104 research outputs found
Inhibition of vascular endothelial growth factor with a sequence-specific hypoxia response element antagonist
Vascular endothelial growth factor (VEGF) and its receptors have been implicated as key factors in tumor angiogenesis that are up-regulated by hypoxia. We evaluated the effects of DNA-binding small molecules on hypoxia-inducible transcription of VEGF. A synthetic pyrrole-imidazole polyamide designed to bind the hypoxia response element (HRE) was found to disrupt hypoxia-inducible factor (HIF) binding to HIRE. In cultured HeLa cells, this resulted in a reduction of VEGF mRNA and secreted protein levels. The observed effects were polyamide-specific and dose-dependent. Analysis of genome-wide effects of the HIRE-specific polyamide revealed that a number of hypoxia-inducible genes were down-regulated. Pathway-based regulation of hypoxia-inducible gene expression with DNA-binding small molecules may represent a new approach for targeting angiogenesis
Modulating hypoxia-inducible transcription by disrupting the HIF-1-DNA interface
Transcription mediated by hypoxia-inducible factor (HIF-1) contributes to tumor angiogenesis and metastasis but is also involved in activation of cell-death pathways and normal physiological processes. Given the complexity of HIF-1 signaling, it could be advantageous to target a subset of HIF-1 effectors rather than the entire pathway. We compare the genome-wide effects of three molecules that each interfere with the HIF-1–DNA interaction: a polyamide targeted to the hypoxia response element, small interfering RNA targeted to HIF-1α, and echinomycin, a DNA-binding natural product with a similar but less specific sequence preference than the polyamide. The polyamide affects a subset of hypoxia-induced genes consistent with its binding site preferences. For comparison, HIF-1α siRNA and echinomycin each affect the expression of nearly every gene induced by hypoxia. Remarkably, the total number of genes affected by either polyamide or HIF-1α siRNA over a range of thresholds is comparable. The data show that polyamides can be used to affect a subset of a pathway regulated by a transcription factor. In addition, this study offers a unique comparison of three complementary approaches towards exogenous control of endogenous gene expression
A pyrrole-imidazole polyamide is active against enzalutamide-resistant prostate cancer
The LREX' prostate cancer model is resistant to the antiandrogen enzalutamide via activation of an alternative nuclear hormone receptor (NHR), glucocorticoid receptor (GR), which has similar DNA binding specificity to the androgen receptor (AR). Small molecules that target DNA to interfere with protein-DNA interactions may retain activity against enzalutamide-resistant prostate cancers where ligand binding domain antagonists are ineffective. We reported previously that a pyrrole-imidazole (Py-Im) polyamide designed to bind the consensus androgen response element half-site has antitumor activity against hormone-sensitive prostate cancer. In enzalutamide-resistant LREX' cells, Py-Im polyamide interfered with both androgen receptor- and glucocorticoid receptor-driven gene expression, while enzalutamide interfered with only that of androgen receptor. Genomic analyses indicated immediate interference with the androgen receptor transcriptional pathway. Long-term treatment with Py-Im polyamide demonstrated a global decrease in RNA levels consistent with inhibition of transcription. The polyamide was active against two enzalutamide-resistant xenografts with minimal toxicity. Overall, our results identify Py-Im polyamide as a promising therapeutic strategy in enzalutamide-resistant prostate cancer
Antitumor activity of a pyrrole-imidazole polyamide
Many cancer therapeutics target DNA and exert cytotoxicity through the induction of DNA damage and inhibition of transcription. We report that a DNA minor groove binding hairpin pyrrole-imidazole (Py-Im) polyamide interferes with RNA polymerase II (RNAP2) activity in cell culture. Polyamide treatment activates p53 signaling in LNCaP prostate cancer cells without detectable DNA damage. Genome-wide mapping of RNAP2 binding shows reduction of occupancy, preferentially at transcription start sites, but occupancy at enhancer sites is unchanged. Polyamide treatment results in a time- and dose-dependent depletion of the RNAP2 large subunit RPB1 that is preventable with proteasome inhibition. This polyamide demonstrates antitumor activity in a prostate tumor xenograft model with limited host toxicity
Gene expression changes in a tumor xenograft by a pyrrole-imidazole polyamide
