Depolymerization of microtubules increases the motional freedom of molecular probes in cellular plasma membranes.

Depolymerization of microtubules resulted in an increase in the motional freedom of molecular probes in the plasma membranes of Chinese hamster ovary cells expressed by the order parameter, S, measured with two different lipid-soluble spin label probes, 5-doxyl stearic acid and 16-doxyl methylstearate. Treatment with a variety of microtubule-depolymerizing agents, including Colcemid, colchicine, vinblastine, podophyllotoxin, and griseofulvin, all had similar effects on motional freedom of the probes whereas beta-lumicolchicine was inactive. Several independent lines of evidence suggest that these changes in motional freedom of the probes were not the direct result of the interaction of these relatively hydrophobic drugs with the plasma membrane: the effects of the drugs were not immediate; the dose response of the Colcemid effect was the same as the dose response for depolymerization of microtubules; taxol, which stabilizes microtubules but does not affect motional freedom in the membranes, blocked the effect of Colcemid on motional freedom; a mutant cell line which is resistant to colchicine because of reduced uptake of the drug showed no effects of colchicine on probe motional freedom; and a Colcemid-resistant mutant cell line with an altered beta-tubulin showed no effect of Colcemid on motional freedom in the membrane. These results support the hypothesis that microtubules might affect, directly or indirectly, plasma membrane functions.

Multidrug transporters as drug targets.

Transport molecules can significantly affect the pharmacodynamics and pharmacokinetics of drugs. An important transport molecule, the 170 kDa P-glycoprotein (Pgp), is constitutively expressed at several organ sites in the human body. Pgp is expressed at the blood-brain barrier, in the kidneys, liver, intestines and in certain T cells. Other transporters such as the multidrug resistance protein 1 (MRP1) and MRP2 also contribute to drug distribution in the human body, although to a lesser extent than Pgp. These three transporters, and especially Pgp, are often targets of drugs. Pgp can be an intentional or unintentional target. It is directly targeted when one wants to block its function by a modifier drug so that another drug, also a substrate of Pgp, can penetrate the cell membrane, which would otherwise be impermeable. Unintentional targeting occurs when several drugs are administered to a patient and as a consequence, the physiological function of Pgp is blocked at different organ sites. Like Pgp, MRP1 also has the capacity to mediate transport of many drugs and other compounds. MRP1 has a protective role in preventing accumulation of toxic compounds and drugs in epithelial tissue covering the choroid plexus/cerebrospinal fluid compartment, oral epithelium, sertoli cells, intesticular tubules and urinary collecting duct cells. MRP2 primarily transports weakly basic drugs and bilirubin from the liver to bile. Most compounds that efficiently block Pgp have only low affinity for MRP1 and MRP2. There are only a few effective and specific MRP inhibitors available. Drug targeting of these transporters may play a role in cancer chemotherapy and in the pharmacokinetics of substrate drugs.

Cyclosporin A depolarizes cytoplasmic membrane potential and interacts with Ca2+ ionophores.

Cytoplasmic membrane potential of mouse lymphocytes was determined with flow cytometry and fluorescence spectroscopy using 3,3'-dihexylcarbocyanine iodide (DiOC6(3)). The amount of this lipophilic cation incorporated into the cytoplasmic membrane is dependent upon the transmembrane potential, so the dye is suitable for continuous monitoring of this parameter, under controlled conditions. Membrane potential of the cells was decreased in the presence of cyclosporin A and cyclosporin G in a dose-dependent manner. However, the depolarization caused by Ca2+ ionophores, ionomycin and A23187, was reduced in the presence of cyclosporin A. Electron spin resonance spectroscopy with 5-doxylstearic acid as a probe indicated that cyclosporin A decreased the apparent motional freedom of membrane lipids. These data suggest incorporation of cyclosporin A into the cytoplasmic membrane, causing changes in ion fluxes. The membrane potential change induced by cyclosporin A may have selective biological consequences in certain subpopulations of lymphocytes.

Cytoplasmic membrane potential of mouse lymphocytes is decreased by cyclosporins.

