Inhibitory effect of mitoxantrone on activity of protein kinase C and growth of HL60 cells.

Mitoxantrone, a new anthraquinone, showed inhibitory an effect on protein kinase C (PKC) activity. Its IC50 value was 4.4 micrograms/ml (8.5 microM), which is much lower than those of the well-known anthracyclines daunorubicin and doxorubicin, the IC50 values of which are more than 100 micrograms/ml (> 170 microM). Kinetic studies demonstrated that mitoxantrone inhibited PKC in a competitive manner with respect to histone H1, and its Ki value was 6.3 microM (Ki values of daunorubicin and doxorubicin were 0.89 and 0.15 mM, respectively), and in a non-competitive manner with respect to phosphatidylserine and ATP. Inhibition of phosphorylation by mitoxantrone was observed with various substrates including S6 peptide, myelin basic protein and its peptide substrate derived from the amino-terminal region. Their IC50 values were 0.49 microgram/ml (0.95 microM), 1.8 micrograms/ml (3.5 microM), and 0.82 microgram/ml (1.6 microM), respectively. Mitoxantrone did not markedly inhibit the activity of cyclic AMP-dependent protein kinase, casein kinase I or casein kinase II, at concentrations of less than 10 micrograms/ml. On the other hand, brief exposure (5 min) of HL60 cells to mitoxantrone caused the inhibition of cell growth with an IC50 value of 52 ng/ml (0.1 microM). In HL60 cells, most of the PKC activity (about 90%) was detected in the cytosolic fraction. When HL60 cells exposed to 10 micrograms/ml mitoxantrone for 5 min were observed with fluorescence microscopy, the fluorescence elicited from mitoxantrone was detected in the extranuclear area. These results indicated that mitoxantrone is a potent inhibitor of PKC, and this inhibition may be one of the mechanisms of antitumor activity of mitoxantrone.     

Attenuation of daunorubicin-augmented microsomal lipid peroxidation and oxygen consumption by calcium channel antagonists.

Daunorubicin (20 microM) stimulated NADPH-dependent microsomal lipid peroxidation about 2-fold over control values and enhanced the rate of oxygen utilization by microsomes. The calcium channel blockers tested inhibited daunorubicin-augmented lipid peroxidation and O2 consumption to varying degrees. Inhibition of daunorubicin-stimulated lipid peroxidation was found to be dose dependent; the IC50 (drug concentration producing 50% inhibition of lipid peroxidation) values for verapamil, nifedipine and diltiazem were approximately 150 microM, 200 microM, and 600 microM respectively. Our in vitro studies suggest that calcium channel antagonists may modulate the free radical-mediated, cardiotoxic effects of daunorubicin.     

Redox cycling of anthracyclines by cardiac mitochondria. I. Anthracycline radical formation by NADH dehydrogenase

