Background
Glioblastoma (GBM) is the most malignant and common primary neoplasm in central nervous system. Despite the advances in conventional treatments, comprised of surgical resection, radiotherapy and chemotherapy, the median duration of survival of GBM is 14.6 months after first diagnosis [
1]. Recent studies report that a minority subset of the whole glioma population is glioma initiating cells (GICs), which led to tumorigenesis, because these GICs show enhanced self-renewal ability, multipotent differentiation that GICs can epitomize the original tumor in vivo [
2]. GICs are considered “seed” cells and have strong radio-chemoresistance and tumorigenic potential [
3,
4]. Bao S et al. found primary tumor cells are less radioresistant than GICs, and GICs were responsible for tumor recurrence after radiotherapy [
5]. Although surgery followed by radiation and chemotherapy could eradicate most part of glioma cells, they did not kill GICs. Thus, GICs are the novel cellular targets and their elimination can hinder cancer progression and recurrence.
Autophagy is also named type II programmed cell death pathway, which is different from the apoptotic type I death pathway [
6]. Autophagy is a balance that weights between reconstruction, energy equilibrium, cell death and survival, correspondingly, with the internal or external stimulations accepted. Autophagy has different effects on tumor outcomes: it is essential for some forms of cancer cell death, but it can help cancer growth by assisting cancer cells fight hard cancer environment (e.g., malnutrition, shortage of oxygen) and combat radiotherapy and chemotherapy [
7]. Yao found that ionizing radiation (IR) of human glioma cells led to increased autophagy [
8]. Other studies showed that the induction of autophagy by radiotherapy result in the radioresistance of GICs [
9]. Therefore, discovery of a novel combination treatment, such as radiotherapy or chemotherapy combines autophagy suppression may be a feasible and promising strategy.
Chloroquine is an anti-malaria drug, which has been used for over eighty years. Recent years, choloroquine, as an autophagy inhibitor, is drawing more and more attentions [
10]. Chloroquine-treated tumor cells are not able to exploit autophagy as an substituting source of energy and will die [
11].
Researchers found that chloroquine combined with chemo-radio-therapy, increased the duration of survival of glioblastoma patients [
12,
13]. However, other researchers reported that the chloroquine treatment was found not to be effective for the medical treatment of malignant astrocytomas [
14].
Therefore, the clinical effect of joint treatment with radiation and chloroquine on the radioresistance of malignant glioma and GICs remains uncertain. We investigate the potential radiosensitization effect and its probable mechanisms of chloroquine on GICs.
Methods
Preparation of CD133+ U87 GICs, cell culture, and reagents
The human glioma cell lines U87 were purchased from the Shanghai Institute of Biochemistry (Shanghai, China). To isolate human CD133+ U87 GICs, Glioma cell line U87 was cultured at 37 °C in the presence of 5 % CO2 in the medium containing 90 % Dulbecco’s modified Eagle’s medium (DMEM) and 10 % Fetal Calf Serum (FBS). After having digested with trypsin, resuspended, centrifuged, and purified by magnetic separation using anti-CD133 microbeads (Miltenyi Biotec) per the manufacturer’s instructions, CD133 negative cells were also obtained at the same time and cultured in conditions appropriate for growth. The human CD133+ U87 GICs, were then cultured at 37 °C in the presence of 5 % CO2 in the serum-free DMEM/F12 culture medium, supplemented with B27 (2 %), firoblast growth factor, N2 supplement, and epidermal growth factor. The medium was replaced every 3–5 days. We observed and counted the attachment and suspension of cells twice per day. Chloroquine was purchased from Sigma.
Cell viability assay
Cell viability was measured by MTT assays. Cells were seeded in 96-well microplates (1 × 105 cells or 1 × 104 cells/well). After incubation at 37 °C in the presence of 5 % CO2 in constant temperature and humidity incubator for 3–5 days, the tumor cells were treated using different doses of chloroquine or radiotherapy alone or in combination. We set up a control group and a zero adjustment group. At specified time points, 20ul of MTT solution (5 mg/mL) was added 4 h before the end of the incubation duration, the reaction was stopped by the addition of 150 μL dimethylsulfoxide (DMSO). The optical density (OD) or absorbance was read at 490 nm.
