Multidrug-resistant K562 Cells Research Articles

4-Nerolidylcatechol analogues as promising anticancer agents

4-Nerolidylcatechol (1) is an isolated compound from Pothomorphe umbellata L. (Piperaceae) with promising antitumor cells properties. However it presents lability under light and room temperatures. Many efforts have been directed towards discovering anticancer agents endowed with cytotoxic activities. Here, we evaluated cytotoxic effects of 4-NRC analogues (LQFMs 2–6) and the cell death pathways induced by these compounds in multidrug-resistant K562 cells. Compounds (2–6) exhibited cytotoxic activities in a concentration-dependent manner against leukaemic cells, specially the compounds (3) and (5). Additionally, compounds (1), (3) and (5) promoted marked alterations on the cell morphology, including nuclear changes as demonstrated by Hoescht 33342 staining. Moreover, these compounds promoted apoptosis induction in K562 cells by phosphatidylserine exposure, increase of sub-G1 cells and modulation of the caspases-3/7, -8 and -9 activation. In addition, the pancaspase inhibitor z-VAD-fmk partially reduced the apoptosis induced by the compounds (1) and (5)-induced, suggesting caspase-dependent and caspase-independent cell death pathways. Compounds (1) and (5) also modified the cell cycle progression by G0/G1 and S arrest, respectively. Furthermore, compounds (1), (3) and (5) promoted mitochondrial dysfunction associated to accumulation of cytosolic cytochrome c and modulated the NF-ĸB activation. In addition, unlike their analogues, 4-NRC (1) also promoted a significant cyclin D1 inhibition. Together, these data suggest that the mechanism of cell death of 4-NRC and its analogues (3) and (5) occurs by apoptosis through mitochondrial mechanisms. Considering that LQFMs are biocompatible synthetic analogues produced by molecular simplification of (1) without the chiral centre, which is associated with the instability found in compound (1), we suggest that these compounds are promising candidates for further pre-clinical studies.

Quercetin enhances adriamycin cytotoxicity through induction of apoptosis and regulation of mitogen-activated protein kinase/extracellular signal-regulated kinase/c-Jun N-terminal kinase signaling in multidrug-resistant leukemia K562 cells.

Multidrug resistance (MDR) has become a significant challenge in chemotherapeutic treatment of cancer. Quercetin, a naturally occurring flavonoid, has been found to possess anti-proliferative, anti-inflammatory and immunoregulatory bioactivities. The present study was performed to examine the effect of quercetin on human leukemic MDR K562/adriamycin (ADR) cells. Treatment of K562/ADR cells with a combination of quercetin and ADR resulted in potentiation of cytotoxicity, which was measured using a cell counting kit-8 assay. Flow cytometric analysis revealed that quercetin dose-dependently promoted cell apoptosis and treatment with a combination of quercetin and ADR caused synergistic enhancement of the apoptotic effect. In addition, treatment of K562/ADR cells with quercetin alone or in combination with ADR resulted in loss of mitochondrial membrane potential, activation of caspase-8, -9 and -3, reduced expression of the anti-apoptotic proteins B-cell lymphoma (Bcl)-2 and Bcl-extra large and enhanced expression of the pro-apoptotic proteins Bcl-2-interacting mediator of cell death, Bcl-2-associated death promoter and Bcl-2-associated X protein in the cells. Furthermore, the combined treatment of quercetin and ADR synergistically increased the expression of phosphorylated (p-)c-Jun N-terminal kinase and p-p38 mitogen-activated protein kinase and decreased the expression of p-extracellular signal-regulated kinase in the K562/ADR cells. In addition, the expression of P-glycoprotein was significantly decreased following treatment with quercetin alone or in combination with ADR. These findings demonstrated that quercetin is important in MDR and may be developed into a new reversal agent for cancer chemotherapy.

