The identification of small molecules that inhibit the sequencespecific binding of transcription factors to DNA is an attractive approach for regulation of gene expression. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that controls genes involved in glycolysis, angiogenesis, migration, and invasion, all of which are important for tumor progression and metastasis. To identify inhibitors of HIF-1 DNA-binding activity, we expressed truncated HIF-1A and HIF-1B proteins containing the basichelix-loop-helix and PAS domains. Expressed recombinant HIF-1A and HIF-1B proteins induced a specific DNA-binding activity to a double-stranded oligonucleotide containing a canonical hypoxia-responsive element (HRE). One hundred twenty-eight compounds previously identified in a HIF-1-targeted cell-based high-throughput screen of the National Cancer Institute 140,000 small-molecule library were tested in a 96-well plate ELISA for inhibition of HIF-1 DNA-binding activity. One of the most potent compounds identified, echinomycin (NSC-13502), a smallmolecule known to bind DNA in a sequence-specific fashion, was further investigated. Electrophoretic mobility shift assay experiments showed that NSC-13502 inhibited binding of HIF-1A and HIF-1B proteins to a HRE sequence but not binding of the corresponding proteins to activator protein-1 (AP-1) or nuclear factor-KB (NF-KB) consensus sequences. Interestingly, chromatin immunoprecipitation experiments showed that NSC-13502 specifically inhibited binding of HIF-1 to the HRE sequence contained in the vascular endothelial growth factor (VEGF) promoter but not binding of AP-1 or NF-KB to promoter regions of corresponding target genes. Accordingly, NSC-13502 inhibited hypoxic induction of luciferase in U251-HRE cells and VEGF mRNA expression in U251 cells. Our results indicate that it is possible to identify small molecules that inhibit HIF-1 DNA binding to endogenous promoters. (Cancer Res 2005; 65(19): 9047-55)

A critical feature of Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), is its ability to survive and multiply within macrophages, making these host cells an ideal niche for persisting microbes. Killing the intracellular tubercle bacilli is a key requirement for efficient tuberculosis treatment, yet identifying potent inhibitors has been hampered by labor-intensive techniques and lack of validated targets. Here, we present the development of a phenotypic cell-based assay that uses automated confocal fluorescence microscopy for high throughput screening of chemicals that interfere with the replication of M. tuberculosis within macrophages. Screening a library of 57,000 small molecules led to the identification of 135 active compounds with potent intracellular anti-mycobacterial efficacy and no host cell toxicity. Among these, the dinitrobenzamide derivatives (DNB) showed high activity against M. tuberculosis, including extensively drug resistant (XDR) strains. More importantly, we demonstrate that incubation of M. tuberculosis with DNB inhibited the formation of both lipoarabinomannan and arabinogalactan, attributable to the inhibition of decaprenyl-phospho-arabinose synthesis catalyzed by the decaprenyl-phosphoribose 2′ epimerase DprE1/DprE2. Inhibition of this new target will likely contribute to new therapeutic solutions against emerging XDR-TB. Beyond validating the high throughput/content screening approach, our results open new avenues for finding the next generation of antimicrobials.

No significant differences were found in the 5-year overall, disease-free survival and local recurrence rates between robotic and laparoscopic surgical procedures. We concluded that robotic surgery for rectal cancer failed to offer any oncologic or clinical benefits as compared with laparoscopy despite an increased cost.

Although mammalian SWI/SNF chromatin remodeling complexes have been well established to play important role in transcription, their role in DNA repair has remained largely unexplored. Here we show that inactivation of the SWI/SNF complexes and downregulation of the catalytic core subunits of the complexes both result in inefficient DNA double-strand break (DSB) repair and increased DNA damage sensitivity as well as a large defect in H2AX phosphorylation (c-H2AX) and nuclear focus formation after DNA damage. The expression of most DSB repair genes remains unaffected and DNA damage checkpoints are grossly intact in the cells inactivated for the SWI/SNF complexes. Although the SWI/SNF complexes do not affect the expression of ATM, DNA-PK and ATR, or their activation and/or recruitment to DSBs, they rapidly bind to DSB-surrounding chromatin via interaction with c-H2AX in the manner that is dependent on the amount of DNA damage. Given the crucial role for c-H2AX in efficient DSB repair, these results suggest that the SWI/ SNF complexes facilitate DSB repair, at least in part, by promoting H2AX phosphorylation by directly acting on chromatin.

