Search

Ubiquinol cytochrome c reductase binding protein (UQCRB) is important for mitochondrial complex III stability, electron transport, cellular oxygen sensing and angiogenesis. However, its potential as a prognostic marker in colorectal cancer (CRC) remains unclear. The aim of this study was to determine whether UQCRB can be used as a diagnostic molecular marker for CRC. The correlation between the expression of three genes (UQCRB, UQCRFS1 and MT-CYB) in the mitochondrial respiratory chain complex III and clinico-pathological features was determined. Compared to non-tumor tissues, UQCRB gene expression was upregulated in CRC tissues. Gene and protein expression of the genes were positively correlated. Copy number variation (CNV) differences in UQCRB were observed in CRC tissues (1.32-fold) compared to non-tumor tissues. The CNV of UQCRB in CRC tissues increased proportionally with gene expression and clinical stage. Single-nucleotide polymorphisms in the 3'-untranslated region of UQCRB (rs7836698 and rs10504961) were investigated, and the rs7836698 polymorphism was associated with CRC clinical stage. DNA methylation of the UQCRB promoter revealed that most CRC patients had high methylation levels (12/15 patients) in CRC tissues compared to non-tumor tissues. UQCRB overexpression and CNV gain were correlated with specific CRC clinico-pathological features, indicating clinical significance as a prognostic predictor in CRC. Gene structural factors may be more important than gene transcription repression factors with respect to DNA methylation in UQCRB overexpression. Our results provide novel insights into the critical role of UQCRB in regulating CRC, supporting UQCRB as a new candidate for the development of diagnostics for CRC patients.

// Min Wu 1 , Sahn-Ho Kim 1 , Indrani Datta 2 , Albert Levin 2 , Gregory Dyson 3 , Jing Li 4 , Stephanie Kaypee 5 , M. Mahadeva Swamy 5 , Nilesh Gupta 6 , Ho Jeong Kwon 7 , Mani Menon 1 , Tapas K. Kundu 5 and G. Prem-Veer Reddy 1 1 Vattikuti Urology Institute, Henry Ford Hospital, Detroit, MI, USA 2 Bioinformatics Core, Public Health Sciences, Henry Ford Hospital, Detroit, MI, USA 3 Biostatistics Core, Karmanos Cancer Institute, Wayne State University, Detroit, MI, USA 4 Pharmacology Core, Karmanos Cancer Institute, Wayne State University, Detroit, MI, USA 5 Transcription and Disease Laboratory, Molecular Biology and Genetics Unit, JNCASR, Bangalore, Karnataka, India 6 Department of Pathology, Henry Ford Hospital, Detroit, MI, USA 7 Department of Biotechnology, Translational Research Center for Protein Function Control, Yonsei University, Seoul, Republic of Korea Correspondence: G. Prem Veer Reddy, email: // Keywords : Androgen receptor, calmodulin, Hydrazinobenzoylcurcumin, CTK7A, castration-resistant prostate cancer Received : November 24, 2014 Accepted : January 20, 2015 Published : January 31, 2015 Abstract There is a critical need for therapeutic agents that can target the amino-terminal domain (NTD) of androgen receptor (AR) for the treatment of castration-resistant prostate cancer (CRPC). Calmodulin (CaM) binds to the AR NTD and regulates AR activity. We discovered that Hydrazinobenzoylcurcumin (HBC), which binds exclusively to CaM, inhibited AR activity. HBC abrogated AR interaction with CaM, suppressed phosphorylation of AR Serine81, and blocked the binding of AR to androgen-response elements. RNA-Seq analysis identified 57 androgen-regulated genes whose expression was significantly (p ≤ 0.002) altered in HBC treated cells as compared to controls. Oncomine analysis revealed that genes repressed by HBC are those that are usually overexpressed in prostate cancer (PCa) and genes stimulated by HBC are those that are often down-regulated in PCa, suggesting a reversing effect of HBC on androgen-regulated gene expression associated with PCa. Ingenuity Pathway Analysis revealed a role of HBC affected genes in cellular functions associated with proliferation and survival. HBC was readily absorbed into the systemic circulation and inhibited the growth of xenografted CRPC tumors in nude mice. These observations demonstrate that HBC inhibits AR activity by targeting the AR NTD and suggest potential usefulness of HBC for effective treatment of CRPC.

The term “isolated v-lesion” was proposed at the 2009 Banff conference on allograft pathology. It is still debated whether the isolated v-lesion is a part of the antibody-mediated or T cell–mediated rejection and whether the isolated v-lesion has any prognostic significance of its own. To investigate the characteristics of the isolated v-lesion, we identified infiltrating inflammatory cells in renal allograft biopsy specimens with these lesions. We selected 11 allograft renal biopsy specimens which were compatible with the original definition of the isolated v-lesion (v1 or v2 with i ≤ 1 and t ≤ 1) and had enough paraffin-embedded tissue for immunohistochemistry. We performed immunohistochemistry for markers of T cells (CD3, CD4, and CD8), B cells (CD20), NK/T cells (CD56), and macrophages (CD68). The number of positive cells was counted in each compartment of the renal tissue including the arteries, peritubular capillaries, glomeruli, tubules, and interstitium. Arteries were infiltrated by CD3/CD8-positive T cells and CD68-positive macrophages. Three cases showed T cell–dominant infiltrates and four cases showed macrophage-dominant infiltrates. Glomeruli showed a similar inflammatory cell profile to that of arteries. Tubulitis was composed of CD3/CD8-positive T cells. The components of interstitial inflammation were more variable with the presence of CD20-positive B cells. In six cases, interstitial infiltrates were predominantly composed of CD3/CD8-positive T cells, and two of these cases showed almost exclusive infiltrates of T cells. However, four cases showed co-dominant infiltrates of T and B cells, and one case showed predominant B cell infiltrates. The isolated v-lesion has a heterogeneous pathogenesis, and B cell-predominant infiltrates in some cases suggest that this lesion could be related to an antibody-related process.

