Epidermal Growth Factor Receptor: Key to Selective Intracellular Delivery.

Epidermal growth factor receptor (EGFR) is an integral surface protein mediating cellular response to a number of growth factors. Its overexpression and increased activation due to mutations is one of the most common traits of many types of cancer. Development and clinical use of the agents, which block EGFR activation, became a prime example of the personalized targeted medicine. However, despite the obvious success in this area, cancer cure remains unattainable in most cases. Because of that, as well as the result of the search for possible ways to overcome the difficulties of treatment, a huge number of new treatment methods relying on the use of EGFR overexpression and its changes to destroy cancer cells. Modern data on the structure, functioning, and intracellular transport of EGFR, its natural ligands, as well as signaling cascades triggered by the EGFR activation, peculiarities of the EGFR expression and activation in oncological disorders, as well as applied therapeutic approaches aimed at blocking EGFR signaling pathway are summarized and analyzed in this review. Approaches to the targeted delivery of various chemotherapeutic agents, radionuclides, immunotoxins, photosensitizers, as well as the prospects for gene therapy aimed at cancer cells with EGFR overexpression are reviewed in detail. It should be noted that increasing attention is being paid nowadays to the development of multifunctional systems, either carrying several different active agents, or possessing several environment-dependent transport functions. Potentials of the systems based on receptor-mediated endocytosis of EGFR and their possible advantages and limitations are discussed.

New Recombinant Carriers Binding Specifically to the Epidermal Growth Factor Receptor.

New recombinant carriers-modular nanotransporters (MNTs)-with N-terminal ligand module to the epidermal growth factor receptor (EGFR) were developed and characterized. Human epidermal growth factor (hEGF) and antibody-like protein Z1907 were used as a ligand module. We demonstrated that MNTs are able to internalize in a receptor-specific manner into the target cancer cells and to accumulate in the target cell nuclei. Conjugation of MNTs with the Auger electron emitter 111In significantly enhanced the cytotoxic effect of 111In on the target cells. It was found that the transfer of EGF from the C-terminus to the N-terminus of the MNT enhanced the proliferation of target cells, whereas the use of Z1907 did not have a similar effect.

Stabilization of Modular Nanotransporters by Embedding Hemin in Them in a New Strain with Heme Receptor Expression.

It was found that the use of a new strain-producer Escherichia coli, expressing the heme receptor ChuA, enables obtaining a hemin-containing modular nanotransporter (MNT) for drug delivery into the nuclei of target cells. The hemin-containing MNT becomes stabilized, which leads to an increase in its thermal stability and prevents aggregation of this protein.

Targeted Intracellular Delivery of Antibodies: The State of the Art.

A dominant area of antibody research is the extension of the use of this mighty experimental and therapeutic tool for the specific detection of molecules for diagnostics, visualization, and activity blocking. Despite the ability to raise antibodies against different proteins, numerous applications of antibodies in basic research fields, clinical practice, and biotechnology are restricted to permeabilized cells or extracellular antigens, such as membrane or secreted proteins. With the exception of small groups of autoantibodies, natural antibodies to intracellular targets cannot be used within living cells. This excludes the scope of a major class of intracellular targets, including some infamous cancer-associated molecules. Some of these targets are still not druggable via small molecules because of large flat contact areas and the absence of deep hydrophobic pockets in which small molecules can insert and perturb their activity. Thus, the development of technologies for the targeted intracellular delivery of antibodies, their fragments, or antibody-like molecules is extremely important. Various strategies for intracellular targeting of antibodies via protein-transduction domains or their mimics, liposomes, polymer vesicles, and viral envelopes, are reviewed in this article. The pitfalls, challenges, and perspectives of these technologies are discussed.

MNT Optimization for Intracellular Delivery of Antibody Fragments.

We studied the possibility of optimizing modular nanotransporters (MNTs) for the intracellular delivery of antibody fragments into the nuclei of cells of a specified type. Basic MNT with a reduced size retaining the desired functions was obtained, and the principal possibility of obtaining an MNT carrying an antibody fragment by microbiological synthesis was shown.

Modular Nanotransporter with P21 Fragment Inhibits DNA Repair after Bleomycin Treatment.

Modular nanotransporter (MNT) with C-terminal fragment of the p21 protein was synthesized and characterized, and its effect on DNA lesions was studied. This p21 fragment in MNT can significantly inhibit DNA repair in A431 human carcinoma cells after bleomycin treatment.

Study of Biodistribution of the Modular Nanotransporters after Systemic Administration in Murine Cloudman S91 Melanoma Model.

