Here we demonstrated, by RT-PCR analysis, the expression of both angiotensin II (Ang II) receptor subtypes, AT1 and AT2, in a breast cancer epithelial cell line, MCF-7. Ang II was not able to affect the intracellular Ca 2+ concentration in Fura-2 loaded cells suggesting that AT1-mediated phospholipid hydrolysis is not involved in its intracellular transduction pathway. Ang II modulated the activity of the Na + /K + ATPase in a dose-and timedependent manner and was mitogenic, with a dosedependent (1-1000 nM) proliferative effect and a maximal response at 100 nM. Both Na + /K + ATPase activation and stimulation of proliferation were mediated by binding of Ang II to AT1, as the effects were completely blocked by DuP 753, a specific AT1 antagonist. CGP 42112, an AT2 antagonist, did not affect Ang II actions.The main conclusion of this study is that Ang II exerts its effects on cell proliferation and Na + /K + ATPase in breast cancer epithelial cells, MCF-7, via AT1 activation independently of the Ca 2+ signalling mechanism.

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Angiotensin II (Ang II) induces, through AT1, intracellular Ca(2+) increase in both normal and cancerous breast cells in primary culture (Greco et al., 2002 Cell Calcium 2:1-10). We here show that Ang II stimulated, in a dose-dependent manner, the 24 h-proliferation of breast cancer cells in primary culture, induced translocation of protein kinase C (PKC)-alpha, -beta1/2, and delta (but not -epsilon, -eta, -theta, -zeta, and -iota), and phosphorylated extracellular-regulated kinases 1 and 2 (ERK1/2). The proliferative effects of Ang II were blocked by the AT1 antagonist, losartan. Also epidermal growth factor (EGF) had mitogenic effects on serum-starved breast cancer cells since induced cell proliferation after 24 h and phosphorylation of ERK1/2. The Ang II-induced proliferation of breast cancer cells was reduced by (a) Gö6976, an inhibitor of conventional PKC-alpha and -beta1, (b) AG1478, an inhibitor of the tyrosine kinase of the EGF receptor (EGFR), and (c) downregulation of 1,2-diacylglycerol-sensitive PKCs achieved by phorbol 12-myristate 13-acetate (PMA). A complete inhibition of the Ang II-induced cell proliferation was achieved using the inhibitor of the mitogen activated protein kinase kinase (MAPKK or MEK), PD098059, or using Gö6976 together with AG1478. These results indicate that in human primary cultured breast cancer cells AT1 regulates mitogenic signaling pathways by two simultaneous mechanisms, one involving conventional PKCs and the other EGFR transactivation.

The communication between the tumor cells and the surrounding cells helps drive the process of tumor progression. Since the microenvironment of breast cancer includes CCL20 chemokine, the purpose of this study was to determine whether CCL20 modulates the physiology of healthy breast epithelial cells in areas adjacent to the tumor. Therefore, primary cultures of mammary cells taken from normal peritumoral areas were used. We assessed that breast cells expressed CCR6 CCL20 receptor. Using molecular (siRNA) and pharmacological (inhibitors) techniques, we found multiple signaling kinases to be activated by CCR6 and involved in CCL20-induced breast cell proliferation and migration. The binding of 10 ng/ml CCL20 to CCR6 induced cell migration whilst higher concentrations (from 15 to 25 ng/ml) led to cell proliferation. CCL20 controlled cell migration and MMP-9 expression by PKC-alpha that activated Src, which caused the activation of downstream Akt, JNK, and NF-kB pathways. Furthermore, higher CCL20 concentrations increased cycE and decreased p27Kip expression ending in enhanced cell proliferation. Cell proliferation occurred through PKC-epsilon activation that transactivated EGFR and ERK1/2/MAPK pathway. Although activated by different CCL20 concentrations, these pathways function in parallel and crosstalk to some extent, inasmuch as Akt activation was responsible for ERK1/2 nuclear translocation and enhanced the transcription of of c-fos and c-myc, involved in cell proliferation. In summary, tumor cells exchange signals with the surrounding healthy cells modifying the extracellular matrix through enzyme secretion; thus, CCL20 might be a factor involved in the ontogeny of breast carcinoma.

Background and purpose: We showed previously that a new Pt complex containing an O,O′‐chelated acetylacetonate ligand (acac) and a dimethylsulphide in the Pt coordination sphere, [Pt(O,O′‐acac)(γ‐acac)(DMS)], induces apoptosis in HeLa cells. The objective of this study was to investigate the hypothesis that [Pt(O,O′‐acac)(γ‐acac)(DMS)] is also cytotoxic in a MCF‐7 breast cancer cell line relatively insensitive to cisplatin, and to gain a more detailed analysis of the cell death pathways. Experimental approach: Cells were treated with Pt compounds and cytotoxicity tests were performed, together with Western blotting of various proteins involved in apoptosis. The mitochondrial membrane potential was assessed by fluorescence microscopy and spectrofluorometry and the Pt bound to cell fractions was measured by atomic absorption spectrometry. Key results: In contrast to cisplatin, the cytotoxicity of [Pt(O,O′‐acac)(γ‐acac)(DMS)] correlated with cellular accumulation but not with DNA binding. Also, the Pt content in DNA bases was considerably higher for cisplatin than for [Pt(O,O′‐acac)(γ‐acac)(DMS)], thus excluding DNA as a target of [Pt(O,O′‐acac)(γ‐acac)(DMS)]. [Pt(O,O′‐acac)(γ‐acac)(DMS)] exerted high and fast apoptotic processes in MCF‐7 cells since it provoked: (a) mitochondria depolarization; (b) cytochrome c accumulation in the cytosol; (c) translocation of Bax and truncated‐Bid from cytosol to mitochondria and decreased expression of Bcl‐2; (d) cleavage of caspases ‐7 and ‐9, and PARP degradation; (e) chromatin condensation and DNA fragmentation. Conclusions and implications: [Pt(O,O′‐acac)(γ‐acac)(DMS)] is highly cytotoxic for MCF‐7 cells, cells relatively resistant to many chemotherapeutic agents, as it activates the mitochondrial apoptotic pathway. Hence, [Pt(O,O′‐acac)(γ‐acac)(DMS)] has the potential to provide us with new opportunities for therapeutic intervention. British Journal of Pharmacology (2008) 153, 34–49; doi:; published online 19 November 2007

