oncotomskСибирский онкологический журналSiberian journal of oncology1814-48612312-3168Tomsk National Research Medical Сепtеr of the Russian Academy of Sciences10.21294/1814-4861-2019-18-3-54-63oncotomsk-1096Research ArticleЛАБОРАТОРНЫЕ И ЭКСПЕРИМЕНТАЛЬНЫЕ ИССЛЕДОВАНИЯLABORATORY AND EXPERIMENTAL STUDIESРОЛЬ ЭПИТЕЛИАЛЬНО-МЕЗЕНХИМАЛЬНОГО ПЕРЕХОДА И АУТОФАГИИ В ПРОТИВООПУХОЛЕВОМ ОТВЕТЕ КЛЕТОЧНЫХ ЛИНИЙ МЕЛАНОМЫ НА ТАРГЕТНОЕ ИНГИБИРОВАНИЕ MEK и mTOR КИНАЗTHE ROLE OF EPITHELIAL-TO-MESENCHYMAL TRANSITION AND AUTOPHAGY IN ANTITUMORAL RESPONSE OF MELANOMA CELL LINES TO TARGET INHIBITION OF MEK AND mTOR KINASEShttps://orcid.org/0000-0001-6295-3497РябаяО. О.RyabayaO. O.

Рябая Оксана Олеговна, научный сотрудник

Научно-исследовательский институт экспериментальной диагностики и терапии опухолей

Москва, 115478, Каширское шоссе, 24

SPIN-код: 4865-5850.

Author ID (Scopus): 56974346800

Researcher ID (WOS): G-1529-2017

Oksana O. Ryabaya, Researcher

Department of Experimental Diagnostic and Tumor Therapy

24, Kashirskoe shosse, 115478-Moscow

Author ID (Scopus): 56974346800.

Researcher ID (WOS): G-1529-2017

oxa2601@yandex.ruПрокофьеваА. А.ProkofievaA. A.

Прокофьева Анастасия Алексеевна, лаборант-исследователь

Научно-исследовательский институт экспериментальной диагностики и терапии опухолей

Москва, 115478, Каширское шоссе, 24

Author ID (Scopus): 57202330994

Anastasia A. Prokofieva, Research assistant

Department of Experimental Diagnostic and Tumor Therapy

24, Kashirskoe shosse, 115478-Moscow

Author ID (Scopus): 57202330994

https://orcid.org/0000-0002-5694-3492ХоченковД. А.KhochenkovD. A.

Хоченков Дмитрий Александрович, кандидат биологических наук, заведующий лабораторией

Научно-исследовательский институт экспериментальной диагностики и терапии опухолей

Москва, 115478, Каширское шоссе, 24

SPIN-код: 9588-1183.

Author ID (Scopus): 25930436100.

Researcher ID (WOS): J-6861-2015

Dmitry A. Khochenkov, PhD, Head of the Laboratory

Department of Experimental Diagnostic and Tumor Therapy

24, Kashirskoe shosse, 115478-Moscow

Author ID (Scopus): 57202330994.

Researcher ID (WOS): J-6861-2015

khochenkov@gmail.comhttps://orcid.org/0000-0001-6486-8114АкасовР. А.AkasovR. A.

Акасов Роман Александрович, кандидат химических наук, научный сотрудник, 

Институт молекулярной медицины,Первый Московский государственный медицинский университет имени И.М. Сеченова Министерства здравоохранения Российской Федерации

Отдел биоматериалов и бионанотехнологий, Институт биоорганической химии имени академиков М.М. Шемякина и Ю.А. Овчинникова РАН

Москва, 119991, ул. Трубецкая, 8/2, 

г. Москва, 117997, ГСП-7, ул. Миклухо-Маклая, 16/10, 

г. Москва, 117997, ул. Бутлерова,17a

SPIN-код: 8383-0004.

Author ID (Scopus): 56285575900.

