Cytotoxic gold(iii) complexes incorporating a 2,2':6',2''-terpyridine ligand framework - the impact of the substituent in the 4'-position of a terpy ring.

Two gold(iii) complexes incorporating 2,2':6',2''-terpyridine derivatives have been synthesised and characterized, and the possibility of tuning the cytotoxic activity by structural modifications of a terpy ligand has been examined. Both complexes [AuCl(4'-R1-terpy)](PF6)2 (1) and [AuCl(4'-R2-terpy)](PF6)2 (2), where R1 is 2-pyridyl and R2 is 3-pyridyl, show good anti-proliferative activities against HCT116, which are higher in relation to those of the free ligands, [AuCl(terpy)](PF6)2 and standard anticancer drug cisplatin. The cytotoxic properties of the gold complexes were examined by MTS assay, cell cycle and apoptosis analysis, ROS measurements, determination of mitochondrial membrane potential and mass, and staining of phosphatidylserine with Annexin-V antibody FITC-conjugated together with PI.

In Vitro Evaluation of Doxorubicin Conjugates Based on Sugar Core Nonlinear Polymethacrylates toward Anticancer Drug Delivery.

V-shaped and star-shaped hydroxylamine-functionalized polymethacrylates designed as nanosized conjugates (<120 nm) with anticancer agent, namely, doxorubicin (DOX), were evaluated in vitro toward their potential usage as drug delivery systems in breast cancer (MCF-7) treatment. Statistical analysis of MTS assay results showed that the 4-arm conjugate (n(DOX) = 16) was the most effective polymeric system against MCF-7/W (wild type) and MCF-7/R (DOX resistant) cell lines. Apoptosis assay analysis showed that MCF-7/R cells cultured with nonlinear copolymers died due to necrosis and late apoptotis, whereas MCF-7/W cells were in early and late apoptosis. Among all tested conjugates, the most promising results with induction of apoptosis without inducing necrosis in both MCF-7 cell lines were obtained for conjugate based on 4-arm stars with low content of DOX. The cell cycle assay revealed that increase of MMA units in 4-arm copolymers induced MCF-7/R cell arrest in the SubG1 phase. In the same cell line, the corresponding conjugates triggered S and G2/M arrest. Gradual internalization of the chosen conjugate by MCF-7/R cells was monitored via fluorescence microscopy showing its main localization in the cytoplasm.

Viability and oxidative response of human colorectal HCT-116 cancer cells treated with visfatin/eNampt in vitro.

Visfatin/eNampt is a novel adipokine, secreted by visceral and subcutaneous fat, which could be involved in the development of obesity-associated cancer. Only few studies revealed reactive oxygen species (ROS)-dependent action of visfatin in endothelial cells, myotubes and melanoma cells. The potential pro-apoptotic properties of visfatin/eNampt in human colorectal HCT-116 cells remain unknown. The aim of the study was to examine the effects of visfatin/eNampt on cell viability along with the determination of apoptosis/necrosis extent and ROS level in HCT-116 cells. Additionally antioxidant enzymes' activities (i.e catalase (CAT), gluthatione peroxidase (GSH-Px)), and lipid peroxidation intensity in HCT-116 cells line was evaluated. Viability of HCT-116 cells was decreased after visfatin/eNampt treatment for 24 hours. The number of apoptotic cells in tested cells treated with increasing visfatin/eNampt concentrations (10, 100, 250 ng/ml) was elevated compared to untreated cells (6.4%, 9.7%, 16% vs. 3.2%; respectively). After 24 hours in the visfatin/eNampt treated group (10 - 100 ng/ml) CAT and GSH-Px activities significantly increased and this observation was accompanied by the decrease of ROS level when compared to the control group. Interestingly ROS level (using DCF detection technique) and lipid peroxidation ratio were increased in cells stimulated by visfatin/eNampt in concentration of 250 ng/ml along with the decreased activity of selected antioxidant enzymes when compared to remaining study groups, including control. We concluded that visfatin/eNampt induces decrease of cell viability and apoptosis boost in human colorectal cancer HCT-116 cells line. Visfatin/eNampt affected the level of ROS as well as antioxidant capacity, however the association of ROS level and apoptosis rate was not linear. The role for visfatin/eNampt in cancer redox status in vitro may provide a greater insight into the association between fat derived visfatin/eNampt and its endocrine action in colorectal carcinoma cells.

