Exploring the therapeutic potential of DV-B-120 as an inhibitor of dengue virus infection.

Dengue fever, an infectious disease prevalent in subtropical and tropical regions, currently lacks effective small-molecule drugs as treatment. In this study, we used a fluorescence peptide cleavage assay to screen seven compounds to assess their inhibition of the dengue virus (DENV) NS2B-NS3 protease. DV-B-120 demonstrated superior inhibition of NS2B-NS3 protease activity and lower toxicity compared to ARDP0006. The selectivity index of DV-B-120 was higher than that of ARDP0006. In vivo assessments of the antiviral efficacy of DV-B-120 against DENV replication demonstrated delayed mortality of suckling mice treated with the compound, with 60-80% protection against life-threatening effects, compared to the outcomes of DENV-infected mice treated with saline. The lower clinical scores of DENV-infected mice treated with DV-B-120 indicated a reduction in acute-progressive illness symptoms, underscoring the potential therapeutic impact of DV-B-120. Investigations of DV-B-120's ability to restore the antiviral type I IFN response in the brain tissue of DENV-infected ICR suckling mice demonstrated its capacity to stimulate IFN and antiviral IFN-stimulated gene expression. DV-B-120 not only significantly delayed DENV-2-induced mortality and illness symptoms but also reduced viral numbers in the brain, ultimately restoring the innate antiviral response. These findings strongly suggest that DV-B-120 holds promise as a therapeutic agent against DENV infection and highlight its potential contribution in addressing the current lack of effective treatments for this infectious disease.IMPORTANCEThe prevalence of dengue virus (DENV) infection in tropical and subtropical regions is escalating due to factors like climate change and mosquito vector expansion. With over 300 million annual infections and potentially fatal outcomes, the urgent need for effective treatments is evident. While the approved Dengvaxia vaccine has variable efficacy, there are currently no antiviral drugs for DENV. This study explores seven compounds targeting the NS2B-NS3 protease, a crucial protein in DENV replication. These compounds exhibit inhibitory effects on DENV-2 NS2B-NS3, holding promise for disrupting viral replication and preventing severe manifestations. However, further research, including animal testing, is imperative to assess therapeutic efficacy and potential toxicity. Developing safe and potent treatments for DENV infection is critical in addressing the rising global health threat posed by this virus.

Genetic interaction analysis of TCF7L2 for biochemical recurrence after radical prostatectomy in localized prostate cancer.

UNLABELLED: Backgroud: Accumulated evidence has demonstrated a significant role of the Wnt pathway in human prostate cancer. We hypothesize that genetic variants in the Wnt pathway effector, Transcription factor 7-like 2 (TCF7L2), may influence clinical outcomes in prostate cancer. METHODS: We comprehensively selected 12 tagged single-nucleotide polymorphisms (SNPs) to capture majority of common variants across TCF7L2, and genotyped in 458 localized prostate cancer patients treated with radical prostatectomy (RP). Kaplan-Meier analysis, Cox proportional hazard model, and survival tree analyses were performed to identify significant SNPs that correlated with biochemical recurrence (BCR) after surgery. RESULTS: A higher-order SNP-SNP interaction profile consisting of TCF7L2 rs7094463, rs10749127, and rs11196224 was significantly associated with BCR (P trend = 0.001). After adjusting for possible confounders, the genetic profile remained significant (P trend = 0.007). None of the studied SNPs were individually associated with BCR. CONCLUSIONS: Our results support a genetic interaction in the TCF7L2 SNPs as a predictor of disease recurrence after curative RP in localized prostate cancer patients.

Discovery of specific inhibitors for intestinal E. coli ß-glucuronidase through in silico virtual screening.

