Antitumor Drug Doxorubicin Research Articles

Essential role of GATA-4 in cell survival and drug-induced cardiotoxicity

In recent years, significant progress has been made in understanding cardiomyocyte differentiation. However, little is known about the regulation of myocyte survival despite the fact that myocyte apoptosis is a leading cause of heart failure. Here we report that transcription factor GATA-4 is a survival factor for differentiated, postnatal cardiomyocytes and an upstream activator of the antiapoptotic gene Bcl-X. An early event in the cardiotoxic effect of the antitumor drug doxorubicin is GATA-4 depletion, which in turn causes cardiomyocyte apoptosis. Mouse heterozygotes for a null Gata4 allele have enhanced susceptibility to doxorubicin cardiotoxicity. Genetic or pharmacologic enhancement of GATA-4 prevents cardiomyocyte apoptosis and drug-induced cardiotoxicity. The results indicate that GATA-4 is an antiapoptotic factor required for the adaptive stress response of the adult heart. Modulation of survival/apoptosis genes by tissue-specific transcription factors may be a general paradigm that can be exploited effectively for cell-specific regulation of apoptosis in disease states.

Design, synthesis, and biological evaluation of doxorubicin-formaldehyde conjugates targeted to breast cancer cells.

The anthracycline antitumor drug doxorubicin (DOX) has been utilized for decades as a broad-spectrum chemotherapeutic. Recent literature evidence documents the role of formaldehyde in the cytotoxic mechanism, and anthracycline-formaldehyde conjugates possess substantially enhanced activity in vitro and in vivo. Targeting a doxorubicin-formaldehyde conjugate specifically to cancer cells may provide a more efficacious chemotherapeutic. The design and 11-step synthesis of doxorubicin-formaldehyde conjugates targeted to the estrogen receptor, which is commonly overexpressed in breast cancer cells, are reported. The formaldehyde is incorporated in a masked form as an N-Mannich linkage between doxorubicin and salicylamide. The salicylamide triggering molecule, previously developed to release the doxorubicin-formaldehyde active metabolite, is tethered via derivatized ethylene glycols to an E and Z mixture of 4-hydroxytamoxifen. The targeting group, E/Z-4-hydroxytamoxifen, was selected for its ability to tightly bind the estrogen receptor and antiestrogen binding sites. The targeted doxorubicin-formaldehyde conjugates' estrogen receptor binding and in vitro growth inhibition were evaluated as a function of tether length. The lead compound, DOX-TEG-TAM, bearing a triethylene glycol tether, binds the estrogen receptor with a binding affinity of 2.5% relative to E/Z-4-hydroxytamoxifen and inhibits the growth of four breast cancer cell lines with 4-fold up to 140-fold enhanced activity relative to doxorubicin.

Membrane effects of the antitumor drugs doxorubicin and thaliblastine: comparison to multidrug resistance modulators verapamil and trans-flupentixol

The interactions of the antitumor drugs doxorubicin and thaliblastine with model membranes composed of neutral (phosphatidylcholine) and negatively charged (phosphatidylserine) phospholipids were studied by differential scanning calorimetry and nuclear magnetic resonance. The membrane activities of doxorubicin and thaliblastine were compared to those of the powerful multidrug resistance (MDR) modulators trans-flupentixol and verapamil. The results point out to the potential role of the drug-membrane interactions for the effects of doxorubicin and thaliblastine in resistant tumor cells. They direct also to the artificial membranes as a suitable tool for screening of compounds with potential ability to modulate MDR.

Differential effects of catalase on apoptosis induction in human promonocytic cells. Relationships with heat-shock protein expression.

The administration of the H(2)O(2)-specific scavenger catalase attenuated the generation of apoptosis by the antitumor drugs etoposide, camptothecin, doxorubicin, and cisplatin in U-937 human promonocytic cells. By contrast, the antioxidant potentiated the generation of apoptosis by the inducers of the stress response, heat shock and cadmium, in this and other myeloid cell types. Catalase also increased the heat shock-provoked stimulation of caspase-3 and -9 activities, as well as the release of cytochrome c from mitochondria to the cytosol. The potentiation of cell death by catalase correlated with its capacity to inhibit the stress response, as demonstrated by the suppression of 70- or 27-kDa heat-shock protein expression and the inhibition of heat-shock transcription factor 1 binding activity. Conversely, the toxicity of catalase plus heat shock was attenuated when the cells were preconditioned with a soft heating, which elevated the 70-kDa heat-shock protein levels. By contrast with catalase, the antioxidants superoxide dismutase and probucol did not inhibit heat-shock protein expression or affect apoptosis in U-937 cells. Finally, it was observed that the antitumor drugs did not activate the stress response in U-937 cells and that catalase failed to inhibit HSP expression and to potentiate apoptosis in heat shock-treated RPMI 8866 lymphoblastic cells. Taken together, these results provide the first demonstration of a proapoptotic action of catalase, suggest that H(2)O(2) is a critical regulator of both apoptosis and the stress response, and corroborate the antiapoptotic action of heat-shock proteins in myeloid cells.