Rab4 interacts with the human P‐glycoprotein and modulates its surface expression in multidrug resistant K562 cells

P-glycoprotein (P-gp) is a plasma membrane glycoprotein that has been signaled as a primary cause of multidrug resistance (MDR) in tumors. We performed a yeast 2-hybrid screen using the C-terminal domain of P-gp and identified 2 small GTPases involved in vesicular trafficking, Rab4 and Rab14, which complex with P-gp. The overexpression of GFP-Rab4, either transiently or stably, but not of Rab14, in K562ADR cells decreased the presence of P-gp in the cell surface. As a result, expression of this GTPase reduced the MDR phenotype of K562ADR cells, by augmenting the intracellular accumulation of daunomycin (DNM). This effect was mimicked by the constitutively active Rab4Q72L mutant, but not by the dominant negative Rab4S27N mutant. Rab4 regulated excocytotic P-gp trafficking to the plasma membrane from intracellular compartments, and this modulation required the interaction of both proteins and the GTPase activity. Noteworthy, K562ADR cells exhibited a significant reduction of Rab4 levels, but not of other Rab GTPases, as compared with the sensitive parental cell line, suggesting that the development of the MDR phenotype in these cells involves upregulation of P-gp and a concomitant downregulation of proteins that regulate its surface expression. Attenuation of endogenous Rab4 levels in K562ADR by RNA interference enhanced the expression of P-gp in the cell surface, and reduced the uptake of DNM. Accordingly, these findings substantiate the notion that modulation of the temporal and spatial distribution of P-gp in cancer cells may be a valid therapeutic strategy to alleviate the MDR phenotype, and signal to Rab4 as a potential target.

Nonactin biosynthesis: Setting limits on what can be achieved with precursor-directed biosynthesis

Nonactin, produced by Streptomyces griseus ETH A7796, is a macrotetrolide assembled from nonactic acid. It is an effective inhibitor of drug efflux in multidrug resistant erythroleukemia K562 cells at sub-toxic concentrations and has been shown to possess both antibacterial and antitumor activity. As total synthesis is impractical for the generation of nonactin analogs we have studied precursor-directed biosynthesis as an alternative as it is known that nonactic acid can serve as a nonactin precursor in vivo. To determine the scope of the approach we prepared and evaluated a furan-based nonactic acid derivative, 11. Although no new nonactin analogs were detected when 11 was administered to S. griseus fermentative cultures, a significant inhibition of nonactin biosynthesis was noted (IC 50 ∼ 100 μM). Cell mass, nonactic acid production and the generation of other secondary metabolites in the culture were unaffected by 11 demonstrating that 11 selectively inhibited the assembly of nonactin from nonactic acid. While we were unable to generate new nonactin analogs we have discovered, however, a useful inhibitor that we can use to probe the mechanism of nonactin assembly with the ultimate goal of developing more successful precursor-directed biosynthesis transformations.

Daunorubicin Loaded Magnetic Nanoparticles of Fe3O4 Greatly Enhance the Responses to Chemotherapy and Induce Apoptosis of Multidrug-Resistant K562 Cells in a Human-Nude Mice Xenograft Leukemia Model

Object: To the effect of Fe3O4-magnetic nanoparticle loaded with DNR on multidrugresistant K562 cells in vivo.Methods: K562-n and its MDR counterpart K562-n/VCR cell were inoculated subcutaneously into both sides of the back of nude mice (5×106 cells/each) to establish a human leukemia xenograft model. The mice were randomly divided into group A receiving normal saline every other day for 20days, group B receiving DNR every other day for 20days, group C receiving Fe3O4-magnetic nanoparticle every other day for 20days, group D receiving Fe3O4-magnetic nanoparticle loaded with DNR every other day for 20days, and group E receiving Fe3O4-magnetic nanoparticle containing DNR every other day for 20days with a magnetic field built on the surface of the tumor tissue. The tumor volume was measured on the day 1, 5, 9, 13, 17 and 21d after the first treatment. Tumor tissues were isolated for examination of the expression of mdr-1, bcl-2, bax and caspase-3 by reverse transcription polymerase chain reaction and Western blotting.Results: For K562-n/VCR tumor, the tumor volume was markedly lower in groups D and E than in groups A, B and C (group D or E vs group A, B or C, P < 0.05). The transcription of mdr-1 and Bcl-2 gene was significantly lower in groups D and E than in groups A, B and C (group D or E vs group A, B or C, P < 0.05). So did the protein expression of Bcl-2. However, there were no differences among these groups about the protein expression of P-gp. The protein and mRNA expressions of Bax and Caspase-3 in groups D and E were increased significantly compared with groups A, B and C (group D or E vs group A, B or C, P < 0.05). The tumor volume of K562-n was markedly lower in groups C, D and E than in groups A and B (group C, D or E vs group A or B, P < 0.05).Conclusion: In conclusion, DNR loaded Fe3O4-magnetic nanoparticles can suppress the growth and induce apoptosis further on the MDR K562-n/VCR tumor in vivo compared to DNR alone but not on the K562-n tumor. The external magnetic field failed to improve the antitumor effect.

