Binding and Internalization in Receptor‐Targeted Carriers: The Complex Role of CD44 in the Uptake of Hyaluronic Acid‐Based Nanoparticles (siRNA Delivery)

This paper is about the actual role of CD44 in the perspective of a hyaluronic acid (HA)-based, targeted therapy. CD44 is the main HA receptor: it is present both in healthy and cancerous cells, but is overexpressed in many carcinomas, with important roles in their initiation and malignancy. This, and its endocytic capacities, have encouraged the use of HA to design CD44-targeting carriers. Here, we have used HA-decorated nanoparticles to deliver a siRNA payload to a panel of human cells comprising both tumoral (AsPC-1, PANC-1, HT-29, HCT-116) and non-tumoral (fibroblasts, differently polarized THP-1 macrophages, HUVEC) lines; we have evaluated in a comparative and quantitative fashion the initial binding of the nanoparticles, their internalization rate and the eventual silencing efficiency (cyclophilin B (PPIB) gene).A first result of our study is that, in general, tumoral cells internalized faster and experienced higher silencing than non-tumoral cells. This result is promising as it suggests that, when in a tumor environment, HA nanocarriers may have limited off-target effects.A more far-reaching result comes from the quantitative analysis of the inter-relation between the four parameters of our study (i.e. total CD44 expression, extent of HA cell surface binding, internalization rate and silencing efficiency). Our experiments showed no correlation between extent of binding (an early event) and any of the other parameters. On the contrary, silencing correlated well both with the speed of the internalization process and also with CD44 expression. This study, therefore, confirms on one hand that HA-based carriers can perform a targeted therapeutic action, but on the other it suggests that this may not be due to the selective binding of a cell surface marker, but possibly to a later recognition event leading to selective internalization

Preclinical anti-cancer activity and multiple mechanisms of action of a cationic silver complex bearing N-heterocyclic carbene ligands

Organometallic complexes offer the prospect of targeting multiple pathways that are important in cancer biology. Here, the preclinical activity and mechanism(s) of action of a silver-bis(N-heterocyclic carbine) complex (Ag8) were evaluated. Ag8 induced DNA damage via several mechanisms including topoisomerase I/II and thioredoxin reductase inhibition and induction of reactive oxygen species. DNA damage induction was consistent with cytotoxicity observed against proliferating cells and Ag8 induced cell death by apoptosis. Ag8 also inhibited DNA repair enzyme PARP1, showed preferential activity against cisplatin resistant A2780 cells and potentiated the activity of temozolomide. Ag8 was substantially less active against non-proliferating non-cancer cells and selectively inhibited glycolysis in cancer cells. Ag8 also induced significant anti-tumour effects against cells implanted intraperitoneally in hollow fibres but lacked activity against hollow fibres implanted subcutaneously. Thus, Ag8 targets multiple pathways of importance in cancer biology, is less active against non-cancer cells and shows activity in vivo in a loco-regional settin

Reversing hypoxic cell chemoresistance in vitro using genetic and small molecule approaches targeting hypoxia inducible factor-1

ABSTRACT The resistance of hypoxic cells to conventional chemotherapy is well documented. Using both adenovirus-mediated gene delivery and small molecules targeting hypoxia-inducible factor-1 (HIF-1), we evaluated the impact of HIF-1 inhibition on the sensitivity of hypoxic tumor cells to etoposide. The genetic therapy exploited a truncated HIF-1␣ protein that acts as a dominant-negative HIF-1␣ (HIF-1␣-no-TAD). Its functionality was validated in six human tumor cell lines using HIF-1 reporter assays. An EGFP-fused protein demonstrated that the dominant-negative HIF-1␣ was nucleus-localized and constitutively expressed irrespective of oxygen tension. The small molecules studied were quinocarmycin monocitrate (KW2152), its analog 7-cyanoquinocarcinol (DX-52-1), and topotecan. DX-52-1 and topotecan have been previously established as HIF-1 inhibitors. HT1080 and HCT116 cells were treated with either AdHIF-1␣-no-TAD or nontoxic concentrations (0.1 M; ϽIC 10 ) of KW2152 and DX-52-1 and exposed to etoposide in air or anoxia (Ͻ0.01% oxygen). Topotecan inhibited HIF-1 activity only at cytotoxic concentrations and was not used in the combination study. Etoposide IC 50 values in anoxia were 3-fold higher than those in air for HT1080 (2.2 Ϯ 0.3 versus 0.7 Ϯ 0.2 M) and HCT116 (9 Ϯ 4 versus 3 Ϯ 2 M) cells. KW2152 and DX-52-1 significantly reduced the anoxic etoposide IC 50 in HT1080 cells, whereas only KW2152 yielded sensitization in HCT116 cells. In contrast, AdHIF-1␣-no-TAD (multiplicity of infection 50) ablated the anoxic resistance in both cell lines (IC 50 values: HT1080, 0.7 Ϯ 0.04 M; HCT116, 3 Ϯ 1 M). HIF-1␣-no-TAD expression inhibited HIF-1-mediated down-regulation of the proapoptotic protein Bid under anoxia. These data support the potential development of HIF-1 targeted approaches in combination with chemotherapy, where hypoxic cell resistance contributes to treatment failure

