Exosomes Derived from Breast Cancer Cells, Small Trojan Horses?

Exosomes are small extracellular vesicles secreted to the extracellular environment by several cell types, including tumor cells. It has been demonstrated that exosomes have an important role in intercellular communication, but they have recently been implicated in various tumor processes, including the oncogenic transformation of cells in the tumor microenvironment, tumor drug resistance, and the transport of tumor factors. Tumors appear to use exosomes to dialogue with and transform neighboring cells to create an ideal environment for their growth and expansion. On the other hand, the structure and function of exosomes may make them useful in cancer diagnosis and prognosis, because they contain molecules that could serve as biomarkers, including oncogenes, miRNAs, and certain proteins. They have the ability to travel via body fluids, from which they could be isolated and used to transport drugs to specific targets. This review aims to provide an update on the role of exosomes derived from breast cancer cells.

Cancer stem cells and their implication in breast cancer.

BACKGROUND: The cancer stem cell (CSC) hypothesis on the origin of cancer has recently gained considerable support. CSCs are tumour cells with the capacity for self-renewal and differentiation that direct the origin and progression of the disease and may be responsible for relapse, metastasis and treatment failures. DESIGN: This article reviews breast CSCs (BCSCs) phenotyping, clinical implications and clinical trials focused on BCSCs in breast cancer. Relevant studies were found through PubMed and Clinicaltrials.gov databases. RESULTS: Cancer stem cells are identified and isolated using membrane and cell activity markers; in the case of BCSCs, these are CD44(+) /CD24(low/-) and show aldehyde dehydrogenase activity, alongside their capacity to grow and form mammospheres. The presence of stem cell properties is associated with a worse outcome. Hence, these cells have important clinical implications, and elucidation of the mechanisms underlying their activity will allow the development of novel effective therapies and diagnostic instruments, improving the prognosis of these patients. CONCLUSIONS: Standard treatments are directed against the tumour mass and do not eliminate CSCs. There is therefore a need for specific anti-CSC therapies, and numerous authors are investigating new targets to this end, as reported in this review. It is also necessary for clinical trials to be undertaken to allow this new knowledge to be applied in the clinical setting. However, there have been few trials on anti-BCSCs therapies to date.

ABC transporters as differentiation markers in glioblastoma cells.

UNLABELLED: Glioblastoma multiforme (GBM) is the most common primary malignant brain tumour, characterized by a high aggressivity, a huge heterogeneity attending a hierarchical model and resistance to therapy. Drug resistance has been correlated with the presence of the ABC efflux transporters which are able to exclude drugs for the cellular cytoplasm. In the nucleus of the GBM, initiating cells (ICs) can self-renew and give rise to cancer stem cells, which differ to the side population cells and the different cellular subtypes that form the mass around them. The ICs do not express or express ATP binding cassette (ABC) at very low levels, but this expression may increase with the differentiation process. We suggest that the differentiation process may be responsible of chemoresistance of the GBM cells. We compared three ABC transporters expression: ABCA1, MRP4 and MRP5, in the ICs obtained from 9 patients with GBM and their respective differentiated GBM cells. We show an overexpression of the three ABC transporters in the differentiated GBM cells in comparison to ICs. IMPLICATIONS OF THE HYPOTHESIS: The blockade of these ABC transporters could help to improve the drug effectivity and thus reduce the tumour growth and prevent the tumour recurrence.

Transcriptional profiling of peripheral blood in pancreatic adenocarcinoma patients identifies diagnostic biomarkers.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy associated with poor survival rates. Fast detection of PDAC appears to be the most relevant strategy to improve the long-term survival of patients. AIMS: Our objective was to identify new markers in peripheral blood that differentiates between PDAC patients and healthy controls. METHODS: Peripheral blood samples from PDAC patients (n = 18) and controls (n = 18) were analyzed by whole genome cDNA microarray hybridization. The most relevant genes were validated by quantitative real-time PCR (RT-qPCR) in the same set of samples. Finally, our gene prediction set was tested in a blinded set of new peripheral blood samples (n = 30). RESULTS: Microarray studies identified 87 genes differentially expressed in peripheral blood samples from PDAC patients. Four of these genes were selected for analysis by RT-qPCR, which confirmed the previously observed changes. In our blinded validation study, the combination of CLEC4D and IRAK3 predicted the diagnosis of PDAC with 93 % accuracy, with a sensitivity of 86 % and specificity of 100 %. CONCLUSIONS: Peripheral blood gene expression profiling is an useful tool for the diagnosis of PDAC. We present a validated four-gene predictor set (ANKRD22, CLEC4D, VNN1, and IRAK3) that may be useful in PDAC diagnosis.

