Glucose deprivation-induced endoplasmic reticulum stress response plays a pivotal role in enhancement of TRAIL cytotoxicity - PubMed (original) (raw)

. 2021 Sep;236(9):6666-6677.

doi: 10.1002/jcp.30329. Epub 2021 Feb 15.

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Glucose deprivation-induced endoplasmic reticulum stress response plays a pivotal role in enhancement of TRAIL cytotoxicity

Kalishwaralal Kalimuthu et al. J Cell Physiol. 2021 Sep.

Abstract

Abnormalities of the tumor vasculature result in insufficient blood supply and development of a tumor microenvironment that is characterized by low glucose concentrations, low extracellular pH, and low oxygen tensions. We previously reported that glucose-deprived conditions induce metabolic stress and promote tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cytotoxicity. In this study, we examined whether the metabolic stress-associated endoplasmic reticulum (ER) stress response pathway plays a pivotal role in the enhancement of TRAIL cytotoxicity. We observed no significant cytotoxicity when human colorectal cancer SW48 cells were treated with various doses of TRAIL (2-100 ng/ml) for 4 h or glucose (0-25 mM) for 24 h. However, a combination of TRAIL and low glucose-induced dose-dependent apoptosis through activation of caspases (-8, -9, and -3). Studies with activating transcription factor 4 (ATF4), C/EBP-homologous protein (CHOP), p53 upregulated modulator of apoptosis (PUMA), or death receptor 5 (DR5)-deficient mouse embryonic fibroblasts or HCT116 cells suggest that the ATF4-CHOP-PUMA axis and the ATF4-CHOP-DR5 axis are involved in the combined treatment-induced apoptosis. Moreover, the combined treatment-induced apoptosis was completely suppressed in BH3 interacting-domain death agonist (Bid)- or Bcl-2-associated X protein (Bax)-deficient HCT116 cells, but not Bak-deficient HCT116 cells. Interestingly, the combined treatment-induced Bax oligomerization was suppressed in PUMA-deficient HCT116 cells. These results suggest that glucose deprivation enhances TRAIL-induced apoptosis by integrating the ATF4-CHOP-PUMA axis and the ATF4-CHOP-DR5 axis, consequently amplifying the Bid-Bax-associated mitochondria-dependent pathway.

Keywords: TRAIL cytotoxicity; endoplasmic reticulum stress; glucose deprivation.

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Conflict of interest statement

CONFLICT OF INTERESTS

The authors declare that there are no conflict of interests.

Figures

FIGURE 1

FIGURE 1

Low glucose promotes TRAIL-induced apoptosis through activation of caspases. (a) SW48 cells were treated with various doses of TRAIL (0–100 ng/ml) for 4 h. (b) SW48 cells were treated with various concentrations of glucose (0–25 mM) for 24 h. (c) SW48 cells were pretreated with various concentrations of glucose for 20 h and then treated with 10 ng/ml TRAIL for an additional 4 h. (d) SW48 cells were pretreated with a glucose-free medium for 20 h and then treated with various doses (10 or 50 ng/ml) of TRAIL for an additional 4 h. Cell survival was determined using trypan blue exclusion assay (upper panels). Error bars represent the mean ± SD from triplicate experiments. p values: *, .05; **, .01; ***, .001. Cell lysates were analyzed with an immunoblotting assay using indicated antibodies (lower panels). Immunoblots from two gels were combined in each figure. Actin was used as a protein loading control in each lane. C-Cas3, cleaved caspase-3; C-Cas8, cleaved caspase-8; C-Cas9, cleaved caspase-9; CM, complete medium; GFM, glucose-free medium; PARP-1, poly(ADP-ribose) polymerase-1; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand

FIGURE 2

FIGURE 2

Low glucose induces ER stress in SW48 cells. (a) Cells were treated with various concentrations of glucose (0–25 mM) for 24 h. C, untreated control cells. (b) cells were treated with a glucose-free medium for various times (0–24 h). (c) Cells were pretreated with/without glucose-free medium for 20 h and treated with various doses (10 or 50 ng/ml) of TRAIL for an additional 4 h. Whole-cell lysates were analyzed using an immunoblotting assay with the indicated antibodies. CHOP, CCAAT-enhancer-binding protein homologous protein; CM, complete medium; GFM, glucose-free medium; HSPA5, heat shock protein family A (Hsp70) member 5; PUMA, p53 upregulated modulator of apoptosis; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand

