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Silibinin induces apoptosis via calpain-dependent AIF nuclear translocation in U87MG human glioma cell death.

Jeong JC, Shin WY, Kim TH, Kwon CH, Kim JH, Kim YK, Kim KH - J. Exp. Clin. Cancer Res. (2011)

Bottom Line: Silibinin caused ROS generation and its effect was inhibited by calpain inhibitor, the general PKC inhibitor GF 109203X, the specific PKCδ inhibitor rottlerin, and catalase.Silibinin induced AIF nuclear translocation and its effect was prevented by calpain inhibitor.Transfection of vector expressing microRNA of AIF prevented the silibinin-induced cell death.

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Affiliation: Department of Oriental Medicine, Dongguk University, Kyung Ju, 780-714, Korea.

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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 PKCδ activation. Cells were exposed to 30 μM silibinin for various times and PKCδ phosphorylation was estimated by Western blot analysis. (D) Effect of calpain inhibitor on PKCδ phosphorylation. Cells were exposed to 30 μM silibinin for 10 min in the presence or absence of 0.5 μM calpain inhibitor (CHO) and PKCδ phosphorylation was estimated by Western blot analysis.
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Figure 2: 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 PKCδ activation. Cells were exposed to 30 μM silibinin for various times and PKCδ phosphorylation was estimated by Western blot analysis. (D) Effect of calpain inhibitor on PKCδ phosphorylation. Cells were exposed to 30 μM silibinin for 10 min in the presence or absence of 0.5 μM calpain inhibitor (CHO) and PKCδ phosphorylation was estimated by Western blot analysis.

Mentions: The silibinin-induced cell death was associated with ROS generation mediated by intracellular Ca2+ [8]. To determine therefore whether ROS production by silibinin is attributed to calpain activation, cells were exposed to silibinin in the presence of calpain inhibitor and ROS generation was measured. As shown in Figure 2A, the silibinin-induced ROS generation was blocked by the calpain inhibitor with potency similar to that of catalase.


Silibinin induces apoptosis via calpain-dependent AIF nuclear translocation in U87MG human glioma cell death.

Jeong JC, Shin WY, Kim TH, Kwon CH, Kim JH, Kim YK, Kim KH - J. Exp. Clin. Cancer Res. (2011)

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 PKCδ activation. Cells were exposed to 30 μM silibinin for various times and PKCδ phosphorylation was estimated by Western blot analysis. (D) Effect of calpain inhibitor on PKCδ phosphorylation. Cells were exposed to 30 μM silibinin for 10 min in the presence or absence of 0.5 μM calpain inhibitor (CHO) and PKCδ phosphorylation was estimated by Western blot analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3108340&req=5

Figure 2: 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 PKCδ activation. Cells were exposed to 30 μM silibinin for various times and PKCδ phosphorylation was estimated by Western blot analysis. (D) Effect of calpain inhibitor on PKCδ phosphorylation. Cells were exposed to 30 μM silibinin for 10 min in the presence or absence of 0.5 μM calpain inhibitor (CHO) and PKCδ phosphorylation was estimated by Western blot analysis.
Mentions: The silibinin-induced cell death was associated with ROS generation mediated by intracellular Ca2+ [8]. To determine therefore whether ROS production by silibinin is attributed to calpain activation, cells were exposed to silibinin in the presence of calpain inhibitor and ROS generation was measured. As shown in Figure 2A, the silibinin-induced ROS generation was blocked by the calpain inhibitor with potency similar to that of catalase.

Bottom Line: Silibinin caused ROS generation and its effect was inhibited by calpain inhibitor, the general PKC inhibitor GF 109203X, the specific PKCδ inhibitor rottlerin, and catalase.Silibinin induced AIF nuclear translocation and its effect was prevented by calpain inhibitor.Transfection of vector expressing microRNA of AIF prevented the silibinin-induced cell death.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oriental Medicine, Dongguk University, Kyung Ju, 780-714, Korea.

Show MeSH
Related in: MedlinePlus