Fig. 2

Effects of EVO on cleavages of poly(ADP ribose) polymerase (PARP) and caspase (Casp)-3 protein, mitochondrial membrane potential, and intracellular peroxide production in SW1736 and KAT4B cells. A EVO induced Casp-3 and PARP protein cleavages in SW1736 and KAT-4B cells in a concentration-dependent study. Both cell lines were treated with different concentrations (1, 2, 4, 8, and 16 µM) of EVO for 24 h, and expressions of PARP, Casp-3, and α-tubulin (α-TUB; as the internal control) were examined by Western blotting. B EVO induced Casp-3 protein cleavages in SW1736 and KAT-4B cells in a time-dependent study. Both cell lines were treated with EVO (8 µM) for different times (6, 12, and 24 h), and expressions of Casp-3, and α-tubulin (α-TUB; as the internal control) were examined by Western blotting. The intensity of each cleaved fragment in (A) and (B) was examined by a densitometric analysis (Image J) and expressed as multiples of the control. Each data point was calculated from triplicate groups, and data are shown as the mean ± SD. C Addition of a pan-caspase inhibitor (Ac-VAD-FMK; CAI) significantly inhibited EVO-induced cell death in SW1736 cells, but no significance in KAT4B cells. Both cell lines were incubated with a pan-caspase inhibitor (10 nM; CAI) for 4 h followed by EVO (8 µM) treatment for an additional 24 h. Viability of cells was examined by an MTT assay. D Alternative increment of intracellular peroxide level by EVO in SW1736 and KAT4B cells. Cells were treated with different concentrations of EVO (4, 8, and 16 µM) for 12 h, and intracellular peroxide levels were detected by a flow cytometric analysis using DCFH-DA as a fluorescent dye. E Disruption of the mitochondrial membrane potential (MMP) by EVO in anaplastic thyroid cancer (ATC) SW1736 and KAT4B cells. Cells were treated with different concentrations of EVO (4, 8, and 16 µM) for 12 h, and the MMP was detected by a flow cytometric analysis using DiOC6 as a fluorescent dye. (Upper panel) a representative of flow cytometric data was shown. (Lower panel) data from three-independent experiments was shown. Each data point was calculated from triplicate groups, and data are shown as the mean ± SD. *p < 0.05 and **p < 0.01 denote a significant difference compared to the control (CON)