The ability of verapamil to restore intracellular accumulation of anthracyclines in multidrug resistant cells depends on the kinetics of their uptake

The basic distinguishing feature of all cells expressing functional P-glycoprotein-multidrug resistance is a decrease of steady state drug levels as compared to those in drug-sensitive controls. A variety of small molecules, such as verapamil and cyclosporin A, bind to P-glycoprotein and inhibit its ability to pump out antitumor drugs. The kinetics of P-glycoprotein-mediated efflux of various anthracycline derivatives was measured in multidrug-resistant (MDR) K562 cells in the presence of verapamil. Used for the purpose were daunorubicin, idarubicin and 8-S-fluoro-idarubicin which have the same pKa of deprotonation equal to 8.4, but different lipophilicity, 4'-epi-2'-bromo-daunorubicin which has a lipophilicity which is comparable to that of daunorubicin but a pKa equal to 6.3, pirarubicin with pKa equal to 7.7 and lipophilicity different from that of these derivatives were used. Our data show (1) that verapamil is unable to completely block the P-glycoprotein-mediated efflux of anthracyclines and that 10% of its functionality remains even with high verapamil concentrations, (2) that the ability of verapamil to restore intracellular accumulation of anthracyclines in MDR cells depends on the kinetics of their uptake. With fast kinetics uptake, as is the case for idarubicin, 8-S-fluoro-idarubicin, 4'-epi-2'-bromo-daunorubicin and pirarubicin (which have either a low pKa and/or high lipophilicity), verapamil can restore in multidrug resistant cells an intracellular drug level which is comparable to that observed in sensitive cells. This is not possible when the kinetics of uptake is low as is the case for daunorubicin. Cyclosporin A is a more potent modulator and is able to fully restore daunorubicin accumulation in multidrug resistant cells.     

The multidrug-resistant human lung tumour cell line, DLKP-A10, expresses novel drug accumulation and sequestration systems

Drug accumulation studies with the anticancer agents adriamycin and vincristine were carried out on the MDR variant of the human lung cell lines DLKP, DLKP-A10 which overexpresses the MDR associated P-glycoprotein efflux pump. Reduced cellular accumulation of both agents was observed in the resistant variant. The subsequent addition of verapamil and cyclosporin A resulted in partial restoration of cellular accumulation of both drugs in the DLKP-A10 resistant variant while complete restoration of cellular drug levels was observed in the SKMES-1/ADR cell line. These results suggested that the accumulation defect observed in the SKMES-1/ADR cell line was P-glycoprotein mediated and that accordingly, the cells exhibited characteristics consistent with the classical MDR phenotype. In contrast, while P-glycoprotein also appears to mediate a reduction in cellular drug accumulation in the DLKP-A10 cells, an alternative transport mechanism may also be present. No significant increase in the expression of either the MRP or LRP transport proteins was observed in the resistant cells. Metabolic inhibition by antimycin A (but not sodium azide or 2-deoxy-D-glucose) resulted in complete restoration of drug accumulation suggesting the presence of an alternative energy dependent transport mechanism. Fluorescent microscopy studies indicated different cellular localisation of the drug within the parental and resistant cells despite equivalent intracellular concentrations. These studies also revealed the presence of an ATP-dependent, vesicular sequestration mechanism which may be involved in the reduction of nuclear adriamycin accumulation in the DLKP-A10 cell line. This was indicated by observation of the disruption of cytoplasmic vesicles by antimycin A and also inhibition of cytoplasmic drug sequestration by the carboxylic ionophores, monensin and nigericin, accompanied by increased adriamycin accumulation and redistribution of the drug from the cytoplasm to the nucleus.     

