Fenretinide stimulates redox-sensitive ceramide production in breast cancer cells: potential role in drug-induced cytotoxicity.

The synthetic retinoid N-(4-hydroxphenyl) retinamide (4HPR) has manifold actions, which may contribute to its chemopreventive effects on breast cancer cell growth and progression. A role for ceramide as a stress-response signal is investigated here during the cytotoxic action of 4HPR in MCF-7 cells. N-(4-hydroxphenyl) retinamide induced a dose-dependent decline in cell growth and survival associated with a maximal 10-fold increase in ceramide production at 10 microM. N-(4-hydroxphenyl) retinamide exhibited a greater potency than all-trans retinoic acid (ATRA) on growth inhibition and ceramide production. The synthetic peroxisome proliferator-activated receptors agonist troglitazone (TGZ), but not the native ligand 15-deoxy-delta 12,14-prostaglandin J2, abrogated both these actions of 4HPR but not that of ATRA. The antioxidant N-acetylcysteine mimicked the abrogative effect of TGZ on 4HPR action, while the exogenous oxidant H2O2 also stimulated ceramide production. The inhibitors of de novo ceramide synthesis, fumonisin B1 and myriocin, blocked the ceramide response to 4HPR and partially reversed the apoptotic response, but did not prevent the overall decline in cell survival. The pancaspase inhibitor Z-VAD fmk reduced the decrease in cell survival caused by 4HPR, but did not affect the ceramide response. These findings describe a novel redox-sensitive elevation of ceramide levels associated with the cytotoxic response of breast cancer cells to 4HPR. However, a major mediatory role for this sphingolipid in this context remains equivocal.

Underediting of glutamate receptor GluR-B mRNA in malignant gliomas.

In mammals, RNA editing by site-selective adenosine deamination regulates key functional properties of neurotransmitter receptors in the central nervous system. Glutamate receptor subunit B is nearly 100% edited at one position (the Q/R-site), which is essential for normal receptor function. Its significance is apparent from mouse models in which a slightly reduced rate of Q/R-site editing is associated with early onset epilepsy and premature death. Here we report that in tissues from malignant human brain tumors, this editing position of glutamate receptor subunit B is substantially underedited compared with control tissues. We also observe alterations in editing and alternative splicing of serotonin receptor 5-HT(2C) transcripts. These changes correlate with a decrease in enzymatic activity of the editing enzyme adenosine deaminase acting on RNA (ADAR) 2, as deduced from analysis of ADAR2 self-editing. Our results suggest a role for RNA editing in tumor progression and may provide a molecular model explaining the occurrence of epileptic seizures in association with malignant gliomas.

Differential effects of protein kinase C agonists on prostaglandin production and growth in human breast cancer cells.

A regulatory role for protein kinase C (PKC) and eicosanoids has been implicated in the control of breast cancer cell growth and function. Here we report on the effects of the two PKC agonists 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and bryostatin 1 on arachidonic acid metabolism, prostaglandin E2 (PGE2) production, and growth in MDA MB 231 human breast cancer cells. TPA caused a dose-dependent increase in PGE2 production which was maximal at 100 nM and which was blocked in the presence of an equimolar concentration of bryostatin 1. Bryostatin 1 alone had no effect on PGE2 synthesis. Both TPA and bryostatin 1 stimulated arachidonic acid release and reduced fatty acid incorporation into phosphatidylinositol, their combined effect being less than additive in co-incubation. Interleukin-1beta (IL-1beta) induced a tenfold and twofold synergistic increase in PGE2 production in the presence of TPA (10 nM) and bryostatin 1 (10 nM) respectively. Bryostatin 1 caused a dose-dependent inhibition of the phorbol ester-potentiated IL-1beta response. Treatment of MDA MB 231 cells for 4 days with TPA (10 nM) or bryostatin 1 (10 nM) inhibited cell growth by 74% and 20% respectively. Co-treatment with both PKC agonists reversed the anti-proliferative effect of TPA to that seen with bryostatin 1 alone. In contrast the anti-proliferative action of ceramide, another PKC modulator, was unaffected in the presence of bryostatin 1. TPA also induced morphological changes in MDA MB 231 cells which were prevented by co-treatment with bryostatin 1. This study further supports a regulatory role for PKC in the control of eicosanoid synthesis and growth in human breast cancer cells. Although the findings are consistent with bryostatin 1 acting as an antagonist/weak agonist in relation to TPA action, the mechanistic basis for this differential action of TPA and bryostatin 1 is uncertain.