Site and Strain-Specific Variation in Gut Microbiota Profiles and Metabolism in Experimental Mice

The gastrointestinal tract microbiota (GTM) of mammals is a complex microbial consortium, the composition and activities of which influences mucosal development, immunity, nutrition and drug metabolism. It remains unclear whether the composition of the dominant GTM is conserved within animals of the same strain and whether stable GTMs are selected for by host-specific factors or dictated by environmental variables.The GTM composition of six highly inbred, genetically distinct strains of mouse (C3H, C57, GFEC, CD1, CBA nu/nu and SCID) was profiled using eubacterial -specific PCR-DGGE and quantitative PCR of feces. Animals exhibited strain-specific fecal eubacterial profiles that were highly stable (c. >95% concordance over 26 months for C57). Analyses of mice that had been relocated before and after maturity indicated marked, reproducible changes in fecal consortia and that occurred only in young animals. Implantation of a female BDF1 mouse with genetically distinct (C57 and Agoutie) embryos produced highly similar GTM profiles (c. 95% concordance) between mother and offspring, regardless of offspring strain, which was also reflected in urinary metabolite profiles. Marked institution-specific GTM profiles were apparent in C3H mice raised in two different research institutions.Strain-specific data were suggestive of genetic determination of the composition and activities of intestinal symbiotic consortia. However, relocation studies and uterine implantation demonstrated the dominance of environmental influences on the GTM. This was manifested in large variations between isogenic adult mice reared in different research institutions

Hypoxia and oxidative stress in breast cancer: Tumour hypoxia – therapeutic considerations

Conclusive research has shown that regions of acute/chronic hypoxia, which exist within the majority of solid tumours, have a profound influence on the therapeutic outcome of cancer chemotherapy and radiotherapy and are a strong prognostic factor of disease progression and survival. A strong argument therefore exists for assessing the hypoxic fraction of tumours, prior to patient treatment, and to tailor this treatment accordingly. Tumour hypoxia also provides a powerful physiological stimulus that can be exploited as a tumour-specific condition, allowing for the rationale design of hypoxia-activated anticancer drugs or novel hypoxia-regulated gene therapy strategies

The Back 2 Activity Trial: education and advice versus education and advice plus a structured walking programme for chronic low back pain

<p>Abstract</p> <p>Background</p> <p>Current evidence supports the use of exercise-based treatment for chronic low back pain that encourages the patient to assume an active role in their recovery. Walking has been shown it to be an acceptable type of exercise with a low risk of injury. However, it is not known whether structured physical activity programmes are any more effective than giving advice to remain active.</p> <p>Methods/Design</p> <p>The proposed study will test the feasibility of using a pedometer-driven walking programme, as an adjunct to a standard education and advice session in participants with chronic low back pain. Fifty adult participants will be recruited via a number of different sources. Baseline outcome measures including self reported function; objective physical activity levels; fear-avoidance beliefs and health-related quality of life will be recorded. Eligible participants will be randomly allocated under strict, double blind conditions to one of two treatments groups. Participants in group A will receive a single education and advice session with a physiotherapist based on the content of the 'Back Book'. Participants in group B will receive the same education and advice session. In addition, they will also receive a graded pedometer-driven walking programme prescribed by the physiotherapist. Follow up outcomes will be recorded by the same researcher, who will remain blinded to group allocation, at eight weeks and six months post randomisation. A qualitative exploration of participants' perception of walking will also be examined by use of focus groups at the end of the intervention. As a feasibility study, treatment effects will be represented by point estimates and confidence intervals. The assessment of participant satisfaction will be tabulated, as will adherence levels and any recorded difficulties or adverse events experienced by the participants or therapists. This information will be used to modify the planned interventions to be used in a larger randomised controlled trial.</p> <p>Discussion</p> <p>This paper describes the rationale and design of a study which will test the feasibility of using a structured, pedometer-driven walking programme in participants with chronic low back pain.</p> <p>Trial Registration</p> <p>[ISRCTN67030896]</p