Role of protein kinase C activators and inhibitors, calmodulin antagonists and membrane sialic acids in polyamine transport in murine leukemia cells

The effects of activators and inhibitors of protein kinase C (phorbol esters and H-7) and antagonist to calmodulin (TFP) on polyamine transport in murine leukemia (L1210) cells are investigated. Phorbol esters and H-7 are found to enhance and curtail the uptake of 14C-Spermidine (Spd) respectively in L1210 cells. TFP also inhibits the uptake process. After desialation of cells with neuraminidase, phorbol esters are found to further increase the uptake of 14C-Spd by 35% compared to untreated cells. The sialic acid contents of the cells are regenerated by incubation with 14C-glucosamine for 18 hours. The regenerated cells mimic like untreated cells for the uptake of 14C-Spd i.e. after regeneration of sialic acids, the Spd uptake is curtailed significantly in comparison with desialated cells. Phorbol esters are found to enhance the activity of transglutaminase present in L1210 cells while H-7 and TFP exhibit reverse effects. The possible role of phorbol esters, H-7 and TFP and their effects on transglutaminase activity in relation with Spd transport process are discussed.     

Quantitative structure-activity relationships of phenolic compounds causing apoptosis

A study of a variety of phenolic compounds (simple phenols, estradiol, bisphenol A, diethylstilbesterol) on their action on L1210 leukemia cells led to the formulation of the following QSAR for apoptosis:log 1/C=-3.16 Clog P+2.77 CMR-3.76n=11, r(2)=0.939, s=0.630, q(2)=0.892C is the molar concentration causing 25% apoptosis, Clog P is the calculated octanol/water partition coefficient and CMR is the calculated molecular refractivity. Our results imply the significance of characterization of the phenolic compounds with apoptotic activity and the development of new agents for cancer therapy.     

Inhibitory effect of the alkyl side chain of caffeic acid analogues on lipopolysaccharide-induced nitric oxide production in RAW264.7 macrophages

Caffeic acid esters, one of the components of propolis, are known to show a variety of biological effects such as anti-tumor, anti-oxidant, and anti-inflammatory activities. Although, the anti-inflammatory activities of caffeic acid esters have been studied by analyzing their structure, the detailed mechanisms of their activities remain unclear. Thus, in this study, we examined the function of the ester functional group and the alkyl side chain (alcoholic part) and transformed caffeic acid to several derivatives. The inhibitory effect of these derivatives on NO production in murine macrophage RAW264.7 cells was dependent on the length and size of the alkyl moiety, and undecyl caffeate was the most potent inhibitor of NO production. In addition, individual experiments using undecanol, caffeic acid, undecanol plus caffeic acid, and undecyl caffeate showed that the connection between caffeic acid and the alkyl chain is critical for activity. Amide and ketone derivatives showed that not only the ester functional group but also the amide and ketone functional groups exhibit an inhibitory effect on NO production.     

QSAR of apoptosis induction in various cancer cells

In continuing our QSAR study of apoptosis, we consider in this report the action of phenolic compounds on Ramos cells (non-Hodgkins B-cell lymphoma): the effect of O-8-thapsigargin analogues on human prostate cancer cells, Tsu-Pr-1 and the induction of apoptosis of a complex set of congeners on human fibrosarcoma cells HT 1080. The human prostate cancer cells activity is very similar to that of the Ramos cells. While the QSAR for the fibrosarcoma cells resembles that of our earlier study with L1210 leukemia cells. The two different types of QSAR suggest at least two quite different types of receptors for the induction of apoptosis.     

