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 kappa₁ selectivity (K_D 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 (K_D 24.5 nM, 330,000 sites/cell). No effect of either opioids or somatostatin on the activity of the Na⁺/P_i 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 ion transport, but they might have a role in the modulation of renal cell proliferation either during ontogenesis or in kidney repair. © 1996 Wiley-Liss, Inc.     

Monomeric and oligomeric flavanols are agonists of membrane androgen receptors

The present work reports a new mode of action of the naturally occurring flavanols catechin and epicatechin and their dimers B2 and B5, in the breast cancer T47D cell line, namely, their interaction with membrane androgen receptors. We show that monomeric and dimeric flavanols are complete (B2) or partial displacers of radiolabeled testosterone bound on T47D membranes, with affinities ranging from 1.7 (B5) to 82.2 nM (B2). In addition, they trigger the phosphorylation of the same signaling molecules (FAK, PI3K) as testosterone-BSA, minutes after binding to membrane receptors, leading to actin cytoskeleton polymerization and redistribution, with formation of filopodia and lamellipodia. The PI3K inhibitor wortmannin reverts the effect of polyphenols and testosterone-BSA, providing additional evidence about activation of a similar signaling cascade. Incubation of T47D cells for more than 2 h with polyphenols or testosterone-BSA induces apoptosis, which follows the same time-dependent pattern. We conclude that flavanols (monomers or dimers) are agonists of membrane androgen receptors and could be used as testosterone-protein conjugates for the management of tumors, in which, application of testosterone-BSA induces regression, providing additional data about the mechanism of their antiproliferative action.     

Modulation of the estrogen-regulated proteins cathepsin D and pS2 by opioid agonists in hormone-sensitive breast cancer cell lines (MCF7 and T47D): Evidence for an interaction between the two systems

In many cancer cell lines, including breast, prostate, lung, brain, head and neck, retina, and the gastrointestinal tract, opioids decrease cell proliferation in a dose-dependent and reversible manner. Opioid and/ or other neuropeptide receptors mediate this decrease. We report that only the steroid-hormone-sensitive cell lines MCF7 and T47D respond to opioid growth inhibition in a dose-dependent manner. Therefore, an interaction of the opioid and steroid receptor system might exist, as is the case with insulin. To investigate this interaction, we have assayed two estrogen-inducible proteins (pS2 and the lysosomal enzyme cathepsin D) in MCF7 and T47D cells. When cells were grown in the presence of FBS (in which case a minimal quantity of estrogens and/ or opioids is provided by the serum), we observed either no effect of etorphine or ethylketocyclazocine (EKC) or an increase of secretion and/ or production of pS2 and cathepsin D. However, when cells were cultured in charcoal-stripped serum and in the absence of phenol red, the effect of the two opioids is different: EKC decreased the production and/ or secretion of pS2 and cathepsin D, whereas etorphine increased their synthesis and/ or secretion. The differential effect of the two general opioids was attributed to their different receptor selectivity. Furthermore, the variations of the ratio of secreted/ produced protein and the use of cycloheximide indicate that opioids selectively modify the regulatory pathway of each protein discretely. In conclusion, through the interaction with opioid and perhaps other membrane-receptor sites, opioid agonists modify in a dose-dependent manner the production and the secretion of two estrogen-regulated proteins. Opioids may therefore disturb hormonal signals mediated by the estrogen receptors. Hence, these chemicals may have potential endocrine disrupting activities. J. Cell. Biochem. 71:416–428, 1998. © 1998 Wiley-Liss, Inc.     

Reorganization of the cytoskeleton and morphological changes induced by 1,25-dihydroxyvitamin D3 in C3H/10T1/2 mouse embryo fibroblasts: relation to inhibition of proliferation.

The effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3) on cell morphology, the cytoskeleton, and fibronectin were studied in three lines of C3H/10T1/2 mouse embryo fibroblasts in which the antiproliferative effect of the hormone had previously been investigated. We showed that 1,25(OH)2D3 induced morphological changes in the nontransformed C3H/10T1/2 Cl 8 cells, which flattened and spread out markedly. Visualization of actin and tubulin by immunocytochemistry disclosed a reorganization of the microfilament and microtubular systems. 1,25(OH)2D3 also induced an increase in cell-surface-associated fibronectin. These changes were only slight in the transformed cell line C3H/10T1/2 Cl 16 and absent in the transformed C3H/10T1/2 TPA 482 cell line. These effects were correlated with the growth inhibition induced by the hormone, and this suggests a possible relationship between the 1,25(OH)2D3-induced alterations of cell shape and of the cytoskeleton and the effects of the hormone on cell proliferation.     

