Bis(thiosemicarbazone) copper complexes: mechanism of intracellular accumulation.

The molecular basis of Alzheimer's disease has not been clearly established, but disruption of brain metal ion homeostasis, particularly copper and zinc, might be closely involved in the pathogenesis of this disease and its characteristic ß-amyloid neuropathological features. The use of complexes of copper with bis(thiosemicarbazones) ([Cu(btsc)]) has been proposed for the treatment of Alzheimer's disease. Their mode of action could involve modulation of the concentration of copper or zinc, and it has been suggested that the compounds can modulate the production of ß-amyloid peptide at the neuron level. Furthermore, it has been reported that [Cu(btsc)] complexes can be reduced inside the cells. However, to our knowledge the intracellular reduction of these compounds has never been demonstrated. Thus, the goal of our study was to increase understanding of the mechanism of intracellular accumulation of [Cu(btsc)] complexes. Our results reveal that the intracellular concentration of copper inside the cells is very high and that these compounds are not P-glycoprotein substrates. This protein is a key element of the low permeability properties of the blood-brain barrier. Furthermore, no intracellular reduction of cupric ions was detected. Finally, once inside the cells, the complexes undergo aggregation, strongly suggesting that aggregation of complexes is the driving force responsible for their intracellular accumulation.

Impact of intracellular chloride concentration on cisplatin accumulation in sensitive and resistant GLC4 cells.

Resistance to cisplatin [cis-diamminedichloroplatinum(II), CDDP] chemotherapy is a major problem in the clinic. Understanding the molecular basis of the intracellular accumulation of CDDP and other platinum-based anticancer drugs is of importance in delineating the mechanism of resistance to these clinically important therapies. Different molecular mechanisms may coexist, but defective uptake of CDDP is one of the most consistently identified characteristics of cells selected for CDDP resistance. We have studied the impact of intracellular chloride concentration on platinum-based compound accumulation in the human GLC4, GLC4/CDDP, and K562 tumor cell lines. We show that (1) a decrease of intracellular chloride concentration yielded an increase of CDDP accumulation and vice versa and (2) the intracellular chloride concentration in GLC4/CDDP cells is higher than in sensitive cells, whereas CDDP accumulation shows the opposite behavior. The identification of chloride as a critical determinant of CDDP intracellular accumulation and the molecular mechanisms by which CDDP-resistant cells modulate chloride concentration may allow alternative therapeutic approaches. Our findings indicate that increase of intracellular chloride concentration may be a major determinant of CDDP resistance.

Impact of the carbon chain length of novel platinum complexes derived from N-alkyl-propanediamines on their cytotoxic activity and cellular uptake.

This work describes the synthesis and characterization of four new ligands derived from 1,3-propanediamine in addition to the preparation and characterization of their respective platinum(II) complexes by reaction with K(2)PtCl(4). These ligands were obtained by the reaction of the corresponding alkyl mesylate with 1,3-propanediamine. We have prepared compounds having different carbon chains lengths in an attempt to correlate this factor, which influences the lipophilicity of the compounds, with cytotoxic activity. Octanol/water partition coefficients, the effect of the four complexes on the growth of two tumoral cell lines, and their cellular uptake were investigated. Increasing lipophilicity enhances the rate of cellular uptake and, consequently, the cytotoxic activity.

Perturbation of membrane microdomains in GLC4 multidrug-resistant lung cancer cells--modification of ABCC1 (MRP1) localization and functionality.

The multidrug resistance-associated protein transporter ABCC1 (MRP1) is an integral plasma membrane protein involved in the multidrug resistance phenotype. It actively expels a number of cytotoxic molecules from cells. To gain insight into the modulation of the functional properties of this integral membrane protein by cholesterol, a main component of the lipid bilayer, we used multidrug-resistant GLC4/ADR cells, which overexpress MRP1. Upon altering the plasma membrane cholesterol content of these cells, membrane localization and the activity of MRP1 were analyzed. A detergent-free methodology was used to separate "light" and "heavy" plasma membrane fractions. Our data show that MRP1 was exclusively found in "light" fractions known as L0 phase membrane microdomains, together with 23% of gangliosides GM1 and 40% of caveolin-1. Depletion of the membrane cholesterol level to 40% by treatment with the cholesterol-chelating agent methyl-beta-cyclodextrin did not modify MRP1 activity, as evidenced either by the rate of efflux of pirarubicin or that of glutathione. Further cholesterol depletion below 40% yielded both a partial shift of MRP1 to the high-density fraction and a decrease of its functionality. Taken together, these data suggest that MRP1 functionality depends on its localization in cholesterol-rich membrane microdomains.

