Methotrexate induces apoptosis of human choriocarcinoma cell line JAR via a mitochondrial pathway

ObjectiveTo investigate methotrexate (MTX)-induced apoptosis and the involved pathways in human choriocarcinoma cells.Study designMTX-induced apoptosis of human choriocarcinoma cell line JAR was examined using a PI/Annexin V stain with flow cytometer (FCM). Mitochondrial apoptosis was detected by fluorescence microscopy, and analyzed by FCM using a MitoCapture mitochondrial apoptosis detection kit. The activities of caspase-8 and caspase-9 were quantified by microtiter plate reader at 405 nm using FLICE/Caspase-8 colorimetric assay kit and Caspase-9/Mch6 colorimetric assay kit. The changes in Bax and Bcl-2 expression were detected during apoptosis using immunocytochemistry and Western blot analysis.ResultsJAR cells underwent apoptosis after exposure to 0.1–2.5 μg/ml MTX for 48 h. Decreased mitochondrial membrane potential was observed both by fluorescence microscopy and FCM. The activation of caspase-9 was increased 4.35 ± 0.76-fold in MTX-incubated JAR, while there was no obvious change in the activation of caspase-8. When JAR cells underwent apoptosis, the expression of Bcl-2 was decreased and the expression of Bax was increased; both were detected by immunocytochemistry assay.ConclusionMethotrexate in lower concentrations induces apoptosis of human choriocarcinoma cells via mitochondrial-initiated pathways, including reduction of mitochondrial membrane potential, activation of caspase-9, and up-regulation of Bax/Bcl-2 expression.