The opioid agonist ethylketocyclazocine reverts the rapid, non-genomic effects of membrane testosterone receptors in the human prostate LNCaP cell line

Neuropeptides influence cancer cell replication and growth. Opioid peptides, and opiergic neurons are found in the prostate gland, and they are proposed to exert a role in tumor regulation, influencing cancer cell growth, as opioid agonists inhibit cell growth in several systems, including the human prostate cancer cell line LNCaP. In the same cell line, the existence of membrane testosterone receptors was recently reported, which increase, in a non-genomic manner, the secretion of PSA, and modify actin cytoskeleton dynamics, through the signaling cascade FAK-->PI-3 kinase-->Cdc42/Rac1. In the present work, we present data supporting that the general opioid agonist Ethylketocyclazocine (EKC) decreases testosterone-BSA (a non-internalizable testosterone analog) induced PSA secretion. Furthermore, we report that this opioid affects this non-genomic testosterone action, by modifying the distribution of the actin cytoskeleton in the cells, disrupting the above signaling cascade. In addition, after long (>24 h) incubation, opioids decrease the number of membrane testosterone receptors, and reverse their effect on the signaling molecules. In conclusion, our results provide some new insights of a possible action of opioids in prostate cancer control by interfering with the action and the expression of membrane testosterone receptors and signaling.     

SAR studies on hydropentalene derivatives—Important core units of biologically active tetramic acid macrolactams and ptychanolides

Structurally diverse bicyclo[3.3.0]octanes were prepared and tested for their biological activity. Both the antiproliferative activity and the results of phenotypic characterization varied with the substitution patterns. Two derivatives displayed high inhibitory (IC₅₀ ⩽3 μM) activity against the L-929 cell line, but differed in their mode of action. A cluster analysis with impedance profiling data showed the two compounds in relationship to microtubule interfering compounds. In PtK₂ cells treated with both derivatives a perturbing effect on the microtubular network was observed, whereas the actin cytoskeleton in incubated PtK₂ cells was disturbed only by one compound. The effects on tubulin and actin polymerization could be confirmed by in vitro polymerization experiments.     

Plant actin filament and microtubule interactions during anaphase--telophase transition: effects of antagonist drugs

F-actin and microtubule co-distribution and interaction were studied during anaphase-telophase. Rapid and drastic changes in the cytoskeleton during these particular stages were studied in isolated plant endosperm cells of the blood lily. These wall-free cells can be considered as natural dividing protoplasts. As identified previously, an F-actin cytoskeletal network characterized the plant cortex and formed an elastic cage around the spindle, remaining throughout interphase, mitosis and cytokinesis. Actin was specifically labeled by fluorescent phalloidin and/or monoclonal antibodies. Gold-labelled secondary antibodies were used for ultrastructural observations and silver-enhancement was applied for video-enhanced microscopy. Microtubule and microfilament dynamics and interaction were studied using drug antagonists to actin (cytochalasins B, D) and to tubulin (colchicine). This permitted precise correlations to be made between chromosome movement inhibition and alteration in the actin/tubulin cytoskeleton. During anaphase chromosome migration, the cortical actin network was stretched along the microtubular spindle, while it remained homogeneous when anaphase was inhibited by colchicine. Cytochalasins did not inhibit chromosome movement but altered actin distribution. A new population of actin filaments appeared at the equator in late anaphase before the microtubular phragmoplast was formed and contributed to cell plate formation. Our conclusion is that F-actin-microtubule interaction may contribute to the regulatory mechanism of plant cytokinesis.     

Reorganization of cytoskeletal proteins by Escherichia coli heat-stable enterotoxin (STa)-mediated signaling cascade

Background

IP₃-mediated calcium mobilization from intracellular stores activates and translocates PKC-α from cytosol to membrane fraction in response to STa in COLO-205 cell line. The present study was undertaken to determine the involvement of cytoskeleton proteins in translocation of PKC-α to membrane from cytosol in the Escherichiacoli STa-mediated signaling cascade in a human colonic carcinoma cell line COLO-205.

