Low-dose photon irradiation induces invasiveness through the SDF-1α/CXCR4 pathway in malignant mesothelioma cells

Abstract

Background: Low-dose photon irradiation has repeatedly been suspected to increase a risk of promoting local recurrence of disease or even systemic dissemination. The purpose of this study was to investigate the motility of malignant pleural mesothelioma (MPM) cell lines after low-doses of photon irradiation and to elucidate the mechanism of the detected phenotype.

Methods: H28 and H226 MPM cells were examined in clonogenic survival experiments and migration assays with and without various doses of photon and carbon ion irradiation. C-X-C chemokine receptor type 4 (CXCR4), SDF-1α, β₁ integrin, α₃ integrin, and α₅ integrin expressions were analyzed by quantitative FACS analysis, ELISA and western blots. Apoptosis was assessed via Annexin-V-staining.

Results: The migration of MPM cells was stimulated by both fetal bovine serum and by stromal cell-derived factor 1α (SDF-1α). Low doses of photon irradiation (1 Gy and 2 Gy) suppressed clonogenicity, but promoted migration of both H28 and H226 cells through the SDF-1α/CXCR4 pathway. Hypermigration was inhibited by the administration of CXCR4 antagonist, AMD3100. In contrast, corresponding doses of carbon ion irradiation (0.3 Gy and 1 Gy) suppressed clonogenicity, but did not promote MPM cell migration.

Conclusion: Our findings suggest that the co-administration of photon irradiation and the CXCR4-antagonist AMD3100 or the use of carbon ions instead of photons may be possible solutions to reduce the risk of locoregional tumor recurrence after radiotherapy for MPM.

Keywords: CXCR4; SDF-1α; carbon ion irradiation; mesothelioma; photon irradiation.

Figure 1. MPM cell migration stimulated by FBS and SDF-1α

(A) H226 cells are microscopically analyzed after the 5-hour migration assay using 0.5% FBS (left), 5% FBS (center left), 10% FBS (center right), and SDF-1α (0.1μg/ml) (right) as chemoattractants and Collagen I-coated membranes (magnification x20). (B) H28 cell migration through Collagen I- (left Figure) and Collagen IV- (middle left figure) coated membranes and H226 cell migration through Collagen I- (middle right Figure) and Collagen IV- (right Figure) coated membranes were examined in the modified Boyden chamber experiments. The number of stimulated MPM cells (H28 and H226) transmigrated through collagen I- and collagen IV-coated membranes was counted. In each Figure, the average cell counts after the 5-hour migration assay using 0.5% FBS is defined as a control, and the normalized ratio of each cell count to the control is calculated.

Figure 2. MPM cell migration following photon irradiation

(A) H226 cells are microscopically analyzed without (left) and with irradiation by single doses of 1 Gy (middle) and 2 Gy (right) in modified Boyden chamber experiments using Collagen I-coated membranes (magnification x20). (B) H28 cell migration through Collagen I- (left Figure) and Collagen IV- (middle left Figure) coated membranes and H226 cells through Collagen I- (middle right Figure) and Collagen IV- (right Figure) coated membranes were examined in the modified Boyden chamber experiments. The number of irradiated MPM cells (H28 and H226) transmigrated through collagen I- and collagen IV-coated membranes was counted. The average number of migrated cells in the non-irradiated condition is defined as a control, and the normalized ratio of each cell count to the control is calculated.

Figure 3. FACS analysis of photon-induced stimulation of expression in cell surface markers

(A) FACS analysis of the expression of β1 integrin (left column), α3 integrin (middle column), and α5 integrin (right column) does not reveal any difference among H28 (upper row) or H226 cells (lower row) without irradiation, with 1 Gy irradiation, and with 2 Gy irradiation. Unstained cells were analyzed in the same experiment as a negative control. (B) FACS histogram analysis of CXCR4 expression (upper row) upon a single dose of 1 Gy irradiation and 2 Gy irradiation in H28 cells (left) and H226 cells (right) compared to non-radiated cells. Data are given as the normalized ratio of CXCR4 expression compared to non-irradiated cells.

Figure 4. MPM cell migration following photon irradiation and treatment of cells with AMD3100 (25μg/ml)

The transmigration of H28 cells through Collagen I-coated membrane (left), H28 cells through Collagen IV-coated membrane (middle left), H226 cells through Collagen I-coated membrane (middle right), and H226 cells through Collagen IV-coated membrane (right) is examined in the modified Boyden chamber experiments In each Figure, the average cell count of non-irradiated cells is defined as a control. Results are displayed as normalized ratio of cell counts compared to the control.

Figure 5. MPM cell migration following carbon irradiation

(A) H28 cell migration through Collagen I- (left figure) and Collagen IV- (middle left figure) coated membranes and H226 cells through Collagen I- (middle right figure) and Collagen IV- (right figure) coated membranes are examined in the modified Boyden chamber experiments. Transmigrated MPM cells without irradiation (left bar) with 0.3 Gy carbon ion irradiation (middle bar) and with 1 Gy carbon ion irradiation (right bar) were counted. In each Figure, the average of the cell count without irradiation is defined as the control, and the normalized ratio of each cell count to the control is analyzed. The mean migration rate of corresponding photon irradiation is presented as a dashed line. (B) Analysis of clonogenic survival with and without photon and carbon ion irradiation in H28 and H226 cells. (C) In each Figure, MFI of the CXCR4 expression without irradiation is defined as a control, and the normalized ratio of each MFI to the control is calculated.

Figure 6. CXCR4 regulation of MPM cell migration following irradiation

(A) CXCR4 ELISA measuring the concentration of CXCR4 among the total protein lysates generated from H28 cells (left) and H226 cells (right). Cells were treated with a single dose of 1 Gy and 2 Gy photon-irradiation or 0.3 Gy and 1 Gy carbon ion irradiation prior to protein lysate generation. In each Figure, the concentration of CXCR4 is depicted as normalized ratio of measured CXCR4 concentration in analyzed samples to the CXCR4 concentration in protein lysate generated from non-irradiated cells (defined as control). (B) Western blot analysis using 20μg of total protein from H28 cells (left) and 20μg membrane-extracted protein from H226 cells (right). H28 and H226 cells were irradiated with a single dose of 1 Gy photon, 2 Gy photon, 0.3 Gy carbon ions and 1 Gy carbon ions before generation of protein lysates. Furthermore, protein was extracted from H226 cells following CXCR4 siRNA treatment. Beta-actin and Na⁺-K⁺ ATPase are used as loading controls of total protein and membrane-extracted protein, respectively. Saos-2 cell lysate are included as negative control and Jurkat cell lysate as positive control for CXCR4 protein expression.