Tomasz Piersiak

Temozolomide, quercetin and cell death in the MOGGCCM astrocytoma cell line.

The aim of the present study was to investigate the effect of Temozolomide (an alkylating chemotherapeutic agent) and quercetin (natural flavonoid) on cell death in the human astrocytoma cell line MOGGCCM (WHO grade III). Our results indicate that Temozolomide induces autophagy, while quercetin promotes severe necrosis in the cell line in a manner dependent on the drug concentration. We demonstrated for the first time that combinations of both drugs were much more effective in programmed cell death induction in glioma cells. At a low (5muM) drug concentration, quercetin potentiated a pro-autophagic effect of Temozolomide, while after treatment with a higher drug concentration (30muM), autophagy switched to apoptosis. Temozolomide attenuated the toxic effect of quercetin. Apoptosis was mediated by the mitochondrial pathway and the activation of caspase 3 and cytochrome C release, but no changes in caspase 8 expression was observed. It was accompanied by decreased mitochondrial membrane potential and inhibition of Hsp27 and Hsp72 expression. Autophagy was correlated with an increased level of LC3II. Temozolomide and quercetin also inhibited migratory phenotype of MOGGCCM cells and changed the nuclei morphology from a circular to an irregular shape. Our results indicate that quercetin acts in synergy with Temozolomide and when used in combination rather than in separate pharmacological application, both drugs are more effective in programmed cell death induction. Temozolomide administered with quercetin seems to be a potent and promising combination which might be useful in glioma therapy.

Behavioural and Histological Effects of Preconditioning with Lipopolysaccharide in Epileptic Rats

Sublethal stress stimuli such as systemic endotoxin treatment can induce tolerance of the brain to subsequent ischemic stress, which results in a decreased infarct size. Based on this evidence, we hypothesized that lipopolysaccharide (LPS)-induced preconditioning could protect hippocampal neurons in epileptic rats. To test this hypothesis, the anticonvulsant effect of a low dose of LPS against seizures elicited by pilocarpine hydrochloride was measured. Using the pilocarpine model of temporal lobe epilepsy and LPS-preconditioning, we also investigated hippocampal pathology in the rat brain. Based on the behavioural observations conducted, it can be assumed that the preconditioning procedure used may decrease seizure excitability in epileptic rats. However, determination of the seizure excitability threshold needs to be elaborated. Qualitative and quantitative analyses of histological brain sections in the LPS-preconditioned rats showed markedly decreased intensity of neurodegenerative changes in the CA1, CA3 and DG hippocampal fields. The tendency was observed in all the periods of the pilocarpine model of epilepsy. We suggest that preconditioning with LPS may have neuroprotective effects in the CA1, CA3 and DG hippocampal sectors; however, it has no influence on the course of the seizures in rats in the pilocarpine model of epilepsy.

The effect of Galleria mellonella apolipophorin III on yeasts and filamentous fungi.

Galleria mellonella apolipophorin III (apoLp-III) has been implicated in the innate immune response against bacterial infections. The protein binds components of bacterial cell wall and inhibits growth of selected Gram-positive and Gram-negative bacteria. Interaction of apoLp-III with fungal β-1,3-glucan suggests antifungal properties of the protein. In the present study, the effect of apoLp-III on the growth, metabolic activity and cell surface characteristics of selected yeasts and filamentous fungi was investigated using light, confocal and atomic force microscopy. ApoLp-III bound to the cell surface of different yeasts and filamentous fungi as confirmed by immunoblotting with anti-apoLp-III antibodies. Incubation of the fungi in the presence of apoLp-III induced alterations in growth morphology. Candida albicans underwent transition from yeast-like to hyphal growth with formation of true hyphae, whereas Fusarium oxysporum hyphae exhibited decreased metabolic activity, increased vacuolization and appearance of numerous monophialids with microconidia. Atomic force microscopy imaging demonstrated evident alterations in the fungal cell surface after incubation with apoLp-III, suggesting that the protein affected the cell wall components.

