpose This study aims to develop a novel formulation of nanosome-encapsulated honokiol (NHNK) for intravenous therapy against mouse experimental autoimmune encephalomyelitis (EAE) that mimics human multiple sclerosis. Methods Nanosomes and NHNK were prepared by using an ultra-high pressure homogenization (UHPH) method. Mice were treated with NHNK or empty nanosomes during the peak phase of EAE symptoms. Symptoms of EAE were monitored and samples of the spinal cord were obtained for histopathological examinations. Results The stock of NHNK containing honokiol in the nanosome formulation, which showed the structure of single phospholipid bilayer membranes, was well formulated with the particle size of 48.0 ± 0.1 nm and the encapsulation efficiency 58.1 ± 4.2%. Intravenous administration of NHNK ameliorated the severity of EAE accompanied by a significant reduction of demyelination and inflammation in the spinal cord. Furthermore, NHNK decreased the number of IL-6+, Iba-1+TNF +, Iba-1+IL-12 p40+, and CD3+IFN-γ+ cells infiltrating the spinal cord. Conclusion The UHPH method simplified the preparation of NHNK with uniformly distributed nanosize and high encapsulation efficiency. Intravenous administration of NHNK ameliorated the severity of EAE by suppressing the infiltration of activated microglia and Th1 cells into the spinal cord. Collectively, these results suggest that the formulation of NHNK is a prospective therapeutic approach for inflammatory CNS diseases, such as multiple sclerosis.

Simple Summary TNF-α is considered to be a potential therapeutic drug for cancer, but the systemic toxicity problem still exists in clinical use. MCPIP1 plays a crucial role in anti-inflammatory and anti-viral contexts, as well as in the inhibition of cell growth. This study aims to investigate the roles of MCPIP1 in TNF-α-treated cells and their underlying molecular mechanisms. MCPIP1 was found to enhance TNF-α-induced apoptosis by activating the caspase cascade, and pan-caspase inhibitor and the caspase-1/-4 inhibitor could reverse TNF-α/MCPIP1-mediated apoptosis. MCPIP1 was first identified with cleavage site of caspase-1/-4 and could be cleaved during apoptosis. Downregulation of NF-κB activation and a caspase-8 inhibitor, cFLIP, were associated with TNF-α/MCPIP1-mediated apoptosis. Abstract Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) is rapidly produced under proinflammatory stimuli, thereby feeding back to downregulate excessive inflammation. In this study, we used the stable, inducible expressions of wild-type (WT) MCPIP1 and an MCPIP1-D141N mutant in T-REx-293 cells by means of a tetracycline on (Tet-on) system. We found that WT MCPIP1 but not MCPIP1-D141N mutant expression dramatically increased apoptosis, caspase-3, -7, -8, and -9 activation, and c-Jun N-terminal kinase (JNK) phosphorylation in TNF-α-treated cells. The pan-caspase inhibitor, z-VAD-fmk, and the caspase-1 inhibitor, z-YVAD-fmk, but not the JNK inhibitor, SP600125, significantly reversed apoptosis and caspase activation in TNF-α/MCPIP1-treated cells. Surprisingly, MCPIP1 itself was also cleaved, and the cleavage was suppressed by treatment with the pan-caspase inhibitor and caspase-1 inhibitor. Moreover, MCPIP1 was found to contain a caspase-1/-4 consensus recognition sequence located in residues 234~238. As expected, the WT MCPIP1 but not the MCPIP1-D141N mutant suppressed NF-κB activation, as evidenced by inhibition of IκB kinase (IKK) phosphorylation and IκB degradation using Western blotting, IKK activity using in vitro kinase activity, and NF-κB translocation to nuclei using an immunofluorescence assay. Interestingly, MCPIP1 also significantly inhibited importin α3 and importin α4 expressions, which are major nuclear transporter receptors for NF-κB. Inhibition of NF-κB activation further downregulated expression of the caspase-8 inhibitor, cFLIP. In summary, the results suggest that MCPIP1 could enhance the TNF-α-induced apoptotic pathway through decreasing NF-κB activation and cFLIP expression.

