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Cancer research  2013;73(17):5497-5507.
Werner syndrome (WS) is genetically linked to mutations in WRN that encodes a DNA helicase-nuclease believed to operate at stalled replication forks. Using a newly identified small molecule inhibitor of WRN helicase (NSC 617145), we investigated the role of WRN in the interstrand cross-link (ICL) response in cells derived from patients with Fanconi Anemia (FA), a hereditary disorder characterized by bone marrow failure and cancer. In FA-D2−/− cells, NSC 617145 acted synergistically with very low concentrations of Mitomycin C (MMC) to inhibit proliferation in a WRN-dependent manner, and induce double strand breaks (DSBs) and chromosomal abnormalities. Under these conditions, ATM activation and accumulation of DNA-PKcs pS2056 foci suggested an increased number of DSBs processed by nonhomologous end-joining (NHEJ). Rad51 foci were also elevated in FA-D2−/− cells exposed to NSC 617145 and MMC, suggesting that WRN helicase inhibition interferes with later steps of homologous recombination (HR) at ICL-induced DSBs. Thus, when the FA pathway is defective, WRN helicase inhibition perturbs the normal ICL response, leading to NHEJ activation. Potential implication for treatment of FA-deficient tumors by their sensitization to DNA cross-linking agents is discussed.
PMCID: PMC3766423  PMID: 23867477
Werner syndrome; Fanconi Anemia; helicase; small molecule; DNA repair
2.  Discovery and Preliminary SAR of Bisbenzylisoquinoline Alkaloids as Inducers of C/EBPα 
Bioorganic & medicinal chemistry  2012;20(15):4646-4652.
A high throughput in vitro screen has been developed to identify substances that induce expression of C/EBPα in tumor cells. An extract of the fruit of Gyrocarpus jacquinii showed induction of C/EBPα activity that was attributed by dereplication analysis to the presence of bisbenzylisoquinoline (BBIQ) alkaloids, including the known compound pheanthine. (13). The research project was broadened to assess the effect of other natural BBIQ structural types occurring outside the genus Gyrocarpus. Several of the 28 compounds assayed showed enhancement of C/EBPα induction in U937 cells. The results of this study should encourage future efforts toward obtaining and screening a larger set of both natural and synthetic analogs of this interesting group of alkaloids.
PMCID: PMC3430122  PMID: 22766217
bisbenzylisoquinoline alkaloids; C/EBPα; Gyrocarpus jacquinii; structure-activity relationships
3.  An imprinted rheumatoid arthritis methylome signature reflects pathogenic phenotype 
Genome Medicine  2013;5(4):40.
A DNA methylation signature has been characterized that distinguishes rheumatoid arthritis (RA) fibroblast like synoviocytes (FLS) from osteoarthritis (OA) FLS. The presence of epigenetic changes in long-term cultured cells suggest that rheumatoid FLS imprinting might contribute to pathogenic behavior. To understand how differentially methylated genes (DMGs) might participate in the pathogenesis of RA, we evaluated the stability of the RA signature and whether DMGs are enriched in specific pathways and ontology categories.
To assess the RA methylation signatures the Illumina HumanMethylation450 chip was used to compare methylation levels in RA, OA, and normal (NL) FLS at passage 3, 5, and 7. Then methylation frequencies at CpGs within the signature were compared between passages. To assess the enrichment of DMGs in specific pathways, DMGs were identified as genes that possess significantly differential methylated loci within their promoter regions. These sets of DMGs were then compared to pathway and ontology databases to establish enrichment in specific categories.
Initial studies compared passage 3, 5, and 7 FLS from RA, OA, and NL. The patterns of differential methylation of each individual FLS line were very similar regardless of passage number. Using the most robust analysis, 20 out of 272 KEGG pathways and 43 out of 34,400 GO pathways were significantly altered for RA compared with OA and NL FLS. Most interestingly, we found that the KEGG 'Rheumatoid Arthritis' pathway was consistently the most significantly enriched with differentially methylated loci. Additional pathways involved with innate immunity (Complement and Coagulation, Toll-like Receptors, NOD-like Receptors, and Cytosolic DNA-sensing), cell adhesion (Focal Adhesion, Cell Adhesion Molecule), and cytokines (Cytokine-cytokine Receptor). Taken together, KEGG and GO pathway analysis demonstrates non-random epigenetic imprinting of RA FLS.
