Abstract
Phorbol myristate acetate (PMA) and ionomycin (Io) can induce T cell activation and proliferation. Furthermore, they stimulate activation-induced cell death (AICD) in mature lymphocytes via Fas/Fas ligand (FasL) up-regulation. In this study, we explored the influence of PMA/Io treatment on glioblastoma cells, and found that AICD-like phenomena may also occur in glioma. Using the MTT assay and cell counting, we demonstrated that treatment of PMA/Io significantly inhibited the proliferation of glioma cell lines, U87 and U251. TUNEL assays and transmission electron microscopy revealed that PMA/Io markedly induced U87 and U251 cell apoptosis. Propidium iodide staining and flow cytometry showed that treatment with PMA/Io resulted in an arrestment of cell cycle and an increase in cell death. Using real-time PCR and western blot, we found that PMA/Io up-regulated the expression of Fas and FasL at both mRNA and protein level, which confirmed that PMA/Io induced glioma cell death. Specific knockdown of NFAT1 expression by small hairpin RNA greatly reduced the PMA/Io induced cell death and apoptosis by inhibition of FasL expression. Microarray analysis showed that the expression of NFAT1 significantly correlated with the expression of Fas. The coexistence of Fas with NFAT1 in vivo provides the background for AICD-like phenomena to occur in glioma. These findings demonstrate that PMA/Io can induce glioblastoma cell death through the NFAT1-Fas/FasL pathway. Glioma-related AICD-like phenomena may provide a novel avenue for glioma treatment.
Highlights
Glioblastoma multiforme (GBM) is the most aggressive type of glioma; even with combined therapy, the prognosis of GBM is still very poor [1,2]
We speculate that factors activating NFAT1, such as phorbol myristate acetate (PMA) and ionomycin (Io), will further influence GBM cell growth
PMA and Io inhibit GBM cell proliferation Using the MTT assay, which reflects total number of live cells, we examined the effect of PMA and/or Io treatment on U87 and U251 cell proliferation at indicated time points
Summary
Glioblastoma multiforme (GBM) is the most aggressive type of glioma; even with combined therapy, the prognosis of GBM is still very poor [1,2]. We found that nuclear factor of activated T cells (NFAT)-1 is overexpressed in GBM [3]. NFAT signaling can regulate cell death in many central nervous system diseases, including inflammation, tumors and degenerative diseases [6,7,8,9,10]. Activated by PMA and Io, NFAT1 is dephosphorylated, translocates to the nucleus, binds to its target promoter elements and regulates the transcription of specific responsive genes, such as Fas ligand (FasL), and cyclin A2 [15,5]. There are several studies that show the importance of Fas/FasL pathway in the apoptosis of glia cells and their respective tumor types [22,23,24,9,25,26,27]. We aimed to investigate the effect of PMA/Io administration on GBM cells and the related mechanism
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