ABSTRACT
<p><b>BACKGROUND</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>
Subject(s)
Humans , AC133 Antigen , Antigens, CD , Metabolism , Antineoplastic Agents, Alkylating , Therapeutic Uses , Apoptosis Regulatory Proteins , Metabolism , Autophagy-Related Protein 5 , Beclin-1 , Blotting, Western , Caspase 3 , Metabolism , Cells, Cultured , Dacarbazine , Therapeutic Uses , Drug Resistance, Neoplasm , Physiology , Glioblastoma , Drug Therapy , Pathology , Glycoproteins , Metabolism , Immunohistochemistry , Membrane Proteins , Metabolism , Microtubule-Associated Proteins , Metabolism , Peptides , MetabolismABSTRACT
The present study investigated the relationship between DNA-dependent protein kinase (DNA-PK) and radiosensitivity of nasopharyngeal carcinoma (NPC) cell lines. The dose-survival relationship for NPC cell lines, CNE1 and CNE2, was analyzed using clonogenic formation assay, the activity of DNA-PK of the two cell lines was measured using the Signa TECT DNA-PK assay kit, and the localization and expression of Kus (a heterodimer) and DNA-PKcs protein in CNE1 and CNE2 before irradiation and 15 min, 1 h, 6 h, 12 h, 24 h after 4 Gy irradiation were analyzed by immunofluorescence, laser scanning confocal microscope (LSCM) and Western blot. The results showed that the surviving fraction of CNE1 was higher than that of CNE2 at each dose. The DNA-PK activity of CNE1 was also significantly higher than that of CNE2 before and after irradiation (P<0.05), while the expression of total Ku70/Ku80 in CNE1 and CNE2 had no significant difference. Increasing translocation of Ku70 and Ku80 from the cytoplasm to the nuclei in the two cell lines was observed with increase of irradiation time as detected by Western blot, and the immunofluorescence of the DNA-PK complex subunits showed greater nuclear translocation in CNE1 than CNE2 after irradiation. The results suggest that the relatively higher radio-resistance of CNE1 correlates with the higher activity of DNA-PK as compared to that of more radiosensitive CNE2 (or lower radio-resistance) before and after irradiation. Thus, DNA-PK activity may be a useful predictor of radiosensitivity of NPC.
Subject(s)
Humans , Carcinoma , Cell Line, Tumor , Radiation Effects , DNA-Activated Protein Kinase , Metabolism , Nasopharyngeal Neoplasms , Radiation ToleranceABSTRACT
<p><b>OBJECTIVE</b>To investigate the relationship between DNA-dependent protein kinase (DNA-PK) activity and anti-cancer drug sensitivity in human glioma tissues.</p><p><b>METHODS</b>Human glioma specimens were primarily cultured and its sensitivity to several anti-cancer drugs were evaluated by MTT assay. Nuclear protein was extracted from the glioma sample of the same patient and its DNA-PK activity was determined by a biotinylated DNA-PK assay with p53-derived peptide as a specific substrate.</p><p><b>RESULTS</b>DNA-PK activity varied widely among these glioma samples. Of all 36 samples, 16 showed higher DNA-PK activity (relative activity > or = 0.40) and 20 samples with lower DNA-PK activity (relative activity < 0.40). The gliomas sensitive to DDP and VCR as evaluated by inhibition rate (IR > or = 50%) under plasma peak concentration (PPC) showed lower DNA-PK activity than the resistant ones (IR < 50%) (t = -3.445, P < 0.01). Furthermore, the gliomas with higher DNA-PK activity showed lower inhibition rate (IR < 50%) than those with lower DNA-PK activity ones (t = -2.145, P < 0.05).</p><p><b>CONCLUSION</b>DNA-PK activity is significantly associated with anti-cancer drug sensitivity to DDP and VCR in human gliomas. DNA-PK activity could be used as a new biomarker for the chemotherapy sensitivity of human gliomas.</p>