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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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XBP-1 plays an important role in up-regulation of TRAIL-R2 by 2-DG. A, 2-DG activates the UPR in melanoma cells. Mel-RM and MM200 cells were treated with 2-DG (10 μM) for indicated periods. Upper panel: Whole cell lysates were subjected to Western blot analysis. Lower panel: RT-PCR products of XBP-1 mRNA from the cells were digested with Apa-LI for 90 minutes followed by electrophoresis. The longer fragment derived from the active form of XBP1 mRNA and two shorter bands derived from the inactive form are indicated. B, Mel-RM and MM200 cells were transfected with the control, IRE1α, ATF6, or PERK siRNA. Twenty-four hours later, whole cell lysates were subjected to Western blot analysis. C, Mel-RM (left panel) and MM200 (right panel) cells were transfected with the control, IRE1α, ATF6, or PERK siRNA. Twenty-four hours later, cells were treated with 2-DG (10 μM) for 24 hours. The cell surface expression of TRAIL-R2 was measured using flow cytometry. The data in the y axes represent mean fluorescence intensity (MFI). D, Whole cell lysates from Mel-RM and MM200 cells transduced with the control or XBP-1 shRNA were subjected to Western blot analysis. E, Left panel: Mel-RM and MM200 cells transduced with the control or XBP-1 shRNA were treated 2-DG (10 μM) for 24 hours. The cell surface expression of TRAIL-R2 was measured using flow cytometry. Filled histograms: isotype controls; Thick open histograms: TRAIL-R2 expression before treatment; Thin open histograms: TRAIL-R2 expression after treatment with 2-DG (10 μM) for 24 hours. Right panel: Total RNA from Mel-RM and MM200 cells transduced with the control or XBP-1 shRNA treated with 2-DG (10 μM) for 16 hours was isolated and subjected to Real-time PCR analysis for TRAIL-R2. The relative abundance of mRNA expression before treatment was arbitrarily designated as 1. Deficiency in XBP-1 significantly blocked up-regulation of TRAIL-R2 mRNA by 2-DG (p < 0.05). The data shown are either the mean ± SE (C & the right panel of E), or representative (A, B, D, & the left panel of E), of three individual experiments.
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Figure 6: XBP-1 plays an important role in up-regulation of TRAIL-R2 by 2-DG. A, 2-DG activates the UPR in melanoma cells. Mel-RM and MM200 cells were treated with 2-DG (10 μM) for indicated periods. Upper panel: Whole cell lysates were subjected to Western blot analysis. Lower panel: RT-PCR products of XBP-1 mRNA from the cells were digested with Apa-LI for 90 minutes followed by electrophoresis. The longer fragment derived from the active form of XBP1 mRNA and two shorter bands derived from the inactive form are indicated. B, Mel-RM and MM200 cells were transfected with the control, IRE1α, ATF6, or PERK siRNA. Twenty-four hours later, whole cell lysates were subjected to Western blot analysis. C, Mel-RM (left panel) and MM200 (right panel) cells were transfected with the control, IRE1α, ATF6, or PERK siRNA. Twenty-four hours later, cells were treated with 2-DG (10 μM) for 24 hours. The cell surface expression of TRAIL-R2 was measured using flow cytometry. The data in the y axes represent mean fluorescence intensity (MFI). D, Whole cell lysates from Mel-RM and MM200 cells transduced with the control or XBP-1 shRNA were subjected to Western blot analysis. E, Left panel: Mel-RM and MM200 cells transduced with the control or XBP-1 shRNA were treated 2-DG (10 μM) for 24 hours. The cell surface expression of TRAIL-R2 was measured using flow cytometry. Filled histograms: isotype controls; Thick open histograms: TRAIL-R2 expression before treatment; Thin open histograms: TRAIL-R2 expression after treatment with 2-DG (10 μM) for 24 hours. Right panel: Total RNA from Mel-RM and MM200 cells transduced with the control or XBP-1 shRNA treated with 2-DG (10 μM) for 16 hours was isolated and subjected to Real-time PCR analysis for TRAIL-R2. The relative abundance of mRNA expression before treatment was arbitrarily designated as 1. Deficiency in XBP-1 significantly blocked up-regulation of TRAIL-R2 mRNA by 2-DG (p < 0.05). The data shown are either the mean ± SE (C & the right panel of E), or representative (A, B, D, & the left panel of E), of three individual experiments.

Mentions: We have previously shown that the IRE1α and ATF6 pathways of the UPR are involved in transcriptional up-regulation of TRAIL-R2 by the classic ER stress inducers TM and TG [29,30]. We tested if 2-DG impinges on ER stress and activates the UPR in melanoma cells. As shown in Figure 6A, 2-DG up-regulated glucose-regulated protein 78 (GRP78) and the active form of x-box-binding protein-1 (XBP-1) mRNA, two commonly used markers of activation of the UPR [35,36].