FIGURE 3

FIGURE 3

Glucose-free medium promotes TRAIL-induced apoptosis via the ATF4–CHOP–PUMA pathway. (a) Mouse embryonic fibroblast (MEF) wild-type (WT) or MEF ATF4−/− cells were pretreated with a glucose-free medium for 20 h and then exposed to recombinant mTRAIL (50 ng/ml) for an additional 4 h. (b) MEF WT or MEF CHOP−/− cells were pretreated with a glucose-free medium for 20 h and then exposed to mTRAIL (50 ng/ml) for an additional 4 h. (c) HCT116 WT or HCT116 PUMA−/− cells were pretreated with a glucose-free medium for 20 h and then exposed to TRAIL (1 ng/ml) for an additional 4 h. Cell death was determined using trypan blue exclusion assay and plotted (right panels). Whole-cell lysates were analyzed with an immunoblotting assay using indicated antibodies (left panels). ATF4, activating transcription factor 4; CHOP, CCAAT-enhancer-binding protein homologous protein; CM, complete medium; GFM, glucose-free medium; MEF, mouse embryonic fibroblast; mTRAIL, murine TRAIL; PARP-1, poly(ADP-ribose) polymerase-1; PUMA, p53 upregulated modulator of apoptosis; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; WT, wild type

FIGURE 4

FIGURE 4

The role of the type I pathway in the combined treatment of glucose-free medium and TRAIL-induced apoptosis. (a) HCT116 WT and HCT116 DR5−/− cells were pretreated with a glucose-free medium for 20 h and then exposed to TRAIL (2 ng/ml) for an additional 4 h. (b) SW480 cells were pretreated with a glucose-free medium for 18 h and the caspase inhibitors Z-LEHD-FMK (caspase-9 inhibitor, 20 μM) or Z-IETD-FMK (caspase-8 inhibitor, 20 μM) for 2 h, and then exposed to TRAIL (5 or 10 ng/ml) for an additional 4 h. Whole-cell lysates were analyzed with an immunoblotting assay using indicated antibodies. CM, complete medium; DR4, death receptor 4; DR5, death receptor 5; FLIP, FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein; GFM, glucose-free medium; PARP-1, poly(ADP-ribose) polymerase-1; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; WT, wild type

FIGURE 5

FIGURE 5

The role of Bid in the combined treatment of glucose-free medium and TRAIL-induced apoptosis. HCT116 WT or Bid−/− cells were pretreated with a glucose-free medium for 20 h and then exposed to TRAIL (2 ng/ml) for an additional 4 h. Whole-cell lysates were analyzed with an immunoblotting assay using the indicated antibodies (left panel). Cell survival was analyzed using trypan blue exclusion assay (right panel). CM, complete medium; GFM, glucose-free medium; PARP-1, poly(ADP-ribose) polymerase-1; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; WT, wild type

FIGURE 6

FIGURE 6

The role of BAX and BAK in the combined treatment of glucose-free medium and TRAIL-induced apoptosis. HCT116 WT, BAX−/− single knockout or BAX−/−/BAK−/− double knockout cells (a) or HCT116 WT, BAK−/− or BAX−/−/BAK−/− cells (b) were pretreated with a glucose-free medium for 20 h and then exposed to TRAIL (2 ng/ml) for an additional 4 h. Cell survival was analyzed using trypan blue exclusion assay (right panels). Whole-cell lysates were analyzed with an immunoblotting assay using indicated antibodies (left panels). ATF4, activating transcription factor 4; BAK, Bcl-2 homologous antagonist/killer; Bax, Bcl-2-associated X protein; CHOP, CCAAT-enhancer-binding protein homologous protein; CM, complete medium; GFM, glucose-free medium; PARP-1, poly(ADP-ribose) polymerase-1; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; WT, wild type

FIGURE 7

FIGURE 7

The role of PUMA in BAX oligomerization during the combined treatment of low glucose and TRAIL in HCT116 cells. Cells were pretreated with a glucose-free medium for 20 h and treated with TRAIL (2 ng/ml) for 2 h. After treatment, mitochondrial fractions were isolated and cross-linked and then subjected to immunoblotting with an antibody to Bax. Bax monomers (1 × ) and multimers (2 × –4 × ) are indicated. We used COX IV for a mitochondrial marker. Bax, Bcl-2-associated X protein; COX IV, cytochrome c oxidase; GFM, glucose-free medium; PUMA, p53 upregulated modulator of apoptosis; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; WT, wild type

FIGURE 8

FIGURE 8

Schematic diagram of glucose deprivation-induced ER stress and its role in the promotion of TRAIL-induced apoptosis. ATF4, activating transcription factor 4; BAK, Bcl-2 homologous antagonist/killer; BAX, Bcl-2-associated X protein; BID, BH3 interacting-domain death agonist; Cas-3, caspase-3; CHOP, CCAAT-enhaner-binding protein homologous protein; DISC, death-inducing signaling complex; DR5, death receptor 5; ER, endoplasmic reticulum; FADD, FAS-associated death domain protein; Mcl-1, myeloid cell leukemia-1; PUMA, p53 upregulated modulator of apoptosis; ROS, reactive oxygen species; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand

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