Santo Marsigliante

Angiotensin II activates extracellular signal regulated kinases via protein kinase C and epidermal growth factor receptor in breast cancer cells.

Angiotensin II (Ang II) induces, through AT1, intracellular Ca2+ 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)‐α, ‐β1/2, and δ (but not ‐ε, ‐η, ‐θ, ‐ζ, and ‐ι), 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‐α and ‐β1, (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. J. Cell. Physiol. 196: 370–377, 2003.

Angiotensin II type 1 receptor expression in human breast tissues.

We demonstrate the expression of angiotensin II type 1 (AT1) receptors in normal and diseased human breast tissues. Using monoclonal antibody 6313/G2, directed against a specific sequence in the extracellular domain of the AT1 receptor, immunocytochemical analysis revealed positive immunoreactivity in membrane and cytoplasm of specific cell types. Immunoblotting of solubilized proteins separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) from benign and malignant tumours identified a single immunoreactive species with a molecular mass of approximately 60 kDa, consistent with that of the mature glycosylated receptor. In studies of [125I]angiotensin II binding using breast membrane preparations, concentrations of specific angiotensin II binding sites were found to range from 1.8 to 100 fmol mg(-1) protein, with a K(d) of approximately 60 nM. Most of the specifically bound [125I]angiotensin II was displaced by losartan, a specific angiotensin II type 1 receptor antagonist, while less was displaced by the AT2 receptor type antagonist, CGP42112A, thus confirming the prevalence of AT1 receptors in this tissue type. These data suggest that the renin-angiotensin system may be involved in normal and abnormal breast tissue function.

[Pt(O,O′‐acac)(γ‐acac)(DMS)], a new Pt compound exerting fast cytotoxicity in MCF‐7 breast cancer cells via the mitochondrial apoptotic pathway

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.

PKC-ζ is required for angiotensin II-induced activation of ERK and synthesis of C-FOS in MCF-7 cells

We examined the signalling pathways responsible for the Ang II induction of growth in MCF‐7 human breast cancer cells. Ang II in MCF‐7 cells induced: (a) the translocation from the cytosol to membrane and nucleus of atypical protein kinase C‐ζ (PKC‐ζ) but not of PKC‐α, ‐δ, ‐ε and ‐η; (b) the expression of c‐fos mRNA and protein; (c) the phosphorylation of the extracellular signal‐regulated protein kinases 1 and 2 (ERK1/2). All these effects were due to the activation of the Ang II type I receptor (AT1) since they were blocked by the AT1 antagonist losartan. The Ang II‐stimulated ERK1/2 phosphorylation was blocked by (a) high doses of staurosporine, inhibitor of PKC‐ζ, and by a synthetic myristoylated peptide with sequences based on the endogenous PKC‐ζ pseudosubstrate region (ζ‐PS); (b) PD098059, a mitogen‐activated protein kinase kinase inhibitor (MAPKK/MEK); and, moreover, (c) the inhibitors of phosphoinositide 3‐kinases (PI3K), LY294002 and wortmannin, thus indicating that PI3K may act upstream of ERK1/2. The Ang II‐evoked c‐fos induction was blocked only by high doses of staurosporine and by ζ‐PS whilst PD098059, LY294002 and wortmannin were ineffective, thus indicating that c‐fos induction is not due to ERK1/2 activity. When the epidermal growth factor‐receptor (EGFR) tyrosine kinase activity was inhibited by the use of its inhibitor AG1478, Ang II was still able to induce ERK1/2 phosphorylation and c‐fos expression, therefore proving that the transactivation of EGFR was not required for these Ang II effects in MCF‐7 cells. The previously reported proliferation of MCF‐7 cells induced by Ang II was blocked by PD098059 and by wortmannin in a dose‐dependent manner, thereby indicating that in MCF‐7 cells the PI3K and ERK pathways mediate the mitogenic signalling of AT1. Our results suggest that in MCF‐7 cells Ang II activates multiple signalling pathways involving PKC‐ζ, PI3K and MAPK; of these pathways only PKC‐ζ appears responsible for the induction of c‐fos. J. Cell. Physiol. 197: 61–68, 2003© 2003 Wiley‐Liss, Inc.

