Direct antiangiogenic actions of cadmium on human vascular endothelial cells.

The vascular endothelium is a primary target of cadmium (Cd) toxicity, but little is known regarding a potential mechanism whereby Cd may inhibit angiogenesis. Recent findings showing that Cd can disrupt cadherin-mediated cell-cell adhesion suggested that Cd might inhibit angiogenesis by altering the function of VE-cadherin, a molecule that is essential for angiogenesis. To address this issue, endothelial cells (ECs) were exposed to Cd in the presence of serum and subjected to angiogenesis-related cell migration and tube formation assays. Initial examination of cytotoxicity showed that ECs are rather resistant to the acute cytotoxic effects of Cd even at concentrations up to 1 mM. However, 10 microM Cd decreased migration of ECs. Cd concentrations of 500 nM and greater significantly reduced organization of microvascular ECs into tubes. These antiangiogenic effects were evident even when ECs were preincubated with Cd and then washed to remove free Cd, indicating that Cd acted directly on the cells rather than on the extracellular matrix. Immunolocalization studies showed that Cd caused the redistribution of VE-cadherin from cell to cell contacts. These findings indicate that Cd acts in an angiostatic manner on ECs, and that this effect may involve alterations in the localization and function of VE-cadherin.

Kidney injury molecule-1 is an early biomarker of cadmium nephrotoxicity.

Cadmium (Cd) exposure results in injury to the proximal tubule characterized by polyuria and proteinuria. Kidney injury molecule-1 (Kim-1) is a transmembrane glycoprotein not normally detected in the mature kidney, but is upregulated and shed into the urine following nephrotoxic injury. In this study, we determine if Kim-1 might be a useful early biomarker of Cd nephrotoxicity. Male Sprague-Dawley rats were given daily injections of Cd for up to 12 weeks. Weekly urine samples were analyzed for Kim-1, protein, creatinine, metallothionein, and Clara cell protein CC-16. Significant levels of Kim-1 were detected in the urine by 6 weeks and continued to increase throughout the treatment period. This appearance of Kim-1 occurred 4-5 weeks before the onset of proteinuria, and 1-3 weeks before the appearance of metallothionein and CC-16. Higher doses of Cd gave rise to higher Kim-1 excretion. Reverse transcriptase-polymerase chain reaction (RT-PCR) expression analysis showed that Kim-1 transcript levels were increased after 6 weeks at the low dose of Cd. Immunohistochemical analysis showed that Kim-1 was present in proximal tubule cells of the Cd-treated rats. Our results suggest that Kim-1 may be a useful biomarker of early stages of Cd-induced proximal tubule injury.

Effect of a short-term in vitro exposure to the marine toxin domoic acid on viability, tumor necrosis factor-alpha, matrix metalloproteinase-9 and superoxide anion release by rat neonatal microglia.

BACKGROUND: The excitatory amino acid domoic acid, a glutamate and kainic acid analog, is the causative agent of amnesic shellfish poisoning in humans. No studies to our knowledge have investigated the potential contribution to short-term neurotoxicity of the brain microglia, a cell type that constitutes circa 10% of the total glial population in the brain. We tested the hypothesis that a short-term in vitro exposure to domoic acid, might lead to the activation of rat neonatal microglia and the concomitant release of the putative neurotoxic mediators tumor necrosis factor-alpha (TNF-alpha), matrix metalloproteinases-2 and-9 (MMP-2 and -9) and superoxide anion (O2-). RESULTS: In vitro, domoic acid [10 microM-1 mM] was significantly neurotoxic to primary cerebellar granule neurons. Although neonatal rat microglia expressed ionotropic glutamate GluR4 receptors, exposure during 6 hours to domoic acid [10 microM-1 mM] had no significant effect on viability. By four hours, LPS (10 ng/mL) stimulated an increase in TNF-alpha mRNA and a 2,233 % increase in TNF-alpha protein In contrast, domoic acid (1 mM) induced a slight rise in TNF-alpha expression and a 53 % increase (p < 0.01) of immunoreactive TNF-alpha protein. Furthermore, though less potent than LPS, a 4-hour treatment with domoic acid (1 mM) yielded a 757% (p < 0.01) increase in MMP-9 release, but had no effect on MMP-2. Finally, while PMA (phorbol 12-myristate 13-acetate) stimulated O2- generation was elevated in 6 hour LPS-primed microglia, a similar pretreatment with domoic acid (1 mM) did not prime O2- release. CONCLUSIONS: To our knowledge this is the first experimental evidence that domoic acid, at in vitro concentrations that are toxic to neuronal cells, can trigger a release of statistically significant amounts of TNF-alpha and MMP-9 by brain microglia. These observations are of considerable pathophysiological significance because domoic acid activates rat microglia several days after in vivo administration.

