Analysis of alpha-1 antitrypsin expression in multidrug resistant cell lines

Abstract

Identification of the signature molecular factors and transcriptional profiles involved in therapy resistance is of vital importance in developing new strategies for treatments and disease monitoring. Tumour secretome is a set of macromolecules secreted by tumour cells into the extracellular space as a response to changes in tumour environment that at the same time shapes the microenvironment further promoting specific phenotypes and contributing to cellular plasticity in tumour. Protein alpha-1 antitrypsin (AAT, encoded by SERPINA1 gene) is an acute phase protein that has emerged as one of the key components in tumour secretome involved in crucial stages of tumour development and progression, with recent data also implicating it in therapeutic resistance. However, what exactly leads to SERPINA1 upregulation during development of therapy resistance, as well as its biological significance in this process, is still unclear. Our aim was to analyse SERPINA1 expression in multidrug resistant cell lines and 3D cellular models. Expression of IL-6 was also analysed, as AAT is an acute phase reactant and its levels increase in response to inflammatory cytokines. Patients and methods: We analysed SERPINA1 and IL-6 expression in three different cell lines - human glioblastoma U87, non-small cell lung carcinoma NCI-H460 and colorectal carcinoma DLD1 as well as their multidrug resistant counterparts U87-TxR, NCI-H460/R and DLD1-TxR, respectively. In addition, expression analysis was performed in long-term 3D glioblastoma model of U87 cells cultured in alginate microfibers, and compared to long-term 2D cell culture of U87. Quantitative RTPCR was performed using Taqman gene expression assays and data were normalized to GAPDH. Results: We found that SERPINA1 expression is significantly upregulated in all the multidrug resistant cell lines analysed compared to their sensitive counterparts. Expression of IL-6 was significantly upregulated in U87- TxR and DLD1-TxR compared to their parental lines, however NCI-H460/R cell line had lower IL-6 expression compared to NCI-H460. In 3D glioblastoma model of U87 cells, previously found to exhibit increased therapy resistance compared to 2D cell culture, both SERPINA1 and IL-6 expression were significantly upregulated. Conclusion: Our results indicate that SERPINA1 expression correlates with therapy resistance in analysed cell lines and 3D model of glioblastoma, revealing the potential of utilizing this molecule as a biomarker of therapy resistance. However, transcriptional profiles connected to its expression in therapy resistance still remain to be determined.