Selective targeting of α7 nicotinic acetylcholine receptor by synthetic peptide mimicking loop I of human SLURP-1 provides efficient and prolonged therapy of epidermoid carcinoma in vivo.

α7-Type nicotinic acetylcholine receptor (α7-nAChR) promotes the growth and metastasis of solid tumors. Secreted Ly6/uPAR-Related Protein 1 (SLURP-1) is a specific negative modulator of α7-nAChR produced by epithelial cells. Here, we investigated mechanisms of antiproliferative activity of recombinant SLURP-1 in epidermoid carcinoma A431 cells and activity of SLURP-1 and synthetic 21 a.a. peptide mimicking its loop I (Oncotag) in a xenograft mice model of epidermoid carcinoma. SLURP-1 inhibited the mitogenic pathways and transcription factors in A431 cells, and its antiproliferative activity depended on α7-nAChR. Intravenous treatment of mice with SLURP-1 or Oncotag for 10 days suppressed the tumor growth and metastasis and induced sustained changes in gene and microRNA expression in the tumors. Both SLURP-1 and Oncotag demonstrated no acute toxicity. Surprisingly, Oncotag led to a longer suppression of pro-oncogenic signaling and downregulated expression of pro-oncogenic miR-221 and upregulated expression of KLF4 protein responsible for control of cell differentiation. Affinity purification revealed SLURP-1 interactions with both α7-nAChR and EGFR and selective Oncotag interaction with α7-nAChR. Thus, the selective inhibition of α7-nAChRs by drugs based on Oncotag may be a promising strategy for cancer therapy.

Polymer/magnetite carriers functionalized by HER2-DARPin: Avoiding lysosomes during internalization and controlled toxicity of doxorubicin by focused ultrasound induced release.

Nanomedicine has revolutionized the available treatment options during the last decade, but poor selectivity of targeted drug delivery and release is still poses a challenge. In this study, doxorubicin (DOX) and magnetite nanoparticles were encapsulated by freezing-induced loading, coated with polymeric shell bearing two bi-layers of polyarginine/dextran sulphate and finally modified with HER2-specific DARPin proteins. We demonstrated that the enhanced cellular uptake of these nanocarriers predominantly occurs by SKOV-3 (HER2+) cells, in comparison to CHO (HER2-) cells, together with the controlled DOX release using low intensity focused ultrasound (LIFU). In addition, a good ability of DARPin+ capsules to accumulate in the tumor and the possibility of combination therapy with LIFU were demonstrated. A relatively high sensitivity of the obtained nanocarriers to LIFU and their preferential interactions with mitochondria in cancer cells make these carriers promising candidates for cancer treatment, including novel approaches to overcome drug resistance.

System for Self-excited Targeted Photodynamic Therapy Based on the Multimodal Protein DARP-NanoLuc-SOPP3.

Despite the significant potential of photodynamic therapy (PDT) as a minimally invasive treatment modality, the use of this method in oncology has remained limited due to two serious problems: 1) limited penetration of the excitation light in tissues, which makes it impossible to affect deep-seated tumors and 2) use of chemical photosensitizers that slowly degrade in the body and cause photodermatoses and hyperthermia in patients. To solve these problems, we propose a fully biocompatible targeted system for PDT that does not require an external light source. The proposed system is based on bioluminescent resonance energy transfer (BRET) from the oxidized form of the luciferase substrate to the photosensitizing protein SOPP3. The BRET-activated system is composed of the multimodal protein DARP-NanoLuc-SOPP3, which contains a BRET pair NanoLuc-SOPP3 and a targeting module DARPin. The latter provides the interaction of the multimodal protein with tumors overexpressing tumor-associated antigen HER2 (human epidermal growth factor receptor type II). In vitro experiments in a 2D monolayer cell culture and a 3D spheroid model have confirmed HER2-specific photo-induced cytotoxicity of the system without the use of an external light source; in addition, experiments in animals with subcutaneous HER2-positive tumors have shown selective accumulation of DARP-NanoLuc-SOPP3 on the tumor site. The fully biocompatible system for targeted BRET-induced therapy proposed in this work makes it possible to overcome the following limitations: 1) the need to use an external light source and 2) the side phototoxic effect from aberrant accumulation of chemical photosensitizers. The obtained results demonstrate that the fully protein-based self-excited BRET system has a high potential for targeted PDT.