E-Cadherin, beta -Catenin and cadmium carcinogenesis.

Cadmium (Cd(2+)) is an important industrial and environmental pollutant that has been classified as a human carcinogen. Studies reported in the literature indicate that cadmium may play a role in both the initiation of cancer, by activating oncogenes, and in the progression of cancer, by increasing the metastatic potential of existing cancer cells. However, the mechanisms underlying these effects have yet to be elucidated. Recent studies from our laboratory have shown that cadmium can disrupt the tight junctions between many types of epithelial cells by interfering with the normal function of E-cadherin, a Ca(2+)-dependent cell adhesion molecule that plays a key role in epithelial cell-cell adhesion. This finding may be especially significant because a large volume of evidence indicates that the disruption of E-cadherin-mediated cell adhesion can trigger the beta-catenin-mediated activation of oncogenes in epithelial cells and increase the invasive potential of existing epithelial-derived cancers. The hypothesis that we are proposing is that the cadmium-induced disruption of E-cadherin dependent cell-cell junctions may represent a pivotal step in both the initiation of cancer by cadmium and in the tumor promoting actions of cadmium.

Evidence that E-cadherin may be a target for cadmium toxicity in epithelial cells.

E-cadherin is a Ca(2+)-dependent cell adhesion molecule that plays an important role in the development and maintenance of epithelial polarity and barrier function. This commentary describes the results of recent studies showing that the environmental pollutant Cd(2+) can damage the E-cadherin-dependent junctions between many types of epithelial cells and reviews the evidence indicating that this effect results from the direct interaction of Cd(2+) with the E-cadherin molecule. In addition, the implications of these findings with respect to the mechanisms of Cd(2+) toxicity in specific target organs such as lung, kidney, bone, and the vascular endothelium are discussed.

Interaction of cadmium (Cd(2+)) with a 13-residue polypeptide analog of a putative calcium-binding motif of E-cadherin.

Previous studies from our laboratory have shown that Cd(2+) can selectively damage the tight junctions between epithelial cells in culture. Recently, we have obtained evidence suggesting that this effect may involve the interaction of Cd(2+) with E-cadherin, a Ca(2+)-dependent cell adhesion molecule that is localized at the adhering junctions of epithelial cells. To begin to determine whether or not Cd(2+) might interact directly with the E-cadherin molecule, we studied the binding of Cd(2+) to peptide B, a synthetic, 13-residue polypeptide that corresponds to one of the extracellular Ca(2+) binding regions of mouse E-cadherin (also known as uvomorulin). The binding of Cd(2+) to peptide B was evaluated by using an equilibrium microdialysis technique and the radioactive isotope (109)Cd(2+). The effects of the binding on the conformation of the peptide were evaluated by circular dichroism (CD) spectroscopy. The results showed that Cd(2+) bound to peptide B, with a maximum of one Cd(2+) binding site per molecule and an apparent dissociation constant (K(d)) of 640 microM. The binding of Cd(2+) was reduced in the presence of excess Ca(2+), an effect that was overcome by raising the concentration of Cd(2+). Both Cd(2+) and Ca(2+) caused a shift in the CD spectrum of the peptide. However, the shift produced by Cd(2+) was about 3 times the magnitude of that produced by Ca(2+). These results indicate that Cd(2+) can interact with the Ca(2+) binding site on the peptide B molecule and distort the secondary structure of the peptide. These findings are consistent with the hypothesis that E-cadherin may be a direct molecular target for Cd(2+) toxicity.

Comparison of the cytotoxic effects of cadmium (Cd(2+)) in high and low resistance strains of MDCK cells that express different levels of E-Cadherin.