Some antihistamines have exhibited significant antitumor activity alone or in combination with other therapies in in vitro and clinical studies. However, the underlying mechanisms of how antihistamines inhibit hepatocellular carcinoma proliferation are still unknown. We first screened the antiproliferation activity of 12 benzocycloheptene structural-analogue drugs, and results showed that deptropine was the most potent inhibitor of both Hep3B and HepG2 human hepatoma cells. Deptropine significantly increased light chain 3B-II (LC3B-II) expression but did not induce sequestosome 1 (SQSTM1/p62) degradation in either cell line. Interestingly, other autophagy-related proteins, such as autophagy-related 7 (ATG7), vacuolar protein sorting 34 (VPS34), phosphorylated adenosine 5&prime, monophosphate-activated protein kinase (AMPK), and phosphorylated protein kinase B (PKB, also known as Akt), exhibited no significant change in either deptropine-treated cell line. Deptropine also inhibited the processing of cathepsin L from its precursor form to its mature form. Immunofluorescence microscopy showed an increase of autophagosomes in deptropine-treated cells, but deptropine blocked the fusion between autophagosomes and lysosomes. In a xenograft nude mice model, 2.5 mg/kg deptropine showed a great inhibitory effect on Hep3B tumor growth. These results suggest that deptropine can induce in vitro and in vivo hepatoma cell death, and the underlying mechanisms might be mediated through inhibiting autophagy by blocking autophagosome-lysosome fusion.

Calcium pyrophosphate (CPP) deposition disease (CPPD) is a form of CPP crystal-induced arthritis. A high concentration of extracellular pyrophosphate (ePPi) in synovial fluid is positively correlated with the formation of CPP crystals, and ePPi can be upregulated by ankylosis human (ANKH) and ectonucleotide pyrophosphatase 1 (ENPP1) and downregulated by tissue non-specific alkaline phosphatase (TNAP). However, there is currently no drug that eliminates CPP crystals. We explored the effects of the histone deacetylase (HDAC) inhibitors (HDACis) trichostatin A (TSA) and vorinostat (SAHA) on CPP formation. Transforming growth factor (TGF)-β1-treated human primary cultured articular chondrocytes (HC-a cells) were used to increase ePPi and CPP formation, which were determined by pyrophosphate assay and CPP crystal staining assay, respectively. Artificial substrates thymidine 5′-monophosphate p-nitrophenyl ester (p-NpTMP) and p-nitrophenyl phosphate (p-NPP) were used to estimate ENPP1 and TNAP activities, respectively. The HDACis TSA and SAHA significantly reduced mRNA and protein expressions of ANKH and ENPP1 but increased TNAP expression in a dose-dependent manner in HC-a cells. Further results demonstrated that TSA and SAHA decreased ENPP1 activity, increased TNAP activity, and limited levels of ePPi and CPP. As expected, both TSA and SAHA significantly increased the acetylation of histones 3 and 4 but failed to block Smad-2 phosphorylation induced by TGF-β1. These results suggest that HDACis prevented the formation of CPP by regulating ANKH, ENPP1, and TNAP expressions and can possibly be developed as a potential drug to treat or prevent CPPD.

Carcinogenicity is one of the most critical endpoints for the risk assessment of food contact chemicals (FCCs). However, the carcinogenicity of FCCs remains insufficiently investigated. To fill the data gap, the application of standard experimental methods for identifying chemicals of carcinogenic concerns from a large set of FCCs is impractical due to their resource-intensive nature. In contrast, computational methods provide an efficient way to quickly screen chemicals with carcinogenic potential for subsequent experimental validation. Since every model was developed based on a limited number of training samples, the use of single models for carcinogenicity assessment may not cover the complex mechanisms of carcinogenesis. This study proposed a novel machine learning-based weight-of-evidence (WoE) model for prioritizing chemical carcinogenesis. The WoE model can nonlinearly integrate complementary computational methods of structural alerts, quantitative structure-activity relationship models and in silico toxicogenomics models into a WoE-score. Compared to the best single method, the WoE model gained 8% and 19.7% improvement in the area under the receiver operating characteristic curve (AUC) value and chemical coverage, respectively. The prioritization of 1623 FCCs concludes 44 chemicals of high carcinogenic concern. The machine learning-based WoE approach provides a fast and comprehensive way for prioritizing chemicals of carcinogenic concern.