Membrane potential of mouse lymphocytes was investigated in the presence and absence of cyclosporin A (CsA) and cyclosporin G (CsG) by flow cytometry and fluorescence spectroscopy. A carbocyanine dye, dihexyloxacarbocyanine iodide [DIOC6(3)], was applied as a membrane potential probe. A dose-dependent decrease in the membrane potential of T and B lymphocytes was observed in the presence of CsA and CsG. However, pretreatment of lymphocytes with insulin reduced the effect of the cyclosporins. Mobile ionophores, such as valinomycin, ionomycin and A23187 were less effective in changing the membrane potential of lymphocytes in the presence of CsA. The channel forming ionophore, gramicidin or high extra-cellular potassium concentration (160 mM) strongly reduced the membrane potential regardless of the absence or presence the CsA. These observations suggest incorporation of CsA into the cytoplasmic membrane causing changes in ion fluxes. Other reported biochemical effects of CsA may be secondary to the observed membrane potential changes. The membrane potential change induced by CsA may have selective biological consequences in a certain subpopulation of lymphocytes.

Specific disengagement of cell-bound anti-LAM-1 (anti-L-selectin) antibodies by aurintricarboxylic acid.

Brief treatment of human peripheral blood lymphocytes with the potential anti-HIV compound aurintricarboxylic acid (ATA) prompts the selective release of already bound L-selectin-specific anti-Leu8 and anti-LAM1-1 antibodies from the cells. Two other anti-LAM1 antibodies, anti-LAM1-3 and anti-LAM1-5 stay antigen-bound at the same time. Interestingly, the ATA-sensitive anti-Leu8 strongly competes with the ATA-resistant anti-LAM1-3 for binding. Photobleaching fluorescence resonance energy transfer (pFRET) measurements on flow-sorted cells suggests that these two antibodies compete for the same epitope, while anti-LAM1-5-FITC and anti-Leu8-PE bind to distinct sites, although they also compete for binding. Combining the data on competition, pFRET and ATA effect, we suggest that the ATA sensitive anti-Leu8 and resistant anti-LAM1-3 bind to overlapping but non-identical epitopes. This remarkably specific effect may be exploited for designing anti-inflammatory drugs that modulate leukocyte adhesion.

On the biophysics of transmembrane signalling.

Transmembrane signalling involves a number of physical translocations, changes in proximity of membrane elements like receptor subunits, or sequestration of proteins from the membrane. The monitoring of such changes with flow cytometric energy transfer revealed a new putative subunit of the IL-2 receptor and a possible intermolecular interaction between HLA class I and class II antigens. Lateral diffusion of the components of the multi-subunit IL-2 receptor was also followed. Changes in the intracellular pH were considered as a measure of efficient signal transfer in a number of cases. An overview and critical comparison of data is presented in the paper.

Induction of apoptosis in MDR1 expressing cells by daunorubicin with combinations of suboptimal concentrations of P-glycoprotein modulators.

The application of most agents with the capacity to reverse multidrug resistance (MDR) via modulation of the multidrug transporter P-glycoprotein (Pgp) was shown to be associated with toxic side-effects. For this reason, we have investigated the effect of combinations of suboptimal concentrations of Pgp blockers on the induction of apoptosis and growth arrest in daunorubicin (D) treated, MDR1 gene transfected cells. We used verapamil, PSC833 and Cremophor EL as Pgp modulators, which affect the function of Pgp by different mechanisms. Treatment of NIH3T3/MDR1 cells with combinations of suboptimal concentrations of Pgp modulators in the presence of D caused apoptosis and G(2) arrest to the same extent as optimal concentrations of singly used blockers. We conclude that combinations of suboptimal concentrations of Pgp modulators may cause effective sensitization of resistant tumor cells, and at the same time, may avoid the frequently observed toxic effects experienced in clinical trials with a single modifier applied at the optimal dose.

Modulation of the anti-proliferative signal of interferon-alpha by tamoxifen in U937 cells.

Several clinical protocols are attempting to utilize the combined anti-proliferative signal of interferon-alpha (INF-alpha) and tamoxifen on cancer cells. We demonstrated here that the effect of these two agents on the growth of the premacrophage U937 cells is antagonistic. This antagonistic effect is paralleled by the ability of tamoxifen to modulate the INF-alpha-induced hyperpolarization in these cells. INF-alpha-induced hyperpolarization was shown before to be an integral part of the anti-proliferative signal of this agent. Tamoxifen liberates Ca2+ from intracellular stores of U937 cells but this effect of the drug is not the cause of its antagonistic effect with the anti-proliferative signal of IFN-alpha. We suggest therefore, that the combined effect of these two anti-cancer drugs could also be advantageous for macrophage proliferation.

