, Volume 24, Issue 1–2, pp 21–32 | Cite as

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

  • Sagar Shelake
  • Umesh T. Sankpal
  • Don Eslin
  • W. Paul Bowman
  • Jerry W. Simecka
  • Sangram Raut
  • Anish Ray
  • Riyaz BashaEmail author


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.


Ewing sarcoma Tolfenamic acid Vincristine Sp1 Survivin 



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.

Compliance with Ethical Standards

Conflict of interest

All authors declare that they have no conflict of interest.

Supplementary material

10495_2018_1508_MOESM1_ESM.pdf (376 kb)
Supplementary material 1 (PDF 375 KB)


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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Pediatrics and Women’s Health, Texas College of Osteopathic MedicineUniversity of North Texas Health Science CenterFort WorthUSA
  2. 2.Pre-clinical Services, UNT Systems College of PharmacyUniversity of North Texas Health Science CenterFort WorthUSA
  3. 3.Department of Physiology and AnatomyUniversity of North Texas Health Science CenterFort WorthUSA
  4. 4.Arnold Palmer Hospital for ChildrenOrlandoUSA
  5. 5.Hematology and OncologyCook Children’s Medical CenterFort WorthUSA
  6. 6.Johns Hopkins University School of MedicineBaltimoreUSA

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