The DNA methylation patterns include anomalies in key genes implicated in the pathogenesis of RA and are stable for multiple cell passages. Persistent epigenetic alterations could contribute to the aggressive phenotype of RA synoviocytes and identify potential therapeutic targets that could modulate the pathogenic behavior.
PMCID: PMC3706831  PMID: 23631487
4.  Substituted 3-(5-Imidazo[2,1-b]thiazolylmethylene)-2-indolinones and Analogues: Synthesis, Cytotoxic Activity and Study of the Mechanism of Action1 
Journal of Medicinal Chemistry  2012;55(5):2078-2088.
The synthesis of substituted 3-(5-imidazo[2,1-b]thiazolylmethylene)-2-indolinones and analogues is reported. Their cytotoxic activity was evaluated according to protocols available at the National Cancer Institute (NCI), Bethesda, MD. The action of selected compounds was examined for potential inhibition of tubulin assembly in comparison with the potent colchicine site agent combretastatin A-4. The most potent compounds also strongly and selectively inhibited the phosphorylation of the oncoprotein kinase Akt in cancer cells. The effect of the most interesting compounds was examined on the growth of HT-29 colon cancer cells. These compounds caused the cells to arrest in the G2/M phase of the cell cycle, as would be expected for inhibitors of tubulin assembly.
PMCID: PMC3297693  PMID: 22283430
5.  A Pharmacological Inhibitor of the Protease Taspase1 Effectively Inhibits Breast and Brain Tumor Growth 
Cancer Research  2011;72(3):736-746.
The threonine endopeptidase Taspase1 has a critical role in cancer cell proliferation and apoptosis. In this study, we developed and evaluated small molecule inhibitors of Taspase1 as a new candidate class of therapeutic modalities. Genetic deletion of Taspase1 in the mouse produced no overt deficiencies, suggesting the possibility of a wide therapeutic index for use of Taspase1 inhibitors in cancers. We defined the peptidyl motifs recognized by Taspase1 and conducted a cell-based dual-fluorescent proteolytic screen of the NCI diversity library to identify Taspase1 inhibitors (TASPINs). Based on secondary and tertiary screens the 4-[(4-arsonophenyl)methyl]phenyl]arsonic acid NSC48300 was determined to be the most specific active compound. Structure-activity relationship studies indicated a crucial role for the arsenic acid moiety in mediating Taspase1 inhibition. Additional FRET-based kinetic analysis characterized NSC48300 as a reversible, non-competitive inhibitor of Taspase1 (KI = 4.22 μM). In the MMTV-neu mouse model of breast cancer and the U251 xenograft model of brain cancer NSC48300 produced effective tumor growth inhibition. Our results offer an initial preclinical proof of concept to develop TASPINs for cancer therapy.
PMCID: PMC3325786  PMID: 22166309
6.  Structural Characterization of Inhibitor Complexes with Checkpoint Kinase 2 (Chk2), a Drug Target for Cancer Therapy 
Journal of structural biology  2011;176(3):292-301.
Chk2 (checkpoint kinase 2) is a serine/threonine kinase that participates in a series of signaling networks responsible for maintaining genomic integrity and responding to DNA damage. The development of selective Chk2 inhibitors has recently attracted much interest as a means of sensitizing cancer cells to current DNA-damaging agents used in the treatment of cancer. Additionally, selective Chk2 inhibitors may reduce p53-mediated apoptosis in normal tissues, thereby helping to mitigate adverse side effects from chemotherapy and radiation. Thus far, relatively few selective inhibitors of Chk2 have been described and none have yet progressed into clinical trials. Here, we report crystal structures of the catalytic domain of Chk2 in complex with a novel series of potent and selective small molecule inhibitors. These compounds exhibit nanomolar potencies and are selective for Chk2 over Chk1. The structures reported here elucidate the binding modes of these inhibitors to Chk2 and provide information that can be exploited for the structure-assisted design of novel chemotherapeutics.
PMCID: PMC3210331  PMID: 21963792
structure-based drug design; kinase inhibitor; crystal structure
8.  X-ray Structures of Checkpoint Kinase 2 in Complex with Inhibitors that Target its Gatekeeper-Dependent Hydrophobic Pocket 
FEBS letters  2011;585(20):3245-3249.
