A new unbiased and highly automated approach to find new prognostic markers in preclinical research.

Data acquisition in (pre)clinical studies is often based on a hypothesis. Numerical algorithms, however, may help to find biomarkers from existing data without formulating any hypothesis. By simply assessing whether a statistical relationship exists between two parameters from a (unlimited) database, every (in)conceivable combination of data becomes a hypothesis. The aim was to create an unbiased and highly automated approach for secondary analysis of (pre)clinical research, including the possibility of a non-linear functional relationship. In our example, an almost homogeneous database was formed by overall 45 parameters (vital, blood and plasma parameters) measured in 11 individual experimental studies at 6 different time points using 57 rats without and 63 rats with systemic inflammation following lipopolysaccharide infusion. For each rat, four group classifiers (treatment, survival, study, ID) were used to get valid samples by a later filtering of the statistical base. Any information about the hypothesis leading to the respective studies was suppressed. In order to assess whether a statistical relationship exists, a total of six different functional prototypes (linear and non-linear) were postulated and examined for their regression. Regression quality, correlation and significance were obtained in form of matrices. In our example, ultimately 510 300 regressions were optimized, automatically evaluated and filtered. The developed algorithm is able to reveal statistical relationships from a nearly crude database with low effort by systematic and unbiased analysis. The finding of well-known correlations proves its reliability, whose validity could be increased by clean aggregation of different studies. In addition, new interesting hints for future research could be gained. Thus, unknown markers can be found which are associated with an increased risk of death during systemic inflammation and sepsis. A further development of the program is planned including multiple regressions (more than two parameters could be related to each other) or cluster analysis.

Lipopolysaccharide-Induced Hemolysis Is Abolished by Inhibition of Thrombin Generation but Not Inhibition of Platelet Aggregation.

In human sepsis, hemolysis is an independent predictor of mortality, but the mechanisms evoking hemolysis have not been fully elucidated. Therefore, we tested the hypotheses that (1) lipopolysaccharide (LPS)-induced hemolysis is dependent on thrombin generation or platelet aggregation and (2) red cell membranes are weakened by LPS. Anesthetized male Wistar rats were subjected to LPS or vehicle for 240 min. The effects of hemostasis inhibition on LPS-induced hemolysis were investigated by use of the thrombin inhibitor argatroban or the platelet function inhibitor eptifibatide. Free hemoglobin concentration, red cell membrane stiffness and red cell morphological changes were determined by spectrophotometry, atomic force microscopy, and light microscopy. Efficacy of argatroban and eptifibatide was assessed by rotational thrombelastometry and impedance aggregometry, respectively. LPS markedly increased free hemoglobin concentration (20.8 µmol/l ± 3.6 vs. 3.5 ± 0.3, n = 6, p < 0.0001) and schistocytes, reduced red cell membrane stiffness, and induced disseminated intravascular coagulation. Inhibition of thrombin formation with argatroban abolished the increase in free hemoglobin concentration, schistocyte formation, and disseminated intravascular coagulation in LPS-treated animals. Eptifibatide had no inhibitory effect. The LPS evoked decrease of red cell stiffness that was not affected by argatroban or eptifibatide. LPS causes hemolysis, schistocyte formation, and red cell membrane weakening in rats. The thrombin inhibitor argatroban but not the platelet inhibitor eptifibatide abolished hemolysis and schistocyte formation. Thus, LPS-induced hemolysis depends on disseminated intravascular coagulation, possibly enhanced by red cell membrane weakening. Clinical studies are necessary to investigate whether thrombin antagonists can decrease hemolysis and mortality in sepsis.

Slow as Compared to Rapid Rewarming After Mild Hypothermia Improves Survival in Experimental Shock.