Glucuronidation is a major metabolism process of detoxification for carcinogens, 4-(methylnitrosamino)-1-(3-pyridy)-1-butanone (NNK) and 1,2-dimethylhydrazine (DMH), of reactive oxygen species (ROS). However, intestinal E. coli ß-glucuronidase (eßG) has been considered pivotal to colorectal carcinogenesis. Specific inhibition of eßG may prevent reactivating the glucuronide-carcinogen and protect the intestine from ROS-mediated carcinogenesis. In order to develop specific eßG inhibitors, we found that 59 candidate compounds obtained from the initial virtual screening had high inhibition specificity against eßG but not human ßG. In particular, we found that compounds 7145 and 4041 with naphthalenylidene-benzenesulfonamide (NYBS) are highly effective and selective to inhibit eßG activity. Compound 4041 (IC50 = 2.8 µM) shows a higher inhibiting ability than compound 7145 (IC50 = 31.6 µM) against eßG. Furthermore, the molecular docking analysis indicates that compound 4041 has two hydrophobic contacts to residues L361 and I363 in the bacterial loop, but 7145 has one contact to L361. Only compound 4041 can bind to key residue (E413) at active site of eßG via hydrogen-bonding interactions. These novel NYBS-based eßG specific inhibitors may provide as novel candidate compounds, which specifically inhibit eßG to reduce eßG-based carcinogenesis and intestinal injury.

Discovery of Akt kinase inhibitors through structure-based virtual screening and their evaluation as potential anticancer agents.

Akt acts as a pivotal regulator in the PI3K/Akt signaling pathway and represents a potential drug target for cancer therapy. To search for new inhibitors of Akt kinase, we performed a structure-based virtual screening using the DOCK 4.0 program and the X-ray crystal structure of human Akt kinase. From the virtual screening, 48 compounds were selected and subjected to the Akt kinase inhibition assay. Twenty-six of the test compounds showed more potent inhibitory effects on Akt kinase than the reference compound, H-89. These 26 compounds were further evaluated for their cytotoxicity against HCT-116 human colon cancer cells and HEK-293 normal human embryonic kidney cells. Twelve compounds were found to display more potent or comparable cytotoxic activity compared to compound H-89 against HCT-116 colon cancer cells. The best results were obtained with Compounds a46 and a48 having IC50 values (for HCT-116) of 11.1 and 9.5 µM, respectively, and selectivity indices (IC50 for HEK-293/IC50 for HCT-116) of 12.5 and 16.1, respectively. Through structure-based virtual screening and biological evaluations, we have successfully identified several new Akt inhibitors that displayed cytotoxic activity against HCT-116 human colon cancer cells. Especially, Compounds a46 and a48 may serve as useful lead compounds for further development of new anticancer agents.

Synthesis, anticonvulsant, sedative and anxiolytic activities of novel annulated pyrrolo[1,4]benzodiazepines.

Four new pentacyclic benzodiazepine derivatives (PBDTs 13-16) were synthesized by conventional thermal heating and microwave-assisted intramolecular cyclocondensation. Their anticonvulsant, sedative and anxiolytic activities were evaluated by drug-induced convulsion models, a pentobarbital-induced hypnotic model and an elevated plus maze in mice. PBDT 13, a triazolopyrrolo[2,1-c][1,4]benzodiazepin-8-one fused with a thiadiazolone ring, exhibited the best anticonvulsant, sedative and anxiolytic effects in our tests. There was no significant difference in potency between PBDT 13 and diazepam, and we proposed that the action mechanism of PBDT 13 could be similar to that of diazepam via benzodiazepine receptors.

Topoisomerase I inhibitor evodiamine acts as an antibacterial agent against drug-resistant Klebsiella pneumoniae.

Topoisomerase inhibitors have been developed in a variety of clinical applications. We investigated the inhibitory effect of evodiamine on E. coli topoisomerase I, which may lead to an anti-bacterial effect. Evodiamine inhibits the supercoiled plasmid DNA relaxation that is catalyzed by E. coli topoisomerase I, and computer-aided docking has shown that the Arg161 and Asp551 residues of topoisomerase I interact with evodiamine. We investigated the bactericidal effect of evodiamine against multidrug-resistant Klebsiella pneumoniae. Evodiamine showed a significantly lower minimal inhibitory concentration value (MIC 128 µg/mL) compared with antibiotics (>512 µg/mL) against the clinical isolate of K. pneumoniae. The results suggested that evodiamine is a potential agent against drug-resistant bacteria.

Development of NS2B-NS3 protease inhibitor that impairs Zika virus replication.

