The anti-apoptotic protein BCL2L1/Bcl-xL is neutralized by pro-apoptotic PMAIP1/Noxa in neuroblastoma, thereby determining bortezomib sensitivity independent of prosurvival MCL1 expression

Abstract

Neuroblastoma is the most frequent extracranial solid tumor in children. Here, we report that the proteasome inhibitor bortezomib (PS-341, Velcade) activated the pro-apoptotic BH3-only proteins PMAIP1/Noxa and BBC3/Puma and induced accumulation of anti-apoptotic MCL1 as well as repression of anti-apoptotic BCL2L1/Bcl-xL. Retroviral expression of Bcl-xL, but not of MCL1, prevented apoptosis by bortezomib. Gene knockdown of Noxa by shRNA technology significantly reduced apoptosis, whereas Puma knockdown did not affect cell death kinetics. Immunoprecipitation revealed that endogenous Noxa associated with both, Bcl-xL and MCL1, suggesting that in neuronal cells Noxa can neutralize Bcl-xL, explaining the pronounced protective effect of Bcl-xL. Tetracycline-regulated Noxa expression did not trigger cell death per se but sensitized to bortezomib treatment in a dose-dependent manner. This implies that the induction of Noxa is necessary but not sufficient for bortezomib-induced apoptosis. We conclude that MCL1 steady-state expression levels do not affect sensitivity to proteasome-inhibitor treatment in neuronal tumor cells, and that both the repression of Bcl-xL and the activation of Noxa are necessary for bortezomib-induced cell death.

FIGURE 1.

The proteasome inhibitor bortezomib induces apoptosis in various neuroblastoma cell lines. The neuroblastoma cell lines SH-EP, Lan-1, SKNSH, SH-SY5Y, IMR-32, STA-NB1, STA-NB3, and STA-NB15 were treated with increasing doses of bortezomib (12, 25, 50, 100 nm) for 24 (A) and 48 h (B) and analyzed by PI-FACS analyses. C, proteasomal activity was analyzed using a fluorescence-based proteasome activity assay. SH-EP cells were either treated with 50 nm bortezomib for 8 h and then subjected to proteasome preparation (bort*) or lysates of untreated SH-EP cells were directly incubated with 50 nm bortezomib (bort) or the proteasome inhibitor EGCG. Statistical significance between control and treated samples was assessed by the unpaired Student's t test (*, p < 0.05; ***, p < 0.0001).

FIGURE 2.

Bortezomib-induced apoptosis requires caspase activation and involves mitochondria. A, mitochondrial activity was measured by CMX-Ros staining of SH-EP cells in the presence or absence of 50 nm bortezomib for 48 h. B, cytoplasmic cytochrome c of untreated and 24 h bortezomib-treated SH-EP cells was detected by immunoblot, and the amount of cytoplasmic cytochrome c was measured by densitometry of 16-bit images using LabWorks software. C, STA-NB15 and SH-EP cells were treated with 50 nm bortezomib for 0, 4, 8, 16, and 24 h. Cell lysates were subjected to immunoblot analyses using specific antibodies against caspase-9 (with cleavage products of 35 and 32 kDa) and caspase-8 (cleavage products of 40, 36, and 23 kDa). α-Tubulin was used as loading control. D, active caspase-3 was detected by the fluorogenic FITC-DEVD.fmk substrate of the caspase-3 cleavage assay in the cells SH-EP and STA-NB15 after treatment with 50 nm bortezomib (bort) for 24 and 48 h. E, SH-EP and STA-NB15 cells were maintained for 24, 48, and 72 h in the presence of 50 nm bortezomib alone or in combination with z-VAD, a pan-caspase-inhibitor (20 nm), and subjected to PI-FACS analyses. Diagrams represent mean values of three independent experiments.

FIGURE 3.

Death receptors are not essential for bortezomib-induced cell death. A, SH-EP cells were infected with retroviral vectors coding for dnFADD and CrmA. The transgenic expression was confirmed by immunoblot. B, SH-EP-ctr, SH-EP-dnFADD, and SH-EP-CrmA cells were subjected to PI-FACS analyses after treatment with 50 nm bortezomib for 24, 48, and 72 h. Bars represent the mean of three independent experiments.

FIGURE 4.

