Combinational silencing of components involved in JAK/STAT signaling pathway.

Combinatorial silencing of more than one protein via small interfering RNA (siRNA) is a new strategy that can enhance the effect of RNA interference on cell function. To explore this strategy, we selected JAK/STAT axis as a major signaling pathway that contributes to several mechanisms involved in cancer cell proliferation and survival. We focused on four proteins involved in this pathway to explore the possibility of identifying a combinatorial targeting strategy (as the proof of concept) with enhanced efficiency: gp 130 (a co-receptor for IL6 cytokines), JAK2, STAT3, and importin α3 (the nuclear transporter reportedly involved in translocation of activated STAT3 to nucleus). Selected proteins were targeted by siRNA in two selected Triple Negative Breast Cancer cell lines (MDA-MB-231 and MDA-MB-468). The effect of individual and selected combinations of siRNAs on selected downstream antiapoptotic proteins, pro-apoptosis proteins, and cell-cycle regulating proteins was explored. Combinatorial silencing of JAK2/gp 130 enhanced the effect of RNA interference on downstream proteins significantly, and demonstrated enhanced effect in reducing cell viability, cell migration, and the level of activation of STAT3. In conclusion, the promising results of simultaneous targeting of JAK2 and gp 130 might be an example for potential combinatorial silencing strategies in cancer treatment.

CD44 Receptor Mediates Urate Crystal Phagocytosis by Macrophages and Regulates Inflammation in A Murine Peritoneal Model of Acute Gout.

Gout is a chronic arthritis caused by the deposition of poorly soluble monosodium urate monohydrate (MSU) crystals in peripheral joints. Resident macrophages initiate inflammation in response to MSU mediated by NF-κB nuclear translocation and NLRP3 inflammasome activation. We investigated the role of CD44, a transmembrane receptor, in mediating MSU phagocytosis by macrophages. We used an antibody that sheds the extracellular domain (ECD) of CD44 to study the role of the receptor and its associated protein phosphatase 2A (PP2A) in macrophage activation. We also studied the significance of CD44 in mediating MSU inflammation in-vivo. Cd44-/- BMDMs showed reduced MSU phagocytosis, LDH release, IL-1ß expression and production compared to Cd44+/+ BMDMs. Elevated CD44 staining was detected intracellularly and CD44 colocalized with α-tubulin as a result of MSU exposure and ECD-shedding reduced MSU phagocytosis in murine and human macrophages. Anti-CD44 antibody treatment reduced NF-κB p65 subunit nuclear levels, IL-1ß expression, pro-IL-1ß and IL-8 production in MSU stimulated THP-1 macrophages (p < 0.01). The effect of the antibody was mediated by an enhancement in PP2A activity. CD44 ECD-shedding reduced the conversion of procaspase-1 to active caspase-1, caspase-1 activity and resultant generation of mature IL-1ß in macrophages. Neutrophil and monocyte influx and upregulated production of IL-1ß was evident in wildtype mice. MSU failed to trigger neutrophil and monocyte recruitment in Cd44-/- mice and lower IL-1ß levels were detected in peritoneal lavages from Cd44-/- mice (p < 0.01). Anti-CD44 antibody treatment reduced neutrophil and monocyte recruitment and resulted in reduced lavage IL-1ß levels in the same model. CD44 plays a biologically significant role in mediating phagocytosis of MSU and downstream inflammation and is a novel target in gout treatment.

A systematic comparison of lipopolymers for siRNA delivery to multiple breast cancer cell lines: In vitro studies.

Small interfering RNA (siRNA) therapy is a promising approach for treatment of a wide range of cancers, including breast cancers that display variable phenotypic features. To explore the general utility of siRNA therapy to control aberrant expression of genes in breast cancer, we conducted a detailed analysis of siRNA delivery and silencing response in vitro in 6 separate breast cancer cell models (MDA-MB-231, MDA-MB-231-KRas-CRM, MCF-7, AU565, MDA-MB-435 and MDA-MB-468 cells). Using lipopolymers for siRNA complexation and delivery, we found a large variation in siRNA delivery efficiency depending on the specific lipopolymer used for siRNA complexation and delivery. Some lipopolymers were effective in all cell types used in this study, indicating the possibility of universal carriers for siRNA therapy. The delivery efficiency for effective lipopolymers was not correlated with dextran uptake in the cells tested, which indicated a receptor-mediated internalization for siRNA complexes with lipopolymers, unlike fluid-phase transfer associated with dextran uptake. Consistent with this, specific inhibitors involved in clathrin- and caveolin-mediated endocytosis significantly (>50%) reduced the internalization of siRNA complexes in all cell types. Using JAK2 and STAT3 silencing in MDA-MB-231 and MDA-MB-468 cells, a general correlation between the uptake and silencing efficiency at the mRNA level was evident, but it appeared that the choice of the target rather than the cell type was more critical for consistent silencing. We conclude that siRNA therapy with lipopolymers can be undertaken in multiple breast cancer cell phenotypes with similar efficiency, indicating the general applicability of non-viral RNAi in clinical management of molecularly heterogeneous breast cancers. STATEMENT OF SIGNIFICANCE: The manuscript investigated the efficacy of siRNA carriers across multiple breast cancer cell lines. The lipopolymeric carriers were capable of delivering effective dose of siRNA to a range of breast cancer cells. Despite some differences in uptake efficiency among cell types, the mechanism of delivery was similar, with CME and CvME significantly involved in the internalization of polyplexes, while fluid-phase endocytosis was not significant. Specific target silencing was correlated to delivery efficiency, but we did notice the presence of lipopolymers that achieved high silencing with minimal siRNA delivery. Silencing specific targets in different cell types were more uniformly achieved as compared to targeting different targets in the same cells. Our studies enhance the feasibility of delivering siRNA to different types of breast cancer cells.