Barnase*Barstar-guided two-step targeting approach for drug delivery to tumor cells in vivo.

For precise ligation of a targeting and cytotoxic moiety, the use of Barnase-Barstar pair as a molecular glue is proposed for the first time. Targeting was mediated through the use of a scaffold protein DARPin_9-29 specific for the human epidermal receptor 2 (HER2) antigen that is highly expressed on some types of cancer and Barnase*Barstar native bacterial proteins interacted with each other with Kd 10-14 M. The approach proposed consists of prelabeling a target tumor with hybrid protein DARPin-Barnase prior to administration of cytotoxic component-loaded liposomes that have Barstar covalently attached to their surface. Based on in vivo bioimaging we have proven that DARPin-based Barnase*Barstar-mediated pretargeting possesses precise tumor-targeting capability as well as antitumor activity leading to apparent tumor-growth inhibition of primary tumors and distant metastases in experimental animals. The results obtained indicate that the new system combining DARPin and Barnase*Barstar can be useful both for the drug development and for monitoring the response to treatment in vivo in preclinical studies.

Near-Infrared Activated Cyanine Dyes As Agents for Photothermal Therapy and Diagnosis of Tumors.

Today, it has become apparent that innovative treatment methods, including those involving simultaneous diagnosis and therapy, are particularly in demand in modern cancer medicine. The development of nanomedicine offers new ways of increasing the therapeutic index and minimizing side effects. The development of photoactivatable dyes that are effectively absorbed in the first transparency window of biological tissues (700-900 nm) and are capable of fluorescence and heat generation has led to the emergence of phototheranostics, an approach that combines the bioimaging of deep tumors and metastases and their photothermal treatment. The creation of near-infrared (NIR) light-activated agents for sensitive fluorescence bioimaging and phototherapy is a priority in phototheranostics, because the excitation of drugs and/or diagnostic substances in the near-infrared region exhibits advantages such as deep penetration into tissues and a weak baseline level of autofluorescence. In this review, we focus on NIR-excited dyes and discuss prospects for their application in photothermal therapy and the diagnosis of cancer. Particular attention is focused on the consideration of new multifunctional nanoplatforms for phototheranostics which allow one to achieve a synergistic effect in combinatorial photothermal, photodynamic, and/or chemotherapy, with simultaneous fluorescence, acoustic, and/or magnetic resonance imaging.

Death Mechanism of Breast Adenocarcinoma Cells Caused by BRET-Induced Cytotoxicity of miniSOG Depends on the Intracellular Localization of the NanoLuc-miniSOG Fusion Protein.

Photodynamic therapy (PDT) is widely used in clinical practice to influence neoplasms in the presence of a photosensitizer, oxygen, and light source. The main problem of PDT of deep tumors is the problem of delivering excitation light (without lost of its intensity) inside the body. An alternative to the external light sources can be the internal light sources based on luciferase-substrate bioluminescent systems. In our work, we used the NanoLuc-furimazine system as an internal light source. This system can be successfully used to excite the protein photosensitizer miniSOG and to induce the phototoxicity of this flavoprotein in cancer cells during bioluminescent resonance energy transfer (BRET). It was shown that the mechanism of cell death caused by BRET-induced phototoxicity of mimiSOG in the presence of furimazine depends on the intracellular localization of the NanoLuc-miniSOG fusion protein: BRET-mediated activation of miniSOG in mitochondrial localization causes apoptosis, while the membrane localization of PS causes necrosis of cancer cells.

