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BACKGROUND/AIM: We aimed to evaluate the changes of androgen receptor (AR) signaling-related long non-coding RNAs (lncRNAs) in serum extracellular vesicles (EVs) from prostate cancer (PC) patients, in order to identify novel biomarkers for AR axis-targeted therapy (ARAT)-resistance among castration-resistant PC (CRPC) patients. PATIENTS AND METHODS: EVs were isolated from 2 patients before and after acquiring ARAT-resistance. RNA profiling of EVs was performed by RNA-sequencing. The expression levels of selected lncRNAs in EVs were analyzed by digital droplet PCR (ddPCR) in 58 localized and 14 metastatic PC patients at diagnosis, 7 ARAT-naïve and 6 ARAT-resistant CRPC patients. LncRNA H19 expression in PC tissue was examined using published data. In order to analyze the role of H19, the prognosis was analyzed in PC patients and proteomic analysis was performed in 22Rv1 PC cells. RESULTS: RNA-sequencing revealed that AR-regulated RNAs were most enriched in EVs after acquiring ARAT-resistance. Among them, up-regulation of AR signaling-related lncRNAs (PCAT1, H19, HOXA-11AS, ZEB1-AS1, ARLNC1, PART1, CTBP1-AS and PCA3) was confirmed by ddPCR. H19 contained in EVs (EV-H19) was significantly increased among ARAT-resistant patients compared to ARAT-naïve CRPC or metastatic PC patients. In PC tissue, H19 was negatively correlated with AR protein and AR-activity score and up-regulated in neuroendocrine CRPC tissue with low AR expression. Furthermore, EV-H19 expression was significantly associated with worse outcome to androgen-deprivation therapy. Proteomic analysis demonstrated that H19 knockdown enhanced PC-related protein expression. CONCLUSION: EV-H19 may negatively correlate with AR-signaling activity and could be a marker to diagnose ARAT-resistance among CRPC patients.
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Vesículas Extracelulares , Neoplasias de la Próstata Resistentes a la Castración , ARN Largo no Codificante , Masculino , Humanos , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Antagonistas de Andrógenos , Proteómica , ARN Largo no Codificante/genética , Vesículas Extracelulares/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión GénicaRESUMEN
BACKGROUND: Identifying a biomarker for the decline in cognitive function in patients with diabetes is important. Therefore, we aimed to identify the N-glycopeptides on plasma proteins associated with diabetic cognitive impairment in participants in a longitudinal study using N-glycoproteomics. METHODS: We used samples from the 3-year SONIC (Septuagenarians, Octogenarians, Nonagenarians Investigation with Centenarians) longitudinal cohort study of older Japanese people in the general population. First, we placed the participants with diabetes into two groups: those that did or did not have cognitive decline over a 6-year period. Next, their plasma protein profiles were compared between baseline and the 6-year time point using two-dimensional fluorescence difference gel electrophoresis. Finally, an N-glycoproteomic study of the focused proteins was performed using an enrichment technique and liquid chromatography-tandem mass spectrometry. RESULTS: Approximately 500 N-glycopeptides, derived from 18 proteins, were identified in each sample, from among which we identified the N-glycopeptides that were associated with diabetic cognitive impairment using multivariate analysis. We found that N-glycopeptides with sialylated tri- or tetra-antennary glycans on alpha-2-macroglobulin, clusterin, serum paraoxonase/arylesterase 1, and haptoglobin were less abundant, whereas 3-sialylated tri-antennary N-glycopeptides on serotransferrin were more abundant. CONCLUSION: N-glycopeptides with sialylated multi-antennary glycans comprise a characteristic signature associated with diabetic cognitive impairment. GENERAL SIGNIFICANCE: The characterized N-glycopeptides represent potential biomarker candidates for diabetic cognitive impairment.
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Disfunción Cognitiva , Diabetes Mellitus , Anciano de 80 o más Años , Humanos , Estudios Longitudinales , Glicosilación , Glicopéptidos , Espectrometría de Masas en Tándem/métodos , Estudios de Cohortes , Biomarcadores , PolisacáridosRESUMEN
Protein acylation is a vital post-translational modification that regulates various protein functions. In particular, protein succinylation has attracted significant attention because of its potential relationship with various biological events and diseases. In this report, we establish a new method for the comprehensive detection and analysis of potentially succinylated proteins using a chemical tagging technology. The newly synthesized alkyne-containing succinyl substrate successfully labeled lysine residues of proteins through intracellular metabolic labeling independent of other acylation pathways such as protein malonylation. Furthermore, reporter molecules such as biotin moieties and fluorescent dyes were conjugated to alkyne-tagged succinylated proteins via Click reactions, permitting enrichment for proteomic analysis and fluorescence imaging of the labeled proteins. We successfully analyzed and identified numerous potential succinylated proteins associated with various biological processes using gel electrophoresis, proteomic and bioinformatic analyses, and their visualization in cells.
