The anti-tumoral potential of the saporin-based uPAR-targeting chimera ATF-SAP.

The development of personalized therapies represents an urgent need owing to the high rate of cancer recurrence and systemic toxicity of conventional drugs. So far, targeted toxins have shown promising results as potential therapeutic compounds. Specifically, toxins conjugated to antibodies or fused to growth factors/enzymes have been largely demonstrated to selectively address and kill cancer cells. We investigated the anti-tumor potential of a chimeric recombinant fusion protein formed by the Ribosome Inactivating Protein saporin (SAP) and the amino-terminal fragment (ATF) of the urokinase-type plasminogen activator (uPA), whose receptor has been shown to be over-expressed on the surface of aggressive tumors. ATF-SAP was recombinantly produced by the P. pastoris yeast and its activity was assessed on a panel of bladder and breast cancer cell lines. ATF-SAP resulted to be highly active in vitro, as nano-molar concentrations were sufficient to impair viability on tumor cell lines. In contrast to untargeted toxins, the chimeric fusion protein displayed a significantly improved toxic effect in uPAR-expressing cells, demonstrating that the selective activity was due to the presence of the targeting moiety. Fibroblasts were not sensitive to ATF-SAP despite uPAR expression, indicating that cell-specific receptor-mediated internalization pathway(s) might be considered. The in vivo anti-tumor effect of the chimera was shown in a bladder cancer xenograft model. Current findings indicate ATF-SAP as a suitable anti-tumoral therapeutic option to cope with cancer aggressiveness, as a single treatment or in combination with traditional therapeutic approaches, to appropriately address the intra- and inter- tumor heterogeneity.

Porous biodegradable EW62 medical implants resist tumor cell growth.

Magnesium alloys have been widely investigated for biodegradable medical applications. However, the shielding of harmful cells (eg. bacteria or tumorous cells) from immune surveillance may be compounded by the increased porosity of biodegradable materials. We previously demonstrated the improved corrosion resistance and mechanical properties of a novel EW62 (Mg-6%Nd-2%Y-0.5%Zr)) magnesium alloy by rapid solidification followed by extrusion (RS) compared to its conventional counterpart (CC). The present in vitro study evaluated the influence of rapid solidification on cytotoxicity to murine osteosarcoma cells. We found that CC and RS corrosion extracts significantly reduced cell viability over a 24-h exposure period. Cell density was reduced over 48 h following direct contact on both CC and RS surfaces, but was further reduced on the CC surface. The direct presence of cells accelerated corrosion for both materials. The corroded RS material exhibited superior mechanical properties relative to the CC material. The data show that the improved corrosion resistance of the rapidly solidified EW62 alloy (RS) resulted in a relatively reduced cytotoxic effect on tumorous cells. Hence, the tested alloy in the form of a rapidly solidified substance may introduce a good balance between its biodegradation characteristics and cytotoxic effect towards cancerous and normal cells.

Life is three dimensional-as in vitro cancer cultures should be.

For many decades, fundamental cancer research has relied on two-dimensional in vitro cell culture models. However, these provide a poor representation of the complex three-dimensional (3D) architecture of living tissues. The more recent 3D culture systems, which range from ridged scaffolds to semiliquid gels, resemble their natural counterparts more closely. The arrangement of the cells in 3D systems allows better cell-cell interaction and facilitates extracellular matrix secretion, with concomitant effects on gene and protein expression and cellular behavior. Many studies have reported differences between 3D and 2D systems as regards responses to therapeutic agents and pivotal cellular processes such as cell differentiation, morphology, and signaling pathways, demonstrating the importance of 3D culturing for various cancer cell lines.

The Parkinson-associated protein PINK1 interacts with Beclin1 and promotes autophagy.

Mutations in the PINK1 gene cause autosomal recessive Parkinson's disease. The PINK1 gene encodes a protein kinase that is mitochondrially cleaved to generate two mature isoforms. In addition to its protective role against mitochondrial dysfunction and apoptosis, PINK1 is also known to regulate mitochondrial dynamics acting upstream of the PD-related protein Parkin. Recent data showed that mitochondrial Parkin promotes the autophagic degradation of dysfunctional mitochondria, and that stable PINK1 silencing may have an indirect role in mitophagy activation. Here we report a new interaction between PINK1 and Beclin1, a key pro-autophagic protein already implicated in the pathogenesis of Alzheimer's and Huntington's diseases. Both PINK1 N- and C-terminal are required for the interaction, suggesting that full-length PINK1, and not its cleaved isoforms, interacts with Beclin1. We also demonstrate that PINK1 significantly enhances basal and starvation-induced autophagy, which is reduced by knocking down Beclin1 expression or by inhibiting the Beclin1 partner Vps34. A mutant, PINK1(W437X), interaction of which with Beclin1 is largely impaired, lacks the ability to enhance autophagy, whereas this is not observed for PINK1(G309D), a mutant with defective kinase activity but unaltered ability to bind Beclin1. These findings identify a new function of PINK1 and further strengthen the link between autophagy and proteins implicated in the neurodegenerative process.

