Arkadiusz Orchel

Butyrate-Induced Differentiation of Colon Cancer Cells Is PKC and JNK Dependent

Butyric acid, a short–chain fatty acid physiologically present in human large gut, is derived from bacterial fermentation of complex carbohydrates. It has been shown to reduce the growth and motility of colon cancer cell lines and to induce cell differentiation and apoptosis. Apoptosis is considered a result of normal colonocyte terminal differentiation in vivo. The aim of this study was to characterize the cellular mechanisms regulating differentiation of colon cancer cells stimulated with sodium butyrate (NaB). The two human colon cancer cell lines Caco-2 and HT-29 were treated with NaB at physiologically relevant concentrations. Alkaline phosphatase (ALP) activity, a marker of colonocyte differentiation, was increased 48 hr after treatment with 1 mM NaB. Higher doses of NaB (5 and 10 mM) induced apoptosis of the cells and failed to stimulate the colonocyte differentiation. Therefore, we assumed that butyrate augments cell differentiation and induces apoptosis, acting via various intracellular mechanisms, and butyrate-mediated programmed cell death cannot be considered a consequence of colonocyte terminal differentiation. The effect of NaB on ALP activity was significantly attenuated in the presence of inhibitors of protein kinase C and JNK. Inhibition of MEK–ERK signal transduction pathways augmented the impact of butyrate on colonocyte differentiation. These results suggest that butyrate could influence the colonocyte differentiation via modulation of the activity of cellular protein kinases and signal transduction.

Phytic Acid Modulates In Vitro IL-8 and IL-6 Release from Colonic Epithelial Cells Stimulated with LPS and IL-1β

Phytic acid (PA), a major fiber-associated component of wheat bran and legumes, is physiologically present in the human large gut. The aim of this study was to examine the role of PA in immunologic function of intestinal epithelial cells by analyzing its effect on interleukin (IL)-8 and IL-6 secretion by colonocytes and its role in the response of these cells to bacterial lipopolysaccharides (LPS) and IL-1β. The human colon cell line Caco-2 was exposed to LPS isolated from two strains of Desulfovibrio desulfuricans, wild intestinal and type soil strains, as well as to LPS from E. coli. Cells were also treated with IL-1β and with a combination of LPS and IL-1β. PA had a suppressive effect on IL-8 basal release and it dose dependently reduced IL-8 secretion by colonocytes stimulated with LPS and IL-1β. On the contrary, PA increased constitutive IL-6 secretion and exhibited differentiated effects on LPS responsiveness of cells depending on its concentration and LPS origin. PA was also an efficient down-regulator of IL-6 secretion stimulated by binary actions of LPS and IL-1β. The ability of PA to modulate IL-8 and IL-6 release suggests that PA present in the intestinal milieu may exert immunoregulatory effects on colonic epithelium under physiological conditions or during microbe-induced infection/inflammation in order to maintain the colonic mucosa in a noninflammatory state or to counteract infection.

Scaffolds with shape memory behavior for the treatment of large bone defects

The aim of the presented study was preparation, analysis of properties, and in vitro characterization of porous shape-memory scaffolds, designed for large bone defects treatment using minimally invasive surgery approach. Biodegradable terpolymers of l-lactide/glycolide/trimethylene carbonate (LA/GL/TMC) and l-lactide/glycolide/ε-caprolactone (LA/GL/Cap) were selected for formulation of these scaffolds. Basic parameters of shape memory behavior (i.e. recovery ratio, recovery time) and changes in morphology (SEM, average porosity) and properties (surface topography, water contact angle, compressive strength) during shape memory cycle were characterized. The scaffolds preserved good mechanical properties (compressive strength about 0.7 to 0.9 MPa) and high porosity (more than 80%) both in initial shape as well as after return from compressed shape. Then the scaffolds in temporary shape were inserted into the model defect of bone tissue at 37°C. After 12 min the defect was filled completely as a result of shape recovery process induced by body temperature. The scaffold obtained from LA/GL/TMC terpolymer was found the most prospective for the planned application thanks to its appropriate recovery time, high recovery ratio (more than 90%), and cytocompatibility in contact with human osteoblasts and chondrocytes.

