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Evaluation of phytoestrogens in inducing cell death mediated by decreasing Annexin A1 in Annexin A1-knockdown leukemia cells

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Abstract

Background

Phytoestrogens are plant compounds that are structurally similar to estrogen and that possess anti-cancer properties. Previous studies have reported that coumestrol, daidzein and genistein could induce cell death by reducing Annexin A1 protein in leukemic cell lines. Annexin A1 (ANXA1) is involved in cell progression, metastasis, and apoptosis in several types of cancer cells. The present study sought to investigate if the effects of phytoestrogens on apoptosis, cell cycle arrest and phagocytosis in ANXA1-knockdown leukemic cells are mediated through ANXA1 or occurred independently.

Methods

Transfection of ANXA1 siRNA was conducted to downregulate ANXA1 expression in Jurkat, K562 and U937 cells. Apoptosis and cell cycle assays were conducted using flow cytometry. Western blot was performed to evaluate ANXA1, caspases and Bcl-2 proteins expression. Phagocytosis was determined using hematoxylin and eosin staining.

Results

The expression of ANXA1 after the knockdown was significantly downregulated in all cell lines. Genistein significantly induced apoptosis associated with an upregulation of procaspase-3, −9, and − 1 in Jurkat cells. The Bcl-2 expression showed no significant difference in Jurkat, K562 and U937 cells. Treatment with phytoestrogens increased procaspase-1 expression in Jurkat and U937 cells while no changes were detected in K562 cells. Flow cytometry analysis demonstrated that after ANXA1 knockdown, coumestrol and genistein caused cell cycle arrest at G2/M phase in selected type of cells. The percentage of phagocytosis and phagocytosis index increased after the treatment with phytoestrogens in all cell lines.

Conclusion

Phytoestrogens induced cell death in ANXA1-knockdown leukemia cells, mediated by Annexin A1 proteins.

Graphical abstract

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Abbreviations

ANXA1:

annexinA1

Annexin V-FITC:

annexin V-fluorescein

PI:

propidium iodide

RPMI-1640:

Roswell Park Memorial Institute medium 1640

IMDM:

Iscove’s modified Dulbecco’s medium

FBS:

fetal bovine serum

siRNA:

small interfering RNA

ER:

estrogen receptor

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Acknowledgements

This project has been funded by Ministry of Higher Education for Fundamental Research Grant Scheme (FRGS), FRGS/1/2015/SKK09/UKM/02/3.

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All data generated or analyzed during this study are included in this published article and supplementary data.

Author information

MH and EK planned the experimental phase and MH performed the experiments and carried out the statistical analysis. EK, MJ, JAJ and FR conceptualized the study hypothesis and coordinated the research activity. The manuscript was written by MH, EK and reviewed by JAJ, NA and FR. All authors have read and approved the manuscript.

Correspondence to Endang Kumolosasi.

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Hasan, M., Kumolosasi, E., Jasamai, M. et al. Evaluation of phytoestrogens in inducing cell death mediated by decreasing Annexin A1 in Annexin A1-knockdown leukemia cells. DARU J Pharm Sci (2020) doi:10.1007/s40199-019-00320-0

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Keywords

  • Annexin A1
  • siRNA
  • Apoptosis
  • Caspase cascade
  • Cell cycle arrest
  • Phagocytosis
  • Human leukemic cell lines