Previous studies from our laboratory have shown that cadmium (Cd(2+)) can disrupt the adhering and occluding junctions between MDCK cells. Recently, we have obtained evidence to suggest that Cd(2+) produces this effect by interacting with E-cadherin, a Ca(2+)-dependent cell adhesion molecule that is localized at the adhering junctions of epithelial cells. The objective of the present study was to examine the junctional and cytotoxic effects of Cd(2+) in subcloned strains of MDCK cells that express different levels of E-cadherin. One strain (MDCK I) expresses high levels of E-cadherin and develops a transepithelial electrical resistance of more than 800 Omega.cm(2), whereas the other strain (MDCK II) expresses much lower levels of E-cadherin and develops a transepithelial resistance of less than 100 Omega.cm(2). The results showed that exposure to 20 mum Cd(2+) for 2-4 hours caused a pronounced loss of E-cadherin from the cell borders in both strains of cells. In the MDCK I cells, the loss of E-cadherin coincided with a decrease in the transepithelial electrical resistance and the loss of the tight junction-associated proteins, ZO-1 and occludin, from the cell borders. By contrast, the MDCK II cells first exhibited a significant increase in the transepithelial electrical resistance that did not begin to decline until the cells had been exposed for 4-6 hours, a time that coincided with the loss of ZO-1 and occludin from the cell borders. Additional results showed that the MDCK I cells were slightly more sensitive to the lethal effects of Cd(2+) than were the MDCK II cells. These findings indicate that E-cadherin may be an early target for Cd(2+) toxicity in both high and low resistance strains of MDCK cells. However, they also suggest that the disruption of E-cadherin-dependent cell-cell junctions may trigger somewhat different responses in the two cell lines.

Cadmium (Cd2+) disrupts E-cadherin-dependent cell-cell junctions in MDCK cells.

Previous studies from our laboratory have shown that Cd2+ can selectively disrupt E-cadherin-dependent cell-cell junctions in the porcine renal epithelial cell line, LLC-PK1. The objective of the present studies was to determine whether or not Cd2+ could produce similar effects in Madin-Darby canine kidney (MDCK) cells, an immortal epithelial cell line derived from dog kidney. This is an important issue because MDCK cells have been used extensively as a model system to study the basic mechanisms of E-cadherin-dependent cell-cell adhesion. MDCK cells on permeable membrane supports were exposed to Cd2+ by adding CdCl2 to either the apical or the basolateral compartment. The integrity of cell-cell junctions was assessed by morphologic observation of the cells and by monitoring the transepithelial electrical resistance. The results showed that exposure to 10-40 microM Cd2+ for 15 min-4 h caused the cells to separate from each other without detaching from the growing surface. The separation of the cells was accompanied by a marked drop in the transepithelial electrical resistance, a loss of E-cadherin from the cell-cell contacts, and a reorganization of the actin cytoskeleton. These effects were much more pronounced when Cd2+ was added basolaterally than when it was added apically. Moreover, the effects of Cd2+ were qualitatively similar to those observed when the cells were incubated in Ca(2+)-free medium. These results show that Cd2+ can disrupt E-cadherin-dependent cell-cell junctions in MDCK cells, and they indicate that this cell line would be an appropriate model for further mechanistic studies in this area.

Ultrastructural characterization of the early changes in intercellular junctions in response to cadmium (Cd2+) exposure in LLC-PK1 cells.

Previous studies have shown that Cd2+ can disrupt the Ca2+-dependent junctions between LLC-PK1 cells. The objective of the present studies was to further characterize the early junctional effects of Cd2+ in LLC-PK1 cells and to identify the initial site of injury. LLC-PK1 cells were grown on permeable membrane supports and were exposed to 10 microM Cd2+ from the basolateral compartment. The integrity of cell junctions was assessed by light and electron microscopy and by measuring the transepithelial electrical resistance. After as little as 15 min of Cd2+ exposure, there was an increase in the amount of light transmitted between the cells. The transepithelial resistance began to decline by 30 min and continued to fall until reaching zero after about 6 hr. Ultrastructural analysis showed that the initial disruption of cell-cell junctions coincided with a decrease in the density of intracellular plaques associated with the adhering junctions (zonulae adherens). This effect increased with time and paralleled an increase in the space between cells and a change in the shape of the cells from squamous to rounded. Sectioning the cells horizontally, in a plane parallel to the membrane support, allowed us to see large areas of zonulae adherens. Cd2+ caused the formation of gaps within the zonulae which increased with time of exposure. No significant changes in most occluding junctions (zonulae occludens) were seen until 6-8 hr after Cd2+ exposure. These results indicate that the adhering junctions and associated cytoplasmic components are primary sites of early Cd2+ injury in LLC-PK1 cells and they suggest that these effects may result from the interaction of Cd2+ with target sites associated with the basolateral cell surface.

