Role of epidermal growth factor receptor signaling in a Pt(II)-resistant human breast cancer cell line.

Platinum complexes are currently used for breast cancer therapy, but, as with other drug classes, a series of intrinsic and acquired resistance mechanisms hinder their efficacy. To better understand the mechanisms underlying platinum complexes resistance in breast cancer, we generated a [Pt(O,O'-acac)(γ-acac)(DMS)]-resistant MCF-7, denoted as [Pt(acac)2]R. [Pt(O,O'-acac)(γ-acac)(DMS)] was chosen as previous works showed that it has distinct mechanisms of action from cisplatin, especially with regard to cellular targets. [Pt(acac)2]R cells are characterized by increased proliferation rates and aggressiveness with higher PKC-δ, BCL-2, MMP-9 and EGFR protein expressions and also by increased expression of various genes covering cell cycle regulation, invasion, survival, and hormone receptors. These [Pt(acac)2]R cells also displayed high levels of activated signaling kinases Src, AKT and ERK/2. [Pt(acac)2]R cells incubated with [Pt(O,O'-acac)(γ-acac)(DMS)], showed a relevant EGFR activation due to PKC-δ and Src phosphorylation that provoked proliferation and survival through MERK1/2/ERK1/2 and PI3K/Akt pathways. In addition, EGFR shuttled from the plasma membrane to the nucleus maybe acting as co-transcriptional factor. The data suggest that growth and survival of resistant cells rely upon a remarkable increase in EGFR level which, in collaboration with an enhanced role of PKC-δ and Src kinases support [Pt(acac)2]R cell. It could therefore be assumed that combination treatments targeting both EGFR and PKC-δ/Src can improve therapy for breast cancer patients.

Antitumour and antiangiogenic activities of [Pt(O,O'-acac)(γ-acac)(DMS)] in a xenograft model of human renal cell carcinoma.

BACKGROUND AND PURPOSE: It is thought that the mechanism of action of anticancer chemotherapeutic agents is mainly due to a direct inhibition of tumour cell proliferation. In tumour specimens, the endothelial cell proliferation rate increases, suggesting that the therapeutic effects of anticancer agents could also be attributed to inhibition of tumour angiogenesis. Hence, we investigated the potential effects of [Pt(O,O'-acac)(γ-acac)(DMS)] ([Pt(DMS)]), a new platinum drug for non-genomic targets, on human renal carcinoma and compared them with those of the well-established anticancer drug, cisplatin. EXPERIMENTAL APPROACH: Tumour growth, tumour cell proliferation and microvessel density were investigated in a xenograft model of renal cell carcinoma, developed by injecting Caki-1 cells into BALB/c nude mice. The antiangiogenic potential of compounds was also investigated using HUVECs. KEY RESULTS: Treatment of the Caki-1 cells with cisplatin or [Pt(DMS)] resulted in a dose-dependent inhibition of cell survival, but the cytotoxicity of [Pt(DMS)] was approximately fivefold greater than that of cisplatin. [Pt(DMS)] was much more effective than cisplatin at inhibiting tumour growth, proliferation and angiogenesis in vivo, as well as migration, tube formation and MMP1, MMP2 and MMP9 secretion of endothelial cells in vitro. Whereas, cisplatin exerted a greater cytotoxic effect on HUVECs, but did not affect tube formation or the migration of endothelial cells. In addition, treatment of the xenograft mice with [Pt(DMS)] decreased VEGF, MMP1 and MMP2 expressions in tumours. CONCLUSIONS AND IMPLICATIONS: The antiangiogenic and antitumour activities of [Pt(DMS)] provide a solid starting point for its validation as a suitable candidate for further pharmacological testing.

Paracrine CCL20 loop induces epithelial-mesenchymal transition in breast epithelial cells.