In the present study we have used beef heart submitochondrial preparations (BH-SMP) to demonstrate that a component of mitochondrial Complex I, probably the NADH dehydrogenase flavin, is the mitochondrial site of anthracycline reduction. During forward electron transport, the anthracyclines doxorubicin (Adriamycin) and daunorubicin acted as one-electron acceptors for BH-SMP (i.e. were reduced to semiquinone radical species) only when NADH was used as substrate; succinate and ascorbate were without effect. Inhibitor experiments (rotenone, amytal, piericidin A) indicated that the anthracycline reduction site lies on the substrate side of ubiquinone. Doxorubicin and daunorubicin semiquinone radicals were readily detected by ESR spectroscopy. Doxorubicin and daunorubicin semiquinone radicals (g congruent to 2.004, signal width congruent to 4.5 G) reacted avidly with molecular oxygen, presumably to produce O2-, to complete the redox cycle. The identification of Complex I as the site of anthracycline reduction was confirmed by studies of ATP-energized reverse electron transport using succinate or ascorbate as substrates, in the presence of antimycin A or KCN respiratory blocks. Doxorubicin and daunorubicin inhibited the reduction of NAD+ to NADH during reverse electron transport. Furthermore, during reverse electron transport in the absence of added NAD+, doxorubicin and daunorubicin addition caused oxygen consumption due to reduction of molecular oxygen (to O2-) by the anthracycline semiquinone radicals. With succinate as electron source both thenoyltrifluoroacetone (an inhibitor of Complex II) and rotenone blocked oxygen consumption, but with ascorbate as electron source only rotenone was an effective inhibitor. NADH oxidation by doxorubicin during BH-SMP forward electron transport had a KM of 99 microM and a Vmax of 30 nmol X min-1 X mg-1 (at pH 7.4 and 23 degrees C); values for daunorubicin were 71 microM and 37 nmol X min-1 X mg-1. Oxygen consumption at pH 7.2 and 37 degrees C exhibited KM values of 65 microM for doxorubicin and 47 microM for daunorubicin, and Vmax values of 116 nmol X min-1 X mg-1 for doxorubicin and 114 nmol X min-1 X mg-1 for daunorubicin. In marked contrast with these results, 5-iminodaunodrubicin (a new anthracycline with diminished cardiotoxic potential) exhibited little or no tendency to undergo reduction, or to redox cycle with BH-SMP. Redox cycling of anthracyclines by mitochondrial NADH dehydrogenase is shown, in the accompanying paper (Doroshow, J. H., and Davies, K. J. A. (1986) J. Biol. Chem. 261, 3068-3074), to generate O2-, H2O2, and OH which may underlie the cardiotoxicity of these antitumor agents.     

Inhibition study of rabbit liver cytosolic reductases involved in daunorubicin toxication

Anthracycline cardiotoxicity represents the most unfavorable side effect of these highly efficient anticancer drugs. Several biotransformation enzymes have been described to contribute to their cardiotoxicity. Besides the activities of CYP450 isoforms which lead to the generation of reactive oxygen species (ROS), the cytosolic reductases have attracted attention nowadays. The reductases known to metabolize anthracyclines to C13-hydroxyanthracyclines are carbonyl reductase (CR, 1.1.1.184) and the aldo-keto reductases (AKR1C2, 1.3.1.20; AKR1A1, 1.1.1.2). Their participation in the formation of the toxic C13-hydroxymetabolite has been investigated in rabbit using diagnostic inhibitors of CR and AKR1C2. The kinetics and the type of reductase inhibition exerted by the two inhibitors have been described and it was found that CR was the main daunorubicin reductase at both optimal and physiological pH with the kinetic parameters for daunorubicin reduction of Km = 17.01 +/- 1.98 microM and V(max) = 139.60 +/- 5.64 pcat/mg. The IC50 values for quercitrin and flufenamic acid were 5.45 +/- 1.37 microM and 3.68 +/- 1.58 microM, respectively. The inhibition was uncompetitive for both inhibitors and irreversible in the case of flufenamic acid.     

Synthetic tyrosyl gallate derivatives as potent melanin formation inhibitors

Three tyrosyl gallate derivatives (1-3) with variable hydroxyl substituent at the aromatic ring of tyrosol were synthesized and evaluated as potent inhibitors on tyrosinase activity and melanin formation in melan-a cells. Among three tyrosyl gallate derivatives, 4-hydroxyphenethyl 3,4,5-trihydroxybenote (1) (IC(50)=4.93 microM), 3-hydroxyphenethyl 3,4,5-trihydroxybenote (2) (IC(50)=15.21 microM), and 2-hydroxyphenethyl 3,4,5-trihydroxybenote (3) (IC(50)=14.50 microM) exhibited significant inhibitory effect on tyrosinase activity. Compound 1 was the most active compound, though it did not show the inhibitory effect on melanin formation in melan-a cells. However, compounds 2 (IC(50)=8.94 microM) and 3 (IC(50)=13.67 microM) significantly suppressed the cellular melanin formation without cytotoxicity. This study shows that the position of hydroxyl substituent at the aromatic ring of tyrosol plays an important role in the intracellular regulation of melanin formation in cell-based assay system.     