Influence of betulin and 28-O-propynoylbetulin on proliferation and apoptosis of human melanoma cells (G-361).

INTRODUCTION Pentacyclic triterpenes are a group of compounds known to have anticancer activity. One of the best characterized triterpenes is betulin, which can be isolated from bark of birch trees and modified into new compounds with various interesting medical properties. Betulin is involved in activation of the caspase cascade and promotes cell death. The aim of the study was to investigate the effect of betulin and its acetylenic derivative, 28-O-propynoylbetulin, on proliferation and apoptosis in a human melanoma cell line. MATERIALS AND METHODS The G-361 melanoma cell line was used. To evaluate growth arrest and caspase-3 activity, cells were treated with betulin and its derivative at a wide range of concentrations from 0.1 to 10 μg/mL. RESULTS Betulin and 28-O-propynoylbetulin inhibited cell proliferation in a concentration-dependent manner. The cell cycle analysis revealed an increase of the sub-G1 cell fraction (representing dead cells) after incubation of cells with betulin and 28-O-propynoylbetulin. The observed cytotoxic effects were more pronounced for 28-O-propynoylbetulin. Activity of caspase-3 in 28-O-propynoylbetulin treated cells was nearly 2-fold greater compared to cells incubated with betulin. DISCUSSION Our results show that betulin and 28-O-propynoylbetulin were effective in inhibition of cell growth and induction of apoptosis in a human melanoma cell line. The addition of the propynoyl group at the C-28 hydroxyl group of betulin led to a greater proapoptotic and antiproliferative effect in comparison to unmodified betulin. These observations suggest that the obtained derivative is a potent anti-melanoma agent.

Biological Activity of Desulfovibrio desulfuricans Lipopolysaccharides Evaluated via Interleukin-8 Secretion by Caco-2 Cells

BACKGROUND Although Desulfovibrio desulfuricans species, besides existing in the natural environment, is also found in the human digestive tract, no information is currently available on its role in the intestinal ecosystem and its activity in regard to the intestinal mucosa. Bacterial products (lipopolysaccharides, LPSs) are generally known for their ability to trigger inflammatory response by stimulating cytokine expression, such as interleukin-8 (IL-8). METHODS Colonic Caco-2 cells were exposed to LPSs isolated from the soil type and intestinal wild strains of D. desulfuricans bacteria. The amount of IL-8 secreted was measured by ELISA. The effects of sodium butyrate and cell preincubation with sodium butyrate on the IL-8 secretion in response to LPSs were also analysed. RESULTS LPSs from D. desulfuricans down-regulated IL-8 secretion by the cells. Incubation of these cells with butyrate alone resulted in a dose-dependent stimulation of IL-8 release. Butyrate also modulated IL-8 secretion by cells stimulated with LPSs. CONCLUSIONS Our findings suggest the lack of inflammatory response of intestinal mucosa in the presence of LPSs of D. desulfuricans. This response can be conditioned by the natural bacterial product, butyrate, which exerts a stimulatory effect on the IL-8 secretion and modulates its release in response to LPSs.Background: Although Desulfovibrio desulfuricans species, besides existing in the natural environment, is also found in the human digestive tract, no information is currently available on its role in the intestinal ecosystem and its activity in regard to the intestinal mucosa. Bacterial products (lipopolysaccharides, LPSs) are generally known for their ability to trigger inflammatory response by stimulating cytokine expression, such as interleukin-8 (IL-8). Methods: Colonic Caco-2 cells were exposed to LPSs isolated from the soil type and intestinal wild strains of D. desulfuricans bacteria. The amount of IL-8 secreted was measured by ELISA. The effects of sodium butyrate and cell preincubation with sodium butyrate on the IL-8 secretion in response to LPSs were also analysed. Results: LPSs from D. desulfuricans down-regulated IL-8 secretion by the cells. Incubation of these cells with butyrate alone resulted in a dose-dependent stimulation of IL-8 release. Butyrate also modulated IL-8 secretion by cells stimulated with LPSs. Conclusions: Our findings suggest the lack of inflammatory response of intestinal mucosa in the presence of LPSs of D. desulfuricans. This response can be conditioned by the natural bacterial product, butyrate, which exerts a stimulatory effect on the IL-8 secretion and modulates its release in response to LPSs.