Ubiquinol-cytochrome c reductase binding protein (UQCRB) is one of the subunits of mitochondrial complex III and is a target protein of the natural anti-angiogenic small molecule terpestacin. Previously, the biological role of UQCRB was thought to be limited to the maintenance of complex III. However, the identification and validation of UQCRB as a target protein of terpestacin enabled the role of UQCRB in oxygen sensing and angiogenesis to be elucidated. To explore the biological role of this protein further, UQCRB mutant stable cell lines were generated on the basis of a human case report. We demonstrated that these cell lines exhibited glycolytic and pro-angiogenic activities via mitochondrial reactive oxygen species (mROS)-mediated HIF1 signal transduction. Furthermore, a morphological abnormality in mitochondria was detected in UQCRB mutant stable cell lines. In addition, the proliferative effect of the UQCRB mutants was significantly regulated by the UQCRB inhibitors terpestacin and A1938. Collectively, these results provide a molecular basis for UQCRB-related biological processes and reveal potential key roles of UQCRB in angiogenesis and mitochondria-mediated metabolic disorders.

R-(-)-β-O-methylsynephrine (OMe-Syn) is a naturally occurring small molecule that was identified in a previous screen as an inhibitor of angiogenesis. In this study, we conducted two animal model experiments to investigate the in vivo antiangiogenic activity of OMe-Syn. OMe-Syn significantly inhibited angiogenesis in a transgenic zebrafish model as well as in a mouse retinopathy model. To elucidate the underlying mechanisms responsible for the antiangiogenic activity of OMe-Syn, we used phage display cloning to isolate potential OMe-Syn binding proteins from human cDNA libraries and identified nucleoporin 153 kDa (NUP153) as a primary binding partner of OMe-Syn. OMe-Syn competitively inhibited mRNA binding to the RNA-binding domain of NUP153. Furthermore, depletion of NUP153 in human cells or zebrafish embryos led to an inhibition of angiogenesis, in a manner similar to that seen in response to OMe-Syn treatment. These data suggest that OMe-Syn is a promising candidate for the development of a novel antiangiogenic agent and that inhibition of NUP153 is possibly responsible for the antiangiogenic activity of OMe-Syn.

Mitochondrial respiration is required for hypoxia-inducible factor (HIF)-1α stabilization, which is important for tumor cell survival, proliferation, and angiogenesis. Herein, small molecules that inhibit HIF-1α protein stability by targeting mitochondrial energy production were screened using the Library of Pharmacologically Active Compounds and cell growth assay in galactose or glucose medium. NNC 55-0396, a T-type Ca(2+) channel inhibitor, was selected as a hit from among 1,280 small molecules. NNC 55-0396 suppressed mitochondrial reactive oxygen species-mediated HIF-1α expression as well as stabilization by inhibiting protein synthesis in a dose-dependent manner. NNC 55-0396 inhibited tumor-induced angiogenesis in vitro and in vivo by suppressing HIF-1α stability. Moreover, NNC 55-0396 significantly suppressed glioblastoma tumor growth in a xenograft model. Thus, NNC 55-0396, a small molecule targeting T-type Ca(2+) channel, was identified by the systemic cell-based assay and was shown to have antiangiogenic activity via the suppression of HIF-1α signal transduction. These results provide new insights into the biological network between ion channel and HIF-1α signal transduction.HIF-1α overexpression has been demonstrated in hypoxic cancer cells. NNC 55-0396, a T-type Ca(2+) channel inhibitor, inhibited HIF-1α expression via both proteasomal degradation and protein synthesis pathways. T-type Ca(2+) channel inhibitors block angiogenesis by suppressing HIF-1α stability and synthesis. NNC 55-0396 could be a potential therapeutic drug candidate for cancer treatment.

