Cytotoxicity of natural hydroxyanthraquinones: role of oxidative stress

In order to assess the role of oxidative stress in the cytotoxicity of natural hydroxyanthraquinones, we compared rhein, emodin, danthron, chrysophanol, and carminic acid, and a series of model quinones with available values of single-electron reduction midpoint potential at pH 7.0 (E(1)7), with respect to their reactivity in the single-electron enzymatic reduction, and their mammalian cell toxicity. The toxicity of model quinones to the bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK), and HL-60, a human promyelocytic leukemia cell line, increased with an increase in their E(1)7. A close parallelism was found between the reactivity of hydroxyanthraquinones and model quinones with single-electron transferring flavoenzymes ferredoxin: NADP+ reductase and NADPH:cytochrome P450 reductase, and their cytotoxicity. This points to the importance of oxidative stress in the toxicity of hydroxyanthraquinones in these cell lines, which was further evidenced by the protective effects of desferrioxamine and the antioxidant N,N'-diphenyl-p-phenylene diamine, by the potentiating effects of 1,3-bis-(2-chloroethyl)-1-nitrosourea, and an increase in lipid peroxidation.     

Flavoenzyme-catalyzed redox cycling of hydroxylamino- and amino metabolites of 2,4,6-trinitrotoluene: implications for their cytotoxicity

The toxicity of 2,4,6-trinitrotoluene (TNT), a widespread environmental contaminant, is exerted through its enzymatic redox cycling and/or covalent binding of its reduction products to proteins and DNA. In this study, we examined the possibility of another cytotoxicity mechanism of the amino- and hydroxylamino metabolites of TNT, their flavoenzyme-catalyzed redox cycling. The above compounds acted as redox-cycling substrates for single-electron transferring NADPH:cytochrome P-450 reductase (P-450R) and ferredoxin:NADP(+) reductase (FNR), as well as substrates for the two-electron transferring flavoenzymes rat liver NAD(P)H:quinone oxidoreductase (NQO1) and Enterobacter cloacae NAD(P)H:nitroreductase (NR). Their reactivity in P-450R-, FNR-, and NR-catalyzed reactions increased with an increase in their single-electron reduction potential (E(1)(7)) or the decrease in the enthalpy of free radical formation. The cytotoxicity of the amino- and hydroxylamino metabolites of TNT towards bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) was partly prevented by the antioxidant N,N'-diphenyl-p-phenylene diamine and desferrioxamine, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea, thus pointing to the involvement of oxidative stress. In general, their cytotoxicity increased with an increase in their electron accepting properties, or their reactivity towards the single-electron transferring FNR and P-450R. Thus, our data imply that the flavoenzyme-catalyzed redox cycling of amino and hydroxylamino metabolites of TNT may be an important factor in their cytotoxicity.     

Quantitative structure-activity relationships in enzymatic single-electron reduction of nitroaromatic explosives: implications for their cytotoxicity

The mechanisms of cytotoxicity of polynitroaromatic explosives, an important group of environmental pollutants, remain insufficiently studied so far. We have found that the rate constants of single-electron enzymatic reduction, and the enthalpies of single-electron reduction of nitroaromatic compounds (DeltaHf(ArNO(2)(-*)), obtained by quantum mechanical calculation, may serve as useful tools for the analysis of cytotoxicity of nitroaromatic explosives with respect to the possible involvement of oxidative stress. The single-electron reduction rate constants of a number of explosives including 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrophenyl-N-methylnitramine (tetryl), and model nitroaromatic compounds by ferredoxin:NADP(+) reductase (FNR, EC 1.18.1.2) and NADPH:cytochrome P-450 reductase (P-450R, EC 1.6.2.4) increased with a decrease in DeltaHf(ArNO(2)(-*)). This indicates that the reduction rates are determined by the electron transfer energetics, but not by the particular structure of the explosives. The cytotoxicity of explosives to bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) increased with a corresponding increase in their reduction rate constant by P-450R and FNR, or with a decrease in their DeltaHf(ArNO(2)(-*)). This points to an importance of oxidative stress in the toxicity of explosives in this cell line, which was further evidenced by the protective effects of desferrioxamine and the antioxidant N,N'-diphenyl-p-phenylene diamine, and an increase in lipid peroxidation. DT-diaphorase (EC 1.6.99.2) exerted a minor and equivocal role in the cytotoxicity of explosives to FLK cells.     

