Glioblastoma stem cells resistant to temozolomide-induced autophagy.
BACKGROUND
Recent studies have demonstrated the existence of a small fraction of cells with features of primitive neural progenitor cells and tumor-initiating function in brain tumors. These cells might represent primary therapeutic target for complete eradication of the tumors. This study aimed to determine the resistant phenotype of glioblastoma stem cells (GSCs) to temozolomide (TMZ) and to explore the possible molecular mechanisms underlying TMZ resistance.


METHODS
Freshly resected glioblastoma specimen was collected and magnetic isolation of GSCs was carried out using the Miltenyi Biotec CD133 Cell Isolation kit. The cytotoxic effect of TMZ on CD133(+) and CD133(-) glioblastoma cells was determined by using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Autophagy-related proteins (Beclin-1, LC3 and Atg5) and cleaved caspase-3 (p17) were analyzed by Western blotting. Immunofluorescent staining was used to detect Atg5, glial fibrillary acidic protein (GFAP) and CD133 expression in glioblastoma cells. Statistical analysis was carried out using SPSS 10.0 software. For all tests, the level of statistical significance was set at P < 0.05.


RESULTS
CD133(+) glioblastoma cells exhibited neurosphere-like growth in vitro and high expression of CD133 stem cell marker. The growth-inhibiting rate in CD133(-) glioblastoma cells treated with 5 or 50 micromol/L TMZ was significantly higher than that in CD133(+) glioblastoma cells ((14.36 +/- 3.75)% vs (2.54 +/- 1.36)% or (25.95 +/- 5.25)% vs (2.72 +/- 1.84)%, respectively, P < 0.05). Atg5, LC3-II and Beclin-1 levels were significantly lower in CD133(+) glioblastoma cells than those in autologous CD133(-) cells after TMZ treatment (P < 0.05). Caspase-3 was mildly activated only in CD133(-) glioblastoma cells after exposure to TMZ (P < 0.05). Immunofluorescent staining revealed elevated expression of Atg5 in GFAP(+) cells following TMZ treatment.


CONCLUSIONS
The GSCs display strong capability of tumor's resistance to TMZ. This resistance is probably contributed by the CD133(+) cells with down-regulation of autophagy-related proteins. Future treatment should target this small population of cancer stem cells in tumors to improve survival of patients.
Fu
Jun
J
State Key Laboratory for Cancer Research in Southern China and Department of Neurosurgery/Neuro-oncology, Cancer Center, Sun Yat-sen University, Guangzhou, Guangdong 510060, China.
Liu
Zhi-gang
ZG
Liu
Xiao-mei
XM
Chen
Fu-rong
FR
Shi
Hong-liu
HL
Pangjesse
Chung-sean
CS
Ng
Ho-keung
HK
Chen
Zhong-ping
ZP
D000071916:AC133 Antigen; D015703:Antigens, CD / Q000378:metabolism; D018906:Antineoplastic Agents, Alkylating / Q000627:therapeutic use*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D000071187:Autophagy-Related Protein 5; D000071186:Beclin-1; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D002478:Cells, Cultured; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000627:therapeutic use; D019008:Drug Resistance, Neoplasm / Q000502:physiology; D005909:Glioblastoma / Q000188:drug therapy* / Q000473:pathology; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D000077204:Temozolomide
D000071916:AC133 Antigen; C502778:ATG5 protein, human; D015703:Antigens, CD; D018906:Antineoplastic Agents, Alkylating; D051017:Apoptosis Regulatory Proteins; D000071187:Autophagy-Related Protein 5; C491997:BECN1 protein, human; D000071186:Beclin-1; D006023:Glycoproteins; C477012:MAP1LC3A protein, human; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; C000606023:PROM1 protein, human; D010455:Peptides; D003606:Dacarbazine; D053148:Caspase 3; D000077204:Temozolomide