RESUMEN
BACKGROUND: Detection of circulating lung cancer cells with cancer-stem like characteristics would represent an improved tool for disease prognosis. However, current antibodies based methods have some disadvantages and therefore cell SELEX (Systematic Evolution of Ligands by Exponential Enrichment) was used to develop DNA aptamers, recognizing cell surface markers of non-small lung carcinoma (NSLC) cells. MATERIALS AND METHODS: The human adenocarcinoma cell line A549 was used for selection in seven cell SELEX cycles. We used human blood leukocytes for negative selection, and lung stem cell protein marker CD90 antibody binding A549 cells for positive selection. RESULTS: The obtained oligonucleotide sequences after the seventh SELEX cycle were subjected to in silico selection analysis based on three independent types of bioinformatics approaches, selecting two closely related aptamer candidates in terms of consensus sequences, structural motifs, binding affinity (Kd) and stability (ΔG). We selected and identified the aptamer A155_18 with very good binding characteristics to A459 cells, selected for CD90 antibody binding. The calculated phylogenetic tree showed that aptamers A155_18 and the known A549 cell aptamer S6 have a close structural relationship. MEME sequence analysis showed that they share two unique motifs, not present in other sequences. CONCLUSIONS: The novel aptamer A155_18 has strong binding affinity for A549 lung carcinoma cell line subpopulation that is expressing stem cell marker CD90, indicating a possible stemness, characteristic for the A459 line, or a subpopulation present within this cell line. This aptamer can be applied as diagnostic tool, identifying NSLC circulating cells.
RESUMEN
Oligonucleotide aptamers are selected from libraries typically comprising up to 10(15) different sequences by an iterative process of binding, separation, amplification and purification, called SELEX. During this process, the diversity of the oligonucleotide pool decreases until, presumably, only sequences with highest binding affinities towards chosen targets remain. This selection technique is time-consuming, labor-intensive and expensive. Though well posed in principles, the SELEX procedure is noise sensitive, due to amplification of unspecific-binding sequences, and it is not surprising that aptamer selection is often not successful in practice. In view of that, a follow-up of the progress of selection during its course with simple yet reliable methods is necessary. In this paper, we describe five independent assays to estimate the sequence complexity of SELEX pools including qualitative restriction fragment length polymorphism analysis, melting curve analysis, quantitative fluorescence intensity measurements of bound ssDNA, real time PCR quantification and pool dissociation constant analysis during the progress of aptamer selection against streptavidin. Properties and features of each method are discussed and compared. Pool dissociation constant analysis and sequencing serve as reference methods.
Asunto(s)
Aptámeros de Nucleótidos/química , Oligonucleótidos/química , Técnica SELEX de Producción de Aptámeros/métodos , Estreptavidina/químicaRESUMEN
Aptamers are small nucleic acid molecules capable of binding to a wide range of target molecules with high affinity and specificity. They have been developed and widely used not only as research tools, but also as biosensors, specific antagonists, and diagnostic markers and as protein purification platform for many pharmaceutical and clinical applications. Here, in this paper we will explore biochemical aspects of aptamer-target interactions and show why aptamers rival antibodies in target recognition and purification procedures. This review will focus on strategies of using aptamers as affinity ligands for molecules of therapeutic and pharmaceutical interest including applications in chromatography and capillary electrophoresis for protein and small molecule purification. Moreover, we will also discuss aptamers whose binding parameters can be controlled on demand for diagnostic approaches and used as sensitive receptors in biosensorics. Aptamers have opened up exciting fields in basic and applied research of pharmaceutical and biotechnological interest.
