PMC

Effect of silibinin on Bax expression. Cells were exposed to 30 μM silibinin for various times and Bax expression was estimated by Western blot analysis. Representative (A) and quantitative (B) results of four independent experiments. (C) Cells were exposed to 30 μM silibinin for 24 h in the presence or absence of 0.5 μM calpain inhibitor (CHO) and Bax expression was estimated by Western blot analysis.

Role of calpain in silibinin-induced cell death. (A) Cells were exposed to 30 μM silibinin for 36 h in the presence of various concentrations of calpain inhibitor (Z-CHO). Cell viability was estimated by MTT assay. Data are mean ± SEM of four independent experiments performed in duplicate. *p < 0.05 compared with silibinin alone. (B) Cells were exposed to 30 μM silibinin for 24 h in the presence of 2 mM EGTA and 0.5 μM Z-CHO. Calpain activity was measured by calpain assay kit. Data are mean ± SEM of four independent experiments performed in duplicate. *p < 0.05 compared with silibinin alone.

Effect of silibinin on mitochondrial membrane potential (MMP). Cells were exposed to 30 μM silibinin for 6 h (A) and various times (B). The MMP was estimated by the uptake of a membrane potential-sensitive fluorescence dye DiCO₆(3). The fluorescence intensity was analyzed using FACS analysis. Data in (B) are mean ± SEM of three independent experiments performed in duplicate. *p < 0.05 compared with control. (C) Effect of inhibitors of calpain and PKC and antioxidant on silibinin-induced disruption of MMP. Cells were exposed to 30 μM silibinin for 6 h in the presence or absence of 0.5 μM calpain inhibitor (CHO), 1 μM GF 109203X (GF), 1 μM rottlerin (Ro), and 800 units/ml catalase (Cat). The MMP was measured as described above. Data are mean ± SEM of four independent experiments performed in duplicate. *p < 0.05 compared with silibinin alone.

Role of calpain and PKC in ROS generation and cell death induced by silibinin. (A) Effect of inhibitors of calpain and PKC on silibinin-induced ROS generation. Cells were exposed to 30 μM silibinin in the presence or absence of 0.5 μM calpain inhibitor (CHO), 1 μM GF 109203X (GF), 1 μM rottlerin (Ro), and 800 units/ml catalase (Cat) and ROS generation was estimated by measuring changes in DCF fluorescence using FACS analysis. Data are mean ± SEM of five independent experiments performed in duplicate. *p < 0.05 compared with silibinin alone. (B) Effect of PKC inhibitors on silibinin-induced cell death. Cells were exposed to 30 μM silibinin in the presence or absence of 1 μM GF 109203X (GF) and 1 μM rottlerin (Ro) and cell viability was measured by MTT assay. Data are mean ± SEM of four independent experiments performed in duplicate. *p < 0.05 compared with silibinin alone. (C) Effect of silibinin on PKC_δ activation. Cells were exposed to 30 μM silibinin for various times and PKC_δ phosphorylation was estimated by Western blot analysis. (D) Effect of calpain inhibitor on PKC_δ phosphorylation. Cells were exposed to 30 μM silibinin for 10 min in the presence or absence of 0.5 μM calpain inhibitor (CHO) and PKC_δ phosphorylation was estimated by Western blot analysis.

Role of AIF nuclear translocation in silibinin-induced cell death. (A) Cells were exposed to with 30 μM silibinin for various times and cytosolic and nuclear fractions were prepared. AIF expression was estimated by Western blot using antibodies specific against AIF. (B) Cells were exposed to 30 μM silibinin for 36 h in the presence or absence of 0.5 μM calpain inhibitor (CHO). AIF nuclear translocation was estimated by immunofluorescence using antibody specific against AIF. Nuclei were counterstained with propidium iodide (PI). Images were captured by confocal microscope and presented. Arrows indicate AIF nuclear localization. (C) Cells were transfected with mipcDNA vector for LacZ or AIF micro-RNA (mi-AIF). The expression levels of AIF were determined by Western blotting. (D) Cells transfected with LacZ or mi-AIF were exposed to 30 μM silibinin for 36 h and cell viability was estimated by MTT assay. Data are mean ± SEM of four independent experiments performed in duplicate. *p < 0.05 compared with LacZ control; #p < 0.05 compared with LacZ silibinin.