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.

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.

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.

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.

Opioid agonists modify breast cancer cell proliferation by blocking cells to the G2/M phase of the cycle: involvement of cytoskeletal elements.

Opioids decrease cell proliferation in different systems including breast, prostate, lung, kidney, and intestine, through an interaction with opioid as well as other membrane-receptor systems (somatostatin, cholinergic), through an unidentified mechanism. Recently, we have reported an interaction of taxol with opioid membrane sites (BBRC 235, 201-204, 1997), and an involvement of opioids to the modification of actin cytoskeleton in renal OK cells (J Cell Biochem. [19981 70:60-69), indicating a possible action of the opioid effect. In the present work, we have examined the effect of two general opioid agonists (ethylketocyclazocine and etorphine) on the cell cycle, in human breast cancer T47D cells, as well as a possible modification of the cellular cytoskeleton under their action, in order to explain the antiproliferative effect of these agents. These two opioids produce a dose-dependent and reversible decrease of the proliferation of T47D cells, with a maximum attained at 10(-8) M. The addition of 10(-8) M of either opioid produced a significant increase of the number of cells arrested in the G2/M phase. Confocal laser microscopy revealed a modification of the actin and tubulin microfilaments, with a clear redistribution at the periphery of the cell, reversed by the addition of the general opioid antagonist diprenorphine. Furthermore, differences between the two opioids were obvious, attributed to the different receptor affinity of each agent. The observed redistribution of actin and tubulin cytoskeletal elements gives therefore a possible answer of the antiproliferative action of opioids. The modification of the cytoskeleton, directly involved to cell division, might provoke a "mechanical" obstacle, which could be the reason of the antiproliferative effect of these agonists. Furthermore, the observed tubulin-opioid interaction by opioids provides a possible explanation of the arrest at the G2/M phase of T47D cells under opioid treatment. Nevertheless, although the observed interaction of opioids with cytoskeletal elements gives a plausible answer of the antiproliferative effects of the agents, this might not be the only action of these agents in cell proliferation. Other, direct or indirect, genomic actions, which which remains to be elucidated, might be taken into consideration.

Early alterations of actin cytoskeleton in OK cells by opioids.

Recently we identified and characterized opioid binding sites in OK (opossum kidney) cells and observed decreased proliferation of these cells in response to opioids. In the present study we investigated the effects of opioids on the actin cytoskeleton and explored whether their antiproliferative action may relate to alterations in the distribution or the dynamics of actin microfilaments. Exposure of OK cells to the opioids alphaS1 casomorphin and ethylketocyclazocine resulted in a rapid and substantial actin microfilament reorganization. This was documented by a significant dose-dependent decrease in the amounts of F-actin, determined by measurements of quantitative fluorescence, by immunoblot analysis and by a concomitant increase of the G/total-actin ratio measured by the DNase I inhibition assay. These changes were verified by confocal laser scanning microscopy, which showed marked redistribution of the microfilamentous structures in the presence of the opioids without affecting the organization of microtubules or vimentin intermediate filaments. The effect of opioids on actin polymerization dynamics occurred within 15 min and persisted for at least 2 h, while their restoration to control levels was accomplished 6 h later, indicating a reversible phenomenon. Northern blot analysis showed that the concentration of the actin transcript was unaffected. The addition of diprenorphine, a general opioid antagonist, prevented the effects of opioids on the actin cytoskeleton. The inhibition of OK cell proliferation, induced by ethylketocyclazocine and alphaS1 casomorphin was partially prevented in the presence of phallacidin, which stabilizes microfilaments. Our findings demonstrate that opioids, acting via kappa 1 binding sites, induce rapidly modifications in the dynamics of actin polymerization, and in the organization of microfilaments in OK cells, which may relate to their antiproliferative effect on these cells.

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.

Taxol inhibits opioid binding on T47D human breast cancer cells.

In the T47D human breast cancer cell line, Taxol was found to compete for ethylketocyclazocine opioid binding (IC50 3.3 pM). In contrast, no interaction of the drug with [3H]diprenorphine binding occurred. Binding was multiphasic, in the absence of colchicine (10[-6] M), but monophasic in its presence, indicating an involvement of the cytoskeleton in this process. Alignment of Taxol binding domains on alpha and beta tubulin with the kappa opioid site revealed homology of these sites with the first extracellular loop of the receptor. These results indicate a possible new action of Taxol, indicating for the first time a membrane action of the agent.

Identification and characterization of opioid and somatostatin binding sites in the opossum kidney (OK) cell line and their effect on growth.

Opioids and somatostatin analogs have been implicated in the modulation of renal water handling, but whether their action is accomplished through central and/or peripheral mechanisms remains controversial. In different cell systems, on the other hand, opioids and somatostatin inhibit cell proliferation. In the present study, we have used an established cell line, derived from opossum kidney (OK) proximal tubules, in order to characterize opioid and somatostatin receptors and to investigate the action of opioids and somatostatin on tubular epithelial tissue. Our results show the presence of one class of opioid binding sites with kappa, selectivity (KD 4.6 +/- 0.9 nM, 57,250 sites/cell), whereas delta, mu, or other subtypes of the kappa site were absent. Somatostatin presents also a high affinity site on these cells (KD 24.5 nM, 330,000 sites/cell). No effect of either opioids or somatostatin on the activity of the NA+/Pi cotransporter was observed, indicating that these agents do not affect ion transport mechanisms. However, opioid agonists and somatostatin analogs decrease OK cell proliferation in a dose-dependent manner; in the same nanomolar concentration range, they displayed reversible specific binding for these agents. The addition of diprenorphine, a general opioid antagonist, reversed the effects of opioids, with the exception of morphine. Furthermore, morphine interacts with the somatostatin receptor in this cell line too, as was the case in the breast cancer T47D cell line. Our results indicate that in the proximal tubule opioids and somatostatin do not affect transport, but they might have a role in the modulation of renal cell proliferation either during ontogenesis or in kidney repair.

