In vitro irradiation of basement membrane enhances the invasiveness of breast cancer cells

Abstract

Following removal of the primary breast tumour by conservative surgery, patients may still have additional malignant foci scattered throughout the breast. Radiation treatments are not designed to eliminate all these residual cancer cells. Rather, the radiation dose is calculated to optimise long-term results with minimal complications. In a tumour, cancer cells are surrounded by a basement membrane, which plays an important role in the regulation of gene expression. Using an invasion chamber, we have shown that irradiation before cell plating of a reconstituted basement membrane (Matrigel; Becton Dickinson, Bedford, MA, USA) increased the invasiveness of the breast cancer cells MDA-MB-231. This radiation enhancement of invasion was associated with the upregulation of the pro-invasive gene matrix metalloproteinase (MMP)-2. The expression of membrane type 1 matrix metalloproteinase (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP), which are required to activate the MMP-2, were also increased. Confirming the role of MMP-2 and MT1-MMP, radiation enhancement of cancer cell invasion was prevented by an MMP-2 inhibitor and an anti-MT1-MMP antibody. This study also demonstrated that radiation can potentially enhance the invasion ability by inducing the release of pro-invasive factors stored in the Matrigel. Conversely, no enhancement of invasiveness was observed with the low metastatic cell line MCF-7. This lack of invasiveness correlated with the absence of the MMP-2 activator MT1-MMP in the MCF-7 cells. Radiotherapy is an efficient modality to treat breast cancer which could be further improved by inhibiting the pro-invasive gene upregulated by radiation.

Figure 1

Irradiated Matrigel increased the secretion of matrix metalloproteinase (MMP)-2 from MDA-MB-231 and MCF-7 breast cancer cells. (A) Cancer cells (6 × 10⁵) were added on a layer of Matrigel which was previously irradiated using a ⁶⁰Co source at 20 Gy. Non-irradiated Matrigel was used as control. After 24 h incubation, the culture media was removed, concentrated and analysed by zymogram electrophoresis gel. As controls, proMMP-2 and -9 obtained from Calbiochem were activated with p-aminophenylmercuric acetate (APMA) and the activated and unactivated forms were added to the zymogram gel. (B) MMP-2 released from Matrigel following irradiation. (1) proMMP-2 activated by APMA; (2) concentrated FBS-free culture media; (3) concentrated FBS-free culture media incubated for 24 h on non-irradiated Matrigel; and (4) concentrated FBS-free culture media incubated for 24 h on Matrigel previously irradiated at 20 Gy. These assays were repeated four times.

Figure 2

Enhancement of invasiveness induced by ionising radiation. Matrigel coated in the invasion chambers was irradiated using a ⁶⁰Co source. MDA-MB-231 and MCF-7 cells (A: 4 × 10⁴ or B: 1 × 10⁴) were added on the Matrigel after the irradiation and then incubated for 6 h in MEM 0.1% BSA. MDA-MB-231 cells: ^*n=4; 0 vs 5 Gy: P=0.05; 0 vs 20 Gy: P=0.0004.

Figure 3

A matrix metalloproteinase (MMP)-2 inhibitor or an anti-membrane type 1 matrix metalloproteinase (MT1-MMP) antibody prevented radiation enhancement of MDA-MB-231 breast cancer cell invasion. MDA-MB-231 cancer cells were mixed with either the MMP-2 inhibitor (2R)-[(4-biphenylylsulfonyl)amino]-N-hydroxy-3-phenylpropionamide at 0.1 mM, or an anti-MT1-MMP antibody (20 μg ml⁻¹) before plating in the invasion chamber which was previously irradiated at 20 Gy.^*n=3, P<0.05.

Figure 4

Matrix metalloproteinase (MMP)-2 activity on MDA-MB-231 and MCF-7 cells plated on irradiated Matrigel. Matrigel was irradiated at 0 or 20 Gy and the MDA-MB-231 or MCF-7 cells were plated and incubated for 18 h at 37°C. Then the MEM 0.1% BSA was replaced by the enzymatic buffer A and the MMP-2 activity was measured by adding the fluorogenic peptide MMP Substrate III. (1) MDA-MB-231 cells and 0 Gy; (2) MDA-MB-231 cells and 20 Gy; (3) MCF-7 and 0 Gy and (4) MCF-7 and 20 Gy. ^*n=3, P<0.05.

Figure 5

Inability of ionising radiation to directly activate the proMMP-2. (A) ProMMP-2 obtained from Calbiochem was irradiated at 20 Gy. As positive control, proMMP-2 was activated by p-aminophenylmercuric acetate (APMA). (B) Active MMP-2 was irradiated at 60 Gy. The level of matrix metalloproteinase (MMP) activity was determined using the fluorogenic peptide MMP Substrate III. ^*n=6, P<0.05.