We previously found that CCL20 induced primarily cultured healthy breast cell proliferation and migration. The objective of this study was to investigate the hypothesis that CCL20 modulated the epithelial-mesenchymal transition (EMT) of primarily cultured healthy breast epithelial cells and the angiogenesis in areas adjacent to the tumor. Key results showed that CCL20 (a) down-regulated E-cadherin and ZO-1; (b) up-regulated N-cadherin, vimentin, and Snail expressions; (c) increased mRNA and secretion of VEGF and (d) increased angiogenic micro vessel sprouting. Thus, the signal transduction pathways evoked by CCL20 were investigated. We showed that NF-kB p65 down-regulation (by small interfering RNA, siRNA) reversed CCL20-induced Snail and blocked the up-regulation of vimentin and N-cadherin mRNAs. Furthermore, PI3K/AKT inhibition (by LY294002) completely blocked CCL20-induced Snail and NF-kB activation. Inhibition of JNK1/2 (by SP60125) or PKC-α (by siRNA) or src (by PP1) blocked NF-kB activation and Snail expression suggesting that these kinases are all upstream of NF-kB/Snail. Inhibition of mTOR (by rapamycin) abolished the effects of CCL20 on N-cadherin and vimentin protein synthesis. Furthermore, siRNA of PKC-δ inhibited the phosphorylation of CCL20-induced mTOR and S6, increased vimentin and N-cadherin expressions and, finally, blocked the CCL20 induced-EMT. CCL20 increased mRNA and secretion of VEGF by healthy breast cells by using PKC-α, src, Akt, NF-kB, and Snail signalling. In summary, tumor cells signal to the surrounding healthy cells through CCL20 inducing the modulation of the expression of molecules involved in EMT and promoting angiogenesis directly and indirectly through the secretion of VEGF, a major contributor to angiogenesis. © 2015 Wiley Periodicals, Inc.

Antitumor activity of [Pt(O,O'-acac)(γ-acac)(DMS)] in mouse xenograft model of breast cancer.

The higher and selective cytotoxicity of [Pt(O,O'-acac)(γ-acac)(DMS)] toward cancer cell in both immortalized cell lines and in breast cancer cells in primary cultures, stimulated a pre-clinical study so as to evaluate its therapeutic potential in vivo. The efficacy of [Pt(O,O'-acac)(γ-acac)(DMS)] was assessed using a xenograft model of breast cancer developed by injection of MCF-7 cells in the flank of BALB/c nude mice. Treatment of solid tumor-bearing mice with [Pt(O,O'-acac)(γ-acac)(DMS)] induced up to 50% reduction of tumor mass compared with an average 10% inhibition recorded in cisplatin-treated animals. Thus, chemotherapy with [Pt(O,O'-acac)(γ-acac)(DMS)] was much more effective than cisplatin. We also demonstrated enhanced in vivo pharmacokinetics, biodistribution and tolerability of [Pt(O,O'-acac)(γ-acac)(DMS)] when compared with cisplatin administered in Wistar rats. Pharmacokinetics studies with [Pt(O,O'-acac)(γ-acac)(DMS)] revealed prolonged Pt persistence in systemic blood circulation and decreased nefrotoxicity and hepatotoxicity, major target sites of cisplatin toxicity. Overall, [Pt(O,O'-acac)(γ-acac)(DMS)] turned out to be extremely promising in terms of greater in vivo anticancer activity, reduced nephrotoxicity and acute toxicity compared with cisplatin.

A new platinum(II) compound anticancer drug candidate with selective cytotoxicity for breast cancer cells.