Syncatalytic inactivation of prolyl 4-hydroxylase by anthracyclines.

The anthracyclines doxorubicin and daunorubicin were found to act as irreversible inhibitors of prolyl 4-hydroxylase. The reaction rate for enzyme from both chick and human origin was first order, the concentration of inhibitor giving 50% inhibition being 60 microM for both compounds after 1 h. The effect was dependent on the presence of iron ions in the reaction mixture. Inactivation could be prevented by addition of high concentrations of ascorbate, but not 2-oxoglutarate, before the inactivation period. The same results were obtained with competitive analogues of these cosubstrates. Lysyl hydroxylase from chick embryos was also susceptible to inactivation. Its activity was decreased by 50% after incubation for 1 h with a 150 microM concentration of the inhibitors. When chick-embryo prolyl 4-hydroxylase was incubated with [14-14C]doxorubicin, both enzyme subunits were radioactively labelled, about 70% of the total radioactivity being found in the alpha-subunit. Since the anthracyclines are known to undergo a redox reaction generating semiquinone radicals with Fe3+ only, the results suggest that the enzyme-bound iron ion is oxidized to a tervalent intermediate in uncoupled reaction cycles. The data also suggest that both enzyme subunits contribute to the catalytic site of prolyl 4-hydroxylase.     

Melanin counter act puromycin-induced inhibition of collagen and DNA biosynthesis in human skin fibroblasts

Puromycin is an experimental anti-tumor antibiotic acting through inhibition of protein synthesis. Because of its untoward side effects (as inner ear and renal lesions) the antibiotic was not approved for clinical trials. The mechanism underlying the organ specificity of the side effect is not understood. In view of the fact that a number of drugs form with melanin complexes that affect their pharmacological activity, we determined whether puromycin interacts with melanin and how this process affects biosynthesis of collagen in cultured human skin fibroblasts. Our results indicate that puromycin forms complexes with melanin. The amount of puromycin bound to melanin increases with increase of initial drug concentration. The Scatchard plot analysis of the drug binding to melanin has shown that at least two classes of independent binding sites are implicated in the puromycin-melanin complex formation: one class of strong binding sites with the association constant K1 = 1.84 x 10(6) M(-1), and the second class of weak binding sites with the association constant K2 = 5.26 x 10(3) M(-1). The number of total binding sites were n1 = 0.1260 and n2 = 0.2861 mumol puromycin per 1 mg melanin. We found that puromycin induced inhibition of collagen and DNA biosynthesis (IC50 approximately 2 microM). Melanin at 100 microg/ml produced about 20% inhibition of DNA synthesis, but it had no effect on collagen biosynthesis in cultured fibroblasts. However, the addition of melanin (100 microg/ml) to puromycin - treated cells (2 microM) abolished the inhibitory action of puromycin on collagen and DNA biosynthesis. We have suggested that IGF-I receptor expression, involved in collagen metabolism, may be one of the targets for puromycin - induced inhibition of collagen biosynthesis. It was found that melanin abolished puromycin induced decrease in the expression of IGF-I receptor as well MAP kinases expression: ERK1 and ERK2 as shown by Western immunoblot analysis. These data suggest that tissue specific pharmacological activity of puromycin may depend on the melanin abundance in tissues.     

