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Clotam enhances anti-proliferative effect of vincristine in Ewing sarcoma cells

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Current therapeutic strategies used in Ewing sarcoma (ES) especially for relapsed patients have resulted in modest improvements in survival over the past 20 years. Combination therapeutic approach presents as an alternative to overcoming drug resistance in metastatic ES. This study evaluated the effect of Clotam (tolfenamic acid or TA), a small molecule and inhibitor of Specificity protein1 (Sp1) and survivin for sensitizing ES cell lines to chemotherapeutic agent, vincristine (VCR). ES cells (CHLA-9 and TC-32) were treated with TA or VCR or TA + VCR (combination), and cell viability was assessed after 24/48/72 h. Effect of TA or VCR or TA + VCR treatment on cell cycle arrest and apoptosis were evaluated using propidium iodide, cell cycle assay and Annexin V flow cytometry respectively. The apoptosis markers, caspase 3/7 (activity levels) and cleaved-PARP (protein expression) were measured. Cardiomyocytes, H9C2 were used as non-malignant cells. While, all treatments caused time- and dose-dependent inhibition of cell viability, interestingly, combination treatment caused significantly higher response (~ 80% inhibition, p < 0.05). Cell viability inhibition was accompanied by inhibition of Sp1 and Survivin. TA + VCR treatment significantly (p < 0.05) increased caspase 3/7 activity which strongly correlated with upregulated c-PARP level and Annexin V staining. Cell cycle arrest was observed at G0/G1 (TA) or G2/M (VCR and TA + VCR). All treatments did not cause cytotoxicity in H9C2 cells. These results suggest that TA could enhance the anti-cancer activity of VCR in ES cells. Therefore, TA + VCR combination could be further tested to develop as safe/effective therapeutic strategy for treating ES.

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Acknowledgements

This work is partially supported by HyundaiHopeOnWheels Young Investigator Grant awarded to AR. RB is supported by National Institute for Minority Health and Health Disparities (Grant # 2U54 MD006882-06) and SR is supported by a grant from the Rutledge Cancer Foundation. Authors thank Childhood Cancer Repository at the Texas Tech University Health Sciences Center in Lubbock, TX for providing Ewing sarcoma cell lines.

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Author notes

  1. Sagar Shelake

    Present address: Johns Hopkins University School of Medicine, Baltimore, MD, 21287, USA

Authors and Affiliations

  1. Department of Pediatrics and Women’s Health, Texas College of Osteopathic Medicine, University of North Texas Health Science Center, 3500 Camp Bowie Blvd, Fort Worth, TX, 76107, USA

    Sagar Shelake, Umesh T. Sankpal, W. Paul Bowman & Riyaz Basha

  2. Pre-clinical Services, UNT Systems College of Pharmacy, University of North Texas Health Science Center, Fort Worth, TX, 76107, USA

    Jerry W. Simecka

  3. Department of Physiology and Anatomy, University of North Texas Health Science Center, Fort Worth, TX, 76107, USA

    Sangram Raut

  4. Arnold Palmer Hospital for Children, Orlando, FL, 32806, USA

    Don Eslin

  5. Hematology and Oncology, Cook Children’s Medical Center, Fort Worth, TX, 76104, USA

    W. Paul Bowman, Anish Ray & Riyaz Basha

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Shelake, S., Sankpal, U.T., Eslin, D. et al. Clotam enhances anti-proliferative effect of vincristine in Ewing sarcoma cells. Apoptosis 24, 21–32 (2019). https://doi.org/10.1007/s10495-018-1508-1

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