enic Hras transformed counterparts. These results are consistent with those obta

f a stellate phenotype in MDA MB 231 cells is MMP dependent To confirm that the stellate phenotype induced in the MDA MB 231 cells in Matrigel was indeed dependent on BMP4, the cells were treated with BMP4 together with a BMP antagonist Gremlin, which inhibits the actions of BMP2, 4 and −7,Gremlin alone had no effect on the morphology of the cells,The cells treated with both KU-0063794 臨床試験 Gremlin and BMP4 had similar morphology than vehicle treated cells and thus Gremlin was able to reverse the stellate phenotype,We then speculated that the stellate phenotype may require the action of matrix metalloproteinases,A broad spectrum MMP inhibitor Batimastat was employed to test its potential in inhibiting the BMP4 induced phenotype.<br><br>@ Batimastat alone resulted in a mod erate reduction of growth of the cells as compared to vehicle treated cells,However, Batimastat was able to inhibit the formation of BMP4 induced stel late structures and, somewhat surprisingly, the combin ation of Batimastat and BMP4 resulted in a pronounced reduction in the size of the Lenalidomide 臨床試験 cell structures,As the stellate phenotype was reversed by an MMP inhibitor, we next examined the contribution of individual MMPs to this phenotype. Using quantitative RT PCR, the expression levels of seven MMPs known to be targeted by Batimastat were measured in BMP4 and vehicle treated MDA MB 231 cells grown in Matrigel for 14 days. MMP2, MMP7 and MMP9 were not expressed in the MDA MB 231 cells at a sufficient level to allow accurate measurements and there was no difference in ADAM17 expression between BMP4 and vehicle treated cells,In contrast, there was a dramatic 19 fold increase in MMP3 expres sion and a 3.<br><br>@ 7 fold increase in MMP14 ex pression in BMP4 treated cells as compared to vehicle treated cells. In addition, MMP1 expression was 4. 3 times higher in BMP4 treated cells but the difference was not statistically significant. To further verify that the induction of MMP3 and MMP14 was ex clusively related to the BMP4 induced stellate pheno type buy LY294002 in MDA MB 231 cells, we measured MMP3 and MMP14 mRNA levels in one of the non stellate cell lines, BT 474, under similar conditions and found that in this case BMP4 did not induce the expression of these MMPs,Discussion We have previously shown that BMP4 reduces prolifera tion and increases migration of breast cancer cells in vitro,As these results were derived from cells grown in 2D monolayer culture, we set out to analyze the effect of BMP4 in a more physiological setting by employing 3D culture systems.<br><br>@ We approached this issue by using both a biological gel and a synthetic material with RGD peptides and MMP degradable peptide links,The two materials studied provided dissimilar 3D envi ronments as first evidenced by differences in the morph ology of the normal and cancer cell clusters. The MCF 10A normal mammary epithelial cells had a polarized acini structure in Matrigel, as previously shown,while in PEG gel the cells formed irregular non polarized struc tures. Similarly, the morphology of the different cancer cells varied between the two 3D models, with the struc tures formed in Matrigel again corresponding to those previously reported,On a functional level, the growth response of cells to BMP4 treatment in PEG gel mirrored the 2D data, whereas in Matrigel more diverse effects were observed.<br><br>@ These data could be explained by several factors. Matrigel contains multiple biologically active molecules, such as laminin, collagen IV and many growth factors,that are likely to impact the results obtained. Of these biologically active molecules, e. g. laminin 1 has been shown to be essential for correct polarization of primary luminal epithelial cells in collagen gels,It has also been reported that 50 mM RGD peptide is an optimal concentration for acinar growth of MCF 10A cells in poly ethylene glycol tetravinyl sulfone gel,A lower concentration of RGD was present in the PEG gel used here, possibly explaining the lack of acinar formation.<br><br>@ In addition, the stiffness and elasticity of the matrix is known to influence the cellular phenotype, in cluding proliferation, differentiation and migration, in 3D environments,To summarize, the differences in cell morphology and BMP4 response between the two ma terials tested demonstrate that the mere 3D architecture is not sufficient to mimic the biological effects of tissue en vironment. Based on the morphological characteristics, Matrigel seems to provide a more appropriate milieu for breast epithelial cells. While many synthetic 3D materials are entering the market, they should be used cautiously until their biological properties have been explored.@ Previous data from us and others clearly demon strate that BMP4 reduces the proliferation of breast cancer cells in 2D culture, and similar results have been reported in other tumor types,Here we extend these find ings and first show the same growth suppressive effect of BMP4 in MCF 10A normal immortalized breast epithelial cells both in 2D and 3D e