Binding of cadmium (Cd2+) to E-CAD1, a calcium-binding polypeptide analog of E-cadherin.

Recent studies have shown that Cd2+ can damage the Ca(2+)-dependent junctions between renal epithelial cells in culture, and preliminary evidence suggests that this effect may involve the interaction of Cd2+ with E-cadherin, a Ca(2+)-dependent cell adhesion molecule that is localized at the adhering junctions of epithelial cells. To determine whether or not Cd2+ might bind directly to the E-cadherin molecule, we studied the binding of Cd2+ to E-CAD1, a recombinant, 145-residue polypeptide that corresponds to one of the extracellular Ca(2+)-binding regions of mouse E-cadherin. By using an equilibrium microdialysis technique, we were able to show that Cd2+ could, in fact, bind to E-CAD1. The binding was saturable, with a maximum of one Cd2+ binding site per E-CAD1 molecule. The apparent dissociation constant (KD) for the binding was about 20 microM, a concentration similar to that which has been shown to disrupt the junctions between epithelial cells. Other results showed that the binding of CD2+ was greatly reduced when excess Ca2+ was included in the dialysis solution. These results suggest that Cd2+ can interact with the Ca2+ binding regions on the E-CAD1 molecule, and they provide additional support for the hypothesis that E-cadherin might be a molecular target for Cd2+ toxicity.

Effects of glutathione depletion on the cytotoxic actions of cadmium in LLC-PK1 cells.

Recent studies have shown that exposure of LLC-PK1 cells to micromolar concentrations of Cd2+ for 1-4 hr causes the disruption of the junctions between the cells, whereas exposure to higher concentrations of Cd2+ for longer periods of time causes more severe toxic effects and cell death. Studies suggesting that glutathione may serve a protective role against Cd2+ toxicity in other tissues and cells led us to examine the effects of glutathione depletion on the cytotoxic actions of Cd2+ in the LLC-PK1 cell line. Confluent cells on Falcon cell culture inserts were depleted of glutathione by exposing them to 250 microM buthionine sulfoximine for 18 hr and then exposed to various concentrations of Cd2+ for up to 24 hr. The integrity of cell-cell junctions was assessed by morphologic observation of the cells and by monitoring the transepithelial electrical resistance. Cell viability was evaluated by monitoring the release of lactate dehydrogenase into the medium. The results showed that depleting the cells of glutathione did not alter the early junction-perturbing effects of Cd2+, but greatly enhanced the lethal effects. In both the glutathione-depleted and the normal cells, junctional changes were evident after as little as 1 hr of Cd2+ exposure. While the normal cells did not begin to die until they had been exposed to Cd2+ for 12-24 hr, the glutathione-depleted cells began to die after only 8 hr of Cd2+ exposure. Additional results showed that Cd2+ exposure had no effect on the total levels of glutathione at the time in which the junctional effects were occurring, but caused a marked decrease in glutathione levels at the time the cells were dying. These results indicate that the early junctional effects of Cd2+ do not result from alterations in intracellular glutathione or sulfhydryl metabolism, whereas the more severe cytotoxic effects and cell death may involve glutathione-sensitive mechanisms.

Teratogenic effects and distribution of cadmium (Cd2+) administered via osmotic minipumps to gravid CF-1 mice.