Zika virus (ZIKV) is a mosquito-borne flavivirus that causes severe neurological disorders, such as microcephaly in fetuses. Most recently, an outbreak of ZIKV started in Brazil in 2015. To date, no therapeutic agents have been approved to treat ZIKV infection in the clinic. Here, we screened a small molecule inhibitor that can inhibit the function of ZIKV non-structural protein 2B (NS2B)-NS3 protease (ZIKV NS2B-NS3 protease), thereby interfering with viral replication and spread. First, we identified the half maximal inhibitory concentration (IC50) of compound 3 (14.01 µM), 8 (6.85 µM), and 9 (14.2 µM) and confirmed that they are all non-competitive inhibitors. In addition, we have used the blind molecular docking method to simulate the inhibition area of three non-competitive inhibitors (compound 3, 8, and 9) with the ZIKV NS2B-NS3 protease. The results indicated that the four allosteric binding residues (Gln139, Trp148, Leu150, and Val220) could form hydrogen bonds or non-bonding interactions most frequently with the three compounds. The interaction might induce the reaction center conformation change of NS2B-NS3 protease to reduce catalyzed efficiency. The concentration of compounds required to reduce cell viability by 50% (CC50), and the concentration of compounds required to inhibit virus-induced cytopathic effect by 50% (EC50) of three potential compounds are >200 µM, 2.15 µM (compound 3), > 200 µM, 0.52 µM (compound 8) and 61.48 µM, 3.52 µM (compound 9), and Temoporfin are 61.05 µM, 2 µM, respectively. To select candidate compounds for further animal experiments, we analyzed the selectivity index (SI) of compound 3 (93.02), 8 (384.61), 9 (17.46), and Temoporfin (30.53, FDA-approved drug against cancer). Compound 8 has the highest SI value. Therefore, compound 8 was selected for verification in animal models. In vivo, compound 8 significantly delayed ZIKV-induced lethality and illness symptoms and decreased ZIKV-induced weight loss in a ZIKV-infected suckling mouse model. We conclude that compound 8 is worth further investigation for use as a potential future therapeutic agent against ZIKV infection.

An activity-based near-infrared glucuronide trapping probe for imaging ß-glucuronidase expression in deep tissues.

ß-glucuronidase is an attractive reporter and prodrug-converting enzyme. The development of near-IR (NIR) probes for imaging of ß-glucuronidase activity would be ideal to allow estimation of reporter expression and for personalized glucuronide prodrug cancer therapy in preclinical studies. However, NIR glucuronide probes are not yet available. In this work, we developed two fluorescent probes for detection of ß-glucuronidase activity, one for the NIR range (containing IR-820 dye) and the other for the visible range [containing fluorescein isothiocyanate (FITC)], by utilizing a difluoromethylphenol-glucuronide moiety (TrapG) to trap the fluorochromes in the vicinity of the active enzyme. ß-glucuronidase-mediated hydrolysis of the glucuronyl bond of TrapG generates a highly reactive alkylating group that facilitates the attachment of the fluorochrome to nucleophilic moieties located near ß-glucuronidase-expressing sites. FITC-TrapG was selectively trapped on purified ß-glucuronidase or ß-glucuronidase-expressing CT26 cells (CT26/mßG) but not on bovine serum albumin or non-ß-glucuronidase-expressing CT26 cells used as controls. ß-glucuronidase-activated FITC-TrapG did not interfere with ß-glucuronidase activity and could label bystander proteins near ß-glucuronidase. Both FITC-TrapG and NIR-TrapG specifically imaged subcutaneous CT26/mßG tumors, but only NIR-TrapG could image CT26/mßG tumors transplanted deep in the liver. Thus NIR-TrapG may provide a valuable tool for visualizing ß-glucuronidase activity in vivo.

Design, synthesis and biological evaluation of benzo[1.3.2]dithiazolium ylide 1,1-dioxide derivatives as potential dual cyclooxygenase-2/5-lipoxygenase inhibitors.

