Identification of polyproline II regions derived from the proline-rich nuclear receptor coactivators PNRC and PNRC2: new insights for ERα coactivator interactions.

Protein-protein interactions are crucial for signal transductions required for cell differentiation and proliferation. Their modulation is therefore key to the development of therapeutic alternatives, particularly in the context of cancer. According to literature data, the polyproline-rich nuclear receptor coactivators PNRC and PNRC2 interact with estrogen receptor (ERα) through their PxxP SH3-binding motifs. In a search to identify the molecular features governing this interaction, we explored using electronic circular dichroism (ECD) spectroscopy and molecular dynamics (MD) calculations, the capacity of a range of putative biologically active peptides derived from these proteins and containing this PxxP motif(s) to form polyproline II (PPII) domains. An additional more exhaustive structural study on a lead PPII peptide was also performed using 2D nuclear magnetic resonance (NMR) spectroscopy. With the exception of one of all the investigated peptides (PNRC-D), binding assays failed to detect any affinity for Grb2 SH3 domains, suggesting that PPII motifs issued from Grb2 antagonists have a binding mode distinct from those derived from Grb2 agonists. Instead, the peptides revealed a competitive binding ability against a synthetic peptide (ERα17p) with a putative PPII-cognate domain located within a coregulator recruitment region of ERα (AF-2 site). Our work, which constitutes the first structure-related interaction study concerning PNRC and PNRC2, supports not only the existence of PxxP-induced PPII sequences in these coregulators, but also confirms the presence of a PPII recognition site in the AF-2 of the steroid receptor ERα, a region important for transcription regulation.

The Therapeutic Potential of the Essential Oil of Thymbra capitata (L.) Cav., Origanum dictamnus L. and Salvia fruticosa Mill. And a Case of Plant-Based Pharmaceutical Development.

This review performs a comprehensive assessment of the therapeutic potential of three native herbs of Crete (Thymbra capitata (L.) Cav., Salvia fruticosa Mill. and Origanum dictamnus L.), their phytochemical constituents, health benefits and issues relevant to their safety, within a translational context. Issues discussed comprise: 1) Ethnopharmacological uses of the three herbs, reviewed through an extensive search of the literature; 2) Systematic analysis of the major phytochemical constituents of each plant, and their medicinal properties; 3) To what extent could the existing medicinal properties be combined and produce an additive or synergistic effect; 4) Possible safety issues. We conclude with a specific example of the use of a combination of the essential oils of these plants as an effective anti-viral product and the experience gained in a case of a plant-based pharmaceutical development, by presenting the major steps and the continuum of the translational chain.

Polyphenols and cancer cell growth.

Polyphenols constitute an important group of phytochemicals that gained increased research attention since it was found that they could affect cancer cell growth. Initial evidence came from epidemiologic studies suggesting that a diet that includes regular consumption of fruits and vegetables (rich in polyphenols) significantly reduces the risk of many cancers. In the present work we briefly review the effects of polyphenols on cancer cell fate, leading towards growth, differentiation and apoptosis. Their action can be attributed not only to their ability to act as antioxidants but also to their ability to interact with basic cellular mechanisms. Such interactions include interference with membrane and intracellular receptors, modulation of signaling cascades, interaction with the basic enzymes involved in tumor promotion and metastasis, interaction with oncogenes and oncoproteins, and, finally, direct or indirect interactions with nucleic acids and nucleoproteins. These actions involve almost the whole spectrum of basic cellular machinery--from the cell membrane to signaling cytoplasmic molecules and to the major nuclear components--and provide insights into their beneficial health effects. In addition, the actions justify the scientific interest in this class of compounds, and provide clues about their possible pharmaceutical exploitation in the field of oncology.

Opioids are non-competitive inhibitors of nitric oxide synthase in T47D human breast cancer cells.

