Thymoquinone-induced platelet apoptosis

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. © 2011 Wiley Periodicals, Inc.     

Response of HEK293 and CHO cells overexpressing fusiogenic syncytin-1 to mitochondrion-mediated apoptosis induced by antimycin A

Apoptosis is essential for the regulation of cellular homeostasis in the placenta and is also involved in the pathophysiology of pregnancy-related diseases such as pre-eclampsia and intrauterine growth restriction (IUGR). Syncytin-1, a fusiogenic glycoprotein of endogenous-retroviral origin expressed in human trophoblasts, facilitates placental syncytium formation and is found reduced in pre-eclamptic placentas. We focus here on the mitochondrial apoptotic pathway and investigate whether the overexpression of syncytin-1 in HEK293-52 (human embryonic kidney cells) and CHO-52 cells influences the apoptotic response to the mitochondrial inhibitor antimycin A (AA). After the induction of apoptosis by 5 microM AA and incubation for up to 36 h in the absence of serum, the mean apoptotic rate was reduced by 15-30% in syncytin-1 transfected cells compared with mock-transfectants. After 12 h of challenge with AA we found lower cytochrome c levels in the cytoplasmic protein fraction and higher amounts in the mitochondrial fraction in syncytin-1 transfectants compared with mock-transfectants. We observed a decreased Mitotracker Red staining of mitochondria following AA challenge for 24 h in mock-treated CHO cells, in particular, compared with syncytin-1 transfectants. Moreover, we found a reduced activation of caspase 9 in syncytin-1 transfected HEK293-52 cells after 48 h of apoptotic challenge compared to mock-transfectants. However, a high expression of anti-apoptotic Bcl-x(L) was found in both cell types. Using syncytin-1 transfected HEK293-52 cells and CHO-52 cells, we provide initial evidence that syncytin-1 may exert its anti-apoptotic function at the mitochondrial level. A reduced release of cytochrome c followed by a diminished activation of caspase 9 is a possible mechanism.