Inhibition of autophagy induced by proteasome inhibition increases cell death in human SHG-44 glioma cells.
OBJECTIVE
The ubiquitin-proteasome system (UPS) and lysosome-dependent macroautophagy (autophagy) are two major intracellular pathways for protein degradation. Recent studies suggest that proteasome inhibitors may reduce tumor growth and activate autophagy. Due to the dual roles of autophagy in tumor cell survival and death, the effect of autophagy on the destiny of glioma cells remains unclear. In this study, we sought to investigate whether inhibition of the proteasome can induce autophagy and the effects of autophagy on the fate of human SHG-44 glioma cells.


METHODS
The proteasome inhibitor MG-132 was used to induce autophagy in SHG-44 glioma cells, and the effect of autophagy on the survival of SHG-44 glioma cells was investigated using an autophagy inhibitor 3-MA. Cell viability was measured by MTT assay. Apoptosis and cell cycle were detected by flow cytometry. The expression of autophagy related proteins was determined by Western blot.


RESULTS
MG-132 inhibited cell proliferation, induced cell death and cell cycle arrest at G(2)/M phase, and activated autophagy in SHG-44 glioma cells. The expression of autophagy-related Beclin-1 and LC3-I was significantly up-regulated and part of LC3-I was converted into LC3-II. However, when SHG-44 glioma cells were co-treated with MG-132 and 3-MA, the cells became less viable, but cell death and cell numbers at G(2)/M phase increased. Moreover, the accumulation of acidic vesicular organelles was decreased, the expression of Beclin-1 and LC3 was significantly down-regulated and the conversion of LC3-II from LC3-I was also inhibited.


CONCLUSIONS
Inhibition of the proteasome can induce autophagy in human SHG-44 glioma cells, and inhibition of autophagy increases cell death. This discovery may shed new light on the effect of autophagy on modulating the fate of SHG-44 glioma cells.Acta Pharmacologica Sinica (2009) 30: 1046-1052; doi: 10.1038/aps.2009.71.
Ge
Peng-Fei
PF
[1] Department of Neurosurgery, 1st Hospital of Jilin University, Changchun, Jilin 130021, China [2] Department of Neurosurgery, Xuanwu Hospital of Capital University of Medical Science, Beijing 100053, China.
Zhang
Ji-Zhou
JZ
Wang
Xiao-Fei
XF
Meng
Fan-Kai
FK
Li
Wen-Chen
WC
Luan
Yong-Xin
YX
Ling
Feng
F
Luo
Yi-Nan
YN
D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001343:Autophagy / Q000502:physiology*; D000071186:Beclin-1; D002453:Cell Cycle / Q000502:physiology; D016923:Cell Death / Q000502:physiology*; D045744:Cell Line, Tumor / Q000187:drug effects; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism / Q000494:pharmacology; D005910:Glioma / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure; D006801:Humans; D007976:Leupeptins / Q000378:metabolism / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors
D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; C477012:MAP1LC3A protein, human; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D061988:Proteasome Inhibitors; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde