Fluorescence Imaging of Nanoparticle Uptake into Liquid-Liquid Phase-Separated Droplets.

Liquid-liquid phase separation (LLPS) in living cells has received considerable attention in the biomedical research field. This study is the first to report nanoparticle (NP) uptake into LLPS droplets. Fluorescent dye, Nile red loaded polystyrene NPs (NR-PSt NPs) uptake into model LLPS droplets consisting of adenosine triphosphate (ATP) and poly-L-lysine (PLL) was visualized using fluorescence imaging. Fluorescence imaging showed that the LLPS droplets had a quick NP uptake behavior. Furthermore, temperature changes (4-37 °C) significantly affected the NP uptake behavior of the LLPS droplets. Moreover, the NP-incorporated droplets displayed high stability under strong ionic strength conditions (1 M NaCl). ATP measurements displayed that ATP was released from the NP-incorporated droplets, indicating that the weakly negatively charged ATP molecules and strongly negatively charged NPs were exchanged, which resulted in the high stability of the LLPS droplets. These fundamental findings will contribute to the LLPS studies using various NPs.

[A Case of Xp.11.2 Traslocational Renal Cell Carcinoma Diagnosed by Fluorescence in Situ Hybridization (FISH)].

A 72-year-old woman was referred to our hospital with complaints of macro-hematuria. The radiographic evaluation including computed tomography (CT) and magnetic resonance imaging (MRI) suggested it to be renal cell carcinoma (RCC) in her right kidney. She underwent laparoscopic nephrectomy. We diagnosed her with renal cell carcinoma associated with Xp11.2 translocation/TFE3 gene fusion, based on pathological findings and break apart of transcription factor E3 (TFE3)by fluorescence in situ hybridization. She was free of recurrence at 8 months postoperatively.

Detection of bladder cancer by measuring CD44v6 expression in urine with real-time quantitative reverse transcription polymerase chain reaction.

OBJECTIVE: To examine urinary CD44v6 total ribonucleic acid (RNA) expression in patients with bladder cancer using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and evaluate its potential as a novel marker of bladder cancer. METHODS: We used the bladder cancer cell line T24 and determined CD44v6 expression in cancer cells using in situ hybridization and immunohistochemistry. Subsequently, we obtained urine samples from 21 patients with bladder cancer and 25 patients without bladder cancer (controls). We extracted total RNA from the urine samples, measured CD44v6 total RNA expression in both groups using qRT-PCR, and compared the expression between groups. We also compared the sensitivity, specificity, and concordance rate between CD44v6 total RNA expression analysis by qRT-PCR and cytologic analysis, UroVysion fluorescent in situ hybridization, bladder tumor antigen identification, and nuclear matrix protein 22 measurements. RESULTS: We observed increased CD44v6 expression in bladder cancer cells using in situ hybridization and immunohistochemistry. CD44v6 total RNA expression was significantly higher in the urine samples of patients with bladder cancer than in those of controls. We calculated the cutoff value from the receiver operating characteristic curve and obtained sensitivity and specificity values of 85.7% and 72.0%, respectively, for qRT-PCR analysis. CONCLUSION: Our results suggest that CD44v6 total RNA levels in urine can serve as a potential noninvasive biomarker of bladder cancer.

Cisplatin induces production of reactive oxygen species via NADPH oxidase activation in human prostate cancer cells.

This study aimed to examine the roles of reactive oxygen species (ROS) in cisplatin treatment of human prostate cancer cells; hormone-sensitive LNCaP and hormone-refractory PC3 and DU145 cells. Intracellular levels of ROS and H(2)O(2) were measured and visualized using specific fluorescent probes. NADPH oxidase (NOX) activity was detected by lucigenin chemiluminescence assay. Expression levels of NOX isoforms were determined by semi-quantitative RT-PCR. Cisplatin treatment increased the intracellular levels of ROS and H(2)O(2) in three prostate cancer cell lines. The increase was transient and robust in hormone-sensitive LNCaP cells compared with hormone-refractory PC3 and DU145 cells. Consistent with these findings, the NOX activity induced by cisplatin was higher in LNCaP cells than in PC3 and DU145 cells. Expression pattern of NOX isoforms varied among three cell lines and the NOX activity was independent of NOX expression. Taken together, we have shown that cisplatin induces production of ROS and H(2)O(2) via NOX activation in human prostate cancer cell lines, which is most prominent in hormone-sensitive LNCaP cells.

Dysregulation of microRNA-34a expression causes drug-resistance to 5-FU in human colon cancer DLD-1 cells.