Flavonoids as inhibitors of human carbonyl reductase 1

Human carbonyl reductase 1 (CBR1), that is one of the enzymes responsible for the reduced efficiency of treatments by the antineoplastic agents anthracyclines, was functionally expressed in Saccharomyces cerevisiae. CBR1 was purified and kinetically characterised using daunorubicin as substrate. CBR1-catalysed reduction of daunorubicin followed an apparent Michaelis-Menten kinetics with K(M)=85.2+/-26.7microM and V(max)=3490+/-220micromol/(mingprotein). The type of inhibition for the flavonoid compound rutin was determined by studying initial reaction rates in the presence of rutin. The inhibition kinetics was found to follow an apparent mixed inhibition with K(ic)=1.8+/-1.2microM and K(iu)=2.8+/-1.6microM. IC50-values were also determined for a set of flavonoids in order to identify essential structure for inhibition activity. Computational docking experiments of the four best inhibitors to the catalytic site of CBR1 showed that the flavonoid skeleton structure was the binding part of the molecule. The presence of a sugar moiety in 1 and 2, or a sugar mimicking part in 9, directed the orientation of the flavonoid so that the sugars were pointing outwards, giving rise to a stabilising effect to the binding. Finally, additional binding epitopes that interacted with various parts of the flavonoid ligand were identified and could potentially be targeted for further improvement of inhibition activity. These included; hydrogen-binding sites surrounding Ser139 and Cys226, Met234 and Tyr193 or Trp229; aromatic-aromatic interaction with Tyr193, Trp229 or NADPH; van der Waals interactions with Ile140.     

The effect of new lipophilic chelators on the activities of cytosolic reductases and P450 cytochromes involved in the metabolism of anthracycline antibiotics: studies in vitro

A major obstacle to the therapeutic use of anthracyclines, highly effective anticancer agents, is the fact that their administration results in dose-dependent cardiomyopathy. According to the currently accepted hypothesis, anthracyclines injure the heart by generating oxygen free radicals. The ability of pyridoxal isonicotinoyl hydrazone (PIH) and salicylaldehyde isonicotinoyl hydrazone (SIH) -- new iron chelators -- to protect against peroxidation as well as their suitable biological, physical and chemical properties make the compounds promising candidates for pre-clinical and clinical studies. Activities of carbonyl reductase CR (1.1.1.184), dihydrodiol dehydrogenase DD2 (1.3.1.20), aldehyde reductase ALR1 (1.1.1.2) and P450 isoenzymes (CYP1A1, CYP1A2, CYP2B, CYP3A) involved in the metabolism of daunorubicin, doxorubicin and other drugs or xenobiotics were studied. Various concentrations of the chelators were used either alone or together with daunorubicin or doxorubicin for in vitro studies in isolated hepatocytes. A significant decrease of activity was observed for all enzymes only at PIH and SIH concentrations higher than those presumed to be used for therapy. The results show that PIH and SIH have no effect on the activities of the enzymes studied in vitro and allow us to believe that they will not interfere with the metabolism of co-administered drugs and other xenobiotics. Daunorubicin (Da) and doxorubicin (Dx) significantly reduce cytochrome P450 activity, but the addition of SIH and PIH chelators (50 microM) reverses the reduction and restores the activity to 70-90 % of the activity of relevant controls.     

Calmodulin inhibitor trifluoperazine selectively enhances cytotoxic effects of strong vs weak DNA binding antitumor drugs in doxorubicin-resistant P388 mouse leukemia cells

Doxorubicin-resistant P388 mouse leukemia cells are cross-resistant to anthracycline and non-anthracycline DNA intercalators as well as to natural and semisynthetic anthracyclines which bind weakly or not at all to DNA. In the presence of a non-lethal concentration of 5 microM trifluoperazine cytotoxic effects of the strong DNA binding drugs actinomycin-D, mitoxantrone and m-AMSA were enhanced less than 2 fold in doxorubicin-sensitive cells and up to 50 fold in doxorubicin-resistant cells. Additionally, trifluoperazine induced a greater than 2-fold enhancement in the cytotoxic effects (but not accumulation and retention) of the strong DNA binder N,N-dimethyladriamycin-14-valerate only in doxorubicin resistant cells. In contrast, cell kill, drug accumulation and retention in P388/S and P388/DOX cells treated with the weak DNA binders N-benzyl-adriamycin-14-valerate and 7(R)-O-methylnogarol, and DNA-nonbinding N,N-dibenzyldaunorubicin was similar with or without trifluoperazine treatment. The study demonstrates that the calmodulin inhibitor trifluoperazine induces a specific and marked enhancement in the cytotoxic effects of strong vs weak DNA binding antitumor drugs in doxorubicin-resistant cells.     