DNA methylation plasticity of human adipose-derived stem cells in lineage commitment.

Adult stem cells have an enormous potential for clinical use in regenerative medicine that avoids many of the drawbacks characteristic of embryonic stem cells and induced pluripotent stem cells. In this context, easily obtainable human adipose-derived stem cells offer an interesting option for future strategies in regenerative medicine. However, little is known about their repertoire of differentiation capacities, how closely they resemble the target primary tissues, and the potential safety issues associated with their use. DNA methylation is one of the most widely recognized epigenetic factors involved in cellular identity, prompting us to consider how the analyses of 27,578 CpG sites in the genome of these cells under different conditions reflect their different natural history. We show that human adipose-derived stem cells generate myogenic and osteogenic lineages that share much of the DNA methylation landscape characteristic of primary myocytes and osteocytes. Most important, adult stem cells and in vitro-generated myocytes and osteocytes display a significantly different DNA methylome from that observed in transformed cells from these tissue types, such as rhabdomyosarcoma and osteosarcoma. These results suggest that the plasticity of the DNA methylation patterns plays an important role in lineage commitment of adult stem cells and that it could be used for clinical purposes as a biomarker of efficient and safely differentiated cells.

Four accessory (supernumerary) intrathoracic ribs: a case report.

Accessory (supernumerary) intrathoracic ribs are a very rare congenital disorder. Here, we present the first case of multiple supernumerary intrathoracic ribs in an adult, which are present consecutively between ribs 1 and 4 and without articulation with the vertebrae. Despite this, anatomical variation is usually silent and accidentally discovered; its knowledge can prevent confusion with other structures during imaging diagnostic techniques of thoracic pathologies.

MGMT promoter methylation status and MGMT and CD133 immunohistochemical expression as prognostic markers in glioblastoma patients treated with temozolomide plus radiotherapy.

BACKGROUND: The CD133 antigen is a marker of radio- and chemo-resistant stem cell populations in glioblastoma (GBM). The O6-methylguanine DNA methyltransferase (MGMT) enzyme is related with temozolomide (TMZ) resistance. Our propose is to analyze the prognostic significance of the CD133 antigen and promoter methylation and protein expression of MGMT in a homogenous group of GBM patients uniformly treated with radiotherapy and TMZ. The possible connection between these GBM markers was also investigated. METHODS: Seventy-eight patients with GBM treated with radiotherapy combined with concomitant and adjuvant TMZ were analyzed for MGMT and CD133. MGMT gene promoter methylation was determined by methylation-specific polymerase chain reaction after bisulfite treatment. MGMT and CD133 expression was assessed immunohistochemically using an automatic quantification system. Overall and progression-free survival was calculated according to the Kaplan-Meier method. RESULTS: The MGMT gene promoter was found to be methylated in 34 patients (44.7%) and unmethylated in 42 patients (55.3%). A significant correlation was observed between MGMT promoter methylation and patients' survival. Among the unmethylated tumors, 52.4% showed low expression of MGMT and 47.6% showed high-expression. Among methylated tumors, 58.8% showed low-expression of MGMT and 41.2% showed high-expression. No correlation was found between MGMT promoter methylation and MGMT expression, or MGMT expression and survival. In contrast with recent results, CD133 expression was not a predictive marker in GBM patients. Analyses of possible correlation between CD133 expression and MGMT protein expression or MGMT promoter methylation were negative. CONCLUSIONS: Our results support the hypothesis that MGMT promoter methylation status but not MGMT expression may be a predictive biomarker in the treatment of patients with GBM. In addition, CD133 should not be used for prognostic evaluation of these patients. Future studies will be necessary to determine its clinical utility.