Isomeric N,N-bis(cyclohexanol)amine aryl esters: the discovery of a new class of highly potent P-glycoprotein (Pgp)-dependent multidrug resistance (MDR) inhibitors.

A new series of P-glycoprotein (Pgp)-dependent multidrug resistance (MDR) inhibitors having a N,N-bis(cyclohexanol)amine scaffold have been designed, following the frozen analog approach. With respect to the parent flexible molecules, the new compounds show improved potency and efficacy. Among them, compound 1d, on anthracycline-resistant erythroleukemia K562 cells, is able to completely reverse Pgp-dependent MDR at low nanomolar concentration.

Ability of carbazole salts, inhibitors of Alzheimer beta-amyloid fibril formation, to cross cellular membranes.

Alzheimer's disease is characterized by the presence of beta-amyloid fibril formation. The inhibition of this peptide accumulation may be a prevention method for Alzheimer's disease. Several classes of molecules have been reported to inhibit beta-amyloid fibril formation and among them carbazoles. However, very few studies have been performed to determine the destination of such molecules in vivo and especially if they can pass the blood brain barrier. The aim of this paper is to study whether carbazoles could pass the blood brain barrier, i.e. if they can circumvent ATP Binding Cassette (ABC) transporters such as P-glycoprotein (P-gp) and Multidrug Resistance-associated protein (MRP1) which efficiently limit drug brain uptake. For this purpose we have synthesized a fluorescent derivative of carbazole benzothiazolium iodide 1,2 disubstituted ethylene (referred as carbazole thiazole: CT), which can be easily detected and followed in the pre-trial study phases in cells or in tissue. We use cellular models overexpressing P-gp and MRP1. Our results show that: i) CT is able to cross membranes and to penetrate rapidly inside the cells, ii) CT is a P-gp substrate and consequently its accumulation in P-gp overexpressing cells is very low, iii) CT is a poor MRP1 substrate. In addition once inside the cells, CT rapidly binds to DNA and is then slowly reduced by intracellular reducing agents. In conclusion, the efficiency of carbazole derivatives in inhibiting the beta-amyloid formation in vivo could be highly compromised because, as P-gp substrates, they will probably not cross the blood brain barrier.

Role of residual Sb(III) in meglumine antimoniate cytotoxicity and MRP1-mediated resistance.

Despite the clinical use of pentavalent antimonials for more than half a century, their metabolism in mammals and mechanisms of action and toxicity remain poorly understood. It has been proposed that the more active and toxic trivalent antimony form Sb(III) plays a critical role in their antileishmanial activity and toxicity. The aim of this work was to investigate the role of residual Sb(III) both in the antileishmanial/antitumoral activities of the pentavalent meglumine antimoniate and in the MRP1 (multidrug resistance-associated protein 1)-mediated resistance to this drug. Samples of meglumine antimoniate differing in their amount of residual Sb(III) (meglumine antimoniate synthesized either from SbCl(5) or from KSb(OH)(6) as well as commercially-available meglumine antimoniate) were evaluated in vitro and in vivo on Leishmania amazonensis infections, as well as for their cytotoxicity to normal and MRP1-overexpressing GLC4 cell lines. Although in vitro the two most effective drugs contained the highest levels of Sb(III), no correlation was found in vivo between the antileishmanial activity of meglumine antimoniate and its residual Sb(III) content, suggesting that residual Sb(III) contributes only marginally to the drug antileishmanial activity. On the other hand, the GLC4 cells growth inhibition data strongly suggests a marked contribution of residual Sb(III). Additionally, the potassium salt of antimoniate (non-complexed form of Sb(V)) was found to be more cytotoxic than meglumine antimoniate. Although MRP1-overexpressing GLC4 cells showed a marked resistance to trivalent antimonials, cross-resistance to meglumine antimoniate was observed only for the products that contained relatively high levels of Sb(III) (at least 0.03% by weight), suggesting that MRP1 mediates resistance to Sb(III) but not to Sb(V). In conclusion, our data strongly suggest that residual Sb(III) in pentavalent antimonial drugs does not contribute significantly to their antileishmanial activity, but is responsible for their cytotoxic activity against mammalian cells and the MRP1-mediated resistance to these drugs.

Antimony(V) complex formation with adenine nucleosides in aqueous solution.

Despite the clinical use of pentavalent antimonial drugs for over half a century, their mode of action against leishmaniasis remains poorly understood. In this paper, we investigated the ability of Sb(V) to form in aqueous solution complexes with adenine nucleosides and deoxynucleosides, using circular dichroism (CD) and (1)H and (13)C NMR spectroscopies. We report that the ribonucleosides, adenosine (A) and adenosine monophosphate (AMP), form in water complexes with Sb(V), as evidenced by the changes induced in their CD spectra. On the other hand, 2'-deoxyadenosine (dA) did not show such a change. CD titration of the ribonucleosides with Sb(V) suggests the formation of 1:2 Sb(V)-nucleoside complexes. NMR analysis indicates that Sb(V) binds to the sugar moiety at the 2' position. Furthermore, the incubation of the antimonial drug, meglumine antimonate, with adenosine at 37 degrees C led to the transfer of Sb(V) from its original ligand to the nucleoside molecule, at acidic pH (pH 5), but not at neutral pH (7.2). Our data therefore suggests that the formation of such complexes may take place in vivo within the acidic cell compartments, including the phagolysosome of macrophage in which Leishmania resides.

Exploratory chemistry toward the identification of a new class of multidrug resistance reverters inspired by pervilleine and verapamil models.

On the basis of the present knowledge of the substrate recognition site of ABC transporter proteins and inspired by the structures of verapamil and pervilleine A, a new class of Pgp-mediated multidrug resistance (MDR) reverters has been designed and synthesized. The new compounds are flexible molecules carrying one or two basic nitrogen atoms flanked, at properly modulated distance, by two aromatic moieties. Most of the molecules studied possess MDR inhibitory activity on anthracycline-resistant erythroleukemia K 562 cells, showing a potency that is higher than that of the reference compound verapamil and, in a few cases (7, 12, 13,17, 20, 22, 28), is in the high nanomolar range. These compounds may be useful leads to develop new MDR reverting agents. In fact, the chemical structure of the class is fairly simple and can be implemented in a variety of ways that will allow the synthesis of new compounds that might be useful leads for the development of drugs to control Pgp-dependent MDR.

Mitomycin antitumor compounds. 2. Interaction of transition metal ions with mitomycin C. Solution structure and biological activity of a Pd(2+)-MMC complex.

Interactions of Cu(2+), Zn(2+), and Pd(2+) ions with the antitumor compound mitomycin C (MMC) have been investigated by UV-vis, circular dichroism, and (13)C NMR spectroscopy. While Zn(2+) and Cu(2+) neither interacted with MMC nor catalyzed the formation of mitosenes, Pd(2+) induced strong MMC spectral modifications, suggesting the formation of a major complex, in which MMC acted as a bidentate ligand through N(1) and N(4) atoms. The coordination mode in this complex was solvent dependent: in MeOH, the NH(2) of the carbamate function was also involved as a third coordination site whereas, in H(2)O, Pd(2+) hydrolysis was more effective, leading to the replacement of the carbamoyl NH(2) function with either H(2)O or OH(-) ligands. Although coordination of the indoline nitrogen prevented methanol elimination and consequent aziridino ring opening, Pd(2+) complexation maintained MMC biological activity against cancer cells, as shown by IC(50) values. This suggests that alternative mechanisms in the biological activity of MMC should be explored.

Kinetic analysis of fluorescein and dihydrofluorescein effluxes in tumour cells expressing the multidrug resistance protein, MRP1.

Multidrug resistance (MDR) in tumour cells is often caused by the overexpression of two transporters the P-glycoprotein (P-gp) and the multidrug resistance-associated protein (MRP1) which actively pump out multiple chemically unrelated substrates across the plasma membrane. A clear distinction in the mechanism of translocation of substrates by MRP1 or P-gp is indicated by the finding that, in most of cases, the MRP1-mediated transport of substrates is inhibited by depletion of intracellular glutathione (GSH), which has no effect on their P-gp-mediated transport. The aim of the present study was to quantitatively characterise the transport of anionic compounds dihydrofluorescein and fluorescein (FLU). We took advantage of the intrinsic fluorescence of FLU and performed a flow cytometric analysis of dye accumulation in the wild-type drug sensitive GLC4 that do not express MRP1 and its MDR subline which display high level of MRP1. The measurements were made in real time using intact cells. The kinetics parameters, k(a)=V(M)/K(m), which is a measure of the efficiency of the transporter-mediated efflux of a substrate, was very similar for the two FLU analogues. They were highly comparable with values for k(a) of other negatively charged substrates, such as GSH and calcein. The active efflux of both FLU derivatives was inhibited by GSH depletion.

Preferential efflux by P-glycoprotein, but not MRP1, of compounds containing a free electron donor amine.

Multidrug resistance (MDR) in model systems is known to be conferred by two different integral proteins, the 170-kDa P-glycoprotein (P-gp) and the 190-kDa multidrug resistance-associated protein (MRP1), both of which pump drugs out of MDR cells. The presence of a nitrogen atom, charged at physiological pH, has frequently been considered to be a hallmark of P-gp substrates and inhibitors. The present study was aimed at investigating the role of nitrogen in the ability of the pump to recognise substrate. We measured the kinetics of active efflux of seven new anthracycline derivatives in P-gp-expressing K562/ADR cells and in MRP1-expressing GLC4/ADR cells. Six of these compounds represent analogues of daunorubicin in which the amino sugar nitrogen is bound to an amino- or a nitro-substituted benzyl moiety, the seventh is a doxorubicin derivative in which benzyl group is bound with 4'-oxygen. We found that the compounds with a nitro group on the benzyl ring were poor substrates for P-gp despite the presence of a secondary amine that can be protonated. In contrast, compounds that have a free amino group were very good substrates even though this amine is not protonated in the pH range studied (pK approximately 3). These results show that the nitrogen atom does not interact with P-gp in a charged form but rather as an electron donating group.

The role of structural factors in the kinetics of cellular uptake of pyrazoloacridines and pyrazolopyrimidoacridines: implications for overcoming multidrug resistance towards leukaemia K562/DOX cells.

The appearance of multidrug resistance (MDR) of tumour cells to a wide array of antitumour drugs, structurally diverse and having different mechanisms of action, constitutes the major obstacle to the successful treatment of cancer. Our approach to search for non-cross resistant antitumour agents is based on the rational design of derivatives, which have a high kinetics of passive cellular uptake rendering their active efflux by MDR exporting pumps inefficient. Recently, two families of acridine cytotoxic agents were obtained, pyrazoloacridines (PACs) and pyrazolopyrimidoacridines (PPACs). The aim of this study was to examine molecular basis of the reported differences in retaining cytotoxic activity of these derivatives at cellular level against resistant erythroleukaemia K562/DOX (overexpressing P-glycoprotein) cell line. The study was performed using a spectrofluorometric method, which allows continuous monitoring of the uptake and efflux of fluorescent molecules by living cells. It was demonstrated that the presence of two additional rings, pyrazole and pyrimidine, fused to the acridine chromophore structure (PPAC) favoured more rapid cellular diffusion than the presence of only one additional pyrazole ring (PAC). The presence of hydrophobic substituent OCH3 markedly favoured the cellular uptake of pyrazoloacridines and pyrazolopyrimidoacridines while compounds having hydrophilic substituent OH exhibited very low kinetics of cellular uptake. In contrast, it was found that neither structure of the ring system nor the hydrophobic/hydrophilic character of examined substituents determined the rate of active efflux of these compounds by P-glycoprotein. Our data showed that a nearly linear relation exists between the resistance factor (RF) and lnV+ reflecting the impact of the cellular uptake rate (V+) on the ability of these compounds to overcome MDR.

MRP1 modulation by PAK-104P: detection with technetium-99m-MIBI in cultured lung tumor cells.

