Cisplatin triggers programmed necrosis in KYSE140 cells.

(A) Mitochondrial membrane potentials of KYSE140 and EC0156 cells were determined using Mito Tracker Red CMXRos staining and analyzed by flow cytometry after cisplatin treatment for 24 h. (B) Cellular necrosis was measured as LDH release after cisplatin treatment for 24 h and 48 h. (C) KYSE140 cells were treated as indicated for 12 h. The cells were stained with H2DCF-DA for 20 min, and then ROS levels were measured using flow cytometry. All experiments were performed at least three times. (D) KYSE140 and EC0156 were treated with 10 µM cisplatin for 12 or 24 h. Caspase activation was assessed by western blotting. Arrowheads indicate caspase cleavage fragments. (E) Total cellular protein extracts from each of the eight esophageal cancer cell lines were subjected to western blot analysis for RIPK3. β-Actin as a loading control. (F) Transmission electron microscopy of the KYSE140 cells after cisplatin treatment. Membrane integrity was noted in the control cells, and the collapse of the membrane and the swelling of the cellular organelles were noted in the cells treated with cisplatin.