BACKGROUND: Accidental hypothermia following trauma is an independent risk factor for mortality. However, in most experimental studies, hypothermia clearly improves outcome. We hypothesized that slow rewarming is beneficial over rapid rewarming following mild hypothermia in a rodent model of hemorrhagic shock. MATERIALS AND METHODS: We subjected 32 male Wistar rats to severe hemorrhagic shock (25-30 mmHg for 30 min). Rats were assigned to four experimental groups (normothermia, hypothermia, rapid rewarming [RW], and slow RW). During induction of severe shock, all but the normothermia group were cooled to 34°C. After 60 min of shock, rats were resuscitated with Ringer's solution. The two RW groups were rewarmed at differing rates (6°C/h versus 2°C/h). RESULTS: Slow RW animals exhibit a significantly prolonged survival compared with the rapid RW animals (P < 0.05). Nevertheless, hypothermic animals show a significant survival benefit as compared to all other experimental groups. Whereas seven animals of the hypothermia group survived to the end of the experiment, none of the other animals did (P < 0.001). No significant differences were found regarding acid base status, metabolism, parameters of organ injury, and coagulation. CONCLUSIONS: The results indicate that even slow RW with 2°C/h may be still too fast in the setting of experimental hemorrhage. Too rapid rewarming may result in a loss of the protective effects of hypothermia. As rewarming is ultimately inevitable in patients with trauma, potential effects of rewarming on patient outcome should be further investigated in clinical studies.

Protective Effects of Sodium Pyruvate during Systemic Inflammation Limited to the Correction of Metabolic Acidosis.

Protective effects by exogenous sodium pyruvate already have been described in various experimental models of injury, among others during intestinal ischemia-reperfusion injury, hemorrhagic shock, and shock secondary to systemic inflammation (endotoxemic shock). Low doses of sodium pyruvate reduced signs of inflammation, enhanced systemic blood pressure, and ameliorated metabolic acidosis when administered in a prophylactic manner during endotoxemic shock. In the present study, we investigated whether low-dosed infusions of sodium pyruvate exhibited beneficial effects when applied therapeutically after the induction of systemic inflammation. Lipopolysaccharide was infused at a rate of 0.5 mg/kg × h over a period of 360 min to induce systemic inflammation in male Wistar rats. Sodium pyruvate (single dose 50 mg/kg × 15 min) was administered intravenously 180 and 270 min after starting of the lipopolysaccharide infusion. Systemic/vital parameters (e.g., systemic blood pressure and breathing rate) and blood/plasma parameters (e.g., acid-base parameters; electrolytes; glucose and lactate concentration; hemolysis; aminotransferase activities; and parameters of coagulation) were determined in regular intervals. Lipopolysaccharide infusion led to metabolic acidosis, hypoglycemia, electrolyte as well as hemostatic disturbances, and hemolysis. Except for the acid-base status (amelioration of metabolic acidosis) and the plasma chloride concentration (reduction of hyperchloremia), the additional infusion of sodium pyruvate failed in significantly improving lipopolysaccharide-dependent alterations (e.g. vital, blood and plasma parameters). Protective effects of a delayed administration of the metabolizable anion pyruvate during systemic inflammation, hence, are limited to its function as alkalizer to counteract metabolic acidosis.

Therapeutic effects of physostigmine during systemic inflammation.

INTRODUCTION: Usually, physostigmine is used as antidote for anticholinergic poisons in order to improve hemodynamics and cardiac output. In addition, it causes beneficial effects during sepsis when added timely. Here, we studied whether physostigmine improves hemodynamics when treatment during systemic inflammation was delayed. METHODS: Two series of randomized studies with overall 44 rats were conducted. Systemic inflammation was induced by lipopolysaccharide (LPS) infusion (0.5 mg LPS/kg×h). Physostigmine (PHY) was intravenously applied after an LPS infusion period of 90 minutes (50 µg PHY/kg within 10 minutes) with (series 1) and without (series 2) additional volume loading. Hemodynamic parameters, blood gases, and parameters for tissue damage were periodically determined for up to 180 minutes. RESULTS: Even though volume was additionally administered (series 1), LPS caused a reduction of peripheral blood flow. Treatment with PHY improved hemodynamics in macrocirculation (mean arterial blood pressure) and microcirculation (peripheral blood flow). PHY neither affected alterations in blood gases, electrolyte homeostasis, and glucose metabolism nor prevented intestinal damage induced by LPS. In series 2, without any additional volume loading, PHY likewise resulted in an improvement of the LPS-induced alterations in macro- and microcirculation, but finally worsened the LPS-mediated effects on plasma parameters for tissue damage such as creatine kinase, probably due to the lack of volume and a further damage to the heart. CONCLUSION: The present results demonstrated that hemodynamic responses to PHY may not only be visible in patients with anticholinergic drug overdose but also be visible in septic patients, provided that fluid intake of these patients is adequate.