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Fenómenos Biológicos , Lisina , Lisina/química , Proteómica/métodos , Proteínas de Plantas , Proteoma/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
Perforin secreted from cytotoxic lymphocytes plays a critical role in cancer immunosurveillance. The aim of this study was to investigate the therapeutic potential of liposomes containing perforin expression vector driven by the promotor of prostate-specific antigen (PSA). The anti-tumor effect of perforin was analyzed using prostate cancer (PC) PC-3 cells in which perforin expression was controlled by Tet-on system (PC-3PRF cells). Liposomes encapsulating PSA promoter-driven perforin expression vector (pLipo) were constructed for its specific expression in PC. The anti-tumor effect of pLipo was evaluated in vitro using docetaxel-resistant PC 22Rv1 PC cell line, 22Rv1DR, and PC-3 cells in the presence of human peripheral blood mono nuclear cells (PBMCs) and also in vivo using male nude mice bearing 22Rv1DR cell-derived tumor xenograft. Induction of perforin significantly inhibited growth of PC-3PRF cells. Treatment with pLipo induced perforin expression in 22Rv1DR cells expressing PSA but not in PC-3 cells lacking it. Treatment with pLipo at a low concentration was prone to inhibit growth of both cell lines and significantly inhibited growth of 22Rv1DR cells when co-incubated with PBMCs. The combined use of pLipo at a high concentration with PBMCs showed nearly complete inhibition of 22Rv1DR cell growth. Intravenous administration of pLipo via tail vein increased the level of perforin in tumor and serum and significantly decreased the tumor volume. Our results suggest that liposome-mediated PC-specific expression of perforin could be a novel therapy for advanced PC.
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Terapia Genética/métodos , Vigilancia Inmunológica/genética , Calicreínas/genética , Perforina/genética , Antígeno Prostático Específico/genética , Neoplasias de la Próstata/terapia , Animales , Línea Celular Tumoral , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Humanos , Liposomas , Masculino , Ratones , Perforina/metabolismo , Regiones Promotoras Genéticas/genética , Neoplasias de la Próstata/inmunología , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND/AIM: De-differentiation is a key step for the progression of cancer cells. This study investigated the anti-tumor effect of kartogenin (KGN), which has the ability to differentiate cells, on prostate cancer (PC) cells. MATERIALS AND METHODS: The effects of KGN on androgen receptor (AR) nuclear localization, prostate-specific antigen (PSA) expression, and Smad2 activation as well as the growth of PC cell lines (LNCaP, 22Rv1 and PC-3) were analyzed. RESULTS: KGN significantly inhibited growth of AR-expressing LNCaP and 22Rv1 cells but not of AR-lacking PC-3 cells. KGN decreased AR nuclear localization and PSA expression, but did not enhance the anti-tumor effect of bicalutamide in LNCaP cells. KGN activated Smad2 both in the absence and presence of TGF-ß1. KGN also inhibited growth of docetaxel-resistant PC cells, 22Rv1DR, and re-sensitized them to the agent. CONCLUSION: KGN has a potential as a novel therapeutic for PC patients after treatment failure.
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Anilidas/farmacología , Antineoplásicos/farmacología , Núcleo Celular/metabolismo , Ácidos Ftálicos/farmacología , Receptores Androgénicos/metabolismo , Proteína Smad2/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Masculino , Fosforilación/efectos de los fármacos , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata/patología , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
We aimed to develop a sandwich ELISA to detect prostate-specific membrane antigen (PSMA) on small extracellular vesicles (EVs) using T-cell immunoglobulin domain and mucin domain-containing protein 4 (Tim4) as a capture molecule for EVs and to evaluate its diagnostic potential in urologic malignancies. First, we optimized the conditions for sandwich ELISA measuring the PSMA level on EVs captured from serum by Tim4 and found that the use of highly-purified EVs released from Tim4 that had captured EVs in serum reduced the background. Second, we confirmed its validity by studying mouse xenograft model for prostate cancer (PC). Lastly, we measured PSMA-EVs in serum of patients with urologic malignancies. The PSMA-EV levels were significantly higher in metastatic PC and castration-resistant PC (CRPC) patients than in therapy-naïve PC patients. In renal cell carcinoma (RCC) patients, PSMA-EVs were elevated in those with metastasis compared with those without metastasis, which may reflect the development of the neovasculature positive for PSMA in tumors. In conclusion, we developed a sandwich ELISA for detection of PSMA-EVs using highly-purified EVs isolated from serum by Tim4. Our results suggest that PSMA-EVs may be useful to diagnose and monitor not only PC but also RCC and possibly other hypervascular solid tumors.
