Glioblastoma (GBM) is one of the most common and aggressive types of brain tumor. Due to its highly recurrent rate and poor prognosis, the overall survival time with this type of tumor is only 20–21 months. Recent knowledge suggests that its recurrence is in part due to the presence of cancer stem cells (CSCs), which display radioresistant, chemoresistant, self-renewal and tumorigenic potential. Enhancers of Zeste 2 (EZH2) and AXL receptor tyrosine kinase (AXL) are both highly expressed in GBM. Additionally, they are an essential regulator involved in CSCs maintenance, migration, invasion, epithelial-to-mesenchymal transition (EMT), stemness, metastasis and patient survival. In this study, we used a small molecule, n-butylidenephthalide (BP), to assess the anti-GBM stem-like cells potential, and then tried to find out the associated genes involved with regulation in migration and invasion. We demonstrated that BP reduced the expression of AXL and stemness related genes in a dose-dependent manner. The migratory and invasive capabilities of GBM stem-like cells could be reduced by AXL/EZH2. Finally, in the overexpression of AXL, EZH2 and Sox2 by transfection in GBM stem-like cells, we found that AXL/EZH2/TGF-β1, but not Sox2, might be a key regulator in tumor invasion, migration and EMT. These results might help in the development of a new anticancer compound and can be a target for treating GBM.

Glioblastoma multiforme (GBM) is the most common and aggressive brain tumour. The neoplasms are difficult to resect entirely because of their highly infiltration property and leading to the tumour edge is unclear. Gliadel wafer has been used as an intracerebral drug delivery system to eliminate the residual tumour. However, because of its local low concentration and short diffusion distance, patient survival improves non-significantly. Axl is an essential regulator in cancer metastasis and patient survival. In this study, we developed a controlled-release polyanhydride polymer loading a novel small molecule, n-butylidenephthalide (BP), which is not only increasing local drug concentration and extending its diffusion distance but also reducing tumour invasion, mediated by reducing Axl expression. First, we determined that BP inhibited the expression of Axl in a dose- and time-dependent manner and reduced the migratory and invasive capabilities of GBM cells. In addition, BP downregulated matrix metalloproteinase activity, which is involved in cancer cell invasion. Furthermore, we demonstrated that BP regulated Axl via the extracellular signal-regulated kinases pathway. Epithelial-to-mesenchymal transition (EMT) is related to epithelial cells in the invasive migratory mesenchymal cells that underlie cancer progression; we demonstrated that BP reduced the expression of EMT-related genes. Furthermore, we used the overexpression of Axl in GBM cells to prove that Axl is a crucial target in the inhibition of GBM EMT, migration and invasion. In an in vivo study, we demonstrated that BP inhibited tumour growth and suppressed Axl expression in a dose-dependent manner according to a subcutaneous tumour model. Most importantly, in an intracranial tumour model with BP wafer in situ treatment, we demonstrated that the BP wafer not only significantly increased the survival rate but also decreased Axl expression, and inhibited tumour invasion. These results contribute to the development of a BP wafer for a novel therapeutic strategy for treating GBM invasion and increasing survival in clinical subjects.

We have shown that the natural compound z-butylidenephthalide (Bdph), isolated from the chloroform extract of Angelica sinensis, has antitumor effects. Because of the limitation of the blood-brain barrier, the Bdph dosage required for treatment of glioma is relatively high. To solve this problem, we developed a local-release system with Bdph incorporated into a biodegradable polyanhydride material, p(CPP-SA; Bdph-Wafer), and investigated its antitumor effects. On the basis of in vitro release kinetics, we demonstrated that the Bdph-Wafer released 50% of the available Bdph by the sixth day, and the release reached a plateau phase (90% of Bdph) by the 30th day. To investigate the in situ antitumor effects of the Bdph-Wafer on glioblastoma multiforme (GBM), we used 2 xenograft animal models-F344 rats (for rat GBM) and nude mice (for human GBM)-which were injected with RG2 and DBTRG-05MG cells, respectively, for tumor formation and subsequently treated subcutaneously with Bdph-Wafers. We observed a significant inhibitory effect on tumor growth, with no significant adverse effects on the rodents. Moreover, we demonstrated that the antitumor effect of Bdph on RG2 cells was via the PKC pathway, which upregulated Nurr77 and promoted its translocation from the nucleus to the cytoplasm. Finally, to study the effect of the interstitial administration of Bdph in cranial brain tumor, Bdph-Wafers were surgically placed in FGF-SV40 transgenic mice. Our Bdph-Wafer significantly reduced tumor size in a dose-dependent manner. In summary, our study showed that p(CPP-SA) containing Bdph delivered a sufficient concentration of Bdph to the tumor site and effectively inhibited the tumor growth in the glioma.