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N-(1-pyrenyl) maleimide induces bak oligomerization and mitochondrial dysfunction in Jurkat Cells.

Huang PR, Hung SC, Pao CC, Wang TC - Biomed Res Int (2015)

Bottom Line: Induction of Bak oligomerization appears to play a critical role in NPM-induced apoptosis, as downregulation of Bak by shRNA significantly prevented NPM-induced apoptosis.Inhibition of caspase 8 by Z-IETD-FMK and/or depletion of Bid did not affect NPM-induced oligomerization of Bak.Taken together, these results suggest that NPM-induced apoptosis is mediated through a pathway that is independent of caspase-8 activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan 333, Taiwan.

ABSTRACT
N-(1-pyrenyl) maleimide (NPM) is a fluorescent reagent that is frequently used as a derivatization agent for the detection of thio-containing compounds. NPM has been shown to display a great differential cytotoxicity against hematopoietic cancer cells. In this study, the molecular mechanism by which NPM induces apoptosis was examined. Here, we show that treatment of Jurkat cells with NPM leads to Bak oligomerization, loss of mitochondrial membrane potential (Δψm), and release of cytochrome C from mitochondria to cytosol. Induction of Bak oligomerization appears to play a critical role in NPM-induced apoptosis, as downregulation of Bak by shRNA significantly prevented NPM-induced apoptosis. Inhibition of caspase 8 by Z-IETD-FMK and/or depletion of Bid did not affect NPM-induced oligomerization of Bak. Taken together, these results suggest that NPM-induced apoptosis is mediated through a pathway that is independent of caspase-8 activation.

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Effect of caspase-8 inhibition and Bid downregulation on NPM-induced Bak oligomerization. (a) Effect of caspase-8 inhibition on NPM-induced oligomerization of Bak and tBid formation. Jurkat cells were pretreated with 50 μM of caspase-8 inhibitor Z-IETD-FMK for 1 h before being exposed to 0.5 or 1 μM of NPM for 3 h. The cell lysates were then assayed for the Bak oligomers and tBid as described in Section 2. (b) Effect of Bid downregulation on NPM-induced oligomerization of Bak and tBid formation. The Jurkat cells that stably express shRNA against Bid (shBid) or against luciferase (shLuc) were treated with NPM at 0.5–1 μM for 3 h. The cell lysates of the treated cells were assayed for the Bak oligomers and tBid as described in Section 2. Data shown were from a representative of three independent experiments that gave similar results. Actin served as a loading control.
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fig6: Effect of caspase-8 inhibition and Bid downregulation on NPM-induced Bak oligomerization. (a) Effect of caspase-8 inhibition on NPM-induced oligomerization of Bak and tBid formation. Jurkat cells were pretreated with 50 μM of caspase-8 inhibitor Z-IETD-FMK for 1 h before being exposed to 0.5 or 1 μM of NPM for 3 h. The cell lysates were then assayed for the Bak oligomers and tBid as described in Section 2. (b) Effect of Bid downregulation on NPM-induced oligomerization of Bak and tBid formation. The Jurkat cells that stably express shRNA against Bid (shBid) or against luciferase (shLuc) were treated with NPM at 0.5–1 μM for 3 h. The cell lysates of the treated cells were assayed for the Bak oligomers and tBid as described in Section 2. Data shown were from a representative of three independent experiments that gave similar results. Actin served as a loading control.

Mentions: Since it is known that the ligation of death receptors in the extrinsic pathway can cause the activation of caspase 8 and cleavage of Bid, leading to the activation of Bak or Bax [1], we investigated whether or not activation of caspase 8 and cleavage of Bid is involved in NPM-induced oligomerization of Bak. Inhibition of caspase 8 with Z-IETD-FMK abolished the cleavage of tBid but did not affect NPM-induced oligomerization of Bak (Figure 6(a)). Depletion of Bid by shRNA greatly reduced the level of Bid, but NPM-induced Bak oligomerization was not affected (Figure 6(b)). These results suggest that activation of caspase 8 and cleavage of Bid are not involved in NPM-induced oligomerization of Bak.


