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BackgroundHormone-refractory prostate cancer remains hindered by inevitable progression of resistance to first-line treatment with docetaxel. Recent studies suggest that phenotypic changes associated with cancer may be transferred from cell-to-cell via microvesicles/exosomes. Here we aimed to investigate phenotypic changes associated with docetaxel-resistance in order to help determine the complexity of this problem and to assess the relevance of secreted exosomes in prostate cancer.Methodology/Principal FindingsDocetaxel-resistant variants of DU145 and 22Rv1 were established and characterised in terms of cross-resistance, morphology, proliferation, motility, invasion, anoikis, colony formation, exosomes secretion their and functional relevance. Preliminary analysis of exosomes from relevant serum specimens was also performed. Acquired docetaxel-resistance conferred cross-resistance to doxorubicin and induced alterations in motility, invasion, proliferation and anchorage-independent growth. Exosomes expelled from DU145 and 22Rv1 docetaxel-resistant variants (DU145RD and 22Rv1RD) conferred docetaxel-resistance to DU145, 22Rv1 and LNCap cells, which may be partly due to exosomal MDR-1/P-gp transfer. Exosomes from prostate cancer patients’ sera induced increased cell proliferation and invasion, compared to exosomes from age-matched controls. Furthermore, exosomes from sera of patients undergoing a course of docetaxel treatment compared to matched exosomes from the same patients prior to commencing docetaxel treatment, when applied to both DU145 and 22Rv1 cells, showed a correlation between cellular response to docetaxel and patients’ response to treatment with docetaxel.Conclusions/SignificanceOur studies indicate the complex and multifaceted nature of docetaxel-resistance in prostate cancer. Furthermore, our in vitro observations and preliminary clinical studies indicate that exosomes may play an important role in prostate cancer, in cell-cell communication, and thus may offer potential as vehicles containing predictive biomarkers and new therapeutic targets.

Exosomes (EVs) have relevance in cell-to-cell communication carrying pro-tumorigenic factors that participate in oncogenesis and drug resistance and are proposed to have potential as self-delivery systems. Advancing on our studies of EVs in triple-negative breast cancer, here we more comprehensively analysed isogenic cell line variants and their EV populations, tissues cell line variants and their EV populations, as well as breast tumour and normal tissues. Profiling 384 miRNAs showed EV miRNA content to be highly representative of their cells of origin. miRNAs most substantially down-regulated in aggressive cells and their EVs originated from 14q32. Analysis of miR-134, the most substantially down-regulated miRNA, supported its clinical relevance in breast tumours compared to matched normal breast tissue. Functional studies indicated that miR-134 controls STAT5B which, in turn, controls Hsp90. miR-134 delivered by direct transfection into Hs578Ts(i)8 cells (in which it was greatly down-regulated) reduced STAT5B, Hsp90, and Bcl-2 levels, reduced cellular proliferation, and enhanced cisplatin-induced apoptosis. Delivery via miR-134-enriched EVs also reduced STAT5B and Hsp90, reduced cellular migration and invasion, and enhanced sensitivity to anti-Hsp90 drugs. While the differing effects achieved by transfection or EV delivery are likely to be, at least partly, due to specific amounts of miR-134 delivered by these routes, these EV-based studies identified miRNA-134 as a potential biomarker and therapeutic for breast cancer.

Background: Triple-negative breast cancer (TNBC) accounts for 15-20% of breast cancers but is responsible for a disproportionate number of deaths. We investigated the relevance, in TNBC, of nano-sized exosomes expelled from cells. Specifically, we compared effects of exosomes derived from the claudin-low TNBC cell line Hs578T and its more invasive Hs578Ts(i) 8 variant, as well as exosomes from TNBC patient sera compared to normal sera. Methods: Exosomes were isolated from conditioned media (CM) of Hs578T and Hs578Ts(i) 8 cells and from sera by filtration and ultracentrifugation. Successful isolation was confirmed by transmission electron microscopy and immunoblotting. Subsequent analysis, of secondary/ recipient cells in response to exosomes, included proliferation; motility/migration; invasion; anoikis assays and endothelial tubule formation assays. Results: Hs578Ts(i) 8 -exosomes versus Hs578T-exosomes significantly increased the proliferation, migration and invasion capacity of all three recipient cell lines evaluated i.e. SKBR3, MDA-MB-231 and HCC1954. Exosomes from Hs578Ts(i) 8 cells also conferred increased invasiveness to parent Hs578T cells. Hs578Ts(i) 8 -exosomes increased sensitivity of SKBR3, MDA-MB-231 and HCC1954 to anoikis when compared to the effects of Hs578T-exosomes reflecting the fact that Hs578Ts(i) 8 cells are themselves innately more sensitive to anoikis. In relation to vasculogenesis and subsequent angiogenesis, Hs578Ts(i) 8 -exosomes versus Hs578T-exosomes stimulated significantly more endothelial tubules formation. Finally, our 0959-8049/$ -see front matter Ó

