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NGF sensitizes TrkA SH-SY5Y neuroblastoma cells to TRAIL-induced apoptosis

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ABSTRACT

We report a novel pro-apoptotic function for nerve growth factor (NGF) and its tropomyosin-related kinase A (TrkA) receptor in sensitizing TRAIL (TNF-related apoptotis-inducing ligand)-resistant SH-SY5Y neuroblastoma (NB) cells to TRAIL-induced apoptosis, resulting in the abrogation of anchorage-independent tumourigenic growth in vitro. We show that the TRAIL-resistant SH-SY5Y phenotype is cFLIP (cellular FLICE-like inhibitory protein) dependent and not due to low-level functional TRAIL receptor or caspase expression or an inhibitory equilibrium between functional and decoy TRAIL receptors or B-cell lymphoma 2 (Bcl-2) and BH3-only (Bcl-2 homology domain 3-only) family proteins. NGF sensitization of SH-SY5Y cells to TRAIL-induced apoptosis was dependent upon TrkA expression, activation and subsequent sequestration of cFLIP. This reduces cFLIP recruitment to TRAIL-activated death receptors and increases the recruitment of caspase-8, leading to TRAIL-induced, caspase-dependent, type II apoptosis via the intrinsic mitochondrial pathway. This effect was temporary, inhibited within 6 h by nuclear factor-κ binding (NF-κB)-mediated increase in myeloid cell leukaemia-1 (Mcl-1) expression, abrogated by transient cFLIP or B-cell lymphoma-extra large (Bcl-xL) overexpression and optimized by NF-κB and Mcl-1 inhibitors. This novel mechanism adds an important pro-apoptotic immunological dimension to NGF/TrkA interaction that may not only help to explain the association between TrkA expression, better prognosis and spontaneous remission in NB, but also provides a novel potential pro-apoptotic therapeutic use for NGF, TRAIL and inhibitors of NF-κB and/or Mcl-1 in favourable and unfavourable NBs that express TrkA and exhibit cFLIP-mediated TRAIL resistance.

No MeSH data available.


Related in: MedlinePlus

NGF sensitizes TrkA SH-SY5Y cells to TRAIL-induced apoptosis. (a) Representative phase contrast and fluorescent micrographs (bar=100 μM) and (b) histograms demonstrating the marked induction of TrkA SH-SY5Y but not NT or pcDNA SH-SY5Y cell death following 16 h of incubation with NGF (100 ng/ml) plus TRAIL (200 ng/ml), compared with the lack of cell death in all three cell lines following overnight (16 h) incubation with either NGF (100 ng/ml) or TRAIL (200 ng/ml) alone. Results are displayed as the mean percentage (%) of alive (white) and dead (black) cells (±S.D.) in three independent experiments each performed in duplicate (*statistical significance). (c) Representative phase contrast micrographs demonstrating the effect of NGF plus TRAIL on the morphology of surviving TrkA SH-SY5Y cells. (d) Histogram demonstrating dose-dependent induction of TrkA SH-SY5Y cell death by TRAIL (10–500 ng/ml) in the presence but not absence of NGF (100 ng/m). Results are displayed as the mean percentage (%) (±S.D.) of alive (white) and dead (black) cells in three independent experiments each performed in duplicate. (e) Representative ethidium bromide stained agarose gel demonstrating laddering of DNA purified from TrkA SH-SY5Y cells treated for 16 h with NGF (100 ng/ml) plus TRAIL (200 ng/ml) but not from TrkA SH-SY5Y cells treated with either NGF (100 ng/ml) or TRAIL (200 ng/ml) alone.
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fig1: NGF sensitizes TrkA SH-SY5Y cells to TRAIL-induced apoptosis. (a) Representative phase contrast and fluorescent micrographs (bar=100 μM) and (b) histograms demonstrating the marked induction of TrkA SH-SY5Y but not NT or pcDNA SH-SY5Y cell death following 16 h of incubation with NGF (100 ng/ml) plus TRAIL (200 ng/ml), compared with the lack of cell death in all three cell lines following overnight (16 h) incubation with either NGF (100 ng/ml) or TRAIL (200 ng/ml) alone. Results are displayed as the mean percentage (%) of alive (white) and dead (black) cells (±S.D.) in three independent experiments each performed in duplicate (*statistical significance). (c) Representative phase contrast micrographs demonstrating the effect of NGF plus TRAIL on the morphology of surviving TrkA SH-SY5Y cells. (d) Histogram demonstrating dose-dependent induction of TrkA SH-SY5Y cell death by TRAIL (10–500 ng/ml) in the presence but not absence of NGF (100 ng/m). Results are displayed as the mean percentage (%) (±S.D.) of alive (white) and dead (black) cells in three independent experiments each performed in duplicate. (e) Representative ethidium bromide stained agarose gel demonstrating laddering of DNA purified from TrkA SH-SY5Y cells treated for 16 h with NGF (100 ng/ml) plus TRAIL (200 ng/ml) but not from TrkA SH-SY5Y cells treated with either NGF (100 ng/ml) or TRAIL (200 ng/ml) alone.

