Honokiol Is a Potential Therapeutic Agent and Has a Synergistic Effect With 5-FU in Human Urothelial Cell Carcinoma Cells.

BACKGROUND/AIM: Honokiol is a biphenolic component of the bark of Magnolia, and has been shown to exert several activities, including anti-depressant, anti-emetic, anti-oxidative, anti-thrombotic, anti-angiogenesis, anti-anxiolytic, anti-inflammatory and anti-tumor effects. MATERIALS AND METHODS: The anti-tumor activities of honokiol and its synergistic effect with 5-fluorouracil (5-FU) in human urothelial cell carcinoma (UCC) cells were investigated. RESULTS: Honokiol significantly suppressed the proliferation of UCC cells in a dose- and time-dependent manner. Moreover, honokiol inhibited the tumorigenesis of UCC cells in vitro. In addition, honokiol induced cell cycle arrest at G0/G1 phase and caused apoptosis of UCC cells through the intrinsic pathway. Importantly, we demonstrated that honokiol potentiated the cytotoxic effect of 5-FU, and displayed a synergistic effect with 5-FU in UCC cells. CONCLUSION: Honokiol causes growth inhibition, tumorigenesis suppression, cell cycle arrest, apoptosis, and importantly has a synergistic effect with 5-FU in human UCC cells. Therefore, this agent displays a therapeutic potential for treating human UCC.

Indirubin-3'-oxime suppresses human cholangiocarcinoma through cell-cycle arrest and apoptosis.

Cholangiocarcinoma (CCA) is one of the most serious of all cancers and a major public health problem. CCA is an extremely invasive cancer, and the survival rate for CCA patients is only 24 months after diagnosis. Although surgery and chemotherapy can extend the survival rate to 5 years, < 20-40% of CCA patients will survive this long; therefore, it is crucial to discover an effective chemotherapeutic agent for CCA. Indirubin-3'-oxime (I3O), a derivative of indirubin, has been shown to suppress cell proliferation and induce cell-cycle arrest and cell apoptosis in various human cancers. In this study, four human CCA cell lines-NOZ, HuCCT1, OCUG-1, and OZ-were used to evaluate the anticancer properties of I3O. Cell viability, cell-cycle arrest, and apoptosis were assessed using Western blotting, immunofluorescence, and flow cytometry analysis. The data show that I3O treatment can inhibit cell proliferation and induce cell-cycle arrest, and caspase-dependent apoptosis in CCA cells. These findings suggest that I3O could suppress tumor growth by regulating the cell cycle and inducing apoptosis, and is a potential therapeutic agent for treating human CCA.

Honokiol inhibits in vitro and in vivo growth of oral squamous cell carcinoma through induction of apoptosis, cell cycle arrest and autophagy.

Honokiol, an active natural product derived from Magnolia officinalis, exerted anticancer effects through a variety of mechanisms on multiple types of cancers. In this study, the molecular mechanisms of honokiol in suppressing the human oral squamous cell carcinoma (OSCC) cells were evaluated. Treatment of two OSCC cell lines with honokiol resulted in reducing the cell proliferation and arresting the cell cycle at G1 stage which was correlated with the down-regulation of Cdk2 and Cdk4 and the up-regulation of cell cycle suppressors, p21 and p27. In addition, the caspase-dependent programmed cell death was substantially detected, and the autophagy was induced as the autophagosome formation and autophagic flux proceeded. Modulation of autophagy by autophagic inducer, rapamycin or inhibitors, 3-MA or bafilomycin, potentiated the honokiol-mediated anti-OSCC effects where honokiol exerted multiple actions in suppression of MAPK pathway and regulation of Akt/mTOR or AMPK pathways. As compared to clinical therapeutic agent, 5-FU, honokiol exhibited more potent activity against OSCC cells and synergistically enhanced the cytotoxic effect of 5-FU. Furthermore, orally administrated honokiol exerted effective antitumour activity in vivo in OSCC-xenografted mice. Thus, this study revealed that honokiol could be a promising candidate in preventing human OSCCs.