Prooxidant cytotoxicity of chromate in mammalian cells: the opposite roles of DT-diaphorase and glutathione reductase

The geno- and cytotoxicity of chromate, an important environmental pollutant, is partly attributed to the flavoenzyme-catalyzed reduction with the concomitant formation of reactive oxygen species. The aim of this work was to characterize the role of NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2) and glutathione reductase (GR, EC 1.6.4.2) in the mammalian cell cytotoxicity of chromate, which was evidenced controversially so far. The chromate reductase activity of NQO1 was higher than that of GR, but lower than that of lipoamide dehydrogenase (EC 1.6.4.3), ferredoxin:NADP+ reductase (EC 1.18.1.2), and NADPH: cytochrome P-450 reductase (EC 1.6.2.4). The reduction of chromate by NQO1 was accompanied by the formation of reactive oxygen species. The concentration of chromate for 50% survival of bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) during a 24-h incubation was (22 +/- 4) microM. The cytotoxicity was partly prevented by desferrioxamine, the antioxidant N,N'-diphenyl-p-phenylene diamine and by an inhibitor of NQO1, dicumarol, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), which inactivates GR. The NADPH-dependent chromate reduction by digitonin-permeabilized FLK cells was partly inhibited by dicumarol and not affected by BCNU. Taken together, these data indicate that the oxidative stress-type cytotoxicity of chromate in FLK cells may be partly attributed to its reduction by NQO1, but not by GR. The effect of BCNU on the chromate cytotoxicity may indicate that the general antioxidant action of reduced glutathione is more important than its prooxidant activities arising from the reactions with chromate.     

Cytotoxicity of RH1 and related aziridinylbenzoquinones: involvement of activation by NAD(P)H:quinone oxidoreductase (NQO1) and oxidative stress

It is supposed that the main cytotoxicity mechanism of antitumour aziridinyl-substituted benzoquinones is their two-electron reduction to alkylating products by NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2). However, other possible cytotoxicity mechanisms, e.g., oxidative stress, are studied insufficiently. In the single-electron reduction of quinones including a novel compound RH1 (2,5-diaziridinyl- 3-(hydroxymethyl)-6-methyl-1,4-benzoquinone), by NADPH:cytochrome P-450 reductase (EC 1.6.2.4, P-450R), their reactivity increased with an increase in the redox potential of quinone/semiquinone couple (E(1)7), reaching a limiting value at E(1)7> or =-0.1V. The reactivity of quinones towards NQO1 did not depend on their E(1)7. The cytotoxicity of aziridinyl-unsubstituted quinones in bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) mimics their reactivity in P-450R-catalyzed reactions, exhibiting a parabolic dependence on their E(1)7. The toxicity of aziridinyl-benzoquinones, although being higher, also followed this trend and did not depend on their reactivity towards NQO1. The action of aziridinylbenzoquinones in FLK cells was accompanied by an increase in lipid peroxidation, their toxicity decreased by desferrioxamine and the antioxidant N,N'-diphenyl-p-phenylene diamine, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea. The inhibitor of NQO1, dicumarol, protected against the toxicity of aziridinyl-benzoquinones except of 2,5-bis-(2'-hydroxyethylamino)-3,6-diaziridinyl-1,4-benzoquinone (BZQ), which was almost inactive as NQO1 substrate. The same events except the absence of pronounced effect of dicumarol were characteristic in the cytotoxicity of aziridinyl-unsubstituted quinones. These findings indicate that in addition to the activation by NQO1, the oxidative stress presumably initiated by single-electron transferring enzymes may be an important factor in the cytotoxicity of aziridinylbenzoquinones. The information obtained may contribute to the understanding of the molecular mechanisms of aziridinylquinone cytotoxicity and may be useful in the design of future bioreductive drugs.     