Imatinib resistance in multidrug-resistant K562 human leukemic cells

The multidrug resistance phenotype (MDR) is one of the major causes of failure in cancer chemotherapy and it is associated with the over-expression of P-glycoprotein (P-gp or MDR1) in tumor cell membranes. A constitutive NF-κB activity has been observed in several haematological malignancies and this is associated with its anti-apoptotic role. In the present work, the relationship between NF-κB and MDR phenotype was evaluated in wild type K562 human leukemic cells (K562-WT) and in its vincristine-resistant counterpart, K562-Vinc cells. These data showed that K562-Vinc cells, which express an active P-gp, exhibited MDR phenotype. The resistant indexes (IC 50 K562-Vinc/IC 50 K562-WT) for structurally unrelated drugs like imatinib, doxorubicin and colchicine were 8.0 ± 0.3, 2.8 ± 0.4 and 44.8 ± 8.8, respectively. The imatinib resistance was reversed by P-gp blockade suggesting the involvement of P-gp in imatinib transport. We observed that NF-κB was constitutively activated in both cell lines but in a lesser extent in K562-Vinc. The inhibition of NF-κB with BAY 11-7082 increased the cytotoxicity of imatinib in K562-Vinc cells but not in K562-WT. Further, the co-administration of imatinib and BAY 11-7082 sensitized multidrug-resistant K562 cells to cell death as detected by increased percentage of annexin V positive cells. The induced cell death in K562-Vinc cells was associated with activation of caspases 9 and 3. Finally, we provide data showing that BAY 11-7082 down-regulates the expression of P-gp suggesting that the activity of NF-κB could be functionally associated to this protein in K562 cells. Our results indicate that the vincristine-resistant K562 cells which developed MDR phenotype, exhibited resistance to imatinib associated with a functional P-gp over-expression. This resistance could be partially overcome by the inhibition of NF-κB pathway.

Cell Discrimination by Attenuated Total Reflection—Fourier Transform Infrared Spectroscopy: The Impact of Preprocessing of Spectra

Fourier transform infrared (FT-IR) spectroscopy has become a powerful tool for biodiagnostics and cell line classification. Typical experimental perturbations included in spectra are baseline shift and scale variation between spectra. They have to be removed by data preprocessings to allow further data analysis and classification. In this work, we addressed baseline shift corrections and normalizations in attenuated total reflection (ATR) FT-IR spectra. We compared the efficiency of several preprocessing methods with series of spectra containing typical perturbations (baseline shift, scaling factor, and noise) and a priori known definite spectral difference. Several baseline-correction and normalization possibilities were evaluated. Our results were generally sensitive, selective, and robust with respect to baseline and scaling. Full-range scaling generated more false-positive results. Use of first- and second-derivative spectra was tested. Results obtained on model spectra were confirmed with series of spectra from sensitive and multidrug-resistant leukemia K562 cells. We showed that the use of derived spectra did not provide more efficiency and required additional preprocessing such as smoothing to obtain results similar to those obtained from non-derived ones. On the other hand, results obtained with derivatives were less sensitive to scaling, a useful feature when scaling is problematic.

