Abstract
BackgroundIn addition to physiological events such as fertilisation, placentation, osteoclastogenesis, or tissue regeneration/wound healing, cell fusion is involved in pathophysiological conditions such as cancer. Cell fusion, which applies to both the proteins and conditions that induce the merging of two or more cells, is not a fully understood process. Inflammation/pro-inflammatory cytokines might be a positive trigger for cell fusion. Using a Cre-LoxP-based cell fusion assay we demonstrated that the fusion between human M13SV1-Cre breast epithelial cells and human MDA-MB-435-pFDR1 cancer cells was induced by the pro-inflammatory cytokine tumour necrosis factor-α (TNF-α).MethodsThe gene expression profile of the cells in the presence of TNF-α and under normoxic and hypoxic conditions was analysed by cDNA microarray analysis. cDNA microarray data were verified by qPCR, PCR, Western blot and zymography. Quantification of cell fusion events was determined by flow cytometry. Proteins of interest were either blocked or knocked-down using a specific inhibitor, siRNA or a blocking antibody.ResultsThe data showed an up-regulation of various genes, including claudin-1 (CLDN1), ICAM1, CCL2 and MMP9 in M13SV1-Cre and/or MDA-MB-435-pFDR1 cells. Inhibition of these proteins using a blocking ICAM1 antibody, CLDN1 siRNA or an MMP9 inhibitor showed that only the blockage of MMP9 was correlated with a decreased fusion rate of the cells. Likewise, the tetracycline-based antibiotic minocycline, which exhibits anti-inflammatory properties, was also effective in both inhibiting the TNF-α-induced MMP9 expression in M13SV1-Cre cells and blocking the TNF-α-induced fusion frequency of human M13SV1-Cre breast epithelial cells and human MDA-MB-435-pFDR1 cancer cells.ConclusionsThe matrix metalloproteinase-9 (MMP9) is most likely involved in the TNF-α-mediated fusion of human M13SV1-Cre breast epithelial cells and human MDA-MB-435-pFDR1 cancer cells. Likewise, our data indicate that the tetracycline-based antibiotic minocycline might exhibit anti-fusogenic properties because it inhibits a cell fusion-related mechanism.
Highlights
In addition to physiological events such as fertilisation, placentation, osteoclastogenesis, or tissue regeneration/wound healing, cell fusion is involved in pathophysiological conditions such as cancer
51 genes were found to be significantly up-regulated in tumour necrosis factor-α (TNF-α)-treated cell lines under both normoxia and hypoxia (Fig. 1a, b; Table 2), including adhesion molecules such as Intercellular cell adhesion molecule 1 (ICAM1), Intercellular cell adhesion molecule 2 (ICAM2), and Intercellular cell adhesion molecule 4 (ICAM4), components of the extracellular matrix, including Collagen type XXVII alpha chain (COL27A1), Laminin subunit beta 3 (LAMB3), Laminin subunit gamma 2 (LAMC2), and Mucin 4 (MUC4), chemokines, growth factors, and interleukins, such as CC-chemokine ligand 2 (CCL2), Vascular endothelial growth factor C (VEGFC) and Interleukin 32 (IL32), and proteases including ADAMTS9 and matrix metalloproteinase-9 (MMP9) (Fig. 1; Table 2)
Validation of microarray data by Quantitative polymerase chain reaction (qPCR) and conventional Polymerase chain reaction (PCR) To validate the microarray data, Claudin-1 (CLDN1), ICAM1, and MMP9 expression was analysed by qPCR and ADAMTS9, CCL2, Interleukin 4 induced 1 (IL4I1), Interleukin 7 receptor (IL7R), Tumour necrosis factor-alpha induced protein 3 (TNFAIP3) and VEGFC expression was analysed by conventional PCR
Summary
In addition to physiological events such as fertilisation, placentation, osteoclastogenesis, or tissue regeneration/wound healing, cell fusion is involved in pathophysiological conditions such as cancer. Cell fusion, which applies to both the proteins and conditions that induce the merging of two or more cells, is not a fully understood process. Inflammation/pro-inflammatory cytokines might be a positive trigger for cell fusion. Even though cell fusion has a pivotal role in several physiological and pathophysiological conditions such as fertilisation, placentation, muscle development, osteoclastogenesis, wound healing, tissue regeneration, infection with enveloped viruses, and cancer (for review see: [1,2,3,4]), the conditions that favour and the detailed mechanisms of how the plasma membranes of two or more cells merge are not fully understood. The fusion of mesenchymal stem cells with MDA-MB231 breast cancer cells depends on S100A4 [10]
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