All-trans-retinoic acid inhibits growth of head and neck cancer stem cells by suppression of Wnt/β-catenin pathway Young Chang Lim, Hyun Jung Kang, Young.

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All-trans-retinoic acid inhibits growth of head and neck cancer stem cells by suppression of Wnt/β-catenin pathway Young Chang Lim, Hyun Jung Kang, Young Sook Kim, Eun Chang Choi European Journal of Cancer Volume 48, Issue 17, Pages 3310-3318 (November 2012) DOI: 10.1016/j.ejca.2012.04.013 Copyright © 2012 Elsevier Ltd Terms and Conditions

Fig. 1 HNSC CSCs have cancer stem cell like phenotypes. (a) Single cell suspension from primary specimen form spheroid clusters 2weeks after plating. (scale bar, 10μm). (b) Stem cell or differentiation markers expression in HNSC CSC and differentiated HNSC CSC (HNSC CSC(D)) by foetal bovine serum (FBS). (c) In vivo tumourigenicity of HNSC CSC. European Journal of Cancer 2012 48, 3310-3318DOI: (10.1016/j.ejca.2012.04.013) Copyright © 2012 Elsevier Ltd Terms and Conditions

Fig. 2 RA inhibits proliferation of HNSC CSCs. (a) Photomicrographics and Quantitative analysis of sphere with or without RA treatment (20μM) for 2weeks, (scale bar, 10μm) (n=3). (b) Cell cycle analysis of HNSC CSC with or without RA treatment for 48h (n=3). (c) The effects of RA on cell cycle related proteins of HNSC SCS. European Journal of Cancer 2012 48, 3310-3318DOI: (10.1016/j.ejca.2012.04.013) Copyright © 2012 Elsevier Ltd Terms and Conditions

Fig. 3 RA suppresses stem cell markers expression of HNSC CSCs. HNSC CSCs were treated with RA for 48h and stained with anti-Oct4, Sox2 and Nestin antibodies (a). In addition, Western blot analysis for Sox2, Nestin, CD44 (a), and involucrin (b) after RA treatment for 48h was performed. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). European Journal of Cancer 2012 48, 3310-3318DOI: (10.1016/j.ejca.2012.04.013) Copyright © 2012 Elsevier Ltd Terms and Conditions

Fig. 4 RA augments chemosensitising effects of conventional chemoagents for head and neck squamous carcinoma cancer stem cells (HNSC CSCs). (a) 3-(4,4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed cell survival rates of single-dissociated HNSC CSC and differentiated HNSC CSC grown in serum-free culture conditions in the presence of RA (20Mm) alone, cisplatin (10 or 20μM) alone, combination of RA and cisplatin (n=3), ∗∗p<0.01. (b) The effect of cisplatin alone (10μM), RA alone (20μM), or combination of RA and cisplatin on expression of cleavaged caspase-3 after 48h treatment. European Journal of Cancer 2012 48, 3310-3318DOI: (10.1016/j.ejca.2012.04.013) Copyright © 2012 Elsevier Ltd Terms and Conditions

Fig. 5 RA induces antitumour effects in xenograft model of head and neck squamous carcinoma cancer stem cells (HNSC CSCs). Gross finding of tumour harvested from nude mouse after 21days of RA treatment and bar graph described serial change of tumour volume measured every 3days after RA treatment, n=6, bar 5mm. European Journal of Cancer 2012 48, 3310-3318DOI: (10.1016/j.ejca.2012.04.013) Copyright © 2012 Elsevier Ltd Terms and Conditions

Fig. 6 RA suppresses Wnt/β-catenin signaling of HNSC CSCs. (a) β-catenin expression after 20μM RA treatment for 24h and 48h by Western blotting. (b) β-catenin expression after 20μM RA treatment for 48h by immunoflurorescence. (c) Reporter gene assay of TOP/Flash of HNSC CSC after RA treatment for 48h. Identification of a constitutively stabilised mutant β-catenin plasmid (pcDNA3-S33Y β-catenin) transfection to HNSC CSCs by detection of anti-flag antibody. (e) Reporter gene assay of TOP/Flash of β-catenin overexpressed HNSC CSCs and control cells. (f) Stem cell markers expression of β-catenin overexpressed HNSC CSCs and control cells. (g) Rescue of sphere-forming capacity by β-catenin overexpression after 20μM RA treatment for 48h, ∗p<0.05. (h) Rescue of cytotoxicity of 20μM RA treatment for 48h by β-catenin overexpression using MTT assay, ∗p<0.05. European Journal of Cancer 2012 48, 3310-3318DOI: (10.1016/j.ejca.2012.04.013) Copyright © 2012 Elsevier Ltd Terms and Conditions

Supplementary Figure 1 European Journal of Cancer 2012 48, 3310-3318DOI: (10.1016/j.ejca.2012.04.013) Copyright © 2012 Elsevier Ltd Terms and Conditions