DR5-mediated DISC controls caspase-8 cleavage and initiation of apoptosis in human glioblastomas

Abstract

To explore the molecular mechanisms by which glioblastomas are resistant to tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), we examined TRAIL signalling pathways in the tumours. TRAIL has four membrane-anchored receptors, death receptor 4/5 (DR4/5) and decoy receptor 1/2 (DcR1/2). Of these receptors, only DR5 was expressed consistently in glioblastoma cell lines and tumour tissues, ruling out the role of DcR1/2 in TRAIL resistance. Upon TRAIL binding, DR5 was homotrimerized and recruited Fas-associated death domain (FADD) and caspase-8 for the assembly of death-inducing signalling complex (DISC) in the lipid rafts of the plasma membrane. In the DISC, caspase-8 was cleaved and initiated apoptosis by cleaving downstream caspases in TRAIL-sensitive glioblastoma cells. In TRAIL-resistant cells, however, DR5-mediated DISC was modified by receptor-interacting protein (RIP), cellular FADD-like interleukin-1beta-converting enzyme inhibitory protein (c-FLIP) and phosphoprotein enriched in diabetes or in astrocyte-15 (PED/PEA-15). This DISC modification occurred in the non-raft fractions of the plasma membrane and resulted in the inhibition of caspase-8 cleavage and activation of nuclear factor-kappaB (NF-kappaB). Treatment of resistant cells with parthenolide, an inhibitor of inhibitor of kappaB (I-kappaB), eliminated TRAIL-induced NF-kappaB activity but not TRAIL resistance. In contrast, however, targeting of RIP, c-FLIP or PED/PEA-15 with small interfering RNA (siRNA) led to the redistribution of the DISC from non-rafts to lipid rafts and eliminated the inhibition of caspase-8 cleavage and thereby TRAIL resistance. Taken together, this study indicates that the DISC modification by RIP, c-FLIP and PED/PEA-15 is the most upstream event in TRAIL resistance in glioblastomas.

Fig 1

Expression of TRAIL signalling proteins in correlation with TRAIL sensitivity. (A) Cell death analysis of nine glioblastoma cell lines treated with serial dilutions of TRAIL (starting from 300 ng/ml) by crystal violet cell viability assay (mean ± S.E.M., n= 8). (B) Annexin V/7ADD analysis of TRAIL-induced apoptosis in sensitive LN18 and resistant LN443 and U118MG cell lines treated with 10 ng/ml TRAIL for the times indicated above the panels. The percent annexin V-positive cells are indicated in the right-bottom quadrant and the percent annexin V and 7ADD cells are indicated in the right-top quadrant. (C) The expression of DR5, DR4, RIP, c-FLIP, DcR1 and DcR2 in nine cell lines, Cos7 cells transfected with DcR1 and DcR2 cDNA (left panel) and normal human brain and glioblastoma tumour tissues (right panel), as determined by Western blotting, with the proteins indicated on the left side and molecular weights on the right side of the panel, with actin as a loading control.

Fig 2

DR5 is the TRAIL functional receptor. Glioblastoma cell lines, as indicated above the panel, were immunoprecipitated by Flag-TRAIL/M2 antibody before (+) and after (–) the cells were lysed and subjected to Western blotting with DR5 (A) and DR4 antibody (B). Cell extract (EX) was included as a control of the endogenous proteins. Glioblastoma cell lines were treated (+) or untreated (–) with 300 ng/ml TRAIL for 30 min. and examined by Western blots under reducing and non-reducing conditions for the presence of the homotrimeric complex of DR5 (C) and DR4 (D). * indicates a non-specific band. (E) TRAIL-sensitive LN18 cells were transfected with scramble, DR4 and DR5 siRNA for 72 hrs and examined by Western blot for the expression of DR4 and DR5, with ERK1/2 used as a loading control. (F). LN18 cells were transfected with scramble, DR4 and DR5 siRNA or double-transfected with DR4 and DR5 siRNA cells, treated with 300 ng/ml TRAIL and examined for cell death by the cell viability assay (mean ± S.E.M., n= 4).

Fig 3

bVAD-fmk trapping of activated initiator caspase-8. (A) TRAIL-sensitive U343MG cells and TRAIL-resistant LN443 cells were pre-treated with bVAD-fmk for 2 hrs and stimulated with 300 ng/ml TRAIL for the times indicated above the panel. The cells were lysed and subjected to Western blotting for cleavage of caspase-8 (Casp-8) and caspase-3 (Casp-3). (B) U343MG and LN443 cells were treated with bVAD-fmk and then TRAIL- and bVAD-fmk-trapped activated caspases were pulled down by streptavidin and examined by Western blotting, with acetyl-CoA carboxylase as a control for precipitation and loading.