Studies to identify the mechanisms underlying the teratogenic effects of cadmium (Cd2+) have been complicated by the inherent difficulties of chronically and subchronically administering specific doses of Cd2+ to gravid animals under strictly controlled conditions. The objective of the present study was to develop a relatively simple animal model for examining the teratogenic effects of subchronic Cd2+ exposure. Cd2+ was administered to gravid CF-1 mice by subcutaneously implanted Alzet osmotic minipumps, which released fixed amounts of Cd2+ over a 14-day period between days 5 and 18 of gestation. The results showed that Cd2+ administered in this manner produced fetal anomalies and that the patterns of Cd2+ distribution and the specific developmental defects were similar to those that have been reported for other routes of Cd2+ administration. These findings indicate that osmotic minipumps may serve as useful tools in long-term studies of Cd2+ teratogenicity. They would appear to be especially useful in teratogenic evaluations where minimizing maternal stress and administering precise doses of Cd2+ are important.

The cadmium-induced disruption of tight junctions in LLC-PK1 cells does not result from apoptosis.

Exposure of LLC-PK1 cells to low micromolar concentrations of Cd2+ for 1-4 hours causes the disruption of the adhering and occluding junctions between the cells, whereas exposure to higher concentrations of Cd2+ for longer periods of time causes more severe toxic effects and cell death. The objective of the present studies was to determine whether or not the junctional effects of Cd2+ might be a consequence of apoptotic injury. LLC-PK1 cells on cell culture inserts were exposed to either Cd2+ or tumor necrosis factor (TNF-alpha) plus cycloheximide, a treatment that has recently been shown to cause apoptosis in LLC-PK1 cells. The results showed that at the time the Cd2(+)-induced junctional changes were occurring, there was no increase in the number of apoptotic cells or evidence of DNA fragmentation. By contrast, TNF-alpha plus cycloheximide induced changes that were characteristic of apoptosis. These results indicate that the disruption of intercellular junctions by Cd2+ in the LLC-PK1 cell line occurs independently of apoptosis.

Comparison of the cytotoxic effects of cadmium chloride and cadmium-metallothionein in LLC-PK1 cells.

Recent studies have shown that ionic cadmium (Cd2+) can selectively damage the tight junctions between LLC-PK1 cells. The objective of the present studies was to determine if cadmium that is bound to metallothionein (Cd-Mt) can also damage the junctions between these cells. Cells on Falcon Cell Culture Inserts were exposed to Cd2+ or Cd-Mt from the apical and basolateral compartments. The integrity of cell junctions was assessed by monitoring the transepithelial electrical resistance, and cell viability was evaluated by monitoring the release of lactate dehydrogenase into the medium. Exposure to Cd2+ for 1-4 hours caused a pronounced decrease in the transepithelial resistance without affecting cell viability. By contrast, exposure to Cd-Mt had little effect on the electrical resistance until the cells began to die, which did not occur until 24-48 hours of exposure. Additional results showed that the cells accumulated Cd2+ more rapidly than Cd-Mt. These results indicate that Cd-Mt does not damage the junctions between LLC-PK1 cells, but that it can kill the cells after prolonged exposure.

Surface binding and uptake of cadmium (Cd2+) by LLC-PK1 cells on permeable membrane supports.

Recent studies have shown that Cd2+ has relatively specific damaging effects on cell-cell junctions in the renal epithelial cell line, LLC-PK1. The objective of the present studies was to examine the surface binding and uptake of Cd2+ by LLC-PK1 cells in relation to the disruption of cell-cell junctions. LLC-PK1 cells on Falcon Cell Culture Inserts were exposed to CdCl2 containing trace amounts of 109Cd2+ from either the apical or the basolateral compartments, and the accumulation of 109Cd2+ was monitored for up to 8 h. The integrity of cell-cell junctions was assessed by monitoring the transepithelial electrical resistance. The results showed that the cells accumulated 3-4 times more Cd2+ from the basolateral compartment than from the apical compartment. The accumulation of Cd2+ from the basolateral compartment occurred in two phases: a rapid, exponential phase that occurred in 1-2 h and coincided with a decrease in transepithelial resistance, and a slower, linear phase that continued for 6-8 h. The Cd2+ that accumulated during the rapid phase was easily removed by washing the cells in EGTA, indicating that most of it was bound to sites on the cell surface. By contrast, most of the Cd2+ that accumulated during the slower phase could not be removed by EGTA, indicating that it had been taken up by the cells. Additional studies showed that the rapid phase of Cd2+ accumulation was enhanced when Ca2+ was present at low concentrations (0.1 mM), and was greatly reduced when Ca2+ was present at high concentrations (10 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Cadmium (Cd2+) disrupts Ca(2+)-dependent cell-cell junctions and alters the pattern of E-cadherin immunofluorescence in LLC-PK1 cells.

