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Etoposide sensitizes neuroblastoma cells expressing caspase 8 to TRAIL.

Kim HR, Lee MW, Kim DS, Jo HY, Lee SH, Chueh HW, Jung HL, Yoo KH, Sung KW, Koo HH - Cell Biol Int Rep (2010) (2012)

Bottom Line: Although TRAIL-R2 expression increased in IMR-32 cells in response to etoposide treatment, cell death was not increased by concurrent treatment with TRAIL compared with etoposide alone, because the cells lacked caspase 8 expression.Restoration of caspase 8 expression by exposure to IFNγ (interferon γ) sensitizes IMR-32 cells to TRAIL.Moreover, pretreatment with etoposide increased TRAIL-induced apoptosis in caspase 8 restored IMR-32 cells through activation of a caspase cascade that included caspases 8, 9 and 3.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.

ABSTRACT
TRAIL [TNF (tumour necrosis factor)-related apoptosis-inducing ligand] is a promising agent for clinical use since it kills a wide range of tumour cells without affecting normal cells. We provide evidence that pretreatment with etoposide significantly enhanced TRAIL-mediated apoptosis via up-regulation of DR5 (death receptor 5 or TRAIL-R2) expression in the caspase 8 expressing neuroblastoma cell line, SK-N-MC. In addition, sequential treatment with etoposide and TRAIL increased caspases 8, 9 and 3 activation, Mcl-1 cleavage and Bid truncation, which suggests that the ability of etoposide and TRAIL to induce apoptosis is mediated through activation of an intrinsic signalling pathway. Although TRAIL-R2 expression increased in IMR-32 cells in response to etoposide treatment, cell death was not increased by concurrent treatment with TRAIL compared with etoposide alone, because the cells lacked caspase 8 expression. Restoration of caspase 8 expression by exposure to IFNγ (interferon γ) sensitizes IMR-32 cells to TRAIL. Moreover, pretreatment with etoposide increased TRAIL-induced apoptosis in caspase 8 restored IMR-32 cells through activation of a caspase cascade that included caspases 8, 9 and 3. These results indicate that the etoposide-mediated sensitization of neuroblastoma cells to TRAIL is associated with an increase in TRAIL-R2 expression and requires caspase 8 expression. These observations support the potential use of a combination of etoposide and TRAIL in future clinical trials.

No MeSH data available.


Related in: MedlinePlus

Pretreatment with etoposide enhances TRAIL-induced cell death in SK-N-MC, but not in IMR-32 cells(a) Cell death was analysed 48 h after exposure to 100, 500 or 1000 ng/ml TRAIL. (b) Neuroblastoma cells were pretreated with etoposide (500 ng/ml) followed by exposure to TRAIL (100 ng/ml) treatment for 48 h. (c, d) Cells were cultured in 60 mm plates and exposed to 500 ng/ml etoposide and/or 100 ng/ml of TRAIL. After 12 h incubation, total cell lysates were obtained and analysed for caspase 3 activity and PARP cleavage using a luminex assay. (e, f) Cells were pretreated with the indicated concentrations of the DR5:Fc fusion protein for 1 h to block TRAIL binding to TRAIL-R2. Cell viability was assessed after consecutive treatment with etoposide (Eto; 500 ng/ml) and/or TRAIL (T; 100 ng/ml) for 48 h. The results are presented as cell death as a percentage against vehicle-treated control (means±S.D. from three independent experiments).
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Figure 2: Pretreatment with etoposide enhances TRAIL-induced cell death in SK-N-MC, but not in IMR-32 cells(a) Cell death was analysed 48 h after exposure to 100, 500 or 1000 ng/ml TRAIL. (b) Neuroblastoma cells were pretreated with etoposide (500 ng/ml) followed by exposure to TRAIL (100 ng/ml) treatment for 48 h. (c, d) Cells were cultured in 60 mm plates and exposed to 500 ng/ml etoposide and/or 100 ng/ml of TRAIL. After 12 h incubation, total cell lysates were obtained and analysed for caspase 3 activity and PARP cleavage using a luminex assay. (e, f) Cells were pretreated with the indicated concentrations of the DR5:Fc fusion protein for 1 h to block TRAIL binding to TRAIL-R2. Cell viability was assessed after consecutive treatment with etoposide (Eto; 500 ng/ml) and/or TRAIL (T; 100 ng/ml) for 48 h. The results are presented as cell death as a percentage against vehicle-treated control (means±S.D. from three independent experiments).

Mentions: Treatment with TRAIL induced cell death in SK-N-MC cells in a dose-dependent manner, but had no effect on IMR-32 cells (Figure 2a). When SK-N-MC cells were pre-treated with etoposide for 2 h before TRAIL treatment, a significant increase in cell death occurred at 48 h after etoposide treatment compared with treatment with either etoposide or TRAIL alone. However, treatment with TRAIL, in either the presence or absence of etoposide, had no effect on cell death in IMR-32 cells (Figure 2b). After consecutive treatment with etoposide and TRAIL, the increase in caspase 3 activity and PARP cleavage were observed in SK-N-MC cells, but not in IMR-32 cells (Figures 2c and 2d). Furthermore, treatment with DR5:Fc had no impact on cell viability in IMR-32 cells, but it completely inhibited the increase in cell death caused by consecutive treatment with etoposide and TRAIL in SK-N-MC cells (Figures 2e and 2f).


