РОЛЬ ЭПИТЕЛИАЛЬНО-МЕЗЕНХИМАЛЬНОГО ПЕРЕХОДА И АУТОФАГИИ В ПРОТИВООПУХОЛЕВОМ ОТВЕТЕ КЛЕТОЧНЫХ ЛИНИЙ МЕЛАНОМЫ НА ТАРГЕТНОЕ ИНГИБИРОВАНИЕ MEK и mTOR КИНАЗ

Introduction. Cutaneous melanoma is a challenge to treat due to rapid progression of disease and acquired resistance to therapy. Autophagy and the epithelial-to-mesenchymal transition (EMT) are closely interrelated and play a key role in tumor progression. Targeted co-inhibition of MEK and mTOR kinases is a potential target for melanoma therapy by downregulatoin of the EMT.Objective: to study the effect of MEK and mTOR co-inhibition on cell viability, ability to form 3D-spheroids and migratory capacity of melanoma cell lines, and correlation of these changes with EMTand autophagy-related markers.Material and Methods. Melanoma cell lines Mel Z and Mel MTP were derived from patients, who were treated at the N.N. Blokhin National Medical Research Center of Oncology. The antiproliferative effect of binimetinib and/or rapamycin was studied by the MTT -test. 3D spheroids were formed using RGD peptides. Cell migration and invasion were assessed by a Boyden chamber migration assay. The expression levels of autophagy and EMT markers were investigated by immunocytochemistry or immunoblotting.Results. Rapamycin increased cytotoxicity of binimetinib in both 2D and 3D melanoma cell line cultures. At the same time, binimetinib and rapamycin reduced invasion, but not migration capacity of melanoma cells in vitro. The effectiveness of the combination was associated with a decrease in the EMT markers (N-cadherin and β-catenin) and autophagy markers (Beclin 1, p62/SQST M1 and LC3BII ) in melanoma cells.Conclusion. Inactivation of autophagy and EMT leads to overcoming the resistance to current anti-melanoma therapy and can be considered as a promising target for the treatment of melanoma.Введение. Возникновение резистентности и дальнейшая опухолевая прогрессия остаются актуальной проблемой в лечении меланомы кожи. Процесс аутофагии и эпителиально-мезенхимальный переход (ЭМП) тесно связаны между собой и играют ключевую роль в опухолевой прогрессии. Таргетное коингибирование МЕК и mTOR киназ является потенциальной мишенью для терапии меланомы, нацеленной на блокирование ЭМП.Цель работы – изучение влияния ко-ингибирования МЕК и mTOR киназ на выживаемость, возможность формирования 3D-сфероидов и миграционные способности клеточных линий меланомы, а также взаимосвязь этих изменений с маркерами ЭМП и аутофагии.Материал и методы. Работа проведена на клеточных линиях меланомы Mel Z и Mel MTP, полученных от пациентов, проходивших лечение в НМИЦ онкологии им. Н.Н. Блохина. Оценку антипролиферативной активности биниметиниба и/или рапамицина исследовали МТТ-тестом. 3D-сфероиды получали на основе RGDпептидов, миграционную способность и инвазивность оценивали к камере Бойдена и базальном матриксе. Изменения экспрессии маркеров аутофагии и ЭМП исследованы иммуноцитохимически или иммуноблоттингом.Результаты. Рапамицин усиливал цитотоксичность биниметиниба как в 2D-, так и в 3D-культурах клеточных линий меланомы. При этом биниметиниб и рапамицин снижали инвазию, но не миграцию клеток меланомы in vitro. Эффективность комбинации связана со снижением маркеров ЭМП N-кадхерина и β-катенина и аутофагии в клетках меланомы – Беклин 1, р62/SQST M1 и LC3BII .Заключение. Инактивация аутофагии и ЭМП позволяет преодолевать резистентность к существующей терапии и может быть рассмотрена как перспективная мишень для терапии меланомы

<i>In vitro и in vivo</i> photodynamic therapy of solid tumors with a combination of riboflavin and upconversion nanoparticles