Design and Biological Evaluation of Colchicine-CD44-Targeted Peptide Conjugate in an In Vitro Model of Crystal Induced Inflammation.

Gout is an inflammatory arthritis due to the joint deposition of monosodium urate (MSU) crystals. Phagocytosis of MSU crystals by tissue macrophages results in the generation of reactive oxygen species (ROS) and production of inflammatory cytokines and chemokines. Colchicine use in gout is limited by severe toxicity. CD44 is a transmembrane glycoprotein that is highly expressed in tissue macrophages and may be involved in gout pathogenesis. The P6 peptide is a 20-amino acid residue peptide that binds to CD44. We hypothesized that the conjugation of colchicine to the P6 peptide would reduce its off-target cytotoxicity while preserving its anti-inflammatory effect. A modified version of P6 peptide and colchicine-P6 peptide conjugate were synthesized using Fmoc/tBu solid-phase and solution-phase chemistry, respectively. A glutaryl amide was used as a linker. The P6 peptide was evaluated for its binding to CD44, association, and internalization by macrophages. Cytotoxic effects of P6 peptide, colchicine, and colchicine-P6 peptide on macrophages were compared and the inhibition of ROS generation and interleukin-8 (IL-8) secretion in MSU-stimulated macrophages treated with P6 peptide, colchicine, or colchicine-P6 peptide was studied. We confirmed that the P6 peptide binds to CD44 and its association and internalization by macrophages were CD44-dependent. Colchicine (1, 10, and 25 µM) demonstrated a significant cytotoxic effect on macrophages while the P6 peptide and colchicine-P6 peptide conjugate (1, 10 and 25 µM) did not alter the viability of the macrophages. The P6 peptide (10 and 25 µM) reduced ROS generation and IL-8 secretion mediated by a reduction in MSU phagocytosis by macrophages. The colchicine-P6 peptide significantly reduced ROS generation and IL-8 secretion compared to the P6 peptide alone at 1 and 10 µM concentrations. Conjugation of colchicine to the P6 peptide reduced the cytotoxic effect of colchicine while preserving its anti-inflammatory activity.

Heterogeneity and Plasticity of Human Breast Cancer Cells in Response to Molecularly-Targeted Drugs.

Non-responsive subpopulation of tumor cells, and acquired resistance in initially responsive cells are major challenges for cancer therapy with molecularly-targeted drugs. While point mutations are considered the major contributing factor to acquired resistance, in this study we explored the role of heterogeneity and plasticity of selected human breast cancer cell lines (MDA-MB-231, MDA-MB-468, and AU565) in their initial and adjusted response, respectively, to ruxolitinib, everolimus, and erlotinib. After determination of lethal concentration for 50% cell death (LC50), cells were exposed to selected drugs using three different approaches: single exposure to 4 × LC50 and collection of surviving cells, multiple exposures to 1.5 × LC50 and monitoring the surviving population, and exposure to gradually increasing concentrations of selected drugs (range of concentrations equivalent to 10% of LC50 to 1.5 × LC50). Surviving cells were studied for adjustments in expression level of selected proteins using quantitative PCR and Western Blot. Our data indicated overexpression of a variety of proteins in resistant populations, which included cell membrane receptors EGFR and HER2, anti-apoptotic proteins Bcl-2 and BIRC8, and other proteins involved in cell signaling (e.g., Akt1, MAPK7, and RPS6KA5). Silencing the identified alternative proteins via siRNA resulted in significant drop in the LC50 of the selected molecularly-targeted drugs cells resistant to ruxolitinib (via targeting Akt), everolimus (via targeting EGFR, MAPK7, RPS6KA5, and HER2), and erlotinib (via silencing Bcl2 and BIRC8). Our data indicates that targeting well-selected alternative proteins could potentially sensitize the resistant cells to the effect of the molecularly-targeted treatment.

Amphiphilic Peptides for Efficient siRNA Delivery.

