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. 2017 Apr 18;8(32):52445-52456.
doi: 10.18632/oncotarget.17190. eCollection 2017 Aug 8.

Eribulin alone or in combination with the PLK1 inhibitor BI 6727 triggers intrinsic apoptosis in Ewing sarcoma cell lines

Lilly Magdalena WeiΔ  1   2   3 , Manuela Hugle  1 , Simone Fulda  1   2   3
Affiliations

Eribulin alone or in combination with the PLK1 inhibitor BI 6727 triggers intrinsic apoptosis in Ewing sarcoma cell lines

Lilly Magdalena WeiΔ et al. Oncotarget. .

Abstract

In this study, we investigated the molecular mechanisms of eribulin-induced cell death and its therapeutic potential in combination with the PLK1 inhibitor BI 6727 in Ewing sarcoma (ES). Here, we show that eribulin triggers cell death in a dose-dependent manner in a panel of ES cell lines. In addition, eribulin at subtoxic, low nanomolar concentrations acts in concert with BI 6727 to induce cell death and to suppress long-term clonogenic survival. Mechanistic studies reveal that eribulin monotherapy at cytotoxic concentrations and co-treatment with eribulin at subtoxic concentrations together with BI 6727 arrest cells in the M phase of the cell cycle prior to the onset of cell death. This mitotic arrest is followed by increased phosphorylation of BCL-2 and BCL-xL as well as downregulation of MCL-1, suggesting inactivation of these antiapoptotic BCL-2 family proteins. Consistently, eribulin monotherapy and eribulin/BI 6727 co-treatment trigger activation of BAX, a key proapoptotic BCL-2 family protein, and increase proteolytic activation of caspase-9 and -3. Importantly, overexpression of BCL-2 or addition of the broad-range caspase inhibitor zVAD.fmk significantly rescue eribulin- as well as eribulin/BI 6727-induced cell death. Together, these findings demonstrate that eribulin induces cell death via the intrinsic pathway of apoptosis in ES cells, both alone at cytotoxic concentrations and in combination with BI 6727 at subtoxic concentrations. Thus, our study highlights the therapeutic potential of eribulin for the treatment of ES alone or in rational combination therapies.

Keywords: Ewing sarcoma; PLK1; apoptosis; eribulin.

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Conflict of interest statement

CONFLICTS OF INTEREST None to declare.

