Daniele Caracciolo1, Giada Juli1, Caterina Riillo1, Adriana Coricello2,3, Francesca Vasile4, Sara Pollastri4, Roberta Rocca2,3, Francesca Scionti5, Nicoletta Polerà1, Katia Grillone1, Mariamena Arbitrio6, Nicoletta Staropoli7, Basilio Caparello8, Domenico Britti2, Giovanni Loprete2, Giosuè Costa2,3, Maria Teresa Di Martino1, Stefano Alcaro2,3, Pierosandro Tagliaferri1, Pierfrancesco Tassone9,10. 1. Department of Experimental and Clinical Medicine, Magna Graecia University, Catanzaro, Italy. 2. Department of Health Science, Magna Græcia University, Catanzaro, Italy. 3. Net4Science Academic Spin-Off, Magna Græcia University, Campus "Salvatore Venuta", Catanzaro, Italy. 4. Department of Chemistry, University of Milan, Milan, Italy. 5. Institute of Research and Biomedical Innovation (IRIB), Italian National Council (CNR), Messina, Italy. 6. Institute of Research and Biomedical Innovation (IRIB), Italian National Council (CNR), Catanzaro, Italy. 7. Medical Oncology Unit, AOU Mater Domini, Catanzaro, Italy. 8. Presidio Ospedaliero Giovanni Paolo II Lamezia Terme, Catanzaro, Italy. 9. Department of Experimental and Clinical Medicine, Magna Graecia University, Catanzaro, Italy. tassone@unicz.it. 10. Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, PA, USA. tassone@unicz.it.
Abstract
BACKGROUND: DNA ligases are crucial for DNA repair and cell replication since they catalyze the final steps in which DNA breaks are joined. DNA Ligase III (LIG3) exerts a pivotal role in Alternative-Non-Homologous End Joining Repair (Alt-NHEJ), an error-prone DNA repair pathway often up-regulated in genomically unstable cancer, such as Multiple Myeloma (MM). Based on the three-dimensional (3D) LIG3 structure, we performed a computational screening to identify LIG3-targeting natural compounds as potential candidates to counteract Alt-NHEJ activity in MM. METHODS: Virtual screening was conducted by interrogating the Phenol Explorer database. Validation of binding to LIG3 recombinant protein was performed by Saturation Transfer Difference (STD)-nuclear magnetic resonance (NMR) experiments. Cell viability was analyzed by Cell Titer-Glo assay; apoptosis was evaluated by flow cytometric analysis following Annexin V-7AAD staining. Alt-NHEJ repair modulation was evaluated using plasmid re-joining assay and Cytoscan HD. DNA Damage Response protein levels were analyzed by Western blot of whole and fractionated protein extracts and immunofluorescence analysis. The mitochondrial DNA (mtDNA) copy number was determined by qPCR. In vivo activity was evaluated in NOD-SCID mice subcutaneously engrafted with MM cells. RESULTS: Here, we provide evidence that a natural flavonoid Rhamnetin (RHM), selected by a computational approach, counteracts LIG3 activity and killed Alt-NHEJ-dependent MM cells. Indeed, Nuclear Magnetic Resonance (NMR) showed binding of RHM to LIG3 protein and functional experiments revealed that RHM interferes with LIG3-driven nuclear and mitochondrial DNA repair, leading to significant anti-MM activity in vitro and in vivo. CONCLUSION: Taken together, our findings provide proof of concept that RHM targets LIG3 addiction in MM and may represent therefore a novel promising anti-tumor natural agent to be investigated in an early clinical setting.
BACKGROUND: DNA ligases are crucial for DNA repair and cell replication since they catalyze the final steps in which DNA breaks are joined. DNA Ligase III (LIG3) exerts a pivotal role in Alternative-Non-Homologous End Joining Repair (Alt-NHEJ), an error-prone DNA repair pathway often up-regulated in genomically unstable cancer, such as Multiple Myeloma (MM). Based on the three-dimensional (3D) LIG3 structure, we performed a computational screening to identify LIG3-targeting natural compounds as potential candidates to counteract Alt-NHEJ activity in MM. METHODS: Virtual screening was conducted by interrogating the Phenol Explorer database. Validation of binding to LIG3 recombinant protein was performed by Saturation Transfer Difference (STD)-nuclear magnetic resonance (NMR) experiments. Cell viability was analyzed by Cell Titer-Glo assay; apoptosis was evaluated by flow cytometric analysis following Annexin V-7AAD staining. Alt-NHEJ repair modulation was evaluated using plasmid re-joining assay and Cytoscan HD. DNA Damage Response protein levels were analyzed by Western blot of whole and fractionated protein extracts and immunofluorescence analysis. The mitochondrial DNA (mtDNA) copy number was determined by qPCR. In vivo activity was evaluated in NOD-SCID mice subcutaneously engrafted with MM cells. RESULTS: Here, we provide evidence that a natural flavonoid Rhamnetin (RHM), selected by a computational approach, counteracts LIG3 activity and killed Alt-NHEJ-dependent MM cells. Indeed, Nuclear Magnetic Resonance (NMR) showed binding of RHM to LIG3 protein and functional experiments revealed that RHM interferes with LIG3-driven nuclear and mitochondrial DNA repair, leading to significant anti-MM activity in vitro and in vivo. CONCLUSION: Taken together, our findings provide proof of concept that RHM targets LIG3 addiction in MM and may represent therefore a novel promising anti-tumor natural agent to be investigated in an early clinical setting.
Authors: Elizabeth Cotner-Gohara; In-Kwon Kim; Michal Hammel; John A Tainer; Alan E Tomkinson; Tom Ellenberger Journal: Biochemistry Date: 2010-07-27 Impact factor: 3.162