A filtration-based real-time PCR method for the quantitative detection of viable Salmonella enterica and Listeria monocytogenes in food samples.

We developed a novel filtration-based method that can eliminate dead or severely damaged Salmonella enterica and Listeria monocytogenes in food samples. This new method can recover all viable bacteria in less than 30 min, and can be coupled with a subsequent bacterial DNA extraction and real-time PCR. No statically significant differences (p<0.01) were found between real-time PCR results obtained separately from S. enterica and L. monocytogenes when different ratios of living and dead cells were used. The analytical sensitivity in both cases was 1 genome equivalent (GE), and the quantification was linear (R(2)>0.9969) over a 5-log dynamic range with PCR efficiencies >0.9754. When compared with the standard microbiological methods for the detection of these foodborne pathogens, the relative accuracy was excellent ranging from 95.72% to 104.48%. Finally, we applied the pre-treatment method to the direct detection of viable forms of these foodborne pathogens in food samples using yogurt as a model, the results being similar to those obtained using pure cultures.

miR-155 is up-regulated in primary and secondary glioblastoma and promotes tumour growth by inhibiting GABA receptors.

An altered expression of microRNAs (miRNAs) contributes both to the development of cancer and to the progression of the disease. Malignant tumours and tumour cell lines have widespread deregulated expressions of miRNAs compared to normal tissues. In this study, we investigated the expression profiles of 340 mammalian miRNAs in 93 cases of multiform glioblastoma (primary and secondary glioblastoma tumours), by means of DNA microarrays. We show that the expression profiles of 10 miRNAs can distinguish primary from secondary glioblastoma types. Moreover, we found elevated miR-155 levels in primary and secondary glioblastoma tissues as well as in glioblastoma primary cultures. We hypothesised that γ-aminobutyric acid A receptor 1 (GABRA1) is a miR-155 target, and studied the correlation between miR-155 up-regulation and the GABRA1 protein in cultured glioblastoma cells by miRNA silencing. We show that a decrease in miR-155 expression to normal levels restores the expression of GABRA1, making glioblastoma cells sensitive to signals that inhibit cell proliferation mediated by GABRA1. In conclusion, the expression patterns of different miRNAs characterise primary and secondary glioblastomas. The aberrant overexpression of miR-155 contributes to the malignant phenotype of glioblastoma cells removing growth inhibition.

Mitogenic signalling by B2 bradykinin receptor in epithelial breast cells

The kinin peptides are released during inflammation and are amongst the most potent known mediators of vasodilatation, pain, and oedema. A role in the modulation or induction of healthy breast tissue growth has been postulated for tissue kallikrein present in human milk. Moreover, tissue kallikrein was found in malignant human breast tissue and bradykinin (BK) stimulates the proliferation of immortalised breast cancer cells. Aim of the present article was to investigate whether BK also exerts mitogenic activity in normal breast epithelial cells and partially characterise the signalling machinery involved. Results show that BK increased up to 2‐fold the 24 h proliferation of breast epithelial cells in primary culture, and that the BK B2 receptor (not B1) inhibitor alone fully blocked the BK response. Intracellular effects of B2 stimulation were the following: (a) the increase of free intracellular Ca2+ concentration by a mechanism dependent upon the phospholipase C (PLC) activity; (b) the cytosol‐to‐membrane translocation of conventional (PKC)‐α and ‐β isozymes, novel PKC‐δ, ‐ε, and ‐η isozymes; (c) the phosphorylation of the extracellular‐regulated kinase 1 and 2 (ERK1/2); and (d) the stimulation of the expression of c‐Fos protein. EGF, a well known stimulator of cell proliferation, regulated the proliferative response in human epithelial breast cells to the same extent of BK. The effects of BK on proliferation, ERK1/2 phosphorylation, and c‐Fos expression were abolished by GF109203X, which inhibits PKC‐δ isozyme. Conversely, Gö6976, an inhibitor of PKC‐α and ‐β isozymes, and the 18‐h treatment of cells with PMA, that led to the complete down‐regulation of PKC‐α, ‐β, ‐ε, and ‐η, but not of PKC‐δ, did not have any effect, thereby indicating that the PKC‐δ mediates the mitogenic signalling of BK. Phosphoinositide 3‐kinase (PI3K), tyrosine kinase of the epidermal growth factor receptor (EGFR), and mitogen activated protein kinase kinases (MEK) inhibitors were also tested. The results suggest that EGFR, PI3K, and ERK are required for the proliferative effects of BK. In addition, the BK induced cytosol‐to‐membrane translocation of PKC‐δ was blocked by PI3K inhibition, suggesting that PI3K is upstream to PKC‐δ. In conclusion, BK has mitogenic actions in cultured human epithelial breast cells; the activation of PKC‐δ through B2 receptor acts in concert with ERK and PI3K pathways to induce cell proliferation. J. Cell. Physiol. 201: 84–96, 2004.

