Methotrexate Promotes Platelet Apoptosis via JNK-Mediated Mitochondrial Damage: Alleviation by N-Acetylcysteine and N-Acetylcysteine Amide

Abstract

Thrombocytopenia in methotrexate (MTX)-treated cancer and rheumatoid arthritis (RA) patients connotes the interference of MTX with platelets. Hence, it seemed appealing to appraise the effect of MTX on platelets. Thereby, the mechanism of action of MTX on platelets was dissected. MTX (10 μM) induced activation of pro-apoptotic proteins Bid, Bax and Bad through JNK phosphorylation leading to ΔΨm dissipation, cytochrome c release and caspase activation, culminating in apoptosis. The use of specific inhibitor for JNK abrogates the MTX-induced activation of pro-apoptotic proteins and downstream events confirming JNK phosphorylation by MTX as a key event. We also demonstrate that platelet mitochondria as prime sources of ROS which plays a central role in MTX-induced apoptosis. Further, MTX induces oxidative stress by altering the levels of ROS and glutathione cycle. In parallel, the clinically approved thiol antioxidant N-acetylcysteine (NAC) and its derivative N-acetylcysteine amide (NACA) proficiently alleviate MTX-induced platelet apoptosis and oxidative damage. These findings underpin the dearth of research on interference of therapeutic drugs with platelets, despite their importance in human health and disease. Therefore, the use of antioxidants as supplementary therapy seems to be a safe bet in pathologies associated with altered platelet functions.

Fig 1. MTX altered ROS levels in platelets and its inhibition by NAC/NACA/Mito-TEMPO.

A, FACS analysis of ROS generation in washed platelets treated with MTX in presence or absence of NAC/NACA. B, GSH/GSSG ratio and (C) γ‑Glutamyltransferase activity. D, Effect of Mito-TEMPO on MTX induced ROS generation, expressed as percentage increase in DCF fluorescence. Effect of MTX in presence or absence of NAC/NACA on components of mitochondrial electron transport chain, (E) Complex I-NADH: ubiquinone oxidoreductase activity (F) Complex II-succinate: ubiquinone oxidoreductase activity (G) Complex III-coenzyme Q: cytochrome c-oxidoreductase activity and (H) Complex IV-cytochrome c oxidase activity. Values are presented as mean ± SEM (n = 5). p*/#< 0.05, p**/##< 0.01, p***/###< 0.001; *: significant compared to control. #: significant compared to MTX.

Fig 2. MTX altered ER stress and mitochondrial apoptotic markers and its inhibition by NAC/NACA.

Effect of MTX in presence or absence of NAC/NACA on (A) changes in intracellular calcium levels, (B) phospho-eIF2-α expression and (C) peroxidation of cardiolipin, (D) FACS analysis of ΔΨm depolarization in washed platelets treated with MTX in presence or absence of NAC/NACA. Values are presented as mean ± SEM (n = 5), expressed as percentage increase in (A) fura-2/AM and (C) NAO fluorescence. p*/#< 0.05, p**/##< 0.01, p***/###< 0.001; *: significant compared to control. #: significant compared to MTX. Membrane was cut based on the molecular weight, probed with antibody of interest and band of interest were presented.

Fig 3. MTX induced platelet apoptosis and its inhibition by NAC/NACA.

Effect of MTX in presence or absence of NAC/NACA/Dicumarol on (A) cyt. c release from mitochondria to cytosol, activation of caspase-9 and caspase-3, (B) protein tyrosine phosphorylation, (C) PS externalization, (D) LDH release and (E) MTT cell viability assay. B, Lane I: resting platelets (untreated), Lane II: platelets treated with A23187 (10 μM), Lane III: platelets treated with MTX (50 μM), Lane IV: pre-loaded platelets with MTX (50 μM) and incubated with NAC (500 μM), Lane V: pre-loaded platelets with MTX (50 μM) and incubated with NACA (50 μM). Values are presented as mean ± SEM (n = 5), expressed as percentage increase in (C) Annexin V-FITC fluorescence. COX IV and β-Tubulin were used as loading control. p*/#< 0.05, p**/##< 0.01, p***/###< 0.001; *: significant compared to control. #: significant compared to MTX. Membrane was cut based on the molecular weight, probed with antibody of interest and band of interest were presented.

Fig 4. JNK activation by mitochondrial ROS in MTX-treated platelets and its reversal by NAC/NACA.

A, Immunoblot showing the expression of phospho-JNK in time- and concentration-dependent manner in MTX-treated platelets. B, Immunoblots showing the effect of Dicumarol on the expression levels of phospho-JNK, Bcl-2, Bad, Bax, cyt. c, Cas-3 in MTX treated platelets. C, Immunoblots showing the effect of z-DEVD-fmk on the expression level of Cas-3 in MTX-treated platelets. D, Immunoblots showing the effect of Mito-TEMPO on the expression levels of phospho-JNK in MTX-treated platelets. E, Effect of NAC/NACA on the expression levels of phospho-JNK, Bcl-2, Bad, Bax, tBid and Cas-8 in MTX-treated platelets. F, Representative densitogram of immunoblots present in panel A, B, C, D, and E. JNK and β-Tubulin were used as loading control. Membrane was cut based on the molecular weight, probed with antibody of interest and band of interest were presented.

Fig 5. A, Effect of NAC/NACA on MTX-induced PS externalization.

Effect of (B) Dicumarol (C) z-DEVD-fmk and (D) Mito-TEMPO on MTX induced PS externalization on washed platelets. (E) Effect of microparticle-rich fraction on plasma clotting time obtained from MTX-treated platelets. Values are presented as mean ± SEM (n = 5), expressed as percentage increase in (B-D) Annexin V-FITC fluorescence. p*/#< 0.05, p**/##< 0.01, p***/###< 0.001; *: significant compared to control. #: significant compared to MTX.