Generation of clade- and symbiont-specific antibodies to characterize marker molecules during Cnidaria-Symbiodinium endosymbiosis.

The endosymbiosis between cnidarians and dinoflagellates is responsible for the formation of coral reefs. Changes in molecules have been identified during the process of cnidaria-Symbiodinium endosymbiosis. However, the complexity of the molecular interaction has prevented the establishment of a mechanistic explanation of cellular regulation in this mutualistic symbiosis. To date, no marker molecules have been identified to specifically represent the symbiotic status. Because the endosymbiotic association occurs in the symbiotic gastrodermal cells (SGCs), whole cells of isolated SGCs were used as an antigen to generate monoclonal antibodies (mAb) to screen possible molecular candidates of symbiotic markers. The results showed that one of the generated monoclonal antibodies, 2-6F, specifically recognized clade C symbiotic Symbiodinium but not its free-living counterpart or other Symbiodinium clades. The expression levels of 2-6F mAb-recognized proteins are highly correlated with the symbiotic status, and these proteins were characterized as N-linked glycoproteins via treatment with peptide N-glycosidase F. Furthermore, their glycan moieties were markedly different from those of free-living Symbiodinium, potentially suggesting host regulation of post-translational modification. Consequently, the 2-6F mAb can be used to detect the symbiotic state of corals and investigate the complex molecular interactions in cnidaria-Symbiodinium endosymbiosis.

Discovering Peptide Inhibitors of Human Squalene Synthase Through Screening the Phage-Displayed Cyclic Peptide c7c Library.

Many drugs for the treatment of hypercholesterolemia are targeting the enzymes involved in human cholesterol biosynthesis pathway. Squalene synthase, the rate-limiting enzyme located at the downstream of cholesterol synthesis pathway, has become a better candidate to develop next-generation hypocholesterolemia drugs. In the present study, we cloned and expressed the recombinant human squalene synthase (hSQS) as the lure to isolate potential peptide inhibitors from screening the conformation-constrained phage-displayed cyclic peptide c7c library. Their binding capabilities were further estimated by ELISA. Their pharmaceutical potentials were then analyzed through molecular modeling and the ADMET property evaluations. Four ennea-peptides and nine tetra-peptides were finally synthesized to evaluate their inhibitory potentials toward hSQS. The results indicate that the ennea-peptide CLSPHSMFC, tetra-peptides SMFC, CKTE, and WHQW can effectively inhibit hSQS activities (IC50 values equal to 64, 76, 87, and 90 µM, respectively). These peptides may have potentials to develop future cholesterol-lowering therapeutics. The ligand-protein interaction analysis also reveals that the inner hydrophobic pocket could be a more critical site of hSQS.

Exploration of Peptide Inhibitors of Human Squalene Synthase through Molecular Modeling and Phage Display Technique.

Many studies have demonstrated the role of elevated levels of serum cholesterol in the pathogenesis of atherosclerosis and coronary heart disease. Various drugs targeting the key enzymes involved in the cholesterol biosynthesis pathway have been investigated for the treatment of hypercholesterolemia. Human squalene synthase has been one of the most important targets for therapeutic intervention. In the present study, we used the recombinant human squalene synthase as the lure for screening the peptide inhibitors from phage-displayed random peptide library. The tightly bound phages and their derived peptides were further evaluated based on their potential binding capabilities, molecular modeling characteristics and predicted absorption, distribution, metabolism, excretion, toxicity (ADMET) properties. Several hexa-peptides and tetra-peptides were finally synthesized to assay their inhibitory effects toward the recombinant human squalene synthase. The results demonstrated that the hexa-peptide FTACNW and tetra-peptide VACL can inhibit human squalene synthase effectively (with IC50 values near 100 µM) and may have potential to develop further as future hypocholesterolemia agents.

Honokiol, a Lignan Biphenol Derived from the Magnolia Tree, Inhibits Dengue Virus Type 2 Infection.

