Obatoclax (GX15-070) triggers necroptosis by promoting the assembly of the necrosome on autophagosomal membranes - PubMed (original) (raw)

Obatoclax (GX15-070) triggers necroptosis by promoting the assembly of the necrosome on autophagosomal membranes

F Basit et al. Cell Death Differ. 2013 Sep.

Erratum in

Abstract

Obatoclax (GX15-070), a small-molecule inhibitor of antiapoptotic Bcl-2 proteins, has been reported to trigger cell death via autophagy. However, the underlying molecular mechanisms have remained elusive. Here, we identify GX15-070-stimulated assembly of the necrosome on autophagosomal membranes as a key event that connects GX15-070-stimulated autophagy to necroptosis. GX15-070 predominately induces a non-apoptotic form of cell death in rhabdomyosarcoma cells, as evident by lack of typical apoptotic features such as DNA fragmentation or caspase activation and by insensitivity to the broad-range caspase inhibitor zVAD.fmk. Instead, GX15-070 triggers massive accumulation of autophagosomes, which are required for GX15-070-induced cell death, as blockade of autophagosome formation by silencing of Atg5 or Atg7 abolishes GX15-070-mediated cell death. Co-immunoprecipitation studies reveal that GX15-070 stimulates the interaction of Atg5, a constituent of autophagosomal membranes, with components of the necrosome such as FADD, RIP1 and RIP3. This GX15-070-induced assembly of the necrosome on autophagosomes occurs in a Atg5-dependent manner, as knockdown of Atg5 abrogates formation of this complex. RIP1 is necessary for GX15-070-induced cell death, as both genetic and pharmacological inhibition of RIP1 by shRNA-mediated knockdown or by the RIP1 inhibitor necrostatin-1 blocks GX15-070-induced cell death. Similarly, RIP3 knockdown rescues GX15-070-mediated cell death and suppression of clonogenic survival. Interestingly, RIP1 or RIP3 silencing has no effect on GX15-070-stimulated autophagosome formation, underlining that RIP1 and RIP3 mediate cell death downstream of autophagy induction. Of note, GX15-070 significantly suppresses tumor growth in a RIP1-dependent manner in the chorioallantoic membrane model in vivo. In conclusion, GX15-070 triggers necroptosis by promoting the assembly of the necrosome on autophagosomes. These findings provide novel insights into the molecular mechanisms of GX15-070-induced non-apoptotic cell death.

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Figures

Figure 1

Figure 1

GX15-070-induced cell death in RMS cells. (a) Expression levels of anti- and proapoptotic Bcl-2 family proteins in RMS cells were assessed by western blot analysis. _β_-Actin was used as loading control. (b and c) RMS cells were treated with the indicated concentrations of GX15-070 for 72 h (b) or with the indicated concentrations of GX15-070 for the indicated times (c). Cell viability was determined by MTT assay and is expressed as percentage of untreated controls. Data represent mean+S.D. of three independent experiments performed in triplicate. (d) TE671 and RMS13 cells were treated with 200 nM GX15-070 for 48 h. Apoptosis was determined by fluorescence-activated cell-sorting analysis of DNA fragmentation of propidium iodide-stained nuclei. Representative histograms of three independent experiments are shown

Figure 2

Figure 2

GX15-070 triggers caspase-independent cell death in RMS cells. (a and b) TE671 and RMS13 cells were treated for 72 h with 200 nM GX15-070 in the presence or absence of 20 _μ_M zVAD.fmk. Cell viability was determined by MTT assay and is expressed as percentage of untreated controls (a). Apoptosis was determined by fluorescence-activated cell-sorting analysis of DNA fragmentation of propidium iodide-stained nuclei (b). Data represent mean+S.D. of three independent experiments performed in triplicate. (c) TE671 and RMS13 cells were treated with 200 nM GX15-070 for the indicated time points or with 10 μ_M ABT-737 for 3 h. Caspase activation was assessed by western blotting. Arrowheads indicate caspase cleavage fragments. (d and e) TE671 and RMS13 cells were treated for 72 h with 200 nM GX15-070 (d) or 100 ng/ml TNF_α and 10 _μ_M IAP (inhibitor of apoptosis) inhibitor 3 (e) in the presence or absence of 20 μ_g/ml Enbrel. Cell viability was determined by MTT assay and is expressed as percentage of untreated controls. (f–h) TE671 and RMS13 cells were transfected with control siRNA (siCtrl) or two distinct siRNA sequences against TNFR1 (siTNFR1#1, siTNFR1#2). Expression of TNFR1 was determined by western blotting (f) Cell viability was determined by MTT assay after treatment for 72 h with the indicated concentrations of GX15-070 (g) or with 100 ng/ml TNF_α and 10 _μ_M IAP inhibitor 3 (h) and is expressed as percentage of untreated controls. In panels a, b, d, e, g and h, data represent mean+S.D. of three independent experiments performed in triplicate; **P<0.001

Figure 3

Figure 3

GX15-070 induces autophagy in RMS cells. (a and b) TE671 and RMS13 cells were treated with 200 nM GX15-070 for the indicated time points (a) or with 200 nM GX15-070 for 3 h in the presence or absence of 1 _μ_M BafA1 (b). LC3 lipidation was assessed by western blotting. (c) TE671 cells were transfected with tandem mCherry-GFP-tagged LC3 and were treated with 200 nM GX15-070 for 3 h in the presence or absence of 1 _μ_M BafA1. Fluorescence of LC3 was detected by confocal microscopy

