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2-Deoxy-D-glucose enhances TRAIL-induced apoptosis in human melanoma cells through XBP-1-mediated up-regulation of TRAIL-R2.

Liu H, Jiang CC, Lavis CJ, Croft A, Dong L, Tseng HY, Yang F, Tay KH, Hersey P, Zhang XD - Mol. Cancer (2009)

Bottom Line: This was associated with increased activation of the caspase cascade and mitochondrial apoptotic pathway, and was blocked by inhibition of TRAIL-R2, and to a lesser extent, inhibition of TRAIL-R1.Up-regulation of TRAIL-R2 was due to increased transcription that was not dependent on the transcription factors, p53 and CHOP.Instead, the IRE1 alpha and ATF6 pathways of the unfolded protein response that were activated by 2-DG appeared to be involved.

View Article: PubMed Central - HTML - PubMed

Affiliation: Immunology and Oncology Unit, Calvary Mater Newcastle Hospital, NSW, Australia. liuhao6886@yahoo.com.cn

ABSTRACT

Background: Past studies have shown that sensitivity of melanoma cells to TRAIL-induced apoptosis is largely correlated with the expression levels of TRAIL death receptors on the cell surface. However, fresh melanoma isolates and melanoma tissue sections express generally low levels of death receptors for TRAIL. The clinical potential of TRAIL in the treatment of melanoma may therefore be limited unless given with agents that increase the cell surface expression of TRAIL death receptors. 2-Deoxy-D-glucose (2-DG) is a synthetic glucose analogue that inhibits glycolysis and glycosylation and blocks cell growth. It has been in clinical evaluation for its potential use as an anticancer agent. In this study, we have examined whether 2-DG and TRAIL interact to enhance their cytotoxicity towards melanoma cells.

Results: 2-DG did not kill melanoma cells, but enhanced TRAIL-induced apoptosis in cultured melanoma cells and fresh melanoma isolates. This was associated with increased activation of the caspase cascade and mitochondrial apoptotic pathway, and was blocked by inhibition of TRAIL-R2, and to a lesser extent, inhibition of TRAIL-R1. Treatment with 2-DG up-regulated TRAIL death receptors, in particular, TRAIL-R2, on the melanoma cell surface. Up-regulation of TRAIL-R2 was due to increased transcription that was not dependent on the transcription factors, p53 and CHOP. Instead, the IRE1 alpha and ATF6 pathways of the unfolded protein response that were activated by 2-DG appeared to be involved. Moreover, XBP-1, which is known to be transcriptionally regulated by ATF6 and functionally activated by IRE1 alpha, was found to play an important role in 2-DG-mediated transcriptional up-regulation of TRAIL-R2 in melanoma cells.

Conclusion: These results indicate that 2-DG sensitizes human melanoma cells to TRAIL-induced apoptosis by up-regulation of TRAIL-2 via the ATF6/IRE1 alpha/XBP-1 axis of the unfolded protein response. They suggest that 2-DG is a promising agent to increase the therapeutic response to TRAIL in melanoma.

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Sensitization of melanoma cells to TRAIL-induced apoptosis by 2-DG is largely due to up-regulation of TRAIL-R2. A, Mel-RM and MM200 cells were treated with a TRAIL-R2/Fc chimera (10 μg/ml) before the addition of 2-DG (10 μM) and TRAIL (200 ng/ml) for a further 24 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. B, Mel-RM and M200 cells were treated the caspase-8 specific inhibitor z-IETD-fmk (30 μM) for 1 hour before the addition of 2-DG (10 μM) and TRAIL (200 ng/ml) for a further 24 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. C, Mel-RM and MM200 cells were treated with a TRAIL-R1/Fc chimera (10 μg/ml) before the addition of 2-DG (10 μM) and TRAIL (200 ng/ml) for a further 24 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. D, Mel-RM and MM200 cells were transfected with the control or TRAIL-R2 siRNA. Left panel: Twenty-four hours later, whole cell lysates were subjected to Western blot analysis. Right panel: Twenty-four hours later, the cells were treated with 2-DG (10 μM) and TRAIL (200 ng/ml) for a further 24 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. The data shown are either the mean ± SE (A, B, C, & the right panel of D), or representative (the left panel of D), of three individual experiments.
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Figure 4: Sensitization of melanoma cells to TRAIL-induced apoptosis by 2-DG is largely due to up-regulation of TRAIL-R2. A, Mel-RM and MM200 cells were treated with a TRAIL-R2/Fc chimera (10 μg/ml) before the addition of 2-DG (10 μM) and TRAIL (200 ng/ml) for a further 24 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. B, Mel-RM and M200 cells were treated the caspase-8 specific inhibitor z-IETD-fmk (30 μM) for 1 hour before the addition of 2-DG (10 μM) and TRAIL (200 ng/ml) for a further 24 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. C, Mel-RM and MM200 cells were treated with a TRAIL-R1/Fc chimera (10 μg/ml) before the addition of 2-DG (10 μM) and TRAIL (200 ng/ml) for a further 24 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. D, Mel-RM and MM200 cells were transfected with the control or TRAIL-R2 siRNA. Left panel: Twenty-four hours later, whole cell lysates were subjected to Western blot analysis. Right panel: Twenty-four hours later, the cells were treated with 2-DG (10 μM) and TRAIL (200 ng/ml) for a further 24 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. The data shown are either the mean ± SE (A, B, C, & the right panel of D), or representative (the left panel of D), of three individual experiments.

