Roman Paduch

The inhibitory effect of zinc on cadmium-induced cell apoptosis and reactive oxygen species (ROS) production in cell cultures

The prevention of apoptosis by Zn(2+) is a well-known phenomenon. Both in in vitro and in vivo Zn(2+) supplementation prevents apoptosis induced by a variety of agents, among them by cadmium ions. The target for protective action of Zn ions on cell apoptosis is still unknown. In this paper we have evaluated the effect of in vitro ZnCl(2) supplementation at a concentration corresponding to the physiological level (10 microM) and higher (50 microM), on apoptosis induced with different Cd concentrations in two cell types: HeLa human tumor cell line and bovine aorta endothelial cells (BAECs). We demonstrated that Zn supplementation, especially at 10 microM concentration, significantly inhibited apoptosis in both types of cells. To assess the mechanism involved in the Zn effect we examined the influence of Zn supplementation on Cd accumulation in cells, Cd-induced superoxide anion (O(2)(-)) and hydrogen peroxide (H(2)O(2)) production. Zn caused 1.2-2.0-fold inhibition of Cd accumulation, 1.2-2.0-fold inhibition of Cd-induced apoptotic cell death, 1.1-2.0-fold decrease in reactive oxygen species (ROS) production in HeLa cells and in BAECs. These results indicate that inhibition of Cd-induced apoptosis in cells by Zn might be due, not only by inhibition of Cd accumulation in cells but, at least in part, to inhibition of Cd-induced production of ROS, which in turn are known as strong inducers of apoptosis.

Terpenes: substances useful in human healthcare

Terpenes are naturally occurring substances produced by a wide variety of plants and animals. A broad range of the biological properties of terpenoids is described, including cancer chemopreventive effects, antimicrobial, antifungal, antiviral, antihyperglycemic, anti-inflammatory, and antiparasitic activities. Terpenes are also presented as skin penetration enhancers and agents involved in the prevention and therapy of several inflammatory diseases. Moreover, a potential mechanism of their action against pathogens and their influence on skin permeability are discussed. The major conclusion is that larger-scale use of terpenoids in modern medicine should be taken into consideration.

Role of reactive oxygen species (ROS), metalloproteinase-2 (MMP-2) and interleukin-6 (IL-6) in direct interactions between tumour cell spheroids and endothelial cell monolayer

Metastasis is a multistep process involving a variety of direct cell—cell, cell—matrix and paracrine interactions. In the present study, we examined some consequences of direct interaction between tumour cells and endothelial cells in vitro. When multicellular spheroids of two human tumour cell lines (HeLa and Hep‐2) were transferred onto a human umbilical vein endothelial cell (HUVEC) monolayer, a peri‐spheroidal zone of damaged endothelial cells was observed after 24 h co‐culture. To determine the cause of this damage, the production levels of superoxide anion (O2 −), interleukin‐6 (IL‐6) and metalloproteinase‐2 (MMP‐2) were measured both in co‐culture and in monocultures of the tumour cell spheroids and endothelial cells. Attachment of HeLa and Hep‐2 cellular spheroids to the HUVEC monolayer resulted in 1.6‐fold and 2.1‐fold increases in O2 − release, respectively. Also, the MMP‐2 level was five times greater in the co‐culture than in the tumour spheroid monoculture. The increase of IL‐6 in the co‐culture model, on the other hand, was only slight. However, a 2 h preincubation of endothelial cells with LPS (10 μg/ml) prior to the transfer of spheroids induced a significant increase in the production of this cytokine compared to an appropriate control (an LPS‐activated endothelial cell monolayer). These results strongly suggest that both ROS and MMP‐2 are involved in endothelial cell injury when tumour cells cross the endothelial barrier. Moreover, IL‐6, which participates in the inflammatory response, may also be involved in the extravasation of tumour cells.