Changes in subcellular localization of visfatin in human colorectal HCT-116 carcinoma cell line after cytochalasin B treatment.

The aim of the study was to assess the expression and subcellular localization of visfatin in HCT-116 colorectal carcinoma cells after cytokinesis failure using Cytochalasin B (CytB) and the mechanism of apoptosis of cells after CytB. We observed translocation of visfatin's antigen in cytB treated colorectal carcinoma HCT-116 cells from cytosol to nucleus. Statistical and morphometric analysis revealed significantly higher area-related numerical density visfatin-bound nano-golds in the nuclei of cytB-treated HCT-116 cells compared to cytosol. Reverse relation to visfatin subcellular localization was observed in un-treated HCT-116 cells. The total amount of visfatin protein and visfatin mRNA level in HCT-116 cells was also decreased after CytB treatment. Additionally, CytB significantly decreased cell survival, increased levels of G2/M fractions, induced bi-nuclei formation as well as increased reactive oxygen species (ROS) level in HCT-116 cells. CytB treatment showed cytotoxic effect that stem from oxidative stress and is connected with the changes in the cytoplasmic/nuclear amount of visfatin in HCT-116 cells.

Exogenous administration of visfatin affects cytokine secretion and increases oxidative stress in human malignant melanoma Me45 cells.

Visfatin has recently been established as a novel adipokine that is predominantly expressed in visceral fat. Recombinant visfatin has immunomodulating properties, which can activate human leukocytes in vitro to induce cytokine production (IL-1ß, TNF-α, and IL-6). Only few studies have investigated the effect of visfatin on prostate, breast, ovarian cancer as well as astrocytoma cell biology. There have been no studies on the cytokine secretion in human melanoma cells in response to visfatin stimulation along with intracellular protein kinases inhibitors. ELISA assay was performed in supernatants of Me45 cells stimulated with visfatin in the presence or the absence of specific pharmacological inhibitors of the indicated protein kinases (p38, MEK 1, PI3k and JAK kinase) and nuclear factor kappa B (NK-κB) inhibitor. Intracellular reactive oxygen species level was measured in 2', 7'-dichlorodihydrofluorescein diacetate (H2DCF-DA)-loaded cells using a fluorescent measurement system. For determination of NF-κB activation, activated NF-κB p65 subunit was determined using an EZ-TFA-detect chemiluminescent transcription factor assay. We report that visfatin led to the significant increase in IL-6 and IL-8 level in culture supernatants of human malignant melanoma Me45 cells. Additionally visfatin resulted in the increase of the intracellular reactive oxygen species level. PI3k and NF-κB pathways were activated upon visfatin stimulation. The results may reflect the fact that PI3k pathway stimulation by visfatin may further lead to NF-κB activation and pro-inflammatory response.

Role of allatostatin-like factors from the brain of Tenebrio molitor females.

The effect of brain extract from females of freshly emerged Tenebrio molitor on ovary, oocyte development, total protein content of hemolymph, and ovary was studied in 4-day-old adult mealworm females. Injections of extracts of 2-brain equivalents into intact (unligatured) Tenebrio females did not affect ovarian and oocyte development. Injections of ligated females, however, with 2-brain equivalents on day 1 and 2 after adult emergence strongly inhibited ovarian growth and oocyte development. At day 4, ligated and injected females did not develop their ovaries and pre-vitellogenic oocytes were not found. The changes in ovarian development correlated with an increase in the concentration of soluble proteins in the hemolymph as compared with the saline-injected controls. Additionally, a strong reduction of total protein content in ovarian tissue was observed. Reverse phase HPLC separation of a methanolic brain extract of T. molitor females showed that fraction 5 has a similar retention time to synthetic cockroach allatostatin. Fraction 5 was eluted at 12.88 min, which was closest to the internal standard Dippu-AST I, which eluted at 12.77 min. An ELISA of fraction 5 from the methanolic brain extract using antibodies against allatostatins Grybi-AST A1 and Grybi-AST B1 from cricket Gryllus bimaculatus showed that fraction 5 cross-reacted with Grybi-AST A1 antibodies. The cross-reactivity was similar to the synthetic allatostatin from D. punctata, which was used as a positive control. These observations demonstrate a possible role for allatostatin-like brain factor(s) in regulating the reproductive cycle of Tenebrio molitor.