Alternative splicing (AS) of pre-messenger (m)RNA is a pivotal mechanism in expanding proteomic diversity, which determines the functions of mammalian cells. By conducting transcriptome analyses to profile splicing events in human colorectal cancer (CRC) tissues compared to adjacent normal counterparts, we noted differential splicing profiles of serine/arginine-rich splicing factor 3 (SRSF3) and mitogen-activated protein 4 kinase 4 (MAP4K4) in cancerous tissues of CRC compared to adjacent normal tissues. In addition to SRSF3-mediated autoregulation, RNA-binding motif protein 4 (RBM4) constituted another mechanism in reprogramming the splicing profile of SRSF3. Upregulated expressions of SRSF3 in CRC cells modulated utilization of MAP4K4 exon 16 in a sequence-dependent manner. Alternatively spliced MAP4K4 variants exhibited differential effects on the phosphorylation of c-Jun N-terminal protein kinase 1 (JNK1) which subsequently modulated expression profiles of E-cadherin, N-cadherin, and vimentin, all of which are involved in the migration and invasion of CRC cells. Collectively, RBM4-SRSF3-MAP4K4 constitutes a novel mechanism for manipulating the metastasis of CRC cells through the JNK1 signaling pathway.

ZFP36 family members include ZFP36, ZFP36L1, and ZFP36L2, which belong to CCCH-type zinc finger proteins with two tandem zinc finger (TZF) regions. Whether ZFP36L1 and ZFP36L2 have antiproliferative activities similar to that of ZFP36 is unclear. In this study, when ZFP36L1 or ZFP36L2 was overexpressed in T-REx-293 cells, cell proliferation was dramatically inhibited and the cell cycle was arrested at the G1 phase. The levels of cell-cycle-related proteins, including cyclin B, cyclin D, cyclin A, and p21, decreased; however, p53 increased in ZFP36L1-or ZFP36L2-overexpressing T-REx-293 cells. Forced expression of ZFP36L1 or ZFP36L2 also inhibited cell proliferation and cyclin D gene expression in three human colorectal cancer cell lines: HCT116 p53

// 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.

Dysregulated splicing of pre-messenger (m)RNA is considered a molecular occasion of carcinogenesis. However, the underlying mechanism is complex and remains to be investigated. Herein, we report that the upregulated miR-92a reduced the RNA-binding motif 4 (RBM4) protein expression, leading to the imbalanced expression of the neuronal polypyrimidine tract-binding (nPTB) protein through alternative splicing-coupled nonsense mediated decay (NMD) mechanism. Increase in nPTB protein enhances the relative level of fibroblast growth factor receptor 2 IIIc (FGFR2) and pyruvate kinase M2 (PKM2) transcripts which contribute to the progression and metabolic signature of CRC cells. Expression profiles of RBM4 and downstream alternative splicing events are consistently observed in cancerous tissues compared to adjacent normal tissues. These results constitute a mechanistic understanding of RBM4 on repressing the carcinogenesis of colorectal cells.

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.

// Fat-Moon Suk 1, 2, * , Shyr-Yi Lin 3, 4, * , Ren-Jye Lin 3, 4 , Yung-Hsin Hsine 5 , Yen-Ju Liao 5 , Sheng-Uei Fang 2, 6 , Yu-Chih Liang 5, 7 1 Division of Gastroenterology, Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan 2 Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan 3 Department of Primary Care Medicine, Taipei Medical University Hospital, Taipei, Taiwan 4 Department of General Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan 5 School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan 6 Division of Gastroenterology and Hepatology, Department of Internal Medicine, Taipei Medical University Hospital, Taipei, Taiwan 7 Traditional Herbal Medicine Research Center, Taipei Medical University Hospital, Taipei, Taiwan * These authors have contributed equally to this work Correspondence to: Yu-Chih Liang, e-mail: ycliang@tmu.edu.tw Keywords: Bortezomib, Burkitt’s lymphoma, hnRNP K, sumoylation, c-Myc Received: April 02, 2015 Accepted: July 06, 2015 Published: July 17, 2015 ABSTRACT Bortezomib (Velcal) was the first proteasome inhibitor to be approved by the US Food and Drug Administration to treat patients with relapsed/refractory multiple myelomas. Previous studies have demonstrated that bortezomib inhibits tumor cell proliferation and induces apoptosis by blocking the nuclear factor (NF)-κB pathway. However, the exact mechanism by which bortezomib induces cancer cell apoptosis is still not well understood. In this study, we found that bortezomib significantly inhibited cell proliferation in both human Burkitt’s lymphoma CA46 and Daudi cells. Through proteomic analysis, we found that bortezomib treatment changed the expression of various proteins in distinct functional categories including unfolding protein response (UPS), RNA processing, protein targeting and biosynthesis, apoptosis, and signal transduction. Among the proteins with altered expression, hnRNP K, hnRNP H, Hsp90α, Grp78, and Hsp7C were common to both Daudi and CA46 cells. Interestingly, bortezomib treatment downregulated the expression of high-molecular-weight (HMw) hnRNP K and c-Myc but upregulated the expression of low-molecular-weight (LMw) hnRNP K. Moreover, cell proliferation was significantly correlated with high expression of HMw hnRNP K and c-Myc. HMw and LMw hnRNP K were identified as sumoylated and desumoylated hnRNP K, respectively. Using transient transfection, we found that sumoylated hnRNP K increased c-Myc expression at the translational level and contributed to cell proliferation, and that Lys422 of hnRNP K is the candidate sumoylated residue. Our results suggest that besides inhibiting the ubiquitin-proteasome pathway, bortezomib may inhibit cell proliferation by downregulating sumoylated hnRNP K and c-Myc expression in Burkitt’s lymphoma cells.