Figures

Figure 1
Figure 1. Eribulin induces cell death in ES cells and cooperates with the PLK1 inhibitor BI 6727
(A–B) ES cells were treated for 48 hours with indicated concentrations of eribulin (A) and/or BI 6727 (B). Apoptosis was determined by quantification of DNA fragmentation of PI-stained nuclei using flow cytometry. Results are expressed as mean with SD of at least three independent experiments performed in triplicate; *p < 0.05; **p < 0.01; ***p < 0.001. (C–D) A4573 cells were treated for 15 hours with 2 nM eribulin (ERI) (C) or for 18 hours with 0.4 nM eribulin and/or 15 nM BI 6727 (D), SK-ES-1 cells were treated for 15 hours with 1 nM eribulin (C) or for 18 hours with 0.15 nM eribulin and/or 15 nM BI 6727 (D). Subsequently the treatment was replaced by drug-free medium and ES cells were incubated for additional 9 (A4573) or 12 days (SK-ES-1). Colonies were stained with crystal violet solution and counted by using ImageJ software. The colony count is expressed as percentage of untreated controls (left panels) and representative images are shown (right panels). Results are presented as mean with SD of at least three independent experiments performed in triplicate; *p < 0.05; **p < 0.01.
Figure 2
Figure 2. Caspases contribute to eribulin- and eribulin/BI 6727-induced cell death
(A–B) A4573 cells were treated with 2 nM eribulin (ERI) (A) or 0.4 nM eribulin and/or 15 nM BI 6727 (B), SK-ES-1 cells with 1 nM eribulin (A) or 0.15 nM eribulin and/or 15 nM BI 6727 (B) for 48 hours in the presence or absence of the broad-range caspase inhibitor zVAD.fmk (20 μM). Apoptosis was determined by quantification of DNA fragmentation of PI-stained nuclei using flow cytometry. Results are expressed as mean with SD of at least three independent experiments performed in triplicate; *p < 0.05; **p < 0.01; ***p < 0.001. (C–D) A4573 cells were treated with 2 nM eribulin (ERI) (C) or 0.4 nM eribulin and/or 15 nM BI 6727 (D), SK-ES-1 cells with 1 nM eribulin (C) or 0.15 nM eribulin and/or 15 nM BI 6727 (D) for indicated times. Cleavage of caspase-8, -9 and -3 was examined by Western blotting. A 2 hour treatment of SK-ES-1 cells with 2 μg/mL TRAIL receptor-2 agonistic antibody ETR2 was used as a positive control (PC) for caspase activation for both A4573 and SK-ES-1 cells. Expression of GAPDH was used as loading control. Arrowheads indicate active cleavage fragments, asterisks unspecific bands. Representative blots of two independent experiments are shown.
Figure 3
Figure 3. Eribulin and eribulin/BI 6727 co-treatment cause mitotic arrest prior to the onset of cell death
(A–B) A4573 cells were treated with 2 nM eribulin (ERI) (A) or 0.4 nM eribulin and/or 15 nM BI 6727 (B), SK-ES-1 cells with 1 nM eribulin (A) or 0.15 nM eribulin and/or 15 nM BI 6727 (B) for indicated times. Apoptosis was assessed by quantification of DNA fragmentation of PI-stained nuclei using flow cytometry. Results are expressed as mean with SD of at least three independent experiments performed in triplicate; n.s., not significant; *p < 0.05; **p < 0.01; ***p < 0.001 comparing eribulin-treated to non-treated or eribulin/BI 6727-treated to BI 6727-treated cells. (C–F) A4573 cells were treated with 2 nM eribulin (ERI) (C and E) or 0.4 nM eribulin and/or 15 nM BI 6727 (D and F), SK-ES-1 cells with 1 nM eribulin (C and E) or 0.15 nM eribulin and/or 15 nM BI 6727 (D and F) for 12 hours. Cell cycle analysis was performed by FlowJo software after measurement of PI-stained nuclei using flow cytometry. Results are expressed as representative cell cycle profiles (C and D) or as mean with SD of at least three independent experiments performed in triplicate (E and F); *p < 0.05; **p < 0.01; ***p < 0.001 comparing the frequency of cells arrested in G2/M phase. (G–H) A4573 cells were treated with 2 nM eribulin (ERI) (G) or 0.4 nM eribulin and/or 15 nM BI 6727 (H), SK-ES-1 cells with 1 nM eribulin (G) or 0.15 nM eribulin and/or 15 nM BI 6727 (H) for indicated times. Expression levels of H3 and pH3 were analyzed by Western blotting. Expression of GAPDH, β-actin or α-tubulin served as loading control. Results are shown as representative blots of two independent experiments.
Figure 4
Figure 4. Eribulin and eribulin/BI 6727 co-treatment trigger inactivation of antiapoptotic BCL-2 proteins
(A–B) A4573 cells were treated with 2 nM eribulin (ERI) (A) or 0.4 nM eribulin and/or 15 nM BI 6727 (B), SK-ES-1 cells with 1 nM eribulin (A) or 0.15 nM eribulin and/or 15 nM BI 6727 (B) for 15 hours. Expression levels of BCL-2, BCL-xL and MCL-1 were assessed by Western blotting. GAPDH served as loading control. Results are shown as representative blots of at least two independent experiments.
Figure 5
Figure 5. BCL-2 overexpression partially rescues eribulin- as well as eribulin/BI 6727-induced cell death
(A–C) ES cells were stably transduced with empty vector (EV) or murine BCL-2. Transduction efficiency of murine BCL-2 was analyzed by Western blotting. Expression of β-actin served as loading control. Representative blots of two independent experiments are shown (A). Stably transduced A4573 cells were treated with 2 nM eribulin (ERI) (B) or 0.4 nM eribulin and/or 15 nM BI 6727 (C), stably transduced SK-ES-1 cells with 1 nM eribulin (B) or 0.15 nM eribulin and/or 15 nM BI 6727 (C) for 48 hours. Apoptosis was assessed by quantification of DNA fragmentation of PI-stained nuclei using flow cytometry. Results are shown as mean with SD of at least three independent experiments performed in triplicate (B and C); n.s. not significant; *p < 0.05; ***p < 0.001.
Figure 6
Figure 6. Eribulin and eribulin/BI 6727 co-treatment cause BAX activation
(A–B) A4573 cells were treated with 2 nM eribulin (ERI) (A) or 0.4 nM eribulin and/or 15 nM BI 6727 (B), SK-ES-1 cells with 1 nM eribulin (A) or 0.15 nM eribulin and/or 15 nM BI 6727 (B) for 15 hours. Activation of BAX was assessed by immunoprecipitation of activated BAX using an active conformation-specific antibody (BAX-IP). Expression of β-actin or GAPDH served as loading control. Asterisks indicate unspecific bands. Results are shown as representative blots of at least two independent experiments.
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