Albumin-covered lipid nanocapsules exhibit enhanced uptake performance by breast-tumor cells.

Liquid lipid nanocapsules (LLN) represent a promising new generation of drug-delivery systems. They can carry hydrophobic drugs in their oily core, but the composition and structure of the surrounding protective shell determine their capacity to survive in the circulatory system and to achieve their goal: penetrate tumor cells. Here, we present a study of LLN covered by the protein human serum albumin (HSA) and loaded with curcumin as a hydrophobic model drug. A cross-linking procedure was performed to further strengthen the protective protein layer. Physicochemical properties and release kinetics of the nanocapsules were investigated, and cellular uptake and killing capacity were evaluated on the human breast-cancer line MCF-7. The nanocapsules exhibited a half maximal inhibitory concentration (IC50) capacity similar to that of free curcumin, but avoiding problems associated with excipients, and displayed an outstanding uptake performance, entering cells massively in less than 1 min.

Clinical and therapeutic potential of protein kinase PKR in cancer and metabolism.

The protein kinase R (PKR, also called EIF2AK2) is an interferon-inducible double-stranded RNA protein kinase with multiple effects on cells that plays an active part in the cellular response to numerous types of stress. PKR has been extensively studied and documented for its relevance as an antiviral agent and a cell growth regulator. Recently, the role of PKR related to metabolism, inflammatory processes, cancer and neurodegenerative diseases has gained interest. In this review, we summarise and discuss the involvement of PKR in several cancer signalling pathways and the dual role that this kinase plays in cancer disease. We emphasise the importance of PKR as a molecular target for both conventional chemotherapeutics and emerging treatments based on novel drugs, and its potential as a biomarker and therapeutic target for several pathologies. Finally, we discuss the impact that the recent knowledge regarding PKR involvement in metabolism has in our understanding of the complex processes of cancer and metabolism pathologies, highlighting the translational research establishing the clinical and therapeutic potential of this pleiotropic kinase.

Validation of suitable normalizers for miR expression patterns analysis covering tumour heterogeneity.

Oncogenic microRNAs (miRs) have emerged as diagnostic biomarkers and novel molecular targets for anti-cancer drug therapies. Real-time quantitative PCR (qPCR) is one of the most powerful techniques for analyzing miRs; however, the use of unsuitable normalizers might bias the results. Tumour heterogeneity makes even more difficult the selection of an adequate endogenous normalizer control. Here, we have evaluated five potential referenced small RNAs (U6, rRNA5s, SNORD44, SNORD24 and hsa-miR-24c-3p) using RedFinder algorisms to perform a stability expression analysis in i) normal colon cells, ii) colon and breast cancer cell lines and iii) cancer stem-like cell subpopulations. We identified SNORD44 as a suitable housekeeping gene for qPCR analysis comparing normal and cancer cells. However, this small nucleolar RNA was not a useful normalizer for cancer stem-like cell subpopulations versus subpopulations without stemness properties. In addition, we show for the first time that hsa-miR-24c-3p is the most stable normalizer for comparing these two subpopulations. Also, we have identified by bioinformatic and qPCR analysis, different miR expression patterns in colon cancer versus non tumour cells using the previously selected suitable normalizers. Our results emphasize the importance of select suitable normalizers to ensure the robustness and reliability of qPCR data for analyzing miR expression.

Data supporting the physico-chemical characterization, cellular uptake and cytotoxicity of lipid nanocapsules.

The aim of this data article is to provide data for a basic knowledge of the properties of lipid nanocapsules, a new colloidal system with very promising applications in drug delivery. Firstly, we pay attention on how it is possible to determine their surface composition by means of electrokinetics measurements. On the other hand, we provide experimental evidences for a better understanding of the factors that determine the interactions of these nanoparticles with cells as a necessary step to guide the design of the most effective formulations. Additionally, we supply information about encapsulation efficiency of docetaxel, a potent chemotherapy drug, inside nanocapsules supporting the experimental cytotoxicity results of these nanosystems.