Selective inhibition of P-glycoprotein expression in multidrug-resistant tumor cells by a designed transcriptional regulator.

Selective inhibition of the multidrug resistance 1 (MDR1) gene and its product, the P-glycoprotein, a membrane transporter responsible for multidrug resistance, could be an important approach for enhancing cancer therapeutics. An emerging strategy for selective gene regulation involves designed zinc finger proteins that can recognize specific sequences in the promoter regions of disease-related genes. Herein, we investigate the behavior of clones of multidrug-resistant NCI/ADR-RES breast carcinoma cells displaying ponasterone-inducible expression of a designed transcriptional repressor targeted to the MDR1 promoter. The controlled production of this novel repressor resulted in major reductions in P-glycoprotein levels in these otherwise highly drug-resistant tumor cells. The regulated reduction of MDR1 expression in NCI/ADR-RES cells was accompanied by a marked increase in the rate of uptake of the P-glycoprotein substrate rhodamine 123. In addition, the cytotoxicity profile of the antitumor drug doxorubicin was dramatically altered in the induced cells compared with controls. The expression levels of other genes were examined both by a DNA array analysis of approximately 2000 genes and by biochemical techniques. Although some changes were observed in mRNA levels of nontargeted genes, the most dramatic effect by far was on MDR1, indicating that the action of the designed transcriptional repressor was quite selective. This study suggests that designed transcriptional regulators can be used to strongly and selectively influence expression of cancer-related genes, even under circumstances of extensive amplification of the target gene.

The modulation by xanthines of the DNA-damaging effect of polycyclic aromatic agents: Part II. The stacking complexes of caffeine with doxorubicin and mitoxantrone

Recently accumulated statistical data indicate the protective effect of caffeine consumption against several types of cancer diseases. There are also reports about protective effect of caffeine and other xanthines against tumors induced by polycyclic aromatic hydrocarbons. One of the explanations of this phenomenon is based on biological activation of such carcinogens by cytochromes that are also known for metabolism of caffeine. In the accompanying paper [Kapuscinski et al., this issue] we provide evidence (flow cytometry and the cell cycle analysis) that the cytostatic effects of caffeine (CAF) on two DNA alkylating agents, which do not require the biological activation, depend on their ability to form stacking (π–π) complexes. In this study, we use physicochemical techniques (computer aided light absorption and microcalorimetry), and molecular modeling to examine previously published qualitative data. This is published both by our and other group’s data, indicates that CAF is able to modify the cytotoxic and/or cytostatic action of the two well known antitumor drugs doxorubicin (DOX) and mitoxantrone (MIT). To obtain the quantitative results from the experimental data we used the statistical-thermodynamical model of mixed aggregation, to find the association constants K AC of the CAF–drug interaction (128±10 and 356±21 M −1 for DOX–CAF and MIT–CAF complex formation, respectively). In addition, the favorable enthalpy change of CAF–MIT (Δ H=−11.3 kcal/mol) was measured by microcalorimetry titration. The molecular modeling (semi-empirical and force field method) allowed us to obtain the geometry of these complexes, which indicated the favorable energy (Δ E) of complex formation of the protonated drug’s molecules in aqueous environment (−7.4 and −8.7 kcal/mol for DOX–CAF·5H 2O and MIT–CAF·8H 2O complex, respectively). The molecular modeling calculation indicates the existence of CAF–drug complexes in which the MIT molecules are intercalated between two CAF molecules (Δ E=−29.9 kcal/mol). These results indicate that the attenuating effect of caffeine on cytotoxic or mutagenic effects of some polycyclic aromatic mutagens cannot be the result of metabolic activation in the cells, but simply is the physicochemical process of the sequestering of aromatic molecules (e.g. carcinogens or mutagens) by formation of the stacking complexes. The caffeine may then act as the “interceptor” of potential carcinogens (especially in the upper part of digesting track) where its concentration can reach the mM level). There is, however, no indication, both, in the literature or from our experiments, that the xanthines can reverse the damage to nucleic acids at the point when the damage to DNA has already occurred.