Multidrug PLA-PEG filomicelles for concurrent delivery of anticancer drugs-The influence of drug-drug and drug-polymer interactions on drug loading and release properties.

This study aimed to analyze the influence of drug-drug and drug-polymer interactions on drug loading and release properties of multidrug micelles. Three hydrophobic drugs-paclitaxel (Ptx), 17-AAG and rapamycin (Rap) were incorporated in poly(l-lactide)-poly(ethylene glycol) (PLA-PEG) filomicelles. Double loaded micelles containing Ptx and 17-AAG were used for the sake of comparison. (1)H NMR confirmed the effective incorporation of the various drugs in micelles, and HPLC allowed to determine the drug loading contents. FTIR was used to evaluate interactions between particular drugs and between drugs and copolymer. Ptx and 17-AAG present similar loading efficiencies in double loaded micelles probably due to interactions of drugs with each other and also with the copolymer. In contrast, unequal drug loading properties are observed for triple loaded micelles. Rapamycin shows very weak interactions with the copolymer, and displays the lowest loading efficiency. In vitro release of drugs from micelles was realized in pH 7.4 phosphate buffered saline at 37°C, and monitored by HPLC. Similar release profiles are observed for the three drugs: a strong burst followed by slower release. Nevertheless, Ptx release from micelles is significantly slower as compared to 17-AAG and Rap, probably due to interactions of NH and OH groups of Ptx with the carbonyl group of PLA. In vitro cytotoxicity of Ptx/17-AAG/Rap loaded micelles and a mixture of free drugs was determined. Drug loaded micelles exhibit advantageous effect of prolonged drug release and cytotoxic activity against Caco-2 cells, which makes them a promising solution for simultaneous drug delivery to solid tumors. Therefore, understanding of interactions within multidrug micelles should be a valuable approach for the development of concurrent delivery systems of anticancer drugs with tailored properties.

The Influence of Chain Microstructure of Biodegradable Copolyesters Obtained with Low-Toxic Zirconium Initiator to In Vitro Biocompatibility

Because of the wide use of biodegradable materials in tissue engineering, it is necessary to obtain biocompatible polymers with different mechanical and physical properties as well as degradation ratio. Novel co- and terpolymers of various composition and chain microstructure have been developed and applied for cell culture. The aim of this study was to evaluate the adhesion and proliferation of human chondrocytes to four biodegradable copolymers: lactide-coglycolide, lactide-co-ε-caprolactone, lactide-co-trimethylene carbonate, glycolide-co-ε-caprolactone, and one terpolymer glycolide-colactide-co-ε-caprolactone synthesized with the use of zirconium acetylacetonate as a nontoxic initiator. Chain microstructure of the copolymers was analyzed by means of 1H and 13C NMR spectroscopy and surface properties by AFM technique. Cell adhesion and proliferation were determined by CyQUANT Cell Proliferation Assay Kit. After 4 h the chondrocyte adhesion on the surface of studied materials was comparable to standard TCPS. Cell proliferation occurred on all the substrates; however, among the studied polymers poly(L-lactide-coglycolide) 85 : 15 that characterized the most blocky structure best supported cell growth. Chondrocytes retained the cell membrane integrity evaluated by the LDH release assay. As can be summarized from the results of the study, all the studied polymers are well tolerated by the cells that make them appropriate for human chondrocytes growth.

