Glioblastoma is the most common malignant brain tumor, with over 10,000 new diagnoses made every year in the United States [19, 20]. Significant efforts are required to understand the molecular mechanism of glioblastoma, which led to modification of the World Health Organization Classification of Tumors of the CNS (CNS WHO) in 2016 [21, 22], grading gliomas according to their pathological evaluation based on molecular features.
Understanding the pathogenesis at the molecular level provides information for designing and developing new chemotherapeutic agents. Temozolomide, bevacizumab, and carmustine are FDA-approved and widely used chemotherapeutic agents in patient with glioblastoma [2, 23]. They all act on molecular tergets: Temozolomide alkylates DNA at the N-7 or O-6 positions of guanine residues, thus damaging it in tumor cells. Bevacizumab, a recombinant humanized monoclonal antibody, blocks angiogenesis by inhibiting vascular endothelial growth factor A (VEGF-A), whereas Carmustine acts as an alkylating agent and forms interstrand crosslinks in DNA, preventing DNA replication and DNA transcription.
Rundle et al. reviewed articles about some older drugs which have potential anticancer activity . Some studies have suggested that beta-blockers might inhibit angiogenesis, cellular proliferation, and invasion, as well as increasing apoptosis in several cancer cell lines [25–27]. Another study investigated the usage of propranolol in several cell lines including breast cancer, neuroblastoma, and glioblastoma cell lines . Jing et al. reported that isoproterenol, an agonist of beta-adrenergic receptors, stimulated the proliferation of U251 glioblastoma cells, but not U87-MG cells . This effect was prevented by the beta-adrenergic receptor antagonist propranolol. According to their study, isoproterenol had different effects on different glioblastoma cell lines, and it could not be said that isoproterenol stimulates all types of glioblastomas. In our study, both propranolol and isoproterenol suppressed glioblastoma and neuroblastoma cell lines. Although many studies have been conducted, the exact mechanism by which beta-blockers inhibit angiogenesis and promote apoptosis is not yet fully understood.
Rajaratnam et al. presented several signaling pathways that are involved in pathogenesis of glioblastoma. These include isocitrate dehydrogenase mutation, Notch pathway, ceramide signaling, vascular endothelial growth factor signaling pathway, platelet-derived growth factor signaling, epidermal growth factor receptor pathway, phosphatidylinositol 3-kinase/serine-threonine-specific protein kinase/mammalian target of rapamycin pathway, phosphate and tensin homolog signaling, and sonic hedgehog signaling . Among these pathways, we focused on the Notch pathway. As mentioned above, Notch signaling is involved in cell differentiation, proliferation, migration, self-renewal and apoptosis . It plays a key role in promoting neural stem cell differentiation into glial cells . Contrastingly, it is related to various cancers, including breast cancer, cervical cancer, lymphomas, pancreatic cancer, renal cell cancer, skin tumor, and lung cancer . Some studies report Notch1 acts as oncogene [32–34], while others report it as a tumor suppressor [35, 36].
Protein and mRNA levels of Notch1 and Hes1 are higher in brain tumor cells than normal brain cells . In this study, immunohistochemical staining of primary human glioblastoma tissues showed strong immunoreactivity of Notch1. In contrast, several studies reported a weak expression of Notch1 in glioblastoma [37–39]. In this study, propranolol suppressed glioblastoma cell proliferation (MTT assay), and induced Notch1 expression in both U87-MG and LN229 cells (RT-PCR and WB). There were no remarkable differences in glioblastoma cell proliferation between the cases treated with negative controlled-Notch1 siRNA and active Notch1 siRNA (p = 0.157). These results demonstrate that pathways other than Notch1 exist and play a key role in the proliferation and survival of glioblastoma. In case of Hes1, copy number was increased in real time PCR, but expression was decreased in western blot analysis after treatment with propranolol. Propranolol may block Hes1 expression at the translation step or post-translational modification step, and these results were compatible with decreased glioblastoma cell proliferation. The Hes1 signaling pathway is thought to play an important role in the proliferation and survival of glioblastoma. A previous study demonstrated that nerve growth stimulated glioblastoma proliferation through the Notch1 pathway . They treated U87-MG with nerve growth factor and stimulated cell proliferation. Expression levels of Notch1 and Hes1 were increased simultaneously. These findings are consistent with the results of our study in that Hes1 plays an important role in glioblastoma proliferation.
Several reports using the same type of cell lines as in this paper have been studied regarding glioblastoma proliferation. Kusaczuk et al. reported that phenylbutyrate has a suppressive effect on the proliferation of glioblastoma LN229 cells . Phenylbutyrate is a histone deacetylase inhibitor known to induce differentiation, cell cycle arrest, and apoptosis in various cancer cells. They added phenylbutyrate to LN229 and cell viability showed dose-dependent reduction in the MTT assay. The density of LN229 cells was reduced and morphology was changed as phenylbutyrate was treated. Another reports demonstrated that CKD-602, a camptothecin derivative, inhibited proliferation and induced apoptosis in U87-MG and LN229 glioma cell lines . CKD-602 is a synthetic water-soluble camptothecin derivative and topoisomerase inhibitor that has been shown to have clinical anticancer effect against ovarian and lung cancer. It stabilizes DNA preventing the religation of DNA breaks, which leads to an inhibition of DNA replication and triggers apoptotic cell death . They treated U87-MG and LN229 with 10 mM stock solutions of CKD-602 and dose-dependent cytotoxicity and proliferation inhibition was observed.
This study has some limitations. First, commercialized glioblastoma cell lines were used with in vitro experiments in this study, and there could be some differences with in vivo reactions in the human brain. Second, only three kinds of glioblastoma and neuroblastoma cell lines were used in this study. Different results could be obtained according to different types of cell lines. More diverse types of glioblastoma cell lines should be evaluated in future study. Third, the impact of propranolol on Hes1 was not clearly revealed. Additional mechanisms of the reaction between propranolol and Hes1 should be evaluated at the molecular level in a future study.