Opioids and nitric oxide (NO) interact functionally in different systems. NO-generating agents decrease the activity of opioid agonists, prevent opioid tolerance, and are used in opioid withdrawal syndromes. There exist, however, few reports indicating a direct interaction of the two systems. T47D human breast cancer cells in culture express opioid receptors, and opioid agonists inhibit their growth, while they release high amounts of the NO-related molecules NO(2-)/NO(3-)to the culture medium. We have used this system to assay a possible direct interaction of opiergic and nitric oxide systems. Our results show that delta- or mu-acting opioid agonists do not modify the release of NO(2-)/NO(3-). In contrast, kappa-acting opioid agonists (ethylketocyclazocine, and alpha(S1)-casomorphine) decrease the release of NO(2-)/NO(3-), in a time- and dose-dependent manner. The general opioid antagonist diprenorphine (10(-6) M) produce a similar NO(2-)/NO(3-)release inhibition, indicating a possible non-opioid-receptor mediated phenomenon. In addition, ethylketocyclazocine, alpha(S1)-casomorphin and diprenorphine directly inhibit NOS activity: agonists, interact with both calcium-dependent and independent NOS-isoforms, while the antagonist diprenorphine modifies only the activity of the calcium-dependent fraction of the enzyme. Analysis of this interaction revealed that opioids modify the dimeric active form of NOS, through binding to the reductase part of the molecule, acting as non-competitive inhibitors of the enzyme. This interaction opens interesting new possibilities for tumor biology and breast cancer therapy.

Opioid alkaloids and casomorphin peptides decrease the proliferation of prostatic cancer cell lines (LNCaP, PC3 and DU145) through a partial interaction with opioid receptors.

Opioid agonists (ethylketocyclazocine, etorphine, [D-Ala2,D-Leu5]enkephalin (DADLE), [D-Ala2, N-Me-Phe4-Gly-ol]enkephalin (DAGO), [D-Ser2,Leu5]enkephalin-Thr6 (DSLET) and morphine were found to inhibit the proliferation of human prostate cancer cell lines (LNCaP, DU145, and PC3), in a dose-dependent manner. The 50% inhibitory concentrations (IC50) were in the picomolar range. In many cases, this effect was antagonized by the general opioid antagonist, diprenorphine, indicating the existence of specific opioid binding sites. Saturation binding experiments with selective ligands and effectors showed no opioid sites on the LNCaP cell line, kappa1 and mu sites on the PC3 cell line, and kappa1, kappa3 and mu sites on the DU145 cell line. In other cases, the opioid effect was not antagonized by diprenorphine, indicating that the action of opioids might be mediated through other membrane receptors. Furthermore, casomorphin peptides, issued from bovine alpha- (alpha-casein-90-95 and alpha-casein-90-96) and beta-caseins (beta-casomorphin and beta-casomorphin-1-5), and human alphaS1-casein (alphas -casomorphin and alphaS1-casomorphin amide) inhibited cell proliferation of human prostate cell lines, also by a mechanism partly involving opioid receptors. As opioid neurons can be found in the prostate gland, and casomorphin peptides might reach the gland through the general circulation, the above findings indicate a putative role of opioids in prostate cancer cell growth.

Kappa1-opioid binding sites are the dominant opioid binding sites in surgical specimens of human pheochromocytomas and in a human pheochromocytoma (KAT45) cell line.

The adrenal medulla produces opioids which exert paracrine effects on adrenal cortical and chromaffin cells and on adrenal splanchnic nerves, via specific binding sites. The opioid binding sites in the adrenals are detectable mainly in the medullary part of it and differ in type between species. Thus, the bovine adrenal medulla contains mostly kappa-opioid binding sites and fewer delta- and mu-opioid binding sites while primate adrenals contain mainly delta sites and few kappa-opioid binding sites. Most chromaffin cell tumors, the pheochromocytomas, produce opioids which suppress catecholamine production by the tumor. The aim of the present work was to identify the types of opioid binding sites in human pheochromocytomas. For this purpose, we characterized the opioid binding sites on crude membrane fractions prepared from 14 surgically excised pheohromocytomas and on whole KAT45 cells, a recently characterized human pheochromocytoma cell line. Our data showed that human pheohromocytomas are heterogeneous, as expected, with regard to the production of catecholamines and the distribution and profile of their opioid binding sites. Indeed, only one out of the 14 pheochromocytomas expressed exclusively delta and mu opioid sites, while in the remaining 13 tumors kappa-type binding sites were dominant. The KAT45 cell line possessed a significant number of kappa1 binding sites, fewer kappa2-opioid binding sites and kappa3-opioid binding sites, and minimal binding capacity for delta- and mu-opioid receptor agonists sites. More specifically, the kappa1 sites/cell were approximately 18,000, the kappa2 4500/cell and the kappa3 sites 2000/cell. Our findings for the surgical specimens and the cell line combined with previously published pharmacological data obtained from KAT45 cells suggest that kappa sites appear to be the most prevalent opioid binding sites in pheochromocytomas. Finally, in normal bovine adrenals the profile of opioid binding sites differs in adrenaline and noradrenaline producing chromaffin cells. To test the hypothesis that the type of catecholamine produced by a pheochromocytoma depends on its cell of origin, we compared our binding data with the catecholamine content of each pheochromocytoma examined. We found no correlation between the type of the predominant catecholamine produced and the opioid binding profile of each tumor suggesting that this hypothesis may not be valid.