Mentions: In two-dimensional culture, TRAIL at concentrations from 10 ng to 1 μg/ml did not induce significant death of either nontransfected (NT), duplicate empty pcDNA3.1 vector (pcDNA)-transfected or duplicate TrkA-transfected SH-SY5Y cell lines, confirming their TRAIL-resistant phenotypes (Figures 1a and b). NGF (100 ng/ml) also failed to induce significant death of NT SH-SY5Y, pcDNA SH-SY5Y or TrkA SH-SY5Y cells (Figures 1a and b). In contrast, the combination of NGF (100 ng/ml) and TRAIL (200 ng/ml) induced a mean (±S.D.) of 68.7±13.8 and 66±14.3% death in the two TrkA SH-SY5Y cell lines but did not induce the death of either NT SH-SY5Y or pcDNA SH-SY5Y cells (Figures 1a and b). TrkA SH-SY5Y cells that survived the effects of NGF plus TRAIL exhibited neurite-like projections, suggesting the initiation of neural differentiation (Figure 1c). In concentration studies, the death of TrkA SH-SY5Y cells in the presence of NGF (100 ng/ml) was detected at TRAIL concentrations ≥50 ng/ml and plateaued with a mean (±S.D.) of 68.3±15.3% death at a concentration of 200 ng/ml (Figures 1a and d). The TRAIL-induced death of TrkA SH-SY5Y cells was confirmed to be apoptotic by Tunel assay (Figure 1a), detection of DNA laddering (Figure 1e) and was abrogated by the caspase inhibitors z-DEVD-fmk (caspase-3), z-IETD-fmk (caspase-8) and z-LEHD-fmk (caspase-9) (see Figures 4d and e).


NGF sensitizes TrkA SH-SY5Y neuroblastoma cells to TRAIL-induced apoptosis
NGF sensitizes TrkA SH-SY5Y cells to TRAIL-induced apoptosis. (a) Representative phase contrast and fluorescent micrographs (bar=100 μM) and (b) histograms demonstrating the marked induction of TrkA SH-SY5Y but not NT or pcDNA SH-SY5Y cell death following 16 h of incubation with NGF (100 ng/ml) plus TRAIL (200 ng/ml), compared with the lack of cell death in all three cell lines following overnight (16 h) incubation with either NGF (100 ng/ml) or TRAIL (200 ng/ml) alone. Results are displayed as the mean percentage (%) of alive (white) and dead (black) cells (±S.D.) in three independent experiments each performed in duplicate (*statistical significance). (c) Representative phase contrast micrographs demonstrating the effect of NGF plus TRAIL on the morphology of surviving TrkA SH-SY5Y cells. (d) Histogram demonstrating dose-dependent induction of TrkA SH-SY5Y cell death by TRAIL (10–500 ng/ml) in the presence but not absence of NGF (100 ng/m). Results are displayed as the mean percentage (%) (±S.D.) of alive (white) and dead (black) cells in three independent experiments each performed in duplicate. (e) Representative ethidium bromide stained agarose gel demonstrating laddering of DNA purified from TrkA SH-SY5Y cells treated for 16 h with NGF (100 ng/ml) plus TRAIL (200 ng/ml) but not from TrkA SH-SY5Y cells treated with either NGF (100 ng/ml) or TRAIL (200 ng/ml) alone.
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Related In: Results  -  Collection