A number of amphiphilic cyclic peptides-[FR]4, [WR]5, and [WK]5-containing hydrophobic and positively-charged amino acids were synthesized by Fmoc/tBu solid-phase peptide methods and evaluated for their efficiency in intracellular delivery of siRNA to triple-negative breast cancer cell lines, MDA-MB-231 and MDA-MB-468, in the presence and absence of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Among the peptides, [WR]5, which contains alternate tryptophan (W) and arginine (R) residues, was found to be the most efficient in the delivery of siRNA by improving the delivery by more than 3-fold when compared to other synthesized cyclic peptides that were not efficient. The data also showed that co-formulation of [WR]5 with lipid DOPE significantly enhanced the efficiency of siRNA delivery by up to ~2-fold compared to peptide alone. Based on the data indicating the efficiency of [WR]5 in siRNA delivery, peptides containing arginine residues on the ring and tryptophan residues on the side chain, [R6K]W6 and [R5K]W5, were also evaluated, and demonstrated improved delivery of siRNA. The presence of DOPE again enhanced the siRNA delivery in most cases. [WR]5, [R5K]W5, and [R6K]W6 did not show any significant toxicity in MDA-MB-231, MDA-MB-468, and AU565 WT cells at N/P ratios of 20:1 or less, in the presence and absence of DOPE. Silencing of kinesin spindle protein (KSP) and Janus kinase 2 (JAK2) was evaluated in MDA-MB-231 cells in the presence of the peptides. The addition of DOPE significantly enhanced the silencing efficiency for all selected peptides. In conclusion, peptides containing tryptophan and arginine residues were found to enhance siRNA delivery and to generate silencing of targeted proteins in the presence of DOPE.

"Do We Know Jack" About JAK? A Closer Look at JAK/STAT Signaling Pathway.

Janus tyrosine kinase (JAK) family of proteins have been identified as crucial proteins in signal transduction initiated by a wide range of membrane receptors. Among the proteins in this family JAK2 has been associated with important downstream proteins, including signal transducers and activators of transcription (STATs), which in turn regulate the expression of a variety of proteins involved in induction or prevention of apoptosis. Therefore, the JAK/STAT signaling axis plays a major role in the proliferation and survival of different cancer cells, and may even be involved in resistance mechanisms against molecularly targeted drugs. Despite extensive research focused on the protein structure and mechanisms of activation of JAKs, and signal transduction through these proteins, their importance in cancer initiation and progression seem to be underestimated. This manuscript is an attempt to highlight the role of JAK proteins in cancer biology, the most recent developments in targeting JAKs, and the central role they play in intracellular cross-talks with other signaling cascades.

Alzheimer's Disease: Dawn of a New Era?

Alzheimer's disease (AD) is an irreversible neurodegenerative disease characterized by a progressive decline in cognition and memory, leading to significant impairment in daily activities and ultimately death. It is the most common cause of dementia, the prevalence of which increases with age; however, age is not the only predisposing factor. The pathology of this cognitive impairing disease is still not completely understood, which has limited the development of valid therapeutic options. Recent years have witnessed a wide range of novel approaches to combat this disease, so that they greatly increased our understanding of the disease and of the unique drug development issues associated with this disease. In this paper, we provide a brief overview of the history, the clinical presentation and diagnosis, and we undertake a comprehensive review of the various approaches that have been brought to clinical trials in recent years, including immunotherapeutic approaches, tau-targeted strategies, neurotransmitter-based therapies, neurotropic and hematopoietic growth factors, and antioxidant therapies, trying to highlight the lessons learned from these approaches. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Difatty Acyl-Conjugated Linear and Cyclic Peptides for siRNA Delivery.

A number of amphiphilic difatty acyl linear and cyclic R5K2 peptide conjugates were synthesized by solid-phase peptide methods to enhance the interaction with the hydrophobic cellular phospholipid bilayer and to improve siRNA delivery and silencing. Binding to siRNA molecules was significantly less for the cyclic peptide conjugates. A gradual decrease was observed in the particle size of the complexes with increasing peptide/siRNA ratio for most of the synthesized peptides, suggesting the complex formation. Most of the complexes showed a particle size of less than 200 nm, which is considered an appropriate size for in vitro siRNA delivery. A number of fatty acyl-conjugated peptides, such as LP-C16 and LP-C18, displayed near complete protection against serum degradation. Flow cytometry studies demonstrated significantly higher internalization of fluorescence-labeled siRNA (FAM-siRNA) in the presence of LP-C16, LP-C18, and CP-C16 with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) addition. Confocal microscopy confirmed the cellular internalization of fluorescence-labeled siRNA in the presence of LP-C16 and LP-C18 with DOPE when compared with cells exposed to DOPE/FAM-siRNA. While C16- and C18-conjugated peptides (especially linear peptides) showed silencing against kinesin spindle protein (KSP) and janus kinase 2 (JAK2) proteins, the addition of DOPE enhanced the silencing efficiency significantly for all selected peptides, except for CP-C16. In conclusion, C16 and C18 difatty acyl peptide conjugates were found to enhance siRNA delivery and generate silencing of targeted proteins in the presence of DOPE. This study provides insights for the design and potential application of optimized difatty acyl peptide/lipid nanoparticles for effective siRNA delivery.