Researcher ID (WOS): K-2275-2016

Roman A. Akasov, PhD, Institute of Molecular Medicine, Sechenov First Moscow State Medical University

Department of Biomaterials and Biotechnologies, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry

82, Trubetskaya Street, 119991-Moscow, 

16/10, Miklukho-Maklaya Street, GSP-7, 117997-Moscow,

17a, Butlerova Street, 117997-Moscow

Author ID (Scopus): 56285575900.

Researcher ID (WOS): K-2275-2016

БуровС. В.BurovS. V.

Буров Сергей Владимирович, доктор химических наук

г. Санкт-Петербург, 199004, 4-я линия Васильевского острова, 11

SPIN-код: 6046-3461.

Author ID (Scopus): 7003948349.

Researcher ID (WOS): P-7225-2015

Sergey V. Burov, DSc

11, 4th line of Vasilyevsky Island, 199004-St. Petersburg

Author ID (Scopus): 7003948349.

Researcher ID (WOS): P-72252015

МарквичеваЕ. А.MarkvichevaE. A.

Марквичева Елена Арнольдовна, доктор химических наук

Отдел биоматериалов и бионанотехнологий

г. Москва, 117997, ГСП-7, ул. Миклухо-Маклая, 16/10

Author ID (Scopus): 6603356683

Elena A. Markvicheva, DSc, Institute of Molecular Medicine

Department of Biomaterials and Biotechnologies

16/10, Miklukho-Maklaya Street, GSP-7, 117997-Moscow

Author ID (Scopus): 6603356683

https://orcid.org/0000-0002-7546-1288СтепановаЕ. В.StepanovaE. V.

Степанова Евгения Владиславовна, доктор медицинских наук, ведущий научный сотрудник

Научно-исследовательский институт экспериментальной диагностики и терапии опухолей

Москва, 115478, Каширское шоссе, 24

SPIN-код: 3008-2690.

Author ID (Scopus): 57189217833.

Researcher ID (WOS): B-3206-2014

Evgenia V. Stepanova, MD, DSc, Leading Researcher

Department of Experimental Diagnostic and Tumor Therapy

24, Kashirskoe shosse, 115478-Moscow

Author ID (Scopus): 57189217833.

Researcher ID (WOS): B-3206-2014

ФГБУ «НМИЦ онкологии им. Н.Н. Блохина» Минздрава РоссииРоссияN.N. Blokhin National Medical Research Center of OncologyRussian FederationФГАОУ ВО «Первый МГМУ им. И.М. Сеченова» Минздрава России; Институт биоорганической химии имени академиков М.М. Шемякина и Ю.А. Овчинникова РАН; Федеральный научно-исследовательский центр «Кристаллография и фотоника» РАНРоссияSechenov First Moscow State Medical University; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry; Federal Scientific Research Center «Crystallography and Photonics» Russian Academy of SciencesRussian FederationМедико-биологический научно-производственный комплекс «Цитомед»РоссияCytomed J.S.CoRussian FederationИнститут биоорганической химии имени академиков М.М. Шемякина и Ю.А. Овчинникова РАНРоссияShemyakin-Ovchinnikov Institute of Bioorganic ChemistryRussian Federation2019300620191835463Copyright © Рябая О.О., Прокофьева А.А., Хоченков Д.А., Акасов Р.А., Буров С.В., Марквичева Е.А., Степанова Е.В., 20192019Рябая О.О., Прокофьева А.А., Хоченков Д.А., Акасов Р.А., Буров С.В., Марквичева Е.А., Степанова Е.В.Ryabaya O.O., Prokofieva A.A., Khochenkov D.A., Akasov R.A., Burov S.V., Markvicheva E.A., Stepanova E.V.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.siboncoj.ru/jour/article/view/1096Введение

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

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

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

Заключение. Инактивация аутофагии и ЭМП позволяет преодолевать резистентность к существующей терапии и может быть рассмотрена как перспективная мишень для терапии меланомы.

Introduction

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

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

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

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

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.

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The authors declare that there are no conflicts of interest present.