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Reversal of cisplatin sensitization and abrogation of cisplatin-enriched cancer stem cells in 5-8F nasopharyngeal carcinoma cell line through a suppression of Wnt/β-catenin-signaling pathway

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Abstract

Nasopharyngeal carcinoma (NPC) is one of the rare cancers in western countries but predominant in Southeast Asian countries including Thailand. One major cause for failure of NPC chemotherapeutic treatments is reportedly correlated with the elevation of cancer stem cell (CSC) fractions. Thus, this present study aims to investigate the effect of cisplatin (CDDP) treatment on the enrichment of cancer stem-like cells (CSCs) and its associated signaling pathway in EBV-negative NPC cells. Cisplatin-pretreated 5-8F NPC cells (5-8F CDDP) were first generated by treating the cells with 0.5 μM cisplatin for 48 h. After the instant treatment, 5-8F CDDP showed increased IC50 values, demonstrating a decrease in CDDP sensitization. Besides, the proportion of NPC cells with cancer stem-like phenotypes comprising side population (SP), key stemness-related gene expressions including SOX2, ALDH1, CD24 was significantly enhanced. Additionally, 5-8F CDDP displayed the upregulation of β-catenin gene, suggesting its association with the CSC-initiating mechanism. Furthermore, a tankyrase inhibitor for Wnt/β-catenin pathway, XAV939, substantially reduced CSCs and retrieved the cisplatin sensitivity in 5-8F CDDP. This confirms that the Wnt/β-catenin signaling is accountable for rising of the CSC population in EBV-negative NPC. Finally, the combined treatment of CDDP and XAV939 exhibited lower 5-8F CDDP cell viability compared to the treatment of CDDP alone, suggesting the reversal of cisplatin sensitization. In conclusion, the enhancement of CSCs in 5-8F NPC cells caused by the instant cisplatin treatment is initially mediated through the upregulation of β-catenin and activation of Wnt/β-catenin signaling pathway. As a result, a primary chemotherapeutic treatment with closely monitoring the targeted Wnt/β-catenin signaling pathway could potentially prevent the development of CSCs and improve the treatment efficiency in NPC.

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Abbreviations

CSC:

Cancer stem cell

CDDP:

Cisplatin

DMSO:

Dimethyl sulfoxide

EBV:

Epstein Barr virus

FBS:

Fetal bovine serum

HRP:

Horseradish peroxidase

NPC:

Nasopharyngeal carcinoma

PBS:

Phosphate buffer saline

SP:

Side population

SEM:

Stand error of mean

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Acknowledgements

The authors would like to thank Siriraj Center of Excellence for Stem Cell Research (SiSCR), Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand for kindly providing flow cytometry equipment (BD FACS Canto). We also would like to thank Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok, Thailand for providing laboratory equipment.

Funding

This research is supported by Kasetsart University Research and Development Institute (Grant no. P-3.1(D)41.61) to PK and National Research Council Thailand and Mahidol University to TJ. SS received the 50th Faculty of Science Scholarship from Faculty of Science, Kasetsart University, Thailand.

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Authors and Affiliations

  1. Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok, 10900, Thailand

    Sirorut Sinnung & Pichamon Kiatwuthinon

  2. Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand

    Tavan Janvilisri

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  1. Sirorut Sinnung
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Contributions

Sirorut Sinnung: Methodology, investigation, data curation, writing—original draft. Tavan Janvilisri: Resources, writing—review and editing, funding acquisition. Pichamon Kiatwuthinon: Conceptualization, supervision, writing—review and editing, project administration, funding acquisition

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Correspondence to Pichamon Kiatwuthinon.

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Supplementary Information

Fig. S1

(a) A cell viability of the 5-8F cells treated with 0.5 and 1 μM of CDDP at 48-h incubation. Data is presented as mean ± SEM from three independent experiments. Statistical analysis was performed using an unpaired t-test, *p < 0.05, ns not statistically significant. (b) The time-course relative gene expressions of drug efflux pumps including ABCB1, ABCC1, ABCC2 and ABCG2 in the 5-8F CDDP. The relative gene expressions were significantly upregulated in 5-8F CDDP at 48-hour incubation compared to 5-8F NPC cells. Values are presented as mean ± SEM from three independent biological replicates. An unpaired t-test was used in the statistical analysis between 5-8FCDDP and 5-8F NPC cells, *p < 0.05, **p < 0.01. (c) Time-course western blot analysis of ABCB1 in 5-8F CDDP cells revealed a significant increase in the expression of ABCB1 from 24 to 48 h that confirmed the relative gene expression result. The expression of GAPDH was used as a loading control. Values are presented as mean ± SEM from three independent experiments. An unpaired t-test is used in the statistical analysis between 5-8FCDDP and 5-8F NPC cells, *p < 0.05. The intensities of protein bands were determined by using ImageJ program (Version 1.52r) (https://imagej.nih.gov/) (DOCX 525 kb)

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Sinnung, S., Janvilisri, T. & Kiatwuthinon, P. Reversal of cisplatin sensitization and abrogation of cisplatin-enriched cancer stem cells in 5-8F nasopharyngeal carcinoma cell line through a suppression of Wnt/β-catenin-signaling pathway. Mol Cell Biochem 476, 1663–1672 (2021). https://doi.org/10.1007/s11010-020-04045-6

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