A chemical probe for proteomic analysis and visualization of intracellular localization of lysine-succinylated proteins.

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.

Proteomic analysis of extracellular vesicles identified PI3K pathway as a potential therapeutic target for cabazitaxel-resistant prostate cancer.

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.

Serum Exosomal Gamma-Glutamyltransferase Activity Increased in Patients with Renal Cell Carcinoma with Advanced Clinicopathological Features.

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.

Urinary exosome as a potential biomarker for urinary tract infection.

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.

'Haemophilus quentini' in the urethra of men complaining of urethritis symptoms.

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.

Exosomes expressing carbonic anhydrase 9 promote angiogenesis.

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.

Gamma-glutamyltransferase activity in exosomes as a potential marker for prostate cancer.

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.

miR-130a activates apoptotic signaling through activation of caspase-8 in taxane-resistant prostate cancer cells.

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.

Dietary restriction increases protein acetylation in the livers of aged rats.

BACKGROUND: Dietary restriction (DR) is a well-established biological method for lifespan extension in various organisms by delaying the progression of age-related disorders. With regard to its molecular mechanisms, a family of NAD-dependent protein deacetylases, such as sirtuins, is considered to mediate DR-induced lifespan extension in some lower organisms. Furthermore, the effects of DR on sirtuins (e.g. SIRT1, SIRT2, SIRT3, and SIRT5) have also been reported in mammals. However, the relationship between sirtuins and DR-associated longevity in mammals is still not clear. In addition, ageing and DR-associated changes in cellular protein acetylation have not been fully elucidated, especially in DR-aged animals. OBJECTIVE: We aimed to elucidate the effect of ageing and DR on cellular protein acetylation in young and aged rats. METHODS: Fischer 344 rats were subjected to DR for 7.5 or 25.5 months from 1.5 months of age. Protein acetylation status in tissues was analyzed by Western blotting, subcellular fractionation, and immuno-pull-down assay. We also analyzed the quantitative changes in some related deacetylases and an acetyltransferase. RESULTS: Acetylation of multiple proteins in the liver of young and aged rats decreased slightly with ageing and increased markedly under DR. The results of subcellular fractionation revealed that the DR-induced increase in protein acetylation was more prominent in extranuclear proteins than in nuclear proteins, indicating that acetylation is global, but protein-specific. This was further confirmed in the results of immune-pull-down assays for mitochondrial acetylated proteins. Cellular protein acetylation is regulated by multiple factors, including various deacetylases and acetyltransferases. With regard to the possible mechanisms of DR-induced increases in protein acetylation, we observed that DR increased SIRT3 expression in the liver of young and aged rats. Expression of the mitochondrial protein acetyltransferase GCN5L1 significantly increased with ageing but did not respond to DR. CONCLUSIONS: The increased acetylation of extranuclear proteins may be involved in DR-induced anti-ageing effects including longevity. However, the mechanisms underlying the changes in protein acetylation might not result from quantitative changes in mitochondrial sirtuins and the mitochondrial protein acetyltransferase.

H19 in Serum Extracellular Vesicles Reflects Resistance to AR Axis-targeted Therapy Among CRPC Patients.

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.

A characteristic N-glycopeptide signature associated with diabetic cognitive impairment identified in a longitudinal cohort study.

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.

Dietary restriction increases site-specific histone H3 acetylation in rat liver: possible modulation by sirtuins.

We studied dietary restriction (DR) related changes of site-specific acetylation of histone H3 in rat livers to explore a possible link to histone modifications and sirtuin levels with anti-aging effects of DR. The acetylation at lysine residue 9, 27 and 56 in H3 was 20-30% higher in DR animals compared with ad libitum fed counterparts. SIRT6, one of histone deacetylases, was significantly decreased by DR and thereby may be involved in an increase in the histone acetylation. Our findings suggest that upregulation of chromatin activities through increased histone acetylation is a mechanism of anti-aging effects of DR.

Gene therapy of prostate cancer using liposomes containing perforin expression vector driven by the promoter of prostate-specific antigen gene.

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.

Kartogenin Inhibits Prostate Cancer Cell Growth Through Smad2 Activation and Decreases Androgen Receptor Nuclear Localization.

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.

Diagnostic potential of serum extracellular vesicles expressing prostate-specific membrane antigen in urologic malignancies.

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.

miRNA-93 in Serum Extracellular Vesicles Before and After Low Dose Rate Prostate Brachytherapy.

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.

Profiling of Serum Extracellular Vesicles Reveals miRNA-4525 as a Potential Biomarker for Advanced Renal Cell Carcinoma.

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.

CD44v8-10 mRNA contained in serum exosomes as a diagnostic marker for docetaxel resistance in prostate cancer patients.

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.

Age-related difference of site-specific histone modifications in rat liver.

Aging is associated with decrease in activities of the transcription, replication and DNA repair that can result in deterioration of cellular and tissue functions. Changes of chromatin structures with age are likely major underling mechanisms for the functional decline. Chromatin consists of DNA and histones as well as non-histone proteins. While age-associated change of DNA methylation is well documented, little information is available on site-specific histone modifications in aging. We studied here age-related change of selected modifications of rat liver histone, i.e., histone H3 Lys9 acetylation (H3K9ac), H3 Lys9 methylation (H3K9me), H3 Ser10 phosphorylation (H3S10ph) and H3 Lys14 acetylation (H3K14ac). H3K9ac was decreased and H3S10ph was increased with age significantly. In view of reports indicating that decrease in acetylation and increase in phosphorylation of H3 histones can suppress gene activity, our findings suggest that a mechanism of decreased chromatin functions with age is due to such epigenetic changes.

Exosomes Expressing Thyrotropin Receptor Attenuate Autoantibody-Mediated Stimulation of Cyclic Adenosine Monophosphate Production.

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.