Regulation of ionizing radiation-induced adhesion of breast cancer cells to fibronectin by alpha5beta1 integrin

Abstract

Ionizing radiation (IR) is commonly used for cancer therapy, however, its potential influence on cancer metastatic potential remains controversial. In this study, we elucidated the role of integrins in regulation of IR-altered adhesion between breast cancer cells and extracellular matrix (ECM) proteins, which is a key step in the initial phase of metastasis. Our data suggest that the extent of effect that ionizing radiation had on cell adhesion depended on the genetic background of the breast cancer cells. Ionizing radiation was a better adhesion inducer for p53-mutated cells, such as MDA-MB-231 cells, than for p53 wild-type cells, such as MCF-7 cells. While IR-induced adhesions between MDA-MB-231 cells to fibronectin, laminin, collagen I and collagen IV, only blocking of the adhesion between α5β1 integrin and fibronectin using anti-α5β1 integrin antibody could completely inhibit the radiation-induced adhesion of the cells. A soluble Arg-Gly-Asp peptide, the binding motif for fibronectin binding integrins, could also reduce the adhesion of the cells to fibronectin with or without ionizing radiation exposure. The inhibition of the cell-fibronectin interaction also affected, but did not always correlate with, transwell migration of the cancer cells. In addition, our data showed that the total expression of α5 integrin and surface expression of α5β1 integrin were increased in the cells treated with ionizing radiation. The increased surface expression of α5β1 integrin, along with the adhesion between the cells and fibronectin, could be inhibited by both ataxia telangiectasia mutated (ATM) and Rad3-related (ATR) kinase inhibitors. These results suggested that ATM/ATR-mediated surface expression of α5β1 integrin might play a central role in regulation of ionizing radiation-altered adhesion.

FIG. 1

The effect of IR on adhesion of MDA-MB-231 cells to ECM proteins. The cells were irradiated (10 Gy) or sham irradiated. At 24 h postirradiation, the cells were incubated with each of the function blocking antibodies as indicated and placed on an ECM coated strip. After removing the unattached cells, the cells were stained with crystal violet, resuspended with 2% SDS, and cell adhesion was determined by absorbance at 570 nm. The wells on the ECM strip were coated with FN (panel A), LN (panel B), Col I (panel C) and Col V (panel D). Relative cell adhesion was determined by the absorbance of the treated cells normalized to the absorbance of the control cells adhering to ECM-coated plate, and the value from control cells was arbitrarily set at 1. Data represent the mean ± SEM of three independent experiments, each done in triplicate. *P < 0.05 IR vs. control, **P < 0.05 IR with function blocking vs. IR without function blocking.

FIG. 2

The effect of RGD peptide on ionizing radiation-altered adhesion and migration of MDA-MB-231 cells. Panel A: The cells were irradiated (10 Gy) or sham irradiated. At 24 h postirradiation, the cells were incubated with RGD peptide as indicated. The cells were placed on an FN-coated strip and the attached cells were stained with crystal violet. The cell adhesion was determined by absorbance at 570 nm. Relative cell adhesion was determined by the absorbance of the treated cells normalized to the absorbance of the control cells adhering to ECM-coated plate, and the value from control cells was arbitrarily set at 1. Panel B: The cells were incubated with RGD peptide and then placed on an FN-coated filters. After incubating for 2 h, the cells were irradiated with 10 Gy of ionizing radiation or sham irradiated and incubated for 24 h. Cells that migrated to the lower side of the membrane were stained with crystal violet and counted under a microscope. Data represent the mean ± SEM of three independent experiments, each done in triplicate. *P < 0.05 IR vs. control, **P < 0.05 control vs. RGD, ***IR vs. IR with RGD. Panel C: The cells were preincubated with isotype antibody or antibody against α5β1 integrin for 30 min, and placed on an FN-coated filters. After 2 h, the treated cells were irradiated with 10 Gy of IR or sham irradiated. At 24 h post incubation, cells that migrated to the lower side of the membrane were stained with crystal violet and counted under microscope. Relative cell migration was determined by the number of the treated cells normalized to the number of the control cells adhering to the lower side of the membrane, and the value from control cells was arbitrarily set at 1. Data represent the mean ± SEM of three independent experiments, each done in triplicate. *P < 0.05 IR vs. control, **P < 0.05 IR vs. α5β1, ***α5β1 vs. IR with α5β1.

FIG. 3

The effect of ionizing radiation on MDA-MB-231 cell surface expression of integrins. The cells were irradiated (10 Gy) or sham irradiated and then incubated for 24 h. Panel A: The cells were suspended and incubated with anti-α5, β1, activated β1 (HUT-4), α2β1 or α5β1. Surface expression of each integrin was determined by flow cytometry. Isotype control antibody was used as a negative control antibody. The histograms represent a typical appearance of three independent experiments. Panel B: The average percentage change of cell surface level of integrin after irradiation. Panel C: The irradiated cells were lysed. The expression level of integrin in total lysate was detected by Western blotting analysis.

FIG. 4

The role of ATM kinase on MDA-MB-231 cell adhesion. The cells were pretreated with 10 uM of CGK-733, KU-55933, VE-821 or DMSO for 2 h. The treated cells were exposed to 10 Gy irradiation and incubated for 24 h. Panel A: The cells were suspended and incubated with anti-α5β1 antibody. Surface expression of α5β1 integrin was determined by flow cytometry. Isotype control antibody was used as a negative control antibody. The histograms represent a typical appearance of three independent experiments. Panel B: The cells were placed on an FN-coated strips. The attached cells were stained with crystal violet and cell adhesion was determined by absorbance at 570 nm. Relative cell adhesion was determined by the absorbance of the treated cells normalized to the absorbance of the control cells adhering to FN-coated plate and the value from control cells was arbitrarily set at 1. Data represent the mean ± SEM of three independent experiments, each done in triplicate. *P < 0.001 control vs. IR, **P < 0.001 control vs. CGK-733, ***P < 0.001 IR vs. IR with CGK-733, KU-55933 or VE-821.