Immunohistochemical and transcriptional expression of Matrix Metalloproteinases in full-term human umbilical cord and Human Umbilical Vein Endothelial Cells

Matrix metalloproteinases (MMPs) are extracellular zinc-dependent endopeptidases involved in the degradation and remodelling of extracellular matrix in physiological and pathological processes. MMPs also have a role on cell proliferation, migration, differentiation, angiogenesis and apoptosis. Umbilical cord is a special organ subjected to many changes during pre-natal life and whose cells can maintain a certain degree of plasticity also in post-natal period; for example recently they have been used as a source of stem cells. In this work we investigated the expression of MMPs in human umbilical cord and Human Umbilical Vein Endothelial Cells (HUVEC) though immunohistochemistry, RT-PCR and gelatin zymography. MMP-2 protein is expressed in the amniotic epithelium of human umbilical cord and in few sub-epithelial fibroblasts, while MMP-3 and MMP-10 only in the umbilical epithelium. MMP-8, MMP-9 and MMP-13 immunoreactivity is localised in the epithelium and in Wharton\u2019s jelly mesenchymal cells. Immunocytochemistry also revealed protein expression for MMP-2, 3, 8, 9 and 10 in cultured HUVEC. In agreement with immunohistochemical data, RT-PCR analysis performed on samples of whole umbilical cord confirmed the transcriptional expression for the genes encoding all the six matrix metalloproteinases investigated, while in HUVEC only the expression of MMP-2, 3, 9, 10 and 13 mRNAs was detected. Gelatin zymograpgy showed a clear MMP-2 and MMP-9 enzymatic activity in the conditioned medium of HUVEC at different culture passages, suggesting that HUVEC secrete gelatinases, that afterwards undergo extracellular activation, and this ability is not affected by passage number

Transcription factors that mediate epithelial–mesenchymal transition lead to multidrug resistance by upregulating ABC transporters

Development of multidrug resistance (MDR) is a major deterrent in the effective treatment of metastatic cancers by chemotherapy. Even though MDR and cancer invasiveness have been correlated, the molecular basis of this link remains obscure. We show here that treatment with chemotherapeutic drugs increases the expression of several ATP binding cassette transporters (ABC transporters) associated with MDR, as well as epithelial–mesenchymal transition (EMT) markers, selectively in invasive breast cancer cells, but not in immortalized or non-invasive cells. Interestingly, the mere induction of an EMT in immortalized and non-invasive cell lines increased their expression of ABC transporters, migration, invasion, and drug resistance. Conversely, reversal of EMT in invasive cells by downregulating EMT-inducing transcription factors reduced their expression of ABC transporters, invasion, and rendered them more chemosensitive. Mechanistically, we demonstrate that the promoters of ABC transporters carry several binding sites for EMT-inducing transcription factors, and overexpression of Twist, Snail, and FOXC2 increases the promoter activity of ABC transporters. Furthermore, chromatin immunoprecipitation studies revealed that Twist binds directly to the E-box elements of ABC transporters. Thus, our study identifies EMT inducers as novel regulators of ABC transporters, thereby providing molecular insights into the long-standing association between invasiveness and MDR. Targeting EMT transcription factors could hence serve as novel strategies to curb both metastasis and the associated drug resistance