Biomaterial effects in articular cartilage tissue engineering using polyglycolic acid, a novel marine origin biomaterial, IGF-I, and TGF-beta 1.

Bovine articular chondrocytes were seeded on either polyglycolic acid (PGA) non-woven mesh scaffolds or a biomatrix from the species Porites lutea (POR). These constructs were cultured for 6 weeks in the presence of insulin-like growth factor (IGF)-I (10 ng/ml or 100 ng/ml) or transforming growth factor (TGF)-beta 1 (5 ng/ml or 30 ng/ml) to determine the in-vitro articular cartilage regeneration capacity of each. Histology, deoxyribonucleic acid content, collagen I and II (immunohistochemistry and enzyme-linked immunosorbent assay), and glycosaminoglycan (GAG) contents were measured at 0 weeks, 2 weeks, and 6 weeks to assess the characteristics of chondrogenesis. Both scaffolds supported the maintenance of the chondrocytic phenotype, as evidenced by the predominance of collagen II and the presence of rounded chondrocytes embedded in lacunae. Regardless of growth factor treatment, cells cultured on PGA scaffolds produced more collagen type II than those cultured on POR. Conversely, by 6 weeks, cells cultured on POR scaffolds produced more GAG than those cultured on PGA scaffolds, again regardless of the growth factor used. Across the two groups, 100 ng/ml of IGF-I had the greatest overall effect in GAG content. This work indicates that PGA and the POR scaffolds are both effective growth matrices for articular cartilage, with each scaffold exhibiting different yet desirable profiles of articular cartilage growth.

Aragonite crystalline matrix as an instructive microenvironment for neural development.

The ability to mimic cell-matrix interactions in a way that closely resembles the natural environment is of a great importance for both basic neuroscience and for fabrication of potent scaffolding materials for nervous tissue engineering. Such scaffolding materials should not only facilitate cell attachment but also create a microenvironment that provides essential developmental and survival cues. We previously found that porous aragonite crystalline matrices of marine origin are an adequate and active biomaterial that promotes neural cell growth and tissue development. Here we studied the mechanism underlying these neural cell-material interactions, focusing on the three-dimensional (3D) surface architecture and matrix activity of these scaffolds. We introduced a new cloning technique of the hydrozoan Millepora dichotoma, through which calcein or (45)Ca(2+) were incorporated into the organism's growing skeleton and neuronal cells could then be cultured on the labelled matrices. Herein, we describe the role of matrix 3D architecture on neural cell type composition and survival in culture, and report for the first time on the capacity of neurons and astrocytes to exploit calcium ions from the supporting biomatrix. We found that hippocampal cells growing on the prelabelled aragonite lattice took up aragonite-derived Ca(2+), and even enhanced this uptake when extracellular calcium ions were chelated by EGTA. When the aragonite-derived Ca(2+) uptake was omitted by culturing the cells on coral skeletons coated with gold, cell survival was reduced but not arrested, suggesting a role for matrix architecture in neural survival. In addition, we found that the effects of scaffold architecture and chemistry on cell survival were more profound for neurons than for astrocytes. We submit that translocation of calcium from the biomaterial to the cells activates a variety of membrane-bound signalling molecules and leads to the subsequent cell behaviour. This kind of cell-material interaction possesses great potential for fabricating advanced biomaterials for neural tissue-engineering applications.

Interconnected network of ganglion-like neural cell spheres formed on hydrozoan skeleton.

