Irradiation enhances expression of cxcr4 in murine glioma cells via HIF-1α-independent pathway

Abstract

Objective

To investigate levels of chemokine (C–X–C motif) receptor 4 (cxcr4) mRNA and protein in X-irradiated glioma cells.

Methods

Murine malignant glioma GL261 cells transfected with hypoxia-inducible factor (HIF)-1α miRNA or control miRNA were irradiated with X-radiation. Cxcr4 mRNA and protein were analysed using real-time reverse transcription–polymerase chain reaction and Western blot, respectively.

Results

Levels of cxcr4 protein in GL261 cells increased in a radiation dose-dependent manner 48 h after 0, 5, 10 and 15 Gy X-irradiation. Irradiation of both HIF-1α knockdown cells and control cells resulted in a significant increase in cxcr4 mRNA levels, compared with nonirradiated cells, at 24 h after 5 Gy X-irradiation.

Conclusion

Irradiation enhances expression of cxcr4 in glioma cells via a HIF-1α-independent pathway.

Introduction

The main features of glioma are aggressive invasion and diffuse infiltration of tumour cells into surrounding brain tissue. ¹ Traditional radiotherapy involves the use of large radiation fields to ensure treatment of these diffusely invasive glioma cells, but clinical research has demonstrated that glioma tends to recur within the irradiated field. ² Radioresistance is therefore an important issue in glioma treatment. In glioma, vascular endothelial growth factor (VEGF) is upregulated by irradiation and promotes the motility of the glioma cell in vitro, ³ indicating that cytokines secreted by irradiated tumour cells play a role in radioresistance.

Stromal cell-derived factor 1α (SDF-1α) is the only known ligand for chemokine (C–X–C motif) receptor 4 (cxcr4). In glioma cells, the SDF-1α promoter is activated 24 h after exposure to 8 Gy radiation, ⁴ and cxcr4 is a key determinant of glioma progression. ⁵ Irradiation induces tumour satellite formation by enhancing the glioma tropism of haematopoietic progenitor cells to the tumour bulk, an effect mediated by SDF-1α. ⁴ The role of tumour cells in SDF-1α/cxcr4-mediated radiotherapy resistance is unclear, however.

Hypoxia stimulates CXCR4 production by the activation of hypoxia-inducible factor (HIF)-1α in glioma cells, ⁶ and SDF-1α has been shown to be co-localized with HIF-1α in glioma cells that surround areas of necrosis. ⁶ The aim of the present study was to use micro RNA (miRNA) knockdown targeting HIF-1α to investigate the role of SDF-1α/cxcr4 signalling in radiosensitivity, in a murine malignant glioma cell line.

Materials and methods

Cell culture

The murine malignant glioma cell line GL261 (transfected with HIF-1α miRNA or control miRNA) was provided by the Brain Science Research Institute of Shandong University, Shandong, China. Cells were cultured at 37℃ in a humidified 5% carbon dioxide atmosphere in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO, Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum, 100 IU/ml penicillin and 100 mg/ml streptomycin.

Irradiation

Exponentially growing cells were irradiated at room temperature with an X-ray dose energy of 6 MV at a dose rate of 3.0 Gy/min (Varian® 2300 Linear Accelerator, Palo Alto, CA, USA). Irradiation was delivered as a single dose of 5, 10 or 15 Gy on a 2-cm solid water (bolus), with a source-to-surface distance of 100 cm. Cxcr4 mRNA and protein were analysed at 24 h and 48 h after irradiation, respectively. Experiments were performed in triplicate.

Real-time RT–PCR

The GL261 cells (5 × 10⁶cells/ml) were lysed with TRIzol® reagent (Invitrogen), following the manufacturer’s instructions. RNA was treated with DNase and purified with the RNeasy mini kit (Qiagen, Hilden, Germany). Purified RNA was eluted in 30 µl of RNA storage solution (Ambion, Austin, TX, USA) and stored at –80℃. Total RNA was quantified by UV spectroscopy (U3000 Spectrophotometer, Hitachi, Japan) and cDNA synthesis was conducted using the high-capacity cDNA archive kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions.

Levels of cxcr4 mRNA were analysed via quantitative real-time reverse transcription–polymerase chain reaction (qRT–PCR) using an ABI Prism 7500HT Sequence Detection System (Applied Biosystems). Primers were: cxcr4, forward 5′-TCCAACAAGGAACCCTGCTTC-3′ and reverse 5′-TTGCCGACTATGCCAGTCAAG-3′; Gapdh (internal control), forward 5′-ATGACATCAAGAAGGTGGTG-3′ and reverse 5′-CATACCAGGAAATGAGCTTG-3′. Reactions were conducted in a total volume of 20 µl (1 µl cDNA, 10 µl SYBRGreen® real-time PCR master mix [Roche, Mannheim, Germany] and 0.6 µl of each primer). Cycling conditions included a prevention of carry-over contamination (2 min, 50℃) and a hot start (10 min, 95℃), followed by 40 cycles of 95℃ for 10 s and 60℃ for 45 s. For analysis, a threshold was set for the change in fluorescence at a point in the linear PCR amplification phase. Melt curve analysis was performed to ensure a single product species. All experiments were performed in triplicate for both target gene and internal control, and were repeated three times, independently.

