HA14-1, but not the BH3 mimetic ABT-737, causes Ca2+ dysregulation in platelets and human cell lines

Many Bcl-2-dependent cancer cells are primed to death due to their upregulation of BH3-only proteins in response to ongoing oncogenic stress. BH3-mimetic drugs like ABT-737 compete with Bim for binding to Bcl-2, releasing Bim and triggering Bax/Bak-mediated apoptosis.1 While ABT-737 causes regression of established tumors,2 it also limits platelet survival.3 The mechanisms underlying the observed thrombocytopenia have been the subject of debate. Since Bcl-Xl is essential for platelet life span,4 it was proposed that ABT-737, which does not discriminate between Bcl-2 and Bcl-Xl,5 kills platelets by antagonizing Bcl-Xl.3 However, these devastating effects of ABT-737 on platelet function were also associated with disturbed intracellular Ca2+ homeostasis and dynamics,3 but this remains poorly understood and controversial.6,7 We, therefore, explored the effect of ABT-737 on apoptosis in platelets and on intracellular Ca2+ signaling in platelets and human cell lines. We compared its effects on Ca2+ homeostasis with another Bcl-2 antagonist, the HA14-1 compound, which was reported to exert inhibitory properties on sarco/endoplasmic reticulum Ca2+-ATPases (SERCA).8 Washed platelets were prepared as described.9 For this, venous blood was collected from healthy donors. Permission was given by the Ethical Committee of the Leuven University Hospital to use blood from healthy individuals for further analysis. For apoptosis measurements, platelets were incubated with Annexin-V-FITC and analyzed with an Attune® Acoustic Focusing Flow Cytometer (Applied Biosystems). For the Ca2+ measurements in intact cells, platelets were seeded the day of measurement in poly-L-lysine-coated 96-well plates (Greiner) at a density of approximately 3 × 108 platelets/mL. Ca2+ measurements in intact Hela cells and unidirectional 45Ca2+-flux experiments in permeabilized cells were basically performed as previously described.10 SERCA2b ATPase activity was determined by colorimetric monitoring11 in a Ca2+-buffered solution containing microsomes (10 μg of protein) from HEK-293T cells ectopically expressing SERCA2b. Results are expressed as average ± standard deviation (SD). Significance was determined using two-tailed paired Student's t-test. P<0.05 was considered significant. We confirmed that both HA14-1 (3 and 10 μM; 2 h) and ABT-737 (0.03, 0.1 and 0.3 μM; 2 h) provoked apoptosis in blood platelets (Figure 1A and B). However, only HA14-1, but not ABT-737, triggered a slow and steady increase in the cytosolic [Ca2+] originating from the intracellular Ca2+ stores in Fura2-loaded platelets exposed to extracellular EGTA (Figure 1C). Moreover, pre-treatment (10 μM; 30 min) of HA14-1, but not of ABT-737, reduced the total Ca2+ released from the ER in response to thapsigargin, an irreversible SERCA inhibitor, while not affecting store-operated Ca2+ influx (Figure 1D). To confirm our result in another cell model, we used the human cell line HeLa. Again, HA14-1, but not ABT-737, affected intracellular Ca2+ homeostasis in these cells in a concentration-dependent manner (Figure 2A). To investigate the underlying mechanism, we applied a highly quantitative 45Ca2+-flux assay10 in permeabilized HeLa cells to specifically assess ER 45Ca2+-uptake activity in the absence of plasmalemmal and mitochondrial Ca2+ fluxes and of IP3R-mediated Ca2+-release. Application of HA14-1 during the ER 45Ca2+-loading phase caused a strong decrease in the steady-state 45Ca2+ loading levels, while ABT-737 was much less effective (Figure 2B). The steady-state ER Ca2+ levels are determined by the balance between the ER Ca2+-uptake and ER Ca2+-leak activities. Importantly, application of either ABT-737 or HA14-1 (up to 30 μM) during the efflux phase did not affect the ER Ca2+-leak rate (Figure 2C), suggesting an inhibition of the ER Ca2+-uptake activity. Next, we directly assessed the effect of ABT-737 and HA14-1 on SERCA2b activity, the housekeeping isoform in human platelets,12 which was ectopically expressed in HEK-293T cells (Figure 2D). HA14-1 effectively inhibited SERCA2b Ca2+ ATPase activity (IC50 = 14 μM) while ABT-737 (up to 30 μM) had no effect. Finally, we assessed whether ABT-737 or HA14-1 directly affected IP3R-mediated Ca2+ release (e.g. which occurs in response to thrombin exposure of platelets). We used the 45Ca2+-flux assay to accurately measure IP3-induced Ca2+ release in permeabilized HeLa cells. We found that 30 μM HA14-1, but not ABT-737, inhibited the IP3R (Figure 2E). Performing a dose-response curve, we found that ABT-737 up to 100 μM did not inhibit IP3Rs. In contrast, HA14-1 potently inhibited IP3Rs at concentrations higher than 10 μM with an IC50 of approximately 50 mM and with an IP3R inhibition of approximately 80% at 100 μM (Figure 2F). Figure 1. The effect of HA14-1 and ABT-737 on apoptosis and intracellular Ca2+ signaling in platelets. (A-B) Flow-cytometry analysis of HA14-1 and ABT-737-induced apoptosis in platelets. (A) Histogram overlay representation of Annexin V-FITC staining of platelets ... Figure 2. The effect of HA14-1 and ABT-737 on ER Ca2+-uptake and –release mechanisms. (A) Fluorimetric analysis of the HA14-1 and ABT-737-induced Ca2+ responses in HeLa cells. The ratio of emitted fluorescence of Fura2 (excitation wavelength 340 nm/380 ... Our experiments clearly indicate that disrupted intracellular Ca2+ homeostasis is not a proximal event in ABT-737-induced thrombocytopenia. Thus, earlier studies indicating depleted intracellular Ca2+ homeostasis in platelets exposed for 10 μM ABT-737 for prolonged periods (e.g. 2 h) may reflect a late event that is the consequence of Bcl-Xl inhibition and ongoing cell death in platelets.6 We conclude that the dysregulation of intracellular Ca2+ signaling in platelets and human cell lines by HA14-1 is an off-target effect on SERCA2b and on IP3Rs, since on-target inhibition of Bcl-2/Bcl-Xl by ABT-737 does not disrupt intracellular Ca2+ signaling. Thus, ABT-737 has a safe Ca2+-signaling profile, since ABT-737, at therapeutically relevant concentrations (i.e. below 1 μM), does not affect intracellular Ca2+-transport mechanisms essential for cellular homeostasis.