Identifying scaffolds supporting in vitro reconstruction of active neuronal tissues in their 3-dimensional (3D) conformation is a major challenge in tissue engineering. We have previously shown that aragonite coral exoskeletons support the development of neuronal tissue from hippocampal neurons and astrocytes. Here we show for the first time that the porous aragonite skeleton obtained from bio-fabricated hydrozoan Millepora dichotoma supports the spontaneous organization of dissociated hippocampal cells into highly interconnected 3D ganglion-like tissue formations. The ganglion-like cell spheres expanded hundreds of microns across and included hundreds to thousands of astrocytes and mature neurons, most of them having only cell-cell and no cell-surface interactions. The spheres were linked to the surface directly or through a neck of cells and were interconnected through thick bundles of dendrites, varicosity-bearing axons, and astrocytic processes. Thus, M. dichotoma exoskeleton is a novel scaffold with the unprecedented ability to support a highly ordered organization of neuronal tissue. This unexpected organization opens new opportunities for neuronal tissue regeneration, because the spheres resemble in vivo nervous tissue having high volume of cells associated primarily through cell-cell rather than cell-matrix interactions.

Saporin as a novel suicide gene in anticancer gene therapy.

We used a non-viral gene delivery approach to explore the potential of the plant saporin (SAP) gene as an alternative to the currently employed suicide genes in cancer therapy. Plasmids expressing cytosolic SAP were generated by placing the region encoding the mature plant ribosome-inactivating protein under the control of cytomegalovirus (CMV) or simian virus 40 (SV40) promoters. Their ability to inhibit protein synthesis was first tested in cultured tumor cells co-transfected with a luciferase reporter gene. In particular, SAP expression driven by CMV promoter (pCI-SAP) demonstrated that only 10 ng of plasmid per 1.6 x 10(4) B16 cells drastically reduced luciferase activity to 18% of that in control cells. Direct intratumoral injection of pCI-SAP complexed with either lipofectamine or N-(2,3-dioleoyloxy-1-propyl) trimethylammonium methyl sulfate (DOTAP) in B16 melanoma-bearing mice resulted in a noteworthy attenuation of tumor growth. This antitumor effect was increased in mice that received repeated intratumoral injections. A SAP catalytic inactive mutant (SAP-KQ) failed to exert any antitumor effect demonstrating that this was specifically owing to the SAP N-glycosidase activity. Our overall data strongly suggest that the gene encoding SAP, owing to its rapid and effective action and its independence from the proliferative state of target cells might become a suitable candidate suicide gene for oncologic applications.

Aragonite crystalline biomatrices support astrocytic tissue formation in vitro and in vivo.

Astrocytes play a pivotal role in the development and function of the central nervous system by regulating synaptic activity and supporting and guiding growing axons. It is therefore a central therapeutic and scientific challenge to develop means to control astrocytic survival and growth. We cultured primary hippocampal astrocytes on a crystalline three-dimensional (3D) aragonite biomatrix prepared from the exoskeleton of the coral Porites lutea. Such culturing led to the formation of astrocytic tissue-like 3D structures in which the cells had a higher survival rate than astrocytes grown in conventional cell culture. Within the pore void areas, multiple layers of astrocytic processes formed concave sheet structures that had no physical contact with the surface. The astrocytes attached to the crystalline perpendicular edges of the crystalline template surface extended processes in 3D and expressed glial fibrillary acidic protein. The astrocytes also expressed gap junctions and developed partly synchronized cytosolic Ca2+ oscillations. Preliminary in vivo models showed that astrocytic networks were also developed when the matrices were implanted into cortical areas of postnatal rat brains. Hence, we suggest that the biomatrix is a biocompatible supportive scaffold for astrocytes and may be exploited in applications for neuronal tissue restoration in injured or diseased central nervous system.

Morphology of sol-gel derived nano-coated coralline hydroxyapatite.

Current bone graft materials are mainly produced from coralline hydroxyapatite (HAp). Due to the nature of the conversion process, commercial coralline HAp has retained coral or CaCO(3,) and the structure possesses nanopores within the inter-pore trabeculae, resulting in high dissolution rates. Under certain conditions these features reduce durability and strength and are not utilised where high structural strength is required. To overcome these limitations, a new coral double-conversion technique has been developed. The technique involves a two-stage application route where, in the first stage, complete conversion of coral to pure HAp is achieved. In the second, a new sol-gel-derived HAp nano-coating is directly applied to cover the micro- and nano-pores within the intra-pore material, whilst maintaining the large pores. Biaxial strength was improved two-fold due to this unique double treatment. This application is expected to result in enhanced durability and longevity due to the monophasic hydroxyapatite structure and strength in the physiological environment. It is anticipated that this new material can be applied to load-bearing bone graft applications where high strength requirements are pertinent.