Stimulation or endothelin-1 secretion by human breast cancer cells through protein kinase A activation: a possible novel paracrine loop involving breast fibroblast-derived prostaglandin E2.

Breast cancer cells secrete endothelin-1 (ET-1), which may act as a paracrine mitogen in breast tumours. The paracrine factors and signal transduction pathways responsible for regulating ET-1 production in breast cancer are unknown. In this study we have examined the involvement of the protein kinase A (PKA) signalling pathway in the control of ET-1 secretion in the human breast cancer cell line MCF-7. Treatment of MCF-7 cells with various agents that activate protein kinase A (PKA) through increases in intracellular cAMP levels including forskolin, cholera toxin (ChT), the cAMP analogue 8-Br-cAMP, or the cAMP phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX) all markedly increased ET-1 release. Prostaglandin E2 (PGE2) while stimulating cAMP production, but not inositol lipid hydrolysis also significantly stimulated ET-1 release. Activation of PKC by 2-O-tetradecanoyl phorbol 13-acetate (TPA) also stimulated ET-1 secretion in MCF-7 cells. The PKA inhibitor H-89 attenuated the ET-1 response to PGE2, forskolin and ChT, but not that due to the PKC agonist TPA. The possibility that human breast fibroblasts (HBFs) are a target for ET-1 action with regard to PGE2 production was also investigated, and revealed that while HBFs were unresponsive to ET-1 alone, pretreatment with the cytokine IL-beta greatly potentiated PGE2 release in response to ET-1. In conclusion our results show that activation of either the PKA or PKC signalling pathways in human breast cancer cells increases ET-1 secretion. We also found that HBFs release PGE2 after treatment with ET-1 and that PGE2 itself stimulates ET-1 production in MCF-7 cells. The implication of this potential novel paracrine loop may be significant in view of the high levels of PGE2 and ET-1 found in malignant breast tissues.

Regulation of arachidonic acid metabolism, aromatase activity and growth in human breast cancer cells by interleukin-1beta and phorbol ester: dissociation of a mediatory role for prostaglandin E2 in the autocrine control of cell function.

Prostaglandin E2 (PGE2) levels are elevated in malignant human breast tissue. However, the cellular mechanisms regulating this arachidonate metabolism and the autocrine influence PGE2 production may have on breast cancer cell growth and function are unclear. In the present study, we have investigated the effects of 2 putative cyclo-oxygenase inducers, interleukin-1beta (IL-1beta) and the protein kinase C agonist 12-O-tetradecanoyl-phorbol-13-acetate (TPA), on PGE2 production, growth and aromatase activity in the MDA MB 231 breast cancer cell line. TPA stimulated a dose-dependent increase in PGE2 production, inhibited cell growth and stimulated aromatase activity. Although IL-1beta alone had no effect on any of these breast cancer cell functions, the cytokine greatly potentiated PGE2 production in the presence of TPA. Similarly, growth inhibition and aromatase stimulation in response to TPA were both further enhanced by IL-1beta treatment. Indomethacin and dexamethasone both prevented PGE2 production in response to IL-Ibeta and TPA but had no effect on the anti-proliferative action of the cytokine and phorbol ester. While indomethacin had no effect on induction of aromatase activity by IL-1beta and TPA, dexamethasone exhibited a temporally biphasic action. Dexamethasone alone stimulated aromatase activity and demonstrated a permissive action on aromatase stimulation by IL-1beta and TPA. However, pre-treatment of cells with dexamethasone prevented subsequent induction of aromatase activity by IL-1beta and TPA. Our study describes a novel synergistic interaction in response to protein kinase C activation and IL-1beta during the regulation of arachidonate metabolism, cell growth and aromatase activity in human breast cancer cells. We conclude that the cyclooxygenase pathway does not play a mediatory role during the inhibition of cell growth and the induction of aromatase activity by IL-1beta and TPA.

