ABSTRACT
Advanced maternal age is associated not only with a significant reduction in fertility but also with an additional risk of developing pregnancy-related disorders. Most of these disorders are now believed to be the clinical manifestation of an incorrect placentation, namely deficient transformation of maternal spiral arteries and ineffective trophoblast invasion through uterine stroma. In the present study it was hypothesized that an age-related loss in uterine redox homeostasis interferes with the function of extravillous trophoblasts (EVTs) and placentation. To test this hypothesis, relative levels of oxidatively modified proteins were evaluated in human samples from placenta and placental bed, and the role of specific oxidative modifications to proteins in placentation was studied using a cell culture model of EVTs. In the placental bed, the carbonylation level of a 66 kDa protein (identified as albumin) presented a strong, positive and significant correlation with maternal age. Albumin was immunodetected preferentially in endothelial cells and connective tissue between muscle fascicles. In vitro results showed that carbonylated albumin overload did not alter cell viability, but reduced EVTs motility and triggered cell stress response pathways. Moreover, EVTs presented decreased ability to adhere to and invade a collagen extracellular matrix pre-treated with carbonylated albumin. In conclusion, reproductive ageing is accompanied by an increase in maternal uterine carbonylated albumin, that may have a deleterious role in the modulation of EVTs function.
Subject(s)
Placenta , Trophoblasts , Albumins , Endothelial Cells , Female , Humans , Maternal Age , Oxidation-Reduction , Oxidative Stress , PregnancyABSTRACT
BACKGROUND: The glycan moieties sialyl-Lewis-X and/or -A (sLeX/A) are the primary ligands for E-selectin, regulating subsequent tumor cell extravasation into distant organs. However, the nature of the glycoprotein scaffolds displaying these glycans in breast cancer remains unclear and constitutes the focus of the present investigation. METHODS: We isolated glycoproteins that bind E-selectin from the CF1_T breast cancer cell line, derived from a patient with ductal carcinoma. Proteins were identified using bottom-up proteomics approach by nanoLC-orbitrap LTQ-MS/MS. Data were curated using bioinformatics tools to highlight clinically relevant glycoproteins, which were validated by flow cytometry, Western blot, immunohistochemistry and in-situ proximity ligation assays in clinical samples. RESULTS: We observed that the CF1_T cell line expressed sLeX, but not sLeA and the E-selectin reactivity was mainly on N-glycans. MS and bioinformatics analysis of the targeted glycoproteins, when narrowed down to the most clinically relevant species in breast cancer, identified CD44 glycoprotein (HCELL) and CD13 as key E-selectin ligands. Additionally, the co-expression of sLeX-CD44 and sLeX-CD13 was confirmed in clinical breast cancer tissue samples. CONCLUSIONS: Both CD44 and CD13 glycoforms display sLeX in breast cancer and bind E-selectin, suggesting a key role in metastasis development. Such observations provide a novel molecular rationale for developing targeted therapeutics. GENERAL SIGNIFICANCE: While HCELL expression in breast cancer has been previously reported, this is the first study indicating that CD13 functions as an E-selectin ligand in breast cancer. This observation supports previous associations of CD13 with metastasis and draws attention to this glycoprotein as an anti-cancer target.
Subject(s)
Breast Neoplasms/metabolism , CD13 Antigens/metabolism , Carcinoma, Ductal, Breast/metabolism , E-Selectin/metabolism , Glycoproteins/metabolism , Proteome/metabolism , Proteomics/methods , Breast Neoplasms/pathology , Carcinoma, Ductal, Breast/pathology , Cell Adhesion , Female , Humans , Hyaluronan Receptors/metabolism , Ligands , Tandem Mass Spectrometry , Tumor Cells, CulturedABSTRACT
The major ligands of nontransferrin-bound iron (NTBI) are suggested to be citrate and albumin. The proportion of iron binding to albumin is influenced by the degree of oxidation and glycation of the protein. LC-ICP-MS is demonstrated to be a useful technique for the speciation of NTBI, with unprocessed serum being subjected to analysis. Ferritin iron, citrate iron, and ferrioxamine can be quantified using this technique. This review describes the use of a new fluorescent probe for NTBI quantification.
