title: TRAIL-Based High Throughput Screening Reveals a Link between TRAIL-Mediated Apoptosis and Glutathione Reductase, a Key Component of Oxidative Stress Response.
abstract: A high throughput screen for compounds that induce TRAIL-mediated apoptosis identified ML100 as an active chemical probe, which potentiated TRAIL activity in prostate carcinoma PPC-1 and melanoma MDA-MB-435 cells. Follow-up in silico modeling and profiling in cell-based assays allowed us to identify NSC130362, pharmacophore analog of ML100 that induced 65-95% cytotoxicity in cancer cells and did not affect the viability of human primary hepatocytes. In agreement with the activation of the apoptotic pathway, both ML100 and NSC130362 synergistically with TRAIL induced caspase-3/7 activity in MDA-MB-435 cells. Subsequent affinity chromatography and inhibition studies convincingly demonstrated that glutathione reductase (GSR), a key component of the oxidative stress response, is a target of NSC130362. In accordance with the role of GSR in the TRAIL pathway, GSR gene silencing potentiated TRAIL activity in MDA-MB-435 cells but not in human hepatocytes. Inhibition of GSR activity resulted in the induction of oxidative stress, as was evidenced by an increase in intracellular reactive oxygen species (ROS) and peroxidation of mitochondrial membrane after NSC130362 treatment in MDA-MB-435 cells but not in human hepatocytes. The antioxidant reduced glutathione (GSH) fully protected MDA-MB-435 cells from cell lysis induced by NSC130362 and TRAIL, thereby further confirming the interplay between GSR and TRAIL. As a consequence of activation of oxidative stress, combined treatment of different oxidative stress inducers and NSC130362 promoted cell death in a variety of cancer cells but not in hepatocytes in cell-based assays and in in vivo, in a mouse tumor xenograft model.
authors.affiliation: Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, Oregon, United States of America.
authors.forename: Dmitri
authors.initials: D
authors.lastname: Rozanov
authors.affiliation: Q-MOL LLC, San Diego, CA, United States of America.
authors.forename: Anton
authors.initials: A
authors.lastname: Cheltsov
authors.affiliation: Q-MOL LLC, San Diego, CA, United States of America.
authors.forename: Eduard
authors.initials: E
authors.lastname: Sergienko
authors.affiliation: The Conrad Prebys Center for Chemical Genomics, Sanford-Burnham Medical Research Institute, Orlando, Florida, United States of America.
authors.forename: Stefan
authors.initials: S
authors.lastname: Vasile
authors.affiliation: Inflammatory and Infectious Disease Center, Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America.
authors.forename: Vladislav
authors.initials: V
authors.lastname: Golubkov
authors.affiliation: Inflammatory and Infectious Disease Center, Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America.
authors.forename: Alexander E
authors.initials: AE
authors.lastname: Aleshin
authors.affiliation: Department of Biomedical Engineering, Oregon Health & Science University, Portland, Oregon, United States of America.
authors.forename: Trevor
authors.initials: T
authors.lastname: Levin
authors.affiliation: Knight Cancer Institute, Oregon Health and Science University, Portland, Oregon, United States of America.
authors.forename: Elie
authors.initials: E
authors.lastname: Traer
authors.affiliation: Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, California, United States of America.
authors.forename: Byron
authors.initials: B
authors.lastname: Hann
authors.affiliation: Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, California, United States of America.
authors.forename: Julia
authors.initials: J
authors.lastname: Freimuth
authors.affiliation: Computational Biology Institute, George Washington University, Ashburn, Virginia, United States of America; Department of Mathematics and Mechanics, Saint Petersburg State University, Saint Petersburg, Russia.
authors.forename: Nikita
authors.initials: N
authors.lastname: Alexeev
authors.affiliation: Computational Biology Institute, George Washington University, Ashburn, Virginia, United States of America.
authors.forename: Max A
authors.initials: MA
authors.lastname: Alekseyev
authors.affiliation: Research Department, Shriners Hospital for Children, Portland, Oregon, United States of America; Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon, United States of America.
authors.forename: Sergey P
authors.initials: SP
authors.lastname: Budko
authors.affiliation: Research Department, Shriners Hospital for Children, Portland, Oregon, United States of America; Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon, United States of America.
authors.forename: Hans Peter
authors.initials: HP
authors.lastname: Bächinger
authors.affiliation: Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, Oregon, United States of America.
authors.forename: Paul
authors.initials: P
authors.lastname: Spellman
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology; D055808:Drug Discovery; D005978:Glutathione / Q000378:metabolism; D005980:Glutathione Reductase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D006801:Humans; D051379:Mice; D018384:Oxidative Stress; D017382:Reactive Oxygen Species; D054852:Small Molecule Libraries; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000378:metabolism* / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D017382:Reactive Oxygen Species; D054852:Small Molecule Libraries; D053221:TNF-Related Apoptosis-Inducing Ligand; D004317:Doxorubicin; D005980:Glutathione Reductase; D005978:Glutathione