Necroptosis as a Novel Facet of Mitotic Catastrophe

Abstract

Mitotic catastrophe is a defensive mechanism that promotes elimination of cells with aberrant mitosis by triggering the cell-death pathways and/or cellular senescence. Nowadays, it is known that apoptosis, autophagic cell death, and necrosis could be consequences of mitotic catastrophe. Here, we demonstrate the ability of a DNA-damaging agent, doxorubicin, at 600 nM concentration to stimulate mitotic catastrophe. We observe that the inhibition of caspase activity leads to accumulation of cells with mitotic catastrophe hallmarks in which RIP1-dependent necroptotic cell death is triggered. The suppression of autophagy by a chemical inhibitor or ATG13 knockout upregulates RIP1 phosphorylation and promotes necroptotic cell death. Thus, in certain conditions mitotic catastrophe, in addition to apoptosis and autophagy, can precede necroptosis.

Keywords: autophagy; doxorubicin treatment; mitotic catastrophe; necroptosis.

Figure 1

Mitotic catastrophe in doxorubicin-treated Caov4, HCT116, and U1810 cells. All cell lines were treated with 600 nM doxorubicin for 48 h and stained with MitoTracker Red FM (red) to observe mitochondria and with DAPI (blue) to detect nuclei. The cells were monitored under a confocal microscope. (A) Representative images of mitotic catastrophe-associated nuclear morphology. (B) Quantification of mitotic catastrophe after 48 h treatment with doxorubicin. The numbers of mitotic catastrophe cells examined in each cell line are shown in the bars. Values are the mean (±standard deviation of the mean) of three independent experiments. Control: no treatment; Doxo: Doxorubicin. ** p < 0.01, *** p < 0.001.

Figure 2

Necroptosis execution in cells upon mitotic catastrophe induction. (A) Caov4, HCT116, and U1810 cells were pre-treated with 40 μM zVAD-fmk and/or 30 μM necrostatin-1s then treated for 48 h with 600 nM doxorubicin. Immunoblot analysis using the indicated antibodies is shown. (B) Caov4, HCT116, and U1810 cells were treated with 600 nM doxorubicin and 40 μM zVAD-fmk. Lysates from untreated and treated cells were immunoprecipitated with anti-caspase-8 antibodies. Immunoblot analysis using the indicated antibodies is shown. (C) Immunofluorescence analysis using anti-phospho-MLKL antibodies in Caov4, HCT116, and U1810 cells treated with the indicated agents. Green dots indicate phospho-MLKL. Cell nuclei were counterstained with Hoechst 33342 (blue). Scale bars: 10 μm.

Figure 2

Necroptosis execution in cells upon mitotic catastrophe induction. (A) Caov4, HCT116, and U1810 cells were pre-treated with 40 μM zVAD-fmk and/or 30 μM necrostatin-1s then treated for 48 h with 600 nM doxorubicin. Immunoblot analysis using the indicated antibodies is shown. (B) Caov4, HCT116, and U1810 cells were treated with 600 nM doxorubicin and 40 μM zVAD-fmk. Lysates from untreated and treated cells were immunoprecipitated with anti-caspase-8 antibodies. Immunoblot analysis using the indicated antibodies is shown. (C) Immunofluorescence analysis using anti-phospho-MLKL antibodies in Caov4, HCT116, and U1810 cells treated with the indicated agents. Green dots indicate phospho-MLKL. Cell nuclei were counterstained with Hoechst 33342 (blue). Scale bars: 10 μm.

Figure 3

Crosstalk between necroptosis, autophagy, and apoptosis after mitotic catastrophe induction. (A) Caov4 and HCT116 cells were treated with 600 nM doxorubicin and 40 μM zVAD-fmk, and/or 30 μM necrostatin-1s, and/or 25 nM bafilomycin A1 for 48 h. Immunoblot analysis using the indicated antibodies is shown. (B) Sub-G1 analysis of Caov4 and HCT116 cells treated with 600 nM doxorubicin and 40 μM zVAD-fmk, and/or 30 μM necrostatin-1s, and/or 25 nM bafilomycin A1 for 48 h. Values are the mean (±standard deviation of the mean) of three independent experiments. (C) U1810 wild type and ATG13-knockout cells were treated with 600 nM doxorubicin and 40 μM zVAD-fmk and/or 30 μM necrostatin-1s for 24 h. Immunoblot analysis using the indicated antibodies is shown. * p < 0.05, ** p < 0.01.

Figure 3

Crosstalk between necroptosis, autophagy, and apoptosis after mitotic catastrophe induction. (A) Caov4 and HCT116 cells were treated with 600 nM doxorubicin and 40 μM zVAD-fmk, and/or 30 μM necrostatin-1s, and/or 25 nM bafilomycin A1 for 48 h. Immunoblot analysis using the indicated antibodies is shown. (B) Sub-G1 analysis of Caov4 and HCT116 cells treated with 600 nM doxorubicin and 40 μM zVAD-fmk, and/or 30 μM necrostatin-1s, and/or 25 nM bafilomycin A1 for 48 h. Values are the mean (±standard deviation of the mean) of three independent experiments. (C) U1810 wild type and ATG13-knockout cells were treated with 600 nM doxorubicin and 40 μM zVAD-fmk and/or 30 μM necrostatin-1s for 24 h. Immunoblot analysis using the indicated antibodies is shown. * p < 0.05, ** p < 0.01.

Figure 4

Caspase inhibition increases the population of cells with mitotic catastrophe morphology. (A) Quantification of mitotic catastrophe after 48 h treatment with 600 nM doxorubicin in combination with 40 μM zVAD-fmk, and/or 30 μM necrostatin-1s, and/or 25 nM bafilomycin A1 in Caov4 and HCT116 cells. (B) Quantification of mitotic catastrophe after 24 h of treatment with 600 nM doxorubicin in combination with 40 μM zVAD-fmk, and/or 30 μM necrostatin-1s in wild type and ATG13-knockout U1810 cells. The number of mitotic catastrophe cells examined in each cell line are shown in the bars. Values are the mean (±standard deviation of the mean) of three independent experiments. * p < 0.05, ** p < 0.01, ns—not significant.

Figure 5

Interplay between necroptosis, autophagy, and apoptosis under conditions of mitotic catastrophe upon doxorubicin/doxorubicin-zVAD-fmk treatment. Doxorubicin treatment leads to mitotic catastrophe formation, which can be terminated by apoptosis. If catalytic activity of caspases is blocked, cells accumulate in the state of mitotic catastrophe and die via necroptosis. The development of mitotic catastrophe triggers autophagy modulation inhibition of which promotes apoptosis and necroptosis.