Effect of dihydrocaffeic acid on UV irradiation of human keratinocyte HaCaT cells

Dihydrocaffeic acid, a dietary constituent and a microbial metabolite of flavonoids, is an antioxidant, but few biological effects have been examined. After its production by microflora in the colon, dihydrocaffeic acid is absorbed and found in plasma as a combination of free and metabolized forms. Excess solar UV radiation provokes damage and initiates immune response and inflammation in skin, sometimes leading to cancer. Dihydrocaffeic acid reduced the cytotoxicity and pro-inflammatory cytokine production (interleukin-6 and -8) in HaCaT cells, a keratinocyte model, following UV radiation. The effect of dihydrocaffeic acid may result from a combination of direct radical scavenging of the reactive oxygen species formed or reinforcement of the antioxidant potential of the keratinocytes, as well as a direct interference with the pathway involved in cytokine stimulation. The minimum structure required for such an effect appears to consist of a propionate side chain attached to a catechol moiety, as indicated by the efficacy of caffeic acid, but not of the methyl and glucuronide conjugates of dihydrocaffeic acid. The data obtained suggest that dihydrocaffeic acid is a potential candidate for photo-protection by interfering with the events initiated after UV exposure in keratinocytes.     

Chemical constituents of Lavatera trimestris L.--antioxidant and antimicrobial activities

Nine phenolic compounds, such as cis-/trans-p-coumaric acid, cis-/trans-p-coumaric acid methyl ester, glucose ester of cis-/trans-p-coumaric acid, caffeic acid methyl ester, kaempferol 7-O-beta-D-glucoside and kaempferol 3-O-beta-D-glucoside, were isolated from Lavatera trimestris flowers by chromatographic techniques and their structures were elucidated by spectral means (NMR). All compounds were tested for their antioxidant activity, while the methanolic extract was tested also for its antimicrobial activity. Also several non-polar constituents have been identified using GC and GC/MS methods. This is the first time that phenolic esters and non-polar constituents were identified in the flowers of L. trimestris L.     

Disruption of DNA synthesis and structure of mouse L1210 leukemia cells, sensitive and resistant to 1-methyl-1 nitrosourea and 1,3-bis(2-chloroethyl)-1-nitrosourea in vivo

Leukemia L1210 cells with acquired resistance to 1-methyl-1-nitrosourea (MNU) (L1210/MNU) and 1.3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (L1210/BCNU) were developed from leukemia L1210 cells sensitive to these drugs (L1210/0). The modal chromosome number of leukemia L1210/MNU and L1210/BCNU cells increases from 40 (L1210/0) to 41. It was shown that in leukemia L1210/MNU cells the inhibition of DNA synthesis after MNU administration in a therapeutic dose (80 mg/kg) is lasted within 24 hours, while that in leukemia L1210/0 cell--within 96 hours. After administration of BCNU (20 mg/kg) inhibition of DNA synthesis in leukemia L1210/BCNU cells reached of 50% of control in comparison with practically complete inhibition of DNA synthesis in leukemia L1210/0 cells. Centrifugation on alkaline sucrose density gradients revealed no differences in the rate of sedimentation of leukemia L1210/0, L1210/MNU and L1210/BCNU cell lysates. After 1 hour treatment with MNU of mice bearing L1210/MNU and L1210/0 leukemia cells single-strand breaks in DNA were determined. After 4 hours these strand-breaks retained in leukemia L1210/0 cells, but were eliminated in leukemia L1210/MNU cells. Administration of BCNU to mice with leukemia L1210/0 and L1210/BCNU cells resulted in both cases in the production of DNA aggregates. There is no complete cross-resistance between MNU and BCNU which allows a substitution of these drugs providing for the increase in their therapeutic efficiency.     

Synthesis and evaluation of a DHA and 10-hydroxycamptothecin conjugate

We have synthesized a conjugate of cis-4,7,10,13,16,19-docosahexenoic acid (DHA) and 10-hydroxycamptothecin (HCPT), DHA-HCPT. The antitumor activity of DHA-HCPT was evaluated in vitro against L1210 leukemia cells and in experimental animal tumor models including L1210 leukemia, Lewis lung carcinoma, and colon 38 adenocarcinoma. DHA-HCPT showed a greatly improved antitumor efficacy compared to HCPT.     

Rosmarinic acid: new aspects

Rosmarinic acid (RA) is an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid which is one of the most frequently occurring caffeic acid esters in the plant kingdom besides chlorogenic acid. RA has numerous biological and pharmacological activities. Its occurrence is spread all over the land plant kingdom. Enzymes and genes of its biosynthesis are well investigated. RA can be produced in high amounts in in vitro cultivated plant cells which offers the possibility of an economical exploitation. The review reports about recent findings in the biosynthesis of RA and related caffeic acid esters and discusses some aspects of the evolution of the biosynthetic enzymes.     

