ABSTRACT
PURPOSE: Glioblastoma multiforme (GBM) is a devastating disease. Recent studies suggest that the stem cell properties of GBM contribute to the development of therapy resistance. EXPERIMENTAL DESIGN: The expression of Survivin and Ran was evaluated by immunohistochemistry with GBM tissues, and quantitative reverse transcriptase (qRT)-PCR and immunocytochemistry with patient-derived GBM sphere cultures. With a computational structure-based drug design, 11 small-molecule compounds were designed, synthesized, and evaluated as inhibitor candidates for the molecular interaction of Survivin protein. The molecular mechanism of the lead compound, LLP-3, was determined by Western blot, ELISA, in situ proximity ligation assay, and immunocytochemistry. The effects of LLP-3 treatment on GSCs were evaluated both in vitro and in vivo. Quantitative immunohistochemistry was carried out to compare Survivin expression in tissues from 44 newly diagnosed and 31 recurrent post-chemoradiation GBM patients. Lastly, the sensitivities of temozolomide-resistant GBM spheres to LLP-3 were evaluated in vitro. RESULTS: Survivin and Ran were strongly expressed in GBM tissues, particularly in the perivasculature, and also in patient-derived GSC cultures. LLP-3 treatment disrupted the Survivin-Ran protein complex in cancer cells and abolished the growth of patient-derived GBM spheres in vitro and in vivo. This inhibition was dependent on caspase activity and associated with p53 status of cells. Immunohistochemistry showed that Survivin expression is significantly increased in recurrent GBM compared with newly diagnosed tumors, and temozolomide-resistant GBM spheres exhibited high sensitivities to LLP-3 treatment. CONCLUSIONS: Disruption of the Survivin-Ran complex by LLP-3 abolishes survival and growth of GSCs both in vitro and in vivo, indicating an attractive novel therapeutic approach for GBM.
Subject(s)
Glioblastoma/metabolism , Inhibitor of Apoptosis Proteins/metabolism , Neoplastic Stem Cells/metabolism , ran GTP-Binding Protein/metabolism , Apoptosis , Cell Line, Tumor , Cell Proliferation , Cell Survival , Drug Resistance, Neoplasm , Glioblastoma/mortality , Glioblastoma/surgery , Humans , Inhibitor of Apoptosis Proteins/chemistry , Models, Molecular , Prognosis , Protein Binding/drug effects , Protein Conformation , Protein Multimerization , Signal Transduction , Survivin , Tumor Suppressor Protein p53/metabolism , Xenograft Model Antitumor Assays , ran GTP-Binding Protein/chemistryABSTRACT
Survivin was initially described as an inhibitor of apoptosis and attracted growing attention as one of the most tumor-specific genes in the human genome and a promising target for cancer therapy. Lately, it has been shown that survivin is a multifunctional protein that takes part in several crucial cell processes. At first, it was supposed that survivin functions only as a homodimer, but now data indicate that many processes require monomeric survivin. Moreover, recent studies reveal a special mechanism regulating the balance between monomeric and dimeric forms of the protein. In this paper we studied the mutant form of survivin that was unable to dimerize and investigated its role in apoptosis. We showed that survivin monomer interacts with Smac/DIABLO and X-linked inhibitor of apoptosis protein (XIAP) both in vitro and in vivo. Due to this feature, it protects cells from caspase-dependent apoptosis even more efficiently than the wild-type survivin. We also identified that mutant monomeric survivin prevents apoptosis-inducing factor release from the mitochondrial intermembrane space, protecting human fibrosarcoma HT1080 cells from caspase-independent apoptosis. On the other hand, our results indicate that only wild-type survivin, but not the monomer mutant form, enhances tubulin stability in cells. These findings suggest that survivin partly performs its functions as a monomer and partly as a dimer. The mechanism of dimer-monomer balance regulation may also work as a "switcher" between survivin functions and thereby explain remarkable functional diversities of this protein.