[Pt(O,O'-acac)(γ-acac)(DMS)] (PtAcD) is able to induce apoptosis in various human cancer cells, including the cisplatin-resistant human breast carcinoma MCF-7 cells. Here, to confirm that PtAcD has the potentiality for therapeutic intervention, we studied its effects in primary cultured epithelial breast cells obtained from cancers and also from the corresponding histologically proven non-malignant tissue adjacent to the tumor. We demonstrated that PtAcD (1) is more cytotoxic in cancer than in normal breast cells; (2) activated NAD(P)H oxidase, leading to PKC-ζ and PKC-α translocations; (3) activated antiapoptotic pathways based on the PKC-α, ERK1/2 and Akt kinases; (4) activated PKC-ζ and, only in cancer cell PKC-δ, responsible for the sustained phosphorylation of p38 and JNK1/2, kinases both of which are involved in the mitochondrial apoptotic process. Moreover, crosstalk between ERK/Akt and JNK/p38 pathways affected cell death and survival in PtAcD-treated breast cell. In conclusion, this study adds and extends data that highlight the pharmacological potential of PtAcD as an anti breast cancer drug.

Effects of cisplatin on matrix metalloproteinase-2 in transformed thyroid cells.

We investigated the effects of cisplatin (cisPt) on matrix metalloproteinase-2 (MMP-2) gelatinolitic activity in transformed PC E1Araf rat thyroid cells. Cells incubated with increasing cisPt concentrations showed dose- and time-dependent decrease of the MMP-2 protein and activity. CisPt provoked the translocation from the cytosol to the plasma membrane of atypical protein kinase C-zeta (PKC-zeta) and the activation of PKB/AKT. The effect of cisPt on MMP-2 was dependent on PKC-zeta activation since it was potentiated by a myristoylated PKC-zeta pseudo substrate peptide or by PKC-zeta down-regulation by siRNA. Moreover, MMP-2 activity modulation by cisPt was also dependent on PKB/AKT activation since it was decreased by PKB/AKT down-regulation by siRNA or by pharmacological inhibition of PI3K, thus indicating the importance of the balance of PKB/AKT and PKC-zeta in regulating the cisPt effect on MMP-2 activity. The PC E1Araf cells displayed a migratory capacity that was blocked by MMP-2 down-regulation using siRNA or pharmacological inhibition. The inhibition of cell migration was also obtained with cisPt; in cisPt-treated cells the administration of MMP-2 active protein was able to restore cell migration capacity. In conclusion, the decrease of MMP-2 secretion after cisPt was allowed by PKB/AKT and counteracted by PKC-zeta; the cisPt-provoked inhibition of MMP-2 secretion ended in reduction of cell migration.

PKC-epsilon-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. (125)I pre-loaded cells showed an (125)I 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 microg/ml; 0-40 min) provoked the cytosol-to-membrane translocation of pendrin and a decrease of intracellular I(-) content in (125)I pre-loaded cells. Insulin- or PMA-treated cells also showed a cytosol-to-membrane translocation of PKC-delta and -epsilon. Inhibition of both PKC-delta and -epsilon activities by GF109203X blocked pendrin translocation, whilst the inhibition of PKA did not. The selective inhibition of PKC-delta by rottlerin did not affect the insulin-provoked translocation of pendrin whilst it was inhibited by a PKC-epsilon translocation inhibitor peptide and also by PKC-epsilon downregulation using the small interfering RNA, thus indicating that such translocation was due to PKC-epsilon activity. In conclusion, our study demonstrates that, in PC Cl3 cells, pendrin expression and localisation are regulated by insulin and influenced by a PKC-epsilon-dependent intracellular pathway.

[Pt(O,O'-acac)(gamma-acac)(DMS)], a new Pt compound exerting fast cytotoxicity in MCF-7 breast cancer cells via the mitochondrial apoptotic pathway.

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)(gamma-acac)(DMS)], induces apoptosis in HeLa cells. The objective of this study was to investigate the hypothesis that [Pt(O,O'-acac)(gamma-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)(gamma-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)(gamma-acac)(DMS)], thus excluding DNA as a target of [Pt(O,O'-acac)(gamma-acac)(DMS)]. [Pt(O,O'-acac)(gamma-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)(gamma-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)(gamma-acac)(DMS)] has the potential to provide us with new opportunities for therapeutic intervention.

