Autophagy Inhibition Promotes Gambogic Acid-induced Suppression of Growth and Apoptosis in Glioblastoma Cells

Guo-Xuan Luo,Yong Zhang,Yu-Yang Jiang,Wei-Shi Luo,Jing-Zhi Lin,Heng-Shan Luo,Jun Cai

doi:10.7314/apjcp.2012.13.12.6211

Abstract

To investigate the effects of gambogic acid (GA) on the growth of human malignant glioma cells. U251MG and U87MG human glioma cell lines were treated with GA and growth and proliferation were investigated by MTT and colony formation assays. Cell apoptosis was analyzed by annexin V FITC/PI flow cytometry, mitochondrial membrane potential assays and DAPI nuclear staining. Monodansylcadaverine (MDC) staining and GFP-LC3 localisation were used to detect autophagy. Western blotting was used to investigate the molecular changes that occurred in the course of GA treatment. GA treatment significantly suppressed cell proliferation and colony formation, induced apoptosis in U251 and U87MG glioblastoma cells in a time- and dose-dependent manner. GA treatment also lead to the accumulation of monodansylcadaverine (MDC) in autophagic vacuoles, upregulated expressions of Atg5, Beclin 1 and LC3-II, and the increase of punctate fluorescent signals in glioblastoma cells pre-transfected with GFP-tagged LC3 plasmid. After the combination treatment of autophagy inhitors and GA, GA mediated growth inhibition and apoptotic cell death was further potentiated. Our results suggested that autophagic responses play roles as a self-protective mechanism in GA-treated glioblastoma cells, and autophagy inhibition could be a novel adjunctive strategy for enhancing chemotherapeutic effect of GA as an anti-malignant glioma agent.

Highlights

Glioblastoma multiforme (GBM) is the most common and aggressive malignant central nervous system tumor in adults (Kleihues et al, 2002)
In order to determine whether the cell growth inhibition were treated with different concentrations of gambogic acid (GA) for 12 caused by GA treatment was accompanied with apoptosis, h
The anticancer effects of Gambogic acid have been widely investigated in several types of cancer, including lung carcinoma, hepatocarcinoma, gastric carcinoma and so on (Wu et al, 2004; Zhao et al, 2004)

Summary

Introduction

Glioblastoma multiforme (GBM) is the most common and aggressive malignant central nervous system tumor in adults (Kleihues et al, 2002). Lots of studies showed that Gambogic acid (GA, C38H44O8), a major active ingredient of gamboge, has potent anti-tumor effects against many different types of cancer in vivo and in vitro including lung carcinoma, hepatocarcinoma, gastric carcinoma and so on (Wu et al, 2004; Zhao et al., 2004). An increasing number of studies tried to elucidating the molecular mechanisms of GA anti-tumor effects, and several critical signaling steps in carcinogenesis have been reported to be influenced by GA treatment (Wang and Chen, 2012). GA arrested cell cycle at G2/M phase via disturbing CDK7 mediated phosphorylation of CDC2/P34 (Yu et al, 2007); GA could interact with transferrin receptor and induce rapid apoptosis of tumor cells (Pandey et al, 2007); GA inhibited cell proliferation by repressing hTERT transcriptional activity and by posttranslational modification of hTERT (Zhao et al, 2008); GA can inhibit tumor growth by repressing angiogenesis (Lu et al, 2007). GA treatment can inhibit proliferation and induce apoptosis of many kinds of tumors via a multi-mechanism manner

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Conclusion