Antonella Muscella

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.

[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.

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.

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.

AT1 angiotensin II receptor subtype in the human larynx and squamous laryngeal carcinoma

The presence of the angiotensin type 1 receptor (AT1 Ang II-R) was investigated in normal and diseased human larynx using a specific monoclonal antibody (6313/G2). When tissue AT1 content was studied by SDS electrophoresis with immunoblotting, the receptor was detected in 10/10 laryngeal tumours, and in 7/10 samples of normal tissue from the same patients. Two immunostaining bands, approximately 75 kDa, were present in all cases. Immunocytochemistry performed on sections of 45 formalin-fixed, paraffin-embedded laryngeal tissue samples showed that the receptor was expressed in normal respiratory epithelium only in a perinuclear pattern, above the nucleus toward the cell apex. In addition, the antigen was invariably present in skeletal muscle cells and in the columnar duct epithelium of minor salivary glands. The secretory cells were negative, but the antibody stained the adjacent myoepithelial cell layer. As expected, smooth muscle cells of the vessel walls also expressed Ang II-R. In metaplastic epithelium deriving from respiratory epithelium, the receptors were distributed diffusely throughout the cytoplasm of basal and parabasal cells. In dysplastic epithelium, cells of all layers were strongly positive. Finally, squamous cell tumours showed varying numbers of immunoreactive cells, which stained in a diffuse cytoplasmic and membranous pattern. Computer-assisted image analysis of the stained sections showed that the positivity for Ang II-R dramatically increased in dysplastic and well-differentiated cancer cells (3- and 5.5-fold higher than in normal epithelium, respectively), but there was less in poorly and very poorly differentiated cancer. Receptor abundance was not correlated with tumour size nor lymph node involvement. These results suggest a possible role of Ang II in the growth or function of normal and neoplastic larynx tissue, which is especially significant in early neoplastic change.

Sublethal concentrations of the platinum(II) complex [Pt(O,O′‐acac)(γ‐acac)(DMS)] alter the motility and induce anoikis in MCF‐7 cells

Background and purpose:  We showed previously that a new Pt(II) complex ([Pt(O,O′‐acac)(γ‐acac)(DMS)]) exerted high and fast apoptotic processes in MCF‐7 cells. The objective of this study was to investigate the hypothesis that [Pt(O,O′‐acac)(γ‐acac)(DMS)] is also able to exert anoikis and alter the migration ability of MCF‐7 cells, and to show some of the signalling events leading to these alterations.

PKC‐ε‐dependent cytosol‐to‐membrane translocation of pendrin in rat thyroid PC Cl3 cells

We studied the expression and the hormonal regulation of the PDS gene product, pendrin, which is, in thyrocytes, responsible for the iodide transport out of the cell. We show that PC Cl3 cells, a fully differentiated thyroid cell line, grown without TSH and insulin, express very low level of PDS mRNA; such expression is greatly increased after stimulation with insulin or TSH. 125I pre‐loaded cells showed an 125I efflux accelerated in chloride‐containing buffer with respect to chloride‐free buffer, suggesting that this efflux is chloride dependent. By immunoblotting, pendrin was found in agonists‐stimulated cells, whereas it was barely detectable in un‐stimulated cells. An increase in both PDS mRNA and protein was also obtained using phorbol ester PMA, or using 8‐Br‐cAMP and forskolin. Stimulation with insulin (1 µg/ml; 0–40 min) provoked the cytosol‐to‐membrane translocation of pendrin and a decrease of intracellular I− content in 125I pre‐loaded cells. Insulin‐ or PMA‐treated cells also showed a cytosol‐to‐membrane translocation of PKC‐δ and ‐ε. Inhibition of both PKC‐δ and ‐ε activities by GF109203X blocked pendrin translocation, whilst the inhibition of PKA did not. The selective inhibition of PKC‐δ by rottlerin did not affect the insulin‐provoked translocation of pendrin whilst it was inhibited by a PKC‐ε translocation inhibitor peptide and also by PKC‐ε downregulation using the small interfering RNA, thus indicating that such translocation was due to PKC‐ε activity. In conclusion, our study demonstrates that, in PC Cl3 cells, pendrin expression and localisation are regulated by insulin and influenced by a PKC‐ε‐dependent intracellular pathway. J. Cell. Physiol. 217: 103–112, 2008.

Human larynx expresses isoforms of the oestrogen receptor

Commercially available enzyme immunoassays (EIAs) were used for oestrogen (ER) and progesterone (PR) receptor determination in the cytosol fraction of 118 human larynx cancer specimens and in the corresponding histologically proven non-malignant tissues. Fifty-one ER positive cancerous samples had corresponding non-cancerous tissues also expressing the receptor. A high resolution isoelectric focusing (IEF) technique followed by immunoblotting with the H222 anti-ER monoclonal antibody was used to evaluate the presence of ER isoforms in the 51 ER positive human larynx cancer specimens and in their corresponding non-malignant tissues. In both tissues, four ER isoforms were detected, with isoelectric points (pI) similar to those obtained in breast and endometrium carcinomas (6.1, 6.3, 6.6 and 6.8). A significant difference in the expression of ER isoforms between cancerous and non-cancerous tissue was found; precisely, the 94.1% of the ER positive non-malignant specimens co-expressed the four isoforms while they were detected in only the 35.5% of the malignant specimens (P < 0.0001 by Fishers exact test). In larynx cancer, the concentration values of ER and PR did not correlate, nevertheless tumours co-expressing the four ER isoforms had PR levels significantly higher than those which did not (P = 0.02 by Mann-Whitney Wilcoxon sum rank test). To investigate the possibility that the isoforms of the monomeric 4S form of the ER (those with pI 6.3, 6.6, and 6.8) could dimerise, a cold agarose gel electrophoresis technique was used on IEF-separated ER isoforms. In summary, the evidence shows that all the isoforms are able to form homodimers and that the isoforms at pI 6.3 and 6.8 are able to dimerise with that at pI 6.6 but, under the same experimental conditions, they do not form the 6.3/6.8 heterodimer. It was concluded that: (1) the four isoforms of the ER are co-expressed by the non-malignant human larynx and the cancer loses the capacity to express some of them; (2) the complete complement of ER isoforms (all four) is needed for PR expression; (3) the monomeric 4S isoform with pI 6.6 has the capacity to form homo- and heterodimers, while the remaining two are only able to homodimerise.