Different cellular receptors mediate the biological effects induced by estrogens. In addition to the classical nuclear estrogen receptors (ERs)-alpha and -beta, estrogen also signals through the seven-transmembrane G-protein-coupled receptor (GPR)-30. Using as a model system SkBr3 and BT20 breast cancer cells lacking the classical ER, the regulation of GPR30 expression by 17beta-estradiol, the selective GPR30 ligand G-1, IGF-I, and epidermal growth factor (EGF) was evaluated. Transient transfections with an expression plasmid encoding a short 5'-flanking sequence of the GPR30 gene revealed that an activator protein-1 site located within this region is required for the activating potential exhibited only by EGF. Accordingly, EGF up-regulated GPR30 protein levels, which accumulated predominantly in the intracellular compartment. The stimulatory role elicited by EGF on GPR30 expression was triggered through rapid ERK phosphorylation and c-fos induction, which was strongly recruited to the activator protein-1 site found in the short 5'-flanking sequence of the GPR30 gene. Of note, EGF activating the EGF receptor-MAPK transduction pathway stimulated a regulatory loop that subsequently engaged estrogen through GPR30 to boost the proliferation of SkBr3 and BT20 breast tumor cells. The up-regulation of GPR30 by ligand-activated EGF receptor-MAPK signaling provides new insight into the well-known estrogen and EGF cross talk, which, as largely reported, contributes to breast cancer progression. On the basis of our results, the action of EGF may include the up-regulation of GPR30 in facilitating a stimulatory role of estrogen, even in ER-negative breast tumor cells.
Mesoporous silica nanoparticles (MSNs) have been proposed as DNA and drug delivery carriers, as well as efficient tools for fluorescent cell tracking. The major limitation is that MSNs enter cells regardless of a target-specific functionalization. Here we show that non functionalized MSNs, synthesized using a PEG surfactant-based interfacial synthesis procedure, do not enter cells, while a highly specific, receptor mediated, cellular internalization of folic acid (FOL) grafted MSNs (MSN-FOL), occurs exclusively in folate receptor (FR) expressing cells. Neither the classical clathrin pathway nor macropinocytosis is involved in the MSN endocytic process, while fluorescent MSNs (MSN-FITC) enter cells through aspecific, caveolae-mediated, endocytosis. Moreover, internalized particles seem to be mostly exocytosed from cells within 96 h. Finally, cisplatin (Cp) loaded MSN-FOL were tested on cancerous FR-positive (HeLa) or normal FR-negative (HEK293) cells. A strong growth arrest was observed only in HeLa cells treated with MSN-FOL-Cp. The results presented here show that our mesoporous nanoparticles do not enter cells unless opportunely functionalized, suggesting that they could represent a promising vehicle for drug targeting applications.
Background: Insulin receptor substrate 1 (IRS-1), a cytoplasmic protein transmitting signals from the insulin and insulin-like growth factor 1 receptors, has been implicated in breast cancer. Previously, it was reported that IRS-1 can be translocated to the nucleus and modulate oestrogen receptor a (ERa) activity in vitro. However, the expression of nuclear IRS-1 in breast cancer biopsy specimens has never been examined. Aims: To assess whether nuclear IRS-1 is present in breast cancer and non-cancer mammary epithelium, and whether it correlates with other markers, especially ERa. Parallel studies were carried out for the expression of cytoplasmatic IRS-1. Methods: IRS-1 and ERa expression was assessed by immunohistochemical analysis. Data were evaluated using Pearson's correlation, linear regression and receiver operating characteristic analysis. Results: Median nuclear IRS-1 expression was found to be low in normal mammary epithelial cells (1.6%) and high in benign tumours (20.5%), ductal grade 2 carcinoma (11.0%) and lobular carcinoma (,30%). Median ERa expression in normal epithelium, benign tumours, ductal cancer grade 2 and 3, and lobular cancer grade 2 and 3 were 10.5, 20.5, 65.0, 0.0, 80 and 15%, respectively. Nuclear IRS-1 and ERa positively correlated in ductal cancer (p,0.001) and benign tumours (p,0.01), but were not associated in lobular cancer and normal mammary epithelium. In ductal carcinoma, both nuclear IRS-1 and ERa negatively correlated with tumour grade, size, mitotic index and lymph node involvement. Cytoplasmic IRS-1 was expressed in all specimens and positively correlated with ERa in ductal cancer. Conclusions: A positive association between nuclear IRS-1 and ERa is a characteristic for ductal breast cancer and marks a more differentiated, non-metastatic phenotype.
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