Balancing the effect of corona on therapeutic efficacy and macrophage uptake of lipid nanocapsules.

Several studies have shown the potential of biocompatible lipid nanocapsules as hydrophobic drug delivery systems. Understanding the factors that determine the interactions of these oil-in-water nanoemulsions with cells is a necessary step to guide the design of the most effective formulations. The aim of this study was to probe the ability of two surfactants with a markedly different nature, a non-ionic poloxamer, and a charged phospholipid, to prepare formulations with shells of different composition and different surface properties. Thus we determined their effects on the interaction with biological environments. In particular, we investigated how the shell formulation affected the adsorption of biomolecules from the surrounding biological fluids on the nanocapsule surface (corona formation). A complete physicochemical characterization including an isothermal titration calorimetry (ITC) study revealed that the use of poloxamer led to nanocapsules with a marked reduction in the number of protein-binding sites. Surface hydrophilicity and changes in corona formation strongly correlated to changes in uptake by cancer cells and by macrophages. Our results indicate that the nature and concentration of surfactants in the nanocapsules can be easily manipulated to effectively modulate their surface architecture with the aim of controlling the environmental interactions, thus optimizing functionality for in vivo applications. In particular, addition of surfactants that reduce protein binding can modulate nanoparticle clearance by the immune system, but also screens the desired interactions with cells, leading to lower uptake, thus lower therapeutic efficacy. The two effects need to be balanced in order to obtain successful formulations.

Regulatory systems in bone marrow for hematopoietic stem/progenitor cells mobilization and homing.

Regulation of hematopoietic stem cell release, migration, and homing from the bone marrow (BM) and of the mobilization pathway involves a complex interaction among adhesion molecules, cytokines, proteolytic enzymes, stromal cells, and hematopoietic cells. The identification of new mechanisms that regulate the trafficking of hematopoietic stem/progenitor cells (HSPCs) cells has important implications, not only for hematopoietic transplantation but also for cell therapies in regenerative medicine for patients with acute myocardial infarction, spinal cord injury, and stroke, among others. This paper reviews the regulation mechanisms underlying the homing and mobilization of BM hematopoietic stem/progenitor cells, investigating the following issues: (a) the role of different factors, such as stromal cell derived factor-1 (SDF-1), granulocyte colony-stimulating factor (G-CSF), and vascular cell adhesion molecule-1 (VCAM-1), among other ligands; (b) the stem cell count in peripheral blood and BM and influential factors; (c) the therapeutic utilization of this phenomenon in lesions in different tissues, examining the agents involved in HSPCs mobilization, such as the different forms of G-CSF, plerixafor, and natalizumab; and (d) the effects of this mobilization on BM-derived stem/progenitor cells in clinical trials of patients with different diseases.

How is gene transfection able to improve current chemotherapy? The role of combined therapy in cancer treatment.

Despite advances in cancer treatment, a large number of patients eventually develop metastatic disease that is generally incurable. Systemic chemotherapy remains the standard treatment for these patients. Several chemotherapeutic combinations have proven effective in the management of cancer. Paradoxically, although the purpose of polychemotherapy is to improve the prognosis and prolong the survival of patients, it often carries considerable toxicity that causes substantial adverse symptoms. For this reason, a major goal of cancer research is to improve the effectiveness of these cytotoxic agents and reduce their adverse effects. Gene transfer has been proposed as a new strategy to enhance the efficacy of anti-tumor drugs in the treatment of intractable or metastatic cancers. In fact, the association of gene therapy and drugs (combined therapy) has been reported to increase the anti-proliferative effect of classical treatments in lung, bladder, pancreatic, colorectal and breast cancers, among others. Various especially promising therapies have been proposed in this context, including the use of suicide genes, antisense oligonucleotides, ribozymes and RNA interference. In this chapter, we review recent progress in the development of novel anti-cancer strategies that associate cytotoxic agents with gene transfer to enhance their antitumor effect.