Protein kinase C (PKC)-delta/-epsilon mediate the PKC/Akt-dependent phosphorylation of extracellular signal-regulated kinases 1 and 2 in MCF-7 cells stimulated by bradykinin.

In this paper the signal transduction pathways evoked by bradykinin (BK) in MCF-7 breast cancer cells were investigated. BK activation of the B(2) receptor provoked: (a) the phosphorylation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2); (b) the translocation from the cytosol to the membrane of the conventional protein kinase C-alpha (PKC-alpha) and novel PKC-delta and PKC-epsilon; (c) the phosphorylation of protein kinase B (PKB/ Akt); (d) the proliferation of MCF-7 cells. The BK-induced ERK1/2 phosphorylation was completely blocked by PD98059 (an inhibitor of the mitogen-activated protein kinase kinase (MAPKK or MEK)) and by LY294002 (an inhibitor of phosphoinositide 3-kinase (PI3K)), and was reduced by GF109203X (an inhibitor of both novel and conventional PKCs); Gö6976, a conventional PKCs inhibitor, did not have any effect. The BK-induced phosphorylation of PKB/Akt was blocked by LY294002 but not by PD98059. Furthermore, LY294002 inhibited the BK-provoked translocation of PKC-delta and PKC-epsilon suggesting that PI3K may be upstream to PKCs. Finally, the proliferative effects of BK were blocked by PD98059, GF109203X and LY294002. These observations demonstrate that BK acts as a proliferative agent in MCF-7 cells activating intracellular pathways involving novel PKC-delta/-epsilon, PKB/Akt and ERK1/2.

Differential functions of PKC-delta and PKC-zeta in cisplatin response of normal and transformed thyroid cells.

We investigated the effects of cisplatin (cisPt) in normal PC Cl3 and in transformed and tumourigenic PC E1Araf cells. cisPt cytotoxicity was higher in PC Cl3 than in PC E1Araf cells. In both cell lines, cisPt provoked the ERK1/2 phosphorylation; this was unaltered by Gö6976, a conventional PKC inhibitor, whilst it was blocked by low doses (0.1 microM) or high doses (10 microM) of GF109203X, an inhibitor of all PKC isozymes, in PC Cl3 and in PC E1Araf cells, respectively. In PC E1Araf, the cisPt-provoked ERK phosphorylation was also blocked by the use of a myristoylated PKC-zeta pseudosubstrate peptide. Conversely, in PC Cl3 the cisPt-provoked ERK phosphorylation was blocked by the use of rottlerin, a PKC-delta inhibitor. Results show that cisPt activates both PKC (the -delta and the -zeta isozymes in PC Cl3 and in PC E1Araf cells, respectively) and ERK in association with prolonged survival of thyroid cell lines.

Bradykinin stimulates cell proliferation through an extracellular-regulated kinase 1 and 2-dependent mechanism in breast cancer cells in primary culture.

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)-alpha, -beta, -delta, -epsilon and -eta and phosphorylation of the extracellular-regulated kinases 1 and 2 (ERK1/2). The following compounds blocked the proliferative effects of BK: Hyp3-BK, a B2 receptor subtype inhibitor; U73122, a phospholipase C-beta 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.

Calcium channels are present in the apical plasma membranes of the hepatopancreatic B-cells of Marsupenaeus japonicus.

This study demonstrates the existence of calcium channels in the apical membranes of the hepatopancreatic blister (B) cells of Marsupenaeus japonicus. Using brush-border membrane vesicles we demonstrated that the channel-mediated calcium passive flux was saturable and was stimulated by a transmembrane electrical potential difference and inhibited by barium. We raised a monoclonal antibody (Mab 24A4) against the calcium channel, which allowed us to inhibit the channel-mediated calcium uptake. By immunocytochemistry, using Mab 24A4, we demonstrated that these channels are located at the apical membrane of hepatopancreatic B cells. Finally, by measuring the calcium uptake in R- and B-enriched cell suspensions, we showed that only the plasma membrane of the B cells expresses a channel-mediated calcium uptake inhibited by barium, verapamil and the monoclonal antibody 24A4. The plasma membrane of R cells did not show calcium channels.