Drastic effect of several caffeic acid derivatives on the induction of heme oxygenase-1 expression revealed by quantitative real-time RT-PCR

Among antioxidative polyphenols, caffeic acid esters such as caffeic acid phenethyl ester (CAPE) and chlorogenic acid are contained in propolis, vegetables and coffee. In this study, we compared the efficacy of some polyphenols on the activation level of a cytoprotective heme oxygenase-1 (HO-1) gene in RAW264.7 mouse macrophage cells using quantitative real-time RT-PCR. The quantitative study revealed a variety of activation level of HO-1 gene by the chemicals. CAPE and caffeic acid ethyl ester (CAEE) at the final concentration of 2 muM drastically activated the HO-1 gene to 39.2-fold and 20.1-fold, respectively. Curcumin, structurally related with caffeic acid and an element of turmeric, induced the HO-1 gene to 5.8-fold. In contrast, no activation was observed by other caffeic acid esters such as chlorogenic acid and rosmarinic acid. Higher concentrations were necessary for the activation by an antioxidant cysteamine and the electrophile diethyl maleate. Although the inducible activities of CAPE and chlorogenic acid were distinctly different, they showed similar reductive capacities when determined by cyclic voltammetry. These results show that the drastic activation of HO-1 gene by CAPE and CAEE is dependent upon their chemical structures, rather than the reductive activity of polyphenols, possibly reflecting the physiological effects of the nutritional elements.     

Synthesis and cytotoxic activity of tetracenomycin D and of saintopin analogues.

Regiospecific synthesis of title compounds is based either on cycloaddition of ketene acetals derived from Hagemann's ester or of homophthalic anhydrides. Thus, tetracenomycin D and 3,8-di-O-methyl saintopin have been prepared in few steps. New derivatives of 10-deoxysaintopin have been also obtained. Evaluation of their cytotoxicity against L1210 leukemia cells are reported.     

Design, synthesis and bioevaluation of novel maleamic amino acid ester conjugates of 3,5-bisarylmethylene-4-piperidones as cytostatic agents

A novel series of maleamic amino acid ester conjugates of 3,5-bisarylmethylene-4-piperidones were prepared to investigate the efficacy of micronutrient conjugation in enhancing cytotoxic potency by improving selectivity and delivery. These compounds, prepared as anticancer agents, were expected to demonstrate enhanced selectivity towards malignant cells through the inhibition of topoisomerase IIα via protein thiolation. The cytostatic effects of these compounds were evaluated against three cell lines, namely murine L1210 leukemia cells, human Molt 4/C8 and CEM T-lymphocyte cells. All compounds were found to have greater potency than the reference drug melphalan. Several compounds were found to potently inhibit topoisomerase IIα and displayed cytostatic activity in the nanomolar range.     

Antileishmanial activity, cytotoxicity and QSAR analysis of synthetic dihydrobenzofuran lignans and related benzofurans

A series of synthetic dihydrobenzofuran lignans and related benzofurans were evaluated for their cytotoxicity in a screening panel consisting of various human tumour cell lines, and for their antiprotozoal activity against L. donovani (axenic amastigotes), chloroquine resistant Plasmodium falciparum (strain K1), Trypanosoma brucei rhodesiense and T. cruzi, and for cytotoxicity on L6 cells. No promising cytotoxicities against human tumour cell lines were observed for newly synthesised compounds, but the dimerisation product of some lipophylic esters of caffeic acid, such as compound 2g, showed a high activity against chloroquine-resistant P. falciparum (strain K1) (IC50 0.43 microg/mL) and L. donovani (axenic amastigotes) (IC50 0.12 microg/mL), which was confirmed in an infected macrophage assay (IC50 0.19 microg/mL). QSAR models for the cytotoxic and antileishmanial activity were generated using Quasar receptor surface modelling.     

