Estrogen promotes the brain metastatic colonization of triple negative breast cancer cells via an astrocyte-mediated paracrine mechanism.

Brain metastases (BM) are a devastating consequence of breast cancer. BM occur more frequently in patients with estrogen receptor-negative (ER-) breast cancer subtypes; HER2 overexpressing (HER2+) tumors and triple-negative (TN) (ER-, progesterone receptor-negative (PR-) and normal HER2) tumors. Young age is an independent risk factor for the development of BM, thus we speculated that higher circulating estrogens in young, pre-menopausal women could exert paracrine effects through the highly estrogen-responsive brain microenvironment. Using a TN experimental metastases model, we demonstrate that ovariectomy decreased the frequency of magnetic resonance imaging-detectable lesions by 56% as compared with estrogen supplementation, and that the combination of ovariectomy and letrozole further reduced the frequency of large lesions to 14.4% of the estrogen control. Human BM expressed 4.2-48.4% ER+ stromal area, particularly ER+ astrocytes. In vitro, E2-treated astrocytes increased proliferation, migration and invasion of 231BR-EGFP cells in an ER-dependent manner. E2 upregulated epidermal growth factor receptor (EGFR) ligands Egf, Ereg and Tgfa mRNA and protein levels in astrocytes, and activated EGFR in brain metastatic cells. Co-culture of 231BR-EGFP cells with E2-treated astrocytes led to the upregulation of the metastatic mediator S100 Calcium-binding protein A4 (S100A4) (1.78-fold, P<0.05). Exogenous EGF increased S100A4 mRNA levels in 231BR-EGFP cells (1.40±0.02-fold, P<0.01 compared with vehicle control) and an EGFR/HER2 inhibitor blocked this effect, suggesting that S100A4 is a downstream effector of EGFR activation. Short hairpin RNA-mediated S100A4 silencing in 231BR-EGFP cells decreased their migration and invasion in response to E2-CM, abolished their increased proliferation in co-cultures with E2-treated astrocytes and decreased brain metastatic colonization. Thus, S100A4 is one effector of the paracrine action of E2 in brain metastatic cells. These studies provide a novel mechanism by which estrogens, acting through ER+ astrocytes in the brain microenvironment, can promote BM of TN breast cancers, and suggests existing endocrine agents may provide some clinical benefit towards reducing and managing BM.

Synergistic activity of histone deacetylase and proteasome inhibition against pancreatic and hepatocellular cancer cell lines.

AIM: To determine the phenotypic effects of belinostat (bel) and bortezomib (bor) against pancreatic cancer (PC) and hepatocellular cancer (HCC) cell lines. MATERIALS AND METHODS: Antiproliferative effects were assessed using a sulforhodamine B assay. Synergy was evaluated using the Chou and Talalay method. Apoptosis was measured by caspase-3/-7 activity and PARP cleavage. Downstream effector proteins were detected via immunoblotting. Quantitative nuclear magnetic resonance (NMR)-based metabolomics analysis was performed. RESULTS: There were single-agent antiproliferative effects against PC and HCC cell lines; the combination of bel and bor (bel+bor) had a synergistic effect. There was up to a 45-fold induction of apoptosis over the control. Post-treatment cell death was associated with p21 up-regulation, more pronounced with treatment with bel+bor. Treatment with bel+bor enhanced hyperacetylation of histone H3 over single-agent bel. A metabolic signature was established for treatments with bor and bel+bor. CONCLUSION: The combination of bel+bor displayed significant antiproliferative activity against PC and HCC cell lines, with exhibiting synergistic antiproliferative and proapoptotic patterns even at suboptimal single-agent doses.

Time-dependent effects of imatinib in human leukaemia cells: a kinetic NMR-profiling study.

The goal of this study was to evaluate the time course of metabolic changes in leukaemia cells treated with the Bcr-Abl tyrosine kinase inhibitor imatinib. Human Bcr-Abl(+) K562 cells were incubated with imatinib in a dose-escalating manner (starting at 0.1 microM with a weekly increase of 0.1 microM imatinib) for up to 5 weeks. Nuclear magnetic resonance spectroscopy and liquid-chromatography mass spectrometry were performed to assess a global metabolic profile, including glucose metabolism, energy state, lipid metabolism and drug uptake, after incubation with imatinib. Initially, imatinib treatment completely inhibited the activity of Bcr-Abl tyrosine kinase, followed by the inhibition of cell glycolytic activity and glucose uptake. This was accompanied by the increased mitochondrial activity and energy production. With escalating imatinib doses, the process of cell death rapidly progressed. Phosphocreatine and NAD(+) concentrations began to decrease, and mitochondrial activity, as well as the glycolysis rate, was further reduced. Subsequently, the synthesis of lipids as necessary membrane precursors for apoptotic bodies was accelerated. The concentrations of the Kennedy pathway intermediates, phosphocholine and phosphatidylcholine, were reduced. After 4 weeks of exposure to imatinib, the secondary necrosis associated with decrease in the mitochondrial and glycolytic activity occurred and was followed by a shutdown of energy production and cell death. In conclusion, monitoring of metabolic changes in cells exposed to novel signal transduction modulators supplements molecular findings and provides further mechanistic insights into longitudinal changes of the mitochondrial and glycolytic pathways of oncogenesis.

Controlled aggregation of superparamagnetic iron oxide nanoparticles for the development of molecular magnetic resonance imaging probes.

A method for synthesizing superparamagnetic iron oxide (SPIO) multi-nanoparticle aggregates as molecular magnetic resonance imaging (MRI) contrast agents is described. The approach utilizes organic acid/base interactions in the colloid to induce highly controllable nanoparticle aggregation. Monodisperse aggregates with diameters as large as 100 nm are synthesized by manipulating the interfacial surface chemistry of the SPIO nanoparticles in tetrahydrofuran solvent. Subsequent phospholipid micelle encapsulation yields micellar multi-SPIO (mmSPIO) aggregates with enhanced T(2) relaxivity (368.0 s(-1) mmol(-1) Fe) as compared to micellar single particle SPIO (302.0 s(-1) mmol(-1) Fe). mmSPIO conjugated to anti-CA125 monoclonal antibodies were incubated with ovarian carcinoma cell lines to demonstrate targeted in vitro molecular MRI, resulting in a 66% shortening in T(2) time for CA125 positive NIH:OVCAR-3 cells and a less than 3% change in T(2) time for CA125 negative SK-OV-3 cells. The controllable aggregation of mmSPIO shows potential for the development of molecular MRI contrast agents with optimal sizes for specific diagnostic imaging applications.