Enhanced radiosensitivity and radiation-induced apoptosis in glioma CD133-positive cells by knockdown of SirT1 expression

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Abstract

CD133-expressing glioma cells play a critical role in tumor recovery after treatment and are resistant to radiotherapy. Herein, we demonstrated that glioblastoma-derived CD133-positive cells (GBM-CD133+) are capable of self-renewal and express high levels of embryonic stem cell genes and SirT1 compared to GBM-CD133 cells. To evaluate the role of SirT1 in GBM-CD133+, we used a lentiviral vector expressing shRNA to knock-down SirT1 expression (sh-SirT1) in GBM-CD133+. Silencing of SirT1 significantly enhanced the sensitivity of GBM-CD133+ to radiation and increased the level of radiation-mediated apoptosis. Importantly, knock-down of SirT1 increased the effectiveness of radiotherapy in the inhibition of tumor growth in nude mice transplanted with GBM-CD133+. Kaplan–Meier survival analysis indicated that the mean survival rate of GBM-CD133+ mice treated with radiotherapy was significantly improved by Sh-SirT1 as well. In sum, these results suggest that SirT1 is a potential target for increasing the sensitivity of GBM and glioblastoma-associated cancer stem cells to radiotherapy.

Section snippets

Materials and methods

Isolation of CD133+ cells. This study followed the tenets of the Declaration of Helsinki, and all samples were obtained after patients provided informed consent. The dissociated cells from the samples of glioblastoma patients were labeled with 1 mL CD133/l micromagnetic beads per 1 million cells using the CD133 cell isolation kit (Miltenyi Biotech, Auburn, CA). CD133+ cells were cultured in a medium consisting of serum-free DMEM/F12 (Gibco-BRL, Gaithersburg, MD), N2 supplement (R&D Systems Inc.,

Isolation and characterization of CD133-positive cells from glioblastoma

Using the magnetic bead method, we isolated CD133+ cells from tissue samples of seven GBM patients (Fig. 1A). It has been reported that cancer stem-like cells can be cultured in suspension to generate floating sphere bodies (SBs) under serum-free medium with bFGF and EGF [3], [11]. The CD133+ cells isolated from these seven patients formed SBs in DF-12 serum-free medium with bFGF and EGF (Fig. 1B). The ability to form SBs (Fig. 1B; Supplement-1) and the proliferation rate of CD133+ cells were

Discussions

SIRT1 has been reported to regulate radiosensitivity through its interactions with p-53, Bcl-2, FOXO, BMI1 polycomb and ATM [12], [13], [16]. Chu et al. found that multidrug resistance gene (mdr1) and its ability to promote chemoresistance were controlled by SIRT1 in correlation with the cell cycle inhibitor p21/WAF1 [17]. Kojima et al. showed that down-regulation of SIRT1 may induce apoptosis and increase radiosensitivity in prostate cancer [19]. Recent work on human GBM indicates that CD133+

Acknowledgments

This study was supported by research grants from the National Science Council (NSC-96-3111-B-075-001-MY3), Taipei City Hospital, Taipei Veterans General Hospital (V97B1-006, E1-008, F-001), and National Yang-Ming University (Ministry of Education, Aim for the Top University Plan), Taiwan.

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