Hispidin, a phenolic compound from Phellinus linteus (a medicinal mushroom), has been shown to possess strong anti-oxidant, anti-cancer, anti-diabetic, and anti-dementia properties. However, the cardioprotective efficacy of hispidin has not yet been investigated. In the present study, we investigated the protective effect of hispidin against oxidative stress-induced apoptosis in H9c2 cardiomyoblast cells and neonatal rat ventricular myocytes. While the treatment of H9c2 cardiomyoblast cells with hydrogen peroxide caused a loss of cell viability and an increase in the number of apoptotic cells, hispidin significantly protected the cells against hydrogen peroxide-induced cell death without any cytotoxicity as determined by XTT assay, LDH release assay, Hoechst 33342 assay, and Western blotting of apoptosis proteins such as caspase-3, Bax, and Bcl-2. Our data also shows that hispidin significantly scavenged intracellular ROS, and markedly enhanced the expression of antioxidant enzymes such as heme oxygenase-1 and catalase, which was accompanied by the concomitant activation of Akt/GSK-3β and ERK1/2 phosphorylation in H9c2 cardiomyoblast cells. The effects of hispidin on Akt and ERK phosphorylation were abrogated by LY294002 (a PI3K/Akt inhibitor) and U0126 (an ERK1/2 inhibitor). The effect of hispidin on GSK-3b activities was also blocked by LY294002. Furthermore, inhibiting the Akt/GSK-3β and ERK1/2 pathway by these inhibitors significantly reversed the hispidin-induced Bax and Bcl-2 expression, apoptosis induction, and ROS production. These findings indicate that hispidin protects against apoptosis in H9c2 cardiomyoblast cells exposed to hydrogen peroxide through reducing intracellular ROS production, regulating apoptosis-related proteins, and the activation of the Akt/GSK-3β and ERK1/2 signaling pathways.

Autophagy and apoptosis are important processes that control cellular homeostasis and have been highlighted as promising targets for novel cancer therapies. Here, we identified convallatoxin (CNT), isolated from Antiaris toxicaria, as a dual inducer of autophagy and apoptosis. CNT exerts cytotoxic effects on a number of cancer and normal cell lines and induces apoptosis by increasing caspase-3 and poly ADP ribose polymerase (PARP) cleavage. Moreover, dose- and time-dependent autophagic activity was detected in CNT-treated cells, and mammalian target of rapamycin (mTOR)/p70S6K signal pathway inhibition was observed. Notably, CNT inhibits human umbilical vein endothelial cell (HUVEC) growth and exerts anti-angiogenic activity in vitro and in vivo. Collectively, these results demonstrate that the naturally occurring compound, CNT, is a novel anti-angiogenic compound via dual inducing of autophagy and apoptosis.

FK228 (formerly FR901228) was recently isolated from Chromobacterium violaceum as a potent antitumor agent and its biologic target protein was identified as histone deacetylase (HDAC). Because of its unique chemical structure (i.e., bicyclic depsipeptide) and activity profile in the National Cancer Institute's developmental therapeutics program, FK228 is currently in a phase I clinical trial for cancer therapy. In the present study, we investigated the antiangiogenic activity of FK228 in vivo and in vitro. FK228 potently blocked the hypoxia-stimulated proliferation, invasion, migration, adhesion and tube formation of bovine aortic endothelial cells at the same concentration at which the agent inhibited the HDAC activity of cells. In addition, FK228 inhibited the neovascularization of chick embryo and that of adult mice in the Matrigel plug assay. Interestingly, the expression of angiogenic-stimulating factors such as vascular endothelial growth factor or kinase insert domain receptor were suppressed by FK228, whereas that of angiogenic-inhibiting factors such as von Hippel Lindau and neurofibromin2 were induced, suggesting that a gene-transcription effect was involved in the inhibition of angiogenesis by FK228. These results indicate that FK228 is a novel antiangiogenic agent and may suppress tumor expansion, at least in part, by the inhibition of neovascularization.

Although histone deacetylases (HDACs) appear to play a crucial role in carcinogenesis, the expression status of HDACs in primary human cancer tissues has not yet been reported. In this study, we investigated the expression level of HDAC1 in 25 paired primary human gastric cancer (GC) tissues and corresponding normal tissues through semi-quantitative RT-PCR and immunoblot analysis. The HDAC1 expression pattern was also topologically examined through immunohistochemistry. Overexpression of HDAC1 mRNA was detected in 68% of GC tissues (17 of 25), and the relative density of HDAC1 mRNA in GC tissue was increased 1.8-fold versus the normal counterpart (P < 0.01). Elevated expression of HDAC1 protein was also detected in 61% of GC samples (11 of 18), which also showed an increased mRNA level of HDAC. Immunohistochemically, overexpression of HDAC1 was predominantly localized in the nuclei of most neoplastic cells, including embolic tumor cells, whereas normal glandular epithelial cells revealed only weak HDAC1 expression that was focal in distribution. Thus, the present study clearly demonstrates that HDAC1 is overexpressed in GC and probably plays a significant role in gastric carcinogenesis.

Wondonins A and B, two alkaloids of an unprecedented structural class, along with the previously described dihydroxystyryl sulfates, have been isolated from an association of the sponges Poecillastra wondoensis and Jaspis sp. collected from Keomun Island, Korea. The structures of these compounds have been determined as imidazole alkaloids containing a bis(dihydroxystyrene) moiety by combined spectroscopic methods. The new compounds exhibited antiangiogenic activity against human umbilical vein endothelial cells.

A panel screening using cdc mutants of Schizosaccharomyces pombe identified radicicol as a potent growth inhibitor of certain mutants at the permissive temperature. The strains sensitive to radicicol were cdc7, cdc11, and cdc14, all of which are defective in early septum formation. Cytokinesis but not nuclear division of these mutants was inhibited by radicicol, but that of cells with the wild-type background was not. A biologically active derivative of radicicol with a biotin moiety at the C-11 position bound Swo1, an Hsp90 homologue in S. pombe. Increased Swo1 expression partially suppressed radicicol sensitivity of cdc14 and almost completely rescued morphological abnormalities in cdc14 and cdc7 cells induced by radicicol at the permissive temperature. On the other hand, the increased Swo1 expression did not restore septum formation at the nonpermissive temperature. These results suggest that Swo1, as a molecular chaperone, plays a role in stabilizing these temperature-sensitive proteins at the permissive temperature or in activating the cytokinesis signaling cascade.