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Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Biotecnología/métodos , Cromatografía de Afinidad/métodos , Electroforesis Capilar/métodos , Humanos , LigandosRESUMEN
In the search for novel biomarkers of endometriosis, we selected 152 genes from the GeneLogic database based on results of genome-wide expression analysis of ovarian endometriosis, plus 20 genes related to estrogen metabolism and action. We then performed low-density array analysis of these 172 genes on 11 ovarian endometriosis samples and 9 control endometrium samples. Principal component analysis of the gene expression levels showed clear separation between the endometriosis and control groups. We identified 78 genes as differentially expressed. Based on Ingenuity pathway analysis, these differentially expressed genes were arranged into groups according to biological function. These analyses revealed that 32 differentially expressed genes are estrogen related, 23 of which have not been reported previously in connection with endometriosis. Functional annotation showed that 25 and 22 genes are associated with the biological terms "secreted" and "extracellular region", respectively. Differential expression of 4 out of 5 genes related to estrogen metabolism and action (ESR1, ESR2, PGR and BGN) was also confirmed by immunohistochemistry. Our study thus reveals differential expression of several genes that have not previously been associated with endometriosis and that encode potential novel biomarkers and drug targets.
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Biomarcadores/análisis , Endometriosis/genética , Endometriosis/metabolismo , Adulto , Aromatasa/genética , Aromatasa/metabolismo , Biglicano/genética , Biglicano/metabolismo , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Inmunohistoquímica , Técnicas In Vitro , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Adulto JovenRESUMEN
Endometriosis is a very common disease in pre-menopausal women, where defective metabolism of steroid hormones plays an important role in its development and promotion. In the present study, we have examined the expression of 11 estrogen and progesterone metabolizing enzymes and their corresponding receptors in samples of ovarian endometriomas and control endometrium. Expression analysis revealed significant up-regulation of enzymes involved in estradiol formation (aromatase, sulfatase and all reductive 17beta-hydroxysteroid dehydrogenases) and in progesterone inactivation (AKR1C1 and AKR1C3). Among the estrogen and progesterone receptors, ERalpha was down-regulated, ERbeta was up-regulated, and there was no significant difference in expression of progesterone receptors A and B (PRAB). Our data indicate that several enzymes of estrogen and progesterone metabolism are aberrantly expressed in endometriosis, which can lead to increased local levels of mitogenic estradiol and decreased levels of protective progesterone. Changes in estrogen receptor expression suggest that estradiol may also act via non-estrogen receptor-mediated pathways, while expression of progesterone receptors still needs further investigation.
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Endometriosis/metabolismo , Estrógenos/metabolismo , Ovario/metabolismo , Ovario/patología , Progesterona/metabolismo , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , 20-Hidroxiesteroide Deshidrogenasas/metabolismo , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Adulto , Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas , Aromatasa/metabolismo , Endometriosis/enzimología , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Femenino , Humanos , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Inmunohistoquímica , Persona de Mediana Edad , Ovario/enzimología , Receptores de Progesterona/metabolismo , SulfotransferasasRESUMEN
Endometrial cancer is related to estrogen stimulation not opposed by progesterone. We have examined expression of the pre-receptor regulatory enzymes aromatase, 17beta-hydroxysteroid dehydrogenases (17beta-HSDs), 20alpha-hydroxysteroid dehydrogenases (20alpha-HSDs), sulfatase and sulfotransferase, and estrogen (ERs) and progesterone (PRs) receptors in samples of endometrial cancer and adjacent normal endometrium. No significant gene up-regulation was seen, although aromatase, AKR1C3, a 17beta-HSD and 20alpha-HSD, and AKR1C1, the major 20alpha-HSD, were up-regulated in 50% of samples. Significant down-regulation was seen for 17beta-HSD types 1 and 7, sulfotransferase, ERalpha, ERbeta, PR-AB. Western blotting revealed higher levels of AKR1C3 and PR-B and lower levels of ERalpha in cancerous endometrium, and immunohistochemistry confirmed expression of AKR1C3, PR-B and ERalpha at the cellular level. Up-regulation of aromatase in concert with AKR1C3 can lead to increased levels of estradiol, which acts via ERalpha. Up-regulation of AKR1C1 and AKR1C3 can result in lower levels of the protective progesterone, which acts mainly via PR-B.
