Isolinderalactone suppresses human glioblastoma growth and angiogenic activity in 3D microfluidic chip and in vivo mouse models

Highlights

• Isolinderalactone reduces tumor growth and tumor vessels in vivo.

• Isolinderalactone inhibits VEGF expression in the glioblastoma cell line U-87.

• Isolinderalactone inhibits proliferation, migration, tube formation, and 3D sprouting of endothelial cells.

• Isolinderalactone reduces HIF-1α, HIF-2α in U-87 and tyrosine phosphorylation of VEGFR2 in endothelial cells.

• Isolinderalactone inhibits VEGF-triggered angiogenesis in vivo.

Abstract

Glioblastoma multiforme (GBM) is a lethal and highly vascular type of brain tumor. We previously reported that isolinderalactone enhances GBM apoptosis in vitro and in vivo, but its role in tumor angiogenesis is unknown. Here, we investigated the anti-angiogenic activity of isolinderalactone and its mechanisms. In a human GBM xenograft mouse model, isolinderalactone significantly reduced tumor growth and vessels. Isolinderalactone decreased the expression of vascular endothelial growth factor (VEGF) mRNA, protein, and VEGF secretion in hypoxic U-87 GBM cells and also in xenograft GMB tissue. In addition, we demonstrated that isolinderalactone significantly inhibited the proliferation, migration, and capillary-like tube formation of human brain microvascular endothelial cells (HBMECs) in the presence of VEGF. We also found that isolinderalactone decreased sprout diameter and length in a 3D microfluidic chip, and strongly reduced VEGF-triggered angiogenesis in vivo Matrigel plug assay. Isolinderalactone downregulated hypoxia-inducible factor-1α (HIF-1α) and HIF-2α proteins, decreased luciferase activity driven by the VEGF promoter in U-87 cells under hypoxic conditions, and suppressed VEGF-driven phosphorylation of VEGFR2 in HBMECs. Taken together, our results suggest that isolinderalactone is a promising candidate for GBM treatment through tumor angiogenesis inhibition.

Introduction

Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults, and a mean progression-free survival is just over six months [1,2]. Maximal surgical resection of the primary tumor in combination with radiotherapy and/or chemotherapy is the conventional treatment mechanism for GBM patients, although the median overall survival is only approximately 15 months, indicating poor outcomes for adult GBM patients [1,3]. Glioblastomas are highly vascularized and their vasculature is structurally and functionally abnormal compared to healthy vasculature. GBMs are characterized by remarkable endothelial proliferation and highly disorganized, convoluted vessels with uneven diameter with diminished pericyte coverage and thickened basement membranes [4,5]. In addition, overexpression of vascular endothelial growth factor (VEGF), which is one of the key regulators of tumor angiogenesis, is characteristic of GBM [1,5,6]. New therapeutic approaches targeting tumor vasculature have yielded encouraging results in several types of highly angiogenic solid tumors, including GBM [5].

Tumor angiogenesis is the process by which tumors form new blood vessels from pre-existing blood vessels, which is required for progression of most types of solid tumors [7]. This suggests that tumor growth could effectively be inhibited if angiogenesis is blocked [8]. Angiogenesis is a multistep process; quiescent endothelial cells are activated by signals from ischemic tissues or hypoxic solid tumors. These activated endothelial cells then degrade the extracellular matrix, proliferate, and migrate toward the source of the angiogenic stimuli, forming vascular networks. This angiogenic process is tightly regulated by angiogenic and anti-angiogenic factors [9]. Anti-angiogenic therapies have primarily focused on VEGF signaling, including treatment with bevacizumab, which is a humanized monoclonal antibody against VEGF and the most-studied anti-angiogenic agent with the most promising results against tumor progression [3]. However, bevacizumab trials to date have demonstrated no extension of overall survival even though the drug appears to prolong progression-free survival, improve quality of life, and decrease steroid usage [3]. To improve a survival benefit, additional research is needed to explore alternative therapeutics.

Isolinderalactone is a sesquiterpene isolated from root extracts of Lindera aggregata [10]. Several studies have reported the inhibitory activity of isolinderalactone in cancer cells [[11], [12], [13], [14]]. The compound inhibits nitric oxide production and inflammatory activity in RAW 264.7 cells [11] and the proliferation of non-small cell lung cancer cells [12] and MDA-MB-231 breast cancer cells [13]. Isolinderalactone also inhibits the migration and invasion of lung cancer cells through its inhibition of MMP-2 and β-catenin expression [14]. We recently reported that isolinderalactone suppresses glioblastoma xenograft tumors in vivo and induces U-87 GBM cell apoptosis through the BCL-2/caspase-3/PARP pathway in vitro [15]. In present study, we investigated whether isolinderalactone affects glioblastoma angiogenesis in vitro and in vivo and characterized its underlying mechanisms. We propose that treatment with isolinderalactone could be an effective novel strategy for inhibiting tumor angiogenesis.