Tamoxifen promotes superoxide production in platelets by activation of PI3-Kinase and NADPH oxidase pathways

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 ([Ca2+](i)) in human platelets by a non-genomic mechanism that involved the activation of phospholipase C. Platelets play a pivotal role in thrombosis and Ca2+ elevation is a central event in platelet activation. Therefore the mechanism by which tamoxifen activated Ca2+ entry into platelets was investigated. Methods: [Ca2+](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 [Ca2+](i) and synergizing with ADP and thrombin to increase [Ca2+](i) in platelets. This result suggests that the effects of tamoxifen and E-and Z-4-hydroxytamoxifen to increase [Ca2+](i) are not mediated by the classical genomic estrogen receptor. The effects of tamoxifen to increase [Ca2+](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 [Ca2+](i). Free radical scavengers TEMPO and TEMPOL also inhibited tamoxifen-induced [Ca2+](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 [Ca2+](i). Conclusion: Tamoxifen increases [Ca2+](i) in human platelets by a non-genomic mechanism. Tamoxifen activates phospholipase C gamma as well as PI3-kinase and NADPH oxidase pathway to generate superoxide which causes the release of Ca2+ from the endoplasmic reticulum, and promotes Ca2+ influx into the platelets. (C) 2011 Elsevier Ltd. All rights reserved.