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.

Primary hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and the third leading cause of cancer-related death. It is important to identify new targets for early diagnosis and treatment of HCC. Niemann-Pick type C2 (NPC2) plays an important role in the regulation of intracellular cholesterol homeostasis via direct binding with free cholesterol. However, little is known about the significance of NPC2 in HCC tumorigenesis. In this study, we showed that NPC2 is abundantly expressed in normal liver, but is downregulated in human HCC tissues. The patients with NPC2 downregulation expressed much higher α-fetoprotein, multiple tumor type, vascular invasion, later pathological stage and shorter survival rate. Knockdown NPC2 in liver cancer cell lines promote cell proliferation, migration and xenograft tumorigenesis. In contrast, NPC2 overexpression inhibits HuH7 promoted tumor growth. Furthermore, administration of hepatotropic adeno-associated virus 8 (AAV8) delivered NPC2 decreased the inflammatory infiltration, the expression of two early HCC markers-glypican 3 and survivin and suppressed the spontaneous HCC development in mice. To identify the NPC2-dependent mechanism, we emphasized on the status of MAPK/ERK signaling. MEK1/2 inhibitor treatment demonstrated that the expression of NPC2 affected the activation of ERK1/2 but not MEK1/2. In addition, cholesterol trafficking inhibitor treatment did not alter the cell proliferation and the activation of MEK/ERK. In conclusion, our study demonstrates that NPC2 may play an important role in negatively regulate cell proliferation and ERK1/2 activation that were independent of cholesterol accumulation. AAV-NPC2 may thus represent a new treatment strategy for liver cancer.

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

ZFP36 family members include ZFP36, ZFP36L1, and ZFP36L2, which belong to CCCH-type zinc finger proteins with two tandem zinc finger (TZF) regions. Whether ZFP36L1 and ZFP36L2 have antiproliferative activities similar to that of ZFP36 is unclear. In this study, when ZFP36L1 or ZFP36L2 was overexpressed in T-REx-293 cells, cell proliferation was dramatically inhibited and the cell cycle was arrested at the G1 phase. The levels of cell-cycle-related proteins, including cyclin B, cyclin D, cyclin A, and p21, decreased; however, p53 increased in ZFP36L1-or ZFP36L2-overexpressing T-REx-293 cells. Forced expression of ZFP36L1 or ZFP36L2 also inhibited cell proliferation and cyclin D gene expression in three human colorectal cancer cell lines: HCT116 p53+/+, HCT116 p53−/−, and SW620 (mutated p53) cells. However, it increased p53 and p21 expression only in HCT116 p53+/+ cells. Knockdown of ZFP36L1 or ZFP36L2 increased cell proliferation and cyclin D expression; furthermore, the mutation of the TZF of ZFP36L1 or ZFP36L2 caused them to lose their antiproliferative ability, to the extent that they could not inhibit cyclin D expression in these three cell lines. The results indicated that ZFP36L1 and ZFP36L2 play a negative role in cell proliferation; the underlying mechanisms might be mediated through a cyclin D-dependent and p53-independent pathway.

SERS of R6G absorbed on this developed array exhibits a higher intensity by ca. 30-fold, as compared with that of R6G absorbed on the Au NP-based array without the modification of Ag films.

