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Suppression of a cancer stem-like phenotype mediated by alpha-lipoic acid in human lung cancer cells through down-regulation of β-catenin and Oct-4

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Abstract

Purpose

Cancer stem cells (CSCs) that possess the ability of self-renewal and multi-potency have been shown to drive tumor progression and metastasis. The majority of recent studies has focused on potential molecules targeting CSCs so as to develop novel strategies for efficient cancer treatment or protection. Here, we show how alpha-lipoic acid (LA), an endogenous mitochondrial anti-oxidant, affects the CSC-like phenotypes of human non-small cell lung cancer-derived H23, H292 and H460 cells.

Methods

CSC-like phenotypes were verified by anchorage-independent growth, three-dimensional (3D) spheroid formation and the expression of CSC markers. Enriched CSC populations were used to confirm the effects of LA. Protein ubiquitination and degradation were assessed using immunoprecipitation.

Results

We found that treatment with LA reduced the CSC-like phenotype, as indicated by a decreased expression of known CSC markers (CD133, CD44, ALDH1A1, Oct-4 and Nanog) in H460 cells. In addition, we found that LA reduced the CSC-related abilities of anchorage-independent growth and 3D spheroid formation, and suppressed factors related to epithelial-mesenchymal transition, such as E-cadherin, Vimentin, Slug and Snail. Mechanistically, we found that LA suppresses CSC through depletion of the cellular stemness proteins β-catenin and Oct-4 via decreasing the level of active (phosphorylated) Akt. This resulted in the induction of GSK3β-dependent β-catenin ubiquitin-proteasomal degradation and a decrease in the stabilized (phosphorylated) form of Oct-4. The effects of LA on the CSC-like phenotypes were confirmed in CSC enriched H460, H292 and H23 non-small cell lung cancer-derived cells.

Conclusion

Our data are indicative for a novel regulatory role and underlying mechanism of LA in the negative regulation of a CSC-like phenotype in non-small cell lung cancer-derived cells.

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Acknowledgments

This research was supported by the Rachadapisek Sompote Fund for Postdoctoral Fellowship, Grant for International Research Integration: Chula Research Scholar, Ratchadaphiseksomphot Endowment Fund and Pharmaceutical Sciences Research Fund, Chulalongkorn University, and Mr. Krich Rajprasit.

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Authors and Affiliations

  1. Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Phatumwan, Bangkok, 10330, Thailand

    Preeyaporn Plaimee Phiboonchaiyanan & Pithi Chanvorachote

  2. Cell-Based Drug and Health Product Development Research Unit, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, 10330, Thailand

    Preeyaporn Plaimee Phiboonchaiyanan & Pithi Chanvorachote

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  1. Preeyaporn Plaimee Phiboonchaiyanan
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Correspondence to Pithi Chanvorachote.

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Electronic supplementary material

Supplementary Fig. 1

LA inhibits the expression of CSC markers, self-renewal transcription factors, EMT-associated proteins and up-stream signaling proteins. H460 cells were treated with LA (5 μM) for 12, 24 and 48 h. After treatment, (A, C) the expression levels of CD133, CD44, ALDH1A1, E-cadherin, Vimentin, Slug and Snail were determined by Western blotting and the relative protein levels were quantified by densitometry. (B, D, E, F) Dose-dependent effect of LA on the expression of CSC markers, self-renewal transcription factors, EMT-associated proteins, and up-stream signaling proteins in H460 cells after LA (0–5 μM) treatment for 48 h. After treatment, the expression levels of CD133, CD44, ALDH1A1, Oct-4, p-Oct-4, Nanog, E-cadherin, Vimentin, Slug, Snail, β-catenin, p-Akt (Ser 473), Akt, p-GSK3β (Ser 9) and GSK3β were determined by Western blotting, and the relative protein levels quantified by densitometry. (G) Cell lysate of the parental wild type cells (WT) and CSC-enriched cells were prepared and analyzed for CD133, CD44, ALDH1A1, Oct-4, p-Oct-4 and Nanog by immunoblotting, and the relative protein levels quantified by densitometry. Data represent mean ± SD (n = 3). * p < 0.05 versus non-treated control. # p < 0.05 versus non-treated wild type control. (GIF 123 kb)

High resolution image (TIFF 1083 kb)

Supplementary Fig. 2

LA inhibits CSC-related proteins in H292 and H23 cell lines. The cells were treated with LA (0–5 μM) for 48 h. After treatment, (E-J) the expression levels of CD133, CD44, ALDH1A1, Oct-4, p-Oct-4, Nanog, β-catenin, p-Akt (Ser 473), Akt, p-GSK3β (Ser 9) and GSK3β were determined by Western blotting, and the relative protein levels quantified by densitometry. Data represent mean ± SD (n = 3). * p < 0.05 versus the untreated control. (GIF 72 kb)

High resolution image (TIFF 584 kb)

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Phiboonchaiyanan, P.P., Chanvorachote, P. Suppression of a cancer stem-like phenotype mediated by alpha-lipoic acid in human lung cancer cells through down-regulation of β-catenin and Oct-4. Cell Oncol. 40, 497–510 (2017). https://doi.org/10.1007/s13402-017-0339-3

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