BACKGROUND: Resistance mediated by the MultiDrug Resistance protein (P-glycoprotein and MRP1), results in energy-dependent efflux of drugs and 99mTc-MIBI from the cells. The goal of our investigation was to evaluate the capacity of PAK-104P to lower multidrug resistance by decreasing substrate efflux. MATERIALS AND METHODS: 99mTc-MIBI accumulation was quantified in the leukaemia cell line which expresses the P-glycoprotein (K562/R) or not (K562/S) and the small lung cancer cell line which expresses MRP1 (GLC4/R) or not (GLC4/S). Three experimental protocols were used: 1). cells were treated with increasing concentrations of PAK-104P; 2). the plasma membrane potential was lowered; 3). intracellular reduced glutathione (GSH) was depleted. RESULTS: Exposure of cells to 5 microM PAK-104P affected 99mTc-MIBI accumulation as follow: 1). no effect on K562 cell lines; 2). increased 8-fold in GLC4/R; 3). enhanced in GLC4 after GSH concentration and transmembrane potential reduction. CONCLUSION: Assessed by 99mTc-MIBI, PAK-104P modulated MRP1 activity by the decrease of intracellular GSH concentration.

Synthesis, characterization, cytotoxic activity, and cellular accumulation of dinuclear platinum complexes derived from N,N'-di-(2-aminoethyl)-1,3-diamino-2-propanol, aryl substituted N-benzyl-1,4-butanediamines, and N-benzyl-1,6-hexanediamines.

This work describes the synthesis and characterization of six new dinuclear platinum complexes having N,N'-di-(2-aminoethyl)-1,3-diamino-2-propanol, aryl substituted N-benzyl-1,4-butanediamines and N-benzyl-1,6-hexanediamines as ligands. They were prepared by the reaction of cis-[PtCl(2)(DMSO)(2)] (DMSO=dimethyl sulfoxide) with the appropriate ligand in water, except for one of them, which was prepared from K(2)PtCl(4). We also report the cytotoxic activity and cellular accumulation of three of these complexes in a human small-cell lung carcinoma cell line and its resistant subline. Resistant cells exhibited a lesser degree of cross-resistance to these compounds when compared to cisplatin. The accumulation of platinum in both cell lines followed the same pattern, i.e. approximately the same intracellular platinum concentration yielded the same cytotoxic effect independent of the nature of the platinum complex used.

New findings in the study on the intercalation of bisdaunorubicin and its monomeric analogues with naked and nucleus DNA.

DNA is a target molecule for anthracycline anticancer drugs. We have used new anthracycline derivatives, bisdaunorubicin (WP631) and its monomeric analogues (WP700 serie), and look if there was a relation between the drug binding affinity to naked DNA and to cell nucleus in the cell with its cytotoxicity. Circular dichroism (CD) and fluorescence were used to follow the interaction of anthracycline derivatives with naked DNA and cell nuclei. WP631 interacts with DNA at two distinct stoichiometries, 6:1 and 3:1 base pair (bp)/WP631 molecule (3:1 and 1.5:1 per anthracycline rings). Monomeric daunorubicin (DNR) with its amino sugar N-bound to amino- and nitro-substituted benzyl moiety, representing p-xylenyl linker present in WP631 bisintercalator, is much more binding to DNA than DNR or WP631. These findings are supported by the study of drug binding by nuclei of K562 cells. Around 70% of WP700 intercalate to nucleus DNA in the steady-state, while only 45% of DNR intercalate DNA in the cell. The binding of WP631 by K562 cells is even less effective ( approximately 20%). WP 700 compounds, which are very similar to each other in their binding to DNA, self-association and cell accumulation, differ very distinctly in their cytotoxicity power. The most effective compounds are amino-benzyl derivatives of WP 700 series. The nitro-benzyl compounds have very low toxicity, even if they bind to DNA with similar power with that of the amino derivatives. The comparison of the all data clearly indicates no relation between cytotoxicity of the drug and its ability to intercalate DNA.

Accumulation of Eu3+ chelates in cells expressing or not P-glycoprotein: implications for blood-brain barrier crossing.