Mechanisms of Hemolysis During Sepsis.

Cell-free hemoglobin is increasingly playing a more central role in the pathogenesis of sepsis being proved to be a potent predictor of patient's outcome. It is crucial, hence, to further investigate the mechanisms of sepsis-induced hemolysis with the aim of deriving possible therapeutic principles. Herein, we collected the most important previously known triggers of hemolysis during sepsis, which are (1) transfusion reactions and complement activation, (2) disseminated intravascular coagulation, (3) capillary stopped-flow, (4) restriction of glucose to red blood cells, (5) changes in red blood cell membrane properties, (6) hemolytic pathogens, and (7) red blood cell apoptosis. Graphical Abstract ᅟ.

Moderate glucose supply reduces hemolysis during systemic inflammation.

BACKGROUND: Systemic inflammation alters energy metabolism. A sufficient glucose level, however, is most important for erythrocytes, since erythrocytes rely on glucose as sole source of energy. Damage to erythrocytes leads to hemolysis. Both disorders of glucose metabolism and hemolysis are associated with an increased risk of death. The objective of the study was to investigate the impact of intravenous glucose on hemolysis during systemic inflammation. MATERIALS AND METHODS: Systemic inflammation was accomplished in male Wistar rats by continuous lipopolysaccharide (LPS) infusion (1 mg LPS/kg and h, 300 min). Sham control group rats received Ringer's solution. Glucose was supplied moderately (70 mg glucose/kg and h) or excessively (210 mg glucose/kg and h) during systemic inflammation. Vital parameters (eg, systemic blood pressure) as well as blood and plasma parameters (eg, concentrations of glucose, lactate and cell-free hemoglobin, and activity of lactate dehydrogenase) were measured hourly. Clot formation was analyzed by thromboelastometry. RESULTS: Continuous infusion of LPS led to a so-called post-aggression syndrome with disturbed electrolyte homeostasis (hypocalcemia, hyperkalemia, and hypernatremia), changes in hemodynamics (tachycardia and hypertension), and a catabolic metabolism (early hyperglycemia, late hypoglycemia, and lactate formation). It induced severe tissue injury (significant increases in plasma concentrations of transaminases and lactate dehydrogenase), alterations in blood coagulation (disturbed clot formation), and massive hemolysis. Both moderate and excessive glucose supply reduced LPS-induced increase in systemic blood pressure. Excessive but not moderate glucose supply increased blood glucose level and enhanced tissue injury. Glucose supply did not reduce LPS-induced alterations in coagulation, but significantly reduced hemolysis induced by LPS. CONCLUSION: Intravenous glucose infusion can diminish LPS-related changes in hemodynamics, glucose metabolism, and, more interestingly, LPS-induced hemolysis. Since cell-free hemoglobin is known to be a predictor for patient's survival, a reduction of hemolysis by 35% only by the addition of a small amount of glucose is another step to minimize mortality during systemic inflammation.

Administration of Exogenous Melatonin After the Onset of Systemic Inflammation Is Hardly Beneficial.

Melatonin improves survival and functional impairment including hemolysis, thrombocytopenia, and hypotension when administered in a prophylactic manner or early after initiation of sepsis or endotoxemia. In the present study, melatonin was given not before first symptoms of systemic inflammation became manifest. Lipopolysaccharide was infused at a rate of 0.5 mg/kg × h to induce systemic inflammation in male Wistar rats. Melatonin (single dose 3 mg/kg × 15 min) was intravenously administered 180 and 270 min after starting of the lipopolysaccharide infusion. Systemic and vital parameters (e.g., systemic blood pressure and breathing rate) as well as blood and plasma parameters (acid-base parameters; electrolytes; parameters of tissue injury such as glucose concentration, lactate concentration, hemolysis, and aminotransferase activities; parameters of thromboelastometry; and platelet count) were determined in regular intervals. Infusion of lipopolysaccharide led to characteristic symptoms of severe systemic inflammation including hypotension, metabolic acidosis and hypoglycemia, electrolyte and hemostatic disturbances, thrombocytopenia, and hemolysis. Melatonin neither decreased mortality nor reduced lipopolysaccharide-dependent changes to vital, blood, and plasma parameters. Even though melatonin may have a beneficial effect in early stages of systemic inflammation, it can hardly be an option in therapy of manifest sepsis or endotoxemia in an intensive care unit.

