Arsenic trioxide induces apoptosis in human platelets via C-Jun NH2-terminal kinase activation

Abstract

Arsenic trioxide (ATO), one of the oldest drugs in both Western and traditional Chinese medicine, has become an effective anticancer drug, especially in the treatment of acute promyelocytic leukemia (APL). However, thrombocytopenia occurred in most of ATO-treated patients with APL or other malignant diseases, and the pathogenesis remains unclear. Here we show that ATO dose-dependently induces depolarization of mitochondrial inner transmembrane potential (ΔΨm), up-regulation of Bax and down-regulation of Bcl-2 and Bcl-XL, caspase-3 activation, and phosphotidylserine (PS) exposure in platelets. ATO did not induce surface expression of P-selectin and PAC-1 binding, whereas, obviously reduced collagen, ADP, and thrombin induced platelet aggregation. ATO dose-dependently induced c-Jun NH2-terminal kinase (JNK) activation, and JNK specific inhibitor dicumarol obviously reduced ATO-induced ΔΨm depolarization in platelets. Clinical therapeutic dosage of ATO was intraperitoneally injected into C57 mice, and the numbers of circulating platelets were significantly reduced after five days of continuous injection. The data demonstrate that ATO induces caspase-dependent apoptosis via JNK activation in platelets. ATO does not incur platelet activation, whereas, it not only impairs platelet function but also reduces circulating platelets in vivo, suggesting the possible pathogenesis of thrombocytopenia in patients treated with ATO.

Figure 1. ATO dose-dependently induces ΔΨm depolarization in platelets.

Washed platelets were pre-treated with 2, 4, 8, or 16 μM of ATO or vehicle at 37°C for 5 hrs. JC-1 was added into the pre-treated platelets to a final concentration of 5 μg/mL and then incubated at 37°C in the dark for 20 min. Then the treated samples were detected using flow cytometry. (A) Typical flow cytometric histograms represented of 3 separate experiments are shown. FL1 and FL3 stand for green and red fluorescence, respectively. Dots inside R1 indicate that platelets are out of the main population, bearing ΔΨm depolarization. (B) Quantitation data from 3 separate experiments with different donors are illustrated (mean ± SD). *P<0.05, **P<0.01, compared with vehicle controls.

Figure 2. ATO dose-dependently induces up-regulation of Bax, down-regulation of Bcl-2 and Bcl-X_L, and caspase-3 activation in platelets.

Platelets were incubated with 2, 4, or 8 μM of ATO or vehicle at 37°C for 5 hrs. (A) The treated platelets were subjected to Western blot analysis with anti-Bax, anti-Bcl-2, and anti-Bcl-X_L antibodies. (B) Pre-treated platelets were subjected to Western blot analysis using anti-caspase-3 antibody. The 32 kDa caspase-3 fragment indicates nonactivated caspase-3, the 17 kDa caspase-3 fragment indicates activated caspase-3. GAPDH levels demonstrate similar loading. Results are representative of 3 separate experiments with different donors.

Figure 3. ATO dose-dependently induces PS exposure in platelets.

Platelets were pre-treated with 2, 4, 8, 16 μM of ATO or vehicle at 37°C for 5 hrs. (A) Annexin V binding buffer was mixed with the pre-treated platelets and FITC-annexin V at a ratio of 50: 10: 1. The samples were gently mixed and incubated at RT for 15 min in the dark, then analyzed by flow cytometry. (B) Quantitation data from 3 separate experiments with different donors are illustrated (mean ± SD). **P<0.01, *P<0.05 compared with vehicle controls.

Figure 4. ATO does not incur P-selectin surface expression and PAC-1 binding in platelets.

Washed Platelets were treated with different concentrations of ATO (2, 4, 8, 16 μM) or vehicle at 37°C for 5 hrs. (A) Pre-treated platelets were further incubated with SZ51 or mouse IgG at RT for 30 min, and then incubated with FITC-GAM and subjected to flow cytometry analysis. Quantitation data from three separate experiments with different donors are shown as mean ± SD. (B) Pre-treated platelets were further incubated with FITC-labeled soluble PAC-1 at RT for 20 min in the dark, then the treated platelets were fixed with 1% cold paraformaldehyde, further incubated at 4°C in the dark for 30 min and then subjected to flow cytometry detection. Quantitation data from three separate experiments with different donors are shown as mean ± SD (n = 3). **P<0.01 compared with vehicle controls.

Figure 5. ATO induces JNK activation and dicumarol reduces ATO induced ΔΨm depolarization in platelets.

(A) Washed platelets were pre-treated with or without dicumaol (2 μM) or vehicle at 37°C for 15 min, then further incubated with various concentration of ATO at 37°C for 5 hrs. The treated platelets were subjected to Western blot analysis using anti-nonphosphorylated JNK1 and JNK2 antibody (anti-JNK 1/2) and anti-phosphorylated JNK1 and JNK2 antibody (anti-p-JNK 1/2). GAPDH levels demonstrate similar loading. Results are representative of 3 separate experiments with different donors. (B) Washed platelets were pre-treated with or without dicumarol (2 μM) or vehicle at 37°C for 15 min, then further incubated with or without ATO at 37°C for 5 hrs. JC-1 was added into the treated platelets to a final concentration of 5 μg/mL and then incubated at 37°C in the dark for 20 min. The treated samples were detected using flow cytometry. Dots inside R1 are platelets out of the main population bearing ΔΨm depolarization. (C) Quantitation data from 3 separate experiments with different donors are illustrated (mean ± SD). **P<0.01 compared with vehicle controls.

Figure 6. ATO reduces collagen, ADP, and thrombin induced platelet aggregation, and intraperitoneal injection of ATO reduces circulating platelets.

(A) PRP was incubated with ATO (2 μM) or vehicle for 1 hr, washed platelets was incubated with ATO (16 μM) or vehicle for 2 hrs. Platelet aggregation assay was performed by addition of collagen (5 μg/mL) or ADP (10 μmol/L) into PRP, or thrombin (0.5 U/mL) into washed platelets under stirring condition, and recorded by a turbidometric platelet aggregometer. Results are representative of 3 separate experiments with different donors. (B) Two groups of mice were intraperitoneally injected with ATO (dissolved in 0.9% NS, 5 mg/kg) or vehicle once a day for 5 continuous days. Blood samples were taken from the mice before injection and 24 hrs after the final injection, and then subjected to platelet count assay using Sysmex KX-21N Blood Cell Analyser. Platelet count (% of baseline) equals platelet count after injection/platelet count before injection ×100%. Quantitation data from 12 mice per group are illustrated as mean ± SD. **P<0.01, compared with vehicle controls.