Methods

Western blots and consequent densitometric analysis were used to assess time-dependent redistribution of cytoskeletal proteins. This redistribution was further confirmed by using confocal microscopy. Pharmacological reagents were applied to colonic carcinoma cells to disrupt the microfilaments (cytochalasin D) and microtubules (nocodazole).

Results

STa treatment in COLO-205 cells showed dynamic redistribution and an increase in actin content in the Triton-insoluble fraction, which corresponds to an increase in polymerization within 1 min. Moreover, pharmacological disruption of actin-based cytoskeleton greatly disturbed PKC-α translocation to the membrane.

Conclusions

These results suggested that the organization of actin cytoskeleton is rapidly rearranged following E. coli STa treatment and the integrity of the actin cytoskeleton played a crucial role in PKC-α movement in colonic cells. Depolymerization of tubulin had no effect on the ability of the kinase to be translocated to the membrane.

General significance

In the present study, we have shown for the first time that in colonic carcinoma cells, STa-mediated rapid changes of actin cytoskeleton arrangement might be involved in the translocation of PKC-α to membrane.     

Binding of the GABA(A) receptor-associated protein (GABARAP) to microtubules and microfilaments suggests involvement of the cytoskeleton in GABARAPGABA(A) receptor interaction

GABA(A) receptor-associated protein (GABARAP) was isolated on the basis of its interaction with the gamma2 subunit of GABA(A) receptors. It has sequence similarity to light chain 3 (LC3) of microtubule-associated proteins 1A and 1B. This suggests that GABARAP may link GABA(A) receptors to the cytoskeleton. GABARAP associates with tubulin in vitro. However, little is known about the mechanism for the interaction, and it is not clear whether the interaction occurs in vivo. Here, we report that GABARAP interacts directly with both tubulin and microtubules in a salt-sensitive manner, indicating the association is mediated by ionic interactions. GABARAP coimmunoprecipitates with tubulin and associates with both microtubules and microfilaments in intact cells. The cellular distribution is altered by treatment with taxol, nocodazole, and cytochalasin D. The tubulin binding domain was located at the N terminus of GABARAP by using synthetic peptides and deletion constructs and is marked by a specific arrangement of basic amino acids. The interaction between GABARAP and actin might be mediated by other proteins. These results demonstrate the GABARAP interacts with the cytoskeleton both in vitro and in cells and suggest a role of GABARAP in the interaction between GABA(A) receptors and the cytoskeleton. Such interactions are presumably needed for receptor trafficking, anchoring, and/or synaptic clustering. The structural arrangement of the basic amino acids present in the tubulin binding domain of GABARAP may aid in recognition of the potential of tubulin binding activity in other known proteins.     

Subpopulations of tau interact with microtubules and actin filaments in various cell types

It has been demonstrated that microtubule-associated proteins (MAPs) interact with tubulin in vitro and in vivo. However, there is no clear evidence on the possible roles of the interactions of MAPs in vivo with other cytoskeletal components in maintaining the integrity of the cell architecture. To address this question we extracted the neuronal cytoskeleton from brain cells and studied the selective dissociation of specific molecular isospecies of tau protein under various experimental conditions. Tau, and in some cases MPA-2, were analysed by the use of anti-idiotypic antibodies that recognize epitopes on their tubulin binding sites. Fractions of microtubule-bound tau isoforms were extracted with 0.35 M NaCl or after the addition of nocodazole to allow microtubule depolymerization. Protein eluted with this inhibitor contained most of the assembled tubulin dimer pool and part of the remaining tau and MAP-2. When the remaining cytoskeletal pellet was treated with cytochalasin D to allow depolymerization of actin filaments, only tau isoforms were extracted. Immunoprecipitation studies along with immunolocalization experiments in cell lines containing tau-like components supported the findings on the roles of tau isospecies as linkers between tubulin in the microtubular structure with actin filaments. Interestingly, in certain types of cells, antibody-reactive tau isospecies were detected by immunofluorescence with a discrete distribution pattern along actin filaments, which was affected by cytochalasin disruption of the actin filament network. These results suggest the possible in vivo roles of subsets of tau protein in modulating the interactions between microtubules and actin filaments.     

