Targeting of the Interleukin-13 Receptor (IL-13R)α2 Expressing Prostate Cancer by a Novel Hybrid Lytic Peptide.

The IL-13Rα2 cell surface receptor is highly expressed in tumours such as prostate cancer. In this report, we evaluated the hypothesis that prostate cancer cells with enhanced IL-13Rα2 expression are a suitable target for the hybrid lytic peptide (Pep-1-Phor21) peptide, which is generated by fusing the IL-13Rα2 specific ligand (Pep-1) and a cell membrane disrupting lytic peptide (Phor21). The expression of IL-13Rα2 mRNA and protein in prostate cancer tissues and cell lines was assessed via real-time PCR (RT-PCR) and immunoblotting. The effect of Pep-1-Phor21 on the viability of prostate cancer cells grown in monolayers (2D) and microtissue spheroids (3D) was assessed via CellTox green cytotoxic assay. IL-13Rα2 expression and Pep-1-Phor21-mediated killing were also determined in the cells treated with epigenetic regulators (Trichostatin A (TSA) and 5-aza-2 deoxycytidine (5-Aza-dC)). The hybrid lytic peptide cytotoxic activity correlated with the expression of IL-13Rα2 in prostate cancer cell lines cultured as monolayers (2D) or 3D spheroids. In addition, TSA or 5-Aza-dC treatment of prostate cancer cells, particularly those with low expression of IL-13Rα2, enhanced the cells' sensitivity to the lytic peptide by increasing IL-13Rα2 expression. These results demonstrate that the Pep-1-Phor21 hybrid lytic peptide has potent and selective anticancer properties against IL-13Rα2-expressing prostate cancer cells.

Protocol for faecal microbiota transplantation in ulcerative colitis (FMTUC): a randomised feasibility study.

BACKGROUND: The interaction of the gut microbiota with the human host is implicated in the pathogenesis of inflammatory and immunological diseases including ulcerative colitis (UC). Faecal microbiota transplantation (FMT) as a method of restoring gut microbial diversity is of increasing interest as a therapeutic approach in the management of UC. The current literature lacks consensus about the dose of FMT, route of administration and duration of response. METHODS AND ANALYSIS: This single-blinded randomised trial will explore the feasibility of FMT in 30 treatment-naïve patients with histologically confirmed distal UC limited to the recto-sigmoid region (up to 40 cm from the anal verge). This study aims to estimate the magnitude of treatment response to FMT under controlled conditions. The intervention (FMT) will be administered by rectal retention enema. It will test the feasibility of randomising patients to: (i) single FMT dose, (ii) five daily FMT doses or (iii) control (no FMT dose). All groups will receive standard antibiotic gut decontamination and bowel preparation before FMT. Recruitment will take place over a 24-month period with a 12-week patient follow-up. Trial objectives include evaluation of the magnitude of treatment response to FMT, investigation of the clinical value of metabolic phenotyping for predicting the clinical response to FMT and testing the recruitment rate of donors and patients for a study in FMT. This feasibility trial will enable an estimate of number of patients needed, help determine optimal study conditions and inform the choice of endpoints for a future definitive phase III study. ETHICS AND DISSEMINATION: The trial is approved by the regional ethics committee and is sponsored by Abertawe Bro Morgannwg University's Health Board. Written informed consent from all patients will be obtained. Serious adverse events will be reported to the sponsor. Trial results will be disseminated via peer review publication and shared with trial participants. TRIAL REGISTRATION NUMBER: ISRCTN 58082603; Pre-results.

Comparison of the antibacterial activity of essential oils and extracts of medicinal and culinary herbs to investigate potential new treatments for irritable bowel syndrome.