Figure 4

Figure 4

Atg5 and Atg7 are required for GX15-070-induced autophagy and cell death. (a) TE671 and RMS13 cells were transduced with control vector (shCtrl) or vectors containing two distinct shRNA sequences against Atg5 (shAtg5#1, shAtg5#2). Expression of Atg5 was determined by western blotting. (b) TE671 and RMS13 cells were treated with 100 nM GX15-070 for the indicated time points and LC3 lipidation was assessed by western blotting. (c) TE671 and RMS13 cells were treated with the indicated concentrations of GX15-070 for 72 h. Cell viability was determined by MTT assay and is expressed as percentage of untreated controls. Data represent mean+S.D. of three independent experiments performed in triplicate; *P<0.05; **P<0.001 comparing control with Atg5 knockdown cells. (d) TE671 and RMS13 cells were transfected with control siRNA (siCtrl) or siRNA targeting Atg7 (siAtg7). Expression of RIP1 was determined by western blotting. (e) TE671 and RMS13 cells were treated with 100 nM GX15-070 for the indicated time points and LC3 lipidation was detected by western blotting. (f) TE671 and RMS13 cells were treated with the indicated concentrations of GX15-070 for 72 h. Cell viability was determined by MTT assay and is expressed as percentage of untreated controls. Data represent mean+S.D. of three independent experiments performed in triplicate; *P<0.05; **P<0.001 comparing control with Atg7 knockdown cells

Figure 5

Figure 5

GX15-070 triggers the assembly of the necrosome on autophagosomes. (a) TE671 and RMS13 cells were treated with 200 nM GX15-070 for the indicated time points. RIP1 was immunoprecipitated (IP) using anti-RIP1 antibody. Atg5, FADD, RIP3 and caspase-8 were detected by western blotting. (b) TE671 and RMS13 cells were transduced with control vector (shCtrl) or vectors containing two distinct shRNA sequences against Atg5 (shAtg5#1, shAtg5#2) and treated with 100 nM GX15-070 for 48 h. RIP1 was immunoprecipitated (IP) using anti-RIP1 antibody. Atg5, FADD, RIP3 and caspase-8 were detected by western blotting

Figure 6

Figure 6

RIP1 is required for GX15-070-induced cell death downstream of autophagosome formation. (a) TE671 and RMS13 cells were treated with 200 nM GX15-070 in the presence or absence of 40 _μ_g/ml Nec-1. Cell viability was determined by MTT assay and is expressed as percentage of untreated controls. (b) TE671 and RMS13 cells were transduced with control vector (shCtrl) and vector containing shRNA sequence against RIP1 (shRIP1). Expression of RIP1 was determined by western blotting. (c) TE671 and RMS13 cells were treated with the indicated concentrations of GX15-070 for 72 h. Cell viability was determined by MTT assay and is expressed as percentage of untreated controls. (d) TE671 and RMS13 cells were treated with 100 nM GX15-070 for 72 h before medium was exchanged by fresh drug-free medium. Colonies were stained with crystal violet and counted under the microscope. The percentage of colony numbers in the presence and absence of GX15-070 is shown. (e) TE671 and RMS13 cells were treated with 100 nM GX15-070 for 48 h. FADD was immunoprecipitated (IP) using anti-FADD antibody. Atg5, FADD, RIP1 and RIP3 were detected by western blotting. (f) TE671 and RMS13 cells were treated with 100 nM GX15-070 for the indicated time points and LC3 lipidation was detected by western blotting. In panels a, c and d, data represent mean+S.D. of three independent experiments performed in triplicate; *P<0.05; **P<0.001 comparing control with RIP1 knockdown cells (c and d) or cells treated in the presence and absence of Nec-1 (a)

Figure 7

Figure 7

RIP3 is required for GX15-070-induced cell death downstream of autophagosome formation. (a) TE671 and RMS13 cells were transduced with control vector (shCtrl) and vectors containing shRNA sequences against RIP3 (shRIP3#1, shRIP3#2). Expression of RIP3 was determined by western blotting. (b) TE671 and RMS13 cells were treated with the indicated concentrations of GX15-070 for 72 h. Cell viability was determined by MTT assay and is expressed as percentage of untreated controls. (c) TE671 and RMS13 cells were treated with 100 nM GX15-070 for 72 h before medium was exchanged by fresh drug-free medium. Colonies were stained with crystal violet and counted under the microscope. The percentage of colony numbers in the presence and absence of GX15-070 is shown. (d) TE671 and RMS13 cells were treated with 100 nM GX15-070 for the indicated time points and LC3 lipidation was detected by western blotting; *indicates empty space between samples. In panels b and c, data represent mean+S.D. of three independent experiments performed in triplicate; *P<0.05; **P<0.001 comparing control with RIP3 knockdown cells

Figure 8

Figure 8

GX15-070 suppresses RMS growth in vivo in a RIP1-dependent manner. TE671 cells transduced with shRNA vectors against RIP1 or control shRNA were seeded on the CAM of chicken embryos and treated with 100 _μ_M GX15-070 for 4 days. Tumor growth was analyzed using hematoxylin/eosin-stained paraffin sections of the CAM as described in Materials and Methods. Tumor area as percentage of the untreated control group is shown. Data represent mean+S.D. of 14 samples per group; **P<0.001

Comment in

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