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Breast Cancer Research and Treatment

, Volume 165, Issue 3, pp 517–527 | Cite as

Pro-apoptotic effect of Δ2-TGZ in “claudin-1-low” triple-negative breast cancer cells: involvement of claudin-1

  • Marine Geoffroy
  • Alexandra Kleinclauss
  • Stéphanie Grandemange
  • Sébastien Hupont
  • Michel Boisbrun
  • Stéphane Flament
  • Isabelle Grillier-VuissozEmail author
  • Sandra Kuntz
Preclinical study
  • 236 Downloads

Abstract

Purpose

40% of triple-negative breast cancer (TNBC) do not express claudin-1, a major constituent of tight junction. Patients with these “claudin-1-low” tumors present a higher relapse incidence. A major challenge in oncology is the development of innovative therapies for such poor prognosis tumors. In this context, we study the anticancer effects of ∆2-TGZ, a compound derived from troglitazone (TGZ), on cell models of these tumors.

Methods and results

In MDA-MB-231 and Hs578T “claudin-1-low” TNBC cells, Δ2-TGZ treatment induced claudin-1 protein expression and triggered apoptosis as measured by FACS analysis (annexin V/PI co-staining). Interestingly, in the non-tumorigenic human breast epithelial cell line MCF-10A, the basal level of claudin-1 was not modified following Δ2-TGZ treatment, which did not induce apoptosis. Furthermore, claudin-1-transfected MDA-MB-231 and Hs578T cells displayed a significant increase of cleaved PARP-1 and caspase 7, caspase 3/7 activities, and TUNEL staining. RNA interference was performed in order to inhibit Δ2-TGZ-induced claudin-1 expression in both the cells. In absence of claudin-1, a decrease of cleaved PARP-1 and caspase 7 and caspase 3/7 activities were observed in MDA-MB-231 but not in Hs578T cells.

Conclusion

Claudin-1 overexpression and Δ2-TGZ treatment are associated to apoptosis in MDA-MB-231 and Hs578T “claudin-1-low” TNBC. Moreover, in MDA-MB-231 cells, claudin-1 is involved in the pro-apoptotic effect of Δ2-TGZ. Our results suggest that claudin-1 re-expression could be an interesting therapeutic strategy for “claudin-1-low” TNBC.

Keywords

Triple-negative breast cancer Apoptosis Claudin-1 Troglitazone derivatives 

Notes

Acknowledgements

This work was supported by grants of the “Université de Lorraine,” the “Conseil Régional du Grand Est,” and the “Ligue Contre le Cancer.” Marine Geoffroy was recipient of a PhD grant of the «Ministère de l’Enseignement Supérieur et de la Recherche». We thank Christelle Thibault-Carpentier and Doulaye Dembele from the IGBMC GenomEast platform for the GEO deposit.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10549_2017_4378_MOESM1_ESM.pptx (36 kb)
Supplementary material 1 (PPTX 36 kb)
10549_2017_4378_MOESM2_ESM.pptx (40 kb)
Supplementary data. 1 Changes in expression level of genes related to cell adhesion and associated with cancer progression in Δ2-TGZ-treated MCF-7 cells after microarray analysis. Cells were treated for 12 h with DMSO or 25 µM of Δ2-TGZ Gene-expression microarray data were generated using GeneChip Human Gene 1.0 ST Array (containing 764,885 distinct probes including 28,869 well-annotated genes) as previously described [28]. Microarray data are available from Geo Data set (https://www.ncbi.nlm.nih.gov/geo/info/linking.html, accession number GSE99941). Supplementary material 2 (PPTX 39 kb)
10549_2017_4378_MOESM3_ESM.pptx (2.3 mb)
Supplementary data. 2 Claudin-1 and occludin co-localization in response to Δ2-TGZ in MDA-MB-231 and Hs578T cells. After treatment for 24 h with DMSO (Ctrl) or 20 µM of Δ2-TGZ, immunofluorescence staining for claudin-1 and occludin were performed in MDA-MB-231 and Hs578T cells. DRAQ5 staining was used for visualisation of the nuclei. a–b Cells were observed by confocal microscopy with magnification from ×400 for Hs578T to ×536 for MDA-MB-231 depending of digital confocal zooming combine with ×40 objective (NA 0.8). One confocal z section of cell of each condition is presented. Claudin-1 staining is upregulated and punctiform in both cell lines. Occludin staining is upregulated only in MDA-MB-231 cells and colocalized with claudin-1. Bar represents 50 µm. Supplementary material 3 (PPTX 2351 kb)
10549_2017_4378_MOESM4_ESM.pptx (3.1 mb)
Supplementary data. 3 Increase apoptosis in MDA-MB-231 cells overexpressing claudin-1 with Δ2-TGZ. MDA-MB-231 cells were transiently transfected with pcDNA3.1 or pcDNA3.1 containing a human claudin-1 expression vector (pcDNA3.1-CLDN1) and treated with 20 µM of Δ2-TGZ. After 48 h of transfection and 24 h of treatment, cells were subjected to western blot analysis. a Western blot analysis was performed with antibodies recognizing claudin-1 (CLDN1) cleaved PARP-1 and cleaved caspase 7. α-tubulin was used as loading control. b Relative protein level corresponds to claudin-1, cleaved PARP-1 (PARP) and cleaved caspase 7 band intensity value adjusted to α-tubulin for Δ2-pcDNA3.1 and Δ2-pcDNA3.1-CLDN1. The values represent the mean ± SEM 5 different experiments. Student-t test was used to determine significance difference from control cells, were *p < 0.05, **p < 0.01 and ***p < 0.001. Supplementary material 4 (PPTX 3130 kb)

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Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  • Marine Geoffroy
    • 1
    • 2
  • Alexandra Kleinclauss
    • 1
    • 2
  • Stéphanie Grandemange
    • 1
    • 2
  • Sébastien Hupont
    • 5
  • Michel Boisbrun
    • 3
    • 4
  • Stéphane Flament
    • 1
    • 2
  • Isabelle Grillier-Vuissoz
    • 1
    • 2
    Email author
  • Sandra Kuntz
    • 1
    • 2
  1. 1.Université de Lorraine, CRAN, UMR 7039Vandœuvre-lès-NancyFrance
  2. 2.CNRS, CRAN, UMR 7039Vandœuvre-lès-NancyFrance
  3. 3.Université de Lorraine, SRSMC, UMR 7565Vandœuvre-lès-NancyFrance
  4. 4.CNRS, SRSMC, UMR 7565Vandœuvre-lès-NancyFrance
  5. 5.CNRS, FR3209 Biologie Moléculaire Cellulaire et Thérapeutique (BMCT), Plateforme d’Imagerie Cellulaire et Tissulaire PTIBC-IBISA, Biopôle de l’Université de LorraineVandœuvre-lès-NancyFrance

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