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 Ca²⁺ 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 Ca²⁺ activity utilizing Fluo 3‐AM fluorescence, caspase activity utilizing immunofluorescence and Western blotting of active caspase‐3 and inactive procaspase‐3, mitochondrial potential utilizing DiOC₆ 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 Ca²⁺ activity, mitochondrial depolarization, and ceramide formation. P‐selectin exposure and integrin α_IIbβ₃ activation did not increase in response to TQ. Nominal absence of extracellular Ca²⁺ 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 Ca²⁺ activity, ceramide formation, mitochondrial depolarization, and caspase‐3 activation. J. Cell. Biochem. 112: 3112–3121, 2011.