Betulinic acid inhibits glioma cell viability by downregulation of NF-κB and enhancement of apoptosis

Zhu Yaozu,Peng Peng,Chen Jincao,Yang Liu,Zhang Tingbao,Huang Zhao

doi:10.4314/tjpr.v19i12.9

Abstract

Purpose: To determine the inhibitory potential of betulinic acid on pro-survival signaling pathway in glioblastoma.Methods: Changes in viabilities of glioma cells and primary astrocytes were measured using 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Apoptotic changes were analyzed using Hoechst 33342 staining and Annexin V-FITC/PI kits. Western blotting was used for assaying the protein expressions of various pro-apoptotic and anti-apoptotic factors.Results: The proliferative potential of U87MG and A172 cells were significantly reduced on treatment with betulinic acid in a concentration- and time-dependent manner. Treatment with betulinic acid at a dose of 8.75 µg/mL increased apoptosis in U87MG and A172 cells to 41.8 ± 0.5 and 48.8 ± 0.5%, respectively (p < 0.05). Betulinic acid significantly decreased intracellular levels of NFκB p65 andsuppressed levels of survivin, XIAP and Bcl-2 in U87MG and A172 cells (p < 0.05). However, betulinic acid significantly increased the levels of Bax and activated caspase-9 and caspase-3 in U87MG and A172 cells (p < 0.05).Conclusion: Betulinic acid inhibited the proliferation of U87MG and A172 glioblastoma cells and mediated their apoptosis. There is need for in vivo studies for validation of the therapeutic potential of betulinic acid as an anti-glioblastoma drug. Keywords: Glioblastoma, Betulinic acid, Proliferation, Apoptosis, Chemotherapy, Intracranial malignancy

Highlights

Glioblastoma multiforme accounts for almost 30 % of primary tumors in the brain, and it is a major cause of carcinoma deaths associated with intracranial malignant tumors [1]
The U87MG and A172 cells were treated with gradient concentrations of betulinic acid (1.25, 2.5, 5.0, 10, 20 and 40 μM/ml), and changes in viability were measured at 24 and 48 h (Figure 1)
Shrinkage and detachment from culture flask surface were prominent in U87MG and A172 cells on treatment with 8.75 μg/mL betulinic acid at 24 h (Figure 2 A)

Summary

Introduction

Glioblastoma multiforme accounts for almost 30 % of primary tumors in the brain, and it is a major cause of carcinoma deaths associated with intracranial malignant tumors [1]. Sustained and effective efforts have led to the development of the novel therapeutic strategy of NovoTTF-100A System for glioblastoma [2]. The chemotherapeutic drug temozolomide has been found effective in the treatment of glioblastoma [3]. The U87MG and A172 cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in plates in DMEM at 37 °C in an incubator with humid atmosphere containing 5 % CO2 and 95 % oxygen. The incubation medium contained 10 % newborn calf serum and antibiotics. The primary cultured astrocytes were a gift from Dr Zhang, while betulinic acid was purchased from SigmaAldrich

Results

Discussion

Conclusion