Blood lipoproteins are formed by various amounts of cholesterol (C), triglycerides (TGs), phospholipids, and apolipoproteins (Apos). ApoA1 is the major structural protein of high-density lipoprotein (HDL), accounting for ~70% of HDL protein, and mediates many of the antiatherogenic functions of HDL. Conversely, ApoB is the predominant low-density lipoprotein (LDL) Apo and is an indicator of circulating LDL, associated with higher coronary heart disease (CHD) risk. Thus, the ratio of ApoB to ApoA1 (ApoB/ApoA1) is used as a surrogate marker of the risk of CHD related to lipoproteins. Elevated or abnormal levels of lipids and/or lipoproteins in the blood are a significant CHD risk factor, and several studies support the idea that aerobic exercise decreases CHD risk by partially lowering serum TG and LDL-cholesterol (LDL-C) levels and increasing HDL-C levels. Exercise also exerts an effect on HDL-C maturation and composition and on reverse C transport from peripheral cells to the liver to favor its catabolism and excretion. This process prevents atherosclerosis, and several studies showed that exercise training increases heart lipid metabolism and protects against cardiovascular disease. In these and other ways, it more and more appears that regular exercise, nutrition, and strategies to modulate lipid profile should be viewed as an integrated whole. The purpose of this review is to assess the effects of endurance training on the nontraditional lipid biomarkers, including ApoB, ApoA1, and ApoB/ApoA1, in CHD risk.

Macrophage inflammatory protein-3␣/CCL20 is a recently identified chemokine that binds to CCR6 and acts as a chemoattractant for memory/differentiated T-cells, B-cells, and immature dendritic cells. We have previously reported that CCL20 and CCR6 mRNAs are expressed in the CNS of SJL mice with experimental autoimmune encephalomyelitis (EAE) and that CCL20 is produced by CNS-infiltrating leukocytes at disease onset and, additionally, by intraparenchymal astrocyte-like cells during disease relapses. In this study, we provide further immunohistochemical evidence that astrocytes represent the main CNS source of CCL20 during EAE. Moreover, we show that the proinflammatory cytokines interleukin-1␤ and tumor necrosis factor-␣, but not interferon-␥, induce expression of CCL20 mRNA and secretion of CCL20 protein in cultures of mouse brain-derived astrocytes. We also show that supernatants from cytokine-activated astrocytes stimulate the migration of polarized T helper cells and that this effect is partially inhibited by anti-CCL20 antibody. These findings suggest that, through secretion of CCL20, astrocytes could play an important role in orchestrating the recruitment of specific leukocyte subsets to the inflamed CNS and in regulating CNStargeted immune responses. GLIA 41:290 -300, 2003.

Following peripheral nerve injury, remnant Schwann cells adopt a migratory phenotype and remodel the extracellular matrix allowing axonal regrowth. Although much evidence has demonstrated that TGF-b1 promotes glioma cell motility and induces the expression of extracellular matrix proteins, the effects of TGF-b1 on Schwann cell migration has not yet been studied. We therefore investigated the cellular effects and the signal transduction pathways evoked by TGF-b1 in rattus norvegicus neuronal Schwann RSC96 cell. TGF-b1 significantly increased migration and invasion of Schwann cells assessed by the wound-healing assay and by cell invasion assay. TGF-b1-enhanced migration/invasion was blocked by inhibition of MMP-2 and MMP-9. Consistently, by real-time and western blot analyses, we demonstrated that TGF-b1 increased MMP-2 and MMP-9 mRNA and protein levels. TGF-b1 also increased MMPs activities in cell growth medium, as shown by gelatin zymography. The selective TGF-b Type I receptor inhibitor SB431542 completely abrogated any effects by TGF-b1. Indeed, TGF-b1 Type I receptor activation provoked the cytosol-to-nucleus translocation of SMAD2 and SMAD3. SMAD2 knockdown by siRNA blocked MMP-2 induction and cell migration/invasion due to TGF-b1. TGF-b1 also provoked phosphorylation of MAPKs extracellular regulated kinase 1/2 and JNK1/2. Both MAPKs were upstream to p65/NF-kB inasmuch as both MAPKs' inhibitors PD98059 and SP600125 or their down-regulation by siRNA significantly blocked the TGF-b1-induced nuclear translocation of p65/NF-kB. In addition, p65/NF-jB siRNA knockdown inhibited the effects of TGF-b1 on both MMP-9 and cell migration/invasion. We conclude that TGF-b1 controls RSC96 Schwann cell migration and invasion through MMP-2 and MMP-9 activities. MMP-2 is controlled by SMAD2 whilst MMP-9 is controlled via an ERK1/2-JNK1/2-NF-jB dependent pathway. Abbreviations used: ERK1/2, extracellular regulated kinase 1/2; FBS, fetal bovine serum; MEK, MAPK/ERK kinase; MMP (2-9), matrix metalloproteinases (2-9); MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide; NF-kB, nuclear factor-kappa B; siRNAs, small interfering RNAs; SMAD (2-3), small mother against decapentaplegic (2-3); TGF-b, transforming growth factor beta.