The serine/threonine checkpoint kinase 2 (Chk2) is an attractive molecular target for the development of small molecule inhibitors to treat cancer. Here, we report the rational design of Chk2 inhibitors that target the gatekeeper-dependent hydrophobic pocket located behind the adenine-binding region of the ATP-binding site. These compounds exhibit IC50 values in the low nanomolar range and are highly selective for Chk2 over Chk1. X-ray crystallography was used to determine the structures of the inhibitors in complex with the catalytic kinase domain of Chk2 to verify their modes of binding.
PMCID: PMC3195894  PMID: 21907711
rational drug design; structure-based drug design; kinase inhibitors
9.  Library-free Methylation Sequencing with Bisulfite Padlock Probes 
Nature methods  2012;9(3):270-272.
We previously developed Bisulfite Padlock Probes (BSPPs) for the specific and parallel digital quantification of DNA methylation1. Here we report the second-generation of BSPPs with a design algorithm to generate more efficient padlock probes, a library-free protocol that dramatically reduces the time and cost of sample preparation and is compatible with automation, and an efficient bioinformatics pipeline that accurately obtains both methylation levels and genotypes from bisulfite sequencing data.
PMCID: PMC3461232  PMID: 22306810
10.  A Novel Knowledge-Driven Systems Biology Approach for Phenotype Prediction upon Genetic Intervention 
Deciphering the biological networks underlying complex phenotypic traits, e.g., human disease is undoubtedly crucial to understand the underlying molecular mechanisms and to develop effective therapeutics. Due to the network complexity and the relatively small number of available experiments, data-driven modeling is a great challenge for deducing the functions of genes/ proteins in the network and in phenotype formation. We propose a novel knowledge-driven systems biology method that utilizes qualitative knowledge to construct a Dynamic Bayesian network (DBN) to represent the biological network underlying a specific phenotype. Edges in this network depict physical interactions between genes and/or proteins. A qualitative knowledge model first translates typical molecular interactions into constraints when resolving the DBN structure and parameters. Therefore, the uncertainty of the network is restricted to a subset of models which are consistent with the qualitative knowledge. All models satisfying the constraints are considered as candidates for the underlying network. These consistent models are used to perform quantitative inference. By in silico inference, we can predict phenotypic traits upon genetic interventions and perturbing in the network. We applied our method to analyze the puzzling mechanism of breast cancer cell proliferation network and we accurately predicted cancer cell growth rate upon manipulating (anti)cancerous marker genes/proteins.
PMCID: PMC3211072  PMID: 21282866
Dynamic Bayesian network; genetic network; phenotype prediction; genetic intervention; systems biology; breast cancer; cell proliferation
11.  Identification of CBX3 and ABCA5 as Putative Biomarkers for Tumor Stem Cells in Osteosarcoma 
PLoS ONE  2012;7(8):e41401.
Recently, there has been renewed interest in the role of tumor stem cells (TSCs) in tumorigenesis, chemoresistance, and relapse of malignant tumors including osteosarcoma. The potential exists to improve osteosarcoma treatment through characterization of TSCs and identification of therapeutic targets. Using transcriptome, proteome, immunophenotyping for cell-surface markers, and bioinformatic analyses, heterogeneous expression of previously reported TSC or osteosarcoma markers, such as CD133, nestin, POU5F1 (OCT3/4), NANOG, SOX2, and aldehyde dehydrogenase, among others, was observed in vitro. However, consistently significantly lower CD326, CD24, CD44, and higher ABCG2 expression in TSC-enriched as compared with un-enriched osteosarcoma cultures was observed. In addition, consistently higher CBX3 expression in TSC-enriched osteosarcoma cultures was identified. ABCA5 was identified as a putative biomarker of TSCs and/or osteosarcoma. Lastly, in a high-throughput screen we identified epigenetic (5-azacytidine), anti-microtubule (vincristine), and anti-telomerase (3,11-difluoro-6,8,13-trimethyl- 8H-quino [4,3,2-kl] acridinium methosulfate; RHPS4)-targeted therapeutic agents as candidates for TSC ablation in osteosarcoma.
PMCID: PMC3411700  PMID: 22870217
12.  Global identification of transcriptional regulators of pluripotency and differentiation in embryonic stem cells 
Nucleic Acids Research  2012;40(17):8199-8209.