MiR-34a was identified as one of the down-regulated micro-RNAs (miRs) in human colorectal cancer 5-fluorouracil (5-FU)-resistant DLD-1 cells compared with those in the parental DLD-1 cells. Exposure to 5-FU at 30 µM activated phosphoinositide 3-kinase (PI3K)/Akt signaling markedly from 12h up to 48 h in the 5-FU-resistant cells compared with that in the parental cells and resulted in an overt difference in growth at those times. Furthermore, the expression of miR-34a in the 5-FU-resistant cells was sustained at a low-level, whereas it was up-regulated in the parental cells after the 5-FU treatment. Sirt1, which is one of the target genes for miR-34a and related to drug-resistance, was strikingly up-regulated in the 5-FU-resistant cells. The ectopic expression of miR-34a in the 5-FU-resistant cells inhibited growth, as in the parental cells, and attenuated the resistance to 5-FU through the down-regulation of Sirt1 and E2F3. Moreover, the silencing of Sirt1 significantly canceled the resistance to 5-FU in the 5-FU-resistant cells. These findings suggest that miR-34a targeting the Sirt1 and E2F3 genes could negatively regulate, at least in part, the resistance to 5-FU in human colorectal cancer DLD-1 cells.

MiR-34a attenuates paclitaxel-resistance of hormone-refractory prostate cancer PC3 cells through direct and indirect mechanisms.

BACKGROUND: Patients with hormone-refractory prostate cancer are treated with taxane drugs, but eventually become drug resistant. We aimed to elucidate the molecular mechanisms underlying paclitaxel resistance of hormone-refractory prostate cancer with a special focus on the roles of miR-34a and SIRT1. METHODS: Paclitaxel-resistant cells (PC3PR) were generated from hormone-refractory PC3 cells. The expression levels of mRNA and miRNA were determined by reverse transcriptase PCR and those of protein were by Western blot analysis. Transfection of miRNA precursor or siRNA was performed using the liposome-mediated method. RESULTS: MiR-34a over-expression and SIRT1 knockdown attenuated paclitaxel resistance of PC3PR cells. MiR-34a expression was reduced in PC3PR cells compared with PC3 cells, while the expression levels of HuR and Bcl2 as well as SIRT1 were elevated in PC3PR cells. Luciferase reporter assays revealed that both SIRT1 3'-UTR and promoter activities were higher in PC3PR cells than in PC3 cells. Introduction of miR-34a precursor into PC3PR cells resulted in decreases in HuR, Bcl2, and SIRT1 expression and inhibition of the SIRT1 3'-UTR activity. HuR knockdown reduced SIRT1 and Bcl2 expression. These results suggest that miR-34a not only directly but also indirectly via regulating HuR expression acts on the 3'-UTR of SIRT1 and Bcl2 mRNAs, thereby controlling their expression. Thus, in PC3PR cells, reduced expression of miR-34a confers paclitaxel resistance via up-regulating SIRT1 and Bcl2 expression. CONCLUSIONS: MiR-34a and its downstream targets SIRT1 and Bcl2 play important roles in the development of paclitaxel resistance, all of which can be useful biomarkers and promising therapeutic targets for the drug resistance in hormone-refractory prostate cancer.

MiR-148a attenuates paclitaxel resistance of hormone-refractory, drug-resistant prostate cancer PC3 cells by regulating MSK1 expression.

MicroRNAs are involved in cancer pathogenesis and act as tumor suppressors or oncogenes. It has been recently reported that miR-148a expression is down-regulated in several types of cancer. The functional roles and target genes of miR-148a in prostate cancer, however, remain unknown. In this report, we showed that miR-148a expression levels were lower in PC3 and DU145 hormone-refractory prostate cancer cells in comparison to PrEC normal human prostate epithelial cells and LNCaP hormone-sensitive prostate cancer cells. Transfection with miR-148a precursor inhibited cell growth, and cell migration and invasion, and increased the sensitivity to anti-cancer drug paclitaxel in PC3 cells. Computer-aided algorithms predicted mitogen- and stress-activated protein kinase, MSK1, as a potential target of miR-148a. Indeed, miR-148a overexpression decreased expression of MSK1. Using luciferase reporter assays, we identified MSK1 as a direct target of miR-148a. Suppression of MSK1 expression by siRNA, however, showed little or no effects on malignant phenotypes of PC3 cells. In PC3PR cells, a paclitaxel-resistant cell line established from PC3 cells, miR-148a inhibited cell growth, and cell migration and invasion, and also attenuated the resistance to paclitaxel. MiR-148a reduced MSK1 expression by directly targeting its 3'-UTR in PC3PR cells. Furthermore, MSK1 knockdown reduced paclitaxel-resistance of PC3PR cells, indicating that miR-148a attenuates paclitaxel-resistance of hormone-refractory, drug-resistant PC3PR cells in part by regulating MSK1 expression. Our findings suggest that miR-148a plays multiple roles as a tumor suppressor and can be a promising therapeutic target for hormone-refractory prostate cancer especially for drug-resistant prostate cancer.