Recent findings from our laboratories have shown that Cd2+ has relatively specific damaging effects on the adhering and occluding junctions in the established porcine renal epithelial cell line, LLC-PK1. Results of the present studies show that the junction-perturbing effects of Cd2+ in LLC-PK1 cells are more pronounced when Cd2+ is applied to the basolateral cell surface than when it is applied to the apical surface, and that the severity of the effects is inversely related to the concentration of Ca2+ in the medium. Additional results show that exposure to sublethal concentrations of Cd2+ decreases the amount of E-cadherin that is associated with cell-cell contacts. These results suggest that Cd2+ damages Ca(2+)-dependent cell-cell junctions in LLC-PK1 cells by interacting with E-cadherin or a similar Ca(2+)-sensitive site that is oriented toward the basolateral cell surface.

Cadmium (Cd2+) disrupts intercellular junctions and actin filaments in LLC-PK1 cells.

Studies reported in the literature suggest that cadmium (Cd2+) may disrupt the junctions between cells in some tissues and cell culture systems. In order to examine this possibility in more detail, we have studied the effects of Cd2+ on the integrity of intercellular junctions in the established porcine renal epithelial cell line, LLC-PK1. Junctional integrity was assessed by monitoring the collapse of domes and by measuring changes in the transepithelial electrical resistance in confluent cell monolayers. Exposure to Cd2+ caused a rapid decrease in transepithelial resistance and the concomitant collapse of domes. These effects occurred at Cd2+ concentrations (20-60 microM) and durations of exposure (as little as 1 hr) that did not alter levels of ATP or kill the cells. Electron microscopic studies showed that Cd2+ caused time-dependent changes in adhering and occluding junctional complexes, which eventually resulted in the complete separation of the cells. Additional studies, in which rhodamine-coupled phalloidin was used to visualize F-actin, showed that Cd2+ altered the structure of actin filaments in the cells; there was a significant reduction in the amount of junction-associated F-actin and in the number of stress fibers. These results indicate that Cd2+ has relatively specific damaging effects on the adhering and occluding junctions between LLC-PK1 cells and that these effects may involve the disruption of cytoskeletal actin filaments.

Differential inhibition of calcium-dependent and calmodulin-dependent enzymes by drug-calmodulin adducts.

Most of the currently available calmodulin (CaM) antagonists inhibit the actions of CaM by binding directly to it. These CaM-binding drugs tend to be relatively nonselective, because they inhibit the interaction of CaM with most, if not all, of its target enzymes. In order to develop more selective CaM antagonists, we synthesized covalent adducts of CaM and several drugs, including chlorpromazine (CPZ), fluphenazine-N-mustard (FNM), and phenoxybenzamine (PBZ), and examined the effects of these adducts on various CaM and Ca2(+)-dependent enzymes. One of the adducts (CPZ-CaM) selectively inhibited the CaM-induced activation of phosphodiesterase and myosin light chain kinase, without affecting the basal activity of either enzyme. The inhibition of these enzymes by CPZ-CaM was competitive with respect to CaM. CPZ-CaM did not inhibit CaM-sensitive Ca2(+)-ATPase or CaM-dependent protein kinase or the CaM-insensitive enzyme protein kinase C. The FNM-CaM and PBZ-CaM adducts did not inhibit the effects of CaM on any of the enzymes, but they selectively activated two of the enzymes; FNM-CaM slightly activated the CaM-dependent protein kinase, and PBZ-CaM slightly activated phosphodiesterase. These results show that certain covalently linked drug-CaM adducts can differentially inhibit or activate various CaM-sensitive enzymes, and they provide further evidence that it may be possible to develop new classes of CaM antagonists that are directed against the CaM recognition sites on CaM-sensitive enzymes.