STI571 combined with vincristine greatly suppressed the tumor formation of multidrug-resistant K562 cells in a human-nude mice xenograft model

The development of the targeted signal transduction inhibitor STI571 has prompted us to treat chronic myeloid leukemia in different ways. Since STI571 may reverse multidrug-resistance of K562/MDR cells in vitro, we studied the effect of STI571 on multidrug-resistant K562 cells in vivo. Multidrug-resistant human leukemia cell line K562-n/VCR expresses both bcr/abl fusion gene and multi-drug resistance (mdr1) gene. It is a vincristine resistant cell line subcloned from the vincristine (VCR) sensitive cell line K562-n induced by vincristine in vitro. K562-n and K562-n/VCR cells were inoculated subcutaneously into both sides of nude mice breast (5 x 106 cells/each) to establish a human leukemia xenograft model. The incidence and volume of tumor were observed. In the tumor-bearing nude mice, anti-tumor drugs vincristine, daunorubicin (DNR), STI571, and STI571 plus VCR for the treatment of mdr1 and bcr/abl double positive leukemia were studied respectively. The tumor incidence was 100% in the nude mice inoculated with either K562-n or K562-n/VCR. The transcription of the mdr1 gene and expression of P-gp were negative in K562-n cells but positive in K562-n/VCR cells. The intracellular accumulation of DNR in K562-n cells was higher than that in K562-n/VCR cells (P < 0.05). The tumor incidence of K562-n/VCR cells in nude mice was much higher than that of K562-n cells in chemotherapy groups, and the mean volume of the tumors was also larger (P < 0.05). STI571 combined with VCR significantly suppressed the proliferation of K562-n/VCR cells. The MDR characteristics of K562-n/VCR in vivo were the same as in vitro. STI571 had a significant tumor-suppressing effect on VCR-sensitive leukemia cells and a moderate effect on MDR leukemia cells. VCR combined with STI571 had an excellent tumor-suppressing effect on both K562-n/VCR and K562-n in the human-nude mice xenograft model.

Proton NMR visible mobile lipid signals in sensitive and multidrug-resistant K562 cells are modulated by rafts

BackgroundMost cancer cells are characterized by mobile lipids visible on proton NMR (1H-NMR), these being comprised mainly of methyl and methylene signals from lipid acyl chains. Erythroleukemia K562 cells show narrow signals at 1.3 and 0.9 ppm, corresponding to mobile lipids (methylene and methyl, respectively), which are reduced when K562 cells are multidrug resistant (MDR). While the significance of the mobile lipids is unknown, their subcellular localization is still a matter of debate and may lie in the membrane or the cytoplasm. In this study, we investigate the role of cholesterol in the generation of mobile lipid signals.ResultsThe proportion of esterified cholesterol was found to be higher in K562-sensitive cells than in resistant cells, while the total cholesterol content was identical in both cell lines. Cholesterol extraction in the K562 wild type (K562wt) cell line and its MDR counterpart (K562adr), using methyl-β-cyclodextrin, was accompanied by a rise of mobile lipids in K562wt cells only. The absence of caveolae was checked by searching for the caveolin-1 protein in K562wt and K562adr cells. However, cholesterol was enriched in another membrane microdomain designated as "detergent-insoluble glycosphingomyelin complexes" or rafts. These microdomains were studied after extraction with triton X-100, a mild non-ionic detergent, revealing mobile lipid signals preserved only in the K562wt spectra. Moreover, following perturbation/disruption of these microdomains using sphingomyelinase, mobile lipids increased only in K562wt cells.ConclusionThese results suggest that cholesterol and sphingomyelin are involved in mobile lipid generation via microdomains of detergent-insoluble glycosphingomyelin complexes such as rafts. Increasing our knowledge of membrane microdomains in sensitive and resistant cell lines may open up new possibilities in resistance reversion.

Drug sequestration in cytoplasmic organelles does not contribute to the diminished sensitivity of anthracyclines in multidrug resistant K562 cells.