Anti-psychotic drugs reverse multidrug resistance of tumor cell lines and human AML cells ex-vivo.

Anti-psychotic drugs are used in cancer patients undergoing chemotherapy frequently and the concomitantly used drugs may alter the pharmacokinetics of each other. One reason for the alteration of pharmacokinetics may be the modulation of the function of P-glycoprotein, whose efflux pump occurs in resistant cancer cells, in human intestine and in the blood-brain barrier. For this reason we tested the effect of several anti-psychotic drugs on the multidrug-resistant pump, P-glycoprotein. We found that in the MDR gene transfected L121C MDR, L5178 MDR and in the KB-V-1 cells selected for resistance some antipsychotic drugs block the function of P-glycoprotein. Blood cells of two treatment-resistant leukemic patients also showed increased uptake of daunorubicin if treated ex vivo with the anti-psychotic drugs. Our results suggest that pharmacokinetic studies should be performed prior to concomitant clinical use of such drugs which block P-glycoprotein function.

Polyionic compounds selectively alter availability of CD4 receptors for HIV coat protein rgp120.

We studied the ability of several polyionic compounds, previously shown to have activity in vitro against human immunodeficiency virus (anti-HIV) to block binding of anti-CD4 and recombinant HIV gp120 to the CD4 receptor on human lymphocytes. We found that Evans blue and aurin tricarboxylic acid could completely inhibit binding of anti-CD4 (Leu3a) and rgp120 and have selectivity for the CD4 receptor. A number of other compounds, including dextran sulfate and heparin had no effect on binding of rgp120 and were shown to be nonspecific for inhibition of binding of monoclonal antibodies to different T-cell receptors. Studies using a number of membrane-active drugs showed that changes in membrane potential or ion fluxes were not involved in the inhibition of binding of rgp120 by Evans blue or aurin tricarboxylic acid.

Behavior of N-acylated daunorubicins in MDR1 gene transfected and parental cells.

The substrate specificity of the P-glycoprotein (P-170), a multidrug transporter, was studied using N-acylated daunorubicin derivatives and four MDR1 cDNA transfected cell lines. Results showed that N-acetyl-daunorubicin is a substrate, but the longer fatty acid derivatives, N-octanoyl and N-dodecanoyl daunorubicins, are not. This conclusion was reached by flow cytometric drug uptake assay, cell proliferation assays, and confocal microscopy. It was concluded that the longer fatty acid derivatives interact with plasma membranes in a way that affected P-glycoprotein function.

Prevention of binding of rgp120 by anti-HIV active tannins.

Several tannins with anti-HIV activity have been described previously (Nonaka et al., J Nat Prod 53: 587-595, 1990). We have shown that the tannins chebulinic acid and punicalin were able to block the binding of HIV rgp120 to CD4. These compounds were not toxic to stimulated human peripheral blood lymphocytes at concentrations ten times above their maximal effective concentration.

Studies on the action of nystatin on cultured rat myocardial cells and cell membranes, isolated rat hearts, and intact rats.

The action of nystatin, a polyene antibiotic, was studied in rat myocardial cells, isolated rat hearts, and intact rats. Myocardial cells responded to 10 and 25 micrograms nystatin/ml with arrhythmias that could be minimized by elevated concentrations of K+ and Mg2+ or reversed by washing the cells. Similarly, the isolated heart responded to 100 micrograms nystatin/ml with arrhythmias that could be tempered by addition of elevated concentrations of K+ and Mg2+. The i.v. injection of the drug caused heart failure in intact animals at the 4-mg/kg dose level. At the subcellular level, nystatin made the myocardial cell membranes more rigid, as measured by electron spin resonance spectrometry. These findings indicate a parallel between physiocochemical changes caused by nystatin in the myocardial cell membrane and the biological changes caused by this drug in myocardial cells, isolated heart, and heart of the intact animal.

Cyclosporin elicits a non-responsive state and a shift in K+ fluxes in the early phase of activation of human lymphocytes with anti-CD3.