Mentions: The role of up-regulation of TRAIL-R2 in sensitization of melanoma cells to TRAIL-induced apoptosis by 2-DG was studied by inhibition of the interaction between TRAIL and TRAIL-R2 using a TRAIL-R2/Fc chimeric protein. Figure 4A shows that the TRAIL-R2/Fc chimera significantly inhibited TRAIL-induced apoptosis in both Mel-RM and MM200 cells in the absence or presence of 2-DG (p < 0.05). Similarly, 2-DG-mediated sensitization of melanoma cells to TRAIL-induced apoptosis was blocked by either the general caspase inhibitor z-VAD-fmk, or the caspase-8 specific inhibitor z-IETD-fmk (p < 0.05) (Figure 4B & data not shown). In contrast, a TRAIL-R1/Fc chimeric protein displayed only minimal inhibitory effects on sensitization of Mel-RM and MM200 cells to TRAIL-induced apoptosis (Figure 4C).


2-Deoxy-D-glucose enhances TRAIL-induced apoptosis in human melanoma cells through XBP-1-mediated up-regulation of TRAIL-R2.

Liu H, Jiang CC, Lavis CJ, Croft A, Dong L, Tseng HY, Yang F, Tay KH, Hersey P, Zhang XD - Mol. Cancer (2009)

Sensitization of melanoma cells to TRAIL-induced apoptosis by 2-DG is largely due to up-regulation of TRAIL-R2. A, Mel-RM and MM200 cells were treated with a TRAIL-R2/Fc chimera (10 μg/ml) before the addition of 2-DG (10 μM) and TRAIL (200 ng/ml) for a further 24 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. B, Mel-RM and M200 cells were treated the caspase-8 specific inhibitor z-IETD-fmk (30 μM) for 1 hour before the addition of 2-DG (10 μM) and TRAIL (200 ng/ml) for a further 24 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. C, Mel-RM and MM200 cells were treated with a TRAIL-R1/Fc chimera (10 μg/ml) before the addition of 2-DG (10 μM) and TRAIL (200 ng/ml) for a further 24 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. D, Mel-RM and MM200 cells were transfected with the control or TRAIL-R2 siRNA. Left panel: Twenty-four hours later, whole cell lysates were subjected to Western blot analysis. Right panel: Twenty-four hours later, the cells were treated with 2-DG (10 μM) and TRAIL (200 ng/ml) for a further 24 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. The data shown are either the mean ± SE (A, B, C, & the right panel of D), or representative (the left panel of D), of three individual experiments.
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Figure 4: Sensitization of melanoma cells to TRAIL-induced apoptosis by 2-DG is largely due to up-regulation of TRAIL-R2. A, Mel-RM and MM200 cells were treated with a TRAIL-R2/Fc chimera (10 μg/ml) before the addition of 2-DG (10 μM) and TRAIL (200 ng/ml) for a further 24 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. B, Mel-RM and M200 cells were treated the caspase-8 specific inhibitor z-IETD-fmk (30 μM) for 1 hour before the addition of 2-DG (10 μM) and TRAIL (200 ng/ml) for a further 24 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. C, Mel-RM and MM200 cells were treated with a TRAIL-R1/Fc chimera (10 μg/ml) before the addition of 2-DG (10 μM) and TRAIL (200 ng/ml) for a further 24 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. D, Mel-RM and MM200 cells were transfected with the control or TRAIL-R2 siRNA. Left panel: Twenty-four hours later, whole cell lysates were subjected to Western blot analysis. Right panel: Twenty-four hours later, the cells were treated with 2-DG (10 μM) and TRAIL (200 ng/ml) for a further 24 hours. Apoptosis was measured by the propidium iodide method using flow cytometry. The data shown are either the mean ± SE (A, B, C, & the right panel of D), or representative (the left panel of D), of three individual experiments.
Mentions: The role of up-regulation of TRAIL-R2 in sensitization of melanoma cells to TRAIL-induced apoptosis by 2-DG was studied by inhibition of the interaction between TRAIL and TRAIL-R2 using a TRAIL-R2/Fc chimeric protein. Figure 4A shows that the TRAIL-R2/Fc chimera significantly inhibited TRAIL-induced apoptosis in both Mel-RM and MM200 cells in the absence or presence of 2-DG (p < 0.05). Similarly, 2-DG-mediated sensitization of melanoma cells to TRAIL-induced apoptosis was blocked by either the general caspase inhibitor z-VAD-fmk, or the caspase-8 specific inhibitor z-IETD-fmk (p < 0.05) (Figure 4B & data not shown). In contrast, a TRAIL-R1/Fc chimeric protein displayed only minimal inhibitory effects on sensitization of Mel-RM and MM200 cells to TRAIL-induced apoptosis (Figure 4C).