CCL20 induces migration and proliferation on breast epithelial cells

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. J. Cell. Physiol. 228: 1873–1883, 2013.

Computerised counting of tumour infiltrating lymphocytes in 90 breast cancer specimens

Tumour infiltrating lymphocytes (TILs) implicated in immunologic cytotoxicity were evaluated by immunohistochemistry and digitally counted in serial sections from 90 breast cancers in order to assess their number, the relationships between them and to tumour histology. CD3+, CD4+, CD8+, CD20+, CD25+ and CD56+ lymphocytes were found in 58 (64.4%), 52 (57.7%), 50 (55.5%), 22 (24.4%), 11 (12.2%) and 21 (23.3%) tumours, respectively. There was no difference in the number of TILs between pure infiltrating ductal (NOS) and non-ductal carcinomas, and no relationship between TILs and histological grades was found. CD3+ TILs directly correlated to age, while lymph node negative patients had tumours infiltrated by fewer CD4+ TILs with respect to lymph node positive patients. In 25/90 patients, randomly chosen, the status of peripheral blood lymphocytes was evaluated but no differences with respect to the status found in healthy blood donors was obtained; nonetheless while in some patients CD8+ TILs outnumbered CD4+ TILs in situ, the CD4/CD8 ratio was normal in their peripheral blood. The results show a considerable diversity of TILs among breast tumours, their lack of relationship with the status of the peripheral blood cells, and their potential important relationship with age (CD3+) and lymph node status (CD4+).

Activation of P2Y2 receptor induces c-FOS protein through a pathway involving mitogen-activated protein kinases and phosphoinositide 3-kinases in HeLa cells.

The effects of P2Y2 purinoceptor activation on c‐Fos expression and the signaling pathways evoked by extracellular ATP/UTP in HeLa cells were investigated. We found that P2Y2 activation induced c‐Fos protein and phosphorylated the extracellular signal‐regulated kinases 1 and 2 (ERK1/2). The P2Y2‐stimulated c‐Fos induction was partly blocked (a) by U73122, a phospholipase C inhibitor, (b) by Gö6976, a conventional PKC inhibitor, (c) by PD098059, a mitogen‐activated protein kinase kinase inhibitor, and, moreover, (d) by the inhibitors of phosphoinositide 3‐kinases (PI3K), LY294002 and wortmannin. When Gö6976 and PD098059, or Gö6976 and wortmannin, were combined there was a totally inhibition of P2Y2‐induced c‐Fos increase. Either U73122 or Gö6976 did not inhibit ERK1/2 phosphorylation induced by ATP/UTP, while it was inhibited by LY294002 (or wortmannin) and by staurosporine. Additionally, wortmannin inhibited the cytosol‐to‐membrane translocation of PKC‐ε induced by ATP/UTP. These data indicated that agonist‐induced PI3K and downstream PKC‐ε activation mediated the effect of ATP/UTP on ERK1/2 activation. To test the biological consequences of ERK1/2 activation, the effect of P2Y2 on cell functions were examined. P2Y2 stimulation increased cell proliferation and this effect was attenuated by PD098059 in a dose‐dependent manner, thereby indicating that the ERK pathway mediates mitogenic signaling by P2Y2. In conclusion, the activation of conventional PKCs through P2Y2 receptor acts in concert with ERK and PI3K/PKC‐ε pathways to induce c‐Fos protein and HeLa cell proliferation.