Targeting of Doxorubicin to the Urinary Bladder of the Rat Shows Increased Cytotoxicity in the Bladder Urine Combined With An Absence of Renal Toxicity

Targeting of anti-tumor drugs to the urinary bladder for the treatment of bladder carcinoma may be useful, since these agents generally have a low degree of urinary excretion and are highly toxic elsewhere in the body. The anti-tumor drug doxorubicin was coupled to the low-molecular weight protein lysozyme via the acid-sensitive cis -aconityl linker. All free amino groups of the lysozyme were used for drug attachment to achieve intact excretion of the doxorubicin-aconityl-lysozyme conjugate into the bladder. In the bladder, the cytotoxic drug should be regenerated through acidification of the urine. First, the doxorubicin-aconityl-lysozyme conjugate was tested in rats for its target specificity and general toxicity. Wistar rats were injected intravenously with 2 mg/kg free doxorubicin or 10 mg/kg lysozyme-conjugated doxorubicin. Total urinary excretion of doxorubicin was about 10 times higher if the drug was coupled to lysozyme (39 ± 3% versus 4.4 ± 0.4%) . Free doxorubicin had no detectable toxic effects on heart, liver and lung but caused severe renal damage (proteinuria, N -acetyl-glucosaminidase excretion and glomerulosclerosis). None of the rats injected with doxorubicin-lysozyme conjugate showed such renal toxicity. Second, we tested whether doxorubicin could be released from the conjugate in the bladder through acidification of the urine and if the released doxorubicin could still exert a cytotoxic effect. Doxorubicin-aconityl-lysozyme (2 mg/kg conjugated doxorubicin, i.v.) was administered in rats with acidified urine (pH 6.1 ± 0.1) and in rats with a high urinary pH (8.2 ± 0.4). Ten times more doxorubicin was released from the conjugate in the group with acidified urine (15 ± 7% versus 1.7 ± 0.1%) . In agreement with this, cytotoxicity was also higher in the low pH group (IC 50 of 255 ± 47 nM versus 684 ± 84 nM doxorubicin). In conclusion, a specific delivery of doxorubicin to the urinary bladder combined with a reduced toxicity of doxorubicin in the kidneys can be achieved by coupling this anti-tumor drug to the low-molecular weight protein lysozyme via an acid-labile linker. A release of cytotoxic doxorubicin in the urinary bladder can be achieved by acidification of the urine. This technology, after further optimization, may provide an interesting tool for the treatment of bladder carcinoma.

Doxorubicin-induced Apoptosis Is Associated with Increased Transcription of Endothelial Nitric-oxide Synthase: EFFECT OF ANTIAPOPTOTIC ANTIOXIDANTS AND CALCIUM

The clinical efficacy of the antitumor antibiotic drug doxorubicin (DOX) is severely limited by its dose-limiting cardiotoxicity in cancer patients. DOX-induced generation of reactive oxygen species was proposed to be a major mechanism of its cardiotoxicity. Previously, we showed that DOX undergoes a reductive activation at the reductase domain of endothelial nitric-oxide synthase (eNOS) forming the semiquinone and superoxide (Vásquez-Vivar, J., Martasek, P., Hogg, N., Masters, B. S. S., Pritchard, K. A., Jr., and Kalyanaraman, B. (1997) Biochemistry 36, 11293-11297). In this report, we provide evidence for DOX-induced increase in eNOS transcription and protein expression in bovine aortic endothelial cells (BAEC). We propose that DOX-induced hydrogen peroxide formation is responsible for the increased transcription of eNOS. BAEC treated with antisense eNOS oligonucleotide inhibits DOX-induced endothelial apoptosis. Treatment with antioxidants restored the levels of antiapoptotic proteins (Hsp70 and Bcl-2) in DOX-treated BAEC. DOX-induced intracellular oxidative stress, as measured by oxidation of dichlorodihydrofluorescein diacetate to dichlorofluorescein and hydroethidium to ethidium, was inhibited by antisense eNOS oligonucleotide and antioxidant treatment. Furthermore, antiapoptotic antioxidants (e.g. FeTBAP, ebselen, and alpha-phenyl-tert-butyl nitrone) inhibited DOX-induced eNOS transcription. We conclude that DOX-induced apoptosis is linked to the redox activation of DOX by eNOS.