Fig 4

The DISC modification leads to the inhibition of caspase-8 cleavage. (A) TRAIL-induced DISC was immunoprecipitated by treating LN443 (left panel) and U343MG (right panel) cells with Flag-TRAIL/M2 antibody for 15 min. (+). For the unstimulated control, the cells were lysed first and then incubated with Flag-TRAIL/M2 antibody for 15 min. (–). Cell lysates (EX) were included as controls. The samples were examined by Western blotting for the presence of the proteins indicated on the left panel, with molecular weights on the right panel. (B) The supernatants collected from LN443 and U343MG cells after the DISC depleted, as described above in (A), were examined by Western blots for caspase-8 cleavage products, with the molecular weights indicated on the right side of the panel. (C) Western blot of the DISC analysis with RIP antibody (right third blot in (A)) was overexposed to show RIP in the unstimulated control (–) and the DISC (+) of U343MG cells. (D) LN443 and U343MG cells were treated with 300 ng/ml TRAIL in the absence or presence of caspase-8 inhibitor z-IETD for 6 hrs and subjected to Western blotting for the expression of RIP (p74) and its cleavage product (p43).

Fig 5

TRAIL-induced NF-κB activity does not contribute to TRAIL resistance. (A) U343MG and LN443 cells were untreated (–) or treated (+) with 300 ng/ml TRAIL for 30 min. and examined for NF-κB DNA-binding activity by EMSA. (B) TRAIL-sensitive (LN71 and LN18) and TRAIL-resistant cell lines (U118MG, U138MG, U87MG and LN443) were treated with 300 ng/ml TRAIL for 6 hrs and examined by an NF-κB-responsive luciferase reporter assay. (C) LN443 cell were transfected with either scramble or DR5 siRNA for 72 hrs, treated with 300 ng/ml TRAIL for 6 hrs and examined by the luciferase reporter assay. (D) LN443 cells were treated or untreated with 10 μM parthenolide for 16 hrs and followed with 300 ng/ml TRAIL treatment for the hours as indicated before being subjected to Western blot analysis of the expression of phosphorylated I-κB (p-I-κB), total I-κB, c-FLIP, PED/PEA-15 and RIP, with actin as a loading control. TRAIL-resistant LN443 and U138MG cells were treated with 10 μM parthenolide for 16 hrs and then with 300 ng/ml TRAIL for 3 hrs for the luciferase reporter assay (E) and for 24 hrs for the cell death assay (F).

Fig 6

Selective knockdown of RIP, c-FLIP and PED/PEA-15 sensitizes the resistant cells to TRAIL. (A) D247MG and U118MG cells were transfected with RIP, c-FLIP, PED/PEA-15 siRNA and scramble siRNA for 72 hrs. The expression of RIP, c-FLIP and PED/PEA-15 was examined by Western blots. (B) The cleavage of caspases and DFF45 was examined by Western blotting in the transfected cells following treatment with 300 ng/ml TRAIL for 3 hrs. Proteins are indicated on the left, and the molecular weights of the proteins and cleavage products are on the right side of the panels. (C) Cell death was measured by the cell viability assay in the transfected cells after treatment with 300 ng/ml TRAIL for 24 hrs (mean ± S.E.M., n = 8).

Fig 7

TRAIL treatment leads to the DISC redistribution to lipid rafts. (A) TRAIL-sensitive U343MG cells were subjected to discontinuous sucrose density gradients for separation of lipid raft and non-raft fractions. Lipid raft fractions 4 and 5 were identified by Western blots using lipid raft markers caveolin-1 and Fyn. (B) The concentration of cholesterol was measured in each of the fractions and a higher cholesterol content was observed in fractions 4 and 5. (C) The lipid raft and non-raft fractions from U343MG cells were subjected to Western blot analysis of the expression of DR5, caspase-8 (Casp-8), FADD, RIP and c-FLIPL, with caveolin-1 included as a lipid raft marker. (D) U343MG cells were treated with 300 ng/ml TRAIL for 15 min. and lipid raft and non-raft fractions were generated and examined by Western blot for the expression of the proteins indicated on the left side of the panel.

Fig 8

RIP, c-FLIP and PED/PEA-15 localize the DISC in non-rafts in TRAIL resistance. (A) The lipid raft and non-raft fractions from TRAIL-resistant LN443 cells were subjected to Western blotting of lipid raft markers caveolin-1 and Fyn. (B) Chemical analysis of cholesterol content in the lipid raft and non-raft fractions of LN443 cells. (C) LN443 cells were untreated (left panel) or treated with 300 ng/ml TRAIL for 15 min. (right panel) and lipid raft and non-raft fractions were separated and examined on Western blots for DR5, caspase-8 (Casp-8), FADD, RIP, c-FLIP and caveolin-1. (D) LN443 cells were transfected with RIP, c-FLIP and PED/PEA-15 siRNA, respectively, and then treated with 300 ng/ml TRAIL and subjected to discontinuous sucrose density gradients. In total, 2–10 fractions were examined by Western blot analysis of DR5 and caspase-8.