Dengue is the most widespread arbovirus infection and poses a serious health and economic issue in tropical and subtropical countries. Currently no licensed vaccine or compounds can be used to prevent or manage the severity of dengue virus (DENV) infection. Honokiol, a lignan biphenol derived from the Magnolia tree, is commonly used in Eastern medicine. Here we report that honokiol has profound antiviral activity against serotype 2 DENV (DENV-2). In addition to inhibiting the intracellular DENV-2 replicon, honokiol was shown to suppress the replication of DENV-2 in baby hamster kidney (BHK) and human hepatocarcinoma Huh7 cells. At the maximum non-toxic dose of honokiol treatment, the production of infectious DENV particles was reduced >90% in BHK and Huh7 cells. The underlying mechanisms revealed that the expression of DENV-2 nonstructural protein NS1/NS3 and its replicating intermediate, double-strand RNA, was dramatically reduced by honokiol treatment. Honokiol has no effect on the expression of DENV putative receptors, but may interfere with the endocytosis of DENV-2 by abrogating the co-localization of DENV envelope glycoprotein and the early endosomes. These results indicate that honokiol inhibits the replication, viral gene expression, and endocytotic process of DENV-2, making it a promising agent for chemotherapy of DENV infection.

Exploration of natural product ingredients as inhibitors of human HMG-CoA reductase through structure-based virtual screening.

Cholesterol plays an important role in living cells. However, a very high level of cholesterol may lead to atherosclerosis. HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase is the key enzyme in the cholesterol biosynthesis pathway, and the statin-like drugs are inhibitors of human HMG-CoA reductase (hHMGR). The present study aimed to virtually screen for potential hHMGR inhibitors from natural product to discover hypolipidemic drug candidates with fewer side effects and lesser toxicities. We used the 3D structure 1HWK from the PDB (Protein Data Bank) database of hHMGR as the target to screen for the strongly bound compounds from the traditional Chinese medicine database. Many interesting molecules including polyphenolic compounds, polisubstituted heterocyclics, and linear lipophilic alcohols were identified and their ADMET (absorption, disrtibution, metabolism, excretion, toxicity) properties were predicted. Finally, four compounds were obtained for the in vitro validation experiments. The results indicated that curcumin and salvianolic acid C can effectively inhibit hHMGR, with IC50 (half maximal inhibitory concentration) values of 4.3 µM and 8 µM, respectively. The present study also demonstrated the feasibility of discovering new drug candidates through structure-based virtual screening.

Peptide inhibitors of human HMG-CoA reductase as potential hypocholesterolemia agents.

Hypercholesterolemia may lead to obesity and cardiovascular diseases. To prevent hypercholesterolemia, many drugs have been developed while searching for better drugs to treat hypercholesterolemia has never been ended. Other than small molecule drugs, peptide drugs are gaining more visibilities in many therapeutic areas. In the present study, we employed phage-display techniques to screen peptide inhibitors against human HMG-CoA reductase. The results indicate that the tetrapeptide PMAS inhibits hHMGR effectively (IC50=68 µM), could be a lead compound to develop hypocholesterolemic agents.

Improving anticancer efficacy of (-)-epigallocatechin-3-gallate gold nanoparticles in murine B16F10 melanoma cells.

(-)-Epigallocatechin-3-gallate (EGCG), the major bioactive constituent in green tea, has been reported to effectively inhibit the formation and development of tumors. To maximize the effectiveness of EGCG, we attached it to nanogold particles (EGCG-pNG) in various ratios to examine in vitro cytotoxicity and in vivo anti-cancer activity. EGCG-pNG showed improved anti-cancer efficacy in B16F10 murine melanoma cells; the cytotoxic effect in the melanoma cells treated with EGCG-pNG was 4.91 times higher than those treated with EGCG. The enhancement is achieved through mitochondrial pathway-mediated apoptosis as determined by annexin V assay, JC-10 staining, and caspase-3, -8, -9 activity assay. Moreover, EGCG-pNG was 1.66 times more potent than EGCG for inhibition of tumor growth in a murine melanoma model. In the hemolysis assay, the pNG surface conjugated with EGCG is most likely the key factor that contributes to the decreased release of hemoglobin from human red blood cells.