Gene regulation by DNA binding small molecules could have important therapeutic applications. This study reports the investigation of a DNA-binding pyrrole-imidazole polyamide targeted to bind the DNA sequence 5′-WGGWWW-3′ with reference to its potency in a subcutaneous xenograft tumor model. The molecule is capable of trafficking to the tumor site following subcutaneous injection and modulates transcription of select genes in vivo. An FITC-labeled analogue of this polyamide can be detected in tumor-derived cells by confocal microscopy. RNA deep sequencing (RNA-seq) of tumor tissue allowed the identification of further affected genes, a representative panel of which was interrogated by quantitative reverse transcription-PCR and correlated with cell culture expression levels
Activity of a Py–Im Polyamide Targeted to the Estrogen Response Element
Pyrrole-imidazole (Py–Im) polyamides are a class of programmable DNA minor groove binders capable of modulating the activity of DNA-binding proteins and affecting changes in gene expression. Estrogen receptor alpha (ERα) is a ligand-activated hormone receptor that binds as a homodimer to estrogen response elements (ERE) and is a driving oncogene in a majority of breast cancers. We tested a selection of structurally similar Py–Im polyamides with differing DNA sequence specificity for activity against 17β-estadiol (E2)–induced transcription and cytotoxicity in ERα positive, E2-stimulated T47DKBluc cells, which express luciferase under ERα control. The most active polyamide targeted the sequence 5′-WGGWCW-3′ (W = A or T), which is the canonical ERE half site. Whole transcriptome analysis using RNA-Seq revealed that treatment of E2-stimulated breast cancer cells with this polyamide reduced the effects of E2 on the majority of those most strongly affected by E2 but had much less effect on the majority of E2-induced transcripts. In vivo, this polyamide circulated at detectable levels following subcutaneous injection and reduced levels of ER-driven luciferase expression in xenografted tumors in mice after subcutaneous compound administration without significant host toxicity
Modulating hypoxia-inducible transcription by disrupting the HIF-1-DNA interface
Transcription mediated by hypoxia-inducible factor (HIF-1) contributes to tumor angiogenesis and metastasis but is also involved in activation of cell-death pathways and normal physiological processes. Given the complexity of HIF-1 signaling, it could be advantageous to target a subset of HIF-1 effectors rather than the entire pathway. We compare the genome-wide effects of three molecules that each interfere with the HIF-1–DNA interaction: a polyamide targeted to the hypoxia response element, small interfering RNA targeted to HIF-1α, and echinomycin, a DNA-binding natural product with a similar but less specific sequence preference than the polyamide. The polyamide affects a subset of hypoxia-induced genes consistent with its binding site preferences. For comparison, HIF-1α siRNA and echinomycin each affect the expression of nearly every gene induced by hypoxia. Remarkably, the total number of genes affected by either polyamide or HIF-1α siRNA over a range of thresholds is comparable. The data show that polyamides can be used to affect a subset of a pathway regulated by a transcription factor. In addition, this study offers a unique comparison of three complementary approaches towards exogenous control of endogenous gene expression
Interference with DNA repair after ionizing radiation by a pyrrole-imidazole polyamide
Pyrrole-imidazole (Py–Im) polyamides are synthetic non-genotoxic minor groove-binding small molecules. We hypothesized that Py–Im polyamides can modulate the cellular response to ionizing radiation. Pre-treatment of cells with a Py-Im polyamide prior to exposure to ionizing radiation resulted in a delay in resolution of phosphorylated γ-H2AX foci, increase in XRCC1 foci, and reduced cellular replication potential. RNA-sequencing of cell lines exposed to the polyamide showed induction of genes related to the ultraviolet radiation response. We observed that the polyamide is almost 10-fold more toxic to a cell line deficient in DNA ligase 3 as compared to the parental cell line. Alkaline single cell gel electrophoresis reveals that the polyamide induces genomic fragmentation in the ligase 3 deficient cell line but not the corresponding parental line. The polyamide interferes directly with DNA ligation in vitro. We conclude that Py-Im polyamides may be further explored as sensitizers to genotoxic therapies
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