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 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.

Angiotensin II AT1 receptor stimulates Na+ -K+ATPase activity through a pathway involving PKC-zeta in rat thyroid cells.

Angiotensin II (Ang II) receptor subtype 1, AT1, is expressed by the rat thyroid. A relationship between thyroid function and several components of the renin-angiotensin system has also been established, but the Ang II cellular effects in thyrocytes and its transduction signalling remain undefined. The aim of the present paper was to investigate the modulation of the activity of the Na(+)-K(+)ATPase by Ang II and its intracellular transduction pathway in PC-Cl3 cells, an established epithelial cell line derived from rat thyroid. Here we have demonstrated, by RT-PCR analysis, the expression of mRNA for the Ang II AT1 receptor in PC-Cl3 cells; mRNA for the Ang II AT2 receptor was not detected. Ang II was not able to affect the intracellular Ca(2+) concentration in fura-2-loaded cells, but it stimulated the translocation from the cytosol to the plasma membrane of atypical protein kinase C-zeta (PKC-zeta) and -iota (PKC-) isoforms with subsequent phosphorylation of the extracellular signal-regulated kinases 1 and 2 (ERK1 and 2). Translocated atypical PKCs displayed temporally different activations, the activation of PKC-zeta being the fastest. PC-Cl3 cells stimulated with increasing Ang II concentrations showed dose- and time-dependent activation of the Na(+)-K(+)ATPase activity, which paralleled the PKC-zeta translocation time course. Na(+)-K(+)ATPase activity modulation was dependent on PKC activation since the PKC antagonist staurosporine abolished the stimulatory effect of Ang II. The inhibition of the ERK kinases 1 and 2 (MEK1 and 2) by PD098059 (2'-amino-3'-methoxyflavone) failed to block the effect of Ang II on the Na(+)-K(+)ATPase activity. In conclusion, our results suggest that Ang II modulates Na(+)-K(+)ATPase activity in PC-Cl3 cells through the AT1 receptor via activation of atypical PKC-zeta while the Ang II-activated PKC- appears to have other as yet unknown functions.

Activation of angiotensin II type I receptor promotes protein kinase C translocation and cell proliferation in human cultured breast epithelial cells.

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. Ang II (0.1-1000 nM) induced an intracellular free calcium ([Ca(2+)](i)) transient peak which was unchanged by external Ca(2+ )removal. In Ca(2+)-free medium pretreatment with thapsigargin abolished Ang II-induced Ca(2+ )release. Suppression of 1,4,5-inositol trisphosphate formation by U73122, a phospholipase C inhibitor, blocked the Ang II-induced Ca(2+) response. Losartan (DuP753), an inhibitor of Ang II type I receptor (AT1), decreased the [Ca(2+)](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-alpha, -beta1, -delta and -zeta isozymes, and Ang II provoked translocation from the cytosol to the membranes of PKC-alpha, -beta1, and -delta (but not -zeta). 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-alpha and -beta1, 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.

AT1 angiotensin II receptor mediates intracellular calcium mobilization in normal and cancerous breast cells in primary culture.

Angiotensin II (Ang II) increases intracellular calcium concentration ([Ca2+]i) in both normal and cancerous human breast cells in primary culture. Maximal [Ca2+]i increase is obtained using 100nM Ang II in both cell types; in cancerous breast cells, [Ca2+]i increase (delta[Ca2+]i) is 135+/-10nM, while in normal breast cells it reaches 65+/-5 nM (P<0.0001). In both cell types, Ang II evokes a Ca2+ transient peak mediated by thapsigargin (TG) sensitive stores; neither Ca2+ entry through L-type membrane channels or capacitative Ca2+ entry are involved. Type I Ang II receptor subtype (AT1) mediates Ang II-dependent [Ca2+]i increase, since losartan, an AT1 inhibitor, blunted [Ca2+]i increase induced by Ang II in a dose-dependent manner, while CGP 4221A, an AT2 inhibitor, does not. Phospholipase C (PLC) is involved in this signaling mechanism, as U73122, a PLC inhibitor, decreases Ang II-dependent [Ca2+]i transient peak in a dose-dependent mode.Thus, the present study provides new information about Ca2+ signaling pathways mediated through AT1 in breast cells in which no data were yet available.