Mass spectrometric measurement of formaldehyde generated in breast cancer cells upon treatment with anthracycline antitumor drugs.

Selected ion flow tube-chemical ionization mass spectrometry was used to measure formaldehyde levels in human breast cancer cells in comparison with levels in cells treated with the antitumor drugs doxorubicin (DOX) and daunorubicin (DAU) and the daunorubicin-formaldehyde conjugate Daunoform (DAUF). The measurement was performed on cell lysates and showed only background levels of formaldehyde in untreated cells and drug-treated resistant cells (MCF-7/Adr cells) but levels above background in DOX- and DAU-treated sensitive cells (MCF-7 cells). The level of formaldehyde above background was a function of drug concentration (0.5-50 microM), treatment time (3-24 h), cell density (0.3 x 10(6) to 7 x 10(6) cells/mL), and cell viability (0-100%). Higher levels of formaldehyde were observed in lysates of MCF-7 cells treated at higher drug levels, unless the treatment resulted in low cell viability. Elevated levels were directly related to cell density and were observed even with 0.5 microM drug. A lower limit for excess formaldehyde in MCF-7 cells treated with 0.5 microM DAU for 24 h is 0.3 mM. Control experiments showed that formaldehyde was not produced after cell lysis. Lysates of sensitive and resistant cells treated with 0.5 micromolar equiv of the formaldehyde conjugate (DAUF) for 3 h showed only background levels of formaldehyde. The results support a mechanism for drug cytotoxicity which involves drug induction of metabolic processes leading to formaldehyde production followed by drug utilization of formaldehyde to virtually cross-link DNA.

A two-plasmid system for the glycosylation of polyketide antibiotics: bioconversion of epsilon-rhodomycinone to rhodomycin D.

The biological activity of many microbial products requires the presence of one or more deoxysugar molecules attached to agylcone. This is especially prevalent among polyketides and is an important reason that the antitumor anthracycline antibiotics are avid DNA-binding drugs. The ability to make different deoxyaminosugars and attach them to the same or different aglycones in vivo would facilitate the synthesis of new anthracyclines and the quest for antitumor drugs. This is feasible using the numerous bacterial genes for deoxysugar biosynthesis that are now available. Production of thymidine diphospho (TDP)-L-daunosamine (dnm), the aminodeoxysugar present in the anthracycline antitumor drugs daunorubicin (DNR) and doxorubicin (DXR), and its attachment to epsilon-rhodomycinone to generate rhodomycin D has been achieved by bioconversion with a strain of Streptomyces lividans that bears two plasmids. One contained the Streptomyces peucetius dnmJVUZTQS genes plus dnmW (previously named dpsH and considered to be a polyketide cyclase gene), dnrH, which is not required for the formation of rhodomycin D, and dnrI, a regulatory gene required for expression of the dnm and drr genes. The other plasmid had genes encoding glucose-1-phosphate thymidylyltransferase and TDP-glucose-4,6-dehydratase (dnmL and dnmM, respectively, or mtmDE, their homologs from Streptomyces agrillaceus) plus the drrAB DNR/DXR resistance genes. The high-yielding glycosylation of the aromatic polyketide epsilon-rhodomycinone using plasmid-borne deoxysugar biosynthesis genes proves that the minimal information for L-daunosamine biosynthesis and attachment in the heterologous host is encoded by the dnmLMJVUTS genes. This is a general approach to making both known and new glycosides of anthracyclines, several of which have medically important antitumor activity.

Nuclear targeting and nuclear retention of anthracycline-formaldehyde conjugates implicates DNA covalent bonding in the cytotoxic mechanism of anthracyclines.