Pleiotropic effects of the neuropeptides CCAP and myosuppressin in the beetle, Tenebrio molitor L.

Biological activities of crustacean cardioactive peptide (CCAP; PFCNAFTGCa) and leucomyosuppressin (Lem-MS; pQDVDHVFLRFa) were studied in heterologous bioassays in the larvae and adults of Tenebrio molitor. CCAP exerted a reversible and dose-dependent cardio-stimulatory effect on the semi-isolated heart of the experimental beetles at a concentration of >10(-7) M and induced an effect similar to the endogenic cardio-stimulatory peptide, proctolin. Injections of CCAP (10(-9)-10(-3) M) into 4-day-old adult reproductive females increased the concentration of soluble proteins in hemolymph in comparison to the saline injected controls. Electrophoretic analyses indicated significant increase in the level of two proteins 130 and 170 kDa, and a partial increase of the level of 67-kDa protein. The studies indicated that CCAP increased also free hemolymph sugar concentration in young larvae and adults of the mealworm beetle. The cardio-inhibitory peptide Lem-MS exerted the opposite effect: at concentration 10(-7)-10(-6) M, it significantly decreased the heartbeat frequency. The induced changes were dose-dependent and reversible, but at higher concentrations (>10(-5) M) the stimulatory effect disappeared. Injections of the Lem-MS into young larvae at concentrations 10(-9)-10(-3) M, also increased the free hemolymph sugar level similarly to the CCAP. This work demonstrates the pleiotropic effects of CCAP and Lem-MS in Tenebrio molitor.

The photochemical attachment of the O-glucoside of 3-hydroxykynurenine to alpha-crystallin: a model for lenticular aging.

The young human lens contains a small metabolite from tryptophan called the O-glucoside of 3-hydroxykynurenine (3-HKG). Its function is to absorb most radiation between 295 and 400 nm, preventing it from reaching the retina. With age the concentration of this component decreases while the lens crystallins acquire covalently attached chromophores. This study investigates the photochemical attachment of 3-HKG to lens alpha-crystallin. Initial studies showed that alpha-crystallin photolyzed in the presence of 3-HKG developed a fluorescence (emission, 440 nm) and UV-visible spectrum similar to that found in aged human lens proteins. Extensive studies were then performed on the tryptic HPLC maps as monitored by photodiode array and fluorescent detection. Numerous photoproducts with either blue (emission, > 400 nm) or green (emission, > 500 nm) fluorescence were formed in addition to nonfluorescent compounds with absorption maxima above 300 nm. Comparisons were made between these model photoproducts and peptide maps from alpha-crystallin isolated from old human lenses. In terms of retention time and UV-visible spectra at least two of the peptides that appear in the model system are also present in the human samples. It is concluded that one of the aging processes in the human lens is the photochemically induced attachment of 3-HKG to lens proteins.

[Difficulties in the diagnosis of intracranial expansive processes coexisting with sinusitis and otitis]. / Trudnosci w rozpoznawaniu sródczaszkowych procesów ekspansywnych z wspólistniejacymi zmianami zapalnymi zatok i uszu.

Differential diagnosis between the sino- and otogenic brain complications and tumor is the most difficult task in otology and neurosurgery. These difficulties are even greater in purulent intracranial complications of sinusitis and otitis giving the same symptoms as cerebral tumors. The illustrative case reports were described.

Influence of increased environmental temperature on oxidation processes in rat liver mitochondria.