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Carcinoma de Células Renales/diagnóstico , Vesículas Extracelulares/metabolismo , Neoplasias Renales/diagnóstico , Proteínas de la Membrana/metabolismo , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata/diagnóstico , Animales , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Humanos , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Masculino , Ratones , Trasplante de Neoplasias , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Sensibilidad y EspecificidadRESUMEN
BACKGROUND/AIM: To identify novel biomarkers for prostate cancer (PC), we evaluated changes of miRNAs contained in serum small extracellular vesicles (EVs) in patients who received low dose rate prostate brachytherapy (BT). MATERIALS AND METHODS: EVs were isolated from the pooled serum of 10 PC patients prior to and 1 month after BT. miRNA profiling and quantitation in EVs was performed by microarray analysis and RT-digital PCR, respectively. Expression of miRNA-93 in prostate tissue was evaluated using the TCGA database and its level in EVs was determined in 25 patients before and 1, 3, 6 and 12 months after BT. RESULTS: Profiling and quantitation identified miRNA-93 as significantly down-regulated in EVs after BT. TCGA database analysis showed that miRNA-93 was increased in PC tissue. miRNA-93 in EVs significantly decreased in 3, 6 and 12 months after BT. CONCLUSION: miRNA-93 contained in serum EVs may be a novel diagnostic and monitoring biomarker for PC.
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Braquiterapia/métodos , Vesículas Extracelulares/genética , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , MicroARNs/genética , Neoplasias de la Próstata/radioterapia , Regulación hacia Abajo/efectos de la radiación , Humanos , Masculino , MicroARNs/sangre , Fosfatidilinositol 3-Quinasas/metabolismo , Neoplasias de la Próstata/sangre , Proteínas Proto-Oncogénicas c-akt/metabolismo , Dosificación Radioterapéutica , Transducción de Señal/genética , Transducción de Señal/efectos de la radiación , Factores de Tiempo , Resultado del TratamientoRESUMEN
BACKGROUND: Cabazitaxel (CBZ) is now widely used for prostate cancer (PC) patients resistant to docetaxel (DOC), however, most patients eventually acquire resistance. It will, therefore, be of great benefit to discover novel therapeutic target for the resistance. We aimed to identify candidate therapeutic targets for CBZ-resistance by proteomic analysis of extracellular vesicles (EVs) isolated from serum of DOC-resistant PC patients who later developed CBZ-resistance as well as those harvested from culture medium of DOC- and CBZ-resistant PC cell lines. METHODS: Using T-cell immunoglobulin domain and mucin domain-containing protein 4 (Tim4) conjugated to magnetic beads, EVs were purified from serum of PC patients with DOC-resistance that was collected before and after acquiring CBZ-resistance and conditioned medium of DOC-resistant (22Rv1DR) and CBZ-resistant (22Rv1CR) PC cell lines. Protein analysis of EVs was performed by nanoLC-MS/MS, followed by a comparative analysis of protein expression and network analysis. The cytotoxic effect of a phosphatidylinositol-3-kinase (PI3K) inhibitor, ZSTK474, was evaluated by WST-1 assay. The expression and phosphorylation of PI3K and PTEN were examined by western blot analysis. RESULTS: Among differentially regulated proteins, 77 and 61 proteins were significantly increased in EVs from CBZ-resistant PC cell line and patients, respectively. A comparison between the two datasets revealed that six proteins, fructose-bisphosphate aldolase, cytosolic nonspecific dipeptidase, CD63, CD151, myosin light chain 9, and peroxiredoxin-6 were elevated in EVs from both cell line and patients. Network analysis of the increased EV proteins identified pathways associated with CBZ-resistance including PI3K signaling pathway. ZSTK474 significantly inhibited growth of 22Rv1CR cells and improved their sensitivity to CBZ. In 22Rv1CR cells, PI3K was activated and PTEN that inhibits PI3K was deactivated. CONCLUSIONS: Proteomic analysis of serum EVs was successfully accomplished by using Tim-4 as a tool to isolate highly purified EVs. Our results suggest that the combination use of CBZ and PI3K inhibitor could be a promising treatment option for CBZ-resistant PC patients.