Phototoxicity of flavoprotein miniSOG induced by bioluminescence resonance energy transfer in genetically encoded system NanoLuc-miniSOG is comparable with its LED-excited phototoxicity.

Photodynamic therapy (PDT) is a clinical, minimally invasive method for destroying cancer cells in the presence of a photosensitizer, oxygen, and a light source. The main obstacle for the PDT treatment of deep tumors is a strong reduction of the excitation light intensity as a result of its refraction, reflection, and absorption by biological tissues. Internal light sources based on bioluminescence resonance energy transfer can be a solution of this problem. Here we show that luciferase NanoLuc being expressed as a fusion protein with phototoxic flavoprotein miniSOG in cancer cells in the presence of furimazine (highly specific NanoLuc substrate) induces a photodynamic effect of miniSOG comparable with its LED-excited (Light Emitting Diode) phototoxicity. Luminescence systems based on furimazine and hybrid protein NanoLuc-miniSOG targeted to mitochondria or cellular membranes possess the similar energy transfer efficiencies and similar BRET-induced cytotoxic effects on cancer cells, though the mechanisms of BRET-induced cell death are different. As the main components of the proposed system for BRET-mediated PDT are genetically encoded (luciferase and phototoxic protein), this system can potentially be delivered to any site in the organism and thus may be considered as a promising approach for simultaneous delivery of light source and photosensitizer in deep-lying tumors and metastasis anywhere in the body.

[Bifunctional Toxin DARP-LoPE Based on the HER2-Specific Innovative Module of a Non-Immunoglobulin Scaffold as a Promising Agent for Theranostics].

We have generated and characterized HER2-specific targeted toxin based on the low-immunogenic variant of Pseudomonas exotoxin A (LoPE), in which most of the human immunodominant B-cell epitopes have been inactivated. Nonimmunoglobulin DARPin-based HER2-specific protein was used as a targeting module for toxin delivery to the cellular target. Using confocal microscopy, it has been found that both domains in this hybrid toxin retained their functionality, i.e., the specific interaction with HER2 receptor, as well as the internalization and effective transport to ER typical of the wild-type Pseudomonas exotoxin A. The HER2-dependent cytotoxic effect correlated with receptor expression level at the cell surface, as shown in vitro using cell lines with different levels of HER2 expression. Due to the very high selective cytotoxicity against HER2-positive human tumor cells, as well as expected low immunogenicity, we believe that this new targeted toxin may be promising for future in vivo studies as a therapeutic agent for HER2-positive tumors.

Flavoprotein miniSOG BRET-induced cytotoxicity depends on its intracellular localization.

It is proposed to use the bioluminescent resonance energy transfer to solve the problem of creating the internal light sources in photodynamic therapy of cancer. Energy donor in the developed system is the oxidized form of the luciferase NanoLuc substrate furimamide, and acceptor is the phototoxic fluorescent protein miniSOG. It is shown that, in the proposed system, the photoinduced cytotoxicity of flavoprotein miniSOG in vitro depends on the intracellular localization, and the cytotoxic effect is 48% for the cytoplasmic localization of the fusion protein, 65% for the mitochondrial localization, and 69% for the membrane localization. The obtained data indicate that, for maximization of the photodynamic effect in vivo, it is appropriate to use the NanoLuc-miniSOG fusion protein in the membrane localization.

Flavoprotein miniSOG Cytotoxisity Can Be Induced By Bioluminescence Resonance Energy Transfer.

In this study, we investigated the possibility of phototoxic flavoprotein miniSOG (photosensitizer) excitation in cancer cells by bioluminescence occurring when luciferase NanoLuc oxidizes its substrate, furimazine. We have shown that the phototoxic flavoprotein miniSOG expressed in eukaryotic cells in fusion with NanoLuc luciferase is activated in the presence of its substrate, furimazine. Upon such condition, miniSOG possesses photoinduced cytotoxicity and causes a 48% cell death level in a stably transfected cell line.