Melatonin reduces changes to small intestinal microvasculature during systemic inflammation.

BACKGROUND: Systemic inflammation is known to impair the microcirculation in intestine and other organs as a result of multifactorial events. Here, we show that melatonin selectively reduces changes to the small intestinal microvasculature during systemic inflammation. MATERIALS AND METHODS: Lipopolysaccharide (LPS) was infused at a rate of 0.5 mg/kg × h to induce systemic inflammation in male Wistar rats. Melatonin (single dose: 3 mg/kg × 15 min) was intravenously administered before as well as 120 and 240 min after the beginning of the LPS infusion. Systemic parameters were determined in regular intervals. Small intestine, liver, and kidney were histologically (structure of the microvessels, intravascular blood accumulation, and hemorrhages) and immunohistochemically (mast cells, granulocytes, and macrophages) analyzed. RESULTS: Continuous infusion of LPS resulted in dilated microvessels with intravascular blood accumulation (congestion) in liver and small intestine, the latter being particularly pronounced. Blood vessel walls remained intact, there were no hemorrhages. Melatonin significantly reduced these changes to the microvasculature in small intestine, but not in liver. It further reduced mast cell and granulocytes count in small intestine enhanced by LPS. However, except for the systemic blood pressure, melatonin neither improved LPS-dependent changes to systemic parameters nor mortality. CONCLUSIONS: Changes to the microvasculature during systemic inflammation are most pronounced in small intestine. Melatonin selectively diminishes these changes to small intestinal microvasculature, probably by reducing the local immune cells recruitment. However, changes to the small intestine are not decisive for the survival. We assume that the therapeutic benefit of melatonin is more likely in local intestinal inflammation.

Lipopolysaccharide-induced hemolysis: Evidence for direct membrane interactions.

While hemolysis in patients with sepsis is associated with increased mortality its mechanisms are unknown and Toll-like receptor (TLR)-4 mediated effects, complement-mediated hemolysis, or direct cell membrane effects are all conceivable mechanisms. In this study, we tested the hypotheses that toxic lipopolysaccharide (LPS) as well as non-toxic RS-LPS evokes hemolysis (1) by direct membrane effects, and (2) independent of the complement system and TLR-4 activation. We found, that incubation with LPS resulted in a marked time and concentration dependent increase of free hemoglobin concentration and LDH activity in whole blood and washed red cells. Red cell integrity was diminished as shown by decreased osmotic resistance, formation of schistocytes and rolls, and a decrease in red cell membrane stiffness. Non-toxic RS-LPS inhibited the LPS-evoked increase in TNF-α concentration demonstrating its TLR-4 antagonism, but augmented LPS-induced increase in supernatant hemoglobin concentration and membrane disturbances. Removal of plasma components in washed red cell assays failed to attenuate hemolysis. In summary, this study demonstrates direct physicochemical interactions of LPS with red cell membranes resulting in hemolysis under in vitro conditions. It might thus be hypothesized, that not all effects of LPS are mediated by TLR and may explain LPS toxicity in cells missing TLR.

Melatonin does not affect disseminated intravascular coagulation but diminishes decreases in platelet count during subacute endotoxaemia in rats.

