Biodegradable interstitial release polymer loading a novel small molecule targeting Axl receptor tyrosine kinase and reducing brain tumour migration and invasion

Abstract

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.

Figure 1

BP regulates Axl expression in human GBM cells. The reverse transcriptase–PCR and western blot data demonstrate that BP can downregulate Axl protein expression in DBTRG brain tumour cells in a time- or dose-dependent manner. (a) Time-dependent downregulation in treatment with BP at 100 μg/ml and (b) dose-dependent downregulation in 24- h treatment with BP. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 2

BP inhibits migration and invasion in the GBM cell line DBTRG-05MG in a dose-dependent manner. (A) Wound-healing assay of DBTRG-05MG cells treated with BP at various doses in 24-h treatment. Transwell assay for migration (B) and invasion (C) with BP at various doses in 24-h treatment: (a) 0, (b) 25, (c) 50, (d) 75, (e) 100 and (f) 150 μg/ml. **P<0.01 and ***P<0.001.

Figure 3

BP inhibits MMP activity in a dose-dependent manner in the GBM cell line DBTRG-05MG. The zymography assay results demonstrate that BP reduced the activity of MMPs in a dose-dependent manner in 24-h treatment, and the quantification is on the below. *P<0.05, and ***P<0.001.

Figure 4

BP inhibits MMP2 expression in a dose-dependent manner in the GBM cell line DBTRG-05MG. MMP2 expression of DBTRG brain tumour cells treated with BP at 0, 25, 50, 75, 100 μg/ml, analysed after 24 h.

Figure 5

Cell viability was reversed by Axl overexpression in DBTRG cells. (a) The selection of colony of DBTRG cells transfected with pcDNA3.0-neo and pcDNA3.0-Axl. (b) The cell viability of DBTRG-neo and DBTRG-Axl with BP administered at various doses in 24-h treatment. **P<0.01 and ***P<0.001.

Figure 6

Cell migration and invasion were reversed by Axl overexpression in DBTRG cells. (a) Migration assay of DBTRG-neo and DBTRG-Axl cells performed using the Oris system, with BP administered at various doses in 24-h treatment. (b) Invasion assay of DBTRG-neo and DBTRG-Axl cells with BP administered in a dose-dependent manner in 24-h treatment. *P<0.05.

Figure 7

BP inhibits expression of EMT genes. Reverse transcriptase–PCR and western blot analysis of EMT-related genes in GBM cells administered BP at various doses in 24-h treatment. Downregulation of EMT-related genes. The results suggest that EMT was inhibited by BP in GBM cells (a and c). In addition, when Axl was overexpressed in DBTRG cells (DBTRG-Axl), EMT gene expression was recovered (b and d). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 8

Signalling pathways involved in BP-mediated Axl repression. (a) DBTRG cells were treated with BP for 0–48 h. Western blot analysis was performed for p-Akt, Akt, p-ERK and ERK. β-Actin was used as an internal control. (b) DBTRG cells were pretreated with the ERK inhibitor PD98059 (25 and 50 μM) for 1 h. Western blot analysis was performed for p-ERK, ERK and Axl expression. β-Actin was used as an internal control.

Figure 9

In vivo xenografts and orthtopic animal study demonstrated that BP inhibited tumour growth and suppressed Axl and MMP2 expression in vivo. (a) Tumour sizes were measured using calipers and were monitored until day 30. (b) Tissue sections stained for Axl and MMP9 in the xenograft model. (c) Rat survival rate in the orthtopic animal study. (d) Brain tissue stain for Axl photographed under a light microscope at a magnification of × 200.