Role of single-electron oxidation potential and lipophilicity in the antiplasmodial in vitro activity of polyphenols: comparison to mammalian cells

In spite of extensive studies, the structure-activity relationships in the action of polyphenols against the malaria parasite Plasmodium falciparum are poorly understood so far. As the mammalian cell cytotoxicity of polyphenols shows a negative dependence on the potential of the phenoxyl radical/phenol redox couple (E(2)(7)), due to the involvement of prooxidant events, and a positive dependence on the octanol/water distribution coefficient at pH 7.0 (log D), we examined the role of these parameters in their antiplasmodial in vitro activity. We found that the concentrations of hydroxybenzenes causing 50% inhibition of the growth of P falciparum strain FcB1 (IC50) are described by the regression log IC50 (microM) = 0.36 + 1.81 E(2)(7) (V) - 0.10 log D [n = 11, r2 = 0.760, F(2.8) = 12.03]. The IC50 values of flavonoids (n = 5), comprising a separate less active series, did not depend on their E(2)(7) values, 0.33 V-0.75 V. These findings were similar to the mammalian cell cytotoxicity data. However, the mammalian cell cytotoxicity of hydroxybenzenes showed more pronounced dependence on their E(2)(7) values [delta log CL50/delta E(2)(7) = (6.9 - 5.1) V(-1), where CL50 is the compound concentration for 50% cell survival] than on their antiplasmodial activity. Although it is unclear whether the prooxidant action is the main factor in the antiplasmodial action of polyphenols or not, our data showed that the ease of their oxidation (decrease in E(2)(7)) may enhance their activity. On the other hand, the different sensitivity of the mammalian cell cytotoxicity and the antiplasmodial activity of the hydroxybenzenes to their E(2)(7) values implied that compounds with high oxidation potential may be used as relatively efficient antiplasmodial agents with low mammalian cell cytotoxicity.     

Redox properties of novel antioxidant 5,8-Dihydroxycoumarin: implications for its prooxidant cytotoxicity

The aim of this work was to characterize the redox properties of the new antioxidant 5,8-dihydroxycoumarin (5,8-DHC), isolated from sweet grass (Hierochlo? odorata L.), and to determine its impact on its cytotoxic action. Reversible electrochemical oxidation of 5,8-DHC at pH 7.0 was characterized by the midpoint potential (E(p/2)) of 0.23 V vs. the normal hydrogen electrode. 5,8-DHC was slowly autoxidized at pH 7.0, and it was active as a substrate for peroxidase (POD, EC 1.11.1.7) and tyrosinase (TYR, EC 1.14.18.1). Oxidation of 5,8-DHC by POD/H202 yielded the product(s) which reacted with reduced glutathione and supported the oxidation of NADPH by ferredoxin:NADP+ reductase (FNR, EC 1.18.1.2) and NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2). The concentration of 5,8-DHC for 50% survival of bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) during a 24-h incubation was (60 +/- 5.5) microM. Cytotoxicity of 5,8-DHC was decreased by desferrioxamine, catalase, the antioxidant N,N'-diphenyl-p-phenylene diamine, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea and dicumarol, an inhibitor of NQO1. This shows that 5,8-DHC possesses the oxidative stress-type cytotoxicity, evidently due to the action of quinodal oxidation product(s). The protective effect of isoniazide, an inhibitor of cytochrome P-450 2E1, points to hydroxylation of 5,8-DHC as additional toxification route, whereas the potentiating effect of 3,5-dinitrocatechol, an inhibitor of catechol-o-methyltransferase (COMT, EC 2.1.1.6), points to the o-methylation of hydroxylation products as the detoxification route.     

Cytochrome P-450 cholesterol 7 alpha-hydroxylase: inhibition of enzyme deactivation by structurally diverse calmodulin antagonists and phosphatase inhibitors

