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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

Re-expression of caspase 8 sensitizes IMR-32 cells to TRAIL cytotoxicity(a) Expression of caspase 8 in IMR-32 cells after treatment with the indicated concentrations of IFNγ for 48 h. Caspase 8 expression in SK-N-MC cells was used as a positive control (PC). Equal loading was assessed by staining with anti-β-actin. (b–d) Cells were cultured in 100 mm plates and exposed to 1000 units/ml IFNγ. After a 24 h incubation, cells were replated in 96 well plates, cultured for 24 h, and treated with etoposide (Eto; 500 ng/ml) and/or TRAIL (T; 100, 250, 500, 750, or 1000 ng/ml) in the absence or presence of 10 μM zIETD-fmk or 50 ng/ml DR5:Fc. The results are presented as cell death percentage against vehicle-treated control (means±S.D. from three independent experiments). Statistically significant differences are specified by asterisks (*P<0.05). (e) IMR-32 cells were exposed to 1000 units/ml IFNγ for 48 h, followed by consecutive treatment with etoposide (500 ng/ml) and/or TRAIL (750 ng/ml) for 12 h. Total cell lysates were used to detect the expression and cleavage of caspases 8, 9, 3, Mcl-1 and Bid. Equal loading was assessed by staining with anti-β-actin.
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Figure 4: Re-expression of caspase 8 sensitizes IMR-32 cells to TRAIL cytotoxicity(a) Expression of caspase 8 in IMR-32 cells after treatment with the indicated concentrations of IFNγ for 48 h. Caspase 8 expression in SK-N-MC cells was used as a positive control (PC). Equal loading was assessed by staining with anti-β-actin. (b–d) Cells were cultured in 100 mm plates and exposed to 1000 units/ml IFNγ. After a 24 h incubation, cells were replated in 96 well plates, cultured for 24 h, and treated with etoposide (Eto; 500 ng/ml) and/or TRAIL (T; 100, 250, 500, 750, or 1000 ng/ml) in the absence or presence of 10 μM zIETD-fmk or 50 ng/ml DR5:Fc. The results are presented as cell death percentage against vehicle-treated control (means±S.D. from three independent experiments). Statistically significant differences are specified by asterisks (*P<0.05). (e) IMR-32 cells were exposed to 1000 units/ml IFNγ for 48 h, followed by consecutive treatment with etoposide (500 ng/ml) and/or TRAIL (750 ng/ml) for 12 h. Total cell lysates were used to detect the expression and cleavage of caspases 8, 9, 3, Mcl-1 and Bid. Equal loading was assessed by staining with anti-β-actin.

Mentions: By treating IMR-32 cells with IFNγ for 48 h, the expression of caspase 8 gradually increased (Figure 4a). Restoration of caspase 8 sensitized IMR-32 cells to TRAIL cytotoxicity and etoposide potentiated this TRAIL cytotoxicity. Additional treatment with a caspase 8 inhibitor or the DR5:Fc fusion protein significantly suppressed TRAIL-induced cell death (Figures 4b–4d). Increased caspase activity, Mcl-1 cleavage and Bid truncation were observed in response to consecutive treatment with etoposide and TRAIL in caspase 8 restored IMR-32 cells (Figure 4e).


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)

Re-expression of caspase 8 sensitizes IMR-32 cells to TRAIL cytotoxicity(a) Expression of caspase 8 in IMR-32 cells after treatment with the indicated concentrations of IFNγ for 48 h. Caspase 8 expression in SK-N-MC cells was used as a positive control (PC). Equal loading was assessed by staining with anti-β-actin. (b–d) Cells were cultured in 100 mm plates and exposed to 1000 units/ml IFNγ. After a 24 h incubation, cells were replated in 96 well plates, cultured for 24 h, and treated with etoposide (Eto; 500 ng/ml) and/or TRAIL (T; 100, 250, 500, 750, or 1000 ng/ml) in the absence or presence of 10 μM zIETD-fmk or 50 ng/ml DR5:Fc. The results are presented as cell death percentage against vehicle-treated control (means±S.D. from three independent experiments). Statistically significant differences are specified by asterisks (*P<0.05). (e) IMR-32 cells were exposed to 1000 units/ml IFNγ for 48 h, followed by consecutive treatment with etoposide (500 ng/ml) and/or TRAIL (750 ng/ml) for 12 h. Total cell lysates were used to detect the expression and cleavage of caspases 8, 9, 3, Mcl-1 and Bid. Equal loading was assessed by staining with anti-β-actin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475444&req=5

Figure 4: Re-expression of caspase 8 sensitizes IMR-32 cells to TRAIL cytotoxicity(a) Expression of caspase 8 in IMR-32 cells after treatment with the indicated concentrations of IFNγ for 48 h. Caspase 8 expression in SK-N-MC cells was used as a positive control (PC). Equal loading was assessed by staining with anti-β-actin. (b–d) Cells were cultured in 100 mm plates and exposed to 1000 units/ml IFNγ. After a 24 h incubation, cells were replated in 96 well plates, cultured for 24 h, and treated with etoposide (Eto; 500 ng/ml) and/or TRAIL (T; 100, 250, 500, 750, or 1000 ng/ml) in the absence or presence of 10 μM zIETD-fmk or 50 ng/ml DR5:Fc. The results are presented as cell death percentage against vehicle-treated control (means±S.D. from three independent experiments). Statistically significant differences are specified by asterisks (*P<0.05). (e) IMR-32 cells were exposed to 1000 units/ml IFNγ for 48 h, followed by consecutive treatment with etoposide (500 ng/ml) and/or TRAIL (750 ng/ml) for 12 h. Total cell lysates were used to detect the expression and cleavage of caspases 8, 9, 3, Mcl-1 and Bid. Equal loading was assessed by staining with anti-β-actin.
Mentions: By treating IMR-32 cells with IFNγ for 48 h, the expression of caspase 8 gradually increased (Figure 4a). Restoration of caspase 8 sensitized IMR-32 cells to TRAIL cytotoxicity and etoposide potentiated this TRAIL cytotoxicity. Additional treatment with a caspase 8 inhibitor or the DR5:Fc fusion protein significantly suppressed TRAIL-induced cell death (Figures 4b–4d). Increased caspase activity, Mcl-1 cleavage and Bid truncation were observed in response to consecutive treatment with etoposide and TRAIL in caspase 8 restored IMR-32 cells (Figure 4e).

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