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Docetaxel/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Vesículas Extracelulares/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Neoplasias de la Próstata , Taxoides/farmacología , Antineoplásicos/farmacología , Línea Celular Tumoral , Descubrimiento de Drogas/métodos , Humanos , Masculino , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteómica/métodos , Transducción de Señal/efectos de los fármacosRESUMEN
AIM: To identify novel diagnostic markers for renal cell carcinoma (RCC), we analyzed miRNAs in serum extracellular vesicles (EVs). MATERIALS AND METHODS: EVs were purified from serum of healthy controls and patients with localized and advanced RCC using T-cell immunoglobulin domain and mucin domain-containing protein 4 conjugated to magnetic beads. miRNA profiling of EVs was conducted by microarray analysis. miRNA expression was examined by quantitative reverse transcription-polymerase chain reaction. Lastly, proteomic analysis of RCC cells transfected with a miRNA inhibitor was performed to identify its potential targets. RESULTS: Microarray analysis revealed that nine miRNAs were increased by more than 1.5-fold in EVs from patients with RCC. Among them, miRNA-4525 was significantly elevated; miRNA-4525 expression was higher in RCC tissue than in the adjacent normal tissue. Proteomic analysis identified alpha fetoprotein and albumin as its potential targets. CONCLUSION: These findings suggest the potential of miRNA-4525 in serum EVs as a novel biomarker for advanced RCC.
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Carcinoma de Células Renales/sangre , Vesículas Extracelulares/metabolismo , Neoplasias Renales/sangre , MicroARNs/sangre , Adulto , Biomarcadores de Tumor/sangre , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Femenino , Humanos , Neoplasias Renales/genética , Neoplasias Renales/patología , Masculino , MicroARNs/genética , Persona de Mediana Edad , TransfecciónRESUMEN
BACKGROUND: There has been no clinically useful diagnostic or prognostic biomarker for renal cell carcinoma (RCC). Serum γ-glutamyltransferase (GGT) activity has been reported to be a prognostic marker for several types of cancer including RCC. Exosomes or small extracellular vesicles present in body fluids have potential as a biomarker. We have recently demonstrated that GGT activity on exosomes isolated from serum is useful for the differential diagnosis of prostate cancer and benign prostate hyperplasia. In this study, we aimed to examine if serum exosomal GGT activity could be a marker for RCC. METHODS: We examined GGT1 expression and GGT activity in cell lysates and exosomes from culture medium of HK-2 proximal tubule epithelial and RCC cell lines. GGT activity was measured using a fluorescent probe for GGT, γ-glutamyl hydroxymethyl rhodamine green. Serum and serum exosomal GGT activities were measured in patients with RCC. GGT1 expression in RCC tissues was evaluated by immunohistochemical staining. RESULTS: GGT1 levels in exosomes from KMRC-1, OS-RC-2 and 786-O cells were elevated compared with those from HK-2 cells. In exosomes, GGT1 expression correlated with GGT activity determined using a fluorescent probe for GGT. In RCC patients, serum exosomal GGT activity was elevated in those with advanced stages (III/IV vs. I/II, p = 0.037) and those with microvascular invasion (with vs. without, p = 0.034). Immunohistochemical analysis showed that membranous GGT1 expression was increased in RCC with microvascular invasion. Notably, preoperative serum exosomal GGT activity could predict the likelihood of having microvascular invasion diagnosed by pathological examination of surgically resected specimens. CONCLUSIONS: Our results suggest that serum exosomal GGT activity could be a clinically useful marker for advanced clinicopathological features of RCC patients, and its combined use with conventional diagnostic modalities may improve the diagnosis and treatment of patients.