X-rays
Radiation was performed using a 6-MV X-rays from a linear accelerator (PRIMUS, DE, Siemens A&D LD, Nelson Avenue Concord, USA).
Clonogenic assay
Cells were seeded in six-well plates (2 × 102 cells/well). After 12 h incubation, cells were treated and irradiated. After two weeks, the colonies were fixed with methanol for 15 min and stained with 0.5 % crystal violet. Colonies with at least 50 cells were counted.
Immunofluorescence
The U87 GICs were plated onto coverslips and treated with 20 μM/L concentration of chloroquine and 6 Gy dose of radiation for 24 h. After fixation with 4 % paraformaldehyde, tumor cells were incubated with blocking buffer (0.1 % Triton X-100, 2 % horse serum) for 30 min at 37 °C, and with the rabbit anti–human light chain 3 antibody (LC3) (1:200, Abcam) at 4 °C for overnight. After wash, a secondary antibody (1:200) conjugated to Alexa 488 (Invitrogen) was added and incubated for 1 h at room temperature. Fluorescent cells were examined with a confocal microscope to appraise the formation of autophagosomes.
Western blot analysis
The cells were lysed with a lysis buffer (Beyotime, Haimen, China), and centrifuged at 12,000 × g for 20 min. The total protein concentration was determined using the a BCA protein assay kit (Pierce). Samples were resolved in SDS-PAGE, transferred to 0.45 μm nitrocellulose transfer membranes and analyzed separately. After blocking with 5 % skim milk at room temperature for 60 min, the blots were survey with primary antibodies against mouse anti-Bcl-2 (1:500; Abcam), rabbit anti-LC3 (1:1000; Abcam), mouse anti-GAPDH (1:1000; Cell Signal), and rabbit anti-active caspase-3 (1:1000; Abcam), at 4 °C for overnight. We washed the membranes three times with TBST buffer (20 mmol/L Tris-buffered saline and 0.1 % Tween 20) for 1 h. And we used peroxidase conjugated anti-mouse-IgG/anti rabbit-IgG as secondary antibodies. We did Chemiluminescence with Amersham ECL plus Western blotting detection system (GE healthcare). After washing with the TBST buffer, the membranes were measured with the Odyssey Infrared Imaging System (LI-COR).
Flow cytometric apoptosis assay
The cells in the different treatment groups were measured using an Annexin V FITC Kit (Sigma, St Louis, MO). Earlier stages apoptotic (annexin V+) (PI-) cells were identified by flow cytometry on a BD FACS flow cytometer (San Diego, CA).
Cell cycle analysis
5 × 105 cells per sample were collected after centrifugation at 1000 r.p.m for 5 min. The cells re-suspended with 0.5 ml PBS and fixed with ice-cold 70 % ethanol overnight at 4 °C. Fixed cells were washed with PBS and stained with propidium at room temperature for 1 h. The cells were subsequently measured by FACScan (Becton-Dickinson, San Diego, CA, USA).
DNA damage
A comet assay quantitatively assesses DNA repair in U87 GICs. The detailed procedure of the comet assay performed in this experiment refers to the report of Wang et al. [
15]. The comet assay metric for DNA damage induced by the treatment was tail DNA (%). We analyzed images from 50 cells (25 from each replicate slide).
Neurosphere assay
We trypsinized the U87 GICs and seeded equal numbers of GICs in 48 well plates, and treated as indicated. Five days after treatment, we counted total neurospheres numbers and photographed.
Statistical analysis
The experimental data is showed as the mean ± SD. All experiments were performed three times. Unpaired Student’s t test was performed to analyses the significance between experimental groups. Statistically significant P values are indicated in the figures with asterisks: **, P < 0.01; *, P < 0.05. We used GraphPad software (Inc.; version 5.02) for all statistical analyses.