Heat shock protein 90 (hsp90) is a molecular chaperone responsible for protein folding and maturation in vivo. Interaction of hsp90 with human glutamyl-prolyl-tRNA synthetase (EPRS) was found by genetic screening, co-immunoprecipitation, and in vitro binding experiments. This interaction was sensitive to the hsp90 inhibitor, geldanamycin, and also ATP, suggesting that the chaperone activity of hsp90 is required for interaction with EPRS. Interaction of EPRS with hsp90 was targeted to the region of three tandem repeats linking the two catalytic domains of EPRS that is also responsible for the interaction with isoleucyl-tRNA synthetase (IRS). Interaction of EPRS and IRS also depended on the activity of hsp90, implying that their association was mediated by hsp90. EPRS and IRS form a macromolecular protein complex with at least six other tRNA synthetases and three cofactors. hsp90 preferentially binds to most of the complex-forming enzymes rather than those that are not found in the complex. In addition, inactivation of hsp90 interfered with the in vivo incorporation of the nascent aminoacyl-tRNA synthetases into the multi-ARS complex. Thus, hsp90 appears to mediate protein-protein interactions of mammalian tRNA synthetases.

In studying biological roles of interferon regulatory factor (IRF)-1 tumor suppressor in cervical carcinogenesis, we found that HPV E7 is functionally associated with IRF-1. Binding assays indicate a physical interaction between IRF-1 and HPV E7in vivo and in vitro. The carboxyl-terminal transactivation domain of IRF-1 was required for the interaction. Transient co-expression of E7 significantly inhibits the IRF-1-mediated activation of IFN-β promoter in NIH-3T3 cells. Co-transfection of E7 mutants reveals that the pRb-binding portion of E7 is necessary for the E7-mediated inactivation of IRF-1. It was next determined whether histone deacetylase (HDAC) is involved in the inactivation mechanism as recently suggested, where the carboxyl-terminal zinc finger domain of E7 associates with NURD complex containing HDAC. When trichostatin A, an inhibitor of HDAC, was treated, the repressing activity of E7 was released in a dose-dependent manner. Furthermore, the mutation of zinc finger abrogates such activity without effect on the interaction with IRF-1. These results suggest that HPV E7 interferes with the transactivation function of IRF-1 by recruiting HDAC to the promoter. The immune-promoting role of IRF-1 evokes the idea that our novel finding might be important for the elucidation of the E7-mediated immune evading mechanism that is frequently found in cervical cancer.

Natural products have played a unique role in providing new tools and insights in chemical biology. The tremendous value of natural products was highlighted by scientists from Korea and Japan at the 4(th) Korea-Japan Chemical Biology symposium.

Depudecin is a fungal metabolite that reverts the rounded phenotype of NIH 3T3 fibroblasts transformed with v- ras and v- src oncogenes to the flattened phenotype of the nontransformed parental cells. The mechanism of detransformation induced by this agent had not been determined. Here, we demonstrate that depudecin inhibits histone deacetylase (HDAC) activity effectively both in vivo and in vitro . Depudecin induces similar morphological reversion in v- ras transformed NIH 3T3 cells as do other naturally occurring HDAC inhibitors such as trichostatin A or trapoxin. It competitively inhibits the binding of [ 3 H]trapoxin in vitro and the nuclear binding of a trapoxin–coumarin fluorophore in vivo , suggesting that depudecin shares a nuclear binding protein and site on that protein with trapoxin. Furthermore, depudecin induces hyperacetylation of histones in a dose-dependent manner and at concentrations comparable with that required for detransformation. An in vitro histone deacetylase assay, using purified recombinant HDAC1, reveals that depudecin inhibits 50% of the enzyme activity at a concentration of 4.7 μM. These results demonstrate that depudecin is a novel HDAC inhibitor and suggest that its ability to induce morphological reversion of transformed cells is the result of its HDAC inhibitory activity.

The leptin receptor, OBR, is involved in the regulation of whole-body energy homeostasis. Most obese people are resistant to leptin and do not respond to the hormone. The prevention and reversal of leptin resistance is one of the major current goals of obesity research. We showed previously that increased OBR cell surface expression concomitantly increases cellular leptin signaling and prevents obesity development in mice. Improvement of OBR cell surface expression can thus be considered as an interesting anti-obesity therapeutic strategy. To identify compounds that increase the surface expression of OBR, we developed a cell-based, phenotypic assay to perform a high-content screen (HCS) against a library of 50,000 chemical compounds. We identified 67 compounds that increased OBR cell surface expression with AC50 values in the low micromolar range and no effect on total OBR expression and cellular toxicity. Compounds were classified into 16 chemical clusters, of which 4 potentiated leptin-promoted signaling through the JAK2/STAT3 pathway. In conclusion, development of a robust phenotypic screening approach resulted in the discovery of four new scaffolds that demonstrate the desired biological activity and could constitute an original therapeutic solution against obesity and associated disorders.