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Carcinoma de Células Renales/enzimología , Exosomas/enzimología , Neoplasias Renales/enzimología , gamma-Glutamiltransferasa/sangre , Anciano , Carcinoma de Células Renales/sangre , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Femenino , Humanos , Inmunohistoquímica , Neoplasias Renales/sangre , Neoplasias Renales/patología , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Estadificación de Neoplasias , gamma-Glutamiltransferasa/biosíntesisRESUMEN
BACKGROUND: Docetaxel is first-line chemotherapy for castration-resistant prostate cancer (CRPC), but most patients acquire docetaxel resistance. CD44 has been shown to be involved in drug resistance of cancers including prostate cancer. We hypothesized that CD44 in serum exosomes could be a diagnostic marker for docetaxel resistance in CRPC patients. In this study, we examined CD44 protein and mRNA expression in cell lysates and exosomes isolated from prostate cancer cells, evaluated the effect of CD44v8-10 knockdown on docetaxel sensitivity and measured CD44 mRNA copy numbers contained in serum exosomes in prostate cancer patients. MATERIALS AND METHODS: Docetaxel-sensitive PC-3 prostate cancer cells and docetaxel-resistant PC-3R cells established previously from parental PC-3 cells were used. CD44v8-10 knockdown was performed by siRNA transfection. Blood was collected from 50 docetaxel-naïve and 10 docetaxel-resistant patients and 15 control males. CD44 protein expression was evaluated by Western blotting. CD44 mRNA expression was measured by RT-digital PCR. RESULTS: The levels of CD44v8-10 protein and mRNA in cell lysates and exosomes were higher in PC-3R cells than in PC-3 cells. CD44v8-10 knockdown significantly increased docetaxel sensitivity of PC-3R cells. The CD44v8-10 mRNA copy numbers in serum exosomes were higher in docetaxel-resistant patients than in docetaxel-naïve patients and control males (median 46, 12 and 17 copies/mL serum, respectively, P = 0.032). In contrast, the serum exosomal mRNA copy numbers of CD44 standard isoform (CD44s) were not different among 3 groups (median 25, 14 and 13 copies/mL serum, respectively, P = 0.150). CONCLUSIONS: CD44v8-10 may be involved in docetaxel resistance in prostate cancer and serum exosomal CD44v8-10 mRNA could be a diagnostic marker for docetaxel-resistant CRPC.
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Background: Exosomes or small extracellular vesicles secreted from cells are nanovesicles with a diameter of 40-150 nm, which play a number of roles in both physiologic and pathologic processes. In Graves' disease (GD), autoantibodies bind to the thyrotropin receptor (TSHR) on the surface of thyroid follicular epithelial cells and stimulate thyroid growth and thyroid hormone synthesis and secretion via cyclic adenosine monophosphate (cAMP) production. The present study aimed to confirm the existence of TSHR in exosomes secreted from thyroid cells and to define the role of TSHR exosomes in GD. Methods: Exosomes were isolated by differential centrifugation from the culture medium of the human thyroid follicular epithelial cell line (NTHY-ori 3-1) and thyroid carcinoma cell lines (8305C, 8505C, and FTC-133). TSHR expression in cell lysates and exosomes was evaluated by Western blot analysis. In order to study the function of TSHR exosomes, human embryonic kidney (HEK) 293 cells stably expressing TSHR (HEK/TSHR) were established. Using exosomes isolated from both HEK and HEK/TSHR cells, the binding capacity of the M22 human monoclonal autoantibody to TSHR exosomes and their effect on M22-mediated stimulation of cAMP production in HEK/TSHR cells were evaluated. As a positive control for the functional assay, human recombinant TSHR chimera protein capable of binding to TSH was used. Results: TSHR was detected in exosomes from cancer cells as well as normal epithelial cells. An in vitro binding assay showed that alkaline phosphatase-labeled M22 bound to TSHR exosomes in a dose-dependent manner. M22 dose-dependently stimulated intracellular cAMP production in HEK/TSHR cells. The addition of exosomes from HEK/TSHR cells but not those from parental HEK cells significantly ameliorated cAMP production stimulated by treatment with M22 in HEK/TSHR cells. A decoy effect similar to TSHR exosomes was observed for human recombinant TSHR chimera. Conclusions: The results suggest that exosomes expressing TSHR may be secreted from normal and cancerous thyroid cells. In the thyroid gland of patients with GD, TSHR exosomes may exert a decoy effect by sequestering autoantibody, thereby ameliorating autoantibody-mediated activation of thyroid function.
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AMP Cíclico/metabolismo , Exosomas/metabolismo , Enfermedad de Graves/metabolismo , Inmunoglobulinas Estimulantes de la Tiroides/metabolismo , Receptores de Tirotropina/metabolismo , Células Epiteliales Tiroideas/metabolismo , Neoplasias de la Tiroides/metabolismo , Autoanticuerpos/metabolismo , Línea Celular , Línea Celular Tumoral , Células HEK293 , Humanos , Técnicas In VitroRESUMEN
Unlike urinary tract infection (UTI), asymptomatic bacteriuria (ABU) should not be treated, with some exceptions such as pregnant women and patients who will undergo traumatic urologic interventions. However, there has been no clinically available marker for their differential diagnosis. Exosomes or small extracellular vesicles carry proteins contained in cells from which they are derived, thus having the potential as a biomarker of several diseases. On the basis of the hypothesis that the molecular signature of exosomes in urine may differ between UTI and ABU patients, we examined if urinary exosomes could serve as a marker for their differential diagnosis. Exosomes were isolated by ultracentrifugation or affinity-based method from cell culture medium of monocytic THP-1 and uroepithelial SV-HUC-1 cells and human urine. Protein expression was examined by Western blot analysis, ELISA, and CLEIA. The results showed that the levels of intracellular signalling molecules Akt and ERK and transcription factor NF-κB increased in exosomes isolated from THP-1 and SV-HUC-1 cells cocultured with Escherichia coli and/or treated with lipopolysaccharide. In urinary exosomes of UTI patients, Akt significantly diminished, and an exosomal marker CD9 showed a trend to decrease after treatment with antimicrobial agents. More importantly, Akt and CD9 levels in urinary exosomes were higher in UTI patients than in ABU patients, which was also observed after correction by urine creatinine. Collectively, these results suggest that Akt and CD9 in urinary exosomes could be useful markers for differential diagnosis of UTI and ABU.