Cytochrome P-450 cholesterol 7 alpha-hydroxylase (P-450Ch7 alpha) catalyzes the first and rate-limiting step in the conversion of cholesterol to bile acids. Incubation of rat liver microsomes in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer resulted in a time-dependent deactivation of P-450Ch7 alpha which was markedly accelerated by the nonionic detergent Tween 80. Microsomal NADPH-cytochrome P-450 reductase and cytochrome P-450-dependent 7-ethoxycoumarin O-deethylase activities were unaffected under these conditions, evidencing the selectivity of the deactivation process for P-450Ch7 alpha. The rate (t 1/2 = 15-19 min at 37 degrees C) and maximal extent of P-450Ch7 alpha deactivation (greater than or equal to 90%) were both unaffected by the presence of cytosolic proteins and were also not dependent on the initial enzyme level, as shown using liver microsomes isolated from untreated, cholestyramine-fed, and xenobiotic-induced rats exhibiting an eight-fold range in P-450Ch7 alpha activity. Scavengers for reduced oxygen species were also without effect. P-450Ch7 alpha was stabilized some six- to sevenfold (t 1/2 = 94-143 min) by the phosphatase inhibitor NaF. Of a series of other phosphatase inhibitors examined, including, among others, EDTA, vanadate, and molybdate, only phosphate-containing compounds and the calmodulin antagonist trifluoperazine, and inhibitor of the Ca2+-calmodulin-dependent phosphatase calcineurin, effectively stabilized P-450Ch7 alpha. Modulation of P-450Ch7 alpha deactivation by these inhibitors generally paralleled their effects on isolated calcineurin. A variety of structurally diverse calmodulin antagonists examined were also found to effectively protect P-450Ch7 alpha from deactivation; these include calmidazolium and tamoxifen (IC50 = 25 to 50 microM), chlorpromazine, thioridazine, amitriptyline, imipramine, and the naphthalene sulfonamide compound W-7 (IC50 = 50 to 300 microM). Structure-activity analysis of several phenothiazines and their derivatives indicated that although little activity was exhibited by the sulfoxides, some protection was provided by the corresponding sulfones. On the basis of these observations, various models for the molecular basis of enzyme deactivation are considered, including the hypothesis that a calcineurin-like microsomal phosphatase mediates deactivation of this cytochrome P-450 enzyme.     

Correlation between mammalian cell cytotoxicity of flavonoids and the redox potential of phenoxyl radical/phenol couple

Flavonoids exhibit prooxidant cytotoxicity in mammalian cells due to the formation of free radicals and oxidation products possessing quinone or quinomethide structure. However, it is unclear how the cytotoxicity of flavonoids depends on the ease of their single-electron oxidation in aqueous medium, i.e., the redox potential of the phenoxyl radical/phenol couple. We verified the previously calculated redox potentials for several flavonoids according to their rates of reduction of cytochrome c and ferricyanide, and proposed experimentally-based values of redox potentials for myricetin, fisetin, morin, kaempferol, galangin, and naringenin. We found that the cytotoxicity of flavonoids (n=10) in bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) and murine hepatoma (line MH-22a) increases with a decrease in their redox potential of the phenoxyl radical/phenol couple and an increase in their lipophilicity. Their cytotoxicity was decreased by antioxidants and inhibitors of cytochromes P-450, α-naphthoflavone and isoniazide, and increased by an inhibitor of catechol-O-methyltransferase, 3,5-dinitrocatechol. It shows that although the prooxidant action of flavonoids may be the main factor in their cytotoxicity, the hydroxylation and oxidative demethylation by cytochromes P-450 and O-methylation by catechol-O-methyltransferase can significantly modulate the cytotoxicity of the parent compounds.     

Synthesis and antineoplastic properties of an ether glycerophosphonocholine, and analog of ET-18-OCH3-GPC.

A glycerophosphonocholine analog of the ether-linked lipid, rac-1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine (ET-18-OCH3-GPC), was synthesized in which the head group is nonhydrolyzable by phospholipase C. The phosphonate analog used in this study is rac-3-octadecyloxy-2-methoxy-propyl-phosphonocholine, C18H37OCH2CH(OCH3)CH2P(O)(O)OCH2CH2N+(CH3)3. The activity of the synthetic phosphonate was tested in the human leukemic cell line, HL-60, and the human undifferentiated cervical carcinoma, C-41. The glycerophosphonocholine inhibited [3H]thymidine uptake by HL-60 cells with an EC50 value of 5-7 microM. The glycerophosphate ET-18-OCH3-GPC had an EC50 value of approximately 2 microM against HL-60 cells. The EC50 values estimated from cell viability experiments were similar to that for [3H]thymidine uptake. The EC50 value for C-41 cells was about 10-15 microM. The data demonstrate that the glycerophosphonocholine is a promising anti-cancer drug for the treatment of both leukemia and solid tumors. Furthermore, the data demonstrate that phospholipase C-catalyzed hydrolysis of ET-18-OCH3-GPC does not play an important role in the cytotoxic action of the ether-linked glycerolipids.     