Tumour malignancy loss and cell differentiation are associated with induction of gef gene in human melanoma cells.

BACKGROUND: Gene therapy is a new method used to induce cancer cell differentiation. Our group previously showed that transfection of the gef gene from Escherichia coli, related to cell-killing functions, may be a novel candidate for cancer gene therapy. Its expression leads to cell cycle arrest unrelated to the triggering of apoptosis in MS-36 melanoma cells. OBJECTIVES: To determine the basis of the antiproliferative effect of the gef gene in this cell line. METHODS: Transmission electron microscopy, apoptosis analysis by confocal microscopy, flow cytometry and immunocytochemical analysis were used. RESULTS: Ultrastructural analysis showed a strikingly different morphology after treatment with dexamethasone and expression of the gef gene, with large accumulations of pigment throughout the cell cytoplasm and presence of melanosomes in different stages of development. High mitochondrial turnover and myeloid bodies, characteristics of neurone cells, were also observed. In addition, both immunocytochemical and indirect immunofluorescence analysis demonstrated a significant decrease in HMB-45, Ki-67 and CD44 antigen expression and an increase in S100 and p53 expression in gef gene-transfected MS-36 melanoma cells that were correlated with the duration of dexamethasone treatment. In the present work, we report that gef gene not only reduces cell proliferation in transfected melanoma MS-36TG cell line but also induces morphological changes clearly indicative of melanoma cell differentiation and a reduction in tumour malignancy. CONCLUSIONS: These findings support the hypothesis that the gef gene offers a new approach to differentiation therapy in melanoma.

Methodology in the European higher education area for the Anatomy learning in the Health Sciences

Development of the European Higher Education Area is linked to the adoption of a new educational model. Thus, in the Health Sciences, basic science knowledge must be integrated with the clinical skills that students will require in their professional activity. The Anatomy and Embryology Teaching Investigation Group at our university (UGR-N-40-UCUA) designed a specific questionnaire to analyse specific items that affect Anatomy learning. It also developed a teaching methodology for the acquisition of anatomic knowledge, integrating theory and practice, including individual and collective tasks for students and leading to future self-learning. Analyses were performed in different groups of first-year students at the School of Medicine of Granada University, School of Nursing of Almería University and School of Physiotherapy of Jaén University. It was found that application of this teaching methodology achieved an improvement in two areas considered essential by these students, i.e. a better understanding of several aspects of the study subject, and greater satisfaction with their acquisition of anatomic and embryologic knowledge (AU)

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Role of the organization of subjects and the educational resources used in their teaching in the Anatomy education-learning process

Within the three-year nursing degree course in Spain, the way in which anatomy teaching is organised shows distinct differences between different universities. At the University of Jaén, first-year students have human anatomy (HA) as a separate subject, whereas at the University of Almeria, anatomy is included within a larger module called the structure and function of the human body (SFHB). The aim of this study was to analyze the reaction of students to the organization of their anatomy courses, the resources used in their teaching, their contents, and the tutoring and evaluation system. For this purpose, a 35-item questionnaire was designed to address aspects related to these objectives and administered at the end of the 2005-6 academic year to 82 students of taking human anatomy at the University of Jaén and 52 students taking structure and function of human body at the University of Almeria. Results obtained showed differences in the evaluation of the educational organization of these subjects at the two universities. The approval rating of Jaen students for the relationship between their theoretical and practical education/training was 25% lower than that of Almeria students. This difference appears to be related to the different distributions of credits between the two subjects in the courses surveyed. Students appeared more highly to value the resources that were most frequently used during the course, suggesting that students may value most highly those resources employed most frequently within a course. There were some similarities between the students at the different universities in the importance they assigned to the different thematic units of the respective subjects. Finally, both groups revealed a preference for face-to-face tutorial sessions and for evaluation by written examinations (AU)

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Anatomy teaching to physiotherapy students: Preliminary study in the European higher education area setting