Bioactive small molecules are powerful tools used to evaluate protein function under physiological and pathological conditions. Over recent decades, utilization of a variety of biologically active small molecules in basic research and clinical applications has provided tremendous benefits in understanding the molecular mechanisms of biology and accelerating drug development. This review focuses on recent advances in the identification of new small molecules and their target proteins for exploring angiogenesis at the molecular level. In particular, we focus on the oxygen-sensing role of ubiquinol-cytochrome c reductase binding protein (UQCRB) of mitochondrial Complex III through identification of the protein target and the mode of action of a natural small molecule, terpestacin. The positive feedback approach of chemistry and biology provides a new way to explore functional roles of proteins and to translate this information into practical applications.

Vascular endothelial growth factor (VEGF) signal transduction is involved in tumor angiogenesis, and the inhibition of this pathway is considered to be a powerful strategy for cancer therapy. We previously showed that small molecules targeting the ubiquinol-cytochrome c reductase binding protein (UQCRB), a subunit of mitochondrial complex III, in tumor cells suppress hypoxia-induced tumor angiogenesis. However, the mechanism by which UQCRB functioned remained unknown. In the present study, we demonstrate in endothelial cells (ECs) that UQCRB enhances VEGF receptor type 2 (VEGFR2) signaling by increasing the levels of mitochondrial reactive oxygen species (ROS). By contrast, terpestacin, a UQCRB targeting small molecule, blocked mitochondrial ROS-mediated VEGFR2 signaling pathways in ECs, thereby suppressing VEGF-dependent angiogenesis in vitro and in vivo. Furthermore, treatment with both terpestacin and bevacizumab, a VEGF signaling inhibitor, resulted in additive inhibition of tumor-induced angiogenesis both in vitro and in vivo. These data demonstrate that mitochondrial UQCRB positively regulates VEGFR2 signaling in ECs and the UQCRB targeting agent could be applied in new therapeutic approaches for human cancer.Inhibiting angiogenesis has been a focus for anti-cancer strategies. Mitochondrial UQCRB enhances VEGFR2 signaling by increasing ROS in endothelial cells. UQCRB inhibitor blocks angiogenesis by suppressing mitochondrial ROS. Findings may provide a new therapeutic approach for human cancer.

International audience; Classical target-based, high-throughput screening has been useful for the identification of inhibitors for known molecular mechanisms involved in the HIV life cycle. In this study, the development of a cell-based assay that uses a phenotypic drug discovery approach based on automated high-content screening is described. Using this screening approach, the antiviral activity of 26,500 small molecules from a relevant chemical scaffold library was evaluated. Among the selected hits, one sulfonamide compound showed strong anti-HIV activity against wild-type and clinically relevant multidrug resistant HIV strains. The biochemical inhibition, point resistance mutations and the activity of structural analogs allowed us to understand the mode of action and propose a binding model for this compound with HIV-1 reverse transcriptase.

Background : (−)-Depudecin is a fungal metabolite that reverts the rounded phenotype of NIH3T3 fibroblasts transformed with v- ras and v- src oncogenes to the flat phenotype of the nontransformed parental cells. The mechanism of action of this detransformation agent is unknown. Although depudecin appears to be an excellent molecule for probing signaling pathways that regulate changes in the cytoskeletal architecture, reagents based on depudecin are not available as it has not yet been successfully synthesized. We therefore set out to synthesize (−)-depudecin. Results : An asymmetric synthesis of (−)-depudecin has been developed. A cell staining assay has been used to reveal the ability of synthetic depudecin, but not several structural variants, to induce a flattened morphology in v-Ha- ras -transformed NlH3T3 cells.This assay also shows that depudecin induces an intricate network of actin stress fibers in these cells and in MG63 osteosarcoma cells and reveals the essential role of the epoxide and hydroxyl moieties in depudecin. Cycloheximide and actinomycin D inhibited the ability of depudecin to induce a morphological change, suggesting that both mRNA synthesis and de novo protein synthesis are required for depudecin-mediated suppression of the transformed phenotypes in ras -transformed cells. Conclusions : The synthetic procedure provides access to (−)-depudecin and could be readily modified to produce depudecin-related reagents for the identification of depudecin's cellular target(s). This target appears to be involved in the regulation of the assembly of the actin microfilament component of the cytoskeleton in mammalian cells.

Radicicol, an inhibitor of protein-tyrosine kinase, was found to cause morphological reversion of v-Ha-ras-transformed NIH3T3 fibroblasts and T24 human urinary bladder carcinoma cells that contain an activated ras mutation. The network of actin stress fibers was restored during the treatment with radicicol. A similar morphological change was observed with another protein-tyrosine kinase inhibitor, herbimycin A. Radicicol did not cause any changes in the proportion of the active GTP binding form of p21ras or its subcellular localization. These results rule out the possibility that the morphological reversion by radicicol is due to direct or indirect inhibition of the p21ras function. Cycloheximide and actinomycin D inhibited the morphological change by radicicol, suggesting that the induced transcription of a gene(s) followed by de novo protein synthesis is required for suppression of the transformed phenotype in ras-transformed cells by tyrosine kinase inhibitors.

A FK506 analogue containing a non-natural starter unit was obtained through mutasynthesis by feeding cultures of Streptomyces sp. KCTC 11604BP fkbO deletion mutant with 3-cyclohexene-1-carboxylic acid. The structure of the new compound, 32-dehydroxy-FK506, and its biological activities were determined.