Angiotensin II stimulation of Na+/K+ATPase activity and cell growth by calcium-independent pathway in MCF-7 breast cancer cells.

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 Ca2+ 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 time-dependent manner and was mitogenic, with a dose-dependent (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.

Muscarinic acetylcholine receptor activation induces Ca2+ mobilization and Na+/K+-ATPase activity inhibition in eel enterocytes.

The effect of carbachol (Cch) on intracellular calcium concentration ([Ca2+]i) in eel enterocytes was examined using the fluorescent Ca2+ indicator fura-2. Cch caused a biphasic increase in [Ca2+]i, with an initial spike followed by a progressively decreasing level (over 6 min) to the initial, pre-stimulated, level. The effect of Cch was dose-dependent with a 7.5-fold increase in [Ca2+]i over basal level induced by the maximal dose of Cch (100 microM). In Ca2+-free/EGTA buffer the effect of Cch was less pronounced and the [Ca2+]i returned rapidly to basal levels. The increment of [Ca2+]i was dose-dependently attenuated in cells pre-treated with U73122, a specific inhibitor of phospholipase C, suggesting that the Cch-stimulated increment of [Ca2+]i required inositol triphosphate formation. In the presence of extracellular Ca2+, thapsigargin (TG), a specific microsomal Ca2+-ATPase inhibitor, caused a sustained rise in [Ca2+]i whereas in Ca2+-free medium the increase in [Ca2+]i was transient; in both cases, subsequent addition of Cch was without effect. When 2 mM CaCl2 were added to the cells stimulated with TG or with Cch in Ca2+-free medium, a rapid increase in [Ca2+]i was detected, corresponding to the capacitative Ca2+ entry. Thus, both TG and Cch depleted intracellular Ca2+ stores and stimulated influx of extracellular Ca2+ consistent with capacitative Ca2+ entry. K+ depolarization obtained with increasing concentrations of KCl in the extracellular medium induced a dose-related increase in [Ca2+]i which was blocked by 2 microM nifedipine, a non-specific L-type Ca2+ channel blocker. Nifedipine also changed significantly the height of the Ca2+ transient, and the rate of decrement to the pre-stimulated [Ca2+]i level, indicating that Ca2+ entry into enterocytes also occurs through an L-type voltage-dependent calcium channel pathway. We also show that isolated enterocytes stimulated with increasing Cch concentrations (0.1-1000 microM) showed a dose-dependent inhibition of the Na+/K+-ATPase activity. The threshold decrease was at 1 microM Cch; it reached a maximum at 100 microM (50.5% inhibition) and did not decrease further with the use of higher dose. The effect of Cch on Na+/K+-ATPase activity was dependent on both protein kinase C (PKC) and protein phosphatase calcineurin activation since the PKC inhibitor calphostin C abolished Cch effects, while the calcineurin inhibitor FK506 augmented Cch effect. Collectively, these data establish a functional pathway by which Cch can modulate the activity of the Na+/K+-ATPase through a PKC-dependent (calphostin C-sensitive) pathway and a calcineurin-dependent (FK506-sensitive) pathway.

Activation of muscarinic acetylcholine receptors induces Ca(2+) mobilization in FRT cells.