Prostaglandin E2 production and metabolism in human breast cancer cells and breast fibroblasts. Regulation by inflammatory mediators.

Malignant human breast tumours contain high levels of prostaglandin E2 (PGE2). However, the mechanisms controlling PGE2 production in breast cancer are unknown. This in vitro study investigates the capacity for PGE2 synthesis and metabolism in several human breast cancer cell lines and early passage human breast fibroblasts and seeks to identify potential regulatory factors which may control these pathways. Basal PGE2 production rose up to 30-fold in breast fibroblast lines on addition of exogenous arachidonic acid (10 microM), whereas no such changes were observed in six out of seven cancer cell lines, with the exception of modest increases in MDA-MB-231 cells. Interleukin 1 beta (IL-1 beta) also induced PGE2 production in breast fibroblasts in the presence of excess substrate, consistent with cyclo-oxygenase induction by the cytokine. Under these conditions only Hs578T cells and MDA-MB-231 cells demonstrated large increases in PGE2 in response to IL-1 beta or phorbol ester; no such responses were seen in MCF-7, T47-D, ZR-75-1, BT-20 or CLF-90-1 cells. In the absence of added arachidonate, bradykinin (BK) and endothelin-1 (ET-1), potentiated PGE2 production in IL-1 beta-treated fibroblasts, possibly by mobilising endogenous substrate. PGE2 also stimulated ET-1 production by breast cancer cells. In co-cultures with T47-D cells both basal and stimulated PGE2 production by breast fibroblasts was greatly reduced. This appeared to be due to metabolic inactivation by the cancer cell since T47-D cells readily converted PGE2 to 15-keto-PGE2. This apparent 15-hydroxy-PG dehydrogenase activity was stimulated by TPA and inhibited by cycloheximide. In conclusion, breast fibroblasts, particularly under the influence of inflammatory mediators, provide a potentially rich source for PGE2 production in breast tumours, whereas significant contributions from the epithelial tumour component may be restricted to cancer cells exhibiting an invasive phenotype. Metabolic inactivation by the cancer cells may also play an important role in the regulation of breast tumour PGE2 levels.

Human breast cancer cells contain a phosphoramidon-sensitive metalloproteinase which can process exogenous big endothelin-1 to endothelin-1: a proposed mitogen for human breast fibroblasts.

Endothelin-1 (ET-1) levels are elevated in human breast tumours compared with normal and benign tissues, and in the presence of insulin-like growth factor 1 (IGF-I) ET-1 is a potent mitogen for human breast fibroblasts. In this study we have examined the ability of intact human breast cancer cell lines to process exogenously added big ET-1 (1-38) to the active mature ET-1 peptide by using a specific radioimmunometric assay. In both hormome-dependent (MCF-7, T47-D) and hormone-independent (MDA-MB-231) breast cancer cell lines the putative endothelin-converting enzyme (ECE) exhibited apparent Michaelis-Menten kinetics when converting added big ET-1 to ET-1. Both basal ET-1 production and exogenously added big ET-1 to ET-1 conversion were greatly reduced in all three cell lines in response to the metalloproteinase inhibitor phosphoramidon but were insensitive to other classes of protease inhibitors. Inhibition was also observed when cells were incubated in the presence of the divalent cation chelators 1,10-phenanthroline and EDTA. In MCF-7 cells the optimal pH for the ECE activity using a saponin cell permeabilisation procedure was found to residue within a narrow range of 6.2-7.26. Our results indicate that human breast cancer cells contain a neutral phosphoramidon-sensitive metalloproteinase which can process big ET-1 to ET-1. In the breast this conversion could contribute substantially to the local extracellular levels of this proposed paracrine breast fibroblast mitogen.