Dynamic recycling of ERGIC53 between the endoplasmic reticulum and the Golgi complex is disrupted by nordihydroguaiaretic acid

Nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, has recently been demonstrated to block protein transport from the endoplasmic reticulum (ER) to the Golgi complex. The ER to Golgi transport is primarily operated by the ER-Golgi intermediate compartment (ERGIC). We examined the effect of NDGA on the ERGIC, focusing on the distribution of its marker ERGIC53. In control cells ERGIC53 was distributed to vesicular tubular structures corresponding to the ERGIC as well as to the ER and the cis-Golgi, reflecting its cycling between these compartments. Upon treatment of cells with NDGA, ERGIC53 was rapidly accumulated in the Golgi and undetectable in the ER and the ERGIC. Prolonged incubation of cells with the drug, however, caused redistribution of ERGIC53 and resident Golgi proteins to the ER. Thus, it is likely that NDGA has dual effects on ERGIC53 cycling; the initial accumulation in the Golgi may be caused by blocking its retrieval from the cis-Golgi to the ER/ERGIC, while the delayed redistribution to the ER may occur through a pathway induced by the drug that is different from the COPI-dependent pathway.     

The relationship between modulation of MDR and glutathione in MRP-overexpressing human leukemia cells

Multidrug resistance-associated protein (MRP) causes multidrug resistance (MDR) involving the anthracyclines and epipodophyllotoxins. Many studies show modulation of anthracycline levels and cytotoxicity in MRP-overexpressing cells, but there is limited data on the modulation of etoposide levels and cytotoxicity in MRP-overexpressing or in P-glycoprotein-expressing cells. Etoposide accumulation was 50% reduced in both the CEM/E1000 MRP-overexpressing subline and the CEM/VLB100 P-glycoprotein-expressing subline compared to the parental CEM cells, correlating with similar resistance to etoposide (200-fold) of the two sublines. For the CEM/VLB100 subline, the P-glycoprotein inhibitor SDZ PSC 833, but not verapamil, was able to increase etoposide accumulation and cytotoxicity. For the CEM/E1000 subline, neither SDZ PSC 833 nor verapamil had any effect on etoposide accumulation. However, verapamil caused a 4-fold sensitization to etoposide in this subline, along with an 80% decrease in cellular glutathione (P < 0.05). Buthionine sulfoximine (BSO), which depletes glutathione, also caused a 2.5-fold sensitization to etoposide with no effect on accumulation in the CEM/E1000 subline. In contrast, SDZ PSC 833 was able to increase daunorubicin accumulation in the CEM/E1000 subline (P < 0.05), but had no effect on daunorubicin cytotoxicity, or cellular glutathione. These results show that modulation of etoposide cytotoxicity in MRP-overexpressing cells may be through changes in glutathione metabolism rather than changes in accumulation and confirm that changes in drug accumulation are not related to drug resistance in MRP-overexpressing cells.     

Kinetics of anthracycline accumulation in multidrug-resistant tumor cells: relationship to drug lipophilicity and serum albumin binding

A multidrug-resistant Ehrlich ascites tumor cell line (EHR2/DNR+) was used to examine the membrane transport kinetics of lipophilic anthracycline derivatives in the presence of serum albumin. We present a model for theoretical data analysis with consideration of drug-albumin complex formation. For a set of five derivatives (doxorubicin, daunorubicin, 4-demethoxydaunorubicin, 4'-deoxy-4'-iododoxorubicin, and 13-dihydro-4'-deoxy-4'-iododoxorubicin), data were given on the rates of diffusional drug uptake, and membrane permeability coefficients of the noncharged molecules were estimated. Both the initial rates and the steady-state levels of drug uptake were found to decrease by addition of BSA at concentrations ranging from 5 to 75 mg/mL. For each drug, this effect of serum albumin could be accounted for by the altered distribution between free and protein-bound drug molecules in the bulk aqueous medium. A good fit of theoretical accumulation curves to the experimental data was obtained. It was concluded that a mathematical simulation method makes it possible to predict the uptake characteristics of lipophilic anthracycline compounds into tumor cells under serum conditions.     

Hepatic sequestration and modulation of the canalicular transport of the organic cation, daunorubicin, in the Rat