Bottom Line: This was associated with increased activation of the caspase cascade and mitochondrial apoptotic pathway, and was blocked by inhibition of TRAIL-R2, and to a lesser extent, inhibition of TRAIL-R1.Up-regulation of TRAIL-R2 was due to increased transcription that was not dependent on the transcription factors, p53 and CHOP.Instead, the IRE1 alpha and ATF6 pathways of the unfolded protein response that were activated by 2-DG appeared to be involved.

View Article: PubMed Central - HTML - PubMed

Affiliation: Immunology and Oncology Unit, Calvary Mater Newcastle Hospital, NSW, Australia. liuhao6886@yahoo.com.cn

ABSTRACT

Background: Past studies have shown that sensitivity of melanoma cells to TRAIL-induced apoptosis is largely correlated with the expression levels of TRAIL death receptors on the cell surface. However, fresh melanoma isolates and melanoma tissue sections express generally low levels of death receptors for TRAIL. The clinical potential of TRAIL in the treatment of melanoma may therefore be limited unless given with agents that increase the cell surface expression of TRAIL death receptors. 2-Deoxy-D-glucose (2-DG) is a synthetic glucose analogue that inhibits glycolysis and glycosylation and blocks cell growth. It has been in clinical evaluation for its potential use as an anticancer agent. In this study, we have examined whether 2-DG and TRAIL interact to enhance their cytotoxicity towards melanoma cells.

Results: 2-DG did not kill melanoma cells, but enhanced TRAIL-induced apoptosis in cultured melanoma cells and fresh melanoma isolates. This was associated with increased activation of the caspase cascade and mitochondrial apoptotic pathway, and was blocked by inhibition of TRAIL-R2, and to a lesser extent, inhibition of TRAIL-R1. Treatment with 2-DG up-regulated TRAIL death receptors, in particular, TRAIL-R2, on the melanoma cell surface. Up-regulation of TRAIL-R2 was due to increased transcription that was not dependent on the transcription factors, p53 and CHOP. Instead, the IRE1 alpha and ATF6 pathways of the unfolded protein response that were activated by 2-DG appeared to be involved. Moreover, XBP-1, which is known to be transcriptionally regulated by ATF6 and functionally activated by IRE1 alpha, was found to play an important role in 2-DG-mediated transcriptional up-regulation of TRAIL-R2 in melanoma cells.

Conclusion: These results indicate that 2-DG sensitizes human melanoma cells to TRAIL-induced apoptosis by up-regulation of TRAIL-2 via the ATF6/IRE1 alpha/XBP-1 axis of the unfolded protein response. They suggest that 2-DG is a promising agent to increase the therapeutic response to TRAIL in melanoma.

Show MeSH
Related in: MedlinePlus