Caffeic acid phenethyl ester protects mice from lethal endotoxin shock and inhibits lipopolysaccharide-induced cyclooxygenase-2 and inducible nitric oxide synthase expression in RAW 264.7 macrophages via the p38/ERK and NF-kappaB pathways

Caffeic acid phenethyl ester has been shown to have anti-inflammatory and anti-cancer effects. We examined the effects of caffeic acid phenethyl ester on lipopolysaccharide-induced production of nitric oxide and prostaglandin E(2), and expression of inducible nitric oxide synthase and cyclooxygenase-2 in RAW 264.7 macrophages. We also investigated the effects of caffeic acid phenethyl ester on lipopolysaccharide-induced septic shock in mice. Our results indicate that caffeic acid phenethyl ester inhibits lipopolysaccharide-induced nitric oxide and prostaglandin E(2) production in a concentration-dependent manner and inhibits inducible nitric oxide synthase and cyclooxygenase-2 in RAW 264.7 cells, without significant cytotoxicity. To further examine the mechanism responsible for the inhibition of inducible nitric oxide synthase and cyclooxygenase-2 expression by caffeic acid phenethyl ester, we examined the effect of caffeic acid phenethyl ester on lipopolysaccharide-induced nuclear factor-kappaB activation and the phosphorylation of mitogen-activated protein kinases. Caffeic acid phenethyl ester treatment significantly reduced nuclear factor-kappaB translocation and DNA-binding in lipopolysaccharide-stimulated RAW 264.7 cells. This effect was mediated through the inhibition of the degradation of inhibitor kappaB and by inhibition of both p38 mitogen-activated protein kinase and extracellular signal-regulated kinase phosphorylation, at least in part by inhibiting the generation of reactive oxygen species. Furthermore, caffeic acid phenethyl ester rescued C57BL/6 mice from lethal lipopolysaccharide-induced septic shock, while decreasing serum levels of tumor necrosis factor-alpha and interleukin-1beta. Collectively, these results suggest that caffeic acid phenethyl ester suppresses the induction of cytokines by lipopolysaccharide, as well as inducible nitric oxide synthase and cyclooxygenase-2 expression, by blocking nuclear factor-kappaB and p38/ERK activation. These findings provide mechanistic insights into the anti-inflammatory and chemopreventive actions of caffeic acid phenethyl ester in macrophages.     

Phorbol esters augment polyamine transport by influencing Na(+)-K+ pump in murine leukemia cells.

In this report, we elucidate the role of Na(+)-K+ pump in the regulation of polyamine spermidine (Spd) transport in murine leukemia (L 1210) cells in culture. Ouabain, known to bind extracellularly to the alpha-subunit of the Na(+)-K+ pump, inhibits the pump activity. The L 1210 cells were found to possess ouabain binding sites at 7.5 fmol/10(6) cells. Ouabain significantly inhibited the Spd uptake in a dose-dependent manner. The maximum inhibition of Spd uptake by ouabain was observed beyond 200 microM. Spd transport was inversely correlated with the [3H]ouabain binding to L 1210 cells: an increase in the saturation of ouabain binding to L 1210 cells resulted in a decrease of the Spd uptake process. Treatment of L 1210 cells with protein kinase C activator phorbol esters increased the Spd transport and, also, ouabain-sensitive 86Rb+ uptake, a measure of the activity of the Na(+)-K+ pump. H-7, a protein kinase C inhibitor, significantly inhibited the ouabain-sensitive 86Rb+ uptake by L 1210 cells. Phorbol esters stimulated the level, but not the rate, of 22Na+ influx. Addition of H-7 to L 1210 cells inhibited the 22Na+ influx process. A concomitant phorbol ester-induced increase in 22Na+ influx, [14C]Spd uptake, together with the functioning of Na(+)-K+ pump, indicates the role of the "Na+ cycle" in the regulation of the polyamine transport process.     

Elucidation of structure-activity relationships for 2- or 6-substituted-5,8-dimethoxy-1,4-naphthoquinones

1,4-Naphthoquinones have already been recognized to possess a wide range of biological activities. We have developed quantitative structure activity relationships (QSAR) for different series of 2- or 6-substituted-5,8-dimethoxy-1,4-naphthoquinones to understand the chemical-biological interaction governing antiproliferative/cytotoxic activities against L1210 cells. QSAR results have shown that these activities of 2- or 6-substituted-5,8-dimethoxy-1,4-naphthoquinones depend largely on their hydrophobicity.     