Antrodia cinnamomea profoundly exalted the reversion of activated hepatic stellate cells by the alteration of cellular proteins.

The direct modulation of Antrodia cinnamomea (AC) on the prominent role of liver fibrosis-hepatic stellate cells (HSCs) in situ remains unclear. Firstly, the administration of A. cinnamomea mycelial extract (ACME) could improve liver morphology and histological changes including collagen formation and GPT activity in the liver of thioacetamide (TAA)-injured rats. The morphology and fatty acid restore of TAA-induced HSCs (THSCs) returned to the non-chemical induced HSCs (NHSCs) type as measured by immunofluorescence and Oil Red O staining. PPARγ was upregulated associated with the lowering of α-SMA protein in NHSC-ACME. ACME inhibited the MMP-2 activity in NHSCs by gelatin Zymography. After LC-MS/MS, the cytoskeleton (tubulin, lamin A) and heat shock protein 8 in NHSC-ACME, and guanylate kinase, brain-specific kinase, SG-II and p55 proteins were downregulated in THSC-ACME. Whereas MHC class II, SMC6 protein, and phospholipase D were upregulated in NHSC-ACME. Furthermore, PKG-1 was downregulated in NHSC-ACME and upregulated in THSC-ACME. SG-II and p55 proteins were downregulated in NHSC-ACME and THSC-ACME by Western blotting. Taken together, the beneficial effect of A. cinnamomea on the induction of HSC cellular proteins is potentially applied as an alternative and complementary medicine for the prevention and amelioration of a liver injury.

Immunomodulation of phloretin by impairing dendritic cell activation and function.

Dietary compounds in fruits and vegetables have been shown to exert many biological activities. In addition to antioxidant effects, a number of flavonoids are able to modulate inflammatory responses. Here, we demonstrated that phloretin (PT), a natural dihydrochalcone found in many fruits, suppressed the activation and function of mouse dendritic cells (DCs). Phloretin disturbed the multiple intracellular signaling pathways in DCs induced by the Toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS), including ROS, MAPKs (ERK, JNK, p38 MAPK), and NF-κB, and thereby reducing the production of inflammatory cytokines and chemokines. Phloretin also effectively suppressed the activation of DCs treated with different dosages of LPS or various TLR agonists. The LPS-induced DC maturation was attenuated by phloretin because the expression levels of the MHC class II and the co-stimulatory molecules were down-regulated, which then inhibited the LPS-stimulating DCs and the subsequent naïve T cell activation in a mixed lymphocyte reaction. Moreover, in vivo administration of phloretin suppressed the phenotypic maturation of the LPS-challenged splenic DCs and decreased the IFN-γ production from the activated CD4 T cells. Thus, we suggest that phloretin may potentially be an immunomodulator by impairing the activation and function of DCs and phloretin-contained fruits may be helpful in the improvement of inflammation and autoimmune diseases.

Monoclonal antibodies for diagnosis of enterovirus 71.

Enterovirus 71 (EV71), one of the major causative agents of hand, foot, and mouth disease (HFMD), is now recognized as an emerging neurotropic virus in Asia and may cause severe neurologic complications and mortalities. Laboratory diagnosis of EV71 infection must be efficient and accurate, which could be accomplished by various immunoassays. In this study we use a live EV71 isolate, Tainan/4643/98, with genotype C2 as an immunogen to sensitize BALB/c (H-2(d)) mice and then generate the EV71-specific murine monoclonal antibodies. Five hybridoma clones were established and their monoclonal antibodies were characterized. All five clones are applicable in immunofluorescence staining but with different sensitivities-that is, MAbs 22, 24, and 27 were sensitive in IFA detection, and MAbs 22 and 24 were also confirmed in flow cytometry. None of these cross-reacted with coxsackievirus A16 (CVA16) or Echovirus type 6 (ECHO6), but each varied in binding to different EV71 subgenogroups (B1, B4, B5, C2, and C4). Western blot analysis revealed that all of these MAbs reacted with EV71 VP1 capsid proteins, and in addition MAbs 22 and 24 exhibited potent neutralizing activities against EV71 and protected cells from infection. Further, mapping the epitopes for each MAb revealed that only MAb 27 showed positive for the linear epitope DVIESSIGDSVSRAL, which was located at the N-terminus (a.a. 6-20) of EV71 VP1 and highly conserved among all EV71 subgenotypes. Thus, these MAbs may provide valuable tools for the laboratory diagnosis of EV71 infection and for vaccine development.

