Cellular and Molecular Aspects of Anti-Melanoma Effect of Minocycline-A Study of Cytotoxicity and Apoptosis on Human Melanotic Melanoma Cells.

Minocycline is a tetracycline compound with pleiotropic pharmacological properties. In addition to its antibacterial action, it shows many non-antimicrobial effects, including an anti-cancer activity. The anti-cancer action was confirmed in studies on ovarian carcinoma cells, hepatocellular carcinoma cells, glioma cells, or acute myeloid leukemia cells. Malignant melanoma remains a serious medical problem despite the extensive knowledge of the disease. The low effectiveness of the standard treatment, as well as the resistance to therapy, result in high mortality rates. This work aimed to investigate the potential and mechanisms of anti-melanoma action of minocycline. Human skin melanotic melanoma cell line COLO 829 was used in the study. The obtained results showed that minocycline decreased cell viability and inhibited the growth of melanoma cells, proportional to the drug concentration as well as to the time of incubation. The EC50 values were calculated to be 78.6 µM, 31.7 µM, and 13.9 µM for 24 h, 48 h, and 72 h, respectively. It was observed that treated cells had a disturbed cell cycle and significantly changed morphology. Moreover, minocycline caused a decrease in mitochondrial membrane potential and an increase in cells with a low level of reduced thiols. Finally, it was found that the anti-melanoma effect of minocycline was related to the induction of apoptosis. The drug activated caspases 8, 9, and 3/7 as well as increased the number of annexin V-positive cells. The presented results show that minocycline possesses anti-melanoma potential.

Monitoring and assessment of cadmium, lead, zinc and copper concentrations in arable roadside soils in terms of different traffic conditions.

Quantification of the contributions from traffic source to arable roadside soil heavy metal loadings is a challenge. The contribution depends on: traffic intensity, road type and distance from the road. At a field scale (3.9 ha), 720 topsoil samples were taken. The aim of the study was to monitor and assess the impact of regional/local roads with various conditions of traffic and period of use on the distribution of Cd, Zn, Pb and Cu in the arable roadside topsoil in their vicinity. PCA indicated the occurrence of two main gradients of 11 environmental elements influencing the distribution of heavy metals in the soils of the neighbouring land. The first gradient was associated mainly with the distance from the edge of the road. The second gradient was associated with the degree of contamination of the soils and with the road type, defined by the traffic volume and period of being use. Anova reviled lack of influence of the factors for Cu contents. Unlike Cu, for Cd, Pb and Zn, the significant impact was observed for both factors and interactions between them. The concentrations of Cd, Pb and Zn, regardless of the distance from the road were 0.21-0.58 mg Cd kg-1 d.m., 13.60-41.96 mg Pb kg-1 d.m. and 40.31-63.97 mg Zn kg-1 d.m. In case of increasing distance from the road, generally the contents of Pb, Zn and Cd contents were decreasing. However, only in the case the oldest and carrying the highest traffic road was a clear, statistically significant differences noted for following distances from the road on the content of Cd, Pb and Zn. Analysis of spread gave trend curves, for Pb, Cd and Zn they were parabolas. The curves let reduce sapling distances to 65 m, 45 and 47 m for Cd, Pb and Zn, respectively.

Cobalamin Deficiency: Effect on Homeostasis of Cultured Human Astrocytes.

Cobalamin deficiency is an important health problem. The major non-hematological symptoms of hypocobalaminemia are nervous system disorders, but the molecular and cellular mechanisms underlying this phenomenon have not yet been fully explained. Increasing scientific evidence is stressing the pivotal role of astrocyte dysfunction in the pathogenesis of a wide range of neurological disorders. In light of the above, the aim of this study was to develop an in vitro model of cobalamin deficiency by optimizing the conditions of astrocyte culture in the presence of vitamin B12 antagonist, and then the model was used for multidirectional analysis of astrocyte homeostasis using image cytometry, immunoenzymatic and colorimetric assays, and fluorescence spectroscopy. Our results indicated that long-term incubation of normal human astrocytes with hydroxycobalamin(c-lactam) causes an increase of extracellular homocysteine level, a reduction of cell proliferation, and an accumulation of cells in the G2/M cell cycle phase. Moreover, we observed dramatic activation of caspases and an increase of catalase activity. Interestingly, we excluded extensive apoptosis and oxidative stress. The study provided significant evidence for astrocyte homeostasis disturbance under hypocobalaminemia, thus indicating an important element of the molecular mechanism of nervous system diseases related to vitamin B12 deficiency.

New generation poly(ε-caprolactone)/gel-derived bioactive glass composites for bone tissue engineering: Part I. Material properties.

