A Proteomic-Based Approach to Study the Mechanism of Cytotoxicity Induced by Interleukin-1\u03b1 and Cycloheximide

Katarzyna Macur,Jacek Bigda,Lucyna Konieczna,Caterina Temporini,Jolanta Grzenkowicz-Wydra,Sara Tengattini,Tomasz Bączek

doi:10.1007/s10337-017-3382-3

Katarzyna Macur, Jacek Bigda + Show 5 more

Open Access

https://doi.org/10.1007/s10337-017-3382-3

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Abstract

The exposure of HeLa cells to interleukin-1 alpha (IL-1α) in the presence of cycloheximide (CHX) leads to the release of active tumor necrosis factor alpha (TNF-α), eliciting cytocidal effect on these cells. A mass spectrometry (MS)-based analysis of the qualitative proteomic profiles of the HeLa cells treated only with IL-1α, CHX or simultaneously with IL-1α and CHX, in comparison to an untreated control, enabled to distinguish protein candidates possibly involved in this process. Among them protein disulphide isomerase (PDI) seemed to be particularly interesting for further research. Therefore, we focused on quantitative changes of PDI levels in HeLa cells subjected to IL-1α and CHX. Enzyme-linked immunosorbent assay (ELISA) was employed for determination of PDI concentrations in the investigated, differently treated HeLa cells. The obtained results confirmed up-regulation of PDI only in the cells stimulated with IL-1α alone. In contrary, the PDI levels in HeLa cells exposed to both IL-1α and CHX, where apoptotic process was intensive, did not increase significantly. Finally, we discuss how different expression levels of PDI together with other proteins, which were detected in this study, may influence the induction of cytotoxic effect and modulate sensitivity to cytotoxic action of IL1.Graphical

Highlights

Tumor necrosis factor alpha (TNF-α) and interleukin-1 alpha (IL-1α) are proinflammatory cytokines
The shotgun proteomic approach was employed to investigate changes on the molecular level, in response to IL-1α and CHX that lead to TNF-α release and human cervical carcinoma cell line (HeLa) cells apoptosis
Tryptic digests of HeLa cell lysates were submitted to the HPLC–mass spectrometry (MS)/MS analysis

Summary

Introduction

Tumor necrosis factor alpha (TNF-α) and interleukin-1 alpha (IL-1α) are proinflammatory cytokines. TNF-α induces cytotoxicity in both, tumor and healthy cells. IL-1 can trigger cytostatic or cytotoxic effect, but usually in the conditions of simultaneous treatment with metabolic inhibitors. In the presence of protein synthesis inhibitor—cycloheximide (CHX), cytotoxicity was observed in human cervical carcinoma cell line (HeLa) in response to IL-1α. There, it was dependent on the increased release of soluble and presence of transmembrane TNF-α. The proteins involved in this mechanism remain unknown. This cytocidal effect was not detected after selective IL-1α

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