Evaluation of Melanogenesis in A-375 Cells in the Presence of DMSO and Analysis of Pyrolytic Profile of Isolated Melanin

The increase of a skin malignant melanoma (melanoma malignum) incidence in the world has been observed in recent years. The tumour, especially in advanced stadium with metastases, is highly resistant to conventional treatment. One of the strategies is to modulate melanogenesis using chemical compounds. In this study, the processes of differentiation and melanogenesis induced by dimethylsulfoxide (DMSO) in human melanoma cells (A-375) were investigated. Natural melanin isolated from A-375 melanoma cell line treated with 0.3% DMSO was analyzed by pyrolysis-gas chromatography-mass spectrometry (Py-GC/MS) method. The products derived from pheomelanin have not been stated in the pyrolytic profile of analyzed melanin. Within all products derived from eumelanins, 1,2-benzenediol has been predominated. It has been shown that in the melanoma cells stimulated with 0.3% and 1% DMSO, the increase of transcriptional activity of the tyrosinase gene took place. It was accompanied by the rise of tyrosinase activity and an accumulation of melanin in the cells. The better knowledge about the structure of melanins can contribute to establish the uniform criteria of malignant melanoma morbidity risk.

Evaluation of melanogenesis in A-375 melanoma cells treated with 5,7-dimethoxycoumarin and valproic acid

Malignant melanoma (melanoma malignum) is one of the most dangerous types of tumor. It is very difficult to cure. In recent years, a lot of attention has been given to chemoprevention. This method uses natural and synthetic compounds to interfere with and inhibit the process of carcinogenesis. In this study, a new treatment strategy was proposed consisting of a combination of 5,7-dimethoxycoumarin (DMC), an activator of melanogenesis, and valproic acid (VPA), a well-known drug that is one of the histone deacetylase inhibitors (HDACis). In conjunction with 1 mM VPA, all of the tested concentrations of DMC (10–150 μM) significantly decreased the proliferation of A-375 cells. VPA and DMC also induced the synthesis of melanin and the formation of dendrite and star-shaped cells. Tyrosinase gene expression and tyrosinase activity significantly increased in response to VPA treatment. Pyrolysis with gas chromatography and mass spectrometry (Py-GC/MS) was used to investigate the structure of the isolated melanin. This showed that the quantitative and qualitative components of melanin degradation products are dependent on the type of applied melanogenesis inductor. Products derived from eumelanin were detected in the pyrolytic profile of melanin isolated from A-375 cells stimulated with DMC. Thermal degradation of melanin isolated from melanoma cells after exposure to VPA or a mixture of VPA and DMC revealed the additional presence of products derived from pheomelanin.

Effect of polymer degradation on prolonged release of paclitaxel from filomicelles of polylactide/poly(ethylene glycol) block copolymers

Paclitaxel is one of the most efficient anticancer agents, but the conventional dosage formulations cause many side effects. PLA-PEG filomicelles are promising carriers of paclitaxel because high loading capacity and long term release can be achieved. Slow release of cytostatic drugs is very advantageous due to prolonged exposure of tumor cells to cytostatic over multiple cell cycles. The aim of this study was to evaluate the potential of bioresorbable PLA-PEG filomicelles for prolonged delivery of paclitaxel. Paclitaxel is encapsulated in PLLA-PEG filomicelles and PDLLA-PEG spherical micelles. Drug release was studied in PBS at 37°C at various pH values to elucidate the influence of polymer degradation on drug release. NMR, GPC and HPLC were used to follow polymer degradation and drug release. The release of paclitaxel is strongly dependent on the degradation of micelles. A biphasic drug release profile is observed for both PLLA-PEG and PDLLA-PEG micelles: slow release in the first phase and faster release in the second phase. Degradation is faster at acidic pH than at pH7.4, and PLLA-PEG filomicelles degrade less rapidly than PDLLA-PEG spherical micelles, leading to various rates of drug release. The correlation between degradation and drug release is very helpful for the development of novel drug carriers with tailored properties. Importantly, the cytotoxic activity of PLLA-PEG filomicelles was evidenced, thus showing their potential as carrier of antitumor drugs.