Human embryonic stem cells (hESCs) hold great promise for regenerative medicine because they can undergo unlimited self-renewal and retain the capability to differentiate into all cell types in the body. Although numerous genes/proteins such as Oct4 and Gata6 have been identified to play critical regulatory roles in self-renewal and differentiation of hESC, the majority of the regulators in these cellular processes and more importantly how these regulators co-operate with each other and/or with epigenetic modifications are still largely unknown. We propose here a systematic approach to integrate genomic and epigenomic data for identification of direct regulatory interactions. This approach allows reconstruction of cell-type-specific transcription networks in embryonic stem cells (ESCs) and fibroblasts at an unprecedented scale. Many links in the reconstructed networks coincide with known regulatory interactions or literature evidence. Systems-level analyses of these networks not only uncover novel regulators for pluripotency and differentiation, but also reveal extensive interplays between transcription factor binding and epigenetic modifications. Especially, we observed poised enhancers characterized by both active (H3K4me1) and repressive (H3K27me3) histone marks that contain enriched Oct4- and Suz12-binding sites. The success of such a systems biology approach is further supported by experimental validation of the predicted interactions.
PMCID: PMC3458541  PMID: 22730289
13.  Systematic Search for Recipes to Generate Induced Pluripotent Stem Cells 
PLoS Computational Biology  2011;7(12):e1002300.
Generation of induced pluripotent stem cells (iPSCs) opens a new avenue in regenerative medicine. One of the major hurdles for therapeutic applications is to improve the efficiency of generating iPSCs and also to avoid the tumorigenicity, which requires searching for new reprogramming recipes. We present a systems biology approach to efficiently evaluate a large number of possible recipes and find those that are most effective at generating iPSCs. We not only recovered several experimentally confirmed recipes but we also suggested new ones that may improve reprogramming efficiency and quality. In addition, our approach allows one to estimate the cell-state landscape, monitor the progress of reprogramming, identify important regulatory transition states, and ultimately understand the mechanisms of iPSC generation.
Author Summary
Converting somatic cells back to the stem cell state (called induced pluripotent stem cells or iPSCs) exemplifies the recent advancement of cellular reprogramming that holds great promise for developing regenerative medicine. Generation of iPSCs is often achieved by overexpressing three to four genes in somatic cells that are critical for regulating pluripotency. Developing optimal reprogramming recipe is a non-trivial task that requires significant effort. We present here a computational method that can facilitate discovery of effective recipes to generate iPSCs with high efficiency and better quality. In addition, our approach provides a new way to estimate the landscape in the cell-state space and monitor the trajectory of cellular reprogramming from a differentiated cell to an iPS cell. This work provides not only practical recipes for iPSC generation but also theoretical understanding of the reprogramming process.
PMCID: PMC3245295  PMID: 22215993
14.  S100A4-induced cell motility and metastasis is restricted by the Wnt/β-catenin pathway inhibitor calcimycin in colon cancer cells 
Molecular Biology of the Cell  2011;22(18):3344-3354.
Calcimycin restricts Wnt/β-catenin–regulated S100A4 expression, leading to reduced S100A4-mediated cell migration and invasion in colon cancer cells, as well as to inhibition of metastasis formation in xenografted mice.
The calcium-binding protein S100A4 is a central mediator of metastasis formation in colon cancer. S100A4 is a target gene of the Wnt/β-catenin pathway, which is constitutively active in the majority of colon cancers. In this study a high-throughput screen was performed to identify small-molecule compounds targeting the S100A4-promoter activity. In this screen calcimycin was identified as a transcriptional inhibitor of S100A4. In colon cancer cells calcimycin treatment reduced S100A4 mRNA and protein expression in a dose- and time-dependent manner. S100A4-induced cellular processes associated with metastasis formation, such as cell migration and invasion, were inhibited by calcimycin in an S100A4-specific manner. Calcimycin reduced β-catenin mRNA and protein levels despite the expression of Δ45-mutated β-catenin. Consequently, calcimycin inhibited Wnt/β-catenin pathway activity and the expression of prominent β-catenin target genes such as S100A4, cyclin D1, c-myc, and dickkopf-1. Finally, calcimycin treatment of human colon cancer cells inhibited metastasis formation in xenografted immunodeficient mice. Our results demonstrate that targeting the expression of S100A4 with calcimycin provides a functional strategy to restrict cell motility in colon cancer cells. Therefore calcimycin may be useful for studying S100A4 biology, and these studies may serve as a lead for the development of treatments for colon cancer metastasis.