The anthracycline, antitumor drugs doxorubicin (DOX), daunorubicin (DAU), and epidoxorubicin (EPI) catalyze production of formaldehyde through induction of oxidative stress. The formaldehyde then mediates covalent bonding of the drugs to DNA. Synthetic formaldehyde conjugates of DOX, DAU, and EPI, denoted Doxoform (DOXF), Daunoform (DAUF), and Epidoxoform (EPIF), exhibit enhanced toxicity to anthracycline-sensitive and -resistant tumor cells. Uptake and retention of parent anthracycline antitumor drugs (DOX, DAU, and EPI) relative to those of their formaldehyde conjugates (DOXF, DAUF, and EPIF) were assessed by flow cytometry in both drug-sensitive MCF-7 cells and drug-resistant MCF-7/ADR cells. The MCF-7 cells took up more than twice as much drug as the MCF-7/ADR cells, and both cell lines took up substantially more of the formaldehyde conjugates than the parent drugs. Both MCF-7 and MCF-7/ADR cells retained fluorophore from DOXF, DAUF, and EPIF hours after drug removal, while both cell lines almost completely expelled DOX, DAU, and EPI within 1 h. Longer treatment with DOX, DAU, and EPI resulted in modest drug retention in MCF-7 cells following drug removal but poor retention of DOX, DAU, and EPI in MCF-7/ADR cells. Fluorescence microscopy showed that the formaldehyde conjugates targeted the nuclei of both sensitive and resistant cells, and remained in the nucleus hours after drug removal. Experiments in which [(3)H]Doxoform was used, synthesized from doxorubicin and [(3)H]formaldehyde, also indicated that Doxoform targeted the nucleus. Elevated levels of (3)H were observed in DNA isolated from [(3)H]Doxoform-treated MCF-7 and MCF-7/ADR cells relative to controls. The results implicate drug-DNA covalent bonding in the tumor cell toxicity mechanism of these anthracyclines.

Evaluation of partition coefficients of low molecular weight solutes between water and micelles of block copolymer of ethylene oxide based on dialysis kinetics and fluorescence spectroscopy

Application of micelle-forming surfactants in various fields of technology and use of micelles as carriers for drug targeting requires simple evaluation of partitioning coefficients of different solutes between water solution and micellar microphase. A number of methods of partitioning evaluation have been already proposed, but most of them are applicable only for fluorescent dyes or require complicated and expensive equipment, such as NMR spectrometers. In the present work two methods of the molar fraction of solubilized material determination are compared. One of the techniques used is based on the changes in fluorescence spectra during solubilization. The other approach is based on the dialysis slowing down, which occurs if the solute is associated with large particles (e.g. micelles). Graphic method of partition coefficient (P) evaluation is proposed and P values of a widely used anti-tumor drug doxorubicin and highly hydrophobic fluorophore perylene are determined. The comparison shows that the results of both methods give very close coincidence. The solubilization data were used for the calculation of the important parameter – the ratio of micelle volume to aggregation number, which makes sense of packing density of the detergent molecules in the micelle. The comparison of this parameter calculated from the solubilization data with that obtained from light scattering experiments is discussed. It is assumed that the dialysis technique could be used for partitioning evaluation of all substances, which may be detected in solution.

Molar Mass Distribution of a Polymer-Drug Conjugate Containing the Antitumor Drug Paclitaxel by Size Exclusion Chromatography and Universal Calibration

The development of a Size Exclusion Chromatography (SEC) method for the characterization of the polymer-drug conjugate PNU 166945 is presented. PNU 166945 is a conjugate between poly[N-(2-hydroxypropyl) methacrylamide)] and the antitumor drug paclitaxel. This study follows a previous paper on a similar polymer conjugate, FCE 28068, containing the same polymeric carrier and the antitumor drug doxorubicin. The aim was an accurate and reproducible, yet relatively simple and rapid method for the routine quality control of production batches. Thirteen PNU 166945 narrow fractions were separated, by semi-preparative SEC, and characterized by on-line SEC-Viscometry and off-line Light Scattering. The fractions allowed us to verify that PNU 166945 follows the universal calibration constructed with PEO/PEG narrow standards. So a conventional SEC method that utilizes just a refractive index detector and universal calibration with commercial narrow standards was found suitable. The study also reports data on accuracy and reproducibility of the SEC measurement.

The Streptomyces peucetius dpsY and dnrX genes govern early and late steps of daunorubicin and doxorubicin biosynthesis.