Development and morphogenesis of human wrist joint during embryonic and early fetal period.

The development of the human wrist joint has been studied widely, with the main focus on carpal chondrogenesis, ligaments and triangular fibrocartilage. However, there are some discrepancies concerning the origin and morphogenetic time-table of these structures, including nerves, muscles and vascular elements. For this study we used serial sections of 57 human embryonic (n = 30) and fetal (n = 27) specimens from O'Rahilly stages 17-23 and 9-14 weeks, respectively. The following phases in carpal morphogenesis have been established: undifferentiated mesenchyme (stage 17), condensated mesenchyme (stages 18 and 19), pre-chondrogenic (stages 19 and 20) and chondrogenic (stages 21 and over). Carpal chondrification and osteogenic processes are similar, starting with capitate and hamate (stage 19) and ending with pisiform (stage 22). In week 14, a vascular bud penetrates into the lunate cartilaginous mold, early sign of the osteogenic process that will be completed after birth. In stage 18, median, ulnar and radial nerves and thenar eminence appear in the hand plate. In stage 21, there are indications of the interosseous muscles, and in stage 22 flexor digitorum superficialis, flexor digitorum profundus and lumbrical muscles, transverse carpal ligament and collateral ligaments emerge. In stage 23, the articular disc, radiocarpal and ulnocarpal ligaments and deep palmar arterial arch become visible. Radiate carpal and interosseous ligaments appear in week 9, and in week 10, dorsal radiocarpal ligament and articular capsule are evident. Finally, synovial membrane is observed in week 13. We have performed a complete analysis of the morphogenesis of the structures of the human wrist joint. Our results present new data on nervous and arterial elements and provide the basis for further investigations on anatomical pathology, comparative morphology and evolutionary anthropology.

Novel drug delivery system based on docetaxel-loaded nanocapsules as a therapeutic strategy against breast cancer cells.

In the field of cancer therapy, lipid nanocapsules based on a core-shell structure are promising vehicles for the delivery of hydrophobic drugs such as docetaxel. The main aim of this work was to evaluate whether docetaxel-loaded lipid nanocapsules improved the anti-tumor effect of free docetaxel in breast cancer cells. Three docetaxel-loaded lipid nanocapsules were synthesized by solvent displacement method. Cytotoxic assays were evaluated in breast carcinoma (MCF-7) cells treated by the sulforhodamine B colorimetric method. Cell cycle was studied by flow cytometry and Annexin V-FITC, and apoptosis was evaluated by using propidium iodide assays. The anti-proliferative effect of docetaxel appeared much earlier when the drug was encapsulated in lipid nanoparticles than when it was free. Docetaxel-loaded lipid nanocapsules significantly enhanced the decrease in IC(50) rate, and the treated cells evidenced apoptosis and a premature progression of the cell cycle from G(1) to G(2)-M phase. The chemotherapeutic effect of free docetaxel on breast cancer cells is improved by its encapsulation in lipid nanocapsules. This approach has the potential to overcome some major limitations of conventional chemotherapy and may be a promising strategy for future applications in breast cancer therapy.

Modulation of MDR1 and MRP3 gene expression in lung cancer cells after paclitaxel and carboplatin exposure.

Carboplatin-paclitaxel is a reference regimen in the treatment of locally advanced or disseminated non-small cell lung cancer (NSCLC). This paper discusses the multidrug resistance developed with this drug combination, which is one of the major obstacles to successful treatment. In order to understand and overcome the drug resistance pattern of NSCLC after carboplatin plus paclitaxel exposure, levels of mRNA expression of multidrug resistance 1 (MDR1) and multidrug resistance-associated protein 3 (MRP3) were investigated in primary NSCLC cell lines (A-549 and A-427) and a metastasis-derived NSCLC cell line (NODO). Our results showed that exposure of the three NSCLC lines to plasma concentrations of paclitaxel (5 µM) produced an increase in MDR1 expression, while MRP3 showed no alteration in expression. By contrast, the same cells exposed to carboplatin plasma concentrations (30 µM) showed overexpression of MRP3. In these cells, MDR1 showed no expression changes. Interestingly, the combination of both paclitaxel and carboplatin caused increased expression of the MDR1 drug resistance gene rather than the individual treatments. These results suggest that carboplatin and paclitaxel may induce drug resistance mediated by MDR1 and MRP3, which may be enhanced by the simultaneous use of both drugs.