Hydroxamate-based HDAC inhibitors have promising anticancer activities but metabolic instability and poor pharmacokinetics leading to poor in vivo results. QSAR and PK studies of HDAC inhibitors showed that a γ-lactam core and a modified cap group, including halo, alkyl, and alkoxy groups with various carbon chain linkers, improved HDAC inhibition and metabolic stability. The biological properties of the γ-lactam HDAC inhibitors were evaluated; the compound designated 8f had potent anticancer activity and high oral bioavailability.

Histone deacetylases (HDACs) are important enzymes in epigenetic regulation and are therapeutic targets for cancer. Most zinc-dependent HDACs induce proliferation, dedifferentiation, and anti-apoptotic effects in cancer cells. We designed and synthesized a new series of pyridone-based HDAC inhibitors that have a pyridone ring in the core structure and a conjugated system with an olefin connecting the hydroxamic acid moiety. Consequently, most of the selected pyridone-based HDAC inhibitors showed similar or higher inhibition profiles in addition to remarkable metabolic stability against hydrolysis relative to the corresponding lactam-based HDAC inhibitors. Furthermore, the selectivity of the novel pyridine-based compounds was evaluated across all of the HDAC isoforms. One of these compounds, (E)-N-hydroxy-3-{1-[3-(naphthalen-2-yl)propyl]-2-oxo-1,2-dihydropyridin-3-yl}acrylamide, exhibited the highest level of HDAC inhibition (IC(50) =0.07 μM), highly selective inhibition of class I HDAC1 and class II HDAC6 enzymes, metabolic stability in mouse liver microsomal studies, and effective growth inhibition of various cancer cell lines. Docking studies indicated that a long alkyl linker and bulky hydrophobic cap groups affect in vitro activities. Overall, the findings reported herein regarding pyridone-based HDAC inhibitors can be used to guide future research efforts to develop new and effective anticancer therapeutics.

Verrulactones A (I) and B (II), new dimeric compounds of alternariol class, strongly inhibit Staphylococcus aureus enoyl-ACP reductase and exhibit antibacterial activity against S.

Radicicol, an inhibitor of src or src-like kinases, causes hypophosphorylation and down-regulation of the RB retinoblastoma tumor suppressor protein in HL-60 human promyelocytic leukemia cells. Both of these changes in the RB protein typically are associated with myeloid or monocytic differentiation of these cells induced by retinoic acid or 1,25-dihydroxy vitamin D3. When added with either inducer, radicicol caused the typically induced myeloid or monocytic differentiation of these cells to be accelerated. By itself radicicol caused a transient inhibition of G1 to S transit, but did not cause phenotypic conversion. The down-regulation and dephosphorylation of RB by radicicol may thus facilitate cell differentiation.

To identify specific biomarkers generated upon exposure of L5178Y mouse lymphoma cells to carcinogens, 2-DE and MALDI-TOF MS analysis were conducted using the cellular proteome of L5178Y cells that had been treated with the known carcinogens, 1,2-dibromoethane and O-nitrotoluene and the noncarcinogens, emodin and D-mannitol. Eight protein spots that showed a greater than 1.5-fold increase or decrease in intensity following carcinogen treatment compared with treatment with noncarcinogens were selected. Of the identified proteins, we focused on the candidate biomarker ERM-binding phosphoprotein 50 (EBP50), the expression of which was specifically increased in response to treatment with the carcinogens. The expression level of EBP50 was determined by western analysis using polyclonal rabbit anti-EBP50 antibody. Further, the expression level of EBP50 was increased in cells treated with seven additional carcinogens, verifying that EBP50 could serve as a specific biomarker for carcinogens.

1,2-Dibromoethane and glycidol are well known genotoxic carcinogens, which have been widely used in industry. To identify a specific biomarker for these carcinogens in cells, the cellular proteome of L5178Y mouse lymphoma cells treated with these compounds was analyzed by 2-dimensional gel electrophoresis (2-DE) and MALDI-TOF mass spectrometry (MS). Of 50 protein spots showing a greater than 1.5-fold increase or decrease in intensity compared to control cells on a 2-D gel, we focused on the candidate biomarker moesin. Western analysis using monoclonal rabbit anti-moesin confirmed the identity of the protein and its increased level of expression upon exposure to the carcinogenic compounds. Moesin expression also increased in cells treated with six additional genotoxic carcinogens, verifying that moesin could serve as a biomarker to monitor phenotypic change upon exposure to genotoxic carcinogens in L5178Y mouse lymphoma cells.

New dimeric compounds of alternariol class, verrulactones A and B, were isolated from a culture broth of the fungal strain Penicillium verruculosum F375 and their structure were established by various spectral analysis. Verrulactones A and B strongly inhibited Staphylococcus aureus enoyl-ACP reductase with IC 50 of 0.92 and 1.41 μM, respectively, and also showed antibacterial activity against S. aureus and MRSA with MICs of 8 and 16 μg/mL, respectively.