Acipensins - Novel Antimicrobial Peptides from Leukocytes of the Russian Sturgeon Acipenser gueldenstaedtii.

Antimicrobial peptides (AMPs) play an important role in the innate defense mechanisms in humans and animals. We have isolated and studied a set of antimicrobial peptides from leukocytes of the Russian sturgeon Acipenser gueldenstaedtii belonging to a subclass of chondrosteans, an ancient group of bony fish. Structural analysis of the isolated peptides, designated as acipensins (Ac), revealed in leukocytes of the Russian sturgeon six novel peptides with molecular masses of 5336.2 Da, 3803.0 Da, 5173.0 Da, 4777.5 Da, 5449.4 Da, and 2740.2 Da, designated as Ac1-Ac6, respectively. Complete primary structures of all the isolated peptides were determined, and the biological activities of three major components - Ac1, Ac2, and Ac6 - were examined. The peptides Ac1, Ac2, Ac3, Ac4, and Ac5 were found to be the N-terminal acetylated fragments 1-0, 1-5, 1-9, 1-4, and 1-1 of the histone H2A, respectively, while Ac6 was shown to be the 62-5 fragment of the histone H2A. The peptides Ac1 and Ac2 displayed potent antimicrobial activity towards Gram-negative and Gram-positive bacteria (Escherichia coli ML35p, Listeria monocytogenes EGD, MRSA ATCC 33591) and the fungus Candida albicans 820, while Ac6 proved effective only against Gram-negative bacteria. The efficacy of Ac 1 and Ac2 towards the fungus and MRSA was reduced upon an increase in the ionic strength of the solution. Ac1, Ac2, and Ac6, at concentrations close to their minimum inhibitory concentrations, enhanced the permeability of the E.coli ML35p outer membrane to the chromogenic marker, but they did not affect appreciably the permeability of the bacterial inner membrane in comparison with a potent pore-forming peptide, protegrin 1. Ac1, Ac2, and Ac6 revealed no hemolytic activity against human erythrocytes at concentrations of 1 to 40 µM and had no cytotoxic effect (1 to 20 µM) on K-562 and U-937 cells in vitro. Our findings suggest that histone-derived peptides serve as important anti-infective host defense molecules.

Characteristics of new monomolecular chimeric T-cell receptors to carcinoembryonic antigen.

We described two original genetic constructs encoding chimeric monomolecular T-cell receptors, where the effector T-cell receptor fragment was linked with the antigen-recognizing part consisting of two variable fragments of two different antibodies to carcinoembryonic antigen. Following transfection, these receptors were expressed on the cell surface and bound carcinoembryonic antigen. Human peripheral blood lymphocytes transfected with the above constructs demonstrated high cytotoxic activity against HCT116 cells expressing carcinoembryonic antigen.

[The status of nucleolus organizing regions in hybrids of pluripotent and somatic mouse cells cultured under different conditions].

In the present work we examined the status of nucleolus organizing regions of mitotic chromosomes (NOR) in hybrid cells obtained by fusion of the mouse teratocarcinoma cells PCC4aza1 and adult mouse spleenocytes upon cultivation of hybrid cells under different conditions. We have shown that extended cultivation of hybrid cells in medium supplemented with HAT (hypoxanthine, aminopterin, thymidine) promotes the maintenance of NO-chromosomes, whereas under nonselective conditions elimination of NO-chromosome occurs. In nonselective medium the number of active, i. e. Ag-positive, NORs has been augmented comparatively to that observed under selective conditions. This observation directly indicates that reprogramming of the parental cell genomes in hybrid cells includes changes in the status of chromosomal NORs. The number of active NORs depends on conditions of hybrid cells culturing and may be changed by either of the two major ways--by elimination of NO-chromosomes (under nonselective conditions) or by inactivation of some NORs, when the general number of NO-chromosomes remains unaltered (under selective conditions).