Receptorphin: a conserved peptide derived from the sequence of the opioid receptor, with opioid displacement activity and potent antiproliferative actions in tumor cells.

BACKGROUND: In addition to endogenous opioids, a number of peptide sequences, derived from endogenous (hemorphins, alphaS1-casomorphin), and exogenous proteins (casomorphins, exorphins) have been reported, possessing opioid activity. In the present work, we report the identification of a new peptide, receptorphin (Tyr-Ile-Phe-Asn-Leu), derived from the sequence of the second transmembrane loop of the opioid receptor. This sequence is unique for the opioid receptor, and conserved in all species and receptor-types. RESULTS AND DISCUSSION: Receptorphin competes for opioid binding, presenting a kappa-receptor interaction, while it binds equally to delta- and mu- opioid and somatostatin-binding sites, and inhibits the cell proliferation of a number of human cancer cell lines, in a dose-dependent and reversible manner, at the picomolar or the nanomolar range. Receptorphin shows a preferential action on prostate cancer cells. CONCLUSION: Our work identifies, for the first time a peptide, in a receptor sequence, possessing ligand-agonistic activities. A hypothesis, based on receptorphin liberation after cell death, is presented, which could tentatively explain the time-lag observed during opioid antiproliferative action.

Somatostatin and opioid receptors in mammary tissue. Role in cancer cell growth.

Somatostatin and opioid systems, are the two main inhibitory systems in mammals. Both classes of substances have been identified in normal and malignant mammary gland, as well as their cognitive receptors. They have been implied in the inhibition of cell growth of cancer cells and cell lines, in a dose-dependent and reversible manner. Somatostatin acts through homologous receptors (SSTRs), belonging to five distinct classes (SSTR1-5). We, and others have identified SSTR2 and 3 as been the only SSTRs present in the breast. Furthermore, opioids act through the three classes of opioid receptors (mu, delta,kappa). In the breast, kappa opioid receptor subtypes (kappa 1-kappa 3) are the most widely expressed. We further have shown that opioids, in addition to their binding to opioid receptors, compete for binding to SSTRs. This functional interaction, together with other identified modes of opioid action in the breast (modulation of steroid receptors, proteases' secretion, interaction with cytoskeletal elements), will be discussed, taking into consideration also the possible local production of casomorphins (casein-derived opioids), which are very potent antiproliferative agents.

kappa-opioids induce a reversible inhibition of CFU-GM from CD133(+) cord blood cells.

BACKGROUND: Opioid agonists have been shown to exert an inhibitory action on a number of malignant and non-malignant cell types. However, there are no reports dealing with their effect on hemopoietic progenitors. Based upon our previous experience of opioid agonists we examined whether opioids could interfere with the growth of CFU-GM from CD133(+) cord blood cells. METHODS: Cord blood samples were subjected to CD133(+) column selection, with subsequent exposure to opioid agonists and antagonists or both, in semi-solid cultures for CFU-GM growth. Colonies of day 7 of culture were replated in fresh medium in the absence of opioids. The colonies were evaluated at 7 and 14 days of culture. RT-PCR was performed for the detection of opioid and somatostatin receptors. Apoptosis tests and immunophenotypic evaluations were employed in liquid cultures in conditions identical to those of the semi-solid ones. RESULTS AND DISCUSSION: Our results suggest that opioids can induce a significant inhibition of CFU-GM growth, which is reversible and not mediated through opioid or somatostatin receptors, while apoptosis is not implicated. Whether this finding could be used for clinical intervention remains to be examined.

Identification of a novel opioid peptide (Tyr-Val-Pro-Phe-Pro) derived from human alpha S1 casein (alpha S1-casomorphin, and alpha S1-casomorphin amide).