Human MCP-1–induced protein 1 (MCPIP1, also known as ZC3H12A and Regnase-1) plays important roles in negatively regulating the cellular inflammatory response. Recently, we found that as an RNase, MCPIP1 has broad-spectrum antiviral effects by targeting viral RNA. In this study, we demonstrated that MCPIP1 expression was induced by hepatitis C virus (HCV) infection in Huh7.5 hepatoma cells. MCPIP1 expression was higher in liver tissue from patients with chronic HCV infection compared with those without chronic HCV infection. Knockdown of MCPIP1 increased HCV replication and HCV-mediated expression of proinflammatory cytokines, such as TNF-α, IL-6, and MCP-1. However, overexpression of MCPIP1 significantly inhibited HCV replication and HCV-mediated expression of proinflammatory cytokines. Various mutants of functional domains of MCPIP1 showed disruption of the RNA binding and oligomerization abilities, as well as RNase activity, but not deubiquitinase activity, which impaired the inhibitory activity against HCV replication. On immunocytochemistry, MCPIP1 colocalized with HCV RNA. Use of a replication-defective HCV John Cunningham 1/AAG mutant and in vitro RNA cleavage assay demonstrated that MCPIP1 could directly degrade HCV RNA. MCPIP1 may suppress HCV replication and HCV-mediated proinflammatory responses with infection, which might contribute to the regulation of host defense against the infection and virus-induced inflammation.

Activating transcription factor-(ATF-) 3, a stress-inducible transcription factor, is rapidly upregulated under various stress conditions and plays an important role in inducing cancer cell apoptosis. NBM-TP-007-GS-002 (GS-002) is a Taiwanese propolin G (PPG) derivative. In this study, we examined the antitumor effects of GS-002 in human hepatoma Hep3B and HepG2 cellsin vitro. First, we found that GS-002 significantly inhibited cell proliferation and induced cell apoptosis in dose-dependent manners. Several main apoptotic indicators were found in GS-002-treated cells, such as the cleaved forms of caspase-3, caspase-9, and poly(ADP-ribose) polymerase (PARP). GS-002 also induced endoplasmic reticular (ER) stress as evidenced by increases in ER stress-responsive proteins including glucose-regulated protein 78 (GRP78), growth arrest- and DNA damage-inducible gene 153 (GADD153), phosphorylated eukaryotic initiation factor 2α(eIF2α), phosphorylated protein endoplasmic-reticular-resident kinase (PERK), and ATF-3. The induction of ATF-3 expression was mediated by mitogen-activated protein kinase (MAPK) signaling pathways in GS-002-treated cells. Furthermore, we found that GS-002 induced more cell apoptosis in ATF-3-overexpressing cells. These results suggest that the induction of apoptosis by the propolis derivative, GS-002, is partially mediated through ER stress and ATF-3-dependent pathways, and GS-002 has the potential for development as an antitumor drug.

A growing body of studies has demonstrated that dysregulated splicing profiles constitute pivotal mechanisms for carcinogenesis. In this study, we identified discriminative splicing profiles of colorectal cancer (CRC) cells compared to adjacent normal tissues using deep RNA-sequencing (RNA-seq). The RNA-seq results and cohort studies indicated a relatively high ratio of exon 4-excluded neuro-oncological ventral antigen 1 (Nova1−4) and intron 2-retained SRSF6 (SRSF6+intron 2) transcripts in CRC tissues and cell lines. Nova1 variants exhibited differential effects on eliminating SRSF6 expression in CRC cells by inducing SRSF6+intron 2 transcripts which were considered to be the putative target of alternative splicing-coupled nonsense-mediated decay mechanism. Moreover, the splicing profile of vascular endothelial growth factor (VEGF)165/VEGF165b transcripts was relevant to SRSF6 expression, which manipulates the progression of CRC calls. These results highlight the novel and hierarchical role of an alternative splicing cascade that is involved in the development of CRC.

Multiple sclerosis (MS) commonly affects young adults who may be sexually active, with sexual dysfunction being a significant, but often underestimated, symptom of MS. However, no large-scaled study has investigated the association between erectile dysfunction (ED) and MS in an Asian population to date.The objective of this study is to estimate the association between ED and a prior diagnosis of MS using a population-based dataset with a case-control design in Taiwan.The data were sourced from National Health Insurance Research Database. We identified 38,139 patients with ED as cases and randomly selected 262,848 subjects as controls. We then used conditional logistic regression to compute the odds ratio for having previously received a diagnosis of MS between cases and controls.The prevalence of prior MS was 0.037% and 0.015% for cases and controls, respectively (P 0.001). Conditional logistic regression analysis revealed that cases were 2.23 times (95% confidence interval = 1.15-4.32) more likely to have been previously diagnosed with MS than controls after adjusting for monthly income, geographic location, hypertension, diabetes, coronary heart disease, hyperlipidemia, obesity, and alcohol abuse/alcohol dependence syndromes.This study revealed an association between ED and prior MS even after adjusting for potential confounders.