The integration of the Spanish university system within the European Higher Education Area implies a change in the current educational model towards a more flexible system that establishes the equivalence of degrees and encourages greater competition among courses. In this system, students will be expected to make a greater contribution to real learning in order for it to be more useful in their future professional activity. These changes will involve new student-teacher relationships, new methodologies, new teaching strategies and different evaluation systems. The success of this project will depend on a thorough knowledge of the present state of the courses that we teach. This is the first study to address the current state of human anatomy and embryology learning in the physiotherapy degree course. The analysis was performed in first-year students and focussed on the subject designated the structure and function of the human body, skeletal and muscle system anatomy at the Universities of Almería and Jaén. Student opinions were sought on the appropriateness of these subjects to their degree, on the methods used in practice and theory classes and on the evaluation and tutorial systems. Results obtained were similar between the two universities included in this study and indicated that: 1) students have a good opinion of the usefulness of the subject contents in human anatomy and embryology, 2) students prefer the new technologies to traditional educational systems, and 3) students have a positive appreciation of written examination versus oral examination or continuous continuous assessment. These findings will assist teachers of anatomy and embryology to establish approaches to improve the quality of learning in the setting of the European Higher Education Area (AU)

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Pedagogic evaluation of anatomical teaching for a medical and surgical degree at the University of Granada

Anatomy has been classically considered as a basic foundation for the teaching of medicine, developing a decisive role in medical education and for future professional activity. But, in common with other scientific disciplines, it has grown simultaneously with technology and communication sciences and in a much prescribed manner. The purpose of our study was to estimate different parameters related to the quality of the anatomical teaching at the University of Granada. In trying to achieve this, we have focused on the Human Anatomy I and II courses (given in the first and second years of the medical degree respectively). Once the examinations in these courses were completed, a questionnaire was filled by the students in which they had to estimate, in a one to five range, the adaptation and the adjustment of different aspects related to the development of the course. The results indicated that the students were in favour of practical classes compared to theoretical tuition. On the other hand, the pedagogical organisation of the courses was highly valued by the students, particularly in relation to the adaptation of programme objectives and to the recommended bibliography (both for textbooks and atlases). The best estimated didactic resource for the practical aspect of the subject was the use of human anatomical specimens, and the most favoured procedure in the theoretical classes was the use of the blackboard. For the examinations and assessments, no special preference for any evaluation method was found, but the use of complementary papers (e.g. use of monographs, oral expositions) was considered by the students to be of very little importance (AU)

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Induction of drug resistance in embryonal rhabdomyosarcoma treated with conventional chemotherapy is associated with HLA class I increase.

Effectiveness of conventional cytotoxic treatment of rhabdomyosarcoma (RMS) may be limited by the development of multidrug resistance (MDR) mediated by mdr1 gene. This gene codes for P-glycoprotein (P-gp) which has been related to a immunoregulatory function. Modulation of HLA expression by P-gp has been described in different types of tumor cells including RMS. However, very little is known about biological implications of the P-gp expression in RMS patients treated with conventional chemotherapy. In order to study the problem, we used embryonal RMS tissue samples from treated patients. Our results indicated that positive RMS samples to mdr1 show higher HLA class I expression than those which were negative to mdr1 PCR, what indicates a significant correlation between the expression of both molecules. In addition, we developed two resistant RMS cell lines (A-204-1 and 2) using similar concentrations of actinomycin D as are plasma levels in clinical situation. Both resistant cell lines showed mdr1 expression and an increase of HLA class I expression which was dose-dependent. These results demonstrated that conventional chemotherapy of embryonal RMS is able to induce resistance which can modulate HLA class I expression and suggest that immunological studies of these tumors may be necessary to the design new specific therapeutic strategies.

Release of alpha-actin into serum after skeletal muscle damage.