Rationale: Riboflavin (vitamin B2) is one of the most promising agents for photodynamic therapy (PDT). However, its use is limited by the excitation in the ultraviolet (UV) and visible spectral ranges and, as a result, by a small penetration into biological tissue not exceeding a few millimeters. This problem could be solved by approaches ensuring excitation of riboflavin molecules within tumor tissues by infrared (IR) light. Upconversion nanoparticles (UCNPs) can be potentially considered as mediators able to effectively convert the exciting radiation of the near IR range, penetrating into biological tissue to a 3 cm depth, into the photoluminescence in the UV and visible spectral ranges.Aim: To evaluate the efficacy of UCNPs for IR-mediated riboflavin activation in the depth of tumor tissue during PDT. Materials and methods: The water-soluble riboflavin flavin mononucleotide (FMN, Pharmstandard-UfaVITA, Russia) was used as a photosensitizer in in vitro and in vivo experiments. The in vitro experiments were performed on human breast adenocarcinoma SK-BR-3, human glioblastoma U-87 MG, and rat glioma C6 cell lines. Lewis lung carcinoma (LLC) inoculated to hybrid BDF1 mice was used as a model to demonstrate the delivery of FMN to the tumor. UCNPs with a core/shell structure [NaYF4:Yb3+, Tm3+/NaYF4] were used for photoactivation of FMN in vivo. PDT based on FMN, UCNPs and laser radiation 975 nm (IR) was performed on mouse xenografts of human breast adenocarcinoma SKBR-3.Results: We were able to show that FMN could act as an effective in vitro photosensitizer for SK-BR-3, U-87 MG, and C6 cell lines. FMN IC50 values for glioma cells were ~30 μM, and for SK-BR-3 cell line ~50 μM (24 h incubation, irradiation 4.2 J/cm2). In the LLC model, the appropriate concentration of FMN (30 μM and above) can be achieved in the tumor as a result of systemic administration of FMN (at 2 and 24 hours after injection). The effect of PDT using near IR light for UCNP-mediated excitation of FMN was demonstrated in mouse xenografts SKBR-3, with the tumor growth inhibition of 90±5%.Conclusion: The study has demonstrated the possibility to use riboflavin (vitamin B2) as a photosensitizer for PDT. The photoexcitation of FMN via the anti-Stokes photoluminescence of UCNPs allows for implementation of the PDT technique with the near IR spectral range

THE ROLE OF EPITHELIAL-TO-MESENCHYMAL TRANSITION AND AUTOPHAGY IN ANTITUMORAL RESPONSE OF MELANOMA CELL LINES TO TARGET INHIBITION OF MEK AND mTOR KINASES

Introduction. Cutaneous melanoma is a challenge to treat due to rapid progression of disease and acquired resistance to therapy. Autophagy and the epithelial-to-mesenchymal transition (EMT) are closely interrelated and play a key role in tumor progression. Targeted co-inhibition of MEK and mTOR kinases is a potential target for melanoma therapy by downregulatoin of the EMT.Objective: to study the effect of MEK and mTOR co-inhibition on cell viability, ability to form 3D-spheroids and migratory capacity of melanoma cell lines, and correlation of these changes with EMTand autophagy-related markers.Material and Methods. Melanoma cell lines Mel Z and Mel MTP were derived from patients, who were treated at the N.N. Blokhin National Medical Research Center of Oncology. The antiproliferative effect of binimetinib and/or rapamycin was studied by the MTT -test. 3D spheroids were formed using RGD peptides. Cell migration and invasion were assessed by a Boyden chamber migration assay. The expression levels of autophagy and EMT markers were investigated by immunocytochemistry or immunoblotting.Results. Rapamycin increased cytotoxicity of binimetinib in both 2D and 3D melanoma cell line cultures. At the same time, binimetinib and rapamycin reduced invasion, but not migration capacity of melanoma cells in vitro. The effectiveness of the combination was associated with a decrease in the EMT markers (N-cadherin and β-catenin) and autophagy markers (Beclin 1, p62/SQST M1 and LC3BII ) in melanoma cells.Conclusion. Inactivation of autophagy and EMT leads to overcoming the resistance to current anti-melanoma therapy and can be considered as a promising target for the treatment of melanoma

Biodistribution of x-ray iodinated contrast agent in nano-emulsions is controlled by the chemical nature of the oily core

In this study, we investigated the role of the chemical nature of the oil droplet core of nano-emulsions used as contrast agents for X-ray imaging on their pharmacokinetics and biodistribution. To this end, we formulated PEGylated nano-emulsions with two iodinated oils (i.e., iodinated monoglyceride and iodinated castor oil) and compared them with another iodinated nano-emulsion based on iodinated vitamin E. By using dynamic light scattering and transmission electron microscopy, the three iodinated nano-emulsions were found to exhibit comparable morphologies, size, and surface composition. Furthermore, they were shown to be endowed with very high iodine concentration, which leads to stronger X-ray attenuation properties as compared to the commercial iodinated nano-emulsion Fenestra VC. The three nano-emulsions were i.v. administered in mice and monitored by microcomputed tomography (micro-CT). They showed high contrast enhancement in blood with similar half-life around 6 h but very different accumulation sites. While iodinated monoglycerides exhibited low accumulation in liver and spleen, high accumulation in spleen was observed for iodinated castor oil and in liver for vitamin E. These data clearly highlighted the important role of the oil composition of the nano-emulsion core to obtain strong X-ray contrast enhancement in specific targets such as liver, spleen, or only blood. These differences in biodistribution were partly attributed to differences in the uptake of the nanodroplets by the macrophages in vitro. Another key feature of these nano-emulsions is their long half-elimination time (several weeks), which offers sufficient retention for micro-CT imaging. This work paves the way for the design of nanoparticulate contrast agents for X-ray imaging of selected organs