The Streptomyces peucetius dpsY and dnrX genes govern early and late steps in the biosynthesis of the clinically valuable antitumor drugs daunorubicin (DNR) and doxorubicin (DXR). Although their deduced products resemble those of genes thought to be involved in antibiotic production in several other bacteria, this information could not be used to identify the functions of dpsY and dnrX. Replacement of dpsY with a mutant form disrupted by insertion of the aphII neomycin-kanamycin resistance gene resulted in the accumulation of UWM5, the C-19 ethyl homolog of SEK43, a known shunt product of iterative polyketide synthases involved in the biosynthesis of aromatic polyketides. Hence, DpsY must act along with the other components of the DNR-DXR polyketide synthase to form 12-deoxyaklanonic acid, the earliest known intermediate of the DXR pathway. Mutation of dnrX in the same way resulted in a threefold increase in DXR production and the disappearance of two acid-sensitive, unknown compounds from culture extracts. These results suggest that dnrX, analogous to the role of the S. peucetius dnrH gene (C. Scotti and C. R. Hutchinson, J. Bacteriol. 178:73167321, 1996), may be involved in the metabolism of DNR and/or DXR to acid-sensitive compounds, possibly related to the baumycins found in many DNR-producing bacteria.

Identification of yeast DNA topoisomerase II mutants resistant to the antitumor drug doxorubicin: implications for the mechanisms of doxorubicin action and cytotoxicity.

Doxorubicin is a therapeutically useful anticancer drug that exerts multiple biological effects. Its antitumor and cardiotoxic properties have been ascribed to anthracycline-mediated free radical damage to DNA and membranes. Evidence for this idea comes in part from the selection by doxorubicin from stationary phase yeast cells of mutants (petites) deficient in mitochondrial respiration and therefore defective in free radical generation. However, doxorubicin also binds to DNA topoisomerase II, converting the enzyme into a DNA damaging agent through the trapping of a covalent enzyme-DNA complex termed the 'cleavable complex.' We have used yeast to determine whether stabilization of cleavable complexes plays a role in doxorubicin action and cytotoxicity. A plasmid-borne yeast TOP2 gene was mutagenized with hydroxylamine and used to transform drug-permeable yeast strain JN394t2-4, which carries a temperature-sensitive top2-4 mutation in its chromosomal TOP2 gene. Selection in growth medium at the nonpermissive temperature of 35 degrees in the presence of doxorubicin resulted in the isolation of plasmid-borne top2 mutants specifying functional doxorubicin-resistant DNA topoisomerase II. Single-point changes of Gly748 to Glu or Ala642 to Ser in yeast topoisomerase II, which lie in and adjacent to the CAP-like DNA binding domain, respectively, were identified as responsible for resistance to doxorubicin, implicating these regions in drug action. None of the mutants selected in JN394t2-4, which has a rad52 defect in double-strand DNA break repair, was respiration-deficient. We conclude that topoisomerase II is an intracellular target for doxorubicin and that the genetic background and/or cell proliferation status can determine the relative importance of topoisomerase II- versus free radical-killing.

Doxoform and Daunoform: anthracycline-formaldehyde conjugates toxic to resistant tumor cells.

The recent discovery that the clinically important antitumor drugs doxorubicin and daunorubicin alkylate DNA via catalytic production of formaldehyde prompted the synthesis of derivatives bearing formaldehyde. Reaction of the parent drugs with aqueous formaldehyde at pH 6 produced in 40-50% yield conjugates consisting of two molecules of the parent drug as oxazolidine derivatives bound together at their 3'-nitrogens by a methylene group. The structures were established as bis(3'-N-(3'-N,4'-O-methylenedoxorubicinyl)) methane (Doxoform) and bis(3'-N-(3'-N,4'-O-methylenedaunorubicinyl))methane (Daunoform) from spectroscopic data. Both derivatives are labile with respect to hydrolysis to the parent drugs. 3'-N,4'-O-Methylenedoxorubicin and 3'-N,4'-O-methylenedaunorubicin are intermediates in the hydrolysis. Daunoform reacts with the self-complementary deoxyoligonucleotide (GC)4 faster than the combination of daunorubicin and formaldehyde at an equivalent concentration to given drug-DNA adducts. In spite of hydrolytic instability, Doxoform is 150-fold more toxic to MCF-7 human breast cancer cells and 10000-fold more toxic to MCF-7/ADR resistant cells. Toxicity to resistant cancer cells is interpreted in terms of higher lipophilicity of the derivatives and circumvention of catalytic formaldehyde production.