Bortezomib causes up-regulation of Noxa, Puma, and MCL1 and repression of Bcl-xL. A, SH-EP and STA-NB15 cells were subjected to immunoblot analyses using antibodies against BCL2, Bcl-xL, A1, MCL1, Puma, Bim, and Noxa after treatment with 50 nm bortezomib for 0, 4, 8, 16, and 24 h. α-Tubulin and GAPDH were used as loading controls. B, total RNA was prepared from SH-EP and STA-NB15 cells after treatment with 50 nm bortezomib for 0, 3, 6, and 9 h and the level of Puma and Noxa mRNA was assessed by quantitative RT-PCR (mean values of three independent experiments, each performed in triplicate). C, subcellular fractionation of untreated and 50 nm bortezomib-treated (24 h) SH-EP cells. Mitochondrial (mito) and cytoplasmic (cyto) fractions were subjected to immunoblot analyses of Noxa, Puma, and α-tubulin.

FIGURE 5.

Transgenic Bcl-xL is neutralized by Noxa and inhibits bortezomib-induced apoptosis, whereas MCL1 has no impact on death sensitivity. A, SH-EP and STA-NB15 cells were retrovirally infected with vectors coding for Bcl-xL and MCL1. The ectopic expression was verified by immunoblot analysis. Cells were treated with 50 nm bortezomib for the times indicated and subjected to PI-FACS analyses. Statistical significance between mock-infected controls and Bcl-xL transgenic cells was assessed by the unpaired Student's t test (*, p < 0.05; ***, p < 0.001). B, NB15-BclxL cells were treated for 18 h with 50 nm bortezomib, lysed, and subjected to co-immunopurification using rabbit anti-MCL1 and rabbit anti-Bcl-xL as precipitating antibodies, rabbit IgG was used as negative control. Immunoprecipitates were analyzed for presence of Noxa, Bcl-xL, and MCL1 by immunoblot. C, single cell clones were isolated by limiting dilution from SH-EP-Mcl1 cells. MCL1 expression in mock-infected SH-EP-ctr, SH-EP-Mcl1 clone1, and clone10 cells was determined by immunoblot analysis. SH-EP-ctr, SH-EP-Mcl1 clone1, and clone10 cells were treated with 25 and 50 nm bortezomib and subjected to PI-FACS analysis for 48 and 72 h.

FIGURE 6.

Noxa is rate limiting for bortezomib-induced apoptosis. A, SH-EP cells were infected with retroviruses expressing shRNA specific for Noxa or Puma. Individual clones were isolated from bulk-selected SH-EP-shNoxa and SH-EP-shPuma cells and steady-state expression level of endogenous Noxa (left panel) or Puma (right panel) was verified by immunoblot analysis. B, cell lines SH-EP-shCtr and SH-EP-shNoxa clone 3 and clone 7 as well as SH-EP shPuma clone 3, clone 6, and clone 7 were treated with 50 nm bortezomib for 24 h and subjected to PI-FACS analyses. C, SH-EP-shctr and SH-EP-shNoxa clone 3 cells were treated with 50 nm bortezomib for 24 h. Caspase-9 cleavage was determined using a fluorometric caspase activity assay and flow cytometry. Shown is the mean of three independent experiments. Statistics were calculated with GraphPad Prism software using the unpaired Student's t test (**, p = 0.005; ***, p < 0.001). D, SH-EP cells designed to express Noxa in a tetracycline-dependent manner were treated with 250 ng/ml doxy for 24 h. Transgene expression was verified by immunoblot analysis. SH-EP-tetCtr, and SH-EP-tetNoxa cells were treated with 250 ng/ml doxy, with 25 nm bortezomib or a combination of both for 24 h and were then subjected to PI-FACS analyses. Shown is the mean of three independent experiments. E, to assess a possible dosage-dependent effect of Noxa SH-EP-tetNoxa, cells were treated with 5, 10, 20, 50, and 100 ng/ml doxy and then subjected to immunoblot analysis for Noxa and GAPDH (loading control) expression. SH-EP-tetCtr and SH-EP-tetNoxa cells were treated with 12.5 nm bortezomib in the presence or absence of 10 or 100 ng/ml doxy for 24 h and then subjected to PI-FACS analyses (bars represent the mean of three independent experiments). Statistical significance was assessed by the unpaired Student's t test (**, p < 0.01; ***, p < 0.001).