Cells that acquire multidrug resistance (MDR) are characterized by a decreased accumulation of a variety of drugs. In addition, sequestration of drugs in intracellular vesicles has often been associated with MDR. However, the nature and role of intracellular vesicles in MDR are unclear. We addressed the relationship between MDR and vesicular anthracycline accumulation in the erythroleukemia cell line K562 and a drug-resistant counterpart K562/ADR that overexpresses P-glycoprotein. We used four anthracyclines (all of which are P-glycoprotein substrates): daunorubicin and idarubicin, which have good affinity for DNA and as weak bases can accumulate inside acidic compartments; hydroxyrubicin, which binds to DNA but is uncharged at physiological or acidic pH and thus cannot accumulate in acidic compartments; and WP900, an enantiomer of daunorubicin, which is a weak DNA binder but has the same pKa and lipophilicity as daunorubicin. The intrinsic fluorescence of anthracyclines allowed us to use macro- and micro-spectrofluorescence, flow cytometry, and confocal microscopy to characterize their nuclear or intravesicular accumulation in living cells. We found that vesicular accumulation of daunorubicin, WP900 and idarubicin, containing a basic 3'-amine was predominantly restricted to lysosomes in both cell lines, that pH regulation of acidic compartments was not defective in human K562 cells, and that vesicular drug accumulation was much more pronounced in the parental tumor cell line than in the multidrug-resistant cells. These results indicate that vesicular anthracycline sequestration does not contribute to the diminished sensitivity to anthracyclines in multidrug-resistant K562 cells.

Effect of PSC 833 on the cytotoxicity and pharmacodynamics of mitoxantrone in multidrug-resistant K562 cells

We examined the effect of PSC 833, a nonimmunosuppressive cyclosporin analogue, on the cytotoxicity, accumulation and retention of an anthraquinone antileukemia drug mitoxantrone (MIT). This was done in P-glycoprotein (PGP)-overexpressing multidrug-resistant K562/D1–9 cells and compared with the effect of cyclosporin A (CsA). We also compared MIT with the effect of PSC 833 on the cytotoxicity of daunorubicin (DNR) and doxorubicin (DOX). While PSC 833 and CsA had no effect on the cytotoxicity, accumulation and retention of MIT in the parent K562 cells, PSC 833 and CsA restored accumulation and retention of MIT in K562/D1–9 cells dose-dependently. Consequently, there was increased sensitivity of K562/D1–9 cells to MIT. The reversing activity of PSC 833 on the cytotoxicity of MIT was stronger than that of CsA, and was almost the same as the reversing activity of PSC 833 on the cytotoxicity of DNR and DOX. The resistance index of MIT decreased from 43.9-fold to 2.8-fold by 0.4 μM PSC 833, which is a clinically achievable plasma concentration. These results suggest that the combination of PSC 833 with MIT could be a promising treatment in reversing PGP-mediated MDR in leukemia patients.

Preferential cytotoxicity for multidrug-resistant K562 cells using the combination of a photosensitizer and a cyanine dye

The cyanine dye 1,1′,3,3,3′,3′-hexamethylindodicarbocyanine iodide (HIDC) protects K562 leukemia cells from photodynamic membrane damage caused by cis-di(4-sulfonatophenyl)diphenylporphine (TPPS 2) and 420 nm light. This wavelength of light is chosen because it is absorbed by TPPS 2, but not by HIDC. The photodynamic system studied may be useful as a model for antineoplastic therapy. A subline of K562 leukemia (K562/DOX), expressing the multidrug-resistance (MDR) phenotype, is found to accumulate smaller amounts of HIDC than the parent cell line and thus has less photoprotection. In the absence of added HIDC, the K562/DOX cell line is more resistant to photodynamic cytotoxicity than the K562 cell line. The resistance of the K562/DOX cell line is not due to a smaller accumulation of TPPS 2 than the K562 cell line. However, when both cell lines are incubated with HIDC and TPPS 2, and then exposed to light, the K562/DOX cell line becomes more sensitive to photodynamic cell damage than the K562 cell line. The combination of a photosensitizer with a cationic or lysomorphotropic photoprotector represents a novel strategy for the eradication of malignant cells expressing the MDR phenotype.

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.

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.

