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

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

NGF plus TRAIL abrogates the tumourigenic activity of TrkA SH-SY5Y cells in vitro. (a) Representative photograph and (b) histogram demonstrating significant inhibition of TrkA SH-SY5Y but not NT SH-SY5Y or pcDNA SH-SY5Y tumourigenic activity in vitro by NGF (100 ng) alone or in combination with TRAIL (200 ng). Results are displayed as the mean (±S.D.) number of tumours in 10×10 magnification fields in triplicate experiments each performed in duplicate (*statistical significance). (c) Representative phase contrast micrographs demonstrating tumour spheroid appearance of TrkA SH-SY5Y cells in vitro in the absence (control) or presence of NGF (100 ng/ml), TRAIL (200 ng/ml) plus NGF (100 ng/ml) or TRAIL (200 ng/ml) alone.
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fig2: NGF plus TRAIL abrogates the tumourigenic activity of TrkA SH-SY5Y cells in vitro. (a) Representative photograph and (b) histogram demonstrating significant inhibition of TrkA SH-SY5Y but not NT SH-SY5Y or pcDNA SH-SY5Y tumourigenic activity in vitro by NGF (100 ng) alone or in combination with TRAIL (200 ng). Results are displayed as the mean (±S.D.) number of tumours in 10×10 magnification fields in triplicate experiments each performed in duplicate (*statistical significance). (c) Representative phase contrast micrographs demonstrating tumour spheroid appearance of TrkA SH-SY5Y cells in vitro in the absence (control) or presence of NGF (100 ng/ml), TRAIL (200 ng/ml) plus NGF (100 ng/ml) or TRAIL (200 ng/ml) alone.

Mentions: NT, pcDNA and TrkA SH-SY5Y cells formed similar numbers of similar-sized tumour spheroids over 14 days in soft agar tumourigenesis assays, under basal conditions (Figures 2a and b, identical results were obtained for duplicate cell lines and results are displayed for single NT, pcDNA and TrkA SH-SY5Y cell lines). The addition of TRAIL (200 ng/ml) to soft agar did not significantly reduce the tumourigenic activity of NT SH-SY5Y, pcDNA SH-SY5Y or TrkA SH-SY5Y cells (Figures 2a and b). NGF (100 ng/ml) added to soft agar significantly reduced the number of tumour spheroids formed by TrkA SH-SY5Y cells by a mean (±S.D.) of 58.5±9.1% (P<0.0001, d.f.=10) but did not inhibit the number of tumour spheroids formed by either NT SH-SY5Y or pcDNA SH-SY5Y cells over 14 days (Figures 2a and b). Furthermore, tumour spheroids formed by TrkA SH-SY5Y cells in the presence of NGF (100 ng/ml) appeared to be less well formed (Figure 2c). The addition of NGF (100 ng/ml) plus TRAIL (200 ng/ml) to soft agar abrogated tumour spheroid growth by TrkA SH-SY5Y cells, resulting in a mean (±S.D.) of 96.8% (P<0.0001, d.f.=10) inhibition when compared with untreated TrkA SH-SY5Y cells and 92.5% (P<0.0001, d.f.=10) inhibition when compared with NGF-treated TrkA SH-SY5Y cells (Figures 2a and b). In contrast, the combination of NGF plus TRAIL did not reduce the number of tumour spheroids formed by NT SH-SY5Y or pcDNA SH-SY5Y cells (Figures 2a and b).


NGF sensitizes TrkA SH-SY5Y neuroblastoma cells to TRAIL-induced apoptosis
NGF plus TRAIL abrogates the tumourigenic activity of TrkA SH-SY5Y cells in vitro. (a) Representative photograph and (b) histogram demonstrating significant inhibition of TrkA SH-SY5Y but not NT SH-SY5Y or pcDNA SH-SY5Y tumourigenic activity in vitro by NGF (100 ng) alone or in combination with TRAIL (200 ng). Results are displayed as the mean (±S.D.) number of tumours in 10×10 magnification fields in triplicate experiments each performed in duplicate (*statistical significance). (c) Representative phase contrast micrographs demonstrating tumour spheroid appearance of TrkA SH-SY5Y cells in vitro in the absence (control) or presence of NGF (100 ng/ml), TRAIL (200 ng/ml) plus NGF (100 ng/ml) or TRAIL (200 ng/ml) alone.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4979468&req=5

fig2: NGF plus TRAIL abrogates the tumourigenic activity of TrkA SH-SY5Y cells in vitro. (a) Representative photograph and (b) histogram demonstrating significant inhibition of TrkA SH-SY5Y but not NT SH-SY5Y or pcDNA SH-SY5Y tumourigenic activity in vitro by NGF (100 ng) alone or in combination with TRAIL (200 ng). Results are displayed as the mean (±S.D.) number of tumours in 10×10 magnification fields in triplicate experiments each performed in duplicate (*statistical significance). (c) Representative phase contrast micrographs demonstrating tumour spheroid appearance of TrkA SH-SY5Y cells in vitro in the absence (control) or presence of NGF (100 ng/ml), TRAIL (200 ng/ml) plus NGF (100 ng/ml) or TRAIL (200 ng/ml) alone.
Mentions: NT, pcDNA and TrkA SH-SY5Y cells formed similar numbers of similar-sized tumour spheroids over 14 days in soft agar tumourigenesis assays, under basal conditions (Figures 2a and b, identical results were obtained for duplicate cell lines and results are displayed for single NT, pcDNA and TrkA SH-SY5Y cell lines). The addition of TRAIL (200 ng/ml) to soft agar did not significantly reduce the tumourigenic activity of NT SH-SY5Y, pcDNA SH-SY5Y or TrkA SH-SY5Y cells (Figures 2a and b). NGF (100 ng/ml) added to soft agar significantly reduced the number of tumour spheroids formed by TrkA SH-SY5Y cells by a mean (±S.D.) of 58.5±9.1% (P<0.0001, d.f.=10) but did not inhibit the number of tumour spheroids formed by either NT SH-SY5Y or pcDNA SH-SY5Y cells over 14 days (Figures 2a and b). Furthermore, tumour spheroids formed by TrkA SH-SY5Y cells in the presence of NGF (100 ng/ml) appeared to be less well formed (Figure 2c). The addition of NGF (100 ng/ml) plus TRAIL (200 ng/ml) to soft agar abrogated tumour spheroid growth by TrkA SH-SY5Y cells, resulting in a mean (±S.D.) of 96.8% (P<0.0001, d.f.=10) inhibition when compared with untreated TrkA SH-SY5Y cells and 92.5% (P<0.0001, d.f.=10) inhibition when compared with NGF-treated TrkA SH-SY5Y cells (Figures 2a and b). In contrast, the combination of NGF plus TRAIL did not reduce the number of tumour spheroids formed by NT SH-SY5Y or pcDNA SH-SY5Y cells (Figures 2a and b).

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&thinsp;h by nuclear factor-&kappa; binding (NF-&kappa;B)-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-&kappa;B 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-&kappa;B 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