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Bacteriuria/orina , Exosomas/genética , Proteínas Proto-Oncogénicas c-akt/orina , Tetraspanina 29/orina , Infecciones Urinarias/orina , Bacteriuria/microbiología , Bacteriuria/patología , Biomarcadores/orina , Diagnóstico Diferencial , Escherichia coli/genética , Exosomas/microbiología , Femenino , Regulación de la Expresión Génica/genética , Humanos , Lipopolisacáridos/farmacología , Monocitos/patología , Embarazo , Infecciones Urinarias/genética , Infecciones Urinarias/microbiologíaAsunto(s)
Carcinoma de Células Renales/inmunología , Neoplasias Renales/inmunología , Nivolumab/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Antineoplásicos Inmunológicos/inmunología , Antineoplásicos Inmunológicos/uso terapéutico , Carcinoma de Células Renales/sangre , Carcinoma de Células Renales/terapia , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Inmunoterapia , Neoplasias Renales/sangre , Neoplasias Renales/terapia , Nivolumab/uso terapéutico , Receptor de Muerte Celular Programada 1/sangre , Reproducibilidad de los ResultadosRESUMEN
We isolated a cryptic genospecies of Haemophilus influenzae referred to as 'Haemophilus quentini' in the urethra of 3 men complaining of urethritis symptoms. H. influenzae strains, which had been isolated from the urethra in 77 of 1518 men complaining of urethritis symptoms, identified by the conventional test, and stored, were re-cultured for this study. Sixty-seven strains surviving storage were screened by a PCR-based assay specific for the cryptic genital Haemophilus genospecies. Three strains (HI09003, HI11006, and HI14016) were screened by PCR and identified as 'H. quentini' by 16S rRNA sequencing. The men positive for HI09003 and HI11006 were diagnosed as having non-chlamydial non-gonococcal urethritis (NGU), and their demographic and clinical features were similar to those of NGU caused by other pathogens. The man positive for HI14016 was ultimately diagnosed as having condyloma acuminatum on the glans. The 3 strains of 'H. quentini' produced no ß-lactamase and were susceptible to ampicillin and other antimicrobial agents, including cephalosporins, fluoroquinolones, tetracyclines, and macrolides, recommended for treatment for urethritis. 'H. quentini' would be an uncommon pathogen in men with urogenital infections. Based on the clinical features of the two patients with 'H. quentini'-positive NGU, it would be difficult to predict the presence of 'H. quentini' in the urethra. The 3 strains of 'H. quentini' were susceptible to a variety of antimicrobial agents. Further accumulation of data regarding 'H. quentini' infections is needed to characterize the pathogenic roles of this genospecies in urogenital infections and to establish appropriate management of 'H. quentini' infections.
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Infecciones por Haemophilus/microbiología , Haemophilus influenzae/aislamiento & purificación , Uretritis/microbiología , Infecciones Urinarias/microbiología , Adulto , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Secuencia de Bases , Demografía , Infecciones por Haemophilus/tratamiento farmacológico , Haemophilus influenzae/efectos de los fármacos , Haemophilus influenzae/genética , Humanos , Masculino , ARN Ribosómico 16S/genética , Estudios Retrospectivos , Uretritis/tratamiento farmacológico , Infecciones Urinarias/tratamiento farmacológicoRESUMEN
Exosomes or microvesicles that are secreted from cells are considered to play important roles in tumor microenvironment. Carbonic anhydrase 9 (CA9), which is induced by hypoxia-inducible factor 1 (HIF1) in response to hypoxia, is overexpressed in many types of cancer including renal cell carcinoma (RCC). We examined the expression level of CA9 in several RCC cell lines and found that the basal level of CA9 was much higher in OSRC-2 cells than in Caki-1, KMRC-1 and 786-O cells. Consistent with the intracellular expression levels, CA9 was abundantly detected in exosomes isolated by ultracentrifugation from OSRC-2 cells. Density gradient centrifugation of OSRC-2 and 786-O exosomes confirmed the co-presence of CA9 with exosomal markers. Upon hypoxia and treatment with CoCl2, a hypoxia mimic agent, the CA9 level in exosomes was increased for all cell lines. In order to examine the effects of CA9 exosomes on angiogenesis, we generated stably transfected HEK293 cells expressing CA9. Immunocytochemical staining demonstrated the uptake of CA9 exosomes by human umbilical vein endothelial cells (HUVEC). In vitro angiogenesis assays using HUVEC revealed that CA9 exosomes promoted migration and tube formation. Lastly, MMP2 expression was increased by treatment with CA9 exosomes in HUVEC. Taken together, our results suggest the possibility that CA9 exosomes released from hypoxic RCC may enhance angiogenesis in microenvironment, thereby contributing to cancer progression.