The first total synthesis of the marine fatty acid (+/-)-2-methoxy-13-methyltetradecanoic acid: a cytotoxic fatty acid to leukemia cells

The recently discovered marine fatty acid (+/-)-2-methoxy-13-methyltetradecanoic acid was synthesized for the first time in six steps (26% overall yield) starting from commercially available methyl 12-methyltridecanoate. The synthetic approach provided enough material to corroborate the structure of the acid, which was recently identified in the sponge Amphimedon complanata from Aguadilla, Puerto Rico, and to test its cytotoxicity to three leukemia cell lines. The key step in the synthesis was the addition of trimethylsilyl cyanide to 12-methyltridecanal under triethylamine catalysis. Nuclear magnetic resonance data are provided for the first time for this methoxylated fatty acid and the synthetic approach utilized is of general applicability since it can be used in the synthesis of other methyl-branched 2-methoxylated fatty acids. We also report that the acid (+/-)-2-methoxy-13-methyltetradecanoic acid is cytotoxic to human chronic myelogenous leukemia K-562 (EC50=238 microM), histiocytic lymphoma U-937 (EC50=250 microM), and promielocytic leukemia HL-60 (EC50=476 microM) in RPMI 1640 medium.     

Copper chelation by D-penicillamine generates reactive oxygen species that are cytotoxic to human leukemia and breast cancer cells

Serum and tumor copper levels are significantly elevated in a variety of malignancies including breast, ovarian, gastric, lung, and leukemia. D-Penicillamine (D-pen), a copper-chelating agent, at low concentrations in the presence of copper generates concentration-dependent cytotoxic hydrogen peroxide (H(2)O(2)). The purpose of these studies was to investigate the in vitro cytotoxicity, intracellular reactive oxygen species (ROS) generation, and the reduction in intracellular thiol levels due to H(2)O(2) and other ROS generated from copper-catalyzed D-pen oxidation in human breast cancer cells (BT474, MCF-7) and human leukemia cells (HL-60, HL-60/VCR, HL-60/ADR). D-pen (< or = 400 microM) in the presence of cupric sulfate (10 microM) resulted in concentration-dependent cytotoxicity. Catalase was able to completely protect the cells, substantiating the involvement of H(2)O(2) in cancer cell cytotoxicity. A linear correlation between the D-pen concentration and the intracellular ROS generated was shown in both breast cancer and leukemia cells. D-pen in the presence of copper also resulted in a reduction in intracellular reduced thiol levels. The H(2)O(2)-mediated cytotoxicity was greater in leukemia cells compared to breast cancer cells. These results support the hypothesis that D-pen can be employed as a cytotoxic copper-chelating agent based on its ROS-generating ability.     

Ecto-alkaline phosphatase activity identified at physiological pH range on intact P19 and HL-60 cells is induced by retinoic acid

The activity of membrane-bound alkaline phosphatase (ALP) expressed on the external surface of cultured murine P19 teratocarcinoma and human HL-60 myeloblastic leukemia cells was studied at physiological pH using p-nitrophenylphosphate (pNPP) as substrate. The rate of substrate hydrolysis catalyzed by intact viable cells remained constant for eight successive incubations of 30 min and was optimal at micromolar substrate concentrations over the pH range 7.4-8.5. The value of apparent K(m) for pNPP in P19 and HL-60 cells was 120 microM. Hydrolytic activity of the ecto-enzyme at physiological pH decreased by the addition of levamisole, a specific and noncompetitive inhibitor of ALP (K(i) P19 = 57 microM; K(i) HL-60 = 50 microM). Inhibition of hydrolysis was reversed by removal of levamisole within 30 min. Retinoic acid (RA), which promotes the differentiation of P19 and HL-60 cells, induced levamisole-sensitive ecto-phosphohydrolase activity at pH 7.4. After its autophosphorylation by ecto-kinase activity, a 98-kDa membrane protein in P19 cells was found to be sensitive to ecto-ALP, and protein dephosphorylation increased after incubation of cells with RA for 24 h and 48 h. Orthovanadate, an inhibitor of all phosphatase activities, blocked the levamisole-sensitive dephosphorylation of the membrane phosphoproteins, while (R)-(-)-epinephrine reversed the effect by complexation of the inhibitor. The results demonstrate that the levamisole-sensitive phosphohydrolase activity on the cell surface is consistent with ecto-ALP activity degrading both physiological concentrations of exogenously added substrate and endogenous surface phosphoproteins under physiological pH conditions. The dephosphorylating properties of ecto-ALP are induced by RA, suggesting a specific function in differentiating P19 teratocarcinoma and HL-60 myeloblastic leukemia cells.     