3-(4-Bromophenyl)-6-nitrobenzo[1.3.2]dithiazolium ylide 1,1-dioxide (5) was discovered as a new prototype for dual inhibitors of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX). Thus, the structure-activity relationships of benzo[1.3.2]dithiazolium ylide 1,1-dioxide skeleton were carried out. The 6-NO(2) group played an essential role in the inhibitory activity. In addition, moderate-sized lipophilic substituents at the para-position of the 3-aryl moiety were required for dual COX-2/5-LOX inhibitory activity. Among the identified potent dual inhibitors, 3-(4-tbutylphenyl) derivative 30c (IC(50) values of 0.27 µM and 0.30 µM against COX-2 and 5-LOX, respectively) and 3-(4-biphenyl) derivative 30f (IC(50) values of 0.50 µM and 0.15µM against COX-2 and 5-LOX, respectively) were the most potent dual COX-2/5-LOX inhibitors. Intraperitoneal administration of 30c at 100mg/kg demonstrated potent acute anti-inflammatory activity. As a result, benzo[1.3.2]dithiazolium ylide 1,1-dioxide represented a novel scaffold for the exploitation in developing dual COX-2/5-LOX inhibitors.

Immobilizing topoisomerase I on a surface plasmon resonance biosensor chip to screen for inhibitors.

BACKGROUND: The topoisomerase I (TopI) reaction intermediate consists of an enzyme covalently linked to a nicked DNA molecule, known as a TopI-DNA complex, that can be trapped by inhibitors and results in failure of re-ligation. Attempts at new derivative designs for TopI inhibition are enthusiastically being pursued, and TopI inhibitors were developed for a variety of applications. Surface plasmon resonance (SPR) was recently used in TopI-inhibition studies. However, most such immobilized small molecules or short-sequence nucleotides are used as ligands onto sensor chips, and TopI was used as the analyte that flowed through the sensor chip. METHODS: We established a sensor chip on which the TopI protein is immobilized to evaluate TopI inhibition by SPR. Camptothecin (CPT) targeting the DNA-TopI complex was used as a representative inhibitor to validate this label-free method. RESULTS: Purified recombinant human TopI was covalently coupled to the sensor chip for the SPR assay. The binding of anti-human (h)TopI antibodies and plasmid pUC19, respectively, to the immobilized hTopI was observed with dose-dependent increases in resonance units (RU) suggesting that the immobilized hTopI retains its DNA-binding activity. Neither CPT nor evodiamine alone in the analyte flowing through the sensor chip showed a significant increase in RU. The combination of pUC19 and TopI inhibitors as the analyte flowing through the sensor chip caused increases in RU. This confirms its reliability for binding kinetic studies of DNA-TopI binders for interaction and for primary screening of TopI inhibitors. CONCLUSIONS: TopI immobilized on the chip retained its bioactivities of DNA binding and catalysis of intermediates of the DNA-TopI complex. This provides DNA-TopI binders for interaction and primary screening with a label-free method. In addition, this biochip can also ensure the reliability of binding kinetic studies of TopI.

Discovery of 3-(4-bromophenyl)-6-nitrobenzo[1.3.2]dithiazolium ylide 1,1-dioxide as a novel dual cyclooxygenase/5-lipoxygenase inhibitor that also inhibits tumor necrosis factor-alpha production.

In the present study we have discovered compound 1, a benzo[1.3.2]dithiazolium ylide-based compound, as a new prototype dual inhibitor of cyclooxygenase (COX) and 5-lipoxygenase (5-LOX). Compound 1 was initially discovered as a COX-2 inhibitor, resulting indirectly from the COX-2 structure-based virtual screening that identified compound 2 as a virtual hit. Compounds 1 and 2 inhibited COX-1 and COX-2 in mouse macrophages with IC(50) in the range of 1.5-18.1microM. Both compounds 1 and 2 were also found to be potent inhibitors of human 5-LOX (IC(50)=1.22 and 0.47microM, respectively). Interestingly, compound 1 also had an inhibitory effect on tumor necrosis factor-alpha (TNF-alpha) production (IC(50)=0.44microM), which was not observed with compound 2. Docking studies suggested the (S)-enantiomer of 1 as the biologically active isomer that binds to COX-2. Being a cytokine-suppressive dual COX/5-LOX inhibitor, compound 1 may represent a useful lead structure for the development of advantageous new anti-inflammatory agents.

