title: Thymoquinone-induced platelet apoptosis.
abstract: Thymoquinone (TQ) is a nutrient with anticarcinogenic activity that stimulates suicidal death of tumor cells. Moreover, TQ triggers suicidal death of erythrocytes or eryptosis, an effect at least partially due to increase in cytosolic Ca(2+) activity and ceramide formation. The present experiments explored whether TQ influences apoptosis of blood platelets. Cell membrane scrambling was determined utilizing Annexin V binding to phosphatidylserine exposing platelets, cytosolic Ca(2+) activity utilizing Fluo 3-AM fluorescence, caspase activity utilizing immunofluorescence and Western blotting of active caspase-3 and inactive procaspase-3, mitochondrial potential utilizing DiOC(6) fluorescence and ceramide by FACS analysis of ceramide-binding antibodies. A 30 min exposure to TQ (≥5 µM) was followed by Annexin V binding, paralleled by caspase activation, increase of cytosolic Ca(2+) activity, mitochondrial depolarization, and ceramide formation. P-selectin exposure and integrin α(IIb) β(3) activation did not increase in response to TQ. Nominal absence of extracellular Ca(2+) blunted but did not fully abolish the TQ-induced activation of caspase-3. The effects of TQ on platelets are significantly abolished with phosphoinositide-3 kinase (PI3K) inhibitor wortmannin and G-protein coupled receptor (GPCR) inhibitor pertussis toxin treatment prior to TQ stimulation. In conclusion, TQ triggers suicidal death of blood platelets in a PI3K-dependent manner, possibly through a GPCR family receptor; an effect paralleled by increase of cytosolic Ca(2+) activity, ceramide formation, mitochondrial depolarization, and caspase-3 activation.
authors.affiliation: Department of Physiology, University of Tübingen, Tübingen, Germany.
authors.forename: Syeda Tasneem
authors.initials: ST
authors.lastname: Towhid
authors.forename: Eva-Maria
authors.initials: EM
authors.lastname: Schmidt
authors.forename: Evi
authors.initials: E
authors.lastname: Schmid
authors.forename: Patrick
authors.initials: P
authors.lastname: Münzer
authors.forename: Syed M
authors.initials: SM
authors.lastname: Qadri
authors.forename: Oliver
authors.initials: O
authors.lastname: Borst
authors.forename: Florian
authors.initials: F
authors.lastname: Lang
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D016227:Benzoquinones / Q000494:pharmacology*; D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002518:Ceramides / Q000378:metabolism; D005434:Flow Cytometry; D005453:Fluorescence; D006801:Humans; D039661:Integrin beta3 / Q000378:metabolism; D019007:P-Selectin / Q000378:metabolism; D040201:Platelet Membrane Glycoprotein IIb / Q000378:metabolism*
chemical_list: D016227:Benzoquinones; D002518:Ceramides; D039661:Integrin beta3; D019007:P-Selectin; D040201:Platelet Membrane Glycoprotein IIb; C003466:thymoquinone; D002118:Calcium