In contrast to organic anions, substrates for the canalicular mdr1a and b are usually organic cations and are often sequestered in high concentrations in intracellular acidic compartments. Because many of these compounds are therapeutic agents, we investigated if their sequestration could be regulated. We used isolated perfused rat liver (IPRL), isolated rat hepatocyte couplets (IRHC), and WIF-B cells to study the cellular localization and biliary excretion of the fluorescent cation, daunorubicin (DNR). Despite rapid (within 15 minutes) and efficient (>90%) cellular uptake in the IPRL, only approximately 10% of the dose administered (0.2-20 micromol) was excreted in bile after 85 minutes. Confocal microscopy revealed fluorescence predominantly in vesicles in the pericanalicular region in IPRL, IRHC, and WIF-B cells. Treatment of these cells with chloroquine and bafilomycin A, agents that disrupt the pH gradient across the vesicular membrane, resulted in a loss of vesicular fluorescence, reversible in the case of bafilomycin A. Taurocholate (TC) and dibutyryl cAMP (DBcAMP), stimulators of transcytotic vesicular transport, increased the biliary recovery of DNR significantly above controls, by 70% and 35%, respectively. The microtubule destabilizer, nocodazole, decreased biliary excretion of DNR. No effect on secretion was noted in TR- mutant rats deficient in mrp2. Coadministration of verapamil, an inhibitor of mdr1, also decreased DNR excretion. While TC and DBcAMP did not affect the fluorescent intensity or pattern of distribution in IRHC, nocodazole resulted in redistribution of DNR to peripheral punctuate structures. These findings suggest that the organic cation, DNR, is largely sequestered in cells such as hepatocytes, yet its excretion can still be modulated.     

An investigation of negative affectivity in Australian adolescents

Mistletoe lectin I (ML I) from Viscum album inhibits cell growth and induces apoptosis (programmed cell death) in several cell types. Because increases in cytosolic Ca2+ concentration ([Ca2+]i) constitute a signal for the induction of apoptosis, we studied the effects of ML I on basal [Ca2+]i, receptor-mediated rises in [Ca2+]i and cell viability, using human U-937 promonocytes as model system. Treatment of U-937 cells with ML I (30-100 ng/ml) significantly increased basal [Ca2+]i. ML I (10-30 ng/ml) enhanced histamine-induced rises in [Ca2+]i up to five-fold. The effect of histamine was inhibited by clemastine but not by famotidine, indicative for its mediation via H1-receptors. ML I additionally enhanced the stimulatory effect of complement C5a on [Ca2+]i, whereas the effect of ATP was unaffected. ML I did not induce responsiveness of U-937 cells towards a bacteria-derived chemotactic peptide. ML I up to 10 ng/ml did not affect cell viability and growth of U-937 cells. ML I at 30 ng/ml moderately inhibited cell growth and reduced cell viability. At 100 ng/ml, ML I was strongly cytotoxic. Our data support the view that Ca2+ plays a role as intracellular signal molecule in the induction of apoptosis and point to an accelerating role of H1- and C5a-receptors in the regulation of this process.     

The CrmA- and TPCK-sensitive pathways that trigger oligonucleosome-sized DNA fragmentation in camptothecin-induced apoptosis: relation to caspase activation and high molecular weight DNA fragmentation

Abstract: In human B lymphoma Namalwa variant cells expressing the serpin-like CrmA protein, the kinetics of oligonucleosome-sized DNA fragmentation was retarded compared with that of control Namalwa cells following camptothecin treatment. However, no difference in the kinetics of high molecular weight DNA fragmentation was observed between the two lines after camptothecin treatment. Similar delay and inhibition of the oligonucleosome-sized DNA fragmentation was observed in human B lymphoma Namalwa and monocytic-like leukemia U-937 cells coincubated in the presence of various concentrations of N-tosyl-L-phenylalanyl chloromethylketone and camptothecin. The effect of N-tosyl-L-phenylalanyl chloromethylketone was similar to that of CrmA and did not prevent the appearance of high molecular weight DNA fragments. Similar suppression of camptothecin-induced internucleosomal DNA fragmentation was also observed in a cell-free system when cytosolic extracts obtained from camptothecin-treated Namalwa and U-937 cells were coincubated with untreated nuclei in the presence of N-tosyl-L-phenylalanyl chloromethylketone. Furthermore, N-tosyl-L-phenylalanyl chloromethylketone had no significant effects on caspase-3-like activities in camptothecin-treated Namalwa and U-937 cells. Hydrolysis of Ac-Asp-Glu-Val-Asp-amino-4-methylcoumarin, a fluorogenic substrate with caspase-3-like activities, was detected in extracts prepared from camptothecin-treated Namalwa and U-937 cells with no apparent difference in the time courses of caspase-3-like activation in the absence or presence of N-tosyl-L-phenylalanyl chloromethylketone. Similarly, N-tosyl-L-phenylalanyl chloromethylketone was a weak inhibitor of caspase-3-like activities in vitro. Taken together, these observations suggest that the pathway sensitive to N-tosyl-L-phenylalanyl chloromethylketone is involved in camptothecin-induced oligonucleosome-sized DNA fragmentation. Furthermore, inhibition of this pathway had no effect on caspase-3-like activation and on the occurrence of high molecular weight DNA fragmentation.     