Etoposide sensitizes neuroblastoma cells expressing caspase 8 to TRAIL.

Kim HR, Lee MW, Kim DS, Jo HY, Lee SH, Chueh HW, Jung HL, Yoo KH, Sung KW, Koo HH - Cell Biol Int Rep (2010) (2012)

Pretreatment with etoposide enhances TRAIL-induced cell death in SK-N-MC, but not in IMR-32 cells(a) Cell death was analysed 48 h after exposure to 100, 500 or 1000 ng/ml TRAIL. (b) Neuroblastoma cells were pretreated with etoposide (500 ng/ml) followed by exposure to TRAIL (100 ng/ml) treatment for 48 h. (c, d) Cells were cultured in 60 mm plates and exposed to 500 ng/ml etoposide and/or 100 ng/ml of TRAIL. After 12 h incubation, total cell lysates were obtained and analysed for caspase 3 activity and PARP cleavage using a luminex assay. (e, f) Cells were pretreated with the indicated concentrations of the DR5:Fc fusion protein for 1 h to block TRAIL binding to TRAIL-R2. Cell viability was assessed after consecutive treatment with etoposide (Eto; 500 ng/ml) and/or TRAIL (T; 100 ng/ml) for 48 h. The results are presented as cell death as a percentage against vehicle-treated control (means±S.D. from three independent experiments).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Pretreatment with etoposide enhances TRAIL-induced cell death in SK-N-MC, but not in IMR-32 cells(a) Cell death was analysed 48 h after exposure to 100, 500 or 1000 ng/ml TRAIL. (b) Neuroblastoma cells were pretreated with etoposide (500 ng/ml) followed by exposure to TRAIL (100 ng/ml) treatment for 48 h. (c, d) Cells were cultured in 60 mm plates and exposed to 500 ng/ml etoposide and/or 100 ng/ml of TRAIL. After 12 h incubation, total cell lysates were obtained and analysed for caspase 3 activity and PARP cleavage using a luminex assay. (e, f) Cells were pretreated with the indicated concentrations of the DR5:Fc fusion protein for 1 h to block TRAIL binding to TRAIL-R2. Cell viability was assessed after consecutive treatment with etoposide (Eto; 500 ng/ml) and/or TRAIL (T; 100 ng/ml) for 48 h. The results are presented as cell death as a percentage against vehicle-treated control (means±S.D. from three independent experiments).
Mentions: Treatment with TRAIL induced cell death in SK-N-MC cells in a dose-dependent manner, but had no effect on IMR-32 cells (Figure 2a). When SK-N-MC cells were pre-treated with etoposide for 2 h before TRAIL treatment, a significant increase in cell death occurred at 48 h after etoposide treatment compared with treatment with either etoposide or TRAIL alone. However, treatment with TRAIL, in either the presence or absence of etoposide, had no effect on cell death in IMR-32 cells (Figure 2b). After consecutive treatment with etoposide and TRAIL, the increase in caspase 3 activity and PARP cleavage were observed in SK-N-MC cells, but not in IMR-32 cells (Figures 2c and 2d). Furthermore, treatment with DR5:Fc had no impact on cell viability in IMR-32 cells, but it completely inhibited the increase in cell death caused by consecutive treatment with etoposide and TRAIL in SK-N-MC cells (Figures 2e and 2f).

Bottom Line: Although TRAIL-R2 expression increased in IMR-32 cells in response to etoposide treatment, cell death was not increased by concurrent treatment with TRAIL compared with etoposide alone, because the cells lacked caspase 8 expression.Restoration of caspase 8 expression by exposure to IFNγ (interferon γ) sensitizes IMR-32 cells to TRAIL.Moreover, pretreatment with etoposide increased TRAIL-induced apoptosis in caspase 8 restored IMR-32 cells through activation of a caspase cascade that included caspases 8, 9 and 3.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.

ABSTRACT
TRAIL [TNF (tumour necrosis factor)-related apoptosis-inducing ligand] is a promising agent for clinical use since it kills a wide range of tumour cells without affecting normal cells. We provide evidence that pretreatment with etoposide significantly enhanced TRAIL-mediated apoptosis via up-regulation of DR5 (death receptor 5 or TRAIL-R2) expression in the caspase 8 expressing neuroblastoma cell line, SK-N-MC. In addition, sequential treatment with etoposide and TRAIL increased caspases 8, 9 and 3 activation, Mcl-1 cleavage and Bid truncation, which suggests that the ability of etoposide and TRAIL to induce apoptosis is mediated through activation of an intrinsic signalling pathway. Although TRAIL-R2 expression increased in IMR-32 cells in response to etoposide treatment, cell death was not increased by concurrent treatment with TRAIL compared with etoposide alone, because the cells lacked caspase 8 expression. Restoration of caspase 8 expression by exposure to IFNγ (interferon γ) sensitizes IMR-32 cells to TRAIL. Moreover, pretreatment with etoposide increased TRAIL-induced apoptosis in caspase 8 restored IMR-32 cells through activation of a caspase cascade that included caspases 8, 9 and 3. These results indicate that the etoposide-mediated sensitization of neuroblastoma cells to TRAIL is associated with an increase in TRAIL-R2 expression and requires caspase 8 expression. These observations support the potential use of a combination of etoposide and TRAIL in future clinical trials.

No MeSH data available.


Related in: MedlinePlus