The distribution of modular nanotransporters (MNTs) that are used to deliver drugs into melanoma cell nuclei after their intravenous administration into mice with Cloudman S91 melanoma was studied. The modification of MNTs with polyethylene glycol (PEG) of different length and their administration during the treatment with docetaxel, nitroglycerin, and excess of nonspecific MNTs leads to an improved accumulation of MNTs in the tummor. Among the variants studied, the MNT with attached PEG with Mr 40 kDa exhibited the best properties.

Antitumor efficacy of Auger electron emitter 111In delivered by modular nanotransporter into the nuclei of cells with folate receptor overexpression.

A new modular nanotransporter (MNT) for the delivery of anticancer agents into the nuclei of cells with folate receptor overexpression was created. An effective method for acceding labeling of this MNT with Auger electron emitter 111In has been developed. A significant therapeutic effect was observed after a single intratumoral injection of the new 111In-labeled MNT to mice grafted with human cervical carcinoma characterized by folate receptor overexpression.

Characterization of new modular nanotransporters with albumin-binding domain.

The albumin-binding domain (ABD) with a site for its cleavage by tumor proteases was inserted in the structure of modular nanotransporters (MNTs), chimeric proteins for the delivery of anticancer drugs into the nuclei of cancer cells. The effectiveness of this cleavage was tested in both variants of created construct: "pure" ABD-MNT and the complex with albumin. The introduction of the ABD module into MNTs had no effect on the binding of MNT with receptors on the surface of the target cancer cells and on the preferential accumulation of MNTs in the nuclei of these cells. The use of thermophoresis allowed us to determine the equilibrium dissociation constants of the ABD-MNT complex with bovine and human serum albumins.

MicroRNAs 223-3p and 93-5p in patients with chronic kidney disease before and after renal transplantation.

Chronic kidney disease (CKD) is associated with a multifactorial dysregulation of bone and vascular calcification and closely linked to increased cardiovascular mortality and concomitant bone disease. We aimed to investigate specific microRNA (miRNA) signatures in CKD patients to find indicators for vascular calcification and/or bone mineralization changes during CKD and after kidney transplantation (KT). A miRNA array was used to investigate serum miRNA profiles in CKD patients, then selected miRNAs were quantified in a validation cohort comprising 73 patients in CKD stages 3 to 5, 67 CKD patients after KT, and 36 healthy controls. A spectrum of biochemical parameters including markers for kidney function, inflammation, glucose, and mineral metabolism was determined. The relative expression of miR-223-3p and miR-93-5p was down-regulated in patients with CKD stage 4 and 5 compared to healthy controls. This down-regulation disappeared after kidney transplantation even when lower glomerular filtration rates (eGFR) persisted. MiR-223-3p and miR-93-5p were associated with interleukin-6 (IL-6) and eGFR levels, and by trend with interleukin-8 (IL-8), C-peptide, hematocrit, and parathyroid hormone (PTH). This study contributes new knowledge of serum miRNA expression profiles in CKD, potentially reflecting pathophysiological changes of bone and calcification pathways associated with inflammation, vascular calcification, mineral and glucose metabolism. Identified miRNA signatures can contribute to future risk markers or future therapeutic targets in bone and kidney disease.

Use of intracellular transport processes for targeted drug delivery into a specified cellular compartment.

Targeted drug delivery into the cell compartment that is the most vulnerable to effects of the corresponding drug is a challenging problem, and its successful solution can significantly increase the efficiency and reduce side effects of the delivered therapeutic agents. To accomplish this one can utilize natural mechanisms of cellular specific uptake of macromolecules by receptor-mediated endocytosis and intracellular transport between cellular compartments. A transporting construction combining the components responsible for different steps of intracellular transport is promising for creating multifunctional modular constructions capable of delivering the necessary therapeutic agent into a given compartment of type-specified cells. This review focuses on intracellular transport peculiarities along with approaches for designing such transporting constructions for new, more effective, and safer strategies for treatment of various diseases.

Malignant melanoma and melanocortin 1 receptor.

The conventional chemotherapeutic treatment of malignant melanoma still remains poorly efficient in most cases. Thus the use of specific features of these tumors for development of new therapeutic modalities is highly needed. Melanocortin 1 receptor (MC1R) overexpression on the cell surface of the vast majority of human melanomas, making MC1R a valuable marker of these tumors, is one of these features. Naturally, MC1R plays a key role in skin protection against damaging ultraviolet radiation by regulating eumelanin production. MC1R activation is involved in regulation of melanocyte cell division. This article reviews the peculiarities of regulation and expression of MC1R, melanocytes, and melanoma cells, along with the possible connection of MC1R with signaling pathways regulating proliferation of tumor cells. MC1R is a cell surface endocytic receptor, thus considered perspective for diagnostics and targeted drug delivery. A number of new therapeutic approaches that utilize MC1R, including endoradiotherapy with Auger electron and α- and ß-particle emitters, photodynamic therapy, and gene therapy are now being developed.