Previously, we demonstrated that lovastatin, a HMG-CoA reductase inhibitor, induced apoptosis, differentiation, and inhibition of invasiveness of human anaplastic thyroid carcinoma cells (ATCs). Here, we further examined the effect of lovastatin on the growth of ARO cells. Lovastatin (0-20μM) concentration-dependently decreased cell number in cultured ATC and arrested the cell at the G0/G1 phase of the cell cycle. Western blot analysis revealed that lovastatin caused an increase of the protein level of p27 and cyclin-dependent kinase (CDK)4 and a decrease of the protein level of cyclin A2, cyclin D3, and phosphorylated Rb (pRb), but did not significantly change the protein levels of p21, cyclins D1 and E, and CDK2, in ARO cells. The formation of the CDK2-p27 complex was increased and the CDK2 activity was decreased in the lovastatin-treated ARO cells. Pretreatment of ARO cells with a p27, but not p21, antisense oligonucleotide prevented the lovastatin-induced G0/G1 arrest in ARO cells. The lovastatin-induced growth inhibition and translocation of RhoA and Rac1 in ARO cells were completely prevented by mevalonate and partially by geranylgeranyl pyrophosphate. Treatment of ARO cells with Y27632, an inhibitor of Rho-associated kinase, abolished the GGPP-mediated prevention of lovastatin-induced anti-proliferation and up-regulation and prolonged degradation of p27. Taken together, these data suggest that lovastatin treatment caused a reduction of Rho geranylgeranylation, which in turn increased the expression and stability of p27, and then inhibited ARO cell proliferation. These data suggest that lovastatin merits further investigation as multipotent therapy for treatment ATC.

The influence of physical characteristics of electrospun three-dimensional (3D) fibrous scaffolds based on polybutylene succinate- co-adipate (PBSA) and poly l-lactic acid (PLLA) on the culture of primary human chondrocytes (PHCs) in terms of cell attachment, proliferation, and re-differentiation was investigated. Physical characteristics assessed for two polymers electrospun at two different delivery rates (PBSA-3, PBSA-16, PLLA-3, and PLLA-16) including average fiber diameter, average pore diameter, porosity, and contact angle. Results demonstrated that 3D fibrous scaffolds are better for PHCs’ attachment than two-dimensional (2D) casting films made of the same polymeric materials. It was also found that 3D fibrous scaffolds are appropriate architecture for the proliferation of PHCs than 2D casting films and dependent upon the polymer used. Histological analysis revealed that a significant amount of PHC was found to be growing only within layers of PLLA fibrous scaffolds. The mitochondrial ribonucleic acid (mRNA) expression of both aggrecan and type II collagen by PHCs cultured in tissue culture polystyrene for 28 days decreased significantly. The mRNA expression of both aggrecan and type II collagen by PHCs cultured in PBSA scaffolds increased from 14 to 28 days, whereas only mRNA expression of aggrecan cultured in both PLLA scaffolds increased from 14 to 28 days.

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.

Monocyte chemotactic protein induced protein 3 (MCPIP3) belongs to the Cys–Cys–Cys–His (CCCH)-zinc finger protein family and contains a highly conserved CCCH-zinc finger domain and a Nedd4-BP1 YacP nuclease (NYN) domain. Previous studies showed that MCPIP3 inhibits the expression of proinflammatory genes, such as vascular cell adhesion molecule (VCAM)-1, in human endothelial cells, but the roles and functions of MCPIP3 in cancer cells are still unknown. In human colorectal cancer specimens, we found that the messenger RNA expression of MCPIP3 was significantly downregulated in cancer tissues compared to adjacent normal tissues (18/25; average fold change of 8.18). Two cell models were used to demonstrate the anti-migration activity of MCPIP3. First, Tet-on T-REx-293/HA-MCPIP3 cells were used to examine whether MCPIP3 can change epithelial–mesenchymal transition (EMT)-related gene expressions. Second, we used two human colorectal cancer cell lines, SW620 and HCT116, to prove the role of MCPIP3 in regulating EMT-related gene expressions. We found that overexpression of MCPIP3 inhibited cell migration according to a wound-healing assay and Transwell invasion assay and vimentin expression, and increased E-cadherin expression in these two cell lines. These results suggest that MCPIP3 might play a negative role in cell migration of human colorectal cancer cells.