Study of P-glycoprotein functionality in living resistant K562 cells after photolabeling with a verapamil analogue

To our knowledge, this is the first study to investigate the modification of P-glycoprotein functionality in living resistant cells after photolabeling. For this purpose, four new photoactive verapamil analogues were synthesized. These compounds have the same efficacy as verapamil to increase pirarubicin (pira) incorporation into living multidrug resistant (MDR) K562 cells and to sensitize them to the cytotoxic effect of this anthracycline derivative, indicating that they act as typical MDR modifiers in MDR cells. These compounds were used to photolabel P-glycoprotein (P-gp) in living resistant cells. Irradiation did not result in photodamage to cells, and P-gp functionality was verified by the ability of living cells to incorporate pira. The irradiation of resistant cells, 10 6/mL, in the presence of a verapamil analogue at concentrations equal to or higher than 3 μM yielded 70% inhibition of P-gp functionality. Our data provide the first evidence that the binding of a verapamil analogue to P-gp is not sufficient to completely inhibit the efflux of this anthracycline. The cells were, subsequently, cultured for several days. Resistance was progressively recovered with time, with the treated cells being just as resistant as before photolabeling after 6 days.

Cellular uptake, cytotoxicity, and transport kinetics of anthracyclines in human sensitive and multidrug-resistant K562 cells

Multidrug resistance in tumor cells is often associated with the presence of an ~170 kDa plasma membrane glycoprotein (Pgp) that acts as a drug-efflux pump and decreases intracellular antitumor drug concentration. We measured the uptake of seven anthracyclines (daunorubicin, doxorubicin, 4′-epi-doxorubicin, 4′-deoxy-doxorubicin, iododoxorubicin, 3′-(3-methoxymorpholino)-doxorubicin (FCE23762) and 4-demethoxydaunorubicin) into K562 cells sensitive and resistant (K562/DNR) to daunorubicin. The K562/DNR subline expresses Pgp at the membrane surface, whereas its sensitive counterpart does not. Laser flow cytometry was used to quantitate intracellular anthracycline content. Uptake of daunorubicin, doxorubicin, 4′-epi-doxorubicin, and 4′-deoxy-doxorubicin was minimal in the K562/DNR subline as compared to their uptake in sensitive cells. On the contrary, iododoxorubicin, FCE23762, and 4-demethoxy-daunorubicin accumulate to nearly the same extent into sensitive and resistant K562 cells. Growth inhibition data indicated that the resistance factor for iododoxorubicin, FCE23762, and 4-demethoxy-daunorubicin is markedly decreased as compared to the other drugs. Fluorescence measurements were carried out to determine the kinetic parameters associated with the influx and efflux of the drugs into and out of K562 cells. Kinetic data indicated that iododoxorubicin, FCE23762, and 4-demethoxy-daunorubicin are not actively rejected from resistant cells, suggesting that they are poor substrates for Pgp-mediated transport. This observation is related to their ability to overcome the multidrug-resistant phenotype of K562/DNR cells in vitro.

Energy-dependent accumulation of daunorubicin into subcellular compartments of human leukemia cells and cytoplasts.

Anthracycline accumulation was evaluated by flow cytometry or radiolabeled drug assays in cells and cytoplasts (enucleated cells) prepared from parental and multidrug-resistant human K562 leukemia cells. Treatment with energy inhibitors, such as dinitrophenol (DNP) or sodium azide/deoxyglucose, led to a marked decrease in daunorubicin accumulation in parental cells and cytoplasts. Another ionophore, monensin, also caused a significant decrease in daunorubicin accumulation; however, ATPase inhibitors ouabain, vanadate, and N-ethylamaleimide had little or no effect. The lysosomatropic agents chloroquine and methylamine caused a moderate decrease in anthracycline accumulation. Fluorescence microscopy showed that the DNP-sensitive daunorubicin uptake occurred in a nonnuclear subcellular compartment. Studies using increasing daunorubicin concentrations demonstrated fluorescence quenching that occurred in the nonnuclear, DNP-sensitive compartment. The effect of inhibitors on the accumulation of rhodamine 123 and acridine orange strongly implicated lysosomes as the principal compartment of this inhibitable daunorubicin accumulation. Cytoplasts from P-glycoprotein containing multidrug-resistant K562 cells demonstrated a verapamil-reversible, decreased daunorubicin accumulation that was observed in resistant whole cells. Verapamil pretreatment of cytoplasts from resistant cells revealed the subcellular DNP-sensitive uptake present in parental cytoplasts. These studies demonstrate that cytoplasts are an effective means to study drug transport in mammalian cells without nuclear drug binding. Parental K562 cells and cytoplasts exhibit an energy-dependent accumulation of daunorubicin into cytoplasmic organelles that is also present in resistant cells and cytoplasts when P-glycoprotein mediated efflux is inhibited.

