A Triple Combination of Metformin, Acetylsalicylic Acid, and Oseltamivir Phosphate Impacts Tumour Spheroid Viability and Upends Chemoresistance in Triple-Negative Breast Cancer

Abstract

Introduction

Targeted multimodal approaches need to be strategically developed to control tumour growth and prevent metastatic burden successfully. Breast cancer presents a unique clinical problem because of the variety of cellular subtypes that arise. The tumour stage and cellular subtypes often dictate the appropriate clinical treatment regimen. Also, the development of chemoresistance is a common clinical challenge with breast cancer. Higher doses and additional drug agents can produce additional adverse effects leading to a more aggressive malignancy. Acetylsalicylic acid (ASA), metformin (Met), and oseltamivir phosphate (OP) were investigated for their efficacy to sensitize MDA-MB-231 triple-negative breast cancer and its tamoxifen (Tmx) resistant variant (MDA-MB-231-TmxR) together in combination with Tmx treatment.

Methods

Microscopic imaging, the formation of 3D multicellular tumour spheroids, immunocytochemistry, flow cytometry, Annexin V Assay, Caspase 3/7 Apoptosis Assay, tube formation assay and analysis, and WST-1 cell viability assay evaluated the formation of MCTS, morphologic changes, cell viability, apoptosis activity and the expression levels of ALDH1A1, CD44 and CD24 on the cell surface, MDA-MB231 triple-negative breast cancer, tamoxifen (Tmx) resistant variant (MDA-MB-231-TmxR).

Results

The results using a triple combination of ASA, Met and OP on MDA-MB-231 and MDA-MB-231-TmxR cells and their matrix-free 3D multicellular tumour spheroids (MCTS) formed by using the cyclic Arg-Gly-Asp-D-Phe-Lys peptide modified with 4-carboxybutyl-triphenylphosphonium bromide (cyclo-RGDfK(TPP)) peptide method demonstrate a consistent and significant decrease in cell and tumour spheroid viability and volume with increased apoptotic activity, and increased sensitivity to Tmx therapy. Tmx treatment of MDA-MB-231 cells in combination with ASA, Met and OP markedly reduced the CD44/CD24 ratio by 6.5-fold compared to the untreated control group. Tmx treatment of MDA-MB-231-TmxR cells in combination with ASA, Met and OP markedly reduced the ALDH1A1 by 134-fold compared to the same treatment for the parental cell line. Also, the triple combination treatment of ASA, Met, and OP inhibited vasculogenic endothelial cell tube formation and induced endothelial cell apoptosis.

Conclusion

For the first time, the findings demonstrate that repurposing ASA, Met, and OP provides a novel and promising targeted multimodal approach in the treatment of triple-negative breast cancer and its chemoresistant variant.

Keywords:

• MDA-MB-231
• MDA-MB-231-TmxR
• human umbilical vein endothelial cell lines
• multicellular tumour spheroids
• MCTS
• endothelial cell tube formation
• apoptosis

Acknowledgments

This work was supported in part by grants to M. R. Szewczuk from the Natural Sciences and Engineering Research Council of Canada (NSERC) and by a grant (N 18-04-01087) from the Russian Foundation for Basic Research (RFBR) to S. Burov and E. Markvicheva. M. Sambi is the recipient of the Queen’s University Graduate Award (QGA) and the R. Samuel McLaughlin Fellowship. B. Qorri is a recipient of the QGA, Terry Fox Research Institute Transdisciplinary Training Program in Cancer Research Scholarship, Dean’s Doctoral Award, and the Franklin Bracken Fellowship.

Abbreviations

ALDH1A1, aldehyde dehydrogenase-1 family member A1; ASA, acetylsalicylic Acid; CD, cluster of differentiation; MCTS, multicellular tumour spheroid; MDA-MB-231, human triple-negative breast cancer cell line; MDA-MB-231-R, chemoresistant human triple-negative breast cancer cell line; Met, metformin; OP, oseltamivir phosphate; RGDfK, Arg-Gly-Asp-D-Phe-Lys; Tmx, Tamoxifen; TNBC, triple-negative breast cancer; TPP, 4-carboxybutyl-triphenylphosphonium bromide.

Disclosure

Dr William Harless reports funding from ACOA/NRC and is employed by Encyt Technologies, Inc.. Sergey V Burov is employed by the Laboratory of Novel Peptide Therapeutics, Cytomed J.S.Co.. The authors have no other possible conflicts of interest in this work.