Influence of preinfarction angina on the release kinetics of endothelial progenitor cells and cytokines during the week after infarction.

BACKGROUND: Preinfarction angina, a possible form of ischaemic preconditioning, improves the prognosis in patients who experience a major ischaemic event; though the associated pathophysiology is not yet fully understood. The aim of this study was to determine the possible involvement of endothelial progenitor cells (EPC), the vascular endothelial growth factor (VEGF) and the hepatocyte growth factor (HGF) in the development of preinfarction angina. METHODS AND RESULTS: We studied 41 patients (60·5 ± 12 years; 34% women) and 14 healthy controls; 43·9% of the patients had preinfarction angina. No differences were found in the baseline characteristics of the two groups. Although the EPC, VEGF and HGF were raised as compared with the control group, no significant differences were found according to the presence or absence of preinfarction angina in the levels of EPC (baseline, P = 0·25; day 3, P = 0·11; day 7, P = 0·32), VEGF (baseline, P = 0·96; day 3, P = 0·06; day 7, P = 0·57) or HGF (baseline, P = 0·18; day 3, P = 1; day 7, P = 0·86). An association was seen in the patients who had preinfarction angina between the EPC levels at baseline and on days 3 and 7 and the HGF on admission with the time from the angina to the STEMI (ß = -0·070; ß = -0·066; ß = -0·081; ß = -80·16; P < 0·05), showing a reduction in the level of EPC cells for each hour passed since the event. CONCLUSIONS: No differences were found in the release kinetics of EPC, VEGF or HGF after a first infarction according to whether the patients had angina during the week before the infarction.

Ultrastructural and molecular analyzes of insulin-producing cells induced from human hepatoma cells.

BACKGROUND AIMS: Diabetes type I is an autoimmune disease characterized by the destruction of pancreatic insulin-producing (beta-) cells and resulting in external insulin dependence for life. Islet transplantation represents a potential treatment for diabetes but there is currently a shortage of suitable organs donors. To augment the supply of donors, different strategies are required to provide a potential source of beta-cells. These sources include embryonic and adult stem cells as well as differentiated cell types. The main goal of this study was to induce the transdifferentiation (or conversion of one type cell to another) of human hepatoma cells (HepG2 cells) to insulin-expressing cells based on the exposure of HepG2 cells to an extract of rat insulinoma cells (RIN). METHODS: HepG2 cells were first transiently permeabilized with Streptolysin O and then exposed to a cell extract obtained from RIN cells. Following transient exposure to the RIN extract, the HepG2 cells were cultured for 3 weeks. RESULTS: Acquisition of the insulin-producing cell phenotype was determined on the basis of (i) morphologic and (ii) ultrastructural observations, (iii) immunologic detection and (iv) reverse transcription (RT)-polymerase chain reaction (PCR) analysis. CONCLUSIONS: This study supports the use of cell extract as a feasible method for achieve transdifferentiation of hepatic cells to insulin-producing cells.

Transdifferentiation: why and how?

Cell therapy is based on the replacement of damaged cells in order to restore injured tissues. The first consideration is that an abundant source of cells is needed; second, these cells should be immunologically compatible with the guest and third, there should be no real threat of these cells undergoing malignant transformation in the future. Given these requirements, already differentiated adult cells or adult stem cells obtained from the body of the patient appear to be the ideal candidates to meet all of these demands. The utilization of somatic cells also avoids numerous ethical and political drawbacks and concerns. Transdifferentiation is the phenomenon by which an adult differentiated cell switches to another differentiated cell. This paper reviews the importance of transdifferentiation, discussing the cells that are suitable for this process and the methods currently employed to induce the change in cell type.

