Following removal of the primary breast tumour by conservative surgery patients
March 18, 2017
Following removal of the primary breast tumour by conservative surgery patients may still have additional malignant foci scattered throughout the breast. 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 ABT-751 improved by inhibiting the pro-invasive gene upregulated by radiation. 4 This data also confirmed that proMMP-2 was absent from the FBS-free culture media (Figure 1B lane 2). ProMMP-2 is activated on the surface of breast cancer cells by the MT1-MMP and TIMP-2. Their corresponding mRNA expressed by MDA-MB-231 and MCF-7 cells was quantified using a real-time PCR assay (Table 1). The expression of both MT1-MMP and TIMP-2 by MDA-MB-231 cells plated on irradiated Matrigel were significantly increased by 3.07-fold and 1.59-fold respectively. Conversely MT1-MMP was not detectable in the weakly metastatic cells MCF-7 while the level of TIMP-2 was not significantly increased by the irradiated Matrigel. Regarding the MMP-2 its expression was stimulated in both cell lines plated on irradiated Matrigel supporting the results obtained with the zymography analysis. Table 1 Effects of irradiated Matrigel on MMP-2 MT1-MMP and TIMP-2 expression Enhancement of MDA-MB-231 cells invasion capacity Invasion chambers were then used to determine whether irradiation of Matrigel can increase the invasiveness of breast cancer cells. Invasion chambers contain an 8?2). This enhancement of the invasiveness of the breast cancer cells was further increased when the invasion chambers were covered by a layer of PBS during exposure to radiation that is an 8.5-fold increase compared to non-irradiated control (condition no. 1 3). These data suggest that ionising radiation does induce some modification of the Matrigel which enhances the invasiveness of MDA-MB-231 cells. Table 2 Effect of Matrigel irradiation on the invasiveness of MDA-MB-231 PIK3CG cells ABT-751 The invasion assay was then repeated to determine whether pro-invasive factors stored in the Matrigel could be released by the ionising radiation. To verify this hypothesis invasion chambers covered ABT-751 by a layer of PBS were irradiated. These conditioned PBS were then transferred to new invasion chambers where MDA-MB-231 cells were then added (condition no. 4). As seen in Table 2 conditioned PBS isolated from irradiated Matrigel increased by more than eightfold the number of MDA-MB-231 cells that have crossed the Matrigel compared to non-irradiated invasion chambers (condition no. 1 4). These data suggest that ionising radiation can induce the release of pro-invasive factors stored in Matrigel which can enhance the invasiveness of MDA-MB-231 breast cancer cells. Enhancement of MMP-2 activity on breast cancer cells surface Irradiated Matrigel increases the expression of MMP-2 as shown by an enhancement of its mRNA and the release of proMMP-2 protein in culture media. We have also determined whether the activity of MMP-2 on the cell membrane of MDA-MB-231 ABT-751 and MCF-7 cells was also increased. The two cell lines were plated on irradiated Matrigel and incubated for 18?h. A fluorogenic peptide cleaved by MMP-2 was then added. Our data demonstrate that irradiation of Matrigel leads to a 4.5-fold increase of MMP-2 activity on the surface of MDA-MB-231 cells while no MMP-2 activity was measured on the MCF-7 cells (Figure 4). Figure 4 Matrix metalloproteinase (MMP)-2 activity on MDA-MB-231 ABT-751 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 … Radiation alone did not convert proMMP-2 into active MMP-2 Radiolysis of water by ionising radiation generates the free radicals O2·? and ·OH. Studies in our laboratory and elsewhere have shown that these free radicals can convert proMMP-2 into active MMP-2 (Saari irradiation of human glioma cells increased the expression of MMP-2 and enhanced their invasiveness.