A new casomorphin pentapeptide (alpha S1-casomorphin) has been isolated from the sequence of human alpha S1-casein [alpha S1-casein-(158-162)], with the sequence Tyr-Val-Pro-Phe-Pro. This peptide was found to bind with high affinity to all three subtypes of the kappa-opioid receptor (kappa 1-kappa 2). When amidated at the C-terminus, alpha S1-casomorphin amide binds to the delta- and kappa 3-opioid sites. Both alpha S1-casomorphin and its amide inhibit in a dose-dependent and reversible manner the proliferation of T47D human breast cancer cells. This anti-proliferative activity was greater for alpha S1-casomorphin, which was the most potent opioid in inhibiting T47D cell proliferation. In T47D breast cancer cells, other casomorphins have been found to bind to somatostatin receptors in addition to opioid sites. In contrast, alpha S1-casomorphin and its amide do not interact with somatostatin receptors in our system.

Wine antioxidant polyphenols inhibit the proliferation of human prostate cancer cell lines.

The effect of different wine antioxidant polyphenols (catechin, epicatechin, quercetin, and resveratrol) on the growth of three prostate cancer cell lines (LNCaP, PC3, and DU145) was investigated. A dose- and time-dependent inhibition of cell growth by polyphenols was found at nanomolar concentrations. The proliferation of LNCaP and PC3 cells was preferentially inhibited by flavonoids (catechin, epicatechin, and quercetin), whereas resveratrol was the most potent inhibitor of DU145 cell growth. Possible mechanisms of action were investigated: 1) The competition of polyphenols for androgen binding in LNCaP cells revealed significant interaction only in the case of high concentrations of quercetin, at least at five orders of magnitude higher than the concentrations needed for cell growth inhibition. All other phenols showed low interactions. 2) Oxygen species production after mitogen stimulation and H2O2 sensitivity of these cell lines did not correlate with the observed antiproliferative effects, ruling out such a mode of action. 3) NO production revealed two different patterns: LNCaP and DU145 cells produced high concentrations of NO, whereas PC3 cells produced low concentrations. Phorbol ester stimulation of cells did not reveal any additional effect in LNCaP and DU145 cells, whereas it enhanced the secretion of NO in PC3 cells. Polyphenols decreased NO secretion. This effect correlates with their antiproliferative action and the inhibition of inducible NO synthase. It is therefore proposed that the antiproliferative effect of polyphenols is mediated through the modulation of NO production. In conclusion, our data show a direct inhibitory effect of low concentrations of antioxidant wine phenols on the proliferation of human prostate cancer cell lines mediated by the production of NO, further suggesting potential beneficial effects of wine and other phenol-containing foods or drinks for the control of prostate cancer cell growth.

Potent inhibitory action of red wine polyphenols on human breast cancer cells.

Breast cancer (one of the most common malignancy in Western societies), as well as esophagus, stomach, lung, bladder, and prostate cancer, depend on environmental factors and diet for growth and evolution. Dietary micronutriments have been proposed as effective inhibitory agents for cancer initiation, progression, and incidence. Among them, polyphenols, present in different foods and beverages, have retained attention in recent years. Red wine is a rich source of polyphenols, and their antioxidant and tumor arresting effects have been demonstrated in different in vitro and in vivo systems. In the present study, we have measured the antiproliferative effect of red wine concentrate, its total polyphenolic pool, and purified catechin, epicatechin, quercetin, and resveratrol, which account for more than 70% of the total polyphenols in red wine, on the proliferation of hormone sensitive (MCF7, T47D) and resistant (MDA-MB-231) breast cancer cell lines. Our results indicate that polyphenols, at the picomolar or the nanomolar range, decrease cell proliferation in a dose- and a time-dependant manner. In hormone sensitive cell lines, a specific interaction of each polyphenol with steroid receptors was observed, with IC(50)s lower than previously described. Interaction of polyphenols with steroid receptors cannot fully explain their inhibitory effect on cell proliferation. In addition, discrete antioxidant action on each cell line was detected under the same concentrations, both by modifying the toxic effect of H(2)O(2), and the production of reactive oxygen species (ROS), after phorbol ester stimulation. Our results suggest that low concentrations of polyphenols, and consecutively, consumption of wine, or other polyphenol-rich foods and beverages, could have a beneficial antiproliferative effect on breast cancer cell growth.