Antiproliferative and receptor binding properties of alpha- and beta-casomorphins in the T47D human breast cancer cell line.

In previous studies, we have shown that opioid agonists ([D-Ala2, D-Leu5]enkephalin (DADLE), [D-Ser2, Leu5]enkephalin-Thr6 (DSLET), ethylketocyclazocine and etorphine) bind to opioid binding sites and decrease cell proliferation of human T47D breast cancer cells. Furthermore, we provided evidence about a cross-reaction, also in the T47D human breast cancer cell line, of mu-acting opioids with type-II somatostatin receptors. Since a potential source of opioid activity in the breast might be casomorphin peptides (produced by the enzymatic degradation of alpha-casein and beta-casein), we investigated the antiproliferative action of five different casomorphin peptides: alpha-casein-(90-95), alpha-casein-(90-96), beta-casomorphin, beta-casomorphin-(1-5) and morphiceptin. We show that all five peptides decreased, in a dose-dependent manner, cell proliferation. The general antagonist diprenorphine produced only a partial reversal of their action. Furthermore, we provide evidence that all peptides (except for morphiceptin) bind to delta- and kappa-opioid binding sites of T47D cells with different selectivity. Finally, we show that these peptides are also partial competitors at the somatostatin receptors present in the same cell line.

The antiproliferative effect of opioid receptor agonists on the T47D human breast cancer cell line, is partially mediated through opioid receptors.

In the present study, we investigated the action of opioid receptor agonists on the proliferation of cells of the T47D human breast cancer cell line, grown in the absence of exogenously added steroids and growth factors. We found that the opioid receptor agonists ethylketocyclazocine, morphine, [D-Ala2,D-Leu5]enkephalin (DADLE), [D-Ser2,Leu5]enkephalin-Thr6 (DSLET) and etorphine inhibit dose dependently cell proliferation. The opioid receptor antagonist diprenorphine had no significant effect per se, but it was able to reverse the action of all opioid receptor agonists except morphine. In order to investigate the mechanism of action of opioids on T47D cells, we characterised the opioid receptors present on this cell line, by saturation binding, using radiolabelled [D-Ala2,N-Me-Phe4-Gly5-ol]enkephalin (DAGO, mu-opioid receptor agonist), ethylketocyclazocine (kappa 1-, kappa 2-, mu- and delta-opioid receptor agonist), diprenorphine (kappa 2-, kappa 3-, delta- and mu-opioid receptor antagonist), DADLE (delta- and mu-opioid receptor agonist), and effectors. We identified opioid binding sites belonging mainly to the kappa-type (kappa 1, kappa 2 and kappa 3), a few delta-opioid receptor sites, but no mu-opioid receptors. Our results indicate that the inhibitory effect of opioids on T47D cell growth is mediated through kappa- and delta-opioid receptors. The effect of mu-acting morphine might not be mediated through opioid receptors.

Identification, characterization and localization of corticotropin-releasing hormone receptors in human placenta.

The purpose of this work was to identify, characterise, and localise specific CRH binding sites in whole vaginally delivered term human placental membranes as well as in dispersed and purified trophoblastic membranes. We have found that whole placenta membranes contained specific and saturable CRH-binding sites. Maximum specific binding was obtained at pH 7.2-7.4, 22 C, for 2 h. The order of potency of CRH analogs was h/rCRH > alpha-helCRH > oCRH. Scatchard analysis revealed a single population of high affinity binding sites with a dissociation constant of 1.25 nM and maximum binding capacity of 119 fmol/mg of protein. Purified syncytiotrophoblast membranes contained high affinity CRH binding sites exhibiting the same dissociation constant and maximum binding capacity as whole placental membranes, suggesting that the CRH binding sites are expressed almost exclusively by the syncytiotrophoblast. Our findings indicate that CRH binding sites in the human placenta are exhibit similar characteristics to that described for anterior pituitary, brain and adrenals.

Morphine cross-reacts with somatostatin receptor SSTR2 in the T47D human breast cancer cell line and decreases cell growth.

In a previous study, we found that morphine decreases, in a dose-dependent manner, the cell growth of T47D human breast cancer cells, despite the lack of mu opioid receptors and an interaction of morphine with other opioid sites. We have therefore examined a possible interaction of morphine with other membrane receptor systems of the cell. The present study describes for the first time an interaction between mu-acting opioid drugs and the somatostatinergic system. We have found that [125I]Tyr11-somatostatin binds with high affinity to T47D cells. Analysis of the binding data showed the presence of two components: one with high affinity but low capacity (Kd, 0.145 nM; 1450 sites/cell), and another of lower affinity but higher capacity (Kd, 1.192 nM; 11920 sites/cell). Somatostatin-14 and somatostatin-28 showed multiphasic displacement curves, indicating heterogeneity of binding sites. The latter was confirmed by reverse transcription-PCR, which revealed the existence of the somatostatin receptor subtypes 2 and 3 (SSTR2 and SSTR3), with a relative mRNA concentration of 85 and 15%, respectively. Morphine and the morphinomimetic peptide morphiceptine (Tyr-Pro-Phe-Pro-NH2) displace somatostatin from its binding sites. Further analysis indicated that mu-acting opioids interact with the SSTR2 receptor subtype.