The effect of steroidal and non-steroidal anti-inflammatory drugs on the cellular immunity of calves with experimentally-induced local lung inflammation

We examined the effect of a single intravenous dose of flumetasone (SAID) and meloxicam (NSAID) treatment of calves with experimentally-induced localized lung inflammation on immunological and hematological variables such as total protein, gamma globulin, hemoglobin (Hb) concentrations, alkaline phosphatase activity, packed red cell volume (PCV), red blood cell (RBC) and white blood cell (WBC) counts. The influence of drug treatment on the phagocytic activity of WBC and bronchoalveolar lavage (BAL) cells and their ex vivo ability to produce interferon (IFN) and tumor necrosis factor (TNF) after induction with Newcastle disease virus (NDV), as well as on the development of PHA-induced skin delayed hypersensitivity reaction was also determined. Two days after the treatment of calves with experimentally-induced local lung inflammation with flumetasone (5 mg per calf), we observed a significant increase in WBC count, especially neutrophils, and a decrease in gamma globulin concentration, in the percent of blood phagocytic cells and their random migration. Flumetasone treatment also inhibited the development of skin delayed hypersensitivity reaction. In contrast, the treatment of calves with meloxicam (50 mg per calf) did not influence any hematological parameters or skin reactivity. Both drugs, flumetasone and meloxicam, influenced TNF production in ex vivo cultures of blood and BAL cells, inhibiting excessive TNF production induced by local lung inflammation. Contrary to TNF, the treatment of calves with meloxicam and flumetasone enhanced ex vivo IFN production in blood and BAL cells. Histological examination of lung tissue revealed that in control calves (those not treated with anti-inflammatory drugs) and in calves treated with flumetasone, symptoms of stromo-purulent inflammation of pulmonary tissue developed. However, in calves treated with meloxicam, only interstitial inflammation with a slight thickening of interalveolar septa and infiltration of lymphoid cells was observed. These results suggest that in this model of pneumonia, it is more appropriate to use a single dose of meloxicam, rather than flumetasone, to modulate lung inflammation.

Biological study on carboxymethylated (1→3)-α-D-glucans from fruiting bodies of Ganoderma lucidum.

Water-insoluble, alkali-soluble polysaccharides (ASPs) were isolated from three fruiting bodies of the macromycete fungus Ganoderma lucidum. The structure of ASPs was determined using composition analysis, methylation analysis, Fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy. The analysis of the biological activity of the carboxymethylated (CM) (1→3)-α-D-glucans was based on an assessment of their cytotoxic, mitochondrial metabolism-modulating, and free radical scavenging effects against a tumor cell line (human cervical carcinoma HeLa), and two normal human cell lines (colon myofibroblasts CCD-18Co and epithelial cells CCD 841 CoTr). The chemical and spectroscopic investigations indicated that the ASPs from G. lucidum were (1→3)-α-D-glucans. After carboxymethylation (1→3)-α-D-glucans were tested in the range of 25-250 μg/mL concentrations. All the tested CM-(1→3)-α-D-glucans decreased the cellular metabolism of tumor and normal cells after 24h of incubation. The CM-(1→3)-α-D-glucans had no toxic effects on cervical carcinoma cells but reduced the viability of normal cells. The cytotoxic activity of the CM-(1→3)-α-D-glucans was concentration- and cell-type-dependent with normal cells more sensitive to their action than tumor cells. Generally, the CM-(1→3)-α-D-glucans tested did not have a free radical scavenging effect. It was concluded that the carboxymethylated derivatives of (1→3)-α-D-glucans isolated from the G. lucidum fruiting bodies are biologically active and after further detailed studies may be regarded as a dietary or therapeutic supplements.