OBJECTIVE: The skeletal muscle protein alpha-actin was investigated in the serum of subjects with severe skeletal muscle damage to assess its utility as a reliable and predictive marker of muscle damage. METHODS: Serum samples were obtained from 33 healthy controls and 33 patients with severe skeletal muscle damage, defined by a total creatine kinase value of >500 IU/l (Rosalki method). Troponin I, troponin T, and myoglobin concentrations were determined by immunoassay and alpha-actin concentrations by Western blot and densitometry. RESULTS: The mean serum concentration of alpha-actin in controls and patients with skeletal muscle damage was 600.9 and 1968.51 ng/ml, respectively, a statistically significant difference. Sera of patients with muscle damage showed higher levels of alpha-actin than of troponin or myoglobin. No significant difference in troponin I levels was observed between the groups. CONCLUSIONS: According to these results, alpha-actin was the most significant skeletal muscle damage marker analysed and may be an ideal candidate for the identification of all types of myofibre injury, including sports injuries. Our findings support the use of alpha-actin as a marker alongside other currently used biological proteins.

Non-viral and viral vectors for gene therapy.

Human gene therapy can be defined as the delivery of genetic material into a patient's cells with a therapeutic aim. The success or failure of gene therapy depends on the development and efficiency of the transfection of viral and non-viral vectors. Viral vectors typically offer higher transduction efficiency and long-term gene expression, but may be associated with toxicity, immunogenicity, restricted target cell specificity and high cost. Non-viral methods have become widespread because of their relative safety, capacity to transfer large genes, site-specificity and their non-inflammatory, non-toxic and non-infectious properties. However, the clinical usefulness of non-viral methods is limited by their low transfection efficiency and relatively poor transgene expression. In this review, we describe the progress made in the development of gene delivery technology and its possible application in clinical trials.

Cancer gene therapy: strategies and clinical trials.

In recent years, great advances have been made in developing novel therapeutic systems based on the introduction of genetic material into damaged cells and designed to correct the error underlying the disease or destroy the pathological cell. One of the main applications of this new approach, known as gene therapy, is the treatment of malignant pathological tumours, in which classic treatments with radiotherapy, chemotherapy and surgery are only palliative. Strategies developed to date include the use of suicide genes, immunity-enhancing genes, apoptosis-inducing genes or genes that inhibit the neovascularization of the tumour, and the blocking of mutated tumour suppressor genes or their restoration in the tumour cell. The effectiveness shown in cell culture and animal experiments and some promising results in clinical trials suggest that gene therapy will help to improve the prognosis of cancer patients and may become the treatment of choice.

Modulation of Ki-67 expression and morphological changes induced by gef gene in MCF-7 human breast cancer cells.

New therapeutic strategies are required to overcome the limitations of conventional breast cancer treatment. Suicide gene therapy offers a potential approach to this type of tumour, since systems based on the use of prodrugs may present some drawbacks related to toxicity, drug release and bioavailability. The gef gene has cell-killing functions in Escherichia coli and does not depend on the use of a prodrug for its action, making it an attractive target for suicide gene therapy. We created a gef-overexpressing human breast cancer cell line (MCF-7TG) by transfecting the gef gene under the control of a pMAMneo promotor. Dexamethasone-induction of gef gene expression in MCF-7TG cells produced a significant decrease in Ki-67 expression, which is a known proliferation marker. In addition, annexin-V-FITC and propidium iodide assays showed the presence of apoptotic cell death, which was confirmed by scanning electron microscopy. The most significant finding was the presence of "craters" in the cell membrane, as previously described in other apoptotic breast cancer cells. These results demonstrate the ability of the gef gene to down regulate Ki-67 expression and induce apoptosis in a breast cancer cell line, suggesting its potential application as a new gene therapy strategy for this type of tumor.

Contractile regulatory proteins tropomyosin and troponin-T as indicators of the modulatory role of retinoic acid.