[Clinical usefulness of chlormadinone acetate as an alternative antiandrogen therapy for prostate cancer relapse after combined androgen blockade therapy].

We prospectively studied the usefulness of chlormadinone acetate (CMA) as an alternative therapy for prostate cancer relapse after combined androgen blockade (CAB) therapy. Sixteen patients with relapsed prostate cancer after treatment with CAB, including surgical or medical castration and nonsteroidal antiandrogens, 80 mg bicalutamide daily or 375 mg flutamide daily, were enrolled. After discontinuing the antiandrogen for evaluating the patient for the antiandrogen withdrawal syndrome, we administered 100 mg CMA daily as alternative antiandrogen and estimated its effect. Four patients showed a > or = 50% decline in prostate-specific antigen (PSA) levels and another 4 patients showed a < 50% decline in PSA levels but residual 8 patients showed no decline in PSA levels. In 8 patients with a decline in PSA levels, the median duration of alternative CMA therapy was 11.4 months. Patients with a PSA level of < 1 ng/ml at the start of CMA therapy showed the tendency of decline in PSA levels. In contrast, patients with a nadir PSA level of > or = 0.2 ng/ml during pretreatment showed no effectiveness of the alternative CMA therapy. The alternative CMA therapy may be useful in a part of patients with prostate cancer relapse after CAB therapy.

[Discrepancy between Gleason score of needle biopsies and radical prostatectomy specimens--current situation of general pathologists].

We retrospectively reviewed the discrepancy in Gleason score between needle biopsy and radical prostatectomy specimens. Specimens from 153 patients who underwent radical retropubic prostatectomy at Gifu University Hospital and 9 community-based institutions between January 2001 and December 2005, were studied. Gleason score was determined by the general pathologist at each institution. The coincidence rate of Gleason score between biopsy and prostatectomy specimens was 49.7%. In contrast, 37.4% of biopsy specimens were undergraded. In biopsy specimens given a Gleason score of 5 or less, the Gleason score was coincident or undergraded compared with prostatectomy specimens. In biopsy specimens given a Gleason score of 6, the coincidence rate was 39.6%. In 56% in biopsy specimens of cancers with a Gleason score of 6 the Gleason score was undergraded compared with the prostatectomy specimen. In this group, extra-prostatic extention was found significantly more often than in other groups (p = 0.04). In patients, who underwent extended biopsy, or had a more positive biopsy core (> or = 25%), the coincidence rate was significantly greater (p = 0.03). We should be aware of the limitations of Gleason scores based on biopsy specimens, and give treatment opinions careful consideration.

Effects of miR-34a on cell growth and chemoresistance in prostate cancer PC3 cells.

Tumor suppressor p53 transcriptionally regulates expression of microRNA-34a, which confers translational inhibition and mRNA degradation of genes involved in cell cycle control and apoptosis. In various cancers, miR-34a expression is lost or reduced. Here, we investigated the role of miR-34a in prostate cancer cell lines. MiR-34a expression was markedly reduced in p53-null PC3 cells and p53-mutated DU145 cells compared with LNCaP cells expressing wild-type p53. In PC3 cell, ectopic expression of miR-34a decreased the SIRT1 mRNA and protein levels as well as protein levels of known direct target genes. Reporter assays revealed that miR-34a-induced SIRT1 inhibition occurred at the transcriptional but not post-transcriptional level despite the presence of a potential miR-34a binding site within its 3'-UTR. Ectopic miR-34a expression resulted in cell cycle arrest and growth inhibition and attenuated chemoresistance to anticancer drug camptothecin by inducing apoptosis, suggesting a potential role of miR-34a for the treatment of p53-defective prostate cancer.

A role for SIRT1 in cell growth and chemoresistance in prostate cancer PC3 and DU145 cells.

SIRT1, which belongs to the family of type III histone deacetylase, is implicated in diverse cellular processes. We have determined the expression levels of SIRT1 in human prostate cancer cell lines and have examined the roles of SIRT1 in cell growth and chemoresistance. SIRT1 expression was markedly up-regulated in androgen-refractory PC3 and DU145 cells compared with androgen-sensitive LNCaP cells and its expression level was correlated with cell growth in PC3 cells. Treatment with a SIRT1 inhibitor, sirtinol, inhibited cell growth and increased sensitivity to camptothecin and cisplatin. Silencing of SIRT1 expression by siRNA also suppressed cell proliferation and reduced camptothecin resistance in PC3 cells, mimicking the chemosensitizing effect caused by sirtinol. Also in DU145 cells, sirtinol treatment enhanced sensitivity to camptothecin and cisplatin. These results suggest that up-regulation of SIRT1 expression may play an important role in promoting cell growth and chemoresistance in androgen-refractory PC3 and DU145 cells.