Two new compounds, an ent-isopimarane-type diterpene, 3alpha,12alpha-dihydroxy-ent-8(14),15-isopimaradien-18-al (1), and a dihydrobenzo[b]furan neolignan, (-)-trans-9-acetyl-4,9'-di-O-methyl-3'-de-O-methyldehydrodiconiferyl alcohol (2), along with five known compounds, 7,7'-dihydroxy-6,8'-bicoumarin (bicoumol) (3), 3,4-dimethoxycinnamaldehyde (4), 6-hydroxy-7-methoxycoumarin (isoscopoletin), N-butylaniline, and vanillin, have been isolated from an ethyl acetate-soluble extract of the stem wood of Euphorbiaquinquecostata. The structures of compounds 1 and 2 were elucidated on the basis of spectroscopic data interpretation, and single-crystal X-ray diffraction analysis was used to confirm the structure and relative stereochemistry of 1. The absolute configuration of 1 was established by a convenient Mosher ester procedure in which the sample was treated with MTPA chlorides in deuterated pyridine directly in NMR tubes. All isolates were evaluated for the induction of quinone reductase in Hepa1c1c7 hepatoma cells and for the inhibition of the transformation of murine epidermal JB6 cells.

Curcumin has been strongly implicated as an anti-inflammatory agent, but the precise mechanisms of its action are largely unknown. In this study, we show that the inhibitory action of curcumin on Janus kinase (JAK)-STAT signaling can contribute to its anti-inflammatory activity in the brain. In both rat primary microglia and murine BV2 microglial cells, curcumin effectively suppressed the ganglioside-, LPS-, or IFN-γ-stimulated induction of cyclooxygenase-2 and inducible NO synthase, important enzymes that mediate inflammatory processes. These anti-inflammatory effects appear to be due, at least in part, to the suppression of the JAK-STAT inflammatory signaling cascade. Curcumin markedly inhibited the phosphorylation of STAT1 and 3 as well as JAK1 and 2 in microglia activated with gangliosides, LPS, or IFN-γ. Curcumin consistently suppressed not only NF binding to IFN-γ-activated sequence/IFN-stimulated regulatory element, but also the expression of inflammation-associated genes, including ICAM-1 and monocyte chemoattractant protein 1, whose promoters contain STAT-binding elements. We further show that activation of Src homology 2 domain-containing protein tyrosine phosphatases (SHP)-2, a negative regulator of JAK activity, is likely to be one of the mechanisms underlying the curcumin-mediated inhibition of JAK-STAT signaling. Treatment of microglial cells with curcumin led to an increase in phosphorylation and association with JAK1/2 of SHP-2, which inhibit the initiation of JAK-STAT inflammatory signaling in activated microglia. Taken together, these data suggest curcumin suppresses JAK-STAT signaling via activation of SHP-2, thus attenuating inflammatory response of brain microglial cells.

Five resveratrol sulfate metabolites were synthesized and assessed for activities known to be mediated by resveratrol: inhibition of tumor necrosis factor (TNF)-α-induced NFκB activity, cylcooxygenases (COX-1 and COX-2), aromatase, nitric oxide production in endotoxin-stimulated macrophages, and proliferation of KB or MCF7 cells, induction of quinone reductase 1 (QR1), accumulation in the sub-G 1 phase of the cell cycle, and quenching of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical. Two metabolites showed activity in these assays; the 3-sulfate exhibited QR1 induction, DPPH free radical scavenging, and COX-1 and COX-2 inhibitory activities, and the 4′-sulfate inhibited NFκB induction, as well as COX-1 and COX-2 activities. Resveratrol, as well as its 3′-sulfate and 4-sulfate, inhibit NO production by NO scavenging and down-regulation of iNOS expression in RAW 264.7 cells. Resveratrol sulfates displayed low antiproliferative activity and negligible uptake in MCF7 cells.

Obesity is associated with diverse health risks, but the role of body weight (BMI) as a risk factor for all and site-specific cancers remains controversial and risks for cancer associated with obesity have not been well-characterized in Asians. Body weight and risk for cancer were examined in a 14-year prospective cohort study of 1,213,829 Koreans aged 30-95 years insured by the National Health Insurance Corporation who had a biennial medical evaluation in 1992-1995. Incidence rates for all cancers and site-specific cancers were examined in relation to BMI. Age- and smoking-status adjusted hazard ratios (HR) with 95% confidence intervals (CI) were examined using the Cox proportional hazards model. For both sexes, the average baseline BMI was 23.2 kg/m(2), and the association of risk for all-cancers with BMI was positive. Obese men (BMI >or= 30 kg/m(2)) were at increased risk for developing the following cancers: stomach (1.31, 1.05-1.64), colon (1.42, 1.02-1.98), liver (1.63, 1.27-2.10) and gallbladder (1.65, 1.11-2.44). Obese women (BMI >or= 30 kg/m(2)) were at increased risk for developing liver cancer (1.39, 1.00-1.94), pancreatic cancer (1.80, 1.14-2.86) and breast cancer among women aged >or=50 years old (1.38, 1.00-1.90). The HRs were comparable in never and ever smokers for all cancers and all specific sites except for lung cancer. For all cancers common to both sexes, the association was significantly weaker (p < 0.01) in females. Our study provides further confirmation of the excess cancer risk associated with obesity. Rising obesity in Asian populations raises concern that increasing numbers of avoidable cancer cases will occur among Asians.