Exposure of rats to elevated temperature of 28 degrees C or 35 degrees C for 3 days six hours daily resulted in a decreased rate of oxidation with succinate or glutamate + malate as substrates, by the mitochondria of liver. The higher decrease was observed in environment temperature of 35 degrees C. There was no change in ADP/O ratio. The activities of NADH: cytochrome c reductase and cytochrome oxidase were stimulated but activities of succinate dehydrogenase and succinate cytochrome reductase were decreased.

Influence of repeated exposure to elevated environmental temperature on the activity of respiratory enzymes of rat liver mitochondria.

The paper presents studies of the activity of lipid-dependent enzymes of the respiratory chain of the liver of rats exposed to increased ambient temperature. The animals were heated in a chamber under controlled humidity (45-55% relative humidity), with forcer air flow and regulated temperature of 21 degrees +/- 1 degree C (control group) and 28 degrees +/- 1 degree C or 35 degrees +/- 1 degree C. They were affected by a relevant temperature for 7 or 14 consecutive days, 6 hrs daily. The enzymes activities were determined in a fraction of submitochondrial particles. The studies demonstrated that under the increased ambient temperature (7 X 6 hrs), the activity of the respiratory enzymes is changed. A statistically significant increase in the activity of NADH dehydrogenase, NADH cytochrome c reductase and cytochrome oxidase was found along with a decrease in the activity of succinate cytochrome c reductase and succinate dehydrogenase. On prolongation of thermal exposure (14 X 6 hrs) the activity of succinate dehydrogenase and succinate reductase: cytochrome c was further decreased. The activities of the other test enzymes did not exhibit any statistically significant differences as compared to controls. Kinetic tests of succinate dehydrogenase point to conformational changes of the enzyme when affected by an increased ambient temperature. This confirms the important role of this enzyme in the animals adaptation to thermally varying environmental conditions.

Respiratory activity and oxidative phosphorylation in mitochondria of the rat brain and liver under conditions of elevated temperature of the environment.

The rate of oxygen consumption by mitochondrial fractions of the brain and liver of rats under elevated ambient temperature was investigated. The animals were placed in a thermostatized chamber for 6 hours at 21 degrees, 28 degrees or 37 degrees C. During the experiment, relative humidity in the chamber was controlled. After exposure, the animals were decapitated, mitochondrial fractions isolated and oxygen consumption measured with the Clark electrode. Respiratory substrates of succinate and glutaminate with malate were used. The analysis of the obtained results was based on the t-Student test, assuming the values obtained for the animals staying at 21 degrees as the control values. It was found that the 6 hrs stay under the ambient hyperthermy conditions did not induce any disturbances in the respiratory processes and oxidative phosphorylation in rat's brain and liver mitochondria.

Modification of brain mitochondrial enzymes by the oxygen analogue of ronnel at various stages of development and aging.

This paper describes the effect of the organophosphorus compound, the oxygen analogue of ronnel (OAR), on the activity of some membrane-bound enzyme systems in the brain mitochondria of developing, young-adult, and old rats. Age-related changes were noted in the cholesterol-to-protein ratio, whereas the phospholipid content in mitochondria showed little change during development as well as aging. The results obtained suggest that development of brain succinate dehydrogenase may consist in a decrease of Km and increase of Vmax values. In aged rats an altered, perhaps inhibited form of the enzyme is produced. The oxygen analogue of ronnel caused a mixed-type inhibition of the succinate dehydrogenase derived from brains of 4-day-old, 16-day-old and 2-month-old animals. In the case of enzyme from the brain of 18-month-old rats, a typical competitive-type inhibition was observed. Mechanisms responsible for inhibition of the succinate: cytochrome c reductase from brains of developing animals are similar to those for succinate dehydrogenase. In aged rats (18 months old), however, a noncompetitive mechanism of inhibition of succinate: cytochrome c reductase was revealed. The experiments reported here provide evidence that lipid-soluble molecules of OAR may interact with membrane phospholipids and lead to modification of membrane architecture and also of enzyme kinetic behaviour. It may be also concluded, that the sensitivity of the enzyme systems studied to inhibition by OAR is an age-dependent phenomenon. Modification of membrane by development or aging alters the kinetics as well as the sensitivity of enzymes to inhibitors.