The aim of this study was to investigate the potential applications of 5,5-diphenyl-2-thiohydantoin-N10 (DPTH-N10) in the treatment of human colon cancer. Subcultured human colon cancer cell line, COLO-205, was used for examining the antiproliferation effect of DPTH-N10 on colon cancer. Thymidine incorporation and cell count were conducted to examine the antiproliferation effect of DPTH-N10. Western blot analysis was performed to examine the protein levels of cell cycle-related proteins. DNA fragmentation assay was performed to examine the occurrence of apoptosis. DPTH-N10 at a range of concentrations (0-30 microM) inhibits the proliferation but did not cause the cell death of COLO-205, indicating that it may have an inhibitory effect on the cell proliferation in COLO-205. The apoptosis was observed in COLO-205 when the DPTH-N10 concentrations were higher than 30 muM. Western blot analysis showed that the protein level of the cell cycle inhibitory protein, p21, in COLO-205 increased after DPTH-N10 treatment. Immunoprecipitation showed that the formation of the cyclin-dependent kinase (CDK)2-p21 complex was increased in the DPTH-N10-treated COLO-205. Kinase assay further demonstrated that the CDK2 activity was decreased in the DPTH-N10-treated COLO-205. DPTH-N10 caused growth inhibition in COLO-205 by inhibiting DNA synthesis and activating apoptosis. The findings from our previous in vitro studies in DPTH-N10-induced anti-angiogenic effect and from the present in vitro studies in DPTH-N10-induced antiproliferation effect on colon cancer cell line strongly suggest the potential applications of DPTH-N10 in the treatment of human colon cancer.

Background With respect to interleukin (IL)-1β concentration, the disease-modifying effects of glucosamine HCl (GLN) on IL-1β-treated chondrocytes followed a dose-dependent manner and were traditionally conducted under normoxic conditions not generally encountered by chondrocytes in vivo. Purposes To demonstrate the beneficial effects of GLN on IL-1β-induced matrix metalloproteinase (MMP) expression in chondrosarcoma cells in a dose-dependent manner, but at different concentration ranges of IL-1β; additionally, to determine how hypoxia affects GLN's beneficial effects after dose-dependent application. Methods Under normoxic and hypoxic conditions, human chondrosarcoma cells (SW1353) induced by high and low IL-1β concentrations were respectively treated with low and high concentration ranges of GLN. mRNA and protein expression for MMPs and glucose transporter protein (GLUT)-1 were examined by reverse transcription–polymerase chain reaction and Western blot analysis. Results High concentrations of GLN could reduce mRNA and protein expressions of MMPs induced by low concentration of IL-1β; whereas low concentrations of GLN could reduce the inflammatory response upregulated by high concentrations of IL-1β. The data might substantiate the beneficial effects in clinical application of lower plasma concentrations of GLN achievable by oral administration. Furthermore, the effects were compared under hypoxic conditions to mimic real conditions encountered in human cartilage under normoxia. Upregulation of HIF-1α protein levels under hypoxia was observed. The level of MMP protein expression induced by IL-1β was lower under hypoxic conditions. Instead, inhibition of MMPs by GLN appeared to be more apparent. Finally, GLUT-1 protein expression of SW1353 was upregulated under IL-1β and hypoxia treatment. Conclusion Glucose transporter, most likely GLUT-1, inducible by HIF-1α, might play an important role in the therapeutic efficacy of a lower plasma concentration of GLN on osteoarthritis achievable by oral administration.

Previously, we demonstrated that magnolol isolated from the bark of Magnolia officinalis has anticancer activity in colon, hepatoma, and leukemia cell lines. In this study, we show that magnolol concentration dependently (0-40 microM) decreased the cell number in a cultured human glioblastoma cancer cell line (U373) and arrested the cells at the G0/G1 phase of the cell cycle. Magnolol treatment decreased the protein levels of cyclins A and D1 and increased p21/Cip1, but not cyclins B and D3, cyclin-dependent kinase (CDK)2, CDK4, CDC25C, Weel, p27/Kip1, and p53. The CDK2-p21/Cip1 complex was increased, and the CDK2 kinase activity was decreased in the magnolol-treated U373. Pretreatment of U373 with p21/Cip1 specific antisense oligodeoxynucleotide prevented the magnolol-induced increase of p21/Cip1 protein levels and the decrease of DNA synthesis. Magnolol at a concentration of 100 microM induced DNA fragmentation in U373. Our findings suggest the potential applications of magnolol in the treatment of human brain cancers.

