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.

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.

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-zeta (PKC-zeta) but not of PKC-alpha, -delta, - epsilon and -eta; (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-zeta, and by a synthetic myristoylated peptide with sequences based on the endogenous PKC-zeta pseudosubstrate region (zeta-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 zeta-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-zeta, PI3K and MAPK; of these pathways only PKC-zeta appears responsible for the induction of c-fos.

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 Ca(2+) concentration by a mechanism dependent upon the phospholipase C (PLC) activity; (b) the cytosol-to-membrane translocation of conventional (PKC)-alpha and -beta isozymes, novel PKC-delta, -epsilon, and -eta 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-delta isozyme. Conversely, Gö6976, an inhibitor of PKC-alpha and -beta isozymes, and the 18-h treatment of cells with PMA, that led to the complete down-regulation of PKC-alpha, -beta, -epsilon, and -eta, but not of PKC-delta, did not have any effect, thereby indicating that the PKC-delta 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-delta was blocked by PI3K inhibition, suggesting that PI3K is upstream to PKC-delta. In conclusion, BK has mitogenic actions in cultured human epithelial breast cells; the activation of PKC-delta through B2 receptor acts in concert with ERK and PI3K pathways to induce cell proliferation.

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- epsilon induced by ATP/UTP. These data indicated that agonist-induced PI3K and downstream PKC- epsilon 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- epsilon pathways to induce c-Fos protein and HeLa cell proliferation.

We have previously reported that bradykinin (BK) represents an influential mitogenic agent in normal breast glandular tissue. We here investigated the mitogenic effects and the signalling pathways of BK in primary cultured human epithelial breast cells obtained from a tumour and from the histologically proven non-malignant tissue adjacent to the tumour. BK provoked cell proliferation, increase in cytosolic calcium, activation of protein kinase C (PKC)-, -, -, -´and -and phosphorylation of the extracellular-regulated kinases 1 and 2 (ERK1/2). The following compounds blocked the proliferative effects of BK: Hyp3-BK, a B 2 receptor subtype inhibitor; U73122, a phospholipase C-inhibitor; GF109203X, a protein kinase C (PKC) inhibitor; and PD98059, a mitogen-activated protein kinase kinase inhibitor. Gö6976, a Ca 2+ -dependent PKC inhibitor, did not have any effect. In conclusion, the mitogenic effects of BK are retained in peritumour and tumour cells; hence, it is likely that BK has an important role in cancer endorsement and progression.

The effect of angiotensin II (Ang II) on Ca 2+ signalling in human primary cultured breast epithelial cells was investigated by using fura-2 as the Ca 2+ probe. ] i increase evoked by Ang II, while CGP4221A, an inhibitor of Ang II type II receptor (AT2) did not. AT1 desensitisation was demonstrated with respect to the Ca 2+ response after subsequent exposure of cells to Ang II and also after pretreatment for 25 min with 1000 nM phorbol 12-myristate 13-acetate. Staurosporine, an inhibitor of protein kinases C (PKC), inhibited the AT1 desensitisation.Epithelial breast cells expressed PKC-, -1, -andisozymes, and Ang II provoked translocation from the cytosol to the membranes of PKC-, -1, and -(but not -). Ang II was also able to stimulate cell proliferation in a dose-dependent manner; this effect was blocked by Gö 6976, a specific inhibitor of PKC-and -1, the Ca 2+ -dependent isozymes. The main conclusion of this study is that the the Ang II signalling mechanism in breast epithelial cells is based on the elevation of [Ca 2+ ] i released from intracellular stores through AT1 activation. In addition, Ang II stimulates cell proliferation by the activation of PKC isozymes.

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