The effect of cyclosporin A on the initial phase of activation of human peripheral blood lymphocytes (PBL) by anti-CD3 was studied in a two-step incubation process. Cyclosporin A treatment in the initial phase of activation blocked the second phase activation of a cell population by anti-CD3, IL-2, or concanavalin A (ConA). In contrast, similar treatment with the calcium ionophore, ionomycin, enhanced the activation of anti-CD3. Only a marginal synergistic increase of intracellular [Ca2+] was elicited by cyclosporin A during anti-CD3 stimulation and this drug prevented activation-induced depolarization of lymphocytes by its ability to hyperpolarize cells. The hyperpolarization effect of cyclosporin A is related to the K+ flux but not the Na+ or Ca2+ flux and is unlikely to be mediated through calmodulin and protein kinase C. We postulate that the K+ flux-modulating ability of cyclosporin A renders T cells non-responsive in the initial phase of activation.

Biochemical and clinical aspects of efflux pump related resistance to anti-cancer drugs.

This review paper will focus on the molecular biological, biochemical and biophysical aspects of the following three efflux pumps: P-glycoprotein (Pgp), Multidrug Resistance Protein (MRP) and Lung cancer Resistance-related Protein. Since suppression of the function of these pumps has clinical implications, novel approaches developed in our laboratory for blocking the function of Pgp and MRP are also discussed. The second part of this review will summarize the clinical significance and the results of clinical trials designed to suppress the effects of these efflux pumps.

Influence of beta-adrenergic antagonists, H1-receptor blockers, analgesics, diuretics, and quinolone antibiotics on the cellular accumulation of the anticancer drug, daunorubicin: P-glycoprotein modulation.

BACKGROUND: Treatment of patients with several drugs simultaneously may result in modulation of the naturally expressed P-glycoprotein (Pgp) at different tissues. With this possibility in mind, we have assessed the ability of different classes of drugs to modulate Pgp function in vitro. Modulation of the Pgp function was studied at in vitro drug concentrations comparable to therapeutic blood levels of the drugs. MATERIALS AND METHODS: Human blood brain barrier endothelial cells and human colon adenocarcinoma cells were transduced or transfected with the multidrug resistance gene (MDR1) to express Pgp. The uptake of fluorescent substrates of Pgp, Rhodamine 123 and daunorubicin, into these cells and NIH3T3/MDR1 and MDCK/MDR1 cells was measured by flow cytometry and in monolayers in the presence and absence of the different drugs. RESULTS: From the tested six H1-receptor blockers, seven beta-adrenergic antagonists, four analgesics, ten diuretics and five quinolons, five drugs inhibited Pgp at therapeutic blood levels and two at somewhat higher concentrations. Significant synergism for blocking Pgp could be demonstrated for several drugs. CONCLUSION: We conclude that administration of several drugs which modulate the function of Pgp to patients may adversely affect the natural function of this efflux pump and may cause drug-drug interactions induced side effects.

Combinations of P-glycoprotein blockers, verapamil, PSC833, and cremophor act differently on the multidrug resistance associated protein (MRP) and on P-glycoprotein (Pgp).

Clinical studies are currently in progress to evaluate functional modifiers of P-glycoprotein (Pgp), an efflux pump associated with resistance to cancer chemotherapy. However, the effects of these modifiers on a more recently discovered efflux pump, the multidrug resistance associated protein (MRP), have not yet been fully characterized. MRP is expressed in most human tissues and is overexpressed in several tumor types. For these reasons, we have investigated the effects of three prototype Pgp modifiers, which act by different modes on the function of Pgp, on the function of MRP in two MRP-overexpressing cell lines: UMCC/VP lung and MCF-7/VP breast cancer cells. Clinically optimal plasma levels of verapamil, cremophor, and PSC833 have been shown to completely block the function of Pgp in Pgp-over expressing cells. However, in the two MRP-over expressing cell lines, these modifiers only partially blocked the function of MRP and combinations of these optimal concentrations acted antagonistically. Similar antagonistic effects were seen with combinations of suboptimal concentration levels of these blockers, while these combinations resulted in synergistic effects in Pgp overexpressing cells. For two biophysical parameters measured at the plasma membrane, membrane fluidity and membrane potential, the effects of these modifiers were essentially similar in Pgp and MRP expressing cells. We suggest that the 170 kD Pgp and the 190 kD MRP glycoproteins, imbedded in the plasma membranes, respond differently to simultaneous effects of the investigated prototype resistance modifiers. These results also suggest that the identification of the specific mechanism of drug resistance is important for the selection of chemotherapeutic strategies to block the efflux pump on the cancer cell.