Isolation of apoptosis- and differentiation-inducing substances toward human promyelocytic leukemia HL-60 cells from leaves of Juniperus taxifolia

A chloroform extract of the leaves of Juniperas taxifolia exhibited a marked antiproliferative effect on human promyelocytic leukemia HL-60 cells at a concentration of 2.5 microg/ml. Deoxypodophyllotoxin (4) was identified in the extract as an outstanding antiproliferative compound, and five diterpenes (1-3, 5, and 6) were isolated as known compounds with weak or no cytotoxicity. These compounds were examined for their respective apoptosis- and differentiation-inducing activities toward HL-60 cells by DNA fragmentation and NBT-reducing assays, respectively. Among them, 7alpha-hydroxysandaracopimaric acid (6) was found to have a potent differentiation-inducing activity in a dose-dependent manner at 0.125-2 microg/ml (0.39-6.29 microM), together with apoptosis-inducing activity at concentrations of more than 2.5 microg/ml (7.86 microM). Deoxypodophyllotoxin (4) that exerted cytotoxic and apoptosis-inducing activities at 2 ng/ml (5 nM) did not induce differentiation at the same concentration, and the other diterpenes (1-3 and 5) showed no effect on cell differentiation, even at 5 microg/ml. It was thus demonstrated for the first time that 7alpha-hydroxysandaracopimaric acid was an effective differentiation-inducing compound toward HL-60 cells.     

Quantitative structure–activity relationships in enzymatic single-electron reduction of nitroaromatic explosives: implications for their cytotoxicity

The mechanisms of cytotoxicity of polynitroaromatic explosives, an important group of environmental pollutants, remain insufficiently studied so far. We have found that the rate constants of single-electron enzymatic reduction, and the enthalpies of single-electron reduction of nitroaromatic compounds (ΔHf(ArNO₂^−⋅)), obtained by quantum mechanical calculation, may serve as useful tools for the analysis of cytotoxicity of nitroaromatic explosives with respect to the possible involvement of oxidative stress. The single-electron reduction rate constants of a number of explosives including 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrophenyl-N-methylnitramine (tetryl), and model nitroaromatic compounds by ferredoxin:NADP⁺ reductase (FNR, EC 1.18.1.2) and NADPH:cytochrome P-450 reductase (P-450R, EC 1.6.2.4) increased with a decrease in ΔHf(ArNO₂^−⋅). This indicates that the reduction rates are determined by the electron transfer energetics, but not by the particular structure of the explosives. The cytotoxicity of explosives to bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) increased with a corresponding increase in their reduction rate constant by P-450R and FNR, or with a decrease in their ΔHf(ArNO₂^−⋅). This points to an importance of oxidative stress in the toxicity of explosives in this cell line, which was further evidenced by the protective effects of desferrioxamine and the antioxidant N,N′-diphenyl-p-phenylene diamine, and an increase in lipid peroxidation. DT-diaphorase (EC 1.6.99.2) exerted a minor and equivocal role in the cytotoxicity of explosives to FLK cells.     

Cytotoxic and proapoptotic activity of diterpenoids from in vitro cultivated Salvia sclarea roots. Studies on the leukemia cell lines

Four diterpenoids, ferruginol, salvipisone, aethiopinone and 1-oxoaethiopinone, were isolated from transformed roots of Salvia sclarea. Salvipisone and aethiopinone showed relatively high cytotoxicity against HL-60 and NALM-6 leukemia cells (IC50 range 0.6-7.7 microg/ mL which is equal to 2.0-24.7 microM), whereas 1-oxoaethiopinone and ferruginol were less active in this regard. Moreover, we have found that all four diterpenoids of S. sclarea had equal cytotoxic activity against parental HL-60 and multidrug-resistant HL-60 ADR cells, what indicates that they are poor substrates for transport by multidrug resistance-associated protein (MRP1). Caspase-3 activity determinations showed that salvipisone and aethiopinone were able to induce apoptosis in a time- and concentration-dependent manner. The results obtained in this study show that S. sclarea diterpenoids aethiopinone and salvipisone may be useful in the treatment of human cancers, especially in the case of drug resistance.     