Acetaminophen stimulates the peroxidative metabolism of anthracyclines

Acetaminophen, a common analgesic and antipyretic drug, is frequently administered to individuals undergoing anthracycline chemotherapy. Here, the effect of acetaminophen on the metabolism of daunorubicin and doxorubicin by isolated enzymes lactoperoxidase and myeloperoxidase, and by myeloperoxidase-containing human leukemia HL-60 cells was investigated using spectrophotometric and EPR techniques. We report that at pharmacological concentrations acetaminophen strongly stimulates oxidation of the anthracyclines by lactoperoxidase and myeloperoxidase systems, which results in irreversibly altered (colorless) products. The initial rate and efficacy of daunorubicin oxidation depends on pH. While at pH approximately 7 the oxidation is rapid and extensive, almost no oxidation occurs at pH approximately 5. In the absence of daunorubicin, oxidation of acetaminophen by lactoperoxidase/hydrogen peroxide is only weakly dependent on pH, however, at pH 7.4 it strongly depends on [daunorubicin]. Ascorbate and reduced glutathione strongly inhibited oxidation of anthracyclines by lactoperoxidase and HL-60 systems. Using EPR, a daunorubicin-derived radical was detected in a daunorubicin/acetaminophen/peroxidase/hydrogen peroxide system as a narrow single line (0.175 mT) with g = 2.0047. When daunorubicin was omitted, only an acetaminophen-melanin EPR signal was detected (g = 2.0043, line width approximately 0.5 mT). Similar results were obtained with doxorubicin. We suggest that the stimulation by acetaminophen is primarily due to its preferential oxidation by peroxidases to the corresponding phenoxyl radical, which subsequently reacts with daunorubicin (doxorubicin). Because biological properties of oxidatively transformed anthracyclines will certainly be different from those of their parent compounds, the possible acetaminophen-enhanced degradation of the anthracyclines in vivo is likely to interfere with anticancer and/or cardiotoxic activities of these agents.     

Sequence-selective topoisomerase II inhibition by anthracycline derivatives in SV40 DNA: relationship with DNA binding affinity and cytotoxicity

Topoisomerase II mediated double-strand breaks produced by anthracycline analogues were studied in SV40 DNA. The compounds included doxorubicin, daunorubicin, two doxorubicin stereoisomers (4'-epimer and beta-anomer), and five chromophore-modified derivatives, with a wide range of cytotoxic activity and DNA binding affinity. Cleavage of 32P-end-labeled DNA fragments was visualized by autoradiography of agarose and polyacrylamide gels. Structure-activity relationships indicated that alterations in the chromophore structure greatly affected drug action on topoisomerase II. In particular, removal of substituents on position 4 of the D ring resulted in more active inducers of cleavage with lower DNA binding affinity. The stereochemistry between the sugar and the chromophore was also essential for activity. All the active anthracyclines induced a single region of prominent cleavage in the entire SV40 DNA, which resulted from a cluster of sites between nucleotides 4237 and 4294. DNA cleavage intensity patterns exhibited differences among analogues and were also dependent upon drug concentration. Intensity at a given site depended on both stimulatory and suppressive effects depending upon drug concentration and DNA sequence. A good correlation was found between cytotoxicity and intensity of topoisomerase II mediated DNA breakage.     