Decidual histamine release and amplification of prostaglandin F2 alpha production by histamine in interleukin-1 beta-primed decidual cells: potential interactive role for inflammatory mediators in uterine function at term.

Inflammatory cytokines such as interleukin-1 (IL-1) have been implicated as paracrine mediators of decidual prostaglandin (PG) production during preterm labor. The aim of the present in vitro study was to investigate a similar potential role for the inflammatory autacoid histamine. Histamine action on human primary decidual cell cultures was monitored by measuring both PGF2 alpha production and PG precursor release. Histamine release from decidual cell suspensions was measured in response to a variety of putative mast cell secretagogues. Histamine stimulated a dose-dependent increase in PGF2 alpha production and [14C]arachidonate release from prelabeled decidual cells, with ED50 values around 1 and 2 mumol/L, respectively. Pretreatment of cells with IL-1 beta enhanced maximal PGF2 alpha production in response to histamine by approximately 4-fold. Mepyramine, an H1 receptor antagonist, completely blocked this PGF2 alpha response. The mean histamine content of unpurified decidual cells was approximately 81 pmol/10(6) cells. Upon challenge with antihuman immunoglobulin E, these cells exhibited a dose-dependent release of histamine. Basal release from decidual cell suspensions and release in response to antiimmunoglobulin E (1:400) or A23187 (1 mumol/L) were 7.6 +/- 2.7%, 25.5 +/- 0.9%, and 63.5 +/- 5.6% of the total histamine content, respectively. No significant changes in histamine secretion were seen in response to compound 48/80, substance-P, phorbol ester, or bacterial endotoxin. These in vitro findings support a potential role for histamine as a local regulator of phospholipase-A2 and PGF2 alpha production in human term decidua. The interaction of this autacoid with other inflammatory mediators, such as IL-1, could play a role in the control of PG production during preterm labor.

A parents' perspective: our needs and our message.

This article, written by parents, describes their fears and frustrations as the child they love is taken from the joyful family life that had been expected, to the terror of facing the possibility of death in a critical care situation. The parents want to help the medical team in healing their child, yet find themselves in a totally foreign environment in which they are often ineffective. By sharing the feelings experienced during their child's initial diagnosis and treatment, the authors hope that health care professionals will understand better the emotions parents are feeling and that parents can be helped to overcome their fears in order to better assume their role as part of the critical care treatment process.

Endothelin-1 stimulates phospholipid hydrolysis and prostaglandin F2 alpha production in primary human decidua cell cultures.

The regulation of prostaglandin (PG) production by the uterine decidua may be an important mechanism controlling the onset and maintenance of human parturition. The present in vitro study has evaluated the potential for endothelin-1 (ET-1) to activate cell signalling and PGE2 alpha production in human primary decidua cell cultures. ET-1 stimulated a dose-dependent increase in inositol phospholipid hydrolysis and PG precursor release as evidenced by respective increases in [3H] inositol monophosphate accumulation and [14C] arachidonate release from radiolabelled decidua cells. PGF2 alpha production was increased in some but not all cell preparations in response to ET-1 alone. Pretreatment of decidua cells with interleukin-1 beta (IL-1 beta) enhanced PGF2 alpha production but not arachidonate release in response to ET-1. These in vitro observations support a possible role for ET-1 in the regulation of decidual PG production during parturition.

Evidence for a role for protein kinase C in the modulation of bombesin-activated cellular signalling in human breast cancer cells.