Dietary alpha-ketoglutarate reduces gastrectomy-evoked loss of calvaria and trabecular bone in female rats

Objective. Surgical removal of the stomach (gastrectomy, Gx) leads to osteopenia in animals and in humans. In the rat, Gx adversely affects calvaria and trabecular bone. α-Ketoglutarate (AKG) is a precursor of hydroxyproline – the most abundant amino acid in bone collagen. The purpose of this study was to investigate the effects of dietary AKG on Gx-induced osteopenia. Material and methods. Twenty female Sprague-Dawley rats were subjected to Gx and divided between two groups: Gx+AKG in the drinking water and Gx+Vehicle (i.e. drinking water without AKG). Another 20 rats were sham-operated and divided between two groups: Sham+AKG and Sham+Vehicle. The daily dose of AKG was 0.43 g per 100 g rat. All the rats were killed 8 weeks later and the calvariae, femora and tibiae were collected. The integrity of the calvariae was analysed planimetrically, following transillumination and photography. The bone mineral content (BMC) and bone mineral density (BMD) were measured in the right femorae and tibiae (bone densitometry), leaving the left femorae and tibiae to be analysed histomorphometrically (measurement of trabecular bone volume and trabecular fractal dimension). Results. Gx caused calvarial bone degradation, reduced trabecular bone (femur and tibia) and impaired trabecular architecture. In addition, Gx lowered the femoral/tibial BMC and BMD (mainly cortical bone). Dietary AKG counteracted the Gx-evoked impairment of calvaria and trabecular bone but failed to affect the BMC and the BMD in either sham- operated or Gx rats. Conclusions. Gx resulted in loss of calvarial, trabecular and cortical bone in the rat. AKG counteracted the effect of Gx on calvaria and trabecular bone but not on cortical bone.

Alpha-ketoglutarate protects the liver of piglets exposed during prenatal life to chronic excess of dexamethasone from metabolic and structural changes

Glucocorticoids play a role in the origin of the features of the metabolic diseases. Alpha-ketoglutarate (AKG) is defined as glutamine homologue and derivative, conditionally an essential amino acid. In the liver, glutamine serves as a precursor for ureagenesis, gluconeogenesis and acute phase protein synthesis The aim of the study was to determine the effect of AKG administered to piglets prenatally exposed to dexamethasone, on the structure of the liver and its metabolic function. Sows were administered with dexamethasone (3 mg/sow/48 h) from day 70 of pregnancy to the parturition, and then after the birth, the piglets were divided into the group administered with AKG (0.4 g/kg body weight) or physiological saline. Biochemical markers, lysozyme and ceruloplasmin serum activities, concentrations of selected free amino acids, macro- and microelements and histomorphometry of the liver tissue were determined. The total cholesterol concentrations in the sows and their newborns from the Dex groups were higher by 72% and 64%, respectively, compared with the control groups. Triacylglycerol concentration was higher by 50% in sows from the Dex group and 55% in the new-born piglets. Alpha-ketoglutarate administered to the piglets after prenatal influence of dexamethasone lowered the total cholesterol concentration by 40%, and enhanced aspartate by 41%, serine by 76%, glutamate by 105%, glutamine by 36%, glycine by 53% and arginine by 105%, as well as methionine and cystathionine, but increased the sulphur concentration compared with the control (p < 0.01). Intracellular space D decreased after AKG administration in comparison with the piglets from Dex/Control group not treated with AKG. Postnatal administration of AKG had a protective effect on liver structure, and lowered the total cholesterol concentration in piglets prenatally exposed to dexamethasone, and also influenced selected macro- and microelement serum concentrations and amino acids plasma concentration.

The importance of release of proinflammatory cytokines, ROS, and NO in different stages of colon carcinoma growth and metastasis after treatment with cytotoxic drugs.

In colorectal cancers, the local cytokine network and the levels of nitric oxide (NO) and reactive oxygen species (ROS) are known to be closely related to cancer progression and metastasis, but the influence of the currently administered therapies on the cancer microenvironment is not completely understood. We analyzed the levels of reactive oxygen species (ROS), nitric oxide (NO), and cachexia-mediated cytokines (IL-1beta, IL-6, TNF-alpha) in cocultures of human colon carcinoma spheroids prepared with cells derived from tumors of different grades with human normal colon epithelial and myofibroblast cells and normal endothelial cells. We also analyzed the influence of standard chemotherapy with 5-fluorouracil (5-FU) and leucovorin (LV) combined with camptothecin (CPT-11) (IFL regimen with drug concentrations adjusted to in vitro conditions) on these parameters. The results indicated that adhesion of colon carcinoma spheroids to colon epithelium and myofibroblast monolayers induced O2- anion production but decreased NO levels compared to the sum of the radicals released by monocultures of the two types of cells. Coculture of colon carcinoma spheroids with endothelium was an exception to this rule, as only HT29 cells decreased NO production. In cocultures, anticancer drugs additionally, though only slightly and insignificantly, increased the production of the radicals compared to a nontreated coculture, but in monocultures, the drugs, and especially CPT-11, were ROS inducers and simultaneously NO production inhibitors. However, the levels of released ROS and NO were dependent on the stage of colon carcinoma that the cells were derived from. LS180 cells (grade B) grown in monocultures produced the lowest ROS levels but were the best producers of NO. Adhesion of tumor spheroids to normal cells influenced the microenvironmental cytokine network compared to monocultures, decreasing IL-1beta and TNF-alpha secretion but significantly enhancing L-6 levels. The addition of the drugs had no effect on IL-1beta levels but increased TNF-alpha production and lowered the amounts of IL-6. In conclusion, cytotoxic drugs may, dependent on the stage of tumor growth or the type of chemotherapy regimen administered, significantly influence the proinflammatory cytokine network and local ROS and NO levels. Moreover, in cocultures of tumor cells with normal epithelial, myofibroblast, and endothelial cells, ROS production seems to be involved in local cell injury, which was detected by confocal microscopy. On the other hand, high level of NO seems to facilitate tumor cell interactions with the endothelium and metastasis as NO production was the highest in a monoculture of HUVEC and remained at high levels in cocultures of colon cancer cells with HUVEC. Among the proinflammatory cytokines, only IL-6 seems to significantly influence colon carcinoma development and metastasis. Attenuation of IL-6 production after chemotherapy can be a useful prognostic factor of its effectiveness.