The role of N-nitrosourea-induced changes in the nucleoid structure and activity of repair enzymes in the development of drug resistance in mice with leukemia L1210

Using centrifugation of the nucleoid in a neutral sucrose gradient, the damages in the secondary structure of DNA and the activity of repair enzymes, such as DNA-polymerases alpha and beta and poly(ADP-riboso) polymerase, induced by 1-methyl-nitrosourea (MNU) and 1.3-bis (2-chloroethyl)-1-nitrosourea (BCNU) injected at maximal nonlethal single doses to mice bearing parent leukemia cells (L1210/0) and resistant to MNU and BCNU leukemia L1210 cells (L1210/MNU and L1210/BCNU), were studied. The MNU-induced production of single-strand breaks in L1210/0 and L1210/MNU cells was more conspicuous in newly replicated DNA than in those in preexisting DNA. A more fast repair of the damages in newly replicated DNA was detected in L1210/BCNU and especially in L1210/MNU leukemia cells as compared with L1210/0 cells. The data obtained suggest that there are prone errors in the repair of DNA template, since most of the single-strand breaks were revealed in the newly replicated DNA synthesized on the repaired DNA. The repair of DNA damages in L1210/BCNU and especially in L1210/MNU cells was accompanied by the activation of DNA-polymerases alpha and beta and poly(ADP-riboso)polymerase. Both DNA-polymerases--alpha and beta--were shown to be involved in repair of DNA damages induced by MNU and only DNA-polymerase beta was involved in the repair of damages induced by BCNU.     

Further studies on a novel class of genetic variants of the L1210 cell with increased folate analogue transport inward. Transport properties of a new variant, evidence for increased levels of a specific transport protein, and its partial characterization following affinity labeling

Studies are reported on the characterization of a new isolate within a novel class of variants of the L1210 cell exhibiting markedly increased transport inward of folate analogues. This variant (L1210/R83), which was selected in the presence of the antifolate metoprine, exhibited a 40-fold increase in [3H]aminopterin influx compared to parental cells and a modest (4-5-fold) increase in [3H]aminopterin efflux. The increase in influx was associated with a comparable increase in influx Vmax for the one-carbon, reduced folate transport system and the same increase in the amount of specific binding of [3H]aminopterin on the cell surface. Values for influx Km for [3H]aminopterin and specificity for various folate structures were unchanged. The alteration in influx Vmax and more rapid efflux accounted for the different level of intracellular exchangeable level of drug at steady state in this variant compared with parental L1210 cells. Otherwise, membrane potential was unchanged. The N-hydroxysuccinimide ester of [3H]aminopterin was used to covalently label the specific binding protein for folate compounds in the plasma membrane of variant and parental L1210 cells. Incorporation of label into this protein was stable under a variety of conditions and accounted for 97 and 52% of total cellular labeling, respectively, for membrane derived from R83 and parental L1210 cells at a reagent concentration of 20 nM. Specific affinity labeling on the surface of parental and variant cells was decreased in the presence of aminopterin, methotrexate, or 5-formyltetrahydrofolate, but not in the presence of folic acid. Also, [3H]aminopterin influx in these cells was inhibited by the N-hydroxysuccinimide ester of aminopterin or methotrexate, but not the N-hydroxysuccinimide ester of folic acid. These findings, in addition to the increased affinity labeling of this variant, which corresponds to the increase in influx of [3H] aminopterin also seen, appears to identify the affinity labeled protein as a component of the "classical" one-carbon, reduced folate transport system in these cells. The affinity labeled protein from each cell type was solubilized in sodium dodecyl sulfate or extracted in detergent in the presence of proteinase inhibitors and was found to elute from Sephacryl S-300 and migrate during sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single peak of Mr = 45,000-48,000. Recovery of labeled binding protein in these fractions from R83 variant cells was approximately 40 times greater than that from parental cells.(ABSTRACT TRUNCATED AT 400 WORDS)