Retinoic acid (RA), the active metabolite of vitamin A, plays a significant role in regulating cardiac form and function throughout the life of the organism. Both cardiac morphogenesis and myocardial differentiation are affected by alterations in RA homeostasis. In order to test the effect of all-trans RA and 13-cis RA on cardiomyocyte differentiation, we studied the level and the subcellular compartmentalization of alpha-tropomyosin and troponin-T proteins in cultures of chick embryo cardiomyocytes obtained from Hamburger and Hamilton's (HH) stage 22, 32 and 40 embryos. The retinoids increased the levels of alpha-tropomyosin and troponin-T in the cytoplasmic and cytoskeletal fractions of cells at all three stages of development. The greatest increases in alpha-tropomyosin occurred in the cytoplasmic fraction in HH22 cells cultured for 24 h with all-trans RA or 13-cis RA, whereas the greatest increases in troponin-T were found in the cytoplasmic fraction of HH32 cells exposed to retinoids for 24 h. In cultures treated for 48 h with retinoids, the levels of alpha-tropomyosin and troponin-T showed significant increases in the cytoplasmic compartment of cells treated in HH32-with respect to the control values. These findings are further evidence that RA plays a modulating role in the formation and reorganization of sarcomeric proteins during the process of cardiomyocyte maturation.

Inhibition of growth and induction of apoptosis in human breast cancer by transfection of gef gene.

The gef gene has cell-killing functions in Escherichia coli. To evaluate the feasibility of using this gene as a new strategy for cancer therapy, we transfected it in MCF-7 cells derived from breast cancer (MCF-7TG). The gef gene was cloned in a pMAMneo vector under the control of a mouse mammary tumour virus promoter, inducible by dexamethasone (Dex), and was transfected with liposomes. After selection and induction, expression of the gef gene was confirmed by reverse transcription-polymerase chain reactions (RT-PCR) and Western blot. Cell viability was determined with a haemocytometre and the sulphorodamine B colorimetric assay, and the cell cycle was studied by propidium iodide (PI) staining. Annexin V-FITC and PI assays were used to evaluate apoptosis, which was confirmed by electron microscopy. In comparison with MCF-7 parental cells and MCF-7 cells transfected with an empty vector, MCF-7TG cells induced with Dex showed a significant decrease in proliferation rate, which was associated with evidence of apoptosis. Morphological findings confirmed apoptosis and showed a typical pattern of mitochondrial dilation. Furthermore, the cell cycle was characterised by premature progression from G(1) to S phase and G(2) delay. Our results show that the gef gene was able to decrease proliferation in a breast cancer cell line, and induce apoptosis. These findings suggest that the gef gene is a potential candidate for tumour therapy.

Modulation of HLA class I expression in multidrug-resistant human rhabdomyosarcoma cells.

An abnormal HLA expression has been detected in some tumors including rhabdomyosarcoma (RMS). Classical cytotoxic treatment of these tumors, the most common childhood soft tissue malignancy, may induce multidrug resistance (MDR) associated with the expression of a 170-kDa membrane-associated glycoprotein (P-glycoprotein). In order to analyse the connection between modulation of HLA expression and the development of the MDR phenotype mediated by P-glycoprotein in RMS, we used three resistant RMS cell lines; two of these resistant cell lines (TE.32.7.DAC and RD-DAC) were established by in vitro exposure to actinomycin D, a drug of choice in the treatment of RMS; the resistant RMS- GR cell line was established from an embryonal RMS tumor after polychemotherapy. Our results showed that all the resistant cell lines showed a significant increase in the expression of HLA class I surface antigens in comparison to drug-sensitive cells. Blockade of P-glycoprotein with verapamil led to a decrease in HLA class I expression in RMS resistant cell lines. However, no modulation of HLA class II expression was observed in any of the three analyzed cell lines. These findings support the hypothesis that the development of resistance mediated by mdr 1/P-glycoprotein, directly influences the expression of HLA class I in RMS cells, inducing to upregulation. This effect may be relevant to the application in RMS of immunotherapy against tumor-associated antigens presented by HLA class I molecules.