Induction of differentiation of U-937 cells by 2-chloro-3-amino-1,4-naphthoquinone

Naphthoquinone compounds have various pharmacological effects such as antiviral, antifungal and anticancer activities. We demonstrated the differentiation of the inducing effect of a naphthoquinone derivative, 2-chloro-3-amino-1,4-nahpthoquinone (NQCA) on the human leukemia cell line U-937. When U-937 cells were treated with NQCA for 4 days, phenotypes indicative of differentiation such as nitroblue tetrazolium (NBT)-reducing activity and phagocytosis were induced. To evaluate the route of differentiation of U-937 cells induced by NQCA, we determined naphthol AS-D chloroacetate esterase and alpha-naphthyl acetate esterase activities. Four days treatment of U-937 cells with NQCA increased alpha-naphthyl acetate esterase activity about 63.5% but naphthol AS-D chloroacetate esterase was not detected. These results indicate that NQCA caused differentiation of U-937 cells into macrophage-like cells. Since protein kinase C (PKC) and protein kinase A (PKA) have important roles in cell-differentiation and proliferation, we employed a PKC inhibitor NA-382 and a PKA inhibitor H-89 to examine the effects of each kinase on the differentiation of U-937 cells. The PKC inhibitor NA-382 decreased the effect of NQCA on U-937 cells, while the PKA inhibitor H-89 did not. Also glutathione (GSH) inhibited the effect of NQCA. It is concluded that the differentiation-inducing effect of NQCA on U-937 cells may be attributed to PKC activation followed by production of free radicals.     

A multidrug resistance transporter from human MCF-7 breast cancer cells

MCF-7/AdrVp is a multidrug-resistant human breast cancer subline that displays an ATP-dependent reduction in the intracellular accumulation of anthracycline anticancer drugs in the absence of overexpression of known multidrug resistance transporters such as P glycoprotein or the multidrug resistance protein. RNA fingerprinting led to the identification of a 2.4-kb mRNA that is overexpressed in MCF-7/AdrVp cells relative to parental MCF-7 cells. The mRNA encodes a 655-aa [corrected] member of the ATP-binding cassette superfamily of transporters that we term breast cancer resistance protein (BCRP). Enforced expression of the full-length BCRP cDNA in MCF-7 breast cancer cells confers resistance to mitoxantrone, doxorubicin, and daunorubicin, reduces daunorubicin accumulation and retention, and causes an ATP-dependent enhancement of the efflux of rhodamine 123 in the cloned transfected cells. BCRP is a xenobiotic transporter that appears to play a major role in the multidrug resistance phenotype of MCF-7/AdrVp human breast cancer cells.     

Endosomal recycling of the Na+/H+ exchanger NHE3 isoform is regulated by the phosphatidylinositol 3-kinase pathway

The NHE3 isoform of the Na+/H+ exchanger localizes to both the plasmalemmal and endosomal compartments in polarized epithelial and transfected Chinese hamster ovary (AP-1) cells. It is unclear how the distribution of NHE3 between these compartments is regulated. In this study, we examined the potential involvement of phosphatidylinositol 3'-kinase (PI3-K) in regulating the activity and distribution of NHE3, as this lipid kinase has been implicated in modulating vesicular traffic in the endosomal recycling pathway. Wortmannin and LY294002, both potent inhibitors of PI3-K, markedly inhibited NHE3-mediated H+ extrusion across the plasma membrane in a concentration- and time-dependent manner. The subcellular distribution of the antiporters was monitored by transfecting epitope-tagged NHE3 into AP-1 cells. In parallel with the inhibition of transport, PI3-K antagonists induced a pronounced loss of NHE3 from the cell surface and its accumulation in an intracellular compartment, as assessed by immunofluorescence microscopy and enzyme-linked immunosorbent assays. Further analysis using cells transfected with antiporters bearing an external epitope tag revealed that the redistribution reflected primarily a decrease in the rate of recycling of intracellular NHE3 to the cell surface. The wortmannin-induced inhibition and redistribution of NHE3 were prevented when cells were incubated at 4 degreesC, consistent with the known temperature dependence of the endocytic process. These observations demonstrate that NHE3 activity is controlled by dynamic endocytic and recycling events that are modulated by PI3-K.     

