Rastrejant el càncer

El càncer és una malaltia produïda pel creixement descontrolat. Entrendre'n l'origen i millorar-ne el diagnòstic són dos dels punts clau a tractar per reduir-ne la incidència i la mortalitat. La cerca de marcadors tumorals específics de cada tipus de càncer és l'escenari ideal per assolir aquestes fites. L'estudi de mostres de pacients obtingudes de manera mínimament invasiva o no invasiva comporta la millora del seu diagnòstic, tant per la reducció de les molèsties en el pacient com per la disminució del cost del tractament per a la societat en general. En aquest article s'exposen tres projectes com a exemple d'aquest tipus de recerca: el càncer d'ovari, el càncer d'endometri i el càncer de pròstata

La Biomedicina del segle XXI

En els darrers trenta anys, la nostra societat ha assistit perplexa al desenvolupament d'una revolució que, sens dubte, la història jutjarà com la de més transcendència des que els humans habiten el planeta Terra. Ens referim a l'immens progrés en el coneixement de la ciència i la tècnica i al desenvolupament de les seves aplicacions. La biologia i, en particular, el desenvolupament de la biologia molecular, ocupa un espai majoritari en aquest procés. També la informàtica, l'electrònica, la robòtica o, de manera més general, la física i la química, estan en la primera línia de la seva evolució. Aquest capítol pretén posar totes aquestes troballes juntes en benefici de la nova medicina que ha sorgit d'aquesta barreja. Sense cap pretensió de voler resumir tots els avenços que componen aquesta nova medicina, hem volgut discutir breument el que signifiquen ara mateix i en un futur molt proper aquests canvis que comporten, entre molts altres, el diagnòstic i tractament millors del càncer, la teràpia gènica per a malalties genètiques fins ara incurables, els diferents tipus de diagnòstic per imatge amb un sofisticat suport informàtic, el coneixement del nostre genoma, la producció de proteïnes de propietats terapèutiques per biotecnologia, així com la connexió dels malalts a una xarxa de metges i d'hospitals que els permeti estar adequadament atesos. Nogensmenys, aquestes millores espectaculars d'un món altament tecnificat no ens han de fer ignorar els desafiaments ètics que tot això pot comportar, així com quelcom més elemental i bàsic, que és la solidaritat i caritat envers els febles, oblidats o mancats de recursos.In the last 30 years, we have assisted astonished to the development of a revolution that, with no doubt, history will judge as the most important since humans live in this planet. We refer to the progress in the knowledge of science and technology, and to the development of their applications. Biology, and, in particular, the development of molecular biology, is fundamental in this process. Also computer science, electronics, robotics, or, in a more general way, physics and chemistry are also in the frontline of their growth. This chapter only pretends to put together all these findings in favor of a new medicine. This is not an exhaustive review of these major advances, but just a brief summary of few breakthroughs like the improved diagnosis and treatment of cancer, gene therapy as a solution for genetic diseases, improved image based diagnosis methods, unravelling of the human genome, production of therapeutic recombinant proteins by biotechnology, as well as the new medical care system by having patients connected to physician and hospital networks. Nevertheless, those spectacular improvements in a highly technified world cannot avoid the new ethic challenges emerging in this process. At the same time, solidarity towards weak, poor and discriminated people does not have to be forgotten

Biomarcadors de diagnòstic, pronòstic i predicció de resposta a tractaments oncològics