Nanomedicine: application areas and development prospects.

Nanotechnology, along with related concepts such as nanomaterials, nanostructures and nanoparticles, has become a priority area for scientific research and technological development. Nanotechnology, i.e., the creation and utilization of materials and devices at nanometer scale, already has multiple applications in electronics and other fields. However, the greatest expectations are for its application in biotechnology and health, with the direct impact these could have on the quality of health in future societies. The emerging discipline of nanomedicine brings nanotechnology and medicine together in order to develop novel therapies and improve existing treatments. In nanomedicine, atoms and molecules are manipulated to produce nanostructures of the same size as biomolecules for interaction with human cells. This procedure offers a range of new solutions for diagnoses and "smart" treatments by stimulating the body's own repair mechanisms. It will enhance the early diagnosis and treatment of diseases such as cancer, diabetes, Alzheimer's, Parkinson's and cardiovascular diseases. Preventive medicine may then become a reality.

gef gene expression in MCF-7 breast cancer cells is associated with a better prognosis and induction of apoptosis by p53-mediated signaling pathway.

Breast cancer research has developed rapidly in the past few decades, leading to longer survival times for patients and opening up the possibility of developing curative treatments for advanced breast cancer. Our increasing knowledge of the biological pathways associated with the progression and development of breast cancer, alongside the failure of conventional treatments, has prompted us to explore gene therapy as an alternative therapeutic strategy. We previously reported that gef gene from E. coli has shown considerable cytotoxic effects in breast cancer cells. However, its action mechanism has not been elucidated. Indirect immunofluorescence technique using flow cytometry and immunocytochemical analysis were used to detect breast cancer markers: estrogen (ER) and progesterone (PR) hormonal receptors, human epidermal growth factor receptor-2 proto-oncogene (c-erbB-2), ki-67 antigen and p53 protein. gef gene induces an increase in ER and PR expressions and a decrease in ki-67 and c-erbB-2 gene expressions, indicating a better prognosis and response to treatment and a longer disease-free interval and survival. It also increased p53 expression, suggesting that gef-induced apoptosis is regulated by a p53-mediated signaling pathway. These findings support the hypothesis that the gef gene offers a new approach to gene therapy in breast cancer.

Human cardiac tissue induces transdifferentiation of adult stem cells towards cardiomyocytes.

BACKGROUND AIMS: The goal was to induce the transdifferentiation (or conversion) of human adipose-derived stem cells to cardiomyocytes using an intracellular extract obtained from adult human heart tissue. METHODS: Human adult stem cells from lipoaspirates were transiently permeabilized, exposed to human atrial extracts and allowed to recover in culture. RESULTS: After 21 days, the cells acquired a cardiomyocyte phenotype, as demonstrated by morphologic changes (appearance of binucleate, striated cells and branching fibers), immunofluorescence detection of cardiac-specific markers (connexin-43, sarcomeric alpha-actinin, cardiac troponin I and T, and desmin) and the presence of cardiomyocyte-related genes analyzed by reverse transcription-polymerase chain reaction (cardiac myosin light chain 1, alpha-cardiac actin, cardiac troponin T and cardiac beta-myosin). CONCLUSIONS: We have demonstrated for the first time that adult cardiomyocytes obtained from human donors retain the capacity to induce cardiomyocyte differentiation of mesenchymal stromal cells. The use of autologous extracts for reprogramming adult stem cells may have potential therapeutic implications for treating heart disease.

Regression of established subcutaneous B16-F10 murine melanoma tumors after gef gene therapy associated with the mitochondrial apoptotic pathway.