A benzophenazine derivative,N-β-dimethylaminoethyl 9-carboxy-5-hydroxy-10-methoxy-benzo[a]phenazine-6-carboxamide, as a new antitumor agent against multidrug-resistant and sensitive tumors

NC-190, a benzophenazine derivative (N-beta-dimethylaminoethyl 9-carboxy-5-hydroxy-10-methoxy-benzo[a]phenazine-6-carboxamide), was effective against multidrug-resistant human and mouse tumor cells in vitro and in vivo. When vincristine (VCR)-resistant P388 leukemia-bearing mice were treated with an optimal dose of NC-190, four of six mice were cured, whereas treatment of mice with VCR resulted in only a marginal increase in life span. The compound also showed chemotherapeutic effect against Adriamycin-resistant P388 leukemia-bearing mice and was effective against various multidrug-resistant human and murine tumor cells in vitro. Its cytotoxicity to multidrug-resistant K562 cells was not enhanced by the addition of verapamil. The accumulation of NC-190 in multidrug-resistant K562 cells was slightly lower than that observed in sensitive K562 cells; the compound did not efficiently inhibit the binding of VCR to the plasma membrane of resistant cells, indicating that NC-190 has little affinity for P-glycoprotein. NC-190 inhibited the activity of DNA topoisomerase II. These observations suggest that NC-190 (1) is not transported out of resistant cells by P-glycoprotein and (2) inhibits DNA topoisomerase II activity in the cells, resulting in its likely effectiveness against various multidrug-resistant tumor cells.

ATP/Mg2+-dependent binding of vincristine to the plasma membrane of multidrug-resistant K562 cells.

To study the mechanism of active drug efflux in multidrug-resistant cells, the interaction between [3H] vincristine (VCR) and plasma membrane prepared from an adriamycin (ADM)-resistant variant (K562/ADM) of human myelogenous leukemia K562 cells was examined by filtration method. [3H]VCR bound to the plasma membrane prepared from K562/ADM cells, but not from parental K562 cells, depending on the concentrations of ATP and Mg2+. Adenosine 5'-O-(3-thio)triphosphate was not effective in the binding of [3H]VCR, indicating that ATP hydrolysis is required for this binding. Dissociation constant (Kd) of VCR binding was 0.24 +/- 0.04 microM in the presence of 3 mM ATP. In the absence of ATP, specific binding of VCR to K562/ADM membrane was also observed; however, the affinity (Kd = 9.7 +/- 3.1 microM) was 40 times lower than that observed in the presence of ATP. The high affinity VCR binding to K562/ADM membrane was dependent on temperature. The bound [3H]VCR molecules were rapidly released by unlabeled VCR added to the reaction mixture at 25 degrees C. The high affinity binding of [3H]VCR to K562/ADM membrane was inhibited by VCR, vinblastine, actinomycin D, and ADM, to which K562/ADM cells exhibit cross-resistance, whereas 5-fluorouracil and camptothecin, to which K562/ADM cells are equally sensitive as K562 cells, did not inhibit the [3H]VCR binding. Furthermore, verapamil and other agents, which are known to circumvent drug resistance by inhibiting the active efflux of antitumor agents from resistant cells, could also inhibit the high affinity [3H]VCR binding. These results indicate that ATP/Mg2+-dependent high affinity VCR binding to the membrane of resistant cells closely correlates with the active drug efflux of this resistant cell line.