Inhibition of the transport function of membrane proteins by some substituted phenothiazines in E. coli and multidrug resistant tumor cells.

Efflux-pumps mediated by P-glycoprotein increase the level of resistance to antibiotics in bacteria and to cytostatics in tumor cells due to decreased drug accumulation, and are also involved in the operation of blood brain barrier. Different compounds are able to enhance drug retention in the cells by inhibiting the efflux-pump mechanism of multidrug resistant (mdr) cancer cells and bacteria. The effects of substituted chlorpromazines were studied on a hemolysin producing and antibiotic resistant plasmid carrying E coli, and rhodamine uptake of multidrug resistant (mdr 1 gene expressing) mouse lymphoma cells. Hemolysin transporter protein encoding plasmids were eliminated from E. coli by a representative phenothiazine namely promethazine. Minimal inhibitory concentrations of tetracyclin and promethazine were lower for plasmidless bacteria as compared to the parent, plasmid carrying strains. The antibiotic resistance plasmid was cured of the R-plasmid of E. coli JE 2571, however, the ring substituted derivatives were less effective then parent compounds. The effect of some substituted phenothiazines on P-glycoprotein efflux-pump of mouse lymphoma cells were studied. The majority of ring substituted derivatives reversed the mdr of tumor cells. The 3,7,8-trihydroxy- and 7,8-dihydroxy derivatives of chlorpromazine were effective as P-glycoprotein blockers, however, 7,8-diacetoxy-, 7,8dimetoxy-, 7-semicarbazone-, and 5-oxo-chlorpromazine derivatives had only moderate effect. A tomato lectin, specific for blood brain capillary endothelium was able to modify the activity of P-glycoprotein in tumor cells. Phenothiazine and tomato lectin had some antagonism in tumor cells. Our results suggest that the inhibition of P-glycoprotein function in murine tumor cells and inhibition of transporter protein in E. coli bacteria may depend on pi-electron superdelocalizibility and electrophile binding of the compounds to the transporter proteins. The intracellular accumulation of antibiotics or chemotherapeutics increased as a consequence of decreased drug efflux in both bacterial and tumor cell systems. The inhibition of the drug effux-pump is the same for all individual cells of the population. These results can be realized by combination chemotherapy, however, antiplasmid effect itself cannot be exploited in this respect because the resistance was reversed in a part of the population only. The similarity with mdr P-glycoprotein in tumor cells and brain capillary endothels provides a good model for molecules opening the blood brain barrier.

Quantitation of amphotericins by reverse-phase high-performance liquid chromatography.

A reverse-phase high-performance liquid chromatographic (HPLC) method was developed for simultaneous potency determination of amphotericin A and amphotericin B in bulk amphotericin B preparations. This single, rapid, specific, and simple method can be used for qualitative and quantitative analyses and is proposed to replace a combination of cumbersome official tests presently in use. This new HPLC method employs an isocratic acidic phosphate bufhotericin A and amphotericin B in bulk amphotericin B preparations. This single, rapid, specific, and simple method can be used for qualitative and quantitative analyses and is proposed to replace a combination of cumbersome official tests presently in use. This new HPLC method employs an isocratic acidic phosphate buffer-methanol mixture in a reverse mode on an octade-cylsilane-bonded silica column at ambient temperature, using UV detection at 313 nm. Results obtained by HPLC compare favorably with the official assay results. During these studies, an apparent heptaenic component, not previously described, was detected in commercial amphotericin B preparations at concentrations ranging from approximately 6 to 14%.

Quantitation of daunorubicin, doxorubicin, and their aglycones by ion-pair reversed-phase chromatography.

A fast and sensitive high-pressure liquid chromatographic method was developed for the quantitation of doxorubicin, daunorubicin, and their aglycones in pharmaceutical preparations. Because its higher pH extends column life while permitting determination of impurities, this system represents an improvement over previously published methods. It utilizes a C18 bonded silica gel column and a solvent system consisting of methanol-0.01 M monobasic ammonium phosphate aqueous solution (65:35) at pH 4.0 and 1600 psi of pressure. The accuracy of the doxorubicin and daunorubicin determinations, expressed as the coefficient of variation, is 1.65 and 1.27% respectively. The aglycones can be determined with a precision of less than 1.3%.