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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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A, 2-DG enhances activation of the mitochondrial apoptotic pathway by TRAIL. Upper panel: Mel-RM and MM200 cells treated with the combination of 2-DG (10 μM) and TRAIL (200 ng/ml) for 16 hours were subjected to measurement of ΔΨm by JC-1 staining in flow cytometry. The number in each left bottom quadrant represents the percentage of cells with reduction in ΔΨm. Lower panel: Cytosolic and mitochondrial fractions of Mel-RM and MM200 cells treated with the combination of 2-DG (10 μM) and TRAIL (200 ng/ml) for 16 hours were subjected to Western blot analysis. Western blot analysis of COX IV or β-actin levels was included to show relative purity of the mitochondrial or cytosolic fractions. B, A summary of studies of the effect of 2-DG on TRAIL-induced apoptosis in a panel of melanoma cell lines and a melanocyte line. Cells were treated with 2-DG (10 μM) and TRAIL (200 ng/ml) for 24 hours before apoptosis was measured by the propidium iodide method using flow cytometry. The data shown are either the mean ± SE (A, B, & F), or representative (C, D, & E), of three individual experiments.
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Figure 2: A, 2-DG enhances activation of the mitochondrial apoptotic pathway by TRAIL. Upper panel: Mel-RM and MM200 cells treated with the combination of 2-DG (10 μM) and TRAIL (200 ng/ml) for 16 hours were subjected to measurement of ΔΨm by JC-1 staining in flow cytometry. The number in each left bottom quadrant represents the percentage of cells with reduction in ΔΨm. Lower panel: Cytosolic and mitochondrial fractions of Mel-RM and MM200 cells treated with the combination of 2-DG (10 μM) and TRAIL (200 ng/ml) for 16 hours were subjected to Western blot analysis. Western blot analysis of COX IV or β-actin levels was included to show relative purity of the mitochondrial or cytosolic fractions. B, A summary of studies of the effect of 2-DG on TRAIL-induced apoptosis in a panel of melanoma cell lines and a melanocyte line. Cells were treated with 2-DG (10 μM) and TRAIL (200 ng/ml) for 24 hours before apoptosis was measured by the propidium iodide method using flow cytometry. The data shown are either the mean ± SE (A, B, & F), or representative (C, D, & E), of three individual experiments.

Mentions: Our initial studies on two melanoma cell lines, Mel-RM and MM200, indicated that 2-DG alone did not induce notable apoptosis, although it inhibited cell proliferation (Figures 1A &1B). Nevertheless, studies on its effect on TRAIL-induced apoptosis showed that the combination of 2-DG and TRAIL enhanced sensitivity of the cells to apoptosis-induced by TRAIL (Figures 1B &1C). The increase in TRAIL-induced apoptosis in the presence of 2-DG was observed as early as 16 hours and reached a peak at 36 hours after treatment (Figure 1B). In association with this, co-treatment with 2-DG enhanced TRAIL-induced activation of caspase-8, reduction in ΔΨm, mitochondrial release of cytochrome c, activation of caspase-3 and cleavage of its substrate PARP (Figures 1D & Figure 2A). It is of note that the cleaved products of caspase-8 were hardly detected in MM200 presumably due to relatively low concentrations within the cells (Figure 1D). Increased activation of caspase-3 was shown by both decreased cleavage of the pro-enzyme of caspase-3, and reduced conversion of the larger cleaved fragment to smaller ones (Figure 1D).


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)

A, 2-DG enhances activation of the mitochondrial apoptotic pathway by TRAIL. Upper panel: Mel-RM and MM200 cells treated with the combination of 2-DG (10 μM) and TRAIL (200 ng/ml) for 16 hours were subjected to measurement of ΔΨm by JC-1 staining in flow cytometry. The number in each left bottom quadrant represents the percentage of cells with reduction in ΔΨm. Lower panel: Cytosolic and mitochondrial fractions of Mel-RM and MM200 cells treated with the combination of 2-DG (10 μM) and TRAIL (200 ng/ml) for 16 hours were subjected to Western blot analysis. Western blot analysis of COX IV or β-actin levels was included to show relative purity of the mitochondrial or cytosolic fractions. B, A summary of studies of the effect of 2-DG on TRAIL-induced apoptosis in a panel of melanoma cell lines and a melanocyte line. Cells were treated with 2-DG (10 μM) and TRAIL (200 ng/ml) for 24 hours before apoptosis was measured by the propidium iodide method using flow cytometry. The data shown are either the mean ± SE (A, B, & F), or representative (C, D, & E), of three individual experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2803449&req=5

Figure 2: A, 2-DG enhances activation of the mitochondrial apoptotic pathway by TRAIL. Upper panel: Mel-RM and MM200 cells treated with the combination of 2-DG (10 μM) and TRAIL (200 ng/ml) for 16 hours were subjected to measurement of ΔΨm by JC-1 staining in flow cytometry. The number in each left bottom quadrant represents the percentage of cells with reduction in ΔΨm. Lower panel: Cytosolic and mitochondrial fractions of Mel-RM and MM200 cells treated with the combination of 2-DG (10 μM) and TRAIL (200 ng/ml) for 16 hours were subjected to Western blot analysis. Western blot analysis of COX IV or β-actin levels was included to show relative purity of the mitochondrial or cytosolic fractions. B, A summary of studies of the effect of 2-DG on TRAIL-induced apoptosis in a panel of melanoma cell lines and a melanocyte line. Cells were treated with 2-DG (10 μM) and TRAIL (200 ng/ml) for 24 hours before apoptosis was measured by the propidium iodide method using flow cytometry. The data shown are either the mean ± SE (A, B, & F), or representative (C, D, & E), of three individual experiments.
Mentions: Our initial studies on two melanoma cell lines, Mel-RM and MM200, indicated that 2-DG alone did not induce notable apoptosis, although it inhibited cell proliferation (Figures 1A &1B). Nevertheless, studies on its effect on TRAIL-induced apoptosis showed that the combination of 2-DG and TRAIL enhanced sensitivity of the cells to apoptosis-induced by TRAIL (Figures 1B &1C). The increase in TRAIL-induced apoptosis in the presence of 2-DG was observed as early as 16 hours and reached a peak at 36 hours after treatment (Figure 1B). In association with this, co-treatment with 2-DG enhanced TRAIL-induced activation of caspase-8, reduction in ΔΨm, mitochondrial release of cytochrome c, activation of caspase-3 and cleavage of its substrate PARP (Figures 1D & Figure 2A). It is of note that the cleaved products of caspase-8 were hardly detected in MM200 presumably due to relatively low concentrations within the cells (Figure 1D). Increased activation of caspase-3 was shown by both decreased cleavage of the pro-enzyme of caspase-3, and reduced conversion of the larger cleaved fragment to smaller ones (Figure 1D).

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