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) is a potent activator of protein kinase C (PKC) and is known to affect a variety of biochemical processes in human breast cancer cells. In the present study we have employed MCF-7 cells to investigate the effects of TPA on inositol lipid signalling, the putative pathway leading to PKC activation and intracellular Ca2+ mobilization. Phosphoinositide hydrolysis in MCF-7 cells was stimulated by bombesin (BN) as evidenced by increases in both inositol phosphate production and cytidine diphosphate diacylglycerol (CDP-DG) accumulation. Pretreatment of MCF-7 cells with TPA caused attenuation of both these BN-induced responses. This inhibitory action of TPA on inositol phosphate production was mimicked by diacylglycerol analogues and was reversed by staurosporine, H-7 and tamoxifen, all known inhibitors of PKC. Furthermore, putative down-regulation of PKC by prolonged TPA pretreatment also reversed the inhibitory action of TPA and enhanced BN-induced phosphoinositide hydrolysis. TPA also inhibited BN-induced increases in cytosolic Ca2+ concentration ([Ca2+]i) and caused a dose-dependent inhibition of epidermal growth factor (EGF) binding in MCF-7 cells. However, EGF receptor occupancy was unaffected by BN. These data support an inhibitory role for PKC in the regulation of phosphoinositide hydrolysis and [Ca2+]i in breast cancer cells and provide a potential mechanism for feedback regulation of this signalling pathway in these cells.

Bombesin and glucocorticoids stimulate human breast cancer cells to produce endothelin, a paracrine mitogen for breast stromal cells.

Human breast cancer cells have been recently reported to produce endothelin (ET) 1. To investigate the potential regulation of ET production in breast cancer cells, we have measured the release of ET-like immunoreactivity from the T47D cell line in response to various paracrine/endocrine factors. Bombesin (0.1 microM) and cortisol (1 microM) stimulated maximal respective increases in IR-ET release to 580 and 369% of basal values after 6 h. The responses to cortisol and bombesin were additive. The response to bombesin was dose dependent with a median effective dose around 1 nM and was inhibited by the receptor antagonist [Leu13-psi-CH2NH-Leu14]bombesin. Pretreatment of T47D cells with pertussis toxin had no effect on bombesin-induced inositol lipid hydrolysis but inhibited ET-like immunoreactivity release in response to bombesin in the presence of glucocorticoid, by 56%. ET-1 (10 nM) and insulin-like growth factor (10 ng/ml) stimulated modest separate increases in DNA synthesis in human breast fibroblasts of 35 and 71%, respectively, but together exhibited a strong synergistic response to 905% of control values. This in vitro study demonstrates the potential for bombesin and glucocorticoid to regulate ET production in human breast cancer cells, which may in turn have a paracrine influence on neighboring stromal cell function.

Heterologous regulation of inositol lipid hydrolysis in human breast cancer cells by oestradiol 17 beta, bombesin and fluoroaluminate.

Inositol lipid turnover has been implicated in the action of oestradiol 17 beta and bombesin-related peptides on the human breast cancer cell line MCF-7. In the present study, in addition to measuring inositol lipid turnover as indicated by inositol monophosphate (IP) accumulation, we have also monitored the effect of oestradiol on the incorporation of both 3H-inositol and 14C-glycerol into MCF-7 cell phospholipids. Pre-treatment of MCF-7 cells with oestradiol (10 nM) for 48 hr stimulated a 4.3-fold increase in IP production. This was similarly accompanied by a 3.4-fold increase in the incorporation of 3H-inositol into total phosphoinositides and a 40% increase in cell growth. The oestrogen antagonist LYI 17018 completely attenuated these effects. Oestradiol also stimulated 14C-glycerol incorporation into phosphatidyl inositol, -choline and -ethanolamine by 97%, 82% and 99%, respectively. IP production in response to bombesin was potentiated by oestradiol in a dose-dependent fashion. Fluoroaluminate (AlF4-) stimulated a dose-dependent increase in IP production and oestradiol pre-treatment increased the sensitivity of this IP response to AlF4-. Medroxyprogesterone acetate inhibited bombesin-stimulated IP production but had no effect on the response to AlF4-. Our data suggest that the oestrogenic action on basal IP production in MCF-7 cells may reflect an effect on inositol lipid synthesis rather than turnover. However, the potentiation by oestradiol of both bombesin- and AlF4(-)-stimulated inositol lipid hydrolysis suggests the operation of a post-receptor regulatory mechanism(s) which is independent of the inositol lipid pool size.

