Beclin 1 resists temozolomide in malignant glioma cells by autophagy

2013 
Objective To analyze the relations of Beclin 1 with both clinical pathological grades of gliomas and glioma recurrence,and investigate its function and mechanism in drug-resistance of gliomas.Methods Fifty-three glioma specimens (WHO grade Ⅰ in 7,2 cases of recurrence; Ⅱ grade in 9,4 cases of recurrence; Ⅲ grade in 11,6 cases of recurrence; Ⅳ grade in 9,5 cases of recurrence),colleted in our hospitals from April 2012 to April 2013,were chosen in our study.The Beclin 1 expressions in these gliomas specimens were detected by immunohistochemistry and Westem blotting;drug resistant cell line U251AR and non-drug resistant cell line U251were cultured,and Beclin 1 and resistance protein P-gP expressions in these cell lines were detected by Western blotting; double-stranded small molecule Beclin 1 interfering RNA (siBeclin 1) was transfected into the U251AR cells and the Beclin 1 protein expression 48 h after the transfection was detected by Western blotting; the activity of U251,U251AR and U251AR-siBeclin 1 cells was detected by CCK8 after being treated with different concentrations of temozolomide (TMZ); the capase-3 expression in the U251,U251AR and U251AR-siBeclin 1 cells 48 h after being treated by 20 μm TMZ was detected by Western blotting; the capase-3 expression and the number of autophagosome in the U251AR cells after Beclin 1 being knocked down or after being treated with hydroxychloroquine (HCQ) were detected by Western blotting and acridine orange staining,respectively.Results The level of Beclin 1 was proportional to the grades of gliomas.The Beclin 1 level in recurrent gliomas of grade Ⅲ and Ⅳ was higher than that in primary tumors of grade Ⅰ and Ⅱ; the Beclin 1 and P-glycoprotein (P-gP) expression in the U251AR cells were signficantly higher than those in the U251 cells (P<0.05).As compared with interfered control group and blank control group,the interfered group had decreased Beclin 1 protein level 48 h after siBeclin 1 transfection; U251AR cell survival rate was significantly higher than that of U251 and si-Bec1-U251AR cells 24 h after being treated with TMZ (50,100,500 and 1000 μmol/L); 48 h after being treated with 200 μmol/L TMZ,the caspase-3 expression in the U251 cells was significantly higher than that in U251AR and si-Bec1-U251AR cells; both HCQ and Beclin 1 interference can induce decreased formation of autophagosome and increased caspase-3 expression after U251AR cells being treated by TMZ for 48 h (P<0.05).Conclusion The level ofBeclin 1 is correlated to grades,recurrence and drug resistance of gliomas,which may be used for malignancy diagnosis and prognostic evaluation; the drug-resistance role of Beclin 1 may stem from autophagy,and combined use of temozolomide and inhibitors of autophagy or Beclin 1 disrupters may help the therapy. Key words: Beclin 1;  Autophagy;  Glioma;  Drug-resistance;  Temozolomide
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