INTRODUCTION: Inhibitory effects of exogenous melatonin (MLT) on plasma coagulation and platelet aggregation have already been observed in vivo and in vitro under normal conditions. Here, we studied whether MLT also diminishes the lipopolysaccharide (LPS)-induced disseminated intravascular coagulation (DIC) during subacute endotoxaemia. MATERIALS AND METHODS: Subacute endotoxaemia was induced in male Wistar rats by an intravenous infusion of LPS over a period of 300min (0.5mg LPS/kg×h). MLT was administered intravenously 15min before and 120min and 240min after starting of the LPS infusion (3×3mg MLT/kg×15min). The kinetic of clot formation was analysed by thromboelastometry. RESULTS: Infusion of LPS led initially to a significant reduction of clotting time (120min, LPS: 150±21s vs. SHAM: 292±36s), and finally a significant increase of clotting time (300min, LPS: 2768±853s vs. SHAM: 299±67s) and a slight increase of clot formation time (300min, LPS: 1038±657s vs. SHAM: 98±14s) as well as a significant decrease of alpha-angle (300min, LPS: 35±15° vs. SHAM: 72±3°), maximum clot firmness (300min, LPS: 22±6mm vs. SHAM: 68±3mm), and area under the curve (300min, LPS: 1657±552mm×100 vs. SHAM: 6849±307mm×100). Simultaneously, a decrease of platelet count (300min, LPS: 55±8 vs. SHAM: 180±55) and a release of cell-free haemoglobin (240min, LPS: 46±5µmol/L vs. SHAM: 16±2µmol/L) could be observed in the course of subacute endotoxaemia. The additional administration of MLT did not reduce the LPS-induced alterations in parameters of thromboelastometry, but significantly reduced the LPS-induced decrease of platelet count (300min, LPS+MLT: 130±10) and release of cell-free haemoglobin (240min, LPS+MLT: 29±3µmol/L). CONCLUSION: Melatonin does not affect DIC but diminishes thrombocytopenia and haemolysis during endotoxaemia.

Impact of bacterial endotoxin on the structure of DMPC membranes.

Bacterial lipopolysaccharides are believed to have a toxic effect on human cell membranes. In this study, the influence of a lipopolysaccharide (LPS) from Escherichia coli on the structure, the dynamics and the mechanical strength of phospholipid membranes are monitored by nuclear magnetic resonance spectroscopy (NMR) and by atomic force microscopy (AFM). Model membranes are formed from 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and are either prepared as multilamellar bulk samples or multilamellar vesicles. Field gradient NMR data directly prove the rapid integration of LPS into DMPC membranes. Solid state NMR experiments primarily detect decreasing molecular order parameters with increasing LPS content. This is accompanied by a mechanical softening of the membrane bilayers as is shown by AFM indentation measurements. Altogether, the data prove that lipopolysaccharide molecules quickly insert into phospholipid bilayers, increase membrane fluctuation amplitudes and significantly weaken their mechanical stiffness.

Glycine selectively reduces intestinal injury during endotoxemia.

BACKGROUND: Glycine is well known to protect the intestine against ischemia-reperfusion injury and during mechanical manipulation. Here, we studied whether glycine protects the small intestine during endotoxemia, even without being the site of the infection. MATERIALS AND METHODS: Lipopolysaccharide (LPS) was infused at a rate of 1 mg/kg × h over a period of 7 h (subacute endotoxemia) in male Wistar rats. Glycine (single dose: 50 mg/kg × 15 min) was applied intravenously at 180 and 270 min after the beginning of the LPS infusion. Systemic parameters were periodically determined. The small intestine was analyzed for macroscopic (hemorrhages) and histopathologic changes (hematoxylin and eosin staining), and markers of inflammation (myeloperoxidase activity). RESULTS: Glycine neither decreased mortality nor beneficially affected vital parameters (e.g., mean arterial blood pressure and breathing rate), electrolytes, blood gases including pH and base excess, and plasma parameters of tissue injury such as lactate concentration, hemolysis, and aminotransferases activities during experimental endotoxemia. It, however, specifically diminished the LPS-induced small intestinal injury, as indicated by less intestinal accumulation of blood, less intestinal hemorrhages, and reduced intestinal hemoglobin content. CONCLUSIONS: The present results demonstrate that glycine selectively protects the small intestine during subacute endotoxemia, even after manifestation of a severe systemic impairment. Because glycine is non-toxic at low doses, an administration of a moderate glycine dose (50-100 mg/kg) may be suitable to protect from intestinal damage during sepsis. Its true clinical potential, however, needs to be verified in further experimental studies and clinical trials.

Immune stimulation by exogenous melatonin during experimental endotoxemia.