An improved screening model to identify inhibitors targeting zinc-enhanced amyloid aggregation.

Zinc, which is abundant in senile plaques consisting mainly of fibrillar beta-amyloid (Abeta), plays a critical role in the pathogenesis of Alzheimer's disease. Treatment with zinc chelators such as clioquinol has been used to prevent Abeta aggregation in Alzheimer's patients; however, clioquinol produces severe side effects. A simple, easy, inexpensive, and versatile screen to identify zinc chelators for inhibition of Abeta aggregation is currently unavailable. We thus developed a high-throughput screen that identifies zinc chelators with anti-Abeta aggregation activity. The recombinant Abeta peptides, aggregated on solid-phase microplates, formed Abeta-immunopositive beta-sheet-containing structures in the presence of zinc. Formation of these Abeta fibrils was specifically blocked by metal ion chelators. This screening model improves identification of zinc-enhanced Abeta fibrils and anti-Abeta aggregation mediated by zinc chelating. The convenient system could qualitatively and quantitatively assay a large sample pool for Abeta aggregation inhibition and dissolution of Abeta aggregates. This screen is practical, reliable, and versatile for comprehensive detection of amyloid fibrillation and identification of inhibitors of Abeta aggregation.

Hydrophilic ester-bearing chlorogenic acid binds to a novel domain to inhibit xanthine oxidase.

Caffeic acid is a xanthine oxidase (XO) inhibitor that binds to the molybdopterin region of its active site. Caffeic acid phenethyl ester (CAPE) has higher hydrophobicity and exhibits stronger inhibition potency toward XO. Chlorogenic acid is a quinyl ester of caffeic acid that has increased hydrophilicity and also shows stronger XO inhibitory activity compared with caffeic acid. Caffeic acid and CAPE showed competitive inhibition against XO, whereas chlorogenic acid displayed mixed-type inhibition, implying that it binds to sites other than the active site. Structure-based molecular modeling was performed to account for the different binding characteristics of the hydrophobic and hydrophilic esters of caffeic acid. Chlorogenic acid showed weak binding to the molybdopterin region of XO, while it more strongly bound the flavin adenine dinucleotide region than it did the molybdopterin region. These results provide the basis for interactions of caffeic acid analogues with XO via various binding domains.

Benzyl ether-linked glucuronide derivative of 10-hydroxycamptothecin designed for selective camptothecin-based anticancer therapy.

A beta-glucuronidase-activated prodrug approach was applied to 10-hydroxycamptothecin, a Camptotheca alkaloid with promising antitumor activity but poor water solubility. We synthesized a glucuronide prodrug of 10-hydroxycamptothecin ( 7) in which glucuronic acid is connected via a self-immolative 3-nitrobenzyl ether linker to the 10-OH group of 10-hydroxycamptothecin. Compound 7 was 80 times more soluble than 10-hydroxycamptothecin in aqueous solution at pH 4.0 and was stable in human plasma. Prodrug 7 was 10- to 15-fold less toxic than the parent drug to four human tumor cell lines. In the presence of beta-glucuronidase, prodrug 7 could be activated to elicit similar cytotoxicity to the parent drug in tumor cells. Enzyme kinetic studies showed that Escherichia coli beta-glucuronidase had a quite low K m of 0.18 microM for compound 7 and exhibited 520 times higher catalytic efficiency for 7 than for 6 (a glucuronide prodrug of 9-aminocamptothecin). Molecular modeling studies predicted that compound 7 would have a higher binding affinity to human beta-glucuronidase than compound 6. Prodrug 7 may be useful for selective cancer chemotherapy by a prodrug monotherapy (PMT) or antibody-directed enzyme prodrug therapy (ADEPT) strategy.

Structure-activity relationship of C6-C3 phenylpropanoids on xanthine oxidase-inhibiting and free radical-scavenging activities.