Alzheimer's disease (AD) is the most commonly form of dementia in the elderly. The development of molecules able to detect biomarkers characteristic of AD is critical to its understanding and treatment. However, such molecules must be able to pass blood-brain barrier (BBB) which is a major impediment to the entry of many therapeutic drugs into the brain. Such a limitation applies to the development of magnetic resonance imaging molecular neuroimaging agents using biomarkers of AD-like beta-amyloid deposits, as the common extracellular contrast agents (CAs) are not able to cross an intact BBB. In this work, we have studied the ability of a series of simple Eu(3+) complexes to enter cells overexpressing or not the ABCB1 (P-gp or P-glycoprotein) protein, which is expressed at the BBB and in human embryonic astrocytes. The intracellular uptake of the Eu(3+) complexes of linear and macrocyclic polyaminocarboxylate ligands with different charges and lipophilicities was followed by atomic absorption spectrometry. Based on biochemical argument, we propose that lipophilic contrast agents can be efficiently taken up by cells and accumulate inside mitochondria when they are positively charged. The important point is that they are not P-gp substrates, which is one of the major obstacles for them to cross the BBB.

Mechanism of thioflavin T accumulation inside cells overexpressing P-glycoprotein or multidrug resistance-associated protein: role of lipophilicity and positive charge.

Alzheimer's disease is characterized by the presence of amyloid deposition. Thioflavin T (ThT) has been one of the molecules of choice to attempt the detection of these amyloid deposits. However, it has been reported that ThT was unable to cross blood-brain barrier (BBB). Our aim was to understand the mechanism according to which it has been said that ThT is not able to cross the BBB. For this purpose we have used cellular models overexpressing P-glycoprotein (P-gp) or multidrug resistance-associated protein (MRP1), two proteins overexpressed in BBB. Our results show that: (i) ThT is able to cross membranes and to penetrate inside the cells; (ii) ThT is a P-gp substrate; (iii) ThT is poor MRP1 substrate. In conclusion, our results suggest that two factors could be involved in the low accumulation of ThT in the brain: ThT is a P-gp substrate and its lipophilicity is low.

Preferential energy- and potential-dependent accumulation of cisplatin-gutathione complexes in human cancer cell lines (GLC4 and K562): A likely role of mitochondria.

cis-Diamminedichloroplatinum(II) (CDDP) is an important chemotherapeutic agent used in the treatment of a wide variety of solid tumors. We have recently shown that aquated forms of cisplatin (aqua-Pt) rapidly accumulate in K562 and GLC4 cultured cells, in comparison to CDDP. Thus, when cells are incubated with aquated forms of cisplatin a gradient of concentration is observed after a short time, approximately 40 min, with an intracellular concentration of aqua-Pt of 20-30 times higher than that of extracellular aqua-Pt. The same gradient of concentration is observed when cells are incubated with CDDP but it takes a longer time, i.e., about 24 h. Therefore, the question arises as to the identity of the intracellular sites of accumulation of aqua-Pt. Using several agents to modulate membrane potential, acidic compartment pH and/or ATP level, we obtained evidence that aqua-Pt may accumulate rapidly inside mitochondria as this accumulation is energy- and membrane-potential-dependent. However, aqua-Pt complexes are not characterized by a delocalized charge and a lipophilic character that would permit their movement through the inner membrane. Therefore, it is suggested that intracellular aqua-Pt reacts rapidly with glutathione with the resultant complex being transported inside the mitochondria via one of the known glutathione transporters, i.e., dicarboxylate and/or 2-oxoglutarate transporters present in the inner membrane.

Diphenylcyclohexylamine derivatives as new potent multidrug resistance (MDR) modulators.

A series of compounds with a diphenylmethyl cyclohexyl skeleton, loosely related to verapamil, has been synthesized and tested as MDR modulators on anthracycline-resistant erythroleukemia K 562 cells. Their residual cardiovascular action (negative inotropic and chronotropic activity as well as vasorelaxant activity) was evaluated on guinea-pig isolated atria preparations and on guinea-pig aortic strip preparations. Most compounds of the series possess a good MDR-reverting activity together with a low cardiovascular action. Among them, compounds 3a1, 7a, and 8a are more potent than verapamil as MDR reverters and lack any cardiovascular action; they can represent useful leads for the development of new safe MDR reversing drugs.