title: Methotrexate Promotes Platelet Apoptosis via JNK-Mediated Mitochondrial Damage: Alleviation by N-Acetylcysteine and N-Acetylcysteine Amide.
abstract: Thrombocytopenia in methotrexate (MTX)-treated cancer and rheumatoid arthritis (RA) patients connotes the interference of MTX with platelets. Hence, it seemed appealing to appraise the effect of MTX on platelets. Thereby, the mechanism of action of MTX on platelets was dissected. MTX (10 μM) induced activation of pro-apoptotic proteins Bid, Bax and Bad through JNK phosphorylation leading to ΔΨm dissipation, cytochrome c release and caspase activation, culminating in apoptosis. The use of specific inhibitor for JNK abrogates the MTX-induced activation of pro-apoptotic proteins and downstream events confirming JNK phosphorylation by MTX as a key event. We also demonstrate that platelet mitochondria as prime sources of ROS which plays a central role in MTX-induced apoptosis. Further, MTX induces oxidative stress by altering the levels of ROS and glutathione cycle. In parallel, the clinically approved thiol antioxidant N-acetylcysteine (NAC) and its derivative N-acetylcysteine amide (NACA) proficiently alleviate MTX-induced platelet apoptosis and oxidative damage. These findings underpin the dearth of research on interference of therapeutic drugs with platelets, despite their importance in human health and disease. Therefore, the use of antioxidants as supplementary therapy seems to be a safe bet in pathologies associated with altered platelet functions.
authors.affiliation: Department of studies in Biochemistry, University of Mysore, Manasagangothri, Mysore, 570 006, India.
authors.forename: Manoj
authors.initials: M
authors.lastname: Paul
authors.affiliation: Department of studies in Biochemistry, University of Mysore, Manasagangothri, Mysore, 570 006, India; Department of Internal Medicine, Manitoba Centre for Proteomics and Systems Biology, University of Manitoba, Winnipeg, R3E3P4, Canada.
authors.forename: Mahadevappa
authors.initials: M
authors.lastname: Hemshekhar
authors.affiliation: Department of studies in Biochemistry, University of Mysore, Manasagangothri, Mysore, 570 006, India.
authors.forename: Ram M
authors.initials: RM
authors.lastname: Thushara
authors.affiliation: Department of studies in Biochemistry, University of Mysore, Manasagangothri, Mysore, 570 006, India.
authors.forename: Mahalingam S
authors.initials: MS
authors.lastname: Sundaram
authors.affiliation: Department of studies in Biochemistry, University of Mysore, Manasagangothri, Mysore, 570 006, India.
authors.forename: Somanathapura K
authors.initials: SK
authors.lastname: NaveenKumar
authors.affiliation: Applied Nutrition Discipline, Defence Food Research Laboratory, Mysore, 570 011, India.
authors.forename: Shivanna
authors.initials: S
authors.lastname: Naveen
authors.affiliation: Department of Studies and Research in Biochemistry, Tumkur University, Tumkur, 572 103, India.
authors.forename: Sannaningaiah
authors.initials: S
authors.lastname: Devaraja
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore, 560 012, India.
authors.forename: Kumar
authors.initials: K
authors.lastname: Somyajit
authors.affiliation: Organosilicon Research Center, University of Wisconsin, Madison, WI, 53706-1396, United States of America.
authors.forename: Robert
authors.initials: R
authors.lastname: West
authors.affiliation: Laboratory of Chemical Biology, Department of Chemistry, Bangalore University, Bangalore, India.
authors.lastname: Basappa
authors.affiliation: Department of Applied Botany and Biotechnology, University of Mysore, Manasagangothri, Mysore, 570 006, India.
authors.forename: Siddaiah C
authors.initials: SC
authors.lastname: Nayaka
authors.affiliation: Organosilicon Research Center, University of Wisconsin, Madison, WI, 53706-1396, United States of America.
authors.forename: Uzma I
authors.initials: UI
authors.lastname: Zakai
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore, 560 012, India.
authors.forename: Ganesh
authors.initials: G
authors.lastname: Nagaraju
authors.affiliation: Department of Studies in Chemistry, University of Mysore, Manasagangothri, Mysore, 570 006, India.
authors.forename: Kanchugarakoppal S
authors.initials: KS
authors.lastname: Rangappa
authors.affiliation: Department of studies in Biochemistry, University of Mysore, Manasagangothri, Mysore, 570 006, India.
authors.forename: Kempaiah
authors.initials: K
authors.lastname: Kemparaju
authors.affiliation: Department of studies in Biochemistry, University of Mysore, Manasagangothri, Mysore, 570 006, India; Department of Studies and Research in Biochemistry, Tumkur University, Tumkur, 572 103, India.
authors.forename: Kesturu S
authors.initials: KS
authors.lastname: Girish
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D000975:Antioxidants / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051023:BH3 Interacting Domain Death Agonist Protein / Q000235:genetics / Q000378:metabolism; D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D048670:MAP Kinase Kinase 4 / Q000235:genetics* / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial; D008727:Methotrexate / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010766:Phosphorylation; D061251:Primary Cell Culture; D015398:Signal Transduction; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism; D051025:bcl-Associated Death Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D000964:Antimetabolites, Antineoplastic; D000975:Antioxidants; C494819:BAD protein, human; C494826:BAX protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D051028:bcl-2-Associated X Protein; D051025:bcl-Associated Death Protein; C487056:N-Acetylcysteinamide; D048670:MAP Kinase Kinase 4; D000111:Acetylcysteine; D008727:Methotrexate