PMCID: PMC3172260  PMID: 21795396
15.  Cysteine 81 Is Critical for the Interaction of S100A4 and Myosin-IIA 
Biochemistry  2011;50(33):7218-7227.
Overexpression of S100A4, a member of the S100 family of Ca2+-binding proteins, is associated with a number of human pathologies, including fibrosis, inflammatory disorders, and metastatic disease. The identification of small molecules that disrupt S100A4/target interactions provides a mechanism for inhibiting S100A4-mediated cellular activities and their associated pathologies. Using an anisotropy assay that monitors the Ca2+-dependent binding of myosin-IIA to S100A4, NSC 95397 was identified as an inhibitor that disrupts the S100A4/myosin-IIA interaction and inhibits S100A4-mediated depolymerization of myosin-IIA filaments. Mass spectrometry demonstrated that NSC 95397 forms covalent adducts with Cys81 and Cys86, which are located in the canonical target binding cleft. Mutagenesis studies showed that covalent modification of just Cys81 is suffcient to inhibit S100A4 function with respect to myosin-IIA binding and depolymerization. Remarkably, substitution of Cys81 with serine or alanine significantly impaired the ability of S100A4 to promote myosin-IIA filament disassembly. As reversible covalent cysteine modifications have been observed for several S100 proteins, we propose that modification of Cys81 may provide an additional regulatory mechanism for mediating the binding of S100A4 to myosin-IIA.
PMCID: PMC3220277  PMID: 21749055
16.  Identification of polymerase and processivity inhibitors of vaccinia DNA synthesis using a stepwise screening approach 
Antiviral research  2008;80(2):114-123.
Nearly all DNA polymerases require processivity factors to ensure continuous incorporation of nucleotides. Processivity factors are specific for their cognate DNA polymerases. For this reason, the vaccinia DNA polymerase (E9) and the proteins associated with processivity (A20 and D4) are excellent therapeutic targets. In this study, we show the utility of stepwise rapid plate assays that i) screen for compounds that block vaccinia DNA synthesis, ii) eliminate trivial inhibitors, e.g. DNA intercalators, and iii) distinguish whether inhibitors are specific for blocking DNA polymerase activity or processivity. The sequential plate screening of 2,222 compounds from the NCI Diversity Set library yielded a DNA polymerase inhibitor (NSC 55636) and a processivity inhibitor (NSC 123526) that were capable of reducing vaccinia viral plaques with minimal cellular cytotoxicity. These compounds are predicted to block cellular infection by the smallpox virus, variola, based on the very high sequence identity between A20, D4 and E9 of vaccinia and the corresponding proteins of variola.
PMCID: PMC3155815  PMID: 18621425
Poxvirus; vaccinia virus; DNA polymerase; processivity factor, antiviral inhibitors; high throughput screening; rapid plate assay, smallpox
17.  Substituted E-3-(3-indolylmethylene)1,3-dihydroindol-2-ones with Antitumor Activity. In-depth study of the effect on growth of breast cancer cells1 
Journal of medicinal chemistry  2010;53(15):5567-5575.
The synthesis of new substituted E-3-(3-indolylmethylene)1,3-dihydroindol-2-ones is reported. The antitumor activity was evaluated according to protocols available at the National Cancer Institute (NCI), Bethesda, MD. Structure-activity relationships are discussed. The action of selected compounds was investigated in MCF-7 breast cancer cells. The ability of these derivatives to inhibit cellular proliferation was accompanied by increased level of p53 and its transcriptional targets p21 and Bax, interference in the cell cycle progression with cell accumulation in the G2/M phase, and lastly activation of apoptosis.
PMCID: PMC2920599  PMID: 20684599
18.  RSK2 Binding Models Delineate Key Features for Activity 
Due to its overexpression and activation in human cancer cells and tissues, an emerging molecular target in cancer therapeutics is p90 ribosomal s6 kinase 2 (RSK2). While a growing number of RSK2 inhibitors have been reported in the literature, only the crystal structure of RSK2 in complex with an AMP analogue provides a structural basis for understanding RSK2 inhibition. To remedy this, we used our fluorescence polarization assay to determine the RSK2 activity for a set of structurally diverse compounds, and followed this by modeling their binding modes in an all-atom, energy refined crystal structure of RSK2. These binding models reveal that Val131 and Leu147 are key interaction sites for potent RSK2 inhibition. This structure-based pharmacophore is an important tool for new lead discovery and refinement.
