Abstract
Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. Despite concerted efforts to improve current therapies and develop novel clinical approaches, patient survival remains poor. As such, increasing attention has focused on developing new therapeutic strategies that specifically target the apoptotic pathway in order to improve treatment responses. Recently, nutlins, small-molecule antagonists of MDM2, have been developed to inhibit p53-MDM2 interaction and activate p53 signaling in cancer cells. Glioma cell lines and primary cultured glioblastoma cells were treated with nutlin-3a. Nutlin-3a induced p53-dependent G1- and G2-M cell cycle arrest and apoptosis in glioma cell lines with normal TP53 status. In addition, nutlin-arrested glioma cells show morphological features of senescence and persistent induction of p21 protein. Furthermore, senescence induced by nutlin-3a might be depending on mTOR pathway activity. In wild-type TP53 primary cultured cells, exposure to nutlin-3a resulted in variable degrees of apoptosis as well as cellular features of senescence. Nutlin-3a-induced apoptosis and senescence were firmly dependent on the presence of functional p53, as revealed by the fact that glioblastoma cells with knockdown p53 with specific siRNA, or cells with mutated or functionally impaired p53 pathway, were completely insensitive to the drug. Finally, we also found that nutlin-3a increased response of glioma cells to radiation therapy. The results provide a basis for the rational use of MDM2 antagonists as a novel treatment option for glioblastoma patients.
Highlights
The protein p53 is a key regulator of the multiple cellular processes, and depending on the cell type and other factors p53 activation can result in apoptosis, reversible and irreversible cell cycle arrest [1,2]. p53 is negatively regulated by MDM2 through different mechanisms in coordination with HDMX (MDM4)
Results obtained in the mRNA pattern were verified by analysis of protein expression with Western blot. p53, p21, MDM2, PUMA and cleaved caspase 3 proteins increased upon treatment with nutlin-3a in U87MG cells, whereas Survivin protein expression markedly decreased (Figure 1G)
We show that nutlin-3a effectively stabilizes and activates p53 in glioblastoma cell lines as well as in primary glioblastoma cultured cells with wild-type TP53, leading to induction of p53-dependent cell-cycle arrest and apoptosis
Summary
The protein p53 is a key regulator of the multiple cellular processes, and depending on the cell type and other factors p53 activation can result in apoptosis, reversible (quiescence) and irreversible cell cycle arrest [1,2]. p53 is negatively regulated by MDM2 through different mechanisms in coordination with HDMX (MDM4). Potent and selective small-molecule antagonists of MDM2, have been shown to activate the p53 pathway in wild-type p53 cell lines of diverse human malignancies both in vitro and in vivo [8,9]. Nutlins bind to the p53-binding pocket in the MDM2 protein, inhibiting the binding of p53 and activating the p53 pathway in cancer cells with wild-type p53, including solid tumors [10,11] and hematological malignancies [12,13,14,15]. Nutlin-3a, the active enantiomer of nutlin-3, has been shown to inhibit growth of p53 wild-type human tumors grown as xenografts in nude mice and to induce apoptosis and cell cycle arrest in cancer cell lines that express wild-type p53 [16,17,18]
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