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Neoplasias Endometriales/metabolismo , Estrógenos/metabolismo , Progesterona/metabolismo , Receptores de Esteroides/metabolismo , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , 20-Hidroxiesteroide Deshidrogenasas/metabolismo , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas , Aromatasa/metabolismo , Neoplasias Endometriales/enzimología , Neoplasias Endometriales/patología , Endotelio/enzimología , Estradiol/biosíntesis , Estrógenos/química , Femenino , Humanos , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Persona de Mediana Edad , Progesterona/química , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Sulfatasas/metabolismo , Sulfotransferasas/metabolismoRESUMEN
Endometriosis is defined as the presence of endometrial glands and stroma within extrauterine sites, and it is well known that endometriosis is an estrogen-dependent disease. The defective formation and metabolism of steroid hormones is responsible for the promotion and development of endometriosis. In the present study we examined the mRNA levels of six enzymes that are involved in the metabolism of estrogen and progesterone--aromatase, 17beta-hydroxysteroid dehydrogenase (17beta-HSD) types 1, 2 and 7, sulfatase and sulfotransferase--and of the steroid receptors--estrogen receptors alpha and beta (ERalpha, ERbeta) and progesterone receptors A and B (PRAB)--implicated in human ovarian endometriosis. We analyzed 16 samples of ovarian endometriosis and 9 of normal endometrium. The real-time polymerase chain reaction analyses revealed that six of the nine genes investigated are differentially regulated. Aromatase, 17beta-HSD types 1 and 7, sulfatase and ERbeta were statistically significantly upregulated, while ERalpha was significantly downregulated, in the endometriosis group compared with the control group. There were no significant differences in 17beta-HSD type 2, sulfotransferase and PRAB gene expression. Our results indicate that, in addition to the previously reported upregulation of aromatase, upregulation of 17beta-HSD types 1 and 7 and sulfatase can also increase the local estradiol concentration. This could thus be responsible for the estrogen-dependent growth of endometriotic tissue. Surprisingly ERalpha was downregulated.
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17-Hidroxiesteroide Deshidrogenasas/metabolismo , Endometriosis/metabolismo , Receptor alfa de Estrógeno/metabolismo , Perfilación de la Expresión Génica , Enfermedades del Ovario/metabolismo , Esteril-Sulfatasa/metabolismo , 17-Hidroxiesteroide Deshidrogenasas/genética , Adulto , Aromatasa/genética , Aromatasa/metabolismo , Regulación hacia Abajo/genética , Endometriosis/enzimología , Endometriosis/genética , Estradiol/sangre , Receptor alfa de Estrógeno/genética , Femenino , Humanos , Enfermedades del Ovario/enzimología , Enfermedades del Ovario/genética , Esteril-Sulfatasa/genética , Regulación hacia Arriba/genéticaRESUMEN
Phytoestrogens are plant-derived, non-steroidal constituents of our diets. They can act as agonists or antagonists of estrogen receptors, and they can modulate the activities of the key enzymes in estrogen biosynthesis. Much less is known about their actions on the androgen and progesterone metabolizing enzymes. We have examined the inhibitory action of phytoestrogens on the key human progesterone-metabolizing enzyme, 20alpha-hydroxysteroid dehydrogenase (AKR1C1). This enzyme inactivates progesterone and the neuroactive 3alpha,5alpha-tetrahydroprogesterone, to form their less active counterparts, 20alpha-hydroxyprogesterone and 5alpha-pregnane-3alpha,20alpha-diol, respectively. We overexpressed recombinant human AKR1C1 in Escherichia coli, purified it to homogeneity, and examined the selected phytoestrogens as inhibitors of NADPH-dependent reduction of a common AKR substrate, 9,10-phenantrenequinone, and progesterone. The most potent inhibitors were 7-hydroxyflavone, 3,7-dihydroxyflavone and flavanone naringenin with IC(50) values in the low microM range. Docking of the flavones in the active site of AKR1C1 revealed their possible binding modes, in which they are sandwiched between the Leu308 and Trp227 of AKR1C1.