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.

The Notch signaling pathway plays important roles in a variety of cellular processes. Aberrant transduction of Notch signaling contributes to many diseases and cancers in humans. The Notch receptor intracellular domain, the activated form of Notch receptor, is extremely difficult to detect in normal cells. However, it can activate signaling at very low protein concentration to elicit its biological effects. In the present study, a cell based luciferase reporter gene assay was established in K562 cells to screen drugs which could modulate the endogenous CBF1‐dependent Notch signal pathway. Using this system, we found that the luciferase activity of CBF1‐dependent reporter gene was activated by baicalin and baicalein but suppressed by niclosamide in both dose‐ and time‐dependent manners. Treatment with these drugs modulated endogenous Notch signaling and affected mRNA expression levels of Notch1 receptor and Notch target genes in K562 cells. Additionally, erythroid differentiation of K562 cells was suppressed by baicalin and baicalein yet was promoted by niclosamide. Colony‐forming ability in soft agar was decreased after treatment with baicalin and baicalein, but was not affected in the presence of niclosamide. Thus, modulation of Notch signaling after treatment with any of these three drugs may affect tumorigenesis of K562 cells suggesting that these drugs may have therapeutic potential for those tumors associated with Notch signaling. Taken together, this system could be beneficial for screening of drugs with potential to treat Notch signal pathway‐associated diseases. J. Cell. Biochem. 106: 682–692, 2009. © 2009 Wiley‐Liss, Inc.

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.

Previous studies have shown that progesterone inhibits endothelial cell proliferation through a nuclear receptor-mediated mechanism. Here, we further demonstrate that progesterone at physiologic levels (5 - 500 nM) dose- and time-dependently inhibited DNA synthesis of cultured human umbilical vein endothelial cells (HUVEC). The mRNA and protein levels of p21, p27, and p53 in HUVEC were increased by progesterone. The formation of CDK2-p21 and CDK2-p27 were increased and the CDK2 activity was decreased in the progesterone-treated HUVEC. The progesterone-inhibited [3H]thymidine incorporation was completely blocked when the expressions of p21 and p27 were knocked-down together. Transfection of HUVEC with dominant negative p53 cDNA prevented the progesterone-induced increases in p21 and p27 promoter activity and protein level, decreases in thymidine incorporation, and capillary-like tube formation. Matrigel angiogenesis assay in mice demonstrated the antiangiogenic effect of progesterone in vivo. These findings demonstrate for the first time that progesterone inhibited endothelial cell proliferation through a p53-dependent pathway.

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.

Previously, we showed that terbinafine (TB) induces cell-cycle arrest in cultured human umbilical vein endothelial cells (HUVEC) through an up-regulation of the p21 protein. The aim of this study is to delineate the molecular mechanisms underlying TB-induced increase of p21 protein. RT-PCR analysis demonstrated that the mRNA levels of p21 and p53 were increased in the TB-treated HUVEC. The p21 promoter activity was also increased by TB treatment. Transfection of HUVEC with p53 dominant negative (DN) abolished the TB-induced increases of p21 promoter activity and protein level, suggesting that the TB-induced increase of p21 is p53-dependent. Western blot analysis demonstrated that TB decreased the levels of phosphorylated extracellular signal-regulated kinase (ERK). Over-expression of mitogen-activated protein kinase (MEK)-1, the immediate upstream activator kinase of ERK, abolished the TB-induced increases of p21 and p53 protein and decrease of thymidine incorporation. The ERK inhibitor (PD98059) enhanced the TB-induced inhibition of thymidine incorporation into HUVEC. Taken together, these data suggest that the decrease of ERK activity plays a role in the TB-induced up-regulation of p21 in HUVEC. On the other hand, pretreatment of the cells with geranylgeraniol (GGOH), farnesol (FOH), or Ras inhibitor peptide did not affect the TB-induced decrease of thymidine incorporation. Taken together, our results suggest that TB might cause a decrease of MEK, which in turn up-regulates p53 through the inhibition of ERK phosphorylation, and finally causes an increase of p21 expression and cell-cycle arrest.