ABSTRACT
Gastric cancer is one of the most common malignancies and a leading cause of cancer death worldwide. The prognosis of stomach cancer is generally poor as this cancer is not very sensitive to commonly used chemotherapies. Epigenetic modifications play a key role in gastric cancer and contribute to the development and progression of this malignancy. In order to explore new treatment options in this target area we have screened a library of epigenetic inhibitors against gastric cancer cell lines and identified inhibitors for the BET family of bromodomains as potent inhibitors of gastric cancer cell proliferations. Here we show that both the pan-BET inhibitor (+)-JQ1 as well as a newly developed specific isoxazole inhibitor, PNZ5, showed potent inhibition of gastric cancer cell growth. Intriguingly, we found differences in the antiproliferative response between gastric cancer cells tested derived from Brazilian patients as compared to those from Asian patients, the latter being largely resistant to BET inhibition. As BET inhibitors are entering clinical trials these findings provide the first starting point for future therapies targeting gastric cancer.
Subject(s)
Azepines/pharmacology , Cell Proliferation/drug effects , Isoxazoles/pharmacology , Nuclear Proteins/antagonists & inhibitors , Transcription Factors/antagonists & inhibitors , Triazoles/pharmacology , Apoptosis/drug effects , Apoptosis/genetics , Asian People , Azepines/chemistry , Brazil , Cell Cycle Proteins , Cell Line, Tumor , Cell Proliferation/genetics , Gene Expression Profiling , HEK293 Cells , Humans , Isoxazoles/chemistry , Molecular Structure , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Spheroids, Cellular/drug effects , Spheroids, Cellular/metabolism , Stomach Neoplasms/ethnology , Stomach Neoplasms/genetics , Stomach Neoplasms/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Triazoles/chemistryABSTRACT
TRIM24 is a transcriptional regulator as well as an E3 ubiquitin ligase. It is overexpressed in diverse tumors, and high expression levels have been linked to poor prognosis in breast cancer patients. TRIM24 contains a PHD/bromodomain offering the opportunity to develop protein interaction inhibitors that target this protein interaction module. Here we identified potent acetyl-lysine mimetic benzimidazolones TRIM24 bromodomain inhibitors. The best compound of this series is a selective BRPF1B/TRIM24 dual inhibitor that bound with a KD of 137 and 222 nM, respectively, but exerted good selectivity over other bromodomains. Cellular activity of the inhibitor was demonstrated using FRAP assays as well as cell viability data.
Subject(s)
Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Benzimidazoles/chemistry , Benzimidazoles/pharmacology , Carrier Proteins/antagonists & inhibitors , Nuclear Proteins/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Cell Line, Tumor , Crystallography, X-Ray , DNA-Binding Proteins , Humans , Lysine/analogs & derivatives , Models, Molecular , Molecular Docking Simulation , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Protein Structure, Tertiary/drug effectsABSTRACT
Abstract Lung gene therapy is being evaluated for a range of acute and chronic diseases, including cystic fibrosis (CF). As these therapies approach clinical realization, it is becoming increasingly clear that the ability to efficiently deliver gene transfer agents (GTAs) to target cell populations within the lung may prove just as critical as the gene therapy formulation itself in terms of generating positive clinical outcomes. Key to the success of any aerosol gene therapy is the interaction between the GTA and nebulization device. We evaluated the effects of aerosolization on our preferred formulation, plasmid DNA (pDNA) complexed with the cationic liposome GL67A (pDNA/GL67A) using commercially available nebulizer devices. The relatively high viscosity (6.3±0.1 cP) and particulate nature of pDNA/GL67A formulations hindered stable aerosol generation in ultrasonic and vibrating mesh nebulizers but was not problematic in the jet nebulizers tested. Aerosol size characteristics varied significantly between devices, but the AeroEclipse II nebulizer operating at 50 psi generated stable pDNA/GL67A aerosols suitable for delivery to the CF lung (mass median aerodynamic diameter 3.4±0.1 µm). Importantly, biological function of pDNA/GL67A formulations was retained after nebulization, and although aerosol delivery rate was lower than that of other devices (0.17±0.01 ml/min), the breath-actuated AeroEclipse II nebulizer generated aerosol only during the inspiratory phase and as such was more efficient than other devices with 83±3% of generated aerosol available for patient inhalation. On the basis of these results, we have selected the AeroEclipse II nebulizer for the delivery of pDNA/GL67A formulations to the lungs of CF patients as part of phase IIa/b clinical studies.
Subject(s)
Aerosols/chemistry , Cystic Fibrosis/therapy , DNA/metabolism , Liposomes/chemistry , Lung/metabolism , Administration, Inhalation , Animals , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , DNA/chemistry , Female , Genetic Therapy , Mice , Mice, Inbred BALB C , Nebulizers and Vaporizers , Plasmids/genetics , Plasmids/metabolismABSTRACT
Nonviral gene therapy utilizing plasmid DNA (pDNA) complexed with cationic lipids (lipoplexes) or cationic polymers (polyplexes) has demonstrated considerable potential for the treatment of a variety of diseases. However, progress toward clinical application is often delayed by the lack of reliable and scalable mixing of components sufficient to guarantee consistent performance in vivo. Attempts to improve and standardize mixing have been limited by the sensitivity of pDNA to shear-related degradation. Here we describe a simple pneumatic mixing device that enables the rapid and reproducible production of large volumes of nonviral gene therapy formulations and demonstrate its suitability for use with shear-sensitive pDNA.
Subject(s)
DNA/administration & dosage , Genetic Therapy/instrumentation , Plasmids/administration & dosage , Animals , Cations/chemistry , DNA/chemistry , DNA/genetics , Equipment Design , Gene Expression , Lipids/chemistry , Mice , Mice, Inbred BALB C , Plasmids/chemistry , Plasmids/geneticsABSTRACT
Gene therapy is being investigated in the treatment of lung-related aspects of the genetic disease, Cystic fibrosis (CF). Clinical studies have demonstrated CF transmembrane conductance regulator (CFTR) expression in the airways of adults with CF using a variety of gene transfer agents. In utero gene therapy is an alternative approach that facilitates vector transduction of rapidly expanding populations of target cells while avoiding immune recognition of the vector. In CF, in utero gene transfer could potentially delay the onset of disease symptoms in childhood and compensate for the role, if any, that CFTR plays in the developing organs. Previously published studies have suggested that transient expression of CFTR in utero was sufficient to rescue the fatal intestinal defect in S489X Cftr(tm1Unc)/Cftr(tm1Unc) knockout mice. We replicated these studies using an identical CFTR-expressing adenoviral vector and CF mouse strain in sufficiently large numbers to provide robust Kaplan-Meier survival data. Although each step of the procedure was carefully controlled and vector-specific CFTR expression was confirmed in the fetal organs after treatment, there was statistically no significant improvement in the survival of mice treated in utero with AdCFTR, compared with contemporaneous control animals.