Delivery of Barnase to Cells in Liposomes Functionalized by Her2-Specific DARPin Module

Russian Journal of Bioorganic Chemistry - In this work for the first time we demonstrate the possibility of ribonuclease barnase delivery to HER2-positive human cancer cells in a liposomal form....     

Key Molecular Mechanisms of Aging,Biomarkers, and Potential Interventions

Molecular Biology - The mechanisms of aging are described at the molecular, cell, tissue, and systemic levels. Primary age-dependent molecular lesions activate the cell stress response to...     

Efficiency of Bioluminescence Resonance Energy Transfer in the NanoLuc-miniSOG-Furimazine System

Russian Journal of Bioorganic Chemistry - Photodynamic therapy (PDT) is a clinically approved, minimally invasive method for tumor destruction in the presence of a photosensitizer (PS), oxygen, and...     

Anti-HER2 phototoxin based on flavoprotein miniSOG causes the oxidative stress and necrosis of HER2-positive cancer cells

Development and functional characterization of novel, high-affinity protein compounds able to selectively kill human cancer cells is an urgent task of modern biomedical research. In this work, we studied the cytotoxicity of a recombinant phototoxic protein DARPin-miniSOG against the HER2-positive human breast adenocarcinoma cells. It was found that targeted phototoxin DARPin-miniSOG interacts specifically with HER2 receptor and causes the light-induced death of HER2-positive cells by the mechanism of necrosis. Irradiation of the cells in the presence of ascorbic acid eliminates the light-induced cytotoxicity of DARPin-miniSOG, which proves the prooxidant mechanism of phototoxin action.     

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.     

Cytotoxicity of targeted HER2-specific phototoxins based on flavoprotein miniSOG is determined by the rate of their internalization

The concept of targeted therapy implies the development of bifunctional agents complementing the therapeutic module with a targeting one. A promising target for the delivery of imaging and/or toxic modules is the HER2 (ErbB2) receptor. Earlier, we have functionally characterized the targeted photosensitizers 4D5scFv–miniSOG and DARPin–miniSOG, causing the death of HER2-overexpressing cells when irradiated with blue light. However, the cytotoxicity of targeted toxins 4D5scFv–miniSOG and DARPin–miniSOG (both having functionally active targeted and cytotoxic modules in recombinant proteins) against human breast adenocarcinoma cells differs 5 times. The study of the dynamics of internalization of 4D5scFv–miniSOG and DARPin–miniSOG proteins in the complex with HER2 in this work showed that the rate of internalization contributes most significantly to the toxicity of these photosensitizers, because it determines the duration of the presence of the phototoxin in the lipid bilayer of the cell membrane, where its damaging effect is maximum.     

A Highly Specific Substrate for NanoLUC Luciferase Furimazine Is Toxic in vitro and in vivo

The bioluminescent platform based on a genetically engineered luciferase NanoLuc and its synthetic substrate furimazine is widely used in modern molecular biology for optical imaging and therapy of deep tissue tumors. Taking into account the perspectives of using this system in molecular biology research, a detailed study of the toxicity of furimazine in vitro and in vivo is of great interest. In this work, the cytotoxicity of furimazine in vitro was studied using four different cell lines. Systemic toxicity of furimazine in vivo was investigated under conditions of prolonged administration of the substrate in animals. It has been found that seven-day intravenous injection of the substrate in animals causes hydropic dystrophy of liver and necrosis of hepatocytes. Splitting of the dose into several injections reduces hepatotoxicity of furimazine.