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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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2-DG up-regulates TRAIL-R2 and enhances TRAIL-induced apoptosis in fresh melanoma isolates. A, Mel-CA and Mel-MC with (thick open histograms) or without (thin open histograms) treatment with 2-DG (10 μM) for 16 hours were subjected to measurement of the cell surface TRAIL-R2 expression in flow cytomety. The filled histograms are isotype controls. B, Whole cell lysates from Mel-CA and Mel-MC with or without treatment with 2-DG (10 μM) for 16 hours were subjected to Western blot analysis. C, Freshly isolated melanoma cells were treated with 2-DG (10 μM), TRAIL (200 ng/ml), or the combination of both for 24 hours were subjected to measurement of apoptosis by the propidium iodide method using flow cytometry. D, Freshly isolated melanoma cells were treated with a TRAIL-R2/Fc chimera (10 μg/ml) 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. The data shown are either the mean ± SE (C & D), or representative (A & B), of three individual experiments.
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Figure 7: 2-DG up-regulates TRAIL-R2 and enhances TRAIL-induced apoptosis in fresh melanoma isolates. A, Mel-CA and Mel-MC with (thick open histograms) or without (thin open histograms) treatment with 2-DG (10 μM) for 16 hours were subjected to measurement of the cell surface TRAIL-R2 expression in flow cytomety. The filled histograms are isotype controls. B, Whole cell lysates from Mel-CA and Mel-MC with or without treatment with 2-DG (10 μM) for 16 hours were subjected to Western blot analysis. C, Freshly isolated melanoma cells were treated with 2-DG (10 μM), TRAIL (200 ng/ml), or the combination of both for 24 hours were subjected to measurement of apoptosis by the propidium iodide method using flow cytometry. D, Freshly isolated melanoma cells were treated with a TRAIL-R2/Fc chimera (10 μg/ml) 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. The data shown are either the mean ± SE (C & D), or representative (A & B), of three individual experiments.

Mentions: Our previous studies have shown that fresh melanoma isolates, which may reflect more closely the in vivo situation, are relatively resistance to TRAIL-induced apoptosis due to low levels of expression of TRAIL death receptors [11]. We studied if 2-DG can also up-regulate TRAIL-R2 in fresh melanoma isolates. Freshly isolated melanoma cells, Mel-CA and Mel-MC were treated with 2-DG for 24 hours. As shown in Figures 7A and 7B, treatment with 2-DG increased the levels of TRAIL-R2 on the cell surface as measured in flow cytometry, and the TRAIL-R2 total protein levels as detected in Western blot analysis, in both Mel-CA and Mel-MC cells. Figure 7C shows that neither 2-DG nor TRAIL induced significant levels of apoptosis (< 20% apoptotic cells) in a panel of fresh melanoma isolates. However, co-treatment with 2-DG and TRAIL resulted in increases in the percentages of apoptotic cells (p < 0.05). Sensitization of fresh melanoma isolates to TRAIL-induced apoptosis by 2-DG was substantially inhibited by a recombinant TRAIL-R2/Fc chimera (p < 0.05) (Figure 7D), indicating that the effect of 2-DG on TRAIL-induced apoptosis in fresh melanoma isolates is largely accounted for by the increase in TRAIL-R2 expression on the cell surface.


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)

2-DG up-regulates TRAIL-R2 and enhances TRAIL-induced apoptosis in fresh melanoma isolates. A, Mel-CA and Mel-MC with (thick open histograms) or without (thin open histograms) treatment with 2-DG (10 μM) for 16 hours were subjected to measurement of the cell surface TRAIL-R2 expression in flow cytomety. The filled histograms are isotype controls. B, Whole cell lysates from Mel-CA and Mel-MC with or without treatment with 2-DG (10 μM) for 16 hours were subjected to Western blot analysis. C, Freshly isolated melanoma cells were treated with 2-DG (10 μM), TRAIL (200 ng/ml), or the combination of both for 24 hours were subjected to measurement of apoptosis by the propidium iodide method using flow cytometry. D, Freshly isolated melanoma cells were treated with a TRAIL-R2/Fc chimera (10 μg/ml) 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. The data shown are either the mean ± SE (C & D), or representative (A & B), of three individual experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2803449&req=5

Figure 7: 2-DG up-regulates TRAIL-R2 and enhances TRAIL-induced apoptosis in fresh melanoma isolates. A, Mel-CA and Mel-MC with (thick open histograms) or without (thin open histograms) treatment with 2-DG (10 μM) for 16 hours were subjected to measurement of the cell surface TRAIL-R2 expression in flow cytomety. The filled histograms are isotype controls. B, Whole cell lysates from Mel-CA and Mel-MC with or without treatment with 2-DG (10 μM) for 16 hours were subjected to Western blot analysis. C, Freshly isolated melanoma cells were treated with 2-DG (10 μM), TRAIL (200 ng/ml), or the combination of both for 24 hours were subjected to measurement of apoptosis by the propidium iodide method using flow cytometry. D, Freshly isolated melanoma cells were treated with a TRAIL-R2/Fc chimera (10 μg/ml) 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. The data shown are either the mean ± SE (C & D), or representative (A & B), of three individual experiments.
Mentions: Our previous studies have shown that fresh melanoma isolates, which may reflect more closely the in vivo situation, are relatively resistance to TRAIL-induced apoptosis due to low levels of expression of TRAIL death receptors [11]. We studied if 2-DG can also up-regulate TRAIL-R2 in fresh melanoma isolates. Freshly isolated melanoma cells, Mel-CA and Mel-MC were treated with 2-DG for 24 hours. As shown in Figures 7A and 7B, treatment with 2-DG increased the levels of TRAIL-R2 on the cell surface as measured in flow cytometry, and the TRAIL-R2 total protein levels as detected in Western blot analysis, in both Mel-CA and Mel-MC cells. Figure 7C shows that neither 2-DG nor TRAIL induced significant levels of apoptosis (< 20% apoptotic cells) in a panel of fresh melanoma isolates. However, co-treatment with 2-DG and TRAIL resulted in increases in the percentages of apoptotic cells (p < 0.05). Sensitization of fresh melanoma isolates to TRAIL-induced apoptosis by 2-DG was substantially inhibited by a recombinant TRAIL-R2/Fc chimera (p < 0.05) (Figure 7D), indicating that the effect of 2-DG on TRAIL-induced apoptosis in fresh melanoma isolates is largely accounted for by the increase in TRAIL-R2 expression on the cell surface.

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