Poly(ε-caprolactone) (PCL) based composite films containing 12 and 21vol.% bioactive glass (SBG) microparticles were prepared by solvent casting method. Two gel-derived SBGs of SiO2-CaO-P2O5 system differing in SiO2 and CaO contents were applied (mol%): S2: 80SiO2, 16CaO, 4P2O5 and A2: 40SiO2, 54CaO, 6P2O5. The surfaces of the films in contact with Petri dish and exposed to the gas phase during casting were denoted as GS and AS, respectively. Both surfaces of films were characterised in terms of their morphology, micro- and nano-topography as well as wettability. Also mechanical properties (tensile strength, Young's modulus) and PCL matrix crystallinity (degree of crystallinity, crystal size) were evaluated. Degradation behaviour was examined by incubation of materials in UHQ-water at 37°C for 56weeks. The crystallinity, melting temperature and mass loss of incubated materials and pH changes of water were monitored. Furthermore, proliferation of MG-63 osteoblastic cells by direct contact and cytotoxic effect of obtained materials were investigated. Results showed that opposite surfaces of the same polymer and composite films differ in studied surface parameters. The addition of SBG particles into PCL matrix improves nano- and micro-roughness of both surfaces, enhances the hydrophilicity of GS surfaces (~67° for 21A2-PCL compared to ~78° for pure PCL) and also makes AS surface more hydrophobic (~94° for 21S2-PCL compared to ~86° for pure PCL). The nucleation density of PCL was increased with increasing content of SBG particles, which results in the large number of fine spherulites on composite AS surfaces observed using polarized optical (POM), scanning electron (SEM), and atomic force (AFM) microscopies. Higher content of SBG particles causes a notable increase of Young's modulus (from 0.38GPa for pure PCL, 0.90GPa for 12A2-PCL to 1.31GPa for 21A2-PCL), which also depends on SBG chemical composition. After 56-week degradation test, considerably higher crystallinity increase (Δχc ~148% for 21S2-PCL, ~81% for 21A2-PCL) and weight loss (~17% for both) were found for composite materials, depending on SBG composition, in contrast to value variations for pure PCL film (Δχc ~43%, weight loss ~1.6%). Furthermore, it seems that both SBG could neutralize acidic degradation by-products of PCL at later incubation stages. Obtained SBG-PCL composites show excellent biocompatibility, support cell proliferation also may modulate cell response depending on the glass composition. The results indicate the possibility to use different contents and/or chemical compositions of SBG to obtain composite materials with various, but controlled, surface and mechanical properties as well as degradation kinetics.

Effect of the preparation methods on architecture, crystallinity, hydrolytic degradation, bioactivity, and biocompatibility of PCL/bioglass composite scaffolds.

In this study, two different composition gel derived silica-rich (S2) or calcium-rich (A2) bioactive glasses (SBG) from a basic CaO-P2 O5 -SiO2 system were incorporated into poly(ε-caprolactone) (PCL) matrix to obtain novel bioactive composite scaffolds for bone tissue engineering applications. The composites were fabricated in the form of highly porous 3D scaffolds using following preparation methods: solvent casting particulate leaching (SCPL), solid-liquid phase separation, phase inversion (PI). Scaffolds containing 21% vol. of each bioactive glass were characterized for architecture, crystallinity, hydrolytic degradation, surface bioactivity, and cellular response. Results indicated that the use of different preparation methods leads to obtain highly porous (60-90%) materials with differentiated morphology: pore shape, size, and distributions. Thermal analysis (DSC) showed that the preparation method of materials and addition of bioactive glass particles into polymer matrix induced the changes of PCL crystallinity. Composites obtained by SCPL and PI method containing A2 SBG rapidly formed a hydroxyapatite calcium phosphate surface layer after incubation in SBF. Bioactive glasses used as filler in composite scaffolds could neutralize the released acidic by-products of the polymer degradation. Preliminary in vitro biological studies of the composites in contact with osteoblastic cells showed good biocompatibility of the obtained materials. Addition of bioactive glass into the PCL matrix promotes mineralization estimated on the basis of the ALP activity. These results suggest that through a process of selection appropriate methods of preparation and bioglass composition it is possible to design and obtain porous materials with suitable properties for regeneration of bone tissue.

New sol-gel bioactive glass and titania composites with enhanced physico-chemical and biological properties.

We developed TiO2 matrix composites modified by sol-gel bioactive glasses (SBG) of either high CaO content (A2) or high SiO2 content (S2). The latter were mixed with titanium dioxide (TiO2) at 75:25, 50:50, and 25:75 weight ratios and sintered at 1250°C for 2 h. We examined the effects of various types (A2 or S2) and compositional TiO2 :SBG ratios on the mechanical properties of resulting composites, their bioactivity and human bone marrow mesenchymal stem cells (MSC) response. The chemistry of SBGs influenced the phase composition, mechanical and biological properties of the composites. Rutile and titanite prevailed in A2-TiO2 composites, and rutile and crystobalite in S2-TiO2 composites. Compressive strength increased significantly for 25A2-TiO2 composites (140 MPa) compared to matrix TiO2 (58 MPa). Composites containing 50-75 wt % of either SBG displayed bioactive properties as determined by simulated body fluid test. Compared to TiO2, human bone marrow stromal cell (BMSC) viability was enhanced on the composites containing 25 wt % of either SBG, whereas the composites modified by 25 wt % of S2 enhanced alkaline phosphatase activity and mineralization in cultures treated with osteogenic inducers-dexamethasone (Dex) or bone morphogenetic protein. Increasing amounts of A2 in TiO2 matrix decreased cell viability but increased collagen deposition and mineralized matrix production by BMSC. Considering the physico-chemical and biological properties of the presented composites, the modification of TiO2 with SBG may prove useful strategy in several bone tissue related regeneration strategies.