Pterocarpans and flavanones from Sophora flavescens displaying potent neuraminidase inhibition

Pterocarpans (1-3) and flavanones (4-10) were isolated from Sophora flavescens and screened for their ability to inhibit neuraminidase (an enzyme crucial in the proliferation of the influenza virus). The majority of inhibitors were shown to have IC(50) values of 20 microM or below. Interestingly, pterocarpan 1 emerged as the best inhibitor with an IC(50) of 1.4 microM. We were thus able to prove that the pterocarpan skeleton is a new class of lead structure for neuraminidase inhibitors. Our studies reveal that the IC(50) has a marked dependence upon structure in the case of the pterocarpans but much less so for the flavanones. Kinetic analysis disclosed that all inhibitors are noncompetitive. Our molecular docking experiment resulted that the most potent pterocarpan-derived inhibitor 1 may bind to another binding pocket adjacent to the active site.     

An antisense oligodeoxynucleotide-doxorubicin conjugate: preparation and its reversal multidrug resistance of human carcinoma cell line in vitro

An antisense oligodeoxynucleotide-doxorubicin conjugate was synthesized by an aminocaproic acid linker. The synthetic conjugate was identified by HPLC analysis and UV-vis spectra. Properties of the conjugate in vitro conditions were investigated. The results demonstrated that the conjugate was remarkably stabilized by doxorubicin. When incubated in Dulbecco Phosphate-Buflered Saline (pH 7.4) at 37 degrees C, the conjugate was more stable than doxorubicin or the mixture of doxorubicin and antisense oligodeoxynucleotide. When incubated in 10% fetal serum at 37 degrees C, the conjugate showed a remarkable stabilization as compared to the unmodified oligodeoxynucleotide. Melting experiments demonstrated that the covalent attachment of doxorubicin strongly stabilized the binding of the oligodeoxynucleotide to its complementary sequence. In addition, in vitro reversion of multidrug resistance by the conjugate was assayed in a human carcinoma cell line (KB-A-1) resisting to doxorubicin. The result showed that the conjugate displayed very high reversal multdrug resistance activity in KB-A-1 cells in vitro. The conjugate lowered the IC50 value from 21.5 microM to 2.2 microM with a fold-reversal factor of 10. In contrast, a slight decrease of the IC50 value was observed when they combined with the "free" antisense oligodeoxynucleotide: the IC50 value was down from 21.5 microM to 16.8 microM. This study suggested that antisense oligodeoxynucleotide-doxorubicin conjugate might be helpful in multidrug resistance reversal.     

Comparison of anthracycline-induced death of human leukemia cells: programmed cell death versus necrosis

We investigated the mode of cell death induced by the anthracyclines, aclarubicin, doxorubicin and daunorubicin in the human leukemia cell lines, HL60 and Jurkat. The cells were incubated with drug concentrations up to 500 nM for periods between 3 and 24 hours, followed by morphological and biochemical analyses. All three substances induced DNA fragmentation, evident as DNA laddering and appearance of cells with hypodiploid DNA content, externalisation of phosphatidyl serine, activation of caspases and degradation of the apoptosis-specific endonuclease inhibitor DFF45. However, concentrations and times necessary for these effects to occur were different, aclarubicin being the quickest acting drug with a lag phase of 3 h, followed by daunorubicin with 6 h and doxorubicin with 24 h. More importantly, aclarubicin induced these effects while the cell membrane was intact, whereas doxorubicin and daunorubicin led to immediate loss of membrane integrity. Programmed cell death is characterised by preservation of membrane integrity in order to allow removal of apoptotic bodies, whereas cell rupture is an early event in necrosis. We therefore suggest that, in our experimental settings, doxorubicin- and daunorubicin-induced cell death occurs by necrosis, while aclarubicin induces programmed cell death.     

