title: ROCK1 deficiency enhances protective effects of antioxidants against apoptosis and cell detachment.
abstract: We have recently reported that the homologous Rho kinases, ROCK1 and ROCK2, play different roles in regulating stress-induced stress fiber disassembly and cell detachment, and the ROCK1 deficiency in mouse embryonic fibroblasts (MEF) has remarkable anti-apoptotic, anti-detachment and pro-survival effects against doxorubicin, a chemotherapeutic drug. This study investigated the roles of ROCK isoforms in doxorubicin-induced reactive oxygen species (ROS) generation which is believed to be the major mechanism underlying its cytotoxicity to normal cells, and especially to cardiomyocytes. Different antioxidants have been shown to provide a protective role reported in numerous experimental studies, but clinical trials of antioxidant therapy showed insufficient benefit against the cardiac side effect. We found that both ROCK1-/- and ROCK2-/- MEFs exhibited reduced ROS production in response to doxorubicin treatment. Interestingly, only ROCK1 deficiency, but not ROCK2 deficiency, significantly enhanced the protective effects of antioxidants against doxorubicin-induced cytotoxicity. First, ROCK1 deficiency and N-acetylcysteine (an anti-oxidant) treatment synergistically reduced ROS levels, caspase activation and cell detachment. In addition, the reduction of ROS generation in ROCK1-/- MEFs in response to doxorubicin treatment was in part through inhibiting NADPH oxidase activity. Furthermore, ROCK1 deficiency enhanced the inhibitory effects of diphenyleneiodonium (an inhibitor of NADPH oxidase) on ROS generation and caspase 3 activation induced by doxorubicin. Finally, ROCK1 deficiency had greater protective effects than antioxidant treatment, especially on reducing actin cytoskeleton remodeling. ROCK1 deficiency not only reduced actomyosin contraction but also preserved central stress fiber stability, whereas antioxidant treatment only reduced actomyosin contraction without preserving central stress fibers. These results reveal a novel strategy to enhance the protective effect of antioxidant therapy by targeting the ROCK1 pathway to stabilize the actin cytoskeleton and boost the inhibitory effects on ROS production, apoptosis and cell detachment.
authors.affiliation: Riley Heart Research Center, Indiana University, School of Medicine, Indianapolis, Indiana, United States of America; Herman B Wells Center for Pediatric Research, Department of Pediatrics, Indiana University, School of Medicine, Indianapolis, Indiana, United States of America.
authors.forename: Michelle
authors.initials: M
authors.lastname: Surma
authors.affiliation: Riley Heart Research Center, Indiana University, School of Medicine, Indianapolis, Indiana, United States of America; Herman B Wells Center for Pediatric Research, Department of Pediatrics, Indiana University, School of Medicine, Indianapolis, Indiana, United States of America.
authors.forename: Caitlin
authors.initials: C
authors.lastname: Handy
authors.affiliation: Texas A&M University Health Science Center, Institute of Biosciences and Technology, Houston, Texas, United States of America.
authors.forename: Jiang
authors.initials: J
authors.lastname: Chang
authors.affiliation: Herman B Wells Center for Pediatric Research, Department of Pediatrics, Indiana University, School of Medicine, Indianapolis, Indiana, United States of America.
authors.forename: Reuben
authors.initials: R
authors.lastname: Kapur
authors.affiliation: Riley Heart Research Center, Indiana University, School of Medicine, Indianapolis, Indiana, United States of America; Herman B Wells Center for Pediatric Research, Department of Pediatrics, Indiana University, School of Medicine, Indianapolis, Indiana, United States of America.
authors.forename: Lei
authors.initials: L
authors.lastname: Wei
authors.affiliation: Riley Heart Research Center, Indiana University, School of Medicine, Indianapolis, Indiana, United States of America; Herman B Wells Center for Pediatric Research, Department of Pediatrics, Indiana University, School of Medicine, Indianapolis, Indiana, United States of America.
authors.forename: Jianjian
authors.initials: J
authors.lastname: Shi
mesh_terms: D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000009:adverse effects*; D000975:Antioxidants / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002478:Cells, Cultured; D004317:Doxorubicin / Q000009:adverse effects*; D004789:Enzyme Activation / Q000187:drug effects; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D017353:Gene Deletion; D051379:Mice; D019255:NADPH Oxidases / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D054460:rho-Associated Kinases / Q000235:genetics*
chemical_list: D000903:Antibiotics, Antineoplastic; D000975:Antioxidants; D017382:Reactive Oxygen Species; D004317:Doxorubicin; D019255:NADPH Oxidases; C515854:Rock1 protein, mouse; C515855:Rock2 protein, mouse; D054460:rho-Associated Kinases