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.     

Effects of opioids on the immune system

Both therapeutic and chronic uses of opioids compromise the optimal functioning of the immune system. Overwhelming evidence suggests that opioid use affects both innate immunity and adaptive immunity. Chronic administration of opioids decreases the proliferative capacity of macrophage progenitor cells and lymphocytes. Additionally, the differentiated function of immune cells is significantly affected by opioids. These effects are mediated by either a direct action of opioids on the target cells or by indirect centrally mediated pathways. Molecular biological and biochemical characterization suggest that immune cells differentially express classical opioid receptors. Interestingly, these studies also reveal the presence of a novel class of opioid receptors in immune cells. We believe that this low affinity morphine binding site mediates the antiproliferative effects of morphine. Special issue dedicated to Dr. Eric J. Simon.     

Distinct signaling pathways regulate differential opioid effects on actin cytoskeleton in malignant MCF7 and nonmalignant MCF12A human breast epithelial cells

The mechanisms through which opioids regulate the activity of malignant breast epithelial cells are currently unknown. In the present study we report the differential actin cytoskeleton reorganization induced by opioids in malignant (MCF7) and nonmalignant (MCF12A) breast epithelial cells expressing functional opioid receptors. Exposure of MCF7 cells to the opioid agonist alpha(s1) casomorphin induced important actin assembly and reorganization, including the formation of filopodia and lamellipodia. In contrast, incubation of MCF12A cells with alpha(s1) casomorphin revealed a partial but transient disassembly of actin microfilaments. Immunoprecipitation and immunoblot analyses showed rapid phosphorylation of focal adhesion kinase (FAK) and vinculin in opioid-treated MCF7 cells. Moreover, FAK associates with phosphatidylinositol-3 (PI-3 kinase), the latter being subsequently phosphorylated and activated. In addition, a substantial activation of the small GTPase Rac1 was observed. Pretreatment of MCF7 cells with the specific PI-3 kinase inhibitor wortmannin abolished both the activation of Rac1 and actin reorganization, while the opioid-induced phosphorylation of FAK and vinculin remained unaffected. Interestingly, in opioid-treated MCF12A cells this signaling cascade remained inactive, while we identified rapid phosphorylation of actin regulating the protein villin. Finally, opioids differentially inhibited cell motility in each cell line. Our data suggest a distinct, opioid-induced, signaling pathway activated in malignant breast epithelial cells, leading to important actin reorganization. These findings may indicate a potential antineoplastic role of opiates, based on the activation of differential signaling mechanisms.     

Role of ornithine decarboxylase in the regulation of cell growth by IL-1 and tumor necrosis factor

Ornithine decarboxylase (ODC) is a rate-limiting enzyme in polyamine synthesis, and polyamines are required for cell growth. As an approach to clarifying the mechanism of action IL-1, the effects of IL-1 on ODC activity were examined in various cell lines whose proliferation was either suppressed or enhanced by IL-1. The proliferation of all cell types used in these experiments was markedly suppressed by a specific ODC inhibitor, alpha-difluoromethyl ornithine (DFMO), substantiating the crucial role of ODC activity for cell proliferation. ODC activity also was considerably suppressed by IL-1 in those cells on which IL-1 exerts an antiproliferative effect, such as a human melanoma cell line (A375) and malignant human mammary cell lines (MCF-7 and T-47D). On the other hand, ODC activity was stimulated in cells that are stimulated to proliferate in response to IL-1, such as a mouse helper T cell line (D10.G4.1), a NK cell-like cell line (YT), and a human glioblastoma cell line (U373 MG). The effect of IL-1 on ODC activity preceded and directly correlated in a dose-dependent manner with its effect on DNA synthesis. Furthermore, putrescine, a product of the ODC reaction and a precursor of polyamines, was able to overcome most, but not all, the antiproliferative action of IL-1 in A375 melanoma cells, which were the most sensitive to suppression by IL-1. However, putrescine did not reverse the cytostatic effect of IL-1 on MCF-7 and T-47D cell lines. In contrast, putrescine, like IL-1, exhibited some co-mitogenic activity on D10.G4.1 cells. Because the biological activities of TNF and IL-1 show considerable overlap, the effect of TNF on ODC activity also was examined. TNF had an antiproliferative effect on A375 cells and stimulated the proliferation of U373 MG cells. The ODC activity in A375 cells was suppressed by TNF, and the ODC activity in U373 MG cells was stimulated by TNF. Putrescine also partially overcame the inhibitory effect of TNF. These results suggest that the regulation of ODC activity may be a key component in the antiproliferative and proliferative action of IL-1 and TNF in some tumor cell types.     

