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The role of hERG1 ion channels in epithelial-mesenchymal transition and the capacity of riluzole to reduce cisplatin resistance in colorectal cancer cells

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Abstract

Purpose

The transition of cells from the epithelial to the mesenchymal state (EMT) plays an important role in tumor progression. EMT allows cells to acquire mobility, stem-like behavior and resistance to apoptosis and drug treatment. These features turn EMT into a central process in tumor biology. Ion channels are attractive targets for the treatment of cancer since they play critical roles in controlling a wide range of physiological processes that are frequently deregulated in cancer. Here, we investigated the role of ether-a-go-go-related 1 (hERG1) ion channels in the EMT of colorectal cancer cells.

Methods

We studied the epithelial-mesenchymal profile of different colorectal cancer-derived cell lines and the expression of hERG1 potassium channels in these cell lines using real-time PCR. Next, we knocked down hERG1 expression in HCT116 cells using lentivirus mediated RNA interference and characterized the hERG1 silenced cells in vitro and in vivo. Finally, we investigated the capacity of riluzole, an ion channel-modulating drug used in humans to treat amyotrophic lateral sclerosis, to reduce the resistance of the respective colorectal cancer cells to the chemotherapeutic drug cisplatin.

Results

We found that of the colorectal cancer-derived cell lines tested, HCT116 showed the highest mesenchymal profile and a high hERG1 expression. Subsequent hERG1 expression knockdown induced a change in cell morphology, which was accompanied by a reduction in the proliferative and tumorigenic capacities of the cells. Notably, we found that hERG1expression knockdown elicited a reversion of the EMT profile in HCT116 cells with a reacquisition of the epithelial-like profile. We also found that riluzole increased the sensitivity of HCT116 cisplatin-resistant cells to cisplatin.

Conclusions

Our data indicate that hERG1 plays a role in the EMT of colorectal cancer cells and that its knockdown reduces the proliferative and tumorigenic capacities of these cells. In addition, we conclude that riluzole may be used in combination with cisplatin to reduce chemo-resistance in colorectal cancer cells.

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Acknowledgements

I would like to thank Annarosa Arcangeli, Massimo D’Amico, Elena Morelli, Serena Pillozzi, Antonella Mannini, Olivia Crociani, Sara Falsini, Luca Gasparoli and Ivo Noci for advice and support.

Author information

Authors and Affiliations

  1. Department of Experimental and Clinical Biomedical Sciences, University of Florence, Florence, Italy

    Angelo Fortunato

  2. Biodesign Center for Personalized Diagnostic, Arizona State University, PO Box 875001, Tempe, AZ, 85287-5001, USA

    Angelo Fortunato

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  1. Angelo Fortunato
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Correspondence to Angelo Fortunato.

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The author declares no conflict of interest.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors and it was partially supported by the University of Florence, Italy.

Electronic supplementary material

Figure 1S

hERG1 isoforms. Protein coding isoforms of the hERG1 gene. Arrows indicate the shRNAs complementary target sequences. Box: exon; grey box: protein coding sequence; box not filled: untranslated sequence; line between boxes: intron. (PDF 17.6 kb)

Figure 2S

Western blot analysis. Western blot image of total protein analysis of different samples of control (pLKO.1–1, 2) and silenced cells (sh7–1, 2, 3, 4). (PDF 171 kb)

Figure 3S

KCa3.1 gene expression in HCT116, HCT8, HT29 and H630 colorectal cancer cells (PDF 79.1 kb)

Table 1S

Sequences of primers. Sequences of primers (5′-3′). (XLSX 10.6 kb)

Table 2S

Sequences of shRNA. Sequences of shRNA constructs capable of post-transcriptional silence the hERG1 gene. Each shRNA construct (commercially distributed by Open Biosystems) included a hairpin of 21 base pair sense and antisense stem and a 6 base pair loop. (XLSX 10.2 kb)

Table 3S

riluzole has a synergic effect in combination with cisplatin on HCT116 cells. Cisplatin and riluzole have a synergic, additive or antagonistic effect when used in combination, determined by cell viability assay (WST-1) and Calcusyn data analysis software. HCT116 cells were treated with a mixture of cisplatin and riluzole at different concentration but a constant a ratio of the two drugs with a 2-fold serial dilution of their IC50 (1/2, 1/4, 1/8, 1/16, 1/32, 1/64). The concentration of cisplatin and riluzole drugs and the combination index are reported in the Table. CI = 1, indicates an additive effect, CI < 1, indicates a synergistic effect and CI > 1, indicates an antagonistic effect. (DOCX 32.1 kb)

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Fortunato, A. The role of hERG1 ion channels in epithelial-mesenchymal transition and the capacity of riluzole to reduce cisplatin resistance in colorectal cancer cells. Cell Oncol. 40, 367–378 (2017). https://doi.org/10.1007/s13402-017-0328-6

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