Demonstration of peptidoglycan-binding sites on lymphocytes and macrophages by photoaffinity cross-linking

One dominant binding site (70 kDa 6.5 pI protein) for bacterial cell wall peptidoglycan (PGN), a macrophage activator and polyclonal B cell mitogen, was demonstrated on mouse B and T lymphocytes and macrophages by photoaffinity cross-linking and two-dimensional polyacrylamide gel electrophoresis. This binding site was not present on erythrocytes. The binding was specific for polymeric PGN and was competitively inhibited by unlabeled PGN with IC50 = 48 micrograms/ml (0.38 microM). The binding was partially inhibited by O-acetylated PGN monomers (IC50 = 469 micrograms/ml, 521 microM), dextran sulfate (IC50 = 1024 micrograms/ml, 124 microM), and (GlcNAc)3 (IC50 = 6.6 mg/ml, 10 mM), and was not inhibited by non-O-acetylated PGN monomers and dimers, muramyl dipeptide, PGN pentapeptide, GlcNAc, teichoic acid, protein A, and gelatin. The cell surface location of the 70-kDa PGN-binding protein was indicated by the ability of PGN to bind to this protein in intact metabolically inactive cells (at 4 degrees C and in the presence of 0.1% NaN3) and by the ability to extract the 70-kDa PGN-binding protein from viable B lymphocytes by noncytotoxic concentration of n-octyl-beta-D-glucopyranoside.     

Redox cycling of anthracyclines by cardiac mitochondria. II. Formation of superoxide anion, hydrogen peroxide, and hydroxyl radical

In the accompanying paper (Davies, K. J. A., and Doroshow, J. A. (1986) J. Biol. Chem. 261, 3060-3067), we have demonstrated that anthracycline antibiotics are reduced to the semiquinone form at Complex I of the mitochondrial electron transport chain. In the experiments presented in this study we examined the effects of doxorubicin (Adriamycin), daunorubicin, and related quinonoid anticancer agents on superoxide, hydrogen peroxide, and hydroxyl radical production by preparations of beef heart submitochondrial particles. Superoxide anion formation was stimulated from (mean +/- S.E.) 1.6 +/- 0.2 to 69.6 +/- 2.7 or 32.1 +/- 1.5 nmol X min-1 X mg-1 by the addition of 90 microM doxorubicin or daunorubicin, respectively. However, the anthracycline 5-iminodaunorubicin, in which an imine group has been substituted in the C ring quinone moiety, did not increase superoxide production over control levels. In the presence of rotenone, initial rates of oxygen consumption and superoxide formation were identical under comparable experimental conditions. Furthermore, H2O2 production increased from undetectable control levels to 2.2 +/- 0.3 nmol X min-1 X mg-1 after treatment of submitochondrial particles with doxorubicin (200 microM). The hydroxyl radical, or a related chemical oxidant, was also detected after the addition of an anthracycline to this system by both ESR spectroscopy using the spin trap 5,5-dimethylpyrroline-N-oxide and by gas chromatographic quantitation of CH4 produced from dimethyl sulfoxide. Hydroxyl radical production, which was iron-dependent in this system, occurred in a nonlinear fashion with an initial lag phase due to a requirement for H2O2 accumulation. We also found that two quinonoid anti-cancer agents which produce less cardiotoxicity than the anthracyclines, mitomycin C, and mitoxantrone, stimulated significantly less or no hydroxyl radical production by submitochondrial particles. These experiments suggest that injury to cardiac mitochondria which is produced by anthracycline antibiotics may result from the generation of the hydroxyl radical during anthracycline metabolism by NADH dehydrogenase.     

Determination of covalent binding to intact DNA, RNA, and oligonucleotides by intercalating anticancer drugs using high-performance liquid chromatography. Studies with doxorubicin and NADPH cytochrome P-450 reductase.