Antiestrogens induce both cytostasis (cell cycle arrest) and apoptosis, but the relationship between these end points and the signaling that regulates their induction are unclear. We have previously implicated the transcription factor and putative tumor suppressor IFN regulatory factor-1 (

Exosomes are nano-sized, cell membrane surrounded structures that are released from many cell types. These exosomes are believed to transport a range of molecules, including mRNAs, miRNAs, and proteins; the contents depending on their cell of origin. The physiological and pathological relevance of exosomes has yet to be fully elucidated. Exosomes have been implicated in cell-to-cell communication. For example, in relation to the immune system, such exosomes may enable exchange of antigen or major histocompatibility complex-peptide complexes between antigen-bearing cells and antigen-presenting cells; in cancer, they may contain molecules that not only have relevance as biomarkers, but may also be taken up and cause adverse effects on secondary cells. Furthermore, exosomes have been proposed as autologous delivery systems that could be exploited for personalised delivery of therapeutics. In order to explore the contents and functional relevance of exosomes from medium conditioned by culture cells or from other biological fluids, prior to extensive molecular profiling, they must be isolated and purified. Here, we describe differential centrifugation methods suitable for isolating exosomes from conditioned medium and from other biological fluids, including serum, saliva, tumour ascites, and urine. We also detail Western blotting and transmission electron microscopy methods suitable for basic assessment of their presence, size, and purity, prior to progressing to global mRNA, miRNA, or protein profiling.Key words: Exosomes, Multivesicular bodies, Extracellular, Cell line, Conditioned medium, Serum, Plasma, Urine, Saliva Exosomes are membrane-bound nanoparticles (30-100 nm in diameter) that are released by many cell types. These small, rightside-out structures form intracellularly by inward budding of endosome membranes (1), resulting in vesicles-containing endosomes

Introduction Peyronie’s disease (PD) is usually seen in men in their fifth decade of life. Aim In this study, we investigated the characteristics of the disease in young men. Main Outcome Measures The demographics, clinical features, and associated comorbidities of the patients with PD were retrospectively reviewed. Methods The findings were compared between men with the disease who were under 40 years of age with those over 40 years. Statistical analyses were conducted to define differentiating features between these two groups. Results Of the 296 patients, 32 were under the age of 40 years and 264 over 40 years. The mean duration of the disease was 2 ± 4 and 6 ± 8 months in the respective age groups. Fifty-six percent of the patients under the age of 40 years and 75% of the patients over this age presented with curvature (P < 0.01). Thirty-seven percent under 40 years and 12% men over 40 years had more than one plaque at presentation (P < 0.01). Dupuytren’s contracture was seen only in patients over 40 years of age. Pain at presentation was found in 75% under the age of 40 years and in 65% over 40 years (P = 0.03). Trauma history was found in 18% under 40 years and in 5% over this age (P < 0.01). Statistical significant differences were found between the groups under and over the age of 40 years for hypertension (P < 0.01) and dyslipidemia (P < 0.01). Diabetes was noted in 50% of the patients under the age of 40 years and in 18% of the patients over this age (P < 0.001). Multivariate analysis of conditions associated in men with PD under 40 years of age showed statistical significant differences for diabetes (P = 0.015), presentation within 6 months (P = 0.004), and having multiple plaques (P = 0.008). Conclusion Young men with PD are more likely to present at an earlier stage of the disease, to have diabetes, and to have more than one plaque at the time of presentation.

The protein kinase C (PKC) family of genes encode serine/threonine kinases that regulate proliferation, apoptosis, cell survival and migration. Multiple isoforms of PKC have been described, one of which is PKCd. Currently, it is unclear whether PKCd is involved in promoting or inhibiting cancer formation/progression. The aim of this study was therefore to investigate the expression of PKCd in human breast cancer and relate its levels to multiple parameters of tumour progression. Protein kinase Cd expression at the mRNA level was measured using real-time PCR (n ¼ 208) and at protein level by both immunoblotting (n ¼ 94) and ELISA (n ¼ 98). Following immunoblotting, two proteins were identified, migrating with molecular masses of 78 and 160 kDa. The 78 kDa protein is likely to be the mature form of PKCd but the identity of the 160 kDa form is unknown. Levels of both these proteins correlated weakly but significantly with PKCd concentrations determined by ELISA (for the 78 kDa form, r ¼ 0.444, Po0.005, n ¼ 91 and for the 160 kDa form, r ¼ 0.237, P ¼ 0.023, n ¼ 91) and with PKCd mRNA levels (for the 78 kDa form, r ¼ 0.351, P ¼ 0.001, n ¼ 94 and for the 160 kDa form, r ¼ 0.216, P ¼ 0.037, n ¼ 94). Protein kinase Cd mRNA expression was significantly higher in oestrogen receptor (ER)-positive compared with ER-negative tumours (P ¼ 0.007, Mann -Whitney U-test). Increasing concentrations of PKCd mRNA were associated with reduced overall patient survival (P ¼ 0.004). Our results are consistent with a role for PKCd in breast cancer progression.