N-(1-pyrenyl) maleimide induces bak oligomerization and mitochondrial dysfunction in Jurkat Cells.

Huang PR, Hung SC, Pao CC, Wang TC - Biomed Res Int (2015)

Effect of caspase-8 inhibition and Bid downregulation on NPM-induced Bak oligomerization. (a) Effect of caspase-8 inhibition on NPM-induced oligomerization of Bak and tBid formation. Jurkat cells were pretreated with 50 μM of caspase-8 inhibitor Z-IETD-FMK for 1 h before being exposed to 0.5 or 1 μM of NPM for 3 h. The cell lysates were then assayed for the Bak oligomers and tBid as described in Section 2. (b) Effect of Bid downregulation on NPM-induced oligomerization of Bak and tBid formation. The Jurkat cells that stably express shRNA against Bid (shBid) or against luciferase (shLuc) were treated with NPM at 0.5–1 μM for 3 h. The cell lysates of the treated cells were assayed for the Bak oligomers and tBid as described in Section 2. Data shown were from a representative of three independent experiments that gave similar results. Actin served as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig6: Effect of caspase-8 inhibition and Bid downregulation on NPM-induced Bak oligomerization. (a) Effect of caspase-8 inhibition on NPM-induced oligomerization of Bak and tBid formation. Jurkat cells were pretreated with 50 μM of caspase-8 inhibitor Z-IETD-FMK for 1 h before being exposed to 0.5 or 1 μM of NPM for 3 h. The cell lysates were then assayed for the Bak oligomers and tBid as described in Section 2. (b) Effect of Bid downregulation on NPM-induced oligomerization of Bak and tBid formation. The Jurkat cells that stably express shRNA against Bid (shBid) or against luciferase (shLuc) were treated with NPM at 0.5–1 μM for 3 h. The cell lysates of the treated cells were assayed for the Bak oligomers and tBid as described in Section 2. Data shown were from a representative of three independent experiments that gave similar results. Actin served as a loading control.
Mentions: Since it is known that the ligation of death receptors in the extrinsic pathway can cause the activation of caspase 8 and cleavage of Bid, leading to the activation of Bak or Bax [1], we investigated whether or not activation of caspase 8 and cleavage of Bid is involved in NPM-induced oligomerization of Bak. Inhibition of caspase 8 with Z-IETD-FMK abolished the cleavage of tBid but did not affect NPM-induced oligomerization of Bak (Figure 6(a)). Depletion of Bid by shRNA greatly reduced the level of Bid, but NPM-induced Bak oligomerization was not affected (Figure 6(b)). These results suggest that activation of caspase 8 and cleavage of Bid are not involved in NPM-induced oligomerization of Bak.

Bottom Line: Induction of Bak oligomerization appears to play a critical role in NPM-induced apoptosis, as downregulation of Bak by shRNA significantly prevented NPM-induced apoptosis.Inhibition of caspase 8 by Z-IETD-FMK and/or depletion of Bid did not affect NPM-induced oligomerization of Bak.Taken together, these results suggest that NPM-induced apoptosis is mediated through a pathway that is independent of caspase-8 activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan 333, Taiwan.

ABSTRACT
N-(1-pyrenyl) maleimide (NPM) is a fluorescent reagent that is frequently used as a derivatization agent for the detection of thio-containing compounds. NPM has been shown to display a great differential cytotoxicity against hematopoietic cancer cells. In this study, the molecular mechanism by which NPM induces apoptosis was examined. Here, we show that treatment of Jurkat cells with NPM leads to Bak oligomerization, loss of mitochondrial membrane potential (Δψm), and release of cytochrome C from mitochondria to cytosol. Induction of Bak oligomerization appears to play a critical role in NPM-induced apoptosis, as downregulation of Bak by shRNA significantly prevented NPM-induced apoptosis. Inhibition of caspase 8 by Z-IETD-FMK and/or depletion of Bid did not affect NPM-induced oligomerization of Bak. Taken together, these results suggest that NPM-induced apoptosis is mediated through a pathway that is independent of caspase-8 activation.

Show MeSH
Related in: MedlinePlus