oncotomskСибирский онкологический журналSiberian journal of oncology1814-48612312-3168Tomsk National Research Medical Сепtеr of the Russian Academy of Sciences10.21294/1814-4861-2018-17-4-24-29oncotomsk-811Research ArticleЛАБОРАТОРНЫЕ И ЭКСПЕРИМЕНТАЛЬНЫЕ ИССЛЕДОВАНИЯLABORATORY AND EXPERIMENTAL STUDIESРЕОРГАНИЗАЦИЯ МЕЖКЛЕТОЧНЫХ АДГЕЗИОННЫХ КОНТАКТОВ И ПОЯВЛЕНИЕ МИГРАЦИОННОЙ АКТИВНОСТИ У КЛЕТОК MCF-7-SNAI1 ПРИ ИНДУКЦИИ ЭПИТЕЛИАЛЬНО-МЕЗЕНХИМАЛЬНОГО ПЕРЕХОДАINDUCTION OF EPITHELIAL-TO-MESENCHYMAL TRANSITION IN MCF-7-SNAI1 CELLS LEADS TO REORGANIZATION OF ADHERENS JUNCTIONS AND ACQUISITION OF MIGRATORY ACTIVITYhttps://orcid.org/0000-0002-0243-5994ЖитнякИ. Ю.ZhitnyakI. Y.

Россия, 115478, г. Москва, Каширское шоссе, 24

кандидат биологических наук, научный сотрудник лаборатории механизмов канцерогенеза, ФГБУ «НМИЦ онкологии им. Н.Н. Блохина» Минздрава России

SPIN-код: 183041. Author ID (Scopus): 35849650500

24, Kashirskoye Shosse, 115478-Moscow, Russia

PhD, Research scientist in the Laboratory of the Mechanisms of Carcinogenesis, FSBI «N.N. Blokhin National Medical  Research Center of Oncology» of the Ministry of Health of the Russian Federation

Author ID (Scopus): 35849650500.

ЛитовкаН. И.LitovkaN. I.

Россия, 115478, г. Москва, Каширское шоссе, 24

лаборант-исследователь лаборатории механизмов канцерогенеза, ФГБУ «НМИЦ онкологии им. Н.Н. Блохина» Минздрава России

24, Kashirskoye Shosse, 115478-Moscow, Russia

Technician in the Laboratory of the Mechanisms of Carcinogenesis, FSBI «N.N. Blokhin National Medical Research Center of Oncology» of the Ministry of Health of the Russian Federation

https://orcid.org/0000-0003-4620-7851РубцоваС. Н.RubtsovaS. N.

Россия, 115478, г. Москва, Каширское шоссе, 24

кандидат биологических наук, научный сотрудник лаборатории механизмов канцерогенеза, ФГБУ «НМИЦ онкологии им. Н.Н. Блохина» Минздрава России

SPIN-код: 93540. Author ID (Scopus): 15754557800

24, Kashirskoye Shosse, 115478-Moscow, Russia

PhD, Research scientist in the Laboratory of the Mechanisms of Carcinogenesis, FSBI «N.N. Blokhin National Medical  Research Center of Oncology» of the Ministry of Health of the  Russian Federation

Author ID (Scopus): 15754557800

https://orcid.org/0000-0002-2044-2063ГлушанковаН. А.GloushankovaN. A.

Россия, 115478, г. Москва, Каширское шоссе, 24

доктор биологических наук, заведующая лабораторией механизмов канцерогенеза,ФГБУ «НМИЦ онкологии им. Н.Н. Блохина» Минздрава России

SPIN-код: 93541. Author ID (Scopus): 6507200005

24, Kashirskoye Shosse, 115478-Moscow, Russia

DSc, Head of the Laboratory of the Mechanisms of Carcinogenesis, FSBI «N.N. Blokhin National Medical Research Center of Oncology» of the Ministry of Health of the Russian Federation 

Author ID (Scopus): 6507200005

natglu@hotmail.comФГБУ «НМИЦ онкологии им. Н.Н. Блохина» Минздрава РоссииРоссияFSBI «N.N. Blokhin National Medical Research Center of Oncology» of the Ministry of Health of the Russian FederationRussian Federation2018010920181742429Copyright © Житняк И.Ю., Литовка Н.И., Рубцова С.Н., Глушанкова Н.А., 20182018Житняк И.Ю., Литовка Н.И., Рубцова С.Н., Глушанкова Н.А.Zhitnyak I.Y., Litovka N.I., Rubtsova S.N., Gloushankova N.A.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.siboncoj.ru/jour/article/view/811

С помощью DIC-микроскопии и конфокальной микроскопии были проанализированы изменения морфологии,  миграционных характеристик и межклеточных адгезионных контактов в культуре клеток рака молочной железы MCF-7-SNAI1 при активации экспрессии транскрипционного фактора ЭМП – SNAI1. Как показал Вестерн-блот  анализ, экспрессия SNAI1 достигала максимальных значений через 24 часа после переноса клеток в среду без  тетрациклина и поддерживалась на этом уровне в течение семи дней. В клетках в течение семи дней  сохранялась экспрессия Е-кадхерина, при этом тангенциальные межклеточные адгезионные контакты,  характерные для клеток со стабильной межклеточной адгезией, замещались радиальными контактами. В  радиальных контактах в течение 24–72 часов отмывки от тетрациклина продолжалась аккумуляция Е- кадхерина. В результате активации SNAI1 клетки вступали в ЭМП и приобретали миграционную активность. На  двумерном субстрате клетки мигрировали как индивидуально, так и коллективно. С увеличением  продолжительности отмывки от тетрациклина повышался процент клеток, мигрировавших через поры в  миграционных камерах, способность клеток инвазировать эпителиальный монослой, напротив, снижалась.  Полученные данные свидетельствуют о том, что сохранение гибридного эпителиально-мезенхимального  фенотипа и аккумуляция Е-кадхерина в межклеточных адгезионных контактах на ранних этапах ЭМП не  препятствуют разрушению стабильной межклеточной адгезии и приобретению клетками миграционной активности.

Using DIC and confocal microscopy, changes in morphology, migratory characteristics and adherence junctions (AJs) were analyzed in the mammary carcinoma cell line MCF-7-SNAI1  after activation of the EMT transcription factor SNAI1. Western Blot analysis showed that  after removal of tetracycline from the cell culture medium expression of SNAI1 reached its  peak in 24 hours and then plateaued for 7 days. During the 7 days the cells continued to  express E-cadherin; however, tangential AJs typical for cells with stable cell-cell adhesion,  changed into radial AJs. The radial AJs continued to accumulate E-cadherin during 24‑72  hours after tetracycline removal. As a result of SNAI1 activation, the cells underwent  epithelial-mesenchymal transition (EMT) and became migratory. On a two-dimensional  substrate, cells exhibited both individual and collective migration. As the tetracycline  washout period progressed, the fraction of the cells capable of migrating through migration chamber membranes increased; on the contrary, cells’ ability to invade an epithelial  monolayer decreased. These results demonstrate that retaining a hybrid epithelial/mesenchymal  phenotype and accumulation of E-cadherin in AJs during early stages of EMT do not impede  disruption of stable cell-cell adhesion and cells’ acquisition of migratory activity.

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