An HPLC method is described which can determine covalent binding to intact nucleic acid by intercalating anticancer drugs and at the same time remove noncovalently bound intercalated drug. The method uses a column containing a nonporous 2-microns DEAE anion-exchange resin capable of chromatographing nucleic acids greater than 50,000 bases in size in under 1 h. After priming with 1 mg of DNA, the column behaves as an intercalator affinity column, strongly retaining the drug while allowing the nucleic acid to pass through normally. Retained drug is released with an injection of 0.1 M potassium hydroxide. Incubations were performed with the intercalator doxorubicin, which is also believed to bind covalently to DNA. When [14C]doxorubicin was mixed with DNA, at a concentration where all the drug would bind by intercalation, the column retained 82% of the total radioactivity, only 18% migrated with the nucleic acid. If the DNA was mildly denatured by treatment with 2 M sodium chloride at 50 degrees C for 45 min before chromatography, then 99.8% of total radioactivity was retained, only background counts migrated with the nucleic acid, as was the case with single-stranded DNA and RNA without any treatment. Purified NADPH cytochrome P-450 reductase was used to activate doxorubicin. DNA inhibited the metabolism of the drug by the enzyme, no covalent binding occurred with RNA, low levels occurred with single-stranded DNA (34 pmol/100 micrograms), and the highest levels were recorded with oligonucleotides (243 pmol/100 micrograms). The assay was sufficiently sensitive to measure covalent binding to DNA extracted from MCF-7 human breast cancer cells treated with 50 microM [14C]doxorubicin (18.6 pmol/100 micrograms). Thus, covalent binding to DNA, RNA, and oligonucleotides by intercalators can be measured quickly (20 min) without the need to either digest the nucleic acid or subject it to long sample preparation techniques.     

Design, synthesis, and biological evaluation of linear 1-(4-, 3- or 2-methylsulfonylphenyl)-2-phenylacetylenes: a novel class of cyclooxygenase-2 inhibitors

A group of regioisomeric 1-(methylsulfonylphenyl)-2-phenylacetylenes possessing a COX-2 SO(2)Me pharmacophore at the para-, meta- or ortho-position of the C-1 phenyl ring, in conjunction with a C-2 phenyl or substituted-phenyl ring substituent (3-F, 3-OMe, 3-OH, 3-OAc, 4-Me), were designed for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors. These target linear 1,2-diarylacetylenes were synthesized via a palladium-catalyzed Sonogashira cross-coupling reaction followed by oxidation of the respective 1-(methylthiophenyl)-2-phenylacetylene intermediate. In vitro COX-1/COX-2 isozyme inhibition structure-activity studies identified 1-(3-methylsulfonylphenyl)-2-(4-methylphenyl)acetylene (12d) as a potent COX-2 inhibitor (IC(50) = 0.32 microM) with a high COX-2 selectivity index (SI > 320) comparable to the reference compound rofecoxib (COX-2 IC(50) = 0.50 microM; COX-2 SI > 200). A molecular modeling study where (12d) was docked in the binding site of COX-2 showed that the MeSO(2) COX-2 pharmacophore was positioned in the vicinity of the secondary COX-2 binding site near Val(523). The 1-(4-methylsulfonylphenyl)-2-(3-acetoxyphenyl)acetylene (11f, COX-1 IC(50) = 1.00 microM; COX-2 IC(50) = 0.06 microM; COX-2 SI = 16.7) and 1-(3-methylsulfonylphenyl)-2-(3-acetoxyphenyl)acetylene (12f, COX-1 IC(50) = 6.5 microM; COX-2 IC(50) = 0.05 microM; COX-2 SI = 130) regioisomers exhibited comparable COX-2 inhibition, and moderately lower selective COX-2 selectivity, relative to the reference drug celecoxib (COX-1 IC(50) = 33.1 microM; COX-2 IC(50) = 0.07 microM; COX-2 SI = 472). The most potent anti-inflammatory agent 1-(3-methylsulfonylphenyl)-2-(4-methylphenyl)acetylene (12d) exhibited moderate oral anti-inflammatory activity (ED(50)= 129 mg/kg) at 3 h postdrug administration relative to the reference drug celecoxib (ED(50) = 10.8 mg/kg) in a carrageenan-induced rat paw edema assay. The structure-activity data acquired indicate that the acetylene moiety constitutes a suitable scaffold (template) to design novel acyclic 1,2-diarylacetylenes with selective COX-2, or dual COX-1/COX-2, inhibitory activities.