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Activity of a Py-Im polyamide targeted to the estrogen response element.

ABSTRACT: 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 (ER?) 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 17?-estadiol (E2)-induced transcription and cytotoxicity in ER? positive, E2-stimulated T47DKBluc cells, which express luciferase under ER? 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.

SUBMITTER: Nickols NG

PROVIDER: S-EPMC3651785 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Project description: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 5M-bM-^@M-^Y-WGGWWW-3M-bM-^@M-^Y 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. A 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 were interrogated by qRT-PCR and correlated with cell culture expression levels. Xenografts. Grafting with A549-luc-C8. Experiments were performed in female SCID-beige mice (Charles River) between 8 and 12 weeks of age. Cells were injected into the left flank area of the animals as suspensions of 25 x 106 mL-1 in RPMI, 200 M-BM-5L per injection. Treatment and tumor proliferation monitoring. Mice were treated following the schedule delineated in treatment protocol. Tumor proliferation was monitored using the XENOGEN imaging device. The animals were anesthetized with 2 5 % isoflurane and subsequently transferred to the imaging chamber, whereupon the isoflurane levels were reduced to 1-2.5 %. The floor of the imager was heated to +37 M-BM-:C to avoid hypothermia. Breathing frequency was monitored and not allowed to drop below 1 s-1, adjusting the isoflurane levels accordingly at all times. Endpoint criteria and euthanasia. Animal endpoint criteria encompassed weight loss of over 15 %, restriction of motor function by the engrafted tumor, dehydration of over 10 % and moribund behavior. Where appropriate, the animals were euthanized by asphyxiation in a CO2 chamber. Tumor tissue harvest. Animals were resected and tumors excised using standard forceps, scissors and surgical blades. The tumors were combined into one sample per condition and mechanically sheared in TRIZOL, employing a specialized device (tissue tearer, model 985370). Total RNA workup was performed following the standard TRIZOL procedure, followed by a DNAse digest.

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Activity of a Py-Im polyamide targeted to the estrogen response element.

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