Better biomarkers are urgently needed to improve diagnosis, prognosis, and targeted intherapy across a wide range of diseases. Currently, scientists are moving forward to solve this problem. New developments in research help to discover many more biomarkers than ever before. However, although the number of articles that have been published in this area is increasing, only few biomarkers have been implemented in the clinical practice. This is mainly due to two reasons: biomarkers fail to meet the demands of the clinic; and false discoveries fail during the long way that represents the validation phases. The new biomarker pipeline, which currently includes new phases in the middle of the process, has significantly increased the percentage of success. In other words, the chances that the new findings are incorporated into routine clinical practice and improve the patient care are higher than before. This improvement in diagnosis, prognosis and targeted therapy, which is increasingly more specific for each patient who suffers a certain disease, brings us step by step closer to what is known as personalized medicine.La necessitat d'identificar nous biomarcadors que aportin una millora significativa del diagnòstic, pronòstic i del seguiment de teràpia de moltes malalties fa que avui dia la cerca de nous biomarcadors sigui un camp de la ciència increïblement explorat. Alhora, l'avenç tecnològic en el camp de la biomedicina ha fet que avui, més que mai, el nombre de nous candidats a biomarcadors hagi crescut de manera exponencial. No obstant això, en els últims anys, tot i l'elevat nombre de publicacions en aquest àmbit, només uns pocs marcadors han arribat a ser emprats en la pràctica clínica. Això es deu principalment a dues raons: els biomarcadors validats no compleixen amb les exigències de la clínica, i la fallida de molts d'aquests en la llarga i feixuga etapa de validació. El nou pipeline de cerca de biomarcadors, que afegeix etapes intermèdies en aquest procés, comporta que el percentatge d'èxit millori significativament; dit en altres paraules, que les possibilitats que aquestes noves troballes siguin incorporades a la rutina hospitalària i millorin la pràctica clínica siguin més elevades. Aquesta millora en el diagnòstic, pronòstic i seguiment de teràpia, que cada vegada més és específica del pacient que pateix la malaltia, fa que pas a pas ens trobem més a prop del que es coneix com a medicina personalitzada

Acció dels andrògens en el testicle: un paper per a la meiosi

La funció que duen a terme els andrògens en l'espermatogènesi és, encara en certa mesura, enigmàtica: mentre que llur implicació és absolutament vital en la iniciació i en el manteniment del procés espermatogènic normal, la seva funció específica encara no està definida de manera precisa. Els andrògens, com les altres hormones esteroïdals, actuen a través del seu corresponent receptor anomenat receptor d'andrògens (AR). Fins avui, no hi ha gaire evidència que recolzi l'existència de diverses isoformes de l'AR com en el cas del sistema estrògensreceptor d'estrògens. Per tant, la pregunta de com els andrògens duen a terme la seva acció en l'espermatogènesi s'ha d'abordar definint dos processos: en primer lloc, s'han d'identifi- car amb total certesa els tipus cell. ulars testiculars capaços de respondre directament a l'estimulació androgènica. De manera específica, la qüestió per resoldre és quins són els tipus cellulars que expressen l'AR en el testicle. En segon lloc, sabent també que el complex del lligand unit a l'AR actua com a factor de transcripció, caldrà determinar quins són els gens que estaran activats o reprimits en les cèll. ules que tenen AR en resposta a l'estimulació androgènica. Fins que aquestes dues preguntes no estiguin contestades amb tota certesa, el mecanisme pel qual els andrògens regulen l'espermatogènesi serà, en el millor dels casos, especulatiu. En aquesta revisió presentem evidència que els andrògens actuen únicament a les cèll. ules somàtiques del testicle, com són les cèll. ules de Sertoli, les de Leydig, les mioides peritubulars i les cèll. ules del múscul llis que envolten els vasos sanguinis. A més a més, també discutim la possibilitat que els andrògens siguin indispensables per a l'inici de la meiosi, encara que continua essent desconegut el mecanisme pel qual els andrògens actuen en aquest procés.The role that androgens play in spermatogenesis still remains enigmatic: whereas their involvement is absolutely vital to the initiation and maintenance of the normal spermatogenic process, their specific role is yet to be defined. Androgens, like other steroid hormones, act via their corresponding receptor termed the androgen receptor (AR). To date, there is little evidence to support the notion that there are multiple forms of AR as is the case for the estrogen-estrogen receptor system. Thus, the question of how androgens manifest their action on spermatogenesis becomes one of defining two processes: First, the cell types within the testis that are capable of responding directly to androgen stimulation must be identified with absolute certainty. Specifically, this question can be stated as what cell types in the testis express AR. Second, given that the ligand-bound AR serves as a transcription factor, the question then becomes what are the genes turned on or off in AR positive cells in response to androgen stimulation? Until these two questions are unequivocally answered, the mechanism of how androgens regulate spermatogenesis will remain speculative at best. In this review we present evidence that androgens act solely at the level of the somatic cells of the testis, including Sertoli cells, Leydig cells, peritubular myoid cells and smooth muscle cells surrounding blood vessels. In addition, we discuss the likely possibility that androgens are indispensable for the onset of meiosis, albeit how they accomplish this remains a mystery