Melatonin has been shown to enhance the immune response under immune-compromised conditions. However, its immune-modulatory effects under inflammatory conditions are unclear at present. Both pro- and anti-inflammation has been reported. To study time-dependent effects of melatonin on the general immune response during endotoxemia in more detail, we used two models in male rats: per-acute endotoxemia was induced by lipopolysaccharide (LPS) bolus injection (2.5 mg/kg), sub-acute endotoxemia by LPS infusion (2.5 mg/kg × h). Melatonin was applied directly before and 2 h after LPS administration (3 mg/kg, each). The LPS-induced formation of the pro-inflammatory cytokines tumor necrosis factor alpha, interferon-gamma, interleukin (IL)-1α/ß, IL-5, and IL-6 and of the anti-inflammatory cytokine IL-10 was further amplified by melatonin, although it was only significant during per-acute endotoxemia. In both models, melatonin had no effect on the LPS-induced nitric oxide release. These findings show that exogenous melatonin is capable of enhancing the general immune response under inflammatory conditions.

Modification of uptake and subcellular distribution of doxorubicin by N-acylhydrazone residues as visualised by intrinsic fluorescence.

PURPOSE: Doxorubicin (1) is commonly used in the treatment of a wide range of cancers. Some N-acylhydrazones of 1 were previously found to have an improved tumour and organ selectivity. In order to clarify the molecular basis for this effect, the cellular uptake into various cancer cells and the localisation in PtK(2) potoroo kidney cells of 1 and its N-acylhydrazones derived from heptadecanoic acid (2) and 11-(menthoxycarbonyl)undecanoic acid (3) were studied drawing on their intrinsic fluorescence. METHODS: The uptake of compounds 1-3 into human cells of HL-60 leukaemia, 518A2 melanoma, HT-29 colon, and resistant KB-V1/Vbl and MCF-7/Topo breast carcinomas was determined fluorometrically from their residual amounts in the supernatant. Their time-dependent accumulation in PtK(2) potoroo kidney cells was visualised by fluorescence microscopy. RESULTS: The uptake, though not the cytotoxicity, of 2 in multi-drug resistant MCF-7/Topo breast cancer cells was conspicuously greater than that of 1 and 3, probably due to an attractive lipophilic interaction with the lipid-rich membranes of these cells. In non-malignant PtK(2) cells, both 1 and 3 accumulated initially in the nuclei. Upon prolonged incubation, their fluorescent metabolites were visualised in lysosomes neighbouring the nuclei. In contrast, conjugate 2 was not observed in the nuclei at any time. After 2 h, it had accumulated in vesicles scattered all over the cells, and upon prolonged incubation, its fluorescent metabolites were concentrated in the cellular membrane. CONCLUSIONS: Long unbranched fatty acyl residues when attached to doxorubicin via a hydrazone can act as lipophilic membrane anchors. This allows an increased uptake of such derivatives into lipid-rich membranes especially of multi-drug resistant cancer cells, a retarded release from there into the cytosol and the eventual storage of their metabolites again in the cell membrane rather than in lysosomes.

Cancer selective metallocenedicarboxylates of the fungal cytotoxin illudin M.

The diester 2a obtained from 1,1'-ferrocenedicarboxylic acid and the highly and indiscriminately cytotoxic fungal metabolite illudin M (1) displayed antiproliferative activity at submicromolar IC(50) (72 h) values against a panel of eight cancer cell lines. Compound 2a was about 40 times less toxic than 1 to nonmalignant human foreskin fibroblasts (HF). The analogous bis(illudinyl M) 1,1'-ruthenocenedicarboxylate (2b) exhibited submicromolar IC(50) (72 h) values only against MDA-MB-231 and MCF-7/Topo breast carcinoma and HL-60 leukemia cells. Cytotoxicity studies in the presence of inhibitors of c-Jun N-terminal kinase (JNK) or extracellular signal-regulated kinase (ERK) revealed that the high efficacy of 2a, but not that of 2b, against HCT-116 colon cancer cells depends on active JNK/ERK signaling. A new illudin M lactone 5 was of low anticancer activity, but its ruthenocene diester 6b also reached single-digit micromolar IC(50) (72 h) values in HCT-116, MCF-7, and HL-60 leukemia cells while not affecting HF. Compounds 2a and 6b were tolerated by mice symptom-free at single doses as high as 25 mg/kg body weight, which is evidence for them being chemically stable under physiological conditions. Compound 2a displayed a manageable in vivo toxicity profile when given repeatedly in high doses.