Interaction of paracrine factors during labour: interleukin-1 beta causes amplification of decidua cell prostaglandin F2 alpha production in response to bradykinin and epidermal growth factor.

Inflammatory mediators have been implicated in the stimulation of decidual prostaglandin (PG) production during infection-driven preterm labour. The aim of the present study is to investigate a potential interaction between interleukin-1 beta (IL-1) and bradykinin (BK) in the regulation of decidual PGF2 alpha production and PG precursor release. Pretreatment of primary decidua cell cultures with IL-1 significantly enhanced PGF2 alpha production in response to BK. This effect was accompanied by an increase in unesterified arachidonic acid and an enhanced turnover of arachidonoyl phosphatidate. Bradykinin also stimulated arachidonic acid release in decidual fibroblasts, an effect which was potentiated in the presence of epidermal growth factor (EGF), but which was not accompanied by an increase in PGF2 alpha production. Decidual fibroblasts pretreated with IL-1 manifest a 10-fold increase in PGF2 alpha production in response to BK and EGF. These observations provide the first description of synergism between cytokine, kinin and growth factor in the uterine decidua which may partly mediate the in vivo increase in prostaglandin production associated with decidual activation and infection-driven labour.

Human decidua is a target tissue for bradykinin and kallikrein: phosphoinositide hydrolysis accompanies arachidonic acid release in uterine decidua cells in vitro.

The endocrine and intracellular mechanisms regulating prostaglandin precursor release in the uterine decidua during labor are unknown. This in vitro study investigates a potential role for a kallikrein-kinin system in the activation of phospholipid hydrolysis and arachidonic acid release in human decidua cells. Primary cultures of human decidua cells were prelabeled with [3H]inositol or [14C]arachidonic acid to monitor phosphoinositide hydrolysis and prostaglandin precursor release, respectively. Bradykinin (100 nmol/L) stimulated a rapid release of arachidonic acid (within 2 min) associated with an increase in inositol trisphosphate which was detectable after 20 s. Protein kinase C activation by phorbol ester enhanced arachidonic acid release in response to both bradykinin and the Ca++ ionophore A23187 but inhibited bradykinin-stimulated phosphoinositide hydrolysis. Epidermal growth factor also enhanced arachidonate release in response to both bradykinin and A23187. Kallikrein stimulated both phosphoinositide hydrolysis and arachidonic acid release in decidua cells. Kallikrein action was inhibited by the kallikrein protease inhibitor aprotinin and D-Arg[Hyp3Thi5,8,D-Phe7] bradykinin, a B2 receptor antagonist. Bradykinin also stimulated prostaglandin F2 alpha production in both primary decidua cell cultures and fibroblasts in the presence of interleukin-1 beta. These findings are consistent with a mediatory role for bradykinin in the action of kallikrein on decidua cells and suggest that inositol phospholipid hydrolysis is instrumental for arachidonic acid release in response to bradykinin in these cells. This study supports a novel role for a kallikrein-kinin system in the human uterine decidua.

Inhibition of DNA synthesis and growth in human breast stromal cells by bradykinin: evidence for independent roles of B1 and B2 receptors in the respective control of cell growth and phospholipid hydrolysis.