A comparison of cytokine production in 2-dimensional and 3-dimensional cultures of bone marrow stromal cells of multiple myeloma patients in response to RPMI8226 myeloma cells.

We examined cytokine production by bone marrow stromal cells (BMSCs) of patients with multiple myeloma (MM) in response to contact with myeloma RPMI8226 cells in standard 2-dimensional (2D) cultures and in 3-dimensional (3D) cultures on a gelatine sponge scaffold. It was detected that BMSCs in the 3D cultures produced more IL-11 and HGF and less IL-10 than in the 2D cultures. Moreover, RPMI8226 cells after contact with BMSCs in 3D cultures produced more sIL-6R than in the classic 2D cultures. We concluded that 3D cultures of BMSCs with myeloma cells offered a promising model for in vitro examination of interactions between myeloma cells and the bone marrow stroma and for examination of potent antimyeloma agents.

Gut bacterium of Dendrobaena veneta (Annelida: Oligochaeta) possesses antimycobacterial activity

The new bacterial strain with antimycobacterial activity has been isolated from the midgut of Dendrobaena veneta (Annelida). Biochemical and molecular characterization of isolates from 18 individuals identified all as Raoultella ornithinolytica genus with 99% similarity. The bacterium is a possible symbiont of the earthworm D. veneta. The isolated microorganism has shown the activity against four strains of fast-growing mycobacteria: Mycobacterium butiricum, Mycobacterium jucho, Mycobacterium smegmatis and Mycobacterium phlei. The multiplication of the gut bacterium on plates with Sauton medium containing mycobacteria has caused a lytic effect. After the incubation of the cell free extract prepared from the gut bacterium with four strains of mycobacteria in liquid Sauton medium, the cells of all tested strains were deformed and divided to small oval forms and sometimes created long filaments. The effect was observed by the use of light, transmission and scanning microscopy. Viability of all examined species of mycobacteria was significantly decreased. The antimycobacterial effect was probably the result of the antibiotic action produced by the gut bacterium of the earthworm. The application of ultrafiltration procedure allowed to demonstrate that antimicrobial substance with strong antimycobacterial activity from bacterial culture supernatant, is a protein with the molecular mass above 100 kDa.

The use of morphometric and fractal parameters to assess the effects of 5-fluorouracil, interferon and dexamethasone treatment on colonic anastomosis healing: an experimental study in rats.

Adjuvant chemotherapy and steroid therapy have been demonstrated to interfere with the wound healing process. The aim of this study was to evaluate the effects of 5-fluorouracil, interferon, and dexamethasone, on the healing of colon anastomosis by assessing morphometric and fractal parameters of the colonic wall. An experimental anastomosis of the ascending colon was performed in 60 male Wistar rats, which were then randomly assigned to four groups. On the second to sixth post-operative days, the rats were administered 5-fluorouracil, interferon-α, dexamethasone, or 0.9% NaCl solution as a control. Macroscopic, histomorphometric and microbiological evaluation was performed in order to assess healing of the anastomosis. In three animals from the dexamethasone group, there was leakage of anastomosis; adhesion formation was highest in the interferon group, and significantly higher than in the control and 5-fluorouracil groups. Histomorphometric parameter alterations were most pronounced on the seventh and fourteenth post-operative days in all treatment groups, with submucosal thickness the most affected parameter. Connective tissue fractal dimension was significantly decreased in those animals treated with interferon and dexamethasone. All three pharmaceutical agents impaired healing of anastomosis, and promoted infection in the anastomosis and skin wound sites. As dexamethasone induced both morphometric and macroscopic alterations, it was considered the most detrimental in this study.