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)

XBP-1 plays an important role in up-regulation of TRAIL-R2 by 2-DG. A, 2-DG activates the UPR in melanoma cells. Mel-RM and MM200 cells were treated with 2-DG (10 μM) for indicated periods. Upper panel: Whole cell lysates were subjected to Western blot analysis. Lower panel: RT-PCR products of XBP-1 mRNA from the cells were digested with Apa-LI for 90 minutes followed by electrophoresis. The longer fragment derived from the active form of XBP1 mRNA and two shorter bands derived from the inactive form are indicated. B, Mel-RM and MM200 cells were transfected with the control, IRE1α, ATF6, or PERK siRNA. Twenty-four hours later, whole cell lysates were subjected to Western blot analysis. C, Mel-RM (left panel) and MM200 (right panel) cells were transfected with the control, IRE1α, ATF6, or PERK siRNA. Twenty-four hours later, cells were treated with 2-DG (10 μM) for 24 hours. The cell surface expression of TRAIL-R2 was measured using flow cytometry. The data in the y axes represent mean fluorescence intensity (MFI). D, Whole cell lysates from Mel-RM and MM200 cells transduced with the control or XBP-1 shRNA were subjected to Western blot analysis. E, Left panel: Mel-RM and MM200 cells transduced with the control or XBP-1 shRNA were treated 2-DG (10 μM) for 24 hours. The cell surface expression of TRAIL-R2 was measured using flow cytometry. Filled histograms: isotype controls; Thick open histograms: TRAIL-R2 expression before treatment; Thin open histograms: TRAIL-R2 expression after treatment with 2-DG (10 μM) for 24 hours. Right panel: Total RNA from Mel-RM and MM200 cells transduced with the control or XBP-1 shRNA treated with 2-DG (10 μM) for 16 hours was isolated and subjected to Real-time PCR analysis for TRAIL-R2. The relative abundance of mRNA expression before treatment was arbitrarily designated as 1. Deficiency in XBP-1 significantly blocked up-regulation of TRAIL-R2 mRNA by 2-DG (p < 0.05). The data shown are either the mean ± SE (C & the right panel of E), or representative (A, B, D, & the left panel of E), of three individual experiments.
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Related In: Results  -  Collection

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Figure 6: XBP-1 plays an important role in up-regulation of TRAIL-R2 by 2-DG. A, 2-DG activates the UPR in melanoma cells. Mel-RM and MM200 cells were treated with 2-DG (10 μM) for indicated periods. Upper panel: Whole cell lysates were subjected to Western blot analysis. Lower panel: RT-PCR products of XBP-1 mRNA from the cells were digested with Apa-LI for 90 minutes followed by electrophoresis. The longer fragment derived from the active form of XBP1 mRNA and two shorter bands derived from the inactive form are indicated. B, Mel-RM and MM200 cells were transfected with the control, IRE1α, ATF6, or PERK siRNA. Twenty-four hours later, whole cell lysates were subjected to Western blot analysis. C, Mel-RM (left panel) and MM200 (right panel) cells were transfected with the control, IRE1α, ATF6, or PERK siRNA. Twenty-four hours later, cells were treated with 2-DG (10 μM) for 24 hours. The cell surface expression of TRAIL-R2 was measured using flow cytometry. The data in the y axes represent mean fluorescence intensity (MFI). D, Whole cell lysates from Mel-RM and MM200 cells transduced with the control or XBP-1 shRNA were subjected to Western blot analysis. E, Left panel: Mel-RM and MM200 cells transduced with the control or XBP-1 shRNA were treated 2-DG (10 μM) for 24 hours. The cell surface expression of TRAIL-R2 was measured using flow cytometry. Filled histograms: isotype controls; Thick open histograms: TRAIL-R2 expression before treatment; Thin open histograms: TRAIL-R2 expression after treatment with 2-DG (10 μM) for 24 hours. Right panel: Total RNA from Mel-RM and MM200 cells transduced with the control or XBP-1 shRNA treated with 2-DG (10 μM) for 16 hours was isolated and subjected to Real-time PCR analysis for TRAIL-R2. The relative abundance of mRNA expression before treatment was arbitrarily designated as 1. Deficiency in XBP-1 significantly blocked up-regulation of TRAIL-R2 mRNA by 2-DG (p < 0.05). The data shown are either the mean ± SE (C & the right panel of E), or representative (A, B, D, & the left panel of E), of three individual experiments.
Mentions: We have previously shown that the IRE1α and ATF6 pathways of the UPR are involved in transcriptional up-regulation of TRAIL-R2 by the classic ER stress inducers TM and TG [29,30]. We tested if 2-DG impinges on ER stress and activates the UPR in melanoma cells. As shown in Figure 6A, 2-DG up-regulated glucose-regulated protein 78 (GRP78) and the active form of x-box-binding protein-1 (XBP-1) mRNA, two commonly used markers of activation of the UPR [35,36].

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