Cellular localisation of antitumoral 6-alkyl thymoquinones revealed by an alkyne-azide click reaction and the streptavidin-biotin system.

The subcellular distribution and accumulation of thymoquinone 1, a natural anticancer agent, has hitherto been unknown. We prepared 6-(dec-9-ynyl)thymoquinone 3, an alkyne-labelled derivative with anticancer activity similar to that of its parent compound 1. Alkyne 3 was seen, after a Huisgen-type click reaction with 3-azido-7-hydroxycoumarin, to accumulate in distinct compartments of the nuclei of PtK(2) potoroo kidney cells, and in adjoining regions that were stained with an antibody specific for the Golgi apparatus. In contrast, a biotinlabelled thymoquinone 4 seemed to accumulate across the entire cell nucleus upon visualisation with streptavidin; but this was less easily traceable because of co-staining of other structures such as mitochondria. In conclusion, for small drug-like molecules, visualisation by alkyne-azide cycloaddition seems to be superior to conventional visualisation by the biotin-streptavidin system.

Synthesis, anticancer activity, and iron affinity of the Actinoplanes metabolite 7,8-dihydroxy-1-methylnaphtho[2,3-c]furan-4,9-dione.

The first synthesis of 7,8-dihydroxy-1-methylnaphtho[2,3-c]furan-4,9-dione (1), an isofuranonaphthoquinone produced by an Actinoplanes strain is described. Lactone ring opening of 6-methylfuro[3,4-c]furan-1(3H)-one (4) with ortho-lithiated veratrole (3), oxidation of product alcohol 5, and Friedel-Crafts acylation of the resulting aroylcarboxylic acid 7 afforded the mono methyl ether 2 of the target compound. The latter was obtained by demethylation of 2 with BBr(3) in 14% overall yield. While mono ether 2 was distinctly more cytotoxic than catechol 1 against a panel of five cancer cell lines, only the latter showed a siderophore-like binding affinity for Fe(III) with a complex dissociation constant K(D) of approximately 10(-29) M(3) (pM = 25.9).

Combinatorial effects of thymoquinone on the anti-cancer activity of doxorubicin.

PURPOSE: Doxorubicin is a mainstay of cancer chemotherapy despite its clinical limitations that arise from its cardiotoxicity and the high incidence of multi-drug resistance. Recent studies revealed a protective effect of thymoquinone, a non-toxic constituent of the essential oil of Nigella sativa, against doxorubicin-induced cardiotoxicity. We now investigated the influence of thymoquinone on various other effects exerted by doxorubicin in human cancer cells. METHODS: Doxorubicin, thymoquinone and equimolar mixtures of both were tested for cytotoxicity on human cells of HL-60 leukaemia, 518A2 melanoma, HT-29 colon, KB-V1 cervix, and MCF-7 breast carcinomas as well as multi-drug-resistant variants thereof and on non-malignant human fibroblasts (HF). Apoptosis induction was analysed via DNA fragmentation, activity studies of the caspases-3, -8 and -9, determination of changes in the mitochondrial membrane potential and in the ratio of the mRNA expressions of pro- and anti-apoptotic proteins bax and bcl-2. The generation of reactive oxygen species (ROS) was assessed by the NBT assay. RESULTS: Thymoquinone improved the anti-cancer properties of doxorubicin in a cell line-specific manner. We found a significant rise of the growth inhibition by doxorubicin in HL-60 and multi-drug-resistant MCF-7/TOPO cells when thymoquinone had been added. The mode of action of both drugs and of their mixture was mainly apoptotic. In HL-60 cells, the drug mixture caused an additional concentration maximum of effector caspase-3 not observed for either of the pure drugs. The impact of the drug mixture on the mitochondria of HL-60 cells was also greater than those of the individual quinones alone. In addition, the drug mixture led to a higher concentration of reactive oxygen species in HL-60 cells. CONCLUSIONS: In summary, thymoquinone is a booster for the anti-cancer effect of doxorubicin in certain cancer cell lines. Distinct improvements on efficacy, selectivity, and even breaches of multi-drug resistance were observed for equimolar mixtures of doxorubicin and thymoquinone.