Immunomodulatory effect of marine cembrane-type diterpenoids on dendritic cells.

Dendritic cells (DCs) are antigen presenting cells, which can present antigens to T-cells and play an important role in linking innate and adaptive immunity. DC maturation can be induced by many stimuli, including pro-inflammatory cytokines and bacterial products, such as lipopolysaccharides (LPS). Here, we examined the immunomodulatory effects of marine cembrane compounds, (9E,13E)-5-acetoxy-6-hydroxy-9,13-dimethyl-3- methylene-3,3a,4,5,6,7,8,11,12,14a-decahydro-2H-cyclotrideca[b]furan-2-one (1), (9E,13E)- 5-acetoxy-6-acetyl-9,13-dimethyl-3-methylene-3,3a,4,5,6,7,8,11,12,14a-decahydro-2H-cyclotrideca[b]furan-2-one (2), lobocrassin B (3), (-)14-deoxycrassin (4), cembranolide B (5) and 13-acetoxysarcocrassolide (6) isolated from a soft coral, Lobophytum crassum, on mouse bone marrow-derived dendritic cells (BMDCs). The results revealed that cembrane-type diterpenoids, especially lobocrassin B, effectively inhibited LPS-induced BMDC activation by inhibiting the production of TNF-α. Pre-treatment of BMDCs with Lobocrassin B for 1 h is essential to prohibit the following activation induced by various toll-like receptor (TLR) agonists, such as LPS, zymosan, lipoteichoic acid (LTA) and Pam2CSK4. Inhibition of NF-κB nuclear translocation by lobocrassin B, which is a key transcription factor for cytokine production in TLR signaling, was evident as assayed by high-content image analysis. Lobocrassin B attenuated DC maturation and endocytosis as the expression levels of MHC class II and the co-stimulatory molecules were downregulated, which may affect the function of DCs to initiate the T-cell responses. Thus, lobocrassin B may have the potential in treatment of immune dysregulated diseases in the future.

5-Episinuleptolide acetate, a norcembranoidal diterpene from the formosan soft coral Sinularia sp., induces leukemia cell apoptosis through Hsp90 inhibition.

5-Episinuleptolide acetate (5EPA), a cytotoxic norcembranoidal diterpene recently identified from the Formosan soft coral Sinularia sp., exhibited potent activity against the K562, Molt 4 and HL 60 cancer cell lines. The antiproliferative assay, as well as the annexin V-FITC/propidium iodide (PI) apoptotic assay, indicated that the HL 60 cell line is the most sensitive one towards 5EPA. This diterpenoid led to caspases -3, -8, and -9 activation as well as PARP cleavage. It also induced ROS generation, calcium accumulation and disruption of mitochondrial membrane potential. Additionally, the expression levels of Hsp90 protein and several client proteins were downregulated in response to 5EPA treatment. These results suggest that 5EPA's cytotoxic effect on HL 60 cells may be attributed to the inhibition of Hsp90 as well as the induction of mitochondrial stress which finally results in apoptotic cell death.