PMCID: PMC3094916  PMID: 21593990
RSK2; binding modes; structure-based pharmacophores; cancer; kinase; inhibition; interaction sites; indirubins; flavopiridol; SL0101; BI-DI870; Ro31-8220; GF109203x; NSC 356821
19.  Intervening in β-Catenin Signaling by Sulindac Inhibits S100A4-Dependent Colon Cancer Metastasis12 
Neoplasia (New York, N.Y.)  2011;13(2):131-144.
Colon cancer metastasis is often associated with activation of the Wnt/β-catenin signaling pathway and high expression of the metastasis mediator S100A4. We previously demonstrated the transcriptional regulation of S100A4 by β-catenin and the importance of the interconnection of these cellular programs for metastasis. Here we probe the hypothesis that the nonsteroidal anti-inflammatory drug sulindac sulfide can inhibit colon cancer metastasis by intervening in β-catenin signaling and thereby interdicting S100A4. We treated colon cancer cell lines heterozygous for gain-of-function and wild-type β-catenin with sulindac. We analyzed sulindac's effects on β-catenin expression and subcellular localization, β-catenin binding to the T-cell factor (TCF)/S100A4 promoter complex, S100A4 promoter activity, S100A4 expression, cell motility, and proliferation. Mice intrasplenically transplanted with S100A4-overexpressing colon cancer cells were treated with sulindac. Tumor growth and metastasis, and their β-catenin and S100A4 expressions, were determined. We report the expression knockdown of β-catenin by sulindac, leading to its reduced nuclear accumulation. The binding of β-catenin to TCF was clearly lowered, resulting in reduced S100A4 promoter activity and expression. This correlated well with the inhibition of cell migration and invasion, which could be rescued by ectopic S100A4 expression. In mice, sulindac treatment resulted in reduced tumor growth in the spleen (P = .014) and decreased liver metastasis in a human colon cancer xenograft model (P = .025). Splenic tumors and liver metastases of sulindac-treated mice showed lowered β-catenin and S100A4 levels. These results suggest that modulators of β-catenin signaling such as sulindac offer potential as antimetastatic agents by interdicting S100A4 expression.
PMCID: PMC3033592  PMID: 21403839
20.  Therapeutic Vulnerability of an In Vivo Model of Alveolar Soft Part Sarcoma (ASPS) to Anti -Angiogenic Therapy 
In vivo growth of Alveolar Soft Part Sarcoma (ASPS) was achieved using subcutaneous xenografts in sex matched NOD.SCID\NCr mice. One tumor, currently at passage 6, has been maintained in vivo for 32 months and has maintained characteristics consistent with those of the original ASPS tumor including (1) tumor histology and staining with Periodic Acid Schiff/Diastase (2) the presence of the ASPL-TFE3 type 1 fusion transcript (3) nuclear staining with antibodies to the ASPL-TFE3 type 1 fusion protein, (4) maintenance of the t(X;17)(p11;q25) translocation characteristic of ASPS, (5) stable expression of signature ASPS gene transcripts and finally, the development and maintenance of a functional vascular network, a hallmark of ASPS. The ASPS xenograft tumor vasculature encompassing nests of ASPS cells is highly reactive to antibodies against the endothelial antigen CD34 and is readily accessible to intravenously administered FITC-dextran. The therapeutic vulnerability of this tumor model to anti-angiogenic therapy, targeting vascular endothelial growth factor (VEGF) and hypoxiainducible factor-1 alpha (HIF-1 α), was examined utilizing bevacizumab and topotecan alone and in combination. Together, the two drugs produced a 70% growth delay accompanied by a 0.7 net log cell kill which was superior to the antitumor effect produced by either drug alone. In summary, the current study describes a pre-clinical in vivo model for ASPS which will facilitate investigation into the biology of this slow growing soft tissue sarcoma and demonstrates the feasibility of employing an anti-angiogenic approach in the treatment of ASPS.
PMCID: PMC2784654  PMID: 19636271
Alveolar Soft Part Sarcoma (ASPS); In-Vivo Growth Model; Anti-Angiogenic Therapy of ASPS
21.  Adaphostin toxicity in a sensitive non-small cell lung cancer model is mediated through Nrf2 signaling and heme oxygenase 1 
Preclinical toxicity of adaphostin has been related to oxidative stress. This study investigated the regulatory mechanism underlying adaphostin induction of heme oxygenase 1 (HMOX1) which plays a significant role in modulation of drug-induced toxicity in the non-small cell lung cancer cell line model, NCI-H522.