Characterization of the molecular changes associated with the overexpression of a novel epithelial cadherin splice variant mRNA in a breast cancer model using proteomics and bioinformatics approaches: identification of changes in cell metabolism and an increased expression of lactate dehydrogenase B

Breast cancer (BC) is the most common female cancer and the leading cause of cancer death in women worldwide. Alterations in epithelial cadherin (E-cadherin) expression and functions are associated to BC, but the underlying molecular mechanisms have not been fully elucidated. We have previously reported a novel human E-cadherin splice variant (E-cadherin variant) mRNA. Stable transfectants in MCF-7 human BC cells (MCF7Ecadvar) depicted fibroblast-like cell morphology, E-cadherin wild-type downregulation, and other molecular changes characteristic of the epithelial-to-mesenchymal transition process, reduced cell-cell adhesion, and increased cell migration and invasion. In this study, a two-dimensional differential gel electrophoresis (2D-DIGE) combined with mass spectrometry (MS) protein identification and bioinformatics analyses were Results: By 2D-DIGE and MS analysis, 50 proteins were found differentially expressed (≥ Δ1.5) in MCF7Ecadvar compared to control cells. Validation of transcript expression was done in the ten most overexpressed and underexpressed proteins. Bioinformatics analyses revealed that 39 of the 50 proteins identified had been previously associated to BC. Moreover, metabolic processes were the most affected, and glycolysis the canonical pathway most altered. The lactate dehydrogenase B (LDHB) was the highest overexpressed protein, and transcript levels were higher in MCF7Ecadvar than in control cells. In agreement with these findings, MCF7Ecadvar conditioned media had lower glucose and higher lactate levels than control cells. MCF7Ecadvar cell treatment with 5 mM of the glycolytic inhibitor 2-deoxy-glucose led to decreased cell viability, and modulation of LDHB expression in MCF7Ecadvar cells with a specific small interfering RNA resulted in decreased cell proliferation. Finally, a positive association between expression levels of the E-cadherin variant and LDHB transcripts was demonstrated in 21 human breast tumor tissues, and breast tumor samples with higher Ki67 expression showed higher LDHB mRNA levels. Conclusions: Results from this investigation contributed to further characterize molecular changes associated to the novel E-cadherin splice variant expression in BC cells. They also revealed an association between expression of the novel variant and changes related to BC progression and aggressiveness, in particular those associated to cell metabolism. Keywords: Breast cancer, Epithelial cadherin, Alternative splicing, Epithelial to mesenchymal transition, Proteomic analysis, 2D-DIGE, Mass spectrometry, Glycolysis, Lactate dehydrogenase

ETV5 i RUNX1, nous factors de transcripció implicats en la invasió miometrial del carcinoma endometrial