BACKGROUND: Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder, which may result from alteration of the gastrointestinal microbiota following gastrointestinal infection, or with intestinal dysbiosis or small intestinal bacterial overgrowth. This may be treated with antibiotics, but there is concern that widespread antibiotic use might lead to antibiotic resistance. Some herbal medicines have been shown to be beneficial, but their mechanism(s) of action remain incompletely understood. To try to understand whether antibacterial properties might be involved in the efficacy of these herbal medicines, and to investigate potential new treatments for IBS, we have conducted a preliminary study in vitro to compare the antibacterial activity of the essential oils of culinary and medicinal herbs against the bacterium, Esherichia coli. METHODS: Essential oils were tested for their ability to inhibit E. coli growth in disc diffusion assays and in liquid culture, and to kill E. coli in a zone of clearance assay. Extracts of coriander, lemon balm and spearmint leaves were tested for their antibacterial activity in the disc diffusion assay. Disc diffusion and zone of clearance assays were analysed by two-tailed t tests whereas ANOVA was performed for the turbidometric assays. RESULTS: Most of the oils exhibited antibacterial activity in all three assays, however peppermint, lemon balm and coriander seed oils were most potent, with peppermint and coriander seed oils being more potent than the antibiotic rifaximin in the disc diffusion assay. The compounds present in these oils were identified by gas chromatography mass spectrometry. Finally, extracts were made of spearmint, lemon balm and coriander leaves with various solvents and these were tested for their antibacterial activity against E. coli in the disc diffusion assay. In each case, extracts made with ethanol and methanol exhibited potent antibacterial activity. CONCLUSIONS: Many of the essential oils had antibacterial activity in the three assays, suggesting that they would be good candidates for testing in clinical trials. The observed antibacterial activity of ethanolic extracts of coriander, lemon balm and spearmint leaves suggests a mechanistic explanation for the efficacy of a mixture of coriander, lemon balm and mint extracts against IBS in a published clinical trial.

The MIT domain of UBPY constitutes a CHMP binding and endosomal localization signal required for efficient epidermal growth factor receptor degradation.

We have identified and characterized a Microtubule Interacting and Transport (MIT) domain at the N terminus of the deubiquitinating enzyme UBPY/USP8. In common with other MIT-containing proteins such as AMSH and VPS4, UBPY can interact with CHMP proteins, which are known to regulate endosomal sorting of ubiquitinated receptors. Comparison of binding preferences for the 11 members of the human CHMP family between the UBPY MIT domain and another ubiquitin isopeptidase, AMSH, reveals common interactions with CHMP1A and CHMP1B but a distinct selectivity of AMSH for CHMP3/VPS24, a core subunit of the ESCRT-III complex, and UBPY for CHMP7. We also show that in common with AMSH, UBPY deubiquitinating enzyme activity can be stimulated by STAM but is unresponsive to its cognate CHMPs. The UBPY MIT domain is dispensable for its catalytic activity but is essential for its localization to endosomes. This is functionally significant as an MIT-deleted UBPY mutant is unable to rescue its binding partner STAM from proteasomal degradation or reverse a block to epidermal growth factor receptor degradation imposed by small interfering RNA-mediated depletion of UBPY.

The ubiquitin isopeptidase UBPY regulates endosomal ubiquitin dynamics and is essential for receptor down-regulation.

UBPY is a ubiquitin-specific protease that can deubiquitinate monoubiquitinated receptor tyrosine kinases, as well as process Lys-48- and Lys-63-linked polyubiquitin to lower denomination forms in vitro. Catalytically inactive UBPY localizes to endosomes, which accumulate ubiquitinated proteins. We have explored the sequelae of short interfering RNA-mediated knockdown of UBPY. Global levels of ubiquitinated protein increase and ubiquitin accumulates on endosomes, although free ubiquitin levels are unchanged. UBPY-depleted cells have more and larger multivesicular endosomal structures that are frequently associated through extended contact areas, characterized by regularly spaced, electron-dense, bridging profiles. Degradation of acutely stimulated receptor tyrosine kinases, epidermal growth factor receptor and Met, is strongly inhibited in UBPY knockdown cells suggesting that UBPY function is essential for growth factor receptor down-regulation. In contrast, stability of the UBPY binding partner STAM is dramatically compromised in UBPY knockdown cells. The cellular functions of UBPY are complex but clearly distinct from those of the Lys-63-ubiquitin-specific protease, AMSH, with which it shares a binding site on the SH3 domain of STAM.

Activation of the endosome-associated ubiquitin isopeptidase AMSH by STAM, a component of the multivesicular body-sorting machinery.

AMSH is an endosomal ubiquitin isopeptidase that can limit EGF receptor downregulation . It directly binds to the SH3 domain of STAM, which is constitutively associated with Hrs, a component of clathrin-coated structures on endosomes. This clathrin coat has been implicated in the recruitment of ubiquitinated growth factor receptors prior to their incorporation into internal vesicles of the multivesicular body (MVB) , through the concerted action of ESCRT complexes I, II, and III . We now show that AMSH is embedded within a network of interactions with components of the MVB-sorting machinery. AMSH and STAM, like Hrs , both bind directly to clathrin. AMSH also interacts with mVps24/CHMP3, a component of ESCRT III complex, and this interaction is reinforced through simultaneous STAM binding. We have explored the effect of interacting components on the in vitro enzymatic activity of AMSH. The enzyme shows specificity for K63- over K48-linked polyubiquitin chains in vitro and is markedly stimulated by coincubation with STAM, indicating that activation of AMSH is coupled to its association with the MVB-sorting machinery. Other interacting factors do not directly stimulate AMSH but may serve to orient the enzyme with respect to substrates on the endosomal membrane.

