title: Tamoxifen promotes superoxide production in platelets by activation of PI3-kinase and NADPH oxidase pathways.
abstract: BACKGROUND
  Tamoxifen is a selective estrogen receptor antagonist that is widely used for treatment and prevention of breast cancer. However, tamoxifen use can lead to an increased incidence of thrombotic events. The reason for this adverse event remains unknown. Previous studies showed that tamoxifen and its active metabolite Z-4-hydroxytamoxifen rapidly increased intracellular free calcium ([Ca(2+)](i)) in human platelets by a non-genomic mechanism that involved the activation of phospholipase C. Platelets play a pivotal role in thrombosis and Ca(2+) elevation is a central event in platelet activation. Therefore the mechanism by which tamoxifen activated Ca(2+) entry into platelets was investigated.
  
  
  METHODS
  [Ca(2+)](i) was measured using the fluorescent indicator fura-2 and reactive oxygen species were measured using lucigenin in isolated human platelets.
  
  
  RESULTS
  Tamoxifen analogs E-4-hydroxytamoxifen, with weak activity at the nuclear estrogen receptor and Z-4-hydroxytamoxifen, with strong activity at nuclear estrogen receptor, were equally active at increasing [Ca(2+)](i) and synergizing with ADP and thrombin to increase [Ca(2+)](i) in platelets. This result suggests that the effects of tamoxifen and E- and Z-4-hydroxytamoxifen to increase [Ca(2+)](i) are not mediated by the classical genomic estrogen receptor. The effects of tamoxifen to increase [Ca(2+)](i) were strongly inhibited by apocynin and apocynin dimer. This suggests that tamoxifen activates NADPH oxidase which leads to superoxide generation and in turn caused an increase in [Ca(2+)](i). Free radical scavengers TEMPO and TEMPOL also inhibited tamoxifen-induced [Ca(2+)](i) elevation. Inhibition of phosphoinositide-3-kinase (PI3-kinase), an upstream effector of NADPH oxidase with wortmannin and LY-294,002 also caused substantial inhibition of tamoxifen-induced elevation of [Ca(2+)](i).
  
  
  CONCLUSIONS
  Tamoxifen increases [Ca(2+)](i) in human platelets by a non-genomic mechanism. Tamoxifen activates phospholipase Cγ as well as PI3-kinase and NADPH oxidase pathway to generate superoxide which causes the release of Ca(2+) from the endoplasmic reticulum, and promotes Ca(2+) influx into the platelets.
authors.affiliation: Department of Physiological Sciences, Eastern Virginia Medical School, P.O. Box 1980, Norfolk, Virginia 23501, USA.
authors.forename: Vidhi P
authors.initials: VP
authors.lastname: Shah
authors.forename: Hesum A
authors.initials: HA
authors.lastname: Chegini
authors.forename: Susan R
authors.initials: SR
authors.lastname: Vishneski
authors.forename: Ross V
authors.initials: RV
authors.lastname: Weatherman
authors.forename: Peter F
authors.initials: PF
authors.lastname: Blackmore
authors.forename: Yuliya
authors.initials: Y
authors.lastname: Dobrydneva
mesh_terms: D001792:Blood Platelets / Q000187:drug effects* / Q000201:enzymology; D002118:Calcium / Q000097:blood; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000494:pharmacology; D005260:Female; D016166:Free Radical Scavengers / Q000494:pharmacology; D006801:Humans; D008297:Male; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000097:blood*; D019869:Phosphatidylinositol 3-Kinases / Q000097:blood*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051966:Phospholipase C gamma / Q000097:blood; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology; D020845:Selective Estrogen Receptor Modulators / Q000494:pharmacology*; D013481:Superoxides / Q000097:blood*; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000494:pharmacology*; D013997:Time Factors; D015854:Up-Regulation
chemical_list: D002118:Calcium; D004791:Enzyme Inhibitors; D004965:Estrogen Antagonists; D016166:Free Radical Scavengers; D019255:NADPH Oxidases; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051966:Phospholipase C gamma; D020845:Selective Estrogen Receptor Modulators; D013481:Superoxides; D013629:Tamoxifen; C016601:afimoxifene