Discussion
In the present study, we explored the radiosensitizing action of an autophagy blocker chloroquine in GICs, and aimed to further understand the molecular mechanisms of its effect. We found that chloroquine or radiation alone inhibited U87 GICs growth in a dose-dependent manner. The combined inhibitory effect of chloroquine and radiation on the viability of human GICs was stronger than that of chloroquine or radiation alone, in a synergetic way (Fig.
1a-c). The response of GICs to radiation was associated with autophagy and the late autophagy inhibitor chloroquine in combination with radiation caused G0/G1 phase arrest, promoted GICs’ apoptosis and impaired the repair of radiation-induced DNA damage in GICs, and decreased the GICs’ tumorsphere numbers and diameters.
Recent researches demonstrated that cancer stem cells could exploit autophagy to survive and accelerate their renewal [
16,
17]. GICs, as the most radioresistant cell subset and autophagy targeting, may be helpful to cure the disease [
18,
19]. Our data indicates that irradiation of U87 GICs results in enhanced autophagy. Chloroquine in combination with radiation inhibited the autophagy induced by radiation and strongly promoted GICs apoptosis. Apoptosis involved many diseases, especially malignant tumors. There are two main apoptotic pathways: the extrinsic or death receptor pathway and intrinsic or mitochondrial pathways [
20]. Study finds that radiation is a stimulus that initiate the intrinsic pathway. The control and regulation of intrinsic pathway is through members of Bcl-2 family of proteins [
21]. Here, we found that the combination of chloroquine and radiation reduced anti-apoptotic Bcl-2, up-regulated caspase-3, and enhanced the fraction of Annexin V-FITC positive and PI negative cells to 35.1 %. However, about 16.7 and 6.9 % respectively were measured with treatment of radiation or chloroquine alone (Fig.
2). These results may suggest that the combined inhibitory effect cause apoptosis at least in part by the intrinsic pathway. These results demonstrate that chloroquine plays a synergetic role in radiation-induced viability inhibition and apoptosis in U87 MG GICs. The result may be helpful to explaining the results of a clinical trial that chloroquine, added to a comprehensive therapeutic protocol (operation plus radiotherapy and chemotherapy) for glioblastoma, significantly prolonged the median overall survival compared with control groups [
22]. Other study on the combining of radiation with chloroquine for the management of recurrent glioblastoma validated the feasibility of the regimen and the authors reported encouraging therapy outcomes [
23].
Growing evidences suggest that chloroquine is a strong anticancer drug in managing several cancers, such as leukemia and hepatocarcinoma [
24,
25]. Yuan et al. showed that suppression of ATG5 by siRNA or suppression of autophagy using 3-methylademine enhanced the radiosensitisation action of gliomas after STAT3 suppression [
26]. Other research demonstrated that high expression levels of early growth response 1, which induces autophagy, is produced by the resistant clones of GICs [
27]. In addition, mitochondrial isoenzyme of NADP + −dependent isocitrate dehydrogenase siRNA-transfected A172 glioma cells were sensitised after suppression of autophagy [
28]. Firat, E et al. also demonstrated that induced-autophagy effects of PI3K/Akt pathway inhibitors obviously prevent cell death induction in γ-irradiated GICs, however, chloroquine significantly promotes γIR-induced cell death in highly radioresistant GICs [
29]. Our study should be of high clinical significance, one reason is all data presented are focused on GICs, a population thought to be pivotal, because for cancer progression and therapy resistance that must be uproot in order to obtain long-term recurrence-free survival, another reason is that a clinically applicable late autophagy inhibitor was used. However, the present study has two limitations. First, our data presented here have been obtained from cell lines, not from patients’s tumor-derived stem-like cells. Second, the further experimentation with animal models and clinical trials were not investigated in this study.
Conclusion
We have shown that inhibition of autophagy in combination with radiation is a promising therapeutic strategy for targeting GICs. The radiosensitization efficiency of chloroquine is obtained by inhibiting autophagy, weakening the capacity for DNA repair in the early stage and promoting cell-cycle arrest in addition to apoptotic responses.
Acknowledgements
Not applicable.
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