Histone deacetylase (HDAC) is an attractive target for cancer therapy because it plays a key role in gene expression and carcinogenesis. N-hydroxy-7-(2-naphthylthio) heptanomide (HNHA) is a novel synthetic HDAC inhibitor (HDACI) that shows better pharmacological properties than a known HDACI present in the human fibrosarcoma cell: suberoylanilide hydroxamic acid (SAHA). Here, we investigate the anti-cancer activity of HNHA against breast cancer both in vitro and in vivo. HNHA arrested the cell cycle at the G1/S phase via p21 induction, which led to profound inhibition of cancer cell growth in vitro. In addition, HNHA-treated cells showed markedly decreased levels of VEGF and HIF-1α than SAHA and fumagillin (FUMA) when accompanied by increased histone acetylation. HNHA significantly inhibited tumor growth in an in vivo mouse xenograft model. HNHA-treated mice survived significantly longer than SAHA- and FUMA-treated mice. Dynamic MRI showed significantly decreased blood flow in the HNHA-treated mice, implying that HNHA inhibits tumor neovascularization. This finding was accompanied by marked reductions of proangiogenic factors and significant induction of angiogenesis inhibitors in tumor tissues. We have shown that HNHA is an effective anti-tumor agent in breast cancer cells in vitro and in breast cancer xenografts in vivo. Collectively, these findings indicate that HNHA may be a potent anti-cancer agent against breast cancer due to its multi-faceted inhibition of HDAC activity, as well as anti-angiogenesis activity. (Cancer Sci 2011; 102: 343–350)

Mitochondrial reactive oxygen species (ROS) are associated with multiple cellular functions such as cell proliferation, differentiation, and apoptosis. In particular, high levels of mitochondrial ROS in hypoxic cells regulate many angiogenesis-related diseases, including cancer and ischemic disorders. Here we report a new angiogenesis inhibitor, YCG063, which suppressed mitochondrial ROS generation in a phenotypic cell-based screening of a small molecule-focused library with an ArrayScan HCS reader. YCG063 suppressed mitochondrial ROS generation under a hypoxic condition in a dose-dependent manner, leading to the inhibition of in vitro angiogenic tube formation and chemoinvasion as well as in vivo angiogenesis of the chorioallantoic membrane (CAM) at non-toxic doses. In addition, YCG063 decreased the expression levels of HIF-1α and its target gene, VEGF. Collectively, a new antiangiogenic small molecule that suppresses mitochondrial ROS was identified. This new small molecule tool will provide a basis for a better understanding of angiogenesis driven under hypoxic conditions.

Autophagy is the mass degradation system that removes long-lived proteins and malfunctioning organelles within the cell. Dysfunctional autophagic processes can cause various diseases such as cancer and neurodegenerative disorders, but the underlying mechanisms responsible for such events remain undefined. Small molecules that control autophagy could be powerful tools to reveal autophagy mechanisms, and to develop treatments for autophagy-related diseases including Alzheimer’s disease, Parkinson’s disease and various cancer types. This review discusses the small molecules that have been identified to control autophagy and how they can be used to understand signaling pathways important for autophagy in the context of chemical genomics.

R-(-)-beta-O-methylsynephrine (OMe-Syn) is an active compound isolated from a plant of the Rutaceae family. We conducted cell proliferation assays on various cell lines and found that OMe-Syn more strongly inhibited the growth of human umbilical vein endothelial cells (HUVECs) than that of other normal and cancer cell lines tested. In angiogenesis assays, it inhibited vascular endothelial growth factor (VEGF)-induced invasion and tube formation of HUVECs with no toxicity. The anti-angiogenic activity of OMe-Syn was also validated in vivo using the chorioallantonic membrane (CAM) assay in growing chick embryos. Expression of the growth factors VEGF, hepatocyte growth factor, and basic fibroblast growth factor was suppressed by OMe-Syn in a dose-dependent manner. Taken together, our results indicate that this compound could be a novel basis for a small molecule targeting angiogenesis.

Recent reports have shown that Ca(2+)/calmodulin (Ca(2+)/CaM) signaling plays a crucial role in angiogenesis. We previously developed a new Ca(2+)/CaM antagonist, HBC (4-{3,5-bis-[2-(4-hydroxy-3-methoxyphenyl)ethyl]-4,5-dihydropyrazol-1-yl}benzoic acid), from a curcumin-based synthetic chemical library. Here, we investigated its anti-angiogenic activity and mode of action. HBC potently inhibited the proliferation of human umbilical vascular endothelial cells with no cytotoxicity. Furthermore, HBC blocked in vitro characteristics of angiogenesis such as tube formation and chemoinvasion, as well as neovascularization of the chorioallantoic membrane of growing chick embryos in vivo. Notably, HBC markedly inhibited expression of hypoxia-inducible factor-1alpha (HIF-1alpha) at the translational level during hypoxia, thereby reducing HIF-1 transcriptional activity and expression of its major target gene, vascular endothelial growth factor. In addition, combination treatment with HBC and various HIF-1 inhibitors, including suberoylanilide hydroxamic acid, rapamycin, and terpestacin, had greater anti-angiogenic activity than treatment with each single agent. Collectively, our findings indicate that HBC is a new anti-angiogenic agent targeting HIF that can be used to explore the biological role of Ca(2+)/CaM in angiogenesis.

Wondonins A and B, two alkaloids of an unprecedented structural class, along with the previously described dihydroxystyryl sulfates, have been isolated from an association of the sponges Poecillastra wondoensis and Jaspis sp. collected from Keomun Island, Korea. The structures of these compounds have been determined as imidazole alkaloids containing a bis(dihydroxystyrene) moiety by combined spectroscopic methods. The new compounds exhibited antiangiogenic activity against human umbilical vein endothelial cells.