Fe‐EDTA‐Bisamide and Fe‐ADR‐925, The Iron‐Bound Hydrolysis Product of the Cardioprotective Agent Dexrazoxane, Cleave DNA Via the Hydroxyl Radical

Use of the antitumor drug doxorubicin is limited by cardiomyopathic side-effects which are believed to be due to iron-mediated hydroxyl radical generation. Dexrazoxane reduces this cardiotoxicity, possibly by removal of iron from doxorubicin by the EDTA-like hydrolysis product of dexrazoxane, ADR-925. However, EDTA-diimides like dexrazoxane, previously used as antitumor agents, are themselves carcinogenic, and recent studies have found that Fe-ADR-925 can also promote hydroxyl radical production. This study demonstrates that, like Fe-EDTA, Fe-ADR-925 and a related desmethyl complex can cleave plasmid DNA under Fenton conditions, and suggests by radical scavenger study that this cleavage is probably via the hydroxyl radical. Differences in DNA cleavage dependence upon concentrations of Fe-EDTA, Fe-ADR-925 and Fe-EDTA-bisamide can be explained by differences in the solution chemistry of the complexes.

Molecular Characterization of Polymeric Antitumor Drug Carriers by Size Exclusion Chromatography and Universal Calibration

Abstract The development of a Size Exclusion Chromatography (SEC) method for the molecular characterization of FCE 28068, a conjugate between a synthetic polymeric carrier and the antitumor drug doxorubicin is presented. The aim was an accurate and reproducible, yet relatively simple and rapid method for the routine quality control of production batches. A standard SEC method that utilizes just a refractive index detector and relies upon universal calibration with commercial narrow standards was found suitable. The development of this method implied the following steps: test of universal calibration in the chosen experimental conditions with commercial narrow standards; fractionation of FCE 28068; test of compliance for the FCE 28068 fractions to universal calibration conditions; test of accuracy and reproducibility of the SEC measurement. The use of off-line light scattering and viscometry for the molecular characterization of standards and of FCE 28068 is discussed.

Volume-sensitive chloride currents in four epithelial cell lines are not directly correlated to the expression of the MDR-1 gene.

It has been shown recently that heterologous expression of human MDR-1 gene, which is responsible for multidrug resistance during cancer therapy, causes appearance of volume-sensitive Cl- currents, thus suggesting that the product of the MDR-1 gene (the P-glycoprotein) has a Cl- channel activity (Valverde, M. A., Diaz, M., Sepulveda, M. A., Gill, D. R., Hyde, S. C., and Higgins, C. F. (1992) Nature 355, 830-833). In the present work, we have tested four epithelial cell lines both for the expression of MDR-1 gene and for the presence of volume-sensitive Cl- currents. LoVo/H and LoVo/Dx cells derive from a human colon adenocarcinoma, the latter cell line being resistant to high concentrations of the antitumoral drug doxorubicin. 9HTEo- cells were obtained by transformation of human tracheal epithelium. The 9HTEo-/Dx cell line was established from these cells by selection in doxorubicin. As expected, higher levels of P-glycoprotein expression were detected in LoVo/Dx and 9HTEo-/Dx by means of reverse transcriptase polymerase chain reaction technique, indirect immunofluorescence, and Western immunoblot assays. In contrast with these data, the size of swelling-induced Cl- current was the same in the sensitive cell line and in its drug-resistant counterpart. Actually, the Cl- conductance of 9HTEo- and 9HTEo-/Dx was 4-fold higher than that of either LoVo/H or LoVo/Dx cells. This indicates that the amplitude of this conductance is not directly related to the expression of the MDR-1 gene.

Which Liposomes to Bypass Multidrug Resistance? A Study of Doxorubicin-Loaded Vesicles

AbstractLiposomes used as antitumoral drug carriers have recently been designated as potential tools to overcome multidrug resistance. In order to understand better the mechanism of such an effect, we have investigated the capacity of liposomes exhibiting a pH gradient to trap efficiently the antitumoral drug doxorubicin in conditions of high dilution such as those used for cell cultures treatment. A simple calculation described the transmembrane pH gradient and the thermodynamic equilibrium of the neutral form, on both sides of the membrane. It showed that liposomes, even efficiently loaded with doxorubicin in response to the pH gradient will lose most of their content upon dilution because of the neutral form physicochemical gradient. Using fluorescence properties of the drug, we found that liposomes made of egg yolk phosphatidylcholine (EPC), phosphatidyl serine (PS) and cholesterol in the ratio 10:1:4 rather closely fitted the situation predicted by the calculation and that the equilibrium state after...