We employed the techniques of DNA relaxation, DPPH (1,1-diphenyl-2-picrylhydrazyl hydrate), and DMPO (5,5-dimethyl-1-pyrroline-N-oxide)-electron spin resonance (ESR), to study the effects of reactive oxygen species (ROS) suppression by 11 selected C6-C3 phenylpropanoid derivatives under oxidative conditions. We also investigated the effects of the derivatives on the inhibition of xanthine oxidase (XO) activity, and the structure-activity relationships (SARs) of these derivatives against XO activity were further examined using computer-aided molecular modeling. Caffeic acid was the most potent radical scavenger among the 11 test compounds. Our results suggest that the chemical structure and number of hydroxyl groups on the benzene ring of phenylpropanoids are correlated with the effects of ROS suppression. All test derivatives were competitive inhibitors of XO. The results of the structure-based molecular modeling exhibited interactions between phenylpropanoid derivatives and the molybdopterin region of XO. The para-hydroxyl of phenylpropanoid derivatives was pointed toward the guanidinium group of Arg 880. The phenylpropanoid derivatives containing the meta-or ortho-hydroxyl formed hydrogen bonds with Thr 1010. In addition, meta-hydroxyl formed hydrogen bonds with the peptide bond between the residues of Thr1010 and Phe1009. CAPE, the phenylenethyl ester of phenylpropanoids, had the highest affinity toward the binding site of XO, and we speculated that this was due to hydrophobic interactions of the phenylethyl ester with several hydrophobic residues surrounding the active site. The hypoxanthine/XO reaction in the DMPO-ESR technique was used to correlate the effects of these phenylpropanoid derivatives on enzyme inhibition and ROS suppression, and the results showed that caffeic acid and CAPE were the two most potent agents among the tested compounds. We further assessed the effects of the test compounds on living cells, and CAPE was the most potent agent for protecting cells against ROS-mediated damage among the tested phenylpropanoids.

Constituents from the Formosan apple reduce tyrosinase activity in human epidermal melanocytes.

Tyrosinase is a copper-containing monooxygenase that catalyzes melanin synthesis in skin melanocytes. Herein, 13 compounds from the Formosan apple (Malus doumeri var. formosana), an indigenous Taiwanese plant, were isolated and identified. The active constituents were identified as 3-hydroxyphloretin (7) and catechol (9); they exhibited potent hydroxyl radical-scavenging (IC(50) values, 0.6 and 1.1 microM) and cellular tyrosinase-reducing activities (IC(50) values, 32 and 22 microM) in human epidermal melanocytes. In addition, we evaluated the level of several tyrosinase-related proteins by Western blot analysis. In contrast to 3-hydroxyphloretin (7), which showed no effect on the level of these proteins, catechol (9) reduced their activity and the expression of the respective genes, as determined by quantitative real-time PCR. In a kinetic analysis of mushroom tyrosinase, 3-hydroxyphloretin (7) was a competitive inhibitor. These two constituents exhibited metal-coordinating interactions with copper ions in a virtual model of molecular docking with human tyrosinase. Thus, 3-hydroxyphloretin (7) and catechol (9) were the most active constituents from the Formosan apple; they exhibited anti-oxidant and tyrosinase reducing activities, suggesting their possible use as cosmetic agents.

The beta-carboline analog Mana-Hox causes mitotic aberration by interacting with DNA.

Mana-Hox, an analog of beta-carbolines with anticancer activity, induces aberrant mitosis and delays mitotic exit. However, the cellular target is not known. In this study, we visualized the intracellular localization of Mana-Hox. Mana-Hox rapidly penetrated into cells (within 1 min) and concentrated on disorganized metaphase chromosomes after 13 hr of exposure. We demonstrated that Mana-Hox is a noncovalent DNA binder that can interact with DNA through intercalation and/or through minor groove binding. Furthermore, Mana-Hox also inhibits topoisomerase II relaxation activity in vitro, suggesting that Mana-Hox could perturb mitotic chromosome decatenation. Overall, Mana-Hox binding to DNA plays a critical role in the induction of aberrant mitosis and contributes to its anticancer activity.

Em08red, a dual functional antiproliferative emodin analogue, is a downregulator of ErbB2 expression and inducer of intracellular oxidative stress.