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Antígenos de Neoplasias/biosíntesis , Anhidrasa Carbónica IX/biosíntesis , Exosomas/metabolismo , Neovascularización Patológica/metabolismo , Antígenos de Neoplasias/metabolismo , Anhidrasa Carbónica IX/metabolismo , Células Cultivadas , HumanosRESUMEN
BACKGROUND: Exosomes or extracellular vesicles have the potential as a diagnostic marker for various diseases including cancer. In order to identify novel exosomal markers for prostate cancer (PC), we performed proteomic analysis of exosomes isolated from PC cell lines and examined the usefulness of the marker in patients. METHODS: Exosomes isolated by differential centrifugation from the culture medium of androgen-dependent LNCaP prostate cancer cell line and its sublines of partially androgen-independent C4, androgen-independent C4-2 and bone metastatic C4-2B were subjected to iTRAQ-based proteomic analysis. Exosomes were also isolated by immunocapture and separated by size exclusion chromatography and density gradient centrifugation. Protein expression was determined by Western blot analysis. GGT activity was measured using a fluorescent probe, γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG). Immunohistochemical analysis of tissues was performed using anti-GGT1 antibody. RESULTS: Among proteins upregulated in C4-2 and C4-2B cells than in LNCaP cells, we focused on gamma-glutamyltransferase 1 (GGT1), a cell-surface enzyme that regulates the catabolism of extracellular glutathione. The levels of both GGT1 large and small subunits were elevated in exosomes isolated from C4-2 and C4-2B cells by differential centrifugation and by immunocapture with anti-CD9 or -prostate-specific membrane antigen (PSMA) antibody. In cell lysates and exosomes, GGT1 expression correlated with GGT activity. Size exclusion chromatography of human serum demonstrated the presence of GGT activity and GGT1 subunits in fractions positive for CD9. Density gradient centrifugation revealed the co-presence of GGT1 subunits with CD9 in exosomes isolated by differential centrifugation from human serum. Since GGT activity correlated with GGT1 expression in serum exosomes isolated by differential centrifugation, we measured serum exosomal GGT activity in patients. Unexpectedly, we found that serum exosomal GGT activity was significantly higher in PC patients than in benign prostatic hyperplasia (BPH) patients. In support of this finding, immunohistochemical analysis showed increased GGT1 expression in PC tissues compared with BPH tissues. CONCLUSIONS: Our results suggest that serum exosomal GGT activity could be a useful biomarker for PC.
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Biomarcadores de Tumor/sangre , Hiperplasia Prostática/sangre , Neoplasias de la Próstata/sangre , gamma-Glutamiltransferasa/sangre , Antígenos de Superficie/sangre , Línea Celular Tumoral , Exosomas/enzimología , Regulación Neoplásica de la Expresión Génica , Glutamato Carboxipeptidasa II/sangre , Humanos , Masculino , Hiperplasia Prostática/patología , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patologíaRESUMEN
Advanced bladder cancer is treated mainly with gemcitabine and cisplatin, but most patients eventually become resistance. Androgen receptor (AR) signaling has been implicated in bladder cancer as well as other types of cancer including prostate cancer. In this study, we investigated the expression and role of AR in gemcitabine-resistant bladder cancer cells and also the potential of enzalutamide, an AR inhibitor, as a therapeutic for the chemoresistance. First of all, we established gemcitabine-resistant T24 cells (T24GR) from T24 bladder cancer cells and performed gene expression profiling. Microarray analysis revealed upregulation of AR expression in T24GR cells compared with T24 cells. AR mRNA and protein expression was confirmed to be increased in T24GR cells, respectively, by quantitative RT-PCR and western blot analysis, which was associated with more potent AR transcriptional activity as measured by luciferase reporter assay. The copy number of AR gene in T24GR cells determined by PCR was twice as many as that of T24 cells. AR silencing by siRNA transfection resulted in inhibition of proliferation of T24GR cells. Cell culture in charcoal-stripped serum and treatment with enzalutamide inhibited growth of T24GR cells, which was accompanied by cell cycle arrest. AR transcriptional activity was found to be reduced in T24GR cells by enzalutamide treatment. Lastly, enzalutamide also inhibited cell proliferation of HTB5 bladder cancer cells that express AR and possess intrinsic resistance to gemcitabine. Our results suggest that enzalutamide may have the potential to treat patients with advanced gemcitabine-resistant bladder cancer with increased AR expression.
