Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma - PubMed (original) (raw)

Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma

Gentao Liu et al. Mol Cancer. 2006.

Abstract

Background: Recently, a small population of cancer stem cells in adult and pediatric brain tumors has been identified. Some evidence has suggested that CD133 is a marker for a subset of leukemia and glioblastoma cancer stem cells. Especially, CD133 positive cells isolated from human glioblastoma may initiate tumors and represent novel targets for therapeutics. The gene expression and the drug resistance property of CD133 positive cancer stem cells, however, are still unknown.

Results: In this study, by FACS analysis we determined the percentage of CD133 positive cells in three primary cultured cell lines established from glioblastoma patients 10.2%, 69.7% and 27.5%, respectively. We also determined the average mRNA levels of markers associated with neural precursors. For example, CD90, CD44, CXCR4, Nestin, Msi1 and MELK mRNA on CD133 positive cells increased to 15.6, 5.7, 337.8, 21.4, 84 and 1351 times, respectively, compared to autologous CD133 negative cells derived from cell line No. 66. Additionally, CD133 positive cells express higher levels of BCRP1 and MGMT mRNA, as well as higher mRNA levels of genes that inhibit apoptosis. Furthermore, CD133 positive cells were significantly resistant to chemotherapeutic agents including temozolomide, carboplatin, paclitaxel (Taxol) and etoposide (VP16) compared to autologous CD133 negative cells. Finally, CD133 expression was significantly higher in recurrent GBM tissue obtained from five patients as compared to their respective newly diagnosed tumors.

Conclusion: Our study for the first time provided evidence that CD133 positive cancer stem cells display strong capability on tumor's resistance to chemotherapy. This resistance is probably contributed by the CD133 positive cell with higher expression of on BCRP1 and MGMT, as well as the anti-apoptosis protein and inhibitors of apoptosis protein families. Future treatment should target this small population of CD133 positive cancer stem cells in tumors to improve the survival of brain tumor patients.

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Figures

Figure 1

Figure 1

Primary culture of adult glioblastoma cells. (A) Tumor cells looking like neurosphere were found in three glioblastoma primary cultured cell lines in 10% FBS/DMEM/F-12 medium. (B) Neurosphere derived from a single isolated CD133 positive cell cultured in NSC medium. (C) CD133 expression on neurospheres derived from a single isolated CD133 positive cell in NSC medium. Red staining indicates CD133 positive. (Magnification = ×100).

Figure 2

Figure 2

CD133 protein expression on primary cultured cells. Tumor cells were cultured in 10% FBS/DMEM/F-12 medium for 3–6 passages and stained with specific mAb to CD133, and isotype control-matched mAb. Results are given as the percentage of CD133 positive cells in the total population. In the histograms, the thick green line represents staining with CD133 mAb, and the gray lines represent the isotype control-matched mAb.

Figure 3

Figure 3

Drug sensitivity of CD133 positive cancer stem cells derived from No. 66. Both CD133 positive (CD133+) and CD133 negative (CD133-) tumor cells were collected from No. 66 by FACS sorting. 1 × 104 cells/well were plated in 96-well plate and treated with various concentrations of temozolomide, carboplatin, Taxol and VP16 for 48 hours in FBS/DMEM/F-12 medium. * indicates p < 0.05 compared to autologous CD133- cells. Data are representative of two independent experiments.

Figure 4

Figure 4

Drug sensitivity of CD133 positive cancer stem cells derived from No. 377. Both CD133+ and CD133- tumor cells were collected from No. 377 by FACS sorting. 1 × 104 cells/well were plated in 96-well plate and treated with various concentrations of temozolomide, carboplatin, Taxol and VP16 for 48 hours in 10% FBS/DMEM/F-12 medium. * indicates p < 0.05 compared to autologous CD133- cells. Data are representative of two independent experiments.

Figure 5

Figure 5

Drug sensitivity of CD133 positive cancer stem cells derived from No. 1049. Both CD133+ and CD133- tumor cells were collected from No. 1049 by FACS sorting. 1 × 104 cells/well were plated in 96-well plate and treated with various concentrations of temozolomide, carboplatin, Taxol and VP16 for 48 hours in 10% FBS/DMEM/F-12 medium. * indicates p < 0.05 compared to autologous CD133- cells. Data are representative of two independent experiments.

Figure 6

Figure 6

CD133 mRNA expression in primary (P) and recurrent (R) tumor tissue. Total RNA was extracted from both primary and recurrent tumor tissue derived from five patients. CD133 mRNA expression was measured by real-time qPCR. The relative CD133 mRNA level of recurrent tumor was presented as the fold increase compared to that of autologous primary tumor tissue (see Methods). Data are representative of two independent experiments.

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