The transcriptional response of NCI-H522 to adaphostin prominently involved oxidative stress genes, particularly HMOX1. Reactive oxygen species (ROS) involvement was additionally established by generation of ROS prior to modulation of adaphostin-toxicity with antioxidants. To identify up-stream regulatory elements of HMOX1, immunofluorescence was used to evaluate nuclear translocation of the transcription factor, NF-E2-related factor 2 (Nrf2), in the presence of adaphostin. The PI3-kinase inhibitor, wortmannin, was employed as a pharmacological inhibitor of this process.
Generation of ROS provided a substantial foundation for the sensitivity of NCI-H522 to adaphostin. However, in contrast to leukemia cell lines, transcriptional response to oxidative stress was associated with induction of HMOX1, which was dependent on nuclear translocation of the transcription factor, Nrf2. Pretreatment of cells with wortmannin inhibited translocation of Nrf2 and induction of HMOX1. Wortmannin pretreatment was also able to diminish adaphostin induction of HMOX1, and as a consequence, enhance the toxicity of adaphostin to NCI-H522.
Adaphostin-induced oxidative stress in NCI-H522 was mediated through nuclear translocation of Nrf2 leading to upregulation of HMOX1. Inhibition of Nrf2 translocation by wortmannin inhibited this cytoprotective response, and enhanced the toxicity of adaphostin, suggesting that inhibitors of the PI3K pathway, such as wortmannin, might augment the antiproliferative effects of adaphostin in solid tumors that depend on the Nrf2/ARE pathway for protection against oxidative stress.
PMCID: PMC2909968  PMID: 20618971
22.  Increased anti-tumor activity of bevacizumab in combination with HIF-1 inhibition 
Molecular cancer therapeutics  2009;8(7):1867-1877.
Inhibition of HIF-1 is an attractive therapeutic strategy to target the tumor microenvironment. However, HIF-1 inhibitors may have limited activity as single agents and combination therapies may be required. We tested the hypothesis that HIF-1 inhibition in a hypoxic stressed tumor microenvironment, which could be generated by administration of antiangiogenic agents, may result in a more pronounced therapeutic effect. The activity of bevacizumab, either alone or in combination with the HIF-1α inhibitor topotecan, was evaluated in U251-HRE xenografts. Tumor tissue was collected at the end of treatment and changes in tumor oxygenation, angiogenesis, proliferation, apoptosis, HIF-1α levels, HIF-1 target genes and DNA damage were evaluated. Bevacizumab decreased microvessel-density and increased intratumor-hypoxia, but did not induce apoptosis. Moreover, bevacizumab alone caused a significant increase of HIF-1-dependent gene expression in tumor tissue. Addition of a low dose of daily topotecan to bevacizumab significantly inhibited tumor growth, relative to mice treated with topotecan or bevacizumab alone (p<0.01). The addition of topotecan to bevacizumab was also associated with profound inhibition of HIF-1 transcriptional activity, significant inhibition of proliferation and induction of apoptosis. Importantly, DNA damage induced by topotecan alone was not augmented by addition of bevacizumab, suggesting that increased cytotoxic activity did not account for the increased anti-tumor effects observed.
These results strongly suggest that combination of anti-VEGF antibodies with HIF-1 inhibitors is an attractive therapeutic strategy targeting in the hypoxic tumor microenvironment.
PMCID: PMC2745101  PMID: 19584228
HIF-1; topotecan; bevacizumab; hypoxia; angiogenesis; cancer therapeutics
23.  Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming 
Nature biotechnology  2009;27(4):353-360.
Current DNA methylation assays are limited in the flexibility and efficiency of characterizing a large number of genomic targets. We report a method to specifically capture an arbitrary subset of genomic targets for single-molecule bisulfite sequencing for digital quantification of DNA methylation at single-nucleotide resolution. A set of ~30,000 padlock probes was designed to assess methylation of ~66,000 CpG sites within 2,020 CpG islands on human chromosome 12, chromosome 20, and 34 selected regions. To investigate epigenetic differences associated with dedifferentiation, we compared methylation in three human fibroblast lines and eight human pluripotent stem cell lines. Chromosome-wide methylation patterns were similar among all lines studied, but cytosine methylation was slightly more prevalent in the pluripotent cells than in the fibroblasts. Induced pluripotent stem (iPS) cells appeared to display more methylation than embryonic stem cells. We found 288 regions methylated differently in fibroblasts and pluripotent cells. This targeted approach should be particularly useful for analyzing DNA methylation in large genomes.