Proteomics-based identification of plasma biomarkers in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is an aggressive cancer and its occurrence is closely related to betel nut chewing in Taiwan. However, there are few prognostic and diagnostic biomarkers for this disease especially for its association with betel nut chewing. Recent progresses in quantitative proteomics have offered opportunities to discover plasma proteins as biomarkers for tracking the progression and for understanding the molecular mechanisms of OSCC. In present study, plasma samples from OSCC patients with at least 5-year history of betel nut chewing and healthy donors were analyzed by fluorescence 2D-DIGE-based proteomic analysis. Totally, 38 proteins have been firmly identified representing 13 unique gene products. These proteins mainly function in inflammatory responses (such as fibrinogen gamma chain) and transport (Apolipoprotein A-I). Additionally, the current quantitative proteomic approach has identified numerous OSCC biomarkers including fibrinogen (alpha/beta/gamma) chain, haptoglobin, leucine-rich alpha-2-glycoprotein and ribosomal protein S6 kinase alpha-3 (RSK2) which have not been reported and may be associated with the progression and development of the disease. In summary, this study reports a comprehensive patient-based proteomic approach for the identification of potential plasma biomarkers in OSCC. The potential of utilizing these markers for screening and treating OSCC warrants further investigations.

Enterovirus 71 infection of monocytes with antibody-dependent enhancement.

Enterovirus (EV) is an RNA virus that has circulated with different serotypes and genotypes worldwide. Enterovirus 71 (EV71) is a major neurotropic virus that causes severe brain stem encephalitis (BE) in infants and young children. The most vulnerable age for fatal infection is 6 to 11 months. This is associated with the coincident decline in maternal antibodies. The current report describes our finding that EV71 can infect human peripheral blood monocytes. We were able to show that EV71 infection is enhanced in the monocytic cell line THP-1 by the presence of subneutralizing concentrations of anti-EV71 antibodies. We also found that antibody-dependent enhancement (ADE) is mediated in part by Fcγ receptors. These observations support the concept that ADE augments the infectivity of EV71 for human monocytes and contributes to the age-dependent pathogenesis of EV71-induced disease. The ADE phenomenon must be considered during the development of an EV71 vaccine.

Micro flow cytometry utilizing a magnetic bead-based immunoassay for rapid virus detection.

The current study presents a new miniature microfluidic flow cytometer integrated with several functional micro-devices capable of viral sample purification and detection by utilizing a magnetic bead-based immunoassay. The magnetic beads were conjugated with specific antibodies, which can recognize and capture target viruses. Another dye-labeled anti-virus antibody was then used to mark the bead-bound virus for the subsequent optical detection. Several essential components were integrated onto a single chip including a sample incubation module, a micro flow cytometry module and an optical detection module. The sample incubation module consisting of pneumatic micropumps and a membrane-type, active micromixer was used for purifying and enriching the target virus-bound magnetic beads with the aid of a permanent magnet. The micro flow cytometry module and the optical detection module were used to perform the functions of virus counting and collection. Experimental results showed that virus samples with a concentration of 10(3)PFU/ml can be automatically detected successfully by the developed system. In addition, the entire diagnosis procedure including sample incubation and virus detection took only about 40min. Consequently, the proposed micro flow cytometry may provide a powerful platform for rapid diagnosis and future biological applications.

Volume replacement in infants with dengue hemorrhagic fever/dengue shock syndrome.

Volume replacement was studied prospectively in 208 infants with dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The mean volume of intravenous fluid used was 110.4 mL/kg administered over a mean period of 25.8 hours. The mean volumes of intravenous fluid replacement in infants with DSS was significantly higher than in those with non-shock DHF (129.8 mL/kg versus 102.1 mL/kg; P = 0.001). Patients with DSS had significantly higher proportional requirements for dextran and blood transfusions than non-shock infants. Recurrent shock, prolonged shock, and acute respiratory failure were recorded in 8, 6, and 13 patients, respectively. Four patients with DSS died of severe complications. Intravenous fluid replacement with special care to avoid fluid overload requires careful attention to established indications for use of colloidal solutions and blood transfusions. To improve case fatality rates, special efforts need to be directed to infants with DHF/DSS accompanied by severe complications.