The effect of the muscarinic receptors agonist carbachol (Cch) on intracellular calcium concentration ([Ca(2+)](i)) and cAMP level was studied in polarized Fischer rat thyroid (FRT) epithelial cells. Cch provoked a transient increase in [Ca(2+)](i), followed by a lower sustained phase. Thapsigargin, a specific microsomal Ca(2+)-ATPase inhibitor, caused a rapid rise in [Ca(2+)](i) and subsequent addition of Cch was without effect. Removal of extracellular Ca(2+) reduced the initial transient response and completely abolished the plateau phase. Ryanodine, an agent that depletes intracellular Ca(2+) stores through stimulation of ryanodine receptors (RyRs), had no effect on [Ca(2+)](i). However, the transitory activation of [Ca(2+)](i) was dose-dependently attenuated in cells pretreated with U73122, a specific inhibitor of phospholipase C (PLC). These data suggest that the Cch-stimulated increment of [Ca(2+)](i) required IP(3) formation and binding to its specific receptors in Ca(2+) stores. Further studies were performed to investigate whether the effect of Cch on Ca(2+) entry into FRT cells was via L-type voltage-dependent Ca(2+) channels (L-VDCCs). Nicardipine, a nonspecific L-type Ca(2+) channel blocker, decreased Cch-induced increase on [Ca(2+)](i), while Bay K-8644, an L-type Ca(2+) channel agonist, slightly increased [Ca(2+)](i) in FRT cells. These data indicate that Ca(2+) entry into these nondifferentiated thyroid cells occurs through an L-VDCC, and probably through another mechanism such as a capacitative pathway. Cch did not affect the intracellular cAMP levels, but its effects on [Ca(2+)](i) were significantly reduced when cells were pretreated with forskolin, suggesting the existence of an intracellular cross-talk between PLC and cAMP mechanisms in the regulation of intracellular Ca(2+) mobilization in neoplastic FRT cells.

Na+/K+ATPase activity inhibition and isoform-specific translocation of protein kinase C following angiotensin II administration in isolated eel enterocytes.

In the eel, angiotensin II (Ang II) has a role at the level of both gill chloride and kidney tubular cells, regulating sodium balance and therefore osmoregulation. The present study extends these findings to another important osmoregulatory organ - the intestine. Enterocytes were obtained from sea-water (SW)-acclimated eels to investigate the role of Ang II on the intestinal Na+/K+ATPase activity, because in SW-acclimated animals the intestine represents an important site of water and NaCl transport from the mucosal to the serosal side. This paper demonstrates that isolated enterocytes stimulated with increasing Ang II concentrations (0.01-100 nM) showed a dose-dependent inhibition of the Na+/K+ATPase activity. The threshold decrease was at 0.01 nM Ang II; it reached a maximum at 10 nM (81.5% inhibition) and did not decrease further with the use of higher hormone doses. These hormonal effects were blocked by a specific competitive antagonist of the AT1 receptor subtype, DuP-753 (100% inhibition at 10 microM), indicating that these effects are mediated by an AT1-like receptor. Isolated enterocytes stimulated with 10 nM Ang II showed a transient increase in intracellular calcium ([Ca2+]i), followed by a lower sustained phase. Removal of extracellular Ca2+ did not reduce the initial transient response and completely abolished the plateau phase. The inhibition of the Na+/K+ATPase activity was dependent on protein kinase C (PKC) activation since PKC antagonists (calphostin C and staurosporine) abolished the inhibitory effect of Ang II, and the PKC activator phorbol 12-myristate 13-acetate reduced transporter activity. Western blot analysis with antibodies to PKC alpha, beta I, beta II, gamma, delta, epsilon, iota, eta and zeta isoforms showed that eel enterocytes expressed the conventional isoforms (alpha and beta I), the novel isoforms (delta and eta) and the atypical isoforms (zeta and iota). Ang II stimulated the translocation from the cytosol to the plasma membrane of PKC alpha, PKC delta and PKC eta isoforms. In conclusion, our results suggest that Ang II modulates intestinal Na+/K+ATPase in SW-acclimated eels via calcium mobilization and PKC activation.