Aloe vera extract activity on human corneal cells

Context: Ocular diseases are currently an important problem in modern societies. Patients suffer from various ophthalmologic ailments namely, conjunctivitis, dry eye, dacryocystitis or degenerative diseases. Therefore, there is a need to introduce new treatment methods, including medicinal plants usage. Aloe vera [Aloe barbadensis Miller (Liliaceae)] possesses wound-healing properties and shows immunomodulatory, anti-inflammatory or antioxidant activities. Materials and methods: NR uptake, MTT, DPPH• reduction, Griess reaction, ELISA and rhodamine-phalloidin staining were used to test toxicity, antiproliferative activity, reactive oxygen species (ROS) reduction, nitric oxide (NO) and cytokine level, and distribution of F-actin in cells, respectively. Aim: The present study analyzes the effect of Aloe vera extracts obtained with different solvents on in vitro culture of human 10.014 pRSV-T corneal cells. Results: We found no toxicity of ethanol, ethyl acetate and heptane extracts of Aloe vera on human corneal cells. No ROS reducing activity by heptane extract and trace action by ethanol (only at high concentration 125 µg/ml) extract of Aloe vera was observed. Only ethyl acetate extract expressed distinct free radical scavenging effect. Plant extracts decreased NO production by human corneal cells as compared to untreated controls. The cytokine (IL-1β, IL-6, TNF-α and IL-10) production decreased after the addition of Aloe vera extracts to the culture media. Discussion and conclusions: Aloe vera contains multiple pharmacologically active substances which are capable of modulating cellular phenotypes and functions. Aloe vera ethanol and ethyl acetate extracts may be used in eye drops to treat inflammations and other ailments of external parts of the eye such as the cornea.

The effect of moonlighting proteins on the adhesion and aggregation ability of Lactobacillus helveticus.

The goal of this study was to identify moonlighting proteins in Lactobacillus helveticus that play an important role in adhesion and aggregation. The label-free method was used for identification and analysis of expression of cellular proteins. The analysis revealed the presence of eight moonlighting proteins in the cell envelope of Lb. helveticus. The tested strains mainly differed with respect to the presence of S-layer proteins and the level of expression of moonlighting proteins in Lb. helveticus strain T159. These surface proteins give the cell a hydrophobic character and play a role in specific interactions with intestinal epithelium cells and with other bacteria. In Lb. helveticus T159, the S-layer associated with moonlighting proteins could act as adherence factors, which was evidenced by the high capability of adhesion, auto- and coaggregation. The hydrophobicity, adhesion and aggregation abilities provide biological activities in food products and they are regarded as an important criterion for probiotic selection.

Chemical characterization of a water insoluble (1 → 3)-α-d-glucan from an alkaline extract of Aspergillus wentii

The chemical structure of a water insoluble α-glucan isolated from the cell wall of Aspergillus wentii was described on the basis of total acid hydrolysis, methylation analysis, and 1D and 2D NMR studies (TOCSY, DQF-COSY, NOESY and HSQC) as well as other instrumental techniques. It was established that the analyzed preparation contained a linear polymer composed almost exclusively of (1→3)-linked α-d-glucose, with a molecular mass of about 850kDa. The polymer was divided into subunits separated by a short spacers of (1→4)-linked α-d-glucoses. Each subunit contained about 200 glucose residues.

The effect of ursolic and oleanolic acids on human skin fibroblast cells

In this article, we look at how ursolic and oleanolic acids can be used for the purpose of quality control of natural products used in dermatocosmetology as well as of various other therapeutic preparations. Ursolic acid (UA) and oleanolic acid (OA) are pentacyclic triterpenes and they are constituents of many medicinal herbs. In this study, we analyzed the cytotoxic and anti-proliferative activity of OA and UA against normal human skin fibroblasts (HSF). Additionally, the scavenging activity of free radicals of both acids was analyzed. The sensitivity of cells to OA and UA activity was determined using a standard spectrophotometric (MTT) assay. The free radical scavenging activity of OA and UA was measured using the DPPH• test. The F-actin cytoskeletal proteins organization was analyzed using TRITC-phalloidine fluorescent staining. The cytotoxic activity of the analyzed acids was determined using Neutral Red (NR) uptake assay. Of the two isomeric compounds, UA showed a higher cytotoxic activity against HSF cells than did OA. Our investigations showed that OA, in view of its non-toxic nature, may be used as a supplementary factor for dermal preparations.

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.