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fig1: NGF sensitizes TrkA SH-SY5Y cells to TRAIL-induced apoptosis. (a) Representative phase contrast and fluorescent micrographs (bar=100 μM) and (b) histograms demonstrating the marked induction of TrkA SH-SY5Y but not NT or pcDNA SH-SY5Y cell death following 16 h of incubation with NGF (100 ng/ml) plus TRAIL (200 ng/ml), compared with the lack of cell death in all three cell lines following overnight (16 h) incubation with either NGF (100 ng/ml) or TRAIL (200 ng/ml) alone. Results are displayed as the mean percentage (%) of alive (white) and dead (black) cells (±S.D.) in three independent experiments each performed in duplicate (*statistical significance). (c) Representative phase contrast micrographs demonstrating the effect of NGF plus TRAIL on the morphology of surviving TrkA SH-SY5Y cells. (d) Histogram demonstrating dose-dependent induction of TrkA SH-SY5Y cell death by TRAIL (10–500 ng/ml) in the presence but not absence of NGF (100 ng/m). Results are displayed as the mean percentage (%) (±S.D.) of alive (white) and dead (black) cells in three independent experiments each performed in duplicate. (e) Representative ethidium bromide stained agarose gel demonstrating laddering of DNA purified from TrkA SH-SY5Y cells treated for 16 h with NGF (100 ng/ml) plus TRAIL (200 ng/ml) but not from TrkA SH-SY5Y cells treated with either NGF (100 ng/ml) or TRAIL (200 ng/ml) alone.
Mentions: In two-dimensional culture, TRAIL at concentrations from 10 ng to 1 μg/ml did not induce significant death of either nontransfected (NT), duplicate empty pcDNA3.1 vector (pcDNA)-transfected or duplicate TrkA-transfected SH-SY5Y cell lines, confirming their TRAIL-resistant phenotypes (Figures 1a and b). NGF (100 ng/ml) also failed to induce significant death of NT SH-SY5Y, pcDNA SH-SY5Y or TrkA SH-SY5Y cells (Figures 1a and b). In contrast, the combination of NGF (100 ng/ml) and TRAIL (200 ng/ml) induced a mean (±S.D.) of 68.7±13.8 and 66±14.3% death in the two TrkA SH-SY5Y cell lines but did not induce the death of either NT SH-SY5Y or pcDNA SH-SY5Y cells (Figures 1a and b). TrkA SH-SY5Y cells that survived the effects of NGF plus TRAIL exhibited neurite-like projections, suggesting the initiation of neural differentiation (Figure 1c). In concentration studies, the death of TrkA SH-SY5Y cells in the presence of NGF (100 ng/ml) was detected at TRAIL concentrations ≥50 ng/ml and plateaued with a mean (±S.D.) of 68.3±15.3% death at a concentration of 200 ng/ml (Figures 1a and d). The TRAIL-induced death of TrkA SH-SY5Y cells was confirmed to be apoptotic by Tunel assay (Figure 1a), detection of DNA laddering (Figure 1e) and was abrogated by the caspase inhibitors z-DEVD-fmk (caspase-3), z-IETD-fmk (caspase-8) and z-LEHD-fmk (caspase-9) (see Figures 4d and e).

View Article: PubMed Central - PubMed

ABSTRACT

We report a novel pro-apoptotic function for nerve growth factor (NGF) and its tropomyosin-related kinase A (TrkA) receptor in sensitizing TRAIL (TNF-related apoptotis-inducing ligand)-resistant SH-SY5Y neuroblastoma (NB) cells to TRAIL-induced apoptosis, resulting in the abrogation of anchorage-independent tumourigenic growth in vitro. We show that the TRAIL-resistant SH-SY5Y phenotype is cFLIP (cellular FLICE-like inhibitory protein) dependent and not due to low-level functional TRAIL receptor or caspase expression or an inhibitory equilibrium between functional and decoy TRAIL receptors or B-cell lymphoma 2 (Bcl-2) and BH3-only (Bcl-2 homology domain 3-only) family proteins. NGF sensitization of SH-SY5Y cells to TRAIL-induced apoptosis was dependent upon TrkA expression, activation and subsequent sequestration of cFLIP. This reduces cFLIP recruitment to TRAIL-activated death receptors and increases the recruitment of caspase-8, leading to TRAIL-induced, caspase-dependent, type II apoptosis via the intrinsic mitochondrial pathway. This effect was temporary, inhibited within 6 h by nuclear factor-κ binding (NF-κB)-mediated increase in myeloid cell leukaemia-1 (Mcl-1) expression, abrogated by transient cFLIP or B-cell lymphoma-extra large (Bcl-xL) overexpression and optimized by NF-κB and Mcl-1 inhibitors. This novel mechanism adds an important pro-apoptotic immunological dimension to NGF/TrkA interaction that may not only help to explain the association between TrkA expression, better prognosis and spontaneous remission in NB, but also provides a novel potential pro-apoptotic therapeutic use for NGF, TRAIL and inhibitors of NF-κB and/or Mcl-1 in favourable and unfavourable NBs that express TrkA and exhibit cFLIP-mediated TRAIL resistance.

No MeSH data available.


Related in: MedlinePlus