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Proliferación Celular/efectos de los fármacos , Feniltiohidantoína/análogos & derivados , Receptores Androgénicos/biosíntesis , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Andrógenos/genética , Andrógenos/metabolismo , Benzamidas , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Nitrilos , Feniltiohidantoína/administración & dosificación , ARN Interferente Pequeño/genética , Receptores Androgénicos/genética , Transducción de Señal/efectos de los fármacos , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patología , GemcitabinaRESUMEN
BACKGROUND/AIM: Advanced renal cell carcinoma is treated with mammalian target of rapamycin (mTOR) inhibitors or tyrosine kinase inhibitors (TKIs). The effects of these drugs are, however, limited and novel treatment strategies are required. Clear-cell type renal cell carcinoma (ccRCC) is chemo-resistant, in part, due to expression of multidrug resistance proteins such as p-glycoprotein. Cabazitaxel, a tubulin-binding taxane drug used for castration-resistant prostate cancer, has less affinity for p-glycoprotein compared to docetaxel. In the current study, the effects of docetaxel and cabazitaxel on ccRCC cells were investigated. MATERIALS AND METHODS: The expression of p-glycoprotein was evaluated in the ccRCC cell lines, Caki-1, KMRC-1 and OS-RC-2 by western blotting. Cells were treated with cabazitaxel or docetaxel, and growth kinetics and tubulin polymerization were determined by the WST-1 assay and cell-based tubulin polymerization assay, respectively. Intracellular drug concentrations were measured by chromatography. AKT activation after treatment was examined by western blotting. RESULTS: All ccRCC cell lines expressed p-glycoprotein. Cabazitaxel inhibited cell growth and induced tubulin polymerization more potently than docetaxel. The intracellular concentration of cabazitaxel was much higher than docetaxel in all cell lines. Both docetaxel and cabazitaxel inhibit AKT phosphorylation at 5 min among three cells. CONCLUSION: Cabazitaxel inhibits growth of ccRCC cells expressing p-glycoprotein and could thus be possibly used for advanced ccRCC patients in combination with targeted-therapy enhancing their effects.
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Carcinoma de Células Renales/tratamiento farmacológico , Taxoides/uso terapéutico , Línea Celular Tumoral , Proliferación Celular , Humanos , Taxoides/administración & dosificaciónRESUMEN
BACKGROUND: The acquisition of drug resistance is one of the most malignant phenotypes of cancer and identification of its therapeutic target is a prerequisite for the development of novel therapy. MicroRNAs (miRNAs) have been implicated in various types of cancer and proposed as potential therapeutic targets for patients. In the present study, we aimed to identify miRNA that could serve as a therapeutic target for taxane-resistant prostate cancer. METHODS: In order to identify miRNAs related to taxane-resistance, miRNA profiling was performed using prostate cancer PC-3 cells and paclitaxel-resistant PC-3 cell lines established from PC-3 cells. Microarray analysis of mRNA expression was also conducted to search for potential target genes of miRNA. Luciferase reporter assay was performed to examine miRNA binding to the 3'-UTR of target genes. The effects of ectopic expression of miRNA on cell growth, tubulin polymerization, drug sensitivity, and apoptotic signaling pathway were investigated in a paclitaxel-resistant PC-3 cell line. RESULTS: The expression of miR-130a was down-regulated in all paclitaxel-resistant cell lines compared with parental PC-3 cells. Based on mRNA microarray analysis and luciferase reporter assay, we identified SLAIN1 as a direct target gene for miR-130a. Transfection of a miR-130a precursor into a paclitaxel-resistant cell line suppressed cell growth and increased the sensitivity to paclitaxel. Lastly, ectopic expression of miR-130a did not affect the polymerized tubulin level, but activated apoptotic signaling through activation of caspase-8. CONCLUSIONS: Our results suggested that reduced expression of miR-130a may be involved in the paclitaxel-resistance and that miR-130a could be a therapeutic target for taxane-resistant prostate cancer patients.