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. 2017 Sep 16;491(2):463-468.
doi: 10.1016/j.bbrc.2017.07.050. Epub 2017 Jul 14.

ML327 induces apoptosis and sensitizes Ewing sarcoma cells to TNF-related apoptosis-inducing ligand

Eric J Rellinger  1 , Chandrasekhar Padmanabhan  2 , Jingbo Qiao  1 , Andrew Appert  3 , Alex G Waterson  4 , Craig W Lindsley  4 , R Daniel Beauchamp  5 , Dai H Chung  6
Affiliations

ML327 induces apoptosis and sensitizes Ewing sarcoma cells to TNF-related apoptosis-inducing ligand

Eric J Rellinger et al. Biochem Biophys Res Commun. .

Abstract

Ewing sarcomas are rare mesenchymal-derived bone and soft tissue tumors in children. Afflicted children with distant metastases have poor survival despite aggressive therapeutics. Epithelial-to-mesenchymal transition in epithelial carcinomas is associated with loss of E-cadherin and resistance to apoptosis. ML327 is a novel small molecule that we have previously shown to reverse epithelial-to-mesenchymal transition features in both epithelial and neural crest-derived cancers. Herein, we sought to evaluate the effects of ML327 on mesenchymal-derived Ewing sarcoma cells, hypothesizing that ML327 initiates growth arrest and sensitizes to TNF-related apoptosis-inducing ligand. ML327 induced protein expression changes, increased E-cadherin and decreased vimentin, consistent with partial induction of mesenchymal-to-epithelial transition in multiple Ewing Sarcoma cell lines (SK-N-MC, TC71, and ES-5838). Induction of epithelial features was associated with apoptosis, as demonstrated by PARP and Caspase 3 cleavage by immunoblotting. Cell cycle analysis validated these findings by marked induction of the subG0 cell population. In vitro combination treatment with TRAIL demonstrated additive induction of apoptotic markers. Taken together, these findings establish a rationale for further in vivo trials of ML327 in cells of mesenchymal origin both alone and in combination with TRAIL.

Keywords: Apoptosis; Epithelial-to-mesenchymal transition; Ewing sarcoma; Isoxazole; ML327; TRAIL.

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Conflict of interest statement

Conflicts of Interest

The authors declare no potential conflicts of interest.

Figures

Figure 1
Figure 1. ML327 Induces MET Features in ES Cells
A. Immunoblotting demonstrates that ML327 enhanced E-cadherin expression in all tested ES cells and blocked vimentin in ES-5838 and TC71 cells. β-actin served as our protein loading control. B. Light microscopy (10x) demonstrates that ML327 (10 μM) treatment increases the proportion of rounded cells with cytoplasmic shrinking and non-adherent vesicles thought to likely represent apoptotic bodies.
Figure 2
Figure 2. PARP and Caspase 3 Cleavage are Induced in ES Cells by ML327
A–C. Immunoblotting demonstrates the onset of Caspase 3 and PARP cleavage over the course of 4 days with or without ML327 (10 μM). β-actin was our protein loading control.
Figure 3
Figure 3. ML327 Treatment Enhances DNA Fragmentation in ES Cells
Cell cycle analysis with propidium iodide was performed in ES cells after 72h of treatment with ML327 (10 μM). A. Representative cell cycle analysis for TC71 ES cells. B–D. Stacked histograms demonstrate marked enrichment of the sub G0 phase was observed by flow cytometry with propidium iodide staining in all three ES cell lines following 72h of ML327 treatment (10 μM). All cell lines were completed in triplicate with representative replicates displayed.
Figure 4
Figure 4. ML327 Sensitizes ES Cells to TRAIL-mediated Apoptosis
A. Schematic demonstrating the role of cFLIP in antagonizing Trail-mediated apoptosis. B–D. Western blotting demonstrates increased TRAIL-mediated PARP and Caspase 3 cleavage in ES cells pretreated with ML327 (10 μM). Decreased cFLIPs was observed in all tested ES cell lines. β-actin was our protein loading control.
See this image and copyright information in PMC

References

    1. Potratz J, Dirksen U, Jurgens H, Craft A. Ewing sarcoma: clinical state-of-the-art. Pediatr Hematol Oncol. 2012;29:1–11. - PubMed
    1. Amaral AT, Manara MC, Berghuis D, Ordonez JL, Biscuola M, Lopez-Garcia MA, Osuna D, Lucarelli E, Alviano F, Lankester A, Scotlandi K, de Alava E. Characterization of human mesenchymal stem cells from ewing sarcoma patients. Pathogenetic implications. PloS one. 2014;9 - PMC - PubMed
    1. Lin PP, Wang Y, Lozano G. Mesenchymal Stem Cells and the Origin of Ewing’s Sarcoma. Sarcoma. 2011;276463:5. - PMC - PubMed
    1. Gorlick R, Janeway K, Lessnick S, Randall RL, Marina N. Children’s Oncology Group’s 2013 blueprint for research: bone tumors. Pediatr Blood Cancer. 2013;60:1009–1015. - PMC - PubMed
    1. Nieto MA, Huang Ruby Y-J, Jackson Rebecca A, Thiery Jean P EMT. Cell. 2016;166:21–45. - PubMed

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