Growth factors induce differential phosphorylation profiles of the Hrs-STAM complex: a common node in signalling networks with signal-specific properties.

Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and STAM (signal-transducing adaptor molecule) form a heterodimeric complex that associates with endosomal membranes and is tyrosine-phosphorylated in response to a variety of growth factors including EGF (epidermal growth factor), HGF (hepatocyte growth factor) and PDGF (platelet-derived growth factor). Phosphorylation of the Hrs-STAM complex requires receptor endocytosis. We show that an intact UIM (ubiquitin interaction motif) within Hrs is a conserved requirement for Hrs phosphorylation downstream of both EGF and HGF stimulations. Consistent with this, expression of a dominant-negative form of the E3 ubiquitin ligase, c-Cbl, inhibits EGF- and HGF-dependent Hrs phosphorylation. Despite this conservation, kinase inhibitor profiles using PP1 (4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and SU6656 indicate that distinct non-receptor tyrosine kinases couple EGF, HGF and PDGF stimulation with the tyrosine phosphorylation of the Hrs-STAM complex. Crucially, analysis with phospho-specific antibodies indicates that these kinases generate a signal-specific, combinatorial phosphorylation profile of the Hrs-STAM complex, with the potential of diversifying tyrosine kinase receptor signalling through a common element.

Lysosome associated membrane protein 1 (Lamp1) traffics directly from the TGN to early endosomes.

The precise trafficking routes followed by newly synthesized lysosomal membrane proteins after exit from the Golgi are unclear. To study these events we created a novel chimera (YAL) having a lumenal domain comprising two tyrosine sulfation motifs fused to avidin, and the transmembrane and cytoplasmic domains of lysosome associated membrane protein 1 (Lamp1). The newly synthesized protein rapidly transited from the trans- Golgi Network (TGN) to lysosomes (t(1/2) approximately 30 min after a lag of 15-20 min). However, labeled chimera was captured by biotinylated probes endocytosed for only 5 min, indicating that the initial site of entry into the endocytic pathway was early endosomes. Capture required export of YAL from the TGN, and endocytosis of the biotinylated reagent, and was essentially quantitative within 2 h of chase, suggesting that all molecules were following an identical route. There was no evidence of YAL trafficking via the cell surface. Fusion of TGN-derived vesicles with 5 min endosomes could be recapitulated in vitro, but neither late endosomes nor lysosomes could serve as acceptor compartments. This suggests that contrary to previous conclusions, most if not all newly synthesized Lamp1 traffics from the TGN to early endosomes prior to delivery to late endosomes and lysosomes.

The UIM domain of Hrs couples receptor sorting to vesicle formation.

Hepatocyte growth factor regulated tyrosine kinase substrate (Hrs), a main component of the 'bilayered' clathrin coat on sorting endosomes, was originally identified as a substrate of activated tyrosine kinase receptors. We have analysed Hrs phosphorylation in response to epidermal growth factor (EGF) stimulation and show that the evolutionary conserved tyrosines Y329 and Y334 provide the principal phosphorylation sites. Hrs is proposed to concentrate ubiquitinated receptors within clathrin-coated regions via direct interaction with its UIM (ubiquitin interaction motif) domain. We show that the same UIM domain is necessary for EGF-stimulated tyrosine phosphorylation of Hrs. Over-expression of wild-type Hrs or a double mutant, Y329/334F, defective in EGF-dependent phosphorylation, both substantially retard EGF receptor (EGFR) degradation by inhibiting internal vesicle formation and thereby preventing EGFR incorporation into lumenal vesicles of the multivesicular bodies. In contrast, mutation or deletion of the Hrs-UIM domain strongly suppresses this effect. In addition the UIM-deletion and point mutants are also observed on internal membranes, indicating a failure to dissociate from the endosomal membrane prior to incorporation of the receptor complex into lumenal vesicles. Our data suggest a role for the UIM-domain of Hrs in actively retaining EGFR at the limiting membrane of endosomes as a prelude to lumenal vesicle formation.