Rapid mixing in microplates is still an underappreciated challenge in screening assay development, particularly with the use of noncontact nanoliter liquid handlers. In high-content/throughput screening (HC/TS), fast and efficient mixing between compounds and cell culture medium is even more critical as biological kinetics dictates speed of mixing, usually within a few minutes. Moreover, mixing in HC/TS should be gentle enough to avoid any negative disruption in cell layer. Here the authors introduce a method to accurately quantify drop diffusion into a microplate well, independently of buffer, liquid handler, or dispensing protocol. This method was used to determine the effect of various mixing methods on the diffusion of a nanoliter drop of pure DMSO in aqueous buffer in 384-well plates. Rapid plate shaking and additional buffer addition were shown to be the most efficient and effective mixing methods for HC/TS. However, efficient mixing by plate shaking is limited by assay volume. Bulk addition shows fast and efficient mixing, without negative effects on cells. Moreover, this simple, fast, and inexpensive method can be easily adapted on any platform.

Nuclear factor-kappaB (NF-kappaB) is a crucial transcription factor that contributes to cancer development by regulating a number of genes involved in angiogenesis and tumorigenesis. Here, we describe (Z)-N-(3-(7-nitro-3-oxobenzo[d][1,2]selenazol-2(3H)-yl)benzylidene)propan-2-amine oxide (NBBA) as a new anti-angiogenic small molecule that targets NF-kappaB activity. NBBA showed stronger growth inhibition on human umbilical vein endothelial cells (HUVECs) than on the cancer cell lines we tested. Moreover, NBBA inhibited tumor necrosis factor-alpha (TNF-alpha)-induced tube formation and invasion of HUVECs. In addition, NBBA suppressed the neovascularization of chorioallantonic membrane from growing chick embryos in vivo. To address the mode of action of the compound, the effect of NBBA on TNF-alpha-induced NF-kappaB transcription activity was investigated. NBBA suppressed TNF-alpha-induced c-Jun N-terminal kinase phosphorylation, which resulted in suppression of transcription of NF-kappaB and its target genes, including interleukin-8, interleukin-1alpha, and epidermal growth factor. Collectively, these results demonstrated that NBBA is a new anti-angiogenic small molecule that targets the NF-kappaB signaling pathway.

Streptopyrrolidine, a benzyl pyrrolidine derivative, was isolated as an angiogenesis inhibitor from the fermentation broth of a marine Streptomyces sp. isolated from the deep sea sediment. Its structure was elucidated by extensive 2D NMR and mass spectroscopic analyses. Streptopyrrolidine exhibited significant anti-angiogenesis activity.

IHY-153 (2-(2,5-difluorobenzyl)-3,4-dihydro-5-(10-hydroxydecyl)-6-methoxy-1-undecylisoquinolinium bromide) was recently discovered as a small molecule that potently inhibits proliferation of tumor cells by inducing cell-cycle arrest at G0-G1 phase. To investigate the basis of anti-proliferative activity of IHY-153, cellular binding proteins of biotinyl-IHY-153 were screened using T7 phage displayed human cDNA libraries. Calmodulin-expressing phage specifically bound to immobilized IHY-153 in a Ca(2+) -dependent manner. The interaction between IHY-153 and Ca(2+) /CaM was validated through phage competition binding assays, surface plasmon resonance analysis, and molecular modeling. IHY-153 induced sustained phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and subsequently increased p21(WAF1) expression in colon cancer cells. These results demonstrate that IHY-153, a novel small molecule, targets Ca(2+) /CaM and indicate that this compound functions as an anti-proliferative agent by influencing Ca(2+) /CaM-dependent signal transduction.

ADP-ribosyl cyclase (ADPR-cyclase) produces a Ca2+-mobilizing second messenger cyclic ADP-ribose (cADPR) from NAD+. In this study, we investigated the molecular basis of ADPR-cyclase activation and the following cellular events in angiotensin II (ANG II) signaling in mouse mesangial cells (MMCs). Treatment of MMCs with ANG II induced an increase in intracellular Ca2+concentrations through a transient Ca2+release via an inositol 1,4,5-trisphosphate receptor and a sustained Ca2+influx via L-type Ca2+channels. The sustained Ca2+signal, but not the transient Ca2+signal, was blocked by a cADPR antagonistic analog, 8-bromo-cADPR (8-Br-cADPR), and an ADPR-cyclase inhibitor, 4,4′-dihydroxyazobenzene (DHAB). In support of the results, ANG II stimulated cADPR production in a time-dependent manner, and DHAB inhibited ANG II-induced cADPR production. Application of pharmacological inhibitors revealed that activation of ADPR-cyclase by ANG II involved ANG II type 1 receptor, phosphoinositide 3-kinase, protein tyrosine kinase, and phospolipase C-γ1. Moreover, DHAB as well as 8-Br-cADPR abrogated ANG II-mediated Akt phosphorylation, nuclear translocation of nuclear factor of activated T cell, and uptake of [3H]thymidine and [3H]leucine in MMCs. These results demonstrate that ADPR-cyclase in MMCs plays a pivotal role in ANG II signaling for cell proliferation and protein synthesis.