Expression of ErbB2 protein is inversely correlated with the prognosis in cancer patients. Consequently, strategies targeting ErbB2 remain an attractive option in treating several types of malignancies, including oral cancer. In addition, many studies have shown that emodin and emodin derivatives are able to inhibit growth of ErbB2-overexpressing tumor cells. In this study, a series of computer modeling-generated emodin analogues were synthesized and tested for their antiproliferative activity against oral cancer cell lines overexpressing ErbB2. Among these analogues, em08red (1,8-dihydroxy-9(10H)-anthracenone) demonstrated potent antiproliferative activity against all three tested ErbB2-overexpressing cell lines, ie, FaDu, HSC3, and OECM1. Treatment with em08red significantly downregulated activation of ErbB2 as well as the ErbB2 protein expression level in the tested cell lines and induced G2 arrest. Antiapoptosis protein (Bcl-xl and Bcl-2) expression levels were also downregulated, and active caspase-3 and caspase-9 was detected in cells after treatment with em08red. Moreover, treatment with em08red stimulated production of cytotoxic reactive oxygen species in treated cells, and this could be partially reversed by pretreatment with N-acetylcysteine. Overall, we demonstrated inhibition of ErbB2 function and induction of reactive oxygen species in tumor cells by em08red, which prevented proliferation of tumor cells and induced apoptotic cell death.

WTC-01, a novel synthetic oxime-flavone compound, destabilizes microtubules in human nasopharyngeal carcinoma cells in vitro and in vivo.

BACKGROUND AND PURPOSE: Dynamic polymerization of microtubules is essential for cancer cell growth and metastasis, and microtubule-disrupting agents have become the most successful anti-cancer agents in clinical use. Besides their antioxidant properties, flavonoids also exhibit strong microtubule-disrupting activity and inhibit tumour growth. We have designed, synthesized and tested a series of oxime/amide-containing flavone derivatives. Here we report the evaluation of one compound, WTC-01 for its anti-proliferative effects in human cancer cells. EXPERIMENTAL APPROACH: We used a range of cancer cell lines including two human nasopharyngeal carcinoma (NPC) cell lines, measuring proliferation, cell cycle and apoptosis, along with caspase levels and mitochondrial membrane potentials. Assays of tubulin polymerisation in vitro and computer modelling of the colchicine binding site in tubulin were also used. In mice, pharmacokinetics and growth of NPC-derived tumours were studied. KEY RESULTS: WTC-01 was most potent against proliferation of NPC cells (IC50 = 0.45 µM), inducing accumulation of cells in G2 /M and increasing apoptosis, time- and concentration-dependently. The colchicine competition-binding experiments and computer modelling results suggested that WTC-01 causes microtubule disruption via binding to the colchicine-binding site of tubulin resulting in mitochondrial membrane damage and cell apoptosis via activation of caspase-9/-3 without noticeable activation of the caspase-8. Notably, our in vivo studies demonstrated that at doses of 25 and 50 mg·kg(-1) , WTC-01 exhibited good pharmacokinetic properties and completely inhibited the growth of NPC-TW01 cells in a xenograft nude mouse model. CONCLUSIONS AND IMPLICATIONS: WTC-01, a new synthetic oxime-containing flavone, exhibited potent anti-tumour activity against NPC cells and merits further investigation.

Development of Membrane-Bound GM-CSF and IL-18 as an Effective Tumor Vaccine.

The development of effective adjuvant is the key factor to boost the immunogenicity of tumor cells as a tumor vaccine. In this study, we expressed membrane-bound granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-18 (IL-18) as adjuvants in tumor cells to stimulate immune response. B7 transmembrane domain fused GM-CSF and IL-18 was successfully expressed in the cell membrane and stimulated mouse splenocyte proliferation. Co-expression of GM-CSF and IL-18 reduced tumorigenesis (P<0.05) and enhanced tumor protective efficacy (P<0.05) significantly in comparison with GM-CSF alone. These results indicated that the combination of GM-CSF andIL-18 will enhance the immunogenicity of a cell-based anti-tumor vaccine. This membrane-bound approach can be applied to other cytokines for the development of novel vaccine strategies.