PMCID: PMC2715272  PMID: 19330000
24.  Induction of Apoptosis in Human Cancer Cells by Candidaspongiolide, a Novel Sponge Polyketide 
Candidaspongiolide (CAN), a novel polyketide from a marine sponge, is the active component of a mixture that was found to be potently cytotoxic in the National Cancer Institute’s 60-cell-line screen.
Effects of CAN on U251 glioma and HCT116 colorectal cancer cells and on normal fibroblasts were assessed using radiolabeling studies to measure protein synthesis, clonogenic assays to measure cell survival, flow cytometry of annexin V– and propidium iodide–stained cells to measure apoptosis, and western blots in the presence or absence of specific inhibitors to assess accumulation and phosphorylation of potential downstream target proteins.
CAN inhibited protein synthesis and potently induced apoptosis in both U251 and HCT116 cells, the latter in part by a caspase 12–dependent pathway. For example, 25%–30% of U251 or HCT116 cells became apoptotic after 24 hours of treatment with 100 nM CAN. CAN also rapidly induced sustained phosphorylation of eukaryotic translation initiation factor-2 (eIF2)-α at Ser51 and of the translation elongation factor eEF2 at Thr56, which could contribute to its dose-dependent inhibition of protein synthesis. Stable expression of dominant-negative eIF2α was sufficient to prevent CAN-induced eIF2α phosphorylation and induction of apoptosis but insufficient to prevent inhibition of protein synthesis. CAN induction of eIF2α phosphorylation did not occur by a classic endoplasmic reticulum stress pathway. However, an inhibitor of and small-interfering RNAs to the double-stranded RNA–dependent protein kinase PKR prevented CAN-mediated eIF2α phosphorylation and apoptosis, respectively. Although CAN inhibited protein synthesis in both cancer cells and normal human fibroblasts, it induced eIF2α phosphorylation and apoptosis only in cancer cells.
CAN triggers PKR/eIF2α/caspase 12–dependent apoptosis and inhibits protein synthesis in cancer cells but only inhibits protein synthesis in normal cells.
PMCID: PMC2720712  PMID: 18728285
25.  Synergistic antitumor activity of oncolytic reovirus and chemotherapeutic agents in non-small cell lung cancer cells 
Molecular Cancer  2009;8:47.
Reovirus type 3 Dearing strain (ReoT3D) has an inherent propensity to preferentially infect and destroy cancer cells. The oncolytic activity of ReoT3D as a single agent has been demonstrated in vitro and in vivo against various cancers, including colon, pancreatic, ovarian and breast cancers. Its human safety and potential efficacy are currently being investigated in early clinical trials. In this study, we investigated the in vitro combination effects of ReoT3D and chemotherapeutic agents against human non-small cell lung cancer (NSCLC).
ReoT3D alone exerted significant cytolytic activity in 7 of 9 NSCLC cell lines examined, with the 50% effective dose, defined as the initial virus dose to achieve 50% cell killing after 48 hours of infection, ranging from 1.46 ± 0.12 ~2.68 ± 0.25 (mean ± SD) log10 pfu/cell. Chou-Talalay analysis of the combination of ReoT3D with cisplatin, gemcitabine, or vinblastine demonstrated strong synergistic effects on cell killing, but only in cell lines that were sensitive to these compounds. In contrast, the combination of ReoT3D and paclitaxel was invariably synergistic in all cell lines tested, regardless of their levels of sensitivity to either agent. Treatment of NSCLC cell lines with the ReoT3D-paclitaxel combination resulted in increased poly (ADP-ribose) polymerase cleavage and caspase activity compared to single therapy, indicating enhanced apoptosis induction in dually treated NSCLC cells. NSCLC cells treated with the ReoT3D-paclitaxel combination showed increased proportions of mitotic and apoptotic cells, and a more pronounced level of caspase-3 activation was demonstrated in mitotically arrested cells.
These data suggest that the oncolytic activity of ReoT3D can be potentiated by taxanes and other chemotherapeutic agents, and that the ReoT3D-taxane combination most effectively achieves synergy through accelerated apoptosis triggered by prolonged mitotic arrest.
PMCID: PMC2723073  PMID: 19594950

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