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20-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , Fitoestrógenos/farmacología , Progesterona/metabolismo , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas , Sitios de Unión , Simulación por Computador , Cumarinas/farmacología , Inhibidores Enzimáticos/farmacología , Congéneres del Estradiol/farmacología , Moduladores de los Receptores de Estrógeno/farmacología , Flavanonas/farmacología , Flavonas/química , Flavonas/farmacología , Humanos , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Isoflavonas/farmacología , Modelos Biológicos , Modelos Moleculares , Fenantrenos/antagonistas & inhibidores , Progesterona Reductasa/metabolismo , Unión Proteica , Proteínas Recombinantes/aislamiento & purificación , Estilbenos/farmacología , Zearalenona/farmacologíaRESUMEN
Estrogen-dependent endometrial cancer is related to unopposed and prolonged estrogen stimulation. We examined the expression of estrogen-metabolizing enzymes in correlation with the ERalpha and ERbeta estrogen receptors in human endometrial Ishikawa adenocarcinoma cells and in endometrial cancer specimens and adjacent normal endometrium from the same patients. Real-time PCR analysis revealed that both estrogen receptors and selected estrogen-metabolizing enzymes were expressed in the Ishikawa cells and in endometrial tissue. We detected higher expression of ERalpha than ERbeta, higher expression of sulfatase than sulfotransferase and low expression of aromatase in the Ishikawa cells and the tissue, as well as higher levels of type 2 17beta-hydroxysteroid dehydrogenase (17beta-HSD) in normal and diseased tissue than in the Ishikawa cells. When we compared the expression in endometrial cancer samples and in the adjacent normal endometrium, ERalpha and ERbeta, sulfatase and sulfotransferase were seen to be downregulated in the majority of the cancerous tissue specimens.
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17-Hidroxiesteroide Deshidrogenasas/genética , Adenocarcinoma/enzimología , Neoplasias Endometriales/enzimología , Estrógenos/metabolismo , Sulfotransferasas/genética , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , Adenocarcinoma/genética , Neoplasias Endometriales/genética , Estradiol Deshidrogenasas , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/genética , Receptor beta de Estrógeno/metabolismo , Femenino , Humanos , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Sulfotransferasas/metabolismo , Células Tumorales CultivadasRESUMEN
Endometrial cancer is the most common malignancy of the female genital tract. Its incidence correlates with prolonged estrogen stimulation unopposed by progesterone or synthetic progestins. Estrogen and progestin action is regulated at the pre-receptor level, by interconversion of active hormones (estradiol (E2), progesterone (P)) with their inactive counterparts (estrone (E1), 20alpha-hydroxyprogesterone (20alpha-OHP)) in target tissues. Expression of enzymes that control the ratio of E2 and P may thus play role in the disease process. We first confirmed that AKR1C1 (human 20alpha-hydroxysteroid dehydrogenase) in a cellular context inactivates P by forming 20alpha-OHP but does not catalyze the reverse reaction. We next examined the expression of AKR1C1 and AKR1C3 (type 5 17beta-hydroxysteroid dehydrogenase) in 16 paired specimens of endometrial cancer and adjacent normal endometrium. Quantification by isoform specific real-time PCR revealed higher expression of AKR1C1 in nine specimens and higher expression of AKR1C3 in four specimens of endometrial cancer. Importantly, upregulation of both enzymes in the same specimen was observed. Since AKR1C1 inactivates P its elevated expression in diseased endometrium may contribute to diminished protection by P, while elevated expression of AKR1C3 which forms E2 in vivo, may contribute to the enhanced estrogen action. It is suggested that the expression of AKR1C1 and AKR1C3 in endometrial cancer will govern the ratio of P:E2.