Investigating the Potential Role of SIRT1 in Glioblastoma Multiforme: A Comparison Between Glioma and Normal Astrocyte Cells in Culture
Objective Glioblastomas (GBMs) are the most prevalent primary brain tumours in adults and the outcome for this disease remains very poor. With treatment options limited, there is growing interest to find potential differences between normal and malignant molecular signaling pathways for this disease. SIRT1 is a histone deacetylase enzyme with key functions in cellular signaling responses including protecting DNA from damage and changing transcriptional events. SIRT1 can act as a switch to affect cellular senescence or anti-apoptotic responses, or alternatively affect autophagic and pro-apoptotic responses. The role of SIRT1 in GBM is unclear. This study aimed to investigate differences between glioma and normal astrocytes with respect to SIRT1. Research design and methods SIRT1 was analysed in murine normal and glioma astrocyte cell lines, and results compared to a normal human astrocyte cell line and a panel of human primary glioma cells taken from patients with GBM. Analysis of SIRT1 was assessed using Western blots and flow cytometry. Sub-cellular localisation was examined using Western blots and immunofluorescent microscopy. Activity for SIRT1 was assessed using acetylation of a key histone SIRT1 substrate, histone 4 lysine 16, and cell proliferation was measured using the MTT assay. Oxidative stress was induced using H₂O₂ and viability measured using propidium iodide exclusion and flow cytometry. To ascertain that activity was SIRT1 related, inhibition and activation of SIRT1 was done using nicotinamide and resveratrol, respectively. Results SIRT1 was mainly localised in the nucleus and to mitochondria of normal cells but was aberrantly distributed in the cytoplasm of glioma cells, which has not been reported before. This was consistent in both murine and human glioma models. Nicotinamide significantly inhibited cell proliferation more for glioma cells compared to normal, and resveratrol had the opposing effect. Nicotinamide rescued normal but not glioma cells from H₂0₂-induced oxidative stress, and resveratrol had the opposing effect. Western blots revealed secondary protein bands for SIRT1 indicating possible smaller species of SIRT1, and results for nuclear/cytoplasmic extractions suggested FL-SIRT1 and the smaller species of SIRT1 have a dynamic pattern of localisation under oxidative stress, nicotinamide and resveratrol treatments. Conclusions These results indicated SIRT1 is involved in both cell proliferation and oxidative stress responses, with a differential activity between glioma and normal astrocyte cells. Aberrant localisation of SIRT1 to the cytoplasm in glioma cells is a significant finding that needs further exploration. The need for further studies to elucidate changes in localisation for SIRT1 under different conditions is highlighted, as is the possible role of truncated variants of SIRT1. This study suggests there may be some potential for SIRT1 inhibition in patients suffering from glioblastoma.