The role of Arlts1 in Glioblastoma multiforme

Determination of specific Glioblastoma (GBM) biomarkers could be beneficial in developing new therapeutics, monitoring the response to therapy or for early detection of recurrence. Downregulation and tumor suppressor effect of Arlts1 (ADP-ribosylation factor-like tumor suppressor gene) could be shown in different familiar and sporadic cancers but has not yet been examined in GBM. The examination of Arlts1 in GBM cells is the main objective of this study. U87MG glioblastoma cell line was transfected with wildtype-Arlts1. Annexin V and Propidium iodide staining was used to determine cell death by flow cytometry. The expression levels of pro-apoptotic (Caspases), anti-apoptotic (Bcl-2) genes and the Matrix metalloproteinases (MMP) 2 and 9 were examined by qRT-PCR. Western Blot was used to evaluate qRT-PCR results on protein level and zymography to reveal activity of MMPs. A migration assay was performed. Cell death rate of GBM cells transfected with the wildtype Arlts1 was not detected. Furthermore, these cells showed no differences in apoptosis compared to control; there was no significant increase of Caspase 3, 7 or 9. Surprisingly, the expression of the anti-apoptotic gene Bcl-2 was significantly reduced in U87MG cells overexpressing Arlts1. Since Bcl-2 is also involved in migration of tumors, expression of MMP 2 and 9 as markers for migration were also determined. The expression level of these matrix metalloproteinases was significantly decreased. The decrease activity of these enzymes could not be detected either using zymography and migration assay. In summary, as a consequence of Artls1 overexpression, Bcl-2, MMP2 and 9 level decreased. Considering already known connection between Bcl-2 expression and invasiveness of GBM, this result would suggest a role of Arlts1 in migration and invasiveness of the tumor. The further experiments, including measurement of different time points in migration assay using a stable transfected cell line, should be performed.2020-07-2