Differential apoptotic effect of wogonin and nor-wogonin via stimulation of ROS production in human leukemia cells

We investigate the roles of methoxyl (OCH(3)) and hydroxyl (OH) substitutions at C8 of flavonoids on their apoptosis-inducing activities. Wogonin (Wog) and nor-wogonin (N-Wog) are structurally related flavonoids, and respectively contain an OH and OCH(3) at C8. In leukemia HL-60 cells, N-Wog exhibited more-potent cytotoxicity than Wog according to the MTT and LDH release assays, and the IC(50) values of Wog and N-Wog in HL-60 cells were 67.5 +/- 2.1 and 21.7 +/- 1.5 microM, respectively. Apoptotic characteristics including DNA ladders, apoptotic bodies, and hypodiploid cells accompanied by the induction of caspase 3 protein processing appeared in Wog- and N-Wog-treated HL-60 cells. Interestingly, an increase in intracellular peroxide production was detected in N-Wog- but not Wog-treated HL-60 cells by the DCHF-DA assay, and the reduction of intracellular peroxide by catalase (CAT) induced by N-Wog significantly reduced the N-Wog- but not the Wog-induced cytotoxic effect according to the MTT assay in accordance with the blocking of DNA ladder formation and caspase 3 and PARP protein processing elicited by N-Wog. We further analyzed the effect of six structurally related compounds, including 5-OH, 7-OH, 5,7-diOH, 5,7-diOCH(3), 7,8-diOCH(3), and 7-OCH(3)-8-OH flavones, on apoptosis induction in HL-60 cells. Results suggested that OH at C5 and C7 is essential for both the apoptosis-inducing activity of flavonoids, and OH at C8 may contribute to apoptosis induction ability. Evidence to support a distinct structure-activity relationship in apoptosis induction of flavonoids is provided for the first time in this study.     

N-formyl-Met-Leu-Phe-induced oxidative burst in DMSO-differentiated HL-60 cells requires active Hsp90, but not intact microtubules

In this study we examined whether microtubules and heat shock protein 90 (Hsp90) are involved in phorbol myristate acetate (PMA) and N-formyl-Met-Leu-Phe (fMLP)-induced oxidative burst in DMSO-differentiated HL-60 cells. Our results showed that microtubule interfering agents, paclitaxel (1-5 microM), colchicine (1-100 microM), nocodazole (1-20 microM), and vincristine (1-50 microM), did not affect either PMA or fMLP-induced oxidative burst. In contrast, radicicol, an inhibitor of Hsp90, inhibited fMLP-induced oxidative burst in time and concentration-dependent manner where IC50 value for 30 min pre-incubation was 16.5 +/- 3.5 microM radicicol. We conclude that both PMA and fMLP-induced oxidative burst in DMSO-differentiated HL-60 cells is microtubule-independent while the latter requires Hsp90 activity.     

Effect of FMdC on the cell cycle of some leukemia cell lines.

BACKGROUND: (E)-2'-deoxy-2'-(fluoromethylene)-cytidine (FMdC), an irreversible inhibitor of ribonucleotide reductase, displays a strong toxicity towards many cell lines derived from human solid tumors, while its activity on leukemia lines is less well-known. The aim of this study was to assess the effect of FMdC on the cell cycle and cell death of human leukemia lines HL-60 and MOLT-4, and murine leukemia L-1210 in vitro. It has been assumed that a prerequisite of FMdC cytotoxicity is intracellular phosphorylation by deoxycytidine kinase (dCK). METHODS:Cell cultures in the exponential phase of growth were exposed to different concentrations of FMdC (10 nM to 10 microM) for 6 and 24 hours. In a parallel set of experiments 1 mM deoxycytidine was added to prevent phosphorylation of the drug by dCK. The DNA and protein content in the cells, as well as Annexin V/PI binding were assessed by flow cytometry. The cell cycle was analyzed by the MacCycle software. RESULTS: The cytotoxic effects of FMdC, i.e., G(1)/S block and cell death were observed, associated with pronounced changes in the protein content. These effects were of variable intensity among the cell lines studied (HL-60 being the most susceptible), and in some cases, were not completely reversed by deoxycytidine excess. CONCLUSIONS: FMdC is a potent cytotoxic/cytostatic agent against human leukemia cell lines in vitro. It also changes the cellular protein content. Unphosphorylated FMdC may slightly influence the cell cycle of some leukemic lines.