The effect of the depolymerization and disintegration of the microtubular system on the cytoskeleton of untransformed and transformed cells]

How important are the changes of microtubule control for the realization of actin cortex changes during neoplastic transformation? To answer this question we studied the actin cytoskeleton and intermediate filaments condition after colcemid destruction or taxol disintegration of microtubule system in non-transformed cells BALB/c 3T3 and in the same cells transformed by Ha-ras gene. We have come to a conclusion that the differences between non-transformed and transformed cells in the actin cytoskeleton organization remain the same after specific inhibitor action on the microtubules; after the microtubules are destroyed the differences between the two cell types appear in the intermediate filament organization; there are reasons to assume that changes in the actin cortex structure may play the central role in morphological transformation expression.     

Organelle inheritance in plant cell division: the actin cytoskeleton is required for unbiased inheritance of chloroplasts, mitochondria and endoplasmic reticulum in dividing protoplasts

Nuclear inheritance is highly ordered, ensuring stringent, unbiased partitioning of chromosomes before cell division. In plants, however, little is known about the analogous cellular processes that might ensure unbiased inheritance of non-nuclear organelles, either in meristematic cell divisions or those induced during the acquisition of totipotency. We have investigated organelle redistribution and inheritance mechanisms during cell division in cultured tobacco mesophyll protoplasts. Quantitative analysis of organelle repositioning observed by autofluorescence of chloroplasts or green fluorescent protein (GFP), targeted to mitochondria or endoplasmic reticulum (ER), demonstrated that these organelles redistribute in an ordered manner before division. Treating protoplasts with cytoskeleton-disrupting drugs showed that redistribution depended on actin filaments (AFs), but not on microtubules (MTs), and furthermore, that an intact actin cytoskeleton was required to achieve unbiased organelle inheritance. Labelling the actin cytoskeleton with a novel GFP-fusion protein revealed a highly dynamic actin network, with local reorganisation of this network itself, appearing to contribute substantially to repositioning of chloroplasts and mitochondria. Our observations show that each organelle exploits a different strategy of redistribution to ensure unbiased partitioning. We conclude that inheritance of chloroplasts, mitochondria and ER in totipotent plant cells is an ordered process, requiring complex interactions with the actin cytoskeleton.     

The prokaryotic cytoskeleton: a putative target for inhibitors and antibiotics?

In the recent decade, our view on the organization of the bacterial cell has been revolutionized by the identification of cytoskeletal elements. Most bacterial species have structural homologs of actin and tubulin that assemble into dynamic, filamentous structures at precisely defined sub-cellular locations. The essential cell division protein FtsZ forms a dynamic ring at mid-cell and is similar in its structure to tubulin. Proteins of the MreB family, which are structural homologs of actin, assemble into helical or straight filaments in the bacterial cytoplasm. As in eukaryotic cells, the bacterial cytoskeleton drives essential cellular processes such as cell division, cell wall growth, DNA movement, protein targeting, and alignment of organelles. Different high-throughput assays have been developed to search for inhibitors of components of the bacterial cytoskeleton. Cell-based assays for the detection of cell division inhibitors as well as FtsZ GTPase assays led to the identification of several compounds that inhibit the polymerization of FtsZ, by this blocking bacterial cell division. Such inhibitors might not only be valuable tools for basic research, but might also lead to novel therapeutic agents against pathogenic bacteria. For example, the polyphenol dichamanetin, the 2-alkoxycarbonylaminopyridine SRI-3072, and the benzophenanthridine alkaloid sanguinarine inhibit the GTPase activity of FtsZ and exhibit antimicrobial activity.