Novel treatment modalities, including gene therapy, are needed for patients with advanced melanoma. We evaluated whether the gef gene, a suicide gene from Escherichia coli, had a significant cytotoxic impact on melanoma in vivo. First, we used a non-viral gene delivery approach (pcDNA3.1/gef) to study the inhibition of melanoma cells (B16-F10) proliferation in vitro. Secondly, we used direct intra-tumoral injection of pcDNA3.1/gef complexed with jetPEI to deliver gef cDNA to rapidly growing murine melanomas. We demonstrated that gef gene not only has an antiproliferative effect on B16-F10 cells in vitro, but also induces an important decrease in melanoma tumor volume (77.7% in 8 days) in vivo. Interestingly, after gef gene treatment, melanoma showed apoptosis activation associated with the mitochondrial pathway, suggesting that the induction of this death mechanism may be an effective strategy for its treatment. Our in vivo results indicate that gef gene might become a suitable therapeutic strategy for patients with advanced melanoma.

Differentiation of intestinal epithelial cells mediated by cell confluence and/or exogenous nucleoside supplementation.

Nucleotides (NT) and nucleosides (NS) play a key role in gastrointestinal development and in enterocyte healing after tissue damage. Exogenous NT and NS may therefore represent a novel therapy for maintaining gastrointestinal tract integrity. An exogenous NS mixture of thymidine, cytidine, guanosine and inosine (T-CGI) increases the proliferation rate of rat intestinal epithelial cell line 6 (IEC-6) cells, while a mixture of uridine, cytidine, guanosine and inosine (U-CGI) reduces IEC-6 proliferation independently of necrosis or apoptosis. This study aimed to analyze the effects of exogenous NS on IEC-6 differentiation under proliferation and differentiation conditions. To this end, IEC-6 cells were treated with NS T-CGI and NS U-CGI mixtures under low- and high-density conditions. Enterocyte differentiation was also assessed by flow cytometry, Western blotting, and light, fluorescence and transmission electron microscopy. Under proliferative conditions, villin expression was reduced in all cases, but NS-treated cells showed twofold the expression observed in NS-free cultures (controls) and more frequently showed characteristics of mature enterocytes. When cells were grown after confluence, villin expression, total protein production and morphology of NS-treated cultures were more differentiated compared with the control group. Our results demonstrate that T-CGI and U-CGI mixtures promote IEC-6 cell differentiation, with no significant differences between them. Unlike previous authors, we obtained this effect in cultures without an exogenous extracellular matrix such as Matrigel, reducing the variability among independent assays.

Promotion of human adipose-derived stem cell proliferation mediated by exogenous nucleosides.

Adult stem cells are becoming the best option for regenerative medicine because they have low tumourigenic potential and permit autologous transplantation, even without in vitro culture. Our objectives were to evaluate the effects of exogenous nucleosides on the proliferation of hASCs (human adipose-derived stem cells), with or without co-treatment with 5-aza (5-azacytidine), and to analyse the expression of lamin A/C during cardiomyocyte differentiation of these cells. We isolated hASCs from human lipoaspirates that were positive for mesenchymal stem cell markers. We found that 5-aza induces a dose-dependent inhibition of hASC proliferation [IC50 (inhibitory concentration 50): 5.37 microM], whereas exogenous nucleosides significantly promote the proliferation of hASCs and partially revert the antiproliferative effect of the drug. Multipotentiality of isolated hASCs was confirmed by adipogenic, osteogenic and cardiomyogenic induction. 5-Aza-induced cells expressed cardiac troponins I and T and myosin light chain 2, myocardial markers that were directly correlated with lamin A/C expression. Our results support the importance of the nucleoside supplementation of media to improve conditions for the expansion and maintenance of hASCs in culture. In addition, the quantification of lamin A/C expression appears to be a good marker for the characterization of cardiomyocyte differentiation of stem cells that has rarely been used.

Capsular origin of the long head of the biceps brachii tendon: a clinical case.

The biceps brachii tendon arises directly from the superior glenoid labrum with the remainder usually attached to the supraglenoid tubercle. Although some cases of anomalous origin of this tendon have been described, these anomalies are rarely encountered in daily practice. We report a patient with a capsular origin of the LHBT as a congenital anomaly and present the clinical, magnetic resonance and arthroscopic findings. Recognition of this anatomic variation may be important to explain the patient's clinical data and to aid both diagnosis and surgery.