Structural features determining activity of phenothiazines and related drugs for inhibition of cell growth and reversal of multidrug resistance.

Phenothiazines and structurally related compounds inhibit cellular proliferation and sensitize multidrug-resistant (MDR) cells to chemotherapeutic agents. To identify more potent pharmaceuticals, we studied the structure-activity relationships of 30 phenothiazines and related compounds on cellular proliferation and MDR in sensitive MCF-7 and resistant MCF-7/DOX human breast cancer cells. Substitutions on the phenothiazine ring that increased hydrophobicity increased antiproliferative and anti-MDR activities. For example, -Cl and -CF3 groups increased whereas -OH groups decreased potency. Modifying the length of the alkyl bridge and the type of amino side chain also influenced potency. Compounds with increased activity against cellular proliferation and MDR possessed a four-carbon bridge rather than a three- or two-carbon bridge and a piperazinyl amine rather than a noncyclic amino group. Compounds with tertiary amines were better anti-MDR agents than those with secondary or primary amines but were equipotent antiproliferative agents. The effects of these substituents were unrelated to hydrophobicity. The structure-activity relationships suggest that an ideal phenothiazine structure for reversing MDR has a hydrophobic nucleus with a -CF3 ring substitution at position 2, connected by a four-carbon alkyl bridge to a para-methyl-substituted piperazinyl amine. We subsequently studied related compounds having certain of these properties. Substitution of a carbon for a nitrogen at position 10 of the tricyclic ring, with a double bond to the side chain (thioxanthene), further increased activity against MDR. For example, (trans)-flupenthixol, the most potent of these compounds, increased the potency of doxorubicin against MDR cells by 15-fold, as compared with its stereoisomer (cis)-flupenthixol (5-fold) or its phenothiazine homolog fluphenazine (3-fold). (cis)- and (trans)-flupenthixol were equipotent antiproliferative agents. (trans)-flupenthixol was not accumulated more than (cis)-flupenthixol in MDR cells, implying that their stereospecific anti-MDR effects were not the result of selective differences in the access of the drugs to intracellular targets. Both drugs increased the accumulation of doxorubicin in MDR cells, but not in sensitive cells, suggesting that they modulate MDR by interacting with a uniquely overexpressed cellular target in these resistant cells. The apparent lack of clinical toxicity of (trans)-flupenthixol makes it an attractive drug for further investigation.

Differential inhibition of calmodulin-sensitive phosphodiesterase and Ca++-adenosine triphosphatase by chlorpromazine-linked calmodulin.

Upon irradiation with UV light, chlorpromazine binds irreversibly to calmodulin and inactivates it. To determine whether this chlorpromazine-calmodulin (CPZ-CaM) complex can inhibit the actions of native calmodulin, we examined its effects on the activity of calmodulin-sensitive cyclic nucleotide phosphodiesterase from rat brain and on the Ca++-adenosine triphosphatase (ATPase) of human erythrocyte membranes. The CPZ-CaM complex was prepared by irradiating purified bovine brain calmodulin in the presence of chlorpromazine and Ca++. The sample was then dialyzed extensively to remove reversibly bound chlorpromazine and then assayed for its ability to activate calmodulin-sensitive phosphodiesterase and Ca++-ATPase, and for its ability to block the stimulatory effects of native calmodulin on these enzymes. The CPZ-CaM complex had no effect on the basal activity of either enzyme; it neither activated nor inhibited the enzymes when assayed in the absence of calmodulin. However, it affected differentially the activation of the two enzymes by native calmodulin. The CPZ-CaM complex totally inhibited calmodulin-stimulated phosphodiesterase but had no effect on the activation of the ATPase by calmodulin. Other studies showed that CPZ-CaM increased the activation constant (Ka) for the interaction of calmodulin with phosphodiesterase but did not affect the maximal activation (Vmax) of the enzyme by calmodulin. Neither calmodulin nor CPZ-CaM altered the Km for the interaction between phosphodiesterase and cyclic AMP. These results suggest that CPZ-CaM inhibits the calmodulin-induced activation of phosphodiesterase by competing with calmodulin for regulatory sites on the enzyme and not by interacting with calmodulin itself or by blocking the interaction of cyclic AMP with the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)