The protein kinase C activators phorbol esters and phosphatidylserine inhibit neutral sphingomyelinase activation, ceramide generation, and apoptosis triggered by daunorubicin

To address the role of protein kinase C (PKC) in the regulation of ceramide production, we evaluated the impact of the PKC activators 12-O-tetradecanoylphorbol-13-acetate and phosphatidylserine on the apoptotic signaling pathway triggered by the chemotherapeutic drug daunorubicin. Treatment of U937 and HL-60 cells with 0.5-1 microM daunorubicin induced a greater than 30% activation of neutral sphingomyelinase activity within 4-10 min with concomitant sphingomyelin hydrolysis and ceramide generation. Activation of PKC by 12-O-tetradecanoylphorbol-13-acetate and phosphatidylserine inhibited daunorubicin-induced neutral sphingomyelinase activation, sphingomyelin hydrolysis, ceramide generation, and apoptosis. The apoptotic response could be restored by the addition of 25 microM cell-permeant C6-ceramide. In conclusion, PKC emerges as a potentially critical negative regulator of the anthracycline-activated sphingomyelin-ceramide apoptotic pathway.     

Distinct steps in DNA fragmentation pathway during camptothecin-induced apoptosis involved caspase-, benzyloxycarbonyl- and N-tosyl-L-phenylalanylchloromethyl ketone-sensitive activities

Monocytic-like leukemia U-937 cells rapidly undergo morphological changes and DNA fragmentation that is typical of apoptosis following treatment with DNA topoisomerase I inhibitor [20-S-camptothecin lactone (CPT)]. The tripeptide derivative benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone blocks Asp-Glu-Val-Asp-ase (DEVDase) activity and prevents the occurrence of high molecular weight and oligonucleosome-sized DNA fragments associated with apoptosis in CPT-treated cells. In contrast, N-tosyl-L-phenylalanylchloromethyl ketone (TPCK) does not prevent DEVDase activity and high molecular weight DNA fragmentation but completely abrogates the appearance of oligonucleosome-sized DNA fragmentation. These results suggest that caspase 3-like activities are involved with high molecular weight DNA fragmentation pathway, whereas TPCK-sensitive activities are involved in oligonucleosome-sized DNA fragmentation pathway in CPT-treated cells. Electron micrographs reveal that caspase inhibition by benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone also abrogates the typical morphological changes associated with apoptosis, whereas TPCK does not delay these morphological changes that are typical of apoptosis. Caspase inhibition slows passage of the cells through G2 and causes a transient accumulation of these cells at the G0/G1 phase of the cell cycle following CPT treatment. In a cell-free system, when purified nuclei are incubated with apoptotic cytosolic extracts obtained from CPT-treated U-937 cells, TPCK causes a similar effect in abrogating the oligonucleosome-sized DNA fragmentation but does not affect DEVDase activity. Addition of either benzyloxycarbonyl-Val-Ala-Asp-free carboxyl group or acetyl-Asp-Glu-Val-Asp-aldehyde completely inhibits DEVDase activity in these extracts. However, acetyl-Asp-Glu-Val-Asp-aldehyde does not affect the occurrence of oligonucleosome-sized DNA fragmentation in the cell-free system, whereas the benzyloxycarbonyl derivatives benzyloxycarbonyl-Val-Ala-Asp-free carboxyl group, benzyloxycarbonyl-Val-Ala-free hydroxyl group, benzyloxycarbonyl-Val-free hydroxyl group, and benzyloxycarbonyl hydrazide abolish it markedly. Taken together, these observations show the pivotal role of DEVDase activity in triggering the apoptotic process and high molecular weight DNA fragmentation, whereas TPCK- and benzyloxycarbonyl-sensitive activities are involved in the oligonucleosome-sized DNA fragmentation pathway induced by CPT.     