Western blotting

Whole-cell extracts were prepared using lysis buffer (50 mM Tris [pH 7.5], 150 mM sodium chloride, 1% Triton® X-100, 0.01% SDS, 1 mM EDTA [pH 8.0] and protease inhibitor cocktail tablets [Roche Applied Science, Indianapolis, IN, USA]). Total protein (30 µg/lane) was separated by 12% SDS–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Amersham, Little Chalfont, UK). After blocking in 5% nonfat skim milk in TBST (Tris-HCl, pH 7.4, 150 mM sodium chloride with 0.1% Tween 20) for 1 h at room temperature, membranes were incubated with rabbit antimouse cxcr4 antibody (1 : 1000 dilution, Abcam, Cambridge, UK) and rabbit antimouse gapdh antibody (1 : 1000 dilution; Santa Cruz Biotechnologies, Santa Cruz, CA, USA) overnight at 4℃. After washing three times with TBST, membranes were incubated with horseradish peroxidase-conjugated antirabbit immunoglobulin G (1 : 5000 dilution, Santa Cruz Biotechnologies) for 1 h at 37℃, then washed three times with TBST. Proteins were visualized using an ECL Plus kit (Millipore, Billerica, MA, USA) followed by chemiluminescent detection using a Fuji LAS 3000 (FujiFilm Medical Systems, Stamford, CT, USA) system.

Statistical analyses

Data were expressed as mean ± SD and between-group comparisons were made using unpaired two-tailed Student’s t test. Statistical analyses were performed using SPSS® version 12.0 (SPSS Inc., Chicago, IL, USA) for Windows®. P-values <0.05 were considered statistically significant.

Results

In malignant glioma cell line GL261, levels of cxcr4 protein at 48 h after irradiation were visibly increased in a radiation dose-dependent manner (Figure 1).

Figure 1.

Western blot of chemokine (C–X–C motif) receptor 4 (cxcr4) protein in the murine malignant glioma cell line GL261 at 48 h after 0, 5, 10 or 15 Gy X-irradiation. GAPDH, glyceraldehyde 3-phosphate dehydrogenase (internal control).

Data regarding cxcr4 mRNA levels in GL261 control cells and HIF-1α knockdown cells at 24 h after 5 Gy irradiation are shown in Figure 2. Irradiation of both cell types resulted in a significant increase in cxcr4 mRNA levels compared with nonirradiated cells (P < 0.05 for both comparisons).

Figure 2.

Real-time quantitative reverse transcription–polymerase chain reaction quantification of chemokine (C–X–C motif) receptor 4 (cxcr4) mRNA in the murine malignant glioma cell line GL261 (transfected with control micro RNA [miRNA] or HIF-1α miRNA) at 24 h after 5 Gy X-irradiation. Cxcr4 mRNA quantified relative to control (glyceraldehyde 3-phosphate dehydrogenase). *P < 0.05 versus 0 Gy; unpaired two-tailed Student’s t test.

Discussion

Findings of the present study indicate that irradiation enhances the expression of cxcr4 in murine glioma cells in vitro. It has been shown that irradiation enhances the invasive potential of glioma cells via activation of the Rho signalling pathway, ⁷ as well as enhancing VEGF and matrix metalloproteinase (MMP) -2 secretion.^8,9 In addition, glioma cell mobility was enhanced by adding irradiated-conditioned medium to nonirradiated cells. ³ We hypothesize that the enhanced invasive ability of irradiated glioma cells is mediated both directly via glioma cells themselves and indirectly via the increased expression of various mediators.

Hypoxia-inducible factor IF-1α is a key factor in hypoxia-induced CXCR4 production. ⁵ The CXCR4 receptor was found to be co-localized with HIF-1α in glioma cells surrounding areas of necrosis, suggesting a correlation with HIF-1α. ⁶ Hypoxia promotes expansion and inhibits the differentiation of human glioma-derived cancer stem cells through activation of HIF-1α. ¹⁰ The present study focused on the role of HIF-1α in irradiation-induced cxcr4 production. Silencing of HIF-1α by RNA interference attenuates glioma cell growth in vivo. ¹¹ Low-dose irradiation was found to activate endothelial cell migration directly via SDF-1α, independent of HIF-1α induction. ¹² Irradiation-stimulated upregulation of cxcr4 in glioma cells was not inhibited by HIF-1α knockdown in the present study, suggesting that HIF-1α is not the key mediator of irradiation induced SDF-1α/CXCR4 signalling.

Our present study found that irradiation induced cxcr4 expression. The SDF-1α/CXCR4 and VEGF/VEGFR pathways synergistically induce tumour neoangiogenesis and metastasis, ¹³ and interrupting this synergistic axis would be a more efficient antiangiogenesis strategy than inhibiting VEGF alone. Preclinical studies have indicated that the therapeutic effect of anti-VEGF treatment combined with irradiation is transitory, and is followed by rapid regrowth and increased tumour aggressiveness.^3,14 Clinical studies of patients with rectal cancer found that monotherapy with the anti-VEGF antibody bevacizumab upregulates SDF-1α and CXCR4 expression in tumour cells, and higher SDF-1α plasma levels during bevacizumab treatment were significantly associated with distant metastasis. ¹⁵ Simultaneous inhibition of both VEGF and SDF-1α/CXCR4 might enhance the efficacy of radiotherapy. New targets for controlling the invasiveness of irradiation-treated glioma cells are required, thereby facilitating a new strategy in the fight against radioresistance.