Actualment, en càncer d'endometri, està àmpliament acceptat el model dualístic que, atenent a bases morfològiques, diferencia tumors de tipus i o endometrioides dels de tipus ii o no endometrioides. La genètica molecular ha aportat dades que donen suport a aquest model dualístic de la tumorigènesi endometrial i algunes claus per a poder especular sobre la seqüència temporal de les alteracions moleculars que defineixen les rutes tumorigèniques. En els càncers endometrials endometrioides, o de tipus i, es coneixen alteracions majors, com poden ser el silenciament del gen PTEN, la inestabilitat de microsatèll. its associada a defectes en els gens reparadors de DNA, o mutacions al gen K-ras. Aquestes alteracions defineixen la progressió de l'endometri normal cap a la hiperplàsia i posteriorment cap al carcinoma. Recentment, l'ús de la tecnologia de microxips de cDNA per a identificar les diferències en els patrons d'expressió gènica entre els diferents tipus histològics de càncer d'endometri han permès la identificació de gens expressats diferencialment que podrien ajudar-nos a entendre les diferències en la biologia i el pronòstic clínic dels diferents histiotips tumorals. En el nostre laboratori hem aïllat i caracteritzat dos nous factors de transcripció, ETV5 i RUNX1, que estan associats amb els passos inicials de la infiltració miometrial en el càncer d'endometri endometrioide. Aquests estudis, i els d'altres gens implicats en el control de la mitosi com a mecanisme major de carcinogènesi en els càncers d'endometri no endometrioides, representen exemples de la utilitat dels estudis genètics amplis per a comprendre el procés de tumorigènesi i les rutes implicades en la patogènesi molecular del càncer d'endometri.A dualistic model, which has been established on a morphological basis and that differentiates type i endometrioid from type ii non-endometrioid endometrial cancer, is widely accepted. Molecular genetics have provided us with data supporting the dualistic model of endometrial tumorigenesis and with some clues to speculate about the sequence of the molecular alterations defining the tumorigenesis pathways. In type i endometrioid endometrial cancer, PTEN gene silencing, microsatellite instability associated with defects in DNA mismatch repair genes, or mutations in the K-ras gene are the known major alterations defining the progression from normal endometrium to hyperplasia and then on to carcinoma. Recently, cDNA microarray technology for identifying the differences in gene expression patterns between the histological types of endometrial cancer have permitted the identification of differentially expressed genes that could help us to understand differences in the biology and the clinical outcome between histiotypes. In our laboratory, we have recently isolated and characterized two new transcription factors, ETV5 and RUNX1, which expression appears to be associated with initial steps of myometrial infiltration in endometrioid endometrial carcinoma. These studies, as well as those on other genes involved in the mitotic checkpoint as a major mechanism of carcinogenesis in non-endometrioid endometrial cancer, represent examples of how useful large genetic screenings can be for understanding the tumorigenesis process and the future directions in the molecular pathogenesis of endometrial cancer

Exosome-like vesicles in uterine aspirates: a comparison of ultracentrifugation-based isolation protocols

Background: Uterine aspirates are used in the diagnostic process of endometrial disorders, yet further applications could emerge if its complex milieu was simplified. Exosome-like vesicles isolated from uterine aspirates could become an attractive source of biomarkers, but there is a need to standardize isolation protocols. The objective of the study was to determine whether exosome-like vesicles exist in the fluid fraction of uterine aspirates and to compare protocols for their isolation, characterization, and analysis. Methods: We collected uterine aspirates from 39 pre-menopausal women suffering from benign gynecological diseases. The fluid fraction of 27 of those aspirates were pooled and split into equal volumes to evaluate three differential centrifugation-based procedures: (1) a standard protocol, (2) a filtration protocol, and (3) a sucrose cushion protocol. Characterization of isolated vesicles was assessed by electron microscopy, nanoparticle tracking analysis and immunoblot. Specifically for RNA material, we evaluate the effect of sonication and RNase A treatment at different steps of the protocol. We finally confirmed the efficiency of the selected methods in non-pooled samples. Results: All protocols were useful to isolate exosome-like vesicles. However, the Standard procedure was the best performing protocol to isolate exosome-like vesicles from uterine aspirates: nanoparticle tracking analysis revealed a higher concentration of vesicles with a mode of 135 +/- 5 nm, and immunoblot showed a higher expression of exosome-related markers (CD9, CD63, and CD81) thus verifying an enrichment in this type of vesicles. RNA contained in exosome-like vesicles was successfully extracted with no sonication treatment and exogenous nucleic acids digestion with RNaseA, allowing the analysis of the specific inner cargo by Real-Time qPCR. Conclusion: We confirmed the existence of exosome-like vesicles in the fluid fraction of uterine aspirates. They were successfully isolated by differential centrifugation giving sufficient proteomic and transcriptomic material for further analyses. The Standard protocol was the best performing procedure since the other two tested protocols did not ameliorate neither yield nor purity of exosome-like vesicles. This study contributes to establishing the basis for future comparative studies to foster the field of biomarker research in gynecology

Biological properties of extracellular vesicles and their physiological functions

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system