The paracrine and intracellular mechanisms controlling stromal cell growth in the normal or neoplastic breast are unknown. This in vitro study uses human breast fibroblasts to investigate a potential role for the inflammatory peptide mediator bradykinin (BK) in the regulation of DNA synthesis and signal transduction in these cells. Bradykinin stimulated a dose-dependent increase in inositol lipid hydrolysis and cytosolic Ca2+ levels in serum-starved fibroblasts derived from both normal and breast tumor tissue. Bradykinin also caused a dose-dependent decrease in cell growth and [3H]thymidine incorporation into DNA in breast fibroblasts. Epidermal growth factor (EGF) and insulin-like growth factor 1 both stimulated DNA synthesis in breast fibroblasts. Bradykinin inhibited this mitogenic effect of EGF but not that due to insulin-like growth factor 1. The binding of 125I-labeled EGF to fibroblasts was also inhibited by BK. Prostaglandin E2 also inhibited fibroblast DNA synthesis, and the cyclooxygenase inhibitor indomethacin partially reversed the inhibitory action of BK on DNA synthesis. Studies with BK receptor antagonists and agonists indicate that inositol lipid signalling and arachidonic acid mobilization in response to BK are B2 receptor-mediated pathways, whereas the inhibition of DNA synthesis appears to be via B1 receptors. Although these data support a role for prostaglandins and EGF receptor down-modulation in the inhibitory action of BK on DNA synthesis in breast fibroblasts, a B1 receptor-mediated pathway is also implicated. This study highlights a potential pathophysiological role for BK as a negative regulator of breast stromal cell growth.

Modulation of inositol lipid hydrolysis in human breast cancer cells by two classes of bombesin antagonist.

Inositol lipid hydrolysis was monitored in the human breast cancer cell line MCF-7 in response to various bombesin (BN) and substance P (SP) analogues. Both members of the BN family of peptides, i.e. BN and gastrin-releasing peptide (GRP), stimulated a dose-related increase in total inositol phosphate production, with a similar half-maximal effective dose (ED50) around 1 nM. The BN receptor antagonist [Leu13-psi-CH2NH-Leu14]-BN (LLBN) at 1 microM was devoid of agonist activity and displaced the BN dose-response to the right, resulting in a tenfold increase in the ED50 for BN. BN also stimulated a dose-related increase in 45Ca2+ efflux which was also inhibited by LLBN. Two SP analogues [DArg1,D-Pro2,D-Trp7,9,Leu11]-SP and [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-SP ([APheTL]-SP), both antagonized BN-stimulated inositol lipid hydrolysis. [APheTL]-SP (60 and 80 microM) alone also exhibited considerable agonist activity which was not antagonized by LLBN. Indeed, a sub-threshold dose of [APheTL]-SP (40 microM) in the presence of LLBN (10 microM) potentiated the inositol lipid hydrolysis response. BN, GRP, LLBN and [APheTL]-SP all inhibited binding of 125I-labelled GRP to MCF-7 cells, to 50% of that occurring in the absence of the peptides, at concentrations of 150 pM, 150 pM, 150 nM and 600 nM respectively. These data are consistent with the presence of separate but interacting receptors or binding sites for BN and SP analogues, which are coupled to a common signal transduction pathway in human breast cancer cells.

Mitogenic peptides in breast cyst fluid: relationship with intracystic electrolyte ratios.

Women with palpable breast cysts which are lined with apocrine epithelium may be at higher risk of developing breast cancer than women with breast cysts which are lined with flattened epithelium, the former group being characterized by intracystic sodium to potassium ratios below 3, while the latter group has intracystic sodium to potassium ratios above 3. In this study the distribution of intracystic concentrations of the mitogenic peptides, epidermal growth factor, endothelin and gastrin-releasing peptide in the 2 groups of breast cysts were compared to see whether differences in concentrations between the 2 cyst groups might provide an explanation for the higher risk of breast cancer observed in women with "apocrine" breast cysts. The concentrations of epidermal growth factor and gastrin-releasing peptide were significantly higher in the low electrolyte ratio group (p less than 0.001). There was no difference in endothelin concentrations between the 2 groups. Negative correlations were found between epidermal growth factor concentrations and Na+/K+ and between gastrin-releasing peptide concentrations and Na+/K+ (p less than 0.001). A positive correlation was found between gastrin-releasing peptide and epidermal growth factor concentrations in breast cyst fluid (p less than 0.001). The significantly higher intracystic concentrations of both epidermal growth factor and gastrin-releasing peptide in the low-electrolyte-ratio group may provide an explanation for the higher risk of breast cancer which has been observed in women with "apocrine" breast cysts.