The mTOR-inhibitor rapamycin mediates proteinuria in nephrotoxic serum nephritis by activating the innate immune response.

Rapamycin (Rapa) is an immunosuppressant used to prevent rejection in recipients of renal transplants. Its clinical use is limited by de novo onset or exacerbation of preexisting proteinuria. In the present study, Rapa administration was started 14 days after induction of murine nephrotoxic serum nephritis (NTS) to study glomerular effects of this mammalian target of rapamycin (mTOR) inhibitor. Glomeruli were laser-microdissected, and real-time PCR was performed to assess effects on glomerular cells and the expression of inflammatory cytokines. Immunohistochemical stainings were performed to confirm mRNA data on the protein level. Compared with nephritic control animals, Rapa-treated mice developed significantly increased albuminuria. This was accompanied by a more prominent glomerular infiltration by CD4(+) T cells and macrophages. Glomerular mRNA expression profiling revealed increased levels of the proinflammatory cytokines interleukin-6 and tumor necrosis factor-α, and the chemokines monocyte chemoattractant protein-1 and macrophage inflammatory protein-1ß and their cognate macrophage-associated receptors CCR2 and CCR5 in the Rapa-treated animals. Furthermore, there were elevated glomerular transcription levels of the regulatory T cell phenotype transcription factor Foxp3. No differences in the glomerular expression of the podocyte marker nephrin or the endothelial cell marker CD31 were observed on the mRNA or protein level. In conclusion, our data indicate that Rapa-induced proteinuria in NTS is a result of the activation of the innate immune system rather than a direct toxicity to podocytes or glomerular endothelial cells.

The PTH (1-84)/non-PTH (1-84) ratio is a risk factor for cardiovascular events in hemodialysis patients.

BACKGROUND: We hypothesized that the PTH (1-84)/non-PTH (1-84) ratio (PTH ratio) might help to assess cardiovascular risk in hemodialysis patients. METHODS: In this prospective cohort study 70 prevalent hemodialysis patients were followed up to 4 years. The PTH ratio was determined at baseline. Primary outcomes were cardiovascular events (CVE) and all-cause mortality. Cumulative event-free survival was compared between patients with a ratio < 1 and those with a ratio > 1. The risk-association of the PTH ratio with CVE was examined using an adjusted Multiple Cox Proportional Hazards model. RESULTS: A PTH ratio > 1 was found in 34 patients (49%). During follow-up 26 patients suffered a CVE. Patients with a CVE showed a higher ratio than patients with event-free survival (p = 0.033). In patients with a ratio > 1 a significantly higher number of CVE occurred (53 vs. 22%; p = 0.013), and these patients showed a significantly shorter event-free survival (p = 0.032). In an adjusted Cox-proportional hazards model a higher PTH ratio was found to be independently associated with an elevated risk for CVE (HR = 3.2; 95% CI 1.06 - 13.63; p = 0.04). CONCLUSIONS: A higher PTH (1-84)/non-PTH (1-84) ratio is associated with an increased risk for CVE in hemodialysis patients and might therefore be useful for cardiovascular risk estimation in this population.

Haemostatic alterations in overweight children: associations between metabolic syndrome, thrombin generation, and fibrinogen levels.

BACKGROUND: The metabolic syndrome (MetS) is associated with central obesity and leads to increased morbidity and mortality due to cardiovascular disease (CVD). Since obesity is associated with a hypercoagulable state, it has been speculated that hypercoagulation is linking MetS to CVD. METHODS: We prospectively examined 81 overweight children and 32 normal-weight children aged 10-16 years. We analyzed blood pressure, fasting lipids, glucose, insulin, fibrinogen, and thrombin generating test determining time to peak (TTPeak), peak, time preceding the thrombin burst (lag-time), and 'endogenous' thrombin potential (ETP). RESULTS: Overweight children demonstrated significantly higher fibrinogen levels (p<0.001), shorter lag-time (p<0.001), and TTPeak (p=0.038) compared to normal-weight children. Furthermore, ETP (p<0.001) and peak (p<0.001) were significantly higher in overweight than in normal-weight children. Fibrinogen and all parameters of the clotting test correlated significantly (p always <0.05) to body mass index (BMI) but not significantly to insulin resistance index HOMA-IR or occurrence of MetS in multiple linear backward regression analyses adjusted for age and gender. CONCLUSIONS: The increased fibrinogen levels and the changes in the thrombin generation test points towards a haemostatic alteration in overweight children. The parameters of the clotting test were related to the degree of overweight but not to insulin resistance or occurrence of MetS questioning a direct association between MetS and the coagulation system. Longitudinal studies are needed to confirm these findings.