The overall partitioning of anthracyclines into phosphatidyl-containing model membranes depends neither on the drug charge nor the presence of anionic phospholipids

Anthracyclines are potent anticancer agents. Their use is limited by the problem of multidrug resistance (MDR) associated with a decreased intracellular accumulation of drug correlated with the presence, in the membrane of resistant cells, of the P-glycoprotein responsible for an active efflux of the drug. The activity of a drug depends upon its intracellular concentration which itself depends on the kinetics (a) of passive influx (b) of passive efflux and (c) of the P-glycoprotein-mediated efflux of drug across the cell membrane. The ability of an anthracycline to overcome MDR depends largely on the first point. The passive drug uptake is governed by their incorporation into the lipid matrix and both electrostatic and hydrophobic forces seem necessary for the stabilization of anthracyclines into lipid bilayers. The aim of the present study was to determine the relative importance of these two interactions. Using microspectrofluorometry and the observation that the fluorescence of anthracycline is enhanced when the dihydroanthraquinone part is embedded within the lipid bilayer, we have determined the partition coefficient (alternatively, the binding constant) of 12 anthracycline derivatives in large unilamellar vesicles. The anthracyclines were (a) doxorubicin, daunorubicin and idarubicin which, at pH 7.2, bear a single positive charge at the level of the amino group on the sugar, (b) their corresponding neutral 3'-hydroxy derivatives where the amino group in the sugar has been replaced by a hydroxyl, (c) the three 13-hydroxy derivatives, doxorubicinol, daunorubicinol and idarubicinol, (d) pirarubicin and (e) two permanently positively charged derivatives. The large unilamellar vesicles contained phosphatidylcholine with various amounts of phosphatidic acid which is negatively charged and of cholesterol. We came to the conclusion that the efficiency of drug incorporation in the bilayers depends neither on the presence of a positive charge on the drug nor on the presence of anionic phospholipid but on the hydrophobicity of the molecule: the neutral and the positively charged form have the same ability to partition into the bilayer. However, the percentage of each form present should depend on the electrostatic parameters.     

Confocal scanning microspectrofluorometry reveals specific anthracyline accumulation in cytoplasmic organelles of multidrug-resistant cancer cells

We used confocal microspectrofluorometry to investigate intracellular distribution of pirarubicin or THP-DOX in parental K562, CEM, and LR73 tumor cells and their corresponding multidrug-resistant (MDR) strains. Each spectrum of a recorded image was considered as a combination of cell autofluorescence and fluorescence of the drug. In the cytoplasm of parental K562, CEM, and LR73 cells, THP-DOX fluorescence emission profile was similar to that of free drug in aqueous buffer. The (I550nm/I600nm) ratio was 0. 50 +/- 0.1. However, in the cytoplasm of resistant cells the 550-nm band profile was modified. The I550nm/I600nm ratio was 0.85 +/- 0.2 in MDR K562 cells, which is significantly different from the ratio in sensitive cells (p<0.01). This appeared first to correspond to accumulation and self-oligomerization of THP-DOX in cytoplasmic organelles of MDR cells. Transfection of LR73 cells with the mdr1 gene conferred this characteristic on the resistant LR73R cells. Bodipy-ceramide, a trans-Golgi probe, was co-localized with the typical fluorescence emission peak at 550 nm observed in the cytoplasm of MDR cells. This organelle has been shown to be more acidic in MDR cells. Moreover, this specific pattern was similar to that observed when anthracycline is complexed with sphingomyelin. The typical fluorescence emission peak at 550 nm decreased in MDR cells incubated simultaneously in the presence of the drug and quinine, verapamil, or S9788. Growth inhibitory effect and nuclear accumulation of THP-DOX data obtained on LR73R and LR73D cell lines showed that only during reversion of resistance by verapamil and S9788 was an increase of nuclear THP-DOX accumulation observed. Our data suggest that characteristics of molecular environment, such as higher pH gradient or lipid structures, would be potential mechanisms of multidrug-resistance via the sequestration of anthracyclines.