Stimulatory effects of the multi-kinase inhibitor sorafenib on human bladder cancer cells.

BACKGROUND AND PURPOSE: Sorafenib is an inhibitor of several intracellular signalling kinases with anti-proliferative, anti-angiogenic and pro-apoptotic effects in tumour cells. Sorafenib is used in the therapy of advanced renal cell carcinoma, and several phase II clinical trials are being carried out in patients with urothelial carcinomas. EXPERIMENTAL APPROACH: Using a panel of human bladder cancer cell lines (RT4, T24, J82), we characterized systematically the effects of sorafenib on intracellular signalling, migration, proliferation and apoptosis. KEY RESULTS: We demonstrated that at low concentrations (<1 microM), sorafenib is capable of significantly stimulating migration and proliferation of the bladder cancer cells. We hypothesize that these stimulatory effects on tumour cell functions might be explained by an activation of the Ras/ERK-1/2 signal transduction pathway. In addition, the comparison of different bladder cancer cell lines not only revealed a different biology (e.g. cell migration), but also a differential susceptibility to the anti-apoptotic effects of sorafenib. Finally, we confirmed in different bladder cancer cell lines the known inhibitory actions of sorafenib in pharmacological concentrations (> or =3 microM) on ERK-1/2 phosphorylation, migration and proliferation, as well as the pro-apoptotic effects of the compound. CONCLUSIONS AND IMPLICATIONS: Taken together, these findings suggest that although sorafenib has the potential to be used in the treatment of urothelial carcinoma, this compound might also activate bladder cancer cells at low concentrations. This should be relevant for dosing regiments to optimize the treatment with this promising anti-tumour drug.

Calibrated automated thrombin generation in paediatric patients with inflammatory bowel disease.

UNLABELLED: In adults, inflammatory bowel disease (IBD) is associated with an increased risk of thromboembolic complications. The pathogenesis of IBD is not really clear and a high thrombin activity might contribute to disease progression. We wanted to see whether children with IBD have a higher thrombin generation (TG). PATIENTS, MATERIAL, METHODS: Plasma samples were collected of 20 patients with IBD and of 60 healthy controls (age range from 10 to 19). TG was measured by means of Calibrated automated thrombography (CAT). The disease activity was estimated, using the Pediatric Crohn's Disease Activity Index (PCDAI) for Crohn's disease and the Pediatric Ulcerative Colitis Disease Activity Index (PUCAI) for Ulcerative Colitis. In addition, we investigated F1+F2, TAT, TFPI and fibrinogen. RESULTS: There was a significant increase of endogenous thrombin potential (ETP), lag time and time to peak in patients with IBD, while peak showed no difference to healthy controls. ETP and F1+F2 in children with IBD also showed a significant correlation with PCDAI (PUCAI) and fibrinogen. CONCLUSION: IBD in children is associated with high TG, but this seems to be caused mainly by the inflammatory process and not by any individual disposition.

No differences in support of thrombin generation by neonatal or adult platelets.

UNLABELLED: Newborns have, despite low clotting factors and poor in vitro platelet function, a well functioning hemostasis. The reason for this is still not completely clear. The aim of our study was to investigate whether phospholipids in neonatal platelets differ from those in adult platelets in their total amount, in their exposure on the platelet surface, and their effect on thrombin generation (TG). METHODS: Clotting times of newborn and adult platelet-rich plasma were measured. Effect of newborn and adult platelets on TG was measured by means of CAT (calibrated automated thrombography). In addition, the effect of newborn and adult platelets with or without stimulation by ionophor on TG was measured in a purified prothrombinase complex. Phosphatidylserine-exposure (PS) of newborn and adult platelets was measured by flow cytometry of annexin V binding. The amount of phospholipids (PL) was determined by means of mass spectrometry. RESULTS: Clotting times of platelet-rich plasma (PRP) of newborns stimulated with ionophor showed a significant lower reduction of clotting time than in adult PRP. No differences in the support of TG between neonatal and adult platelets were found in neonatal or adult plasma by means of CAT. In the purified system TG was increased by adding ionophor stimulated platelets but no difference was evident between stimulated newborn and adult platelets. Flow cytometric analysis showed no difference in annexin V binding between adult and newborn platelets. The results of mass spectrometry showed a very similar amount and pattern of PL of adult and newborns platelets. CONCLUSION: Our results do not provide any evidence that a different PL content or expression of neonatal platelets may alter TG in neonates.