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ВЫВОДЫ
1.Получена линия мышиных эмбриональных фибробластов, нокаутных по гену Nf2, моделирующая нейрофиброматоз II типа.
2.Сравнение данной линии с исходной (контрольной) показало, что полученные Nf2-отрицательные клетки отличаются от контрольных по морфологии (имеются признаки дезорганизации актинового цитоскелета, отсутствуют плотные клеточные контакты), большей скоростью роста и устойчивостью к неблагоприятным воздействиям среды.
3.На полученных клеточных линиях проведён скрининг коллекции 486 низкомолекулярный биоактивных соединений, который выявил 9 веществ, обладающих селективностью в отношении Nf2-отрицательных клеток.
• Из отобранных по результатам скрининга 9 соединений-кандидатов 2
– ингибитор 3-гидрокси-3-метилглутарил-коА-редуктазы ловастатин и ингибитор синтазы жирных кислот церуленин – прошли первичную валидацию результатов скрининга, проявив стабильную селективность в отношении различных Nf2-отрицательных клеток. Селективность оказалась также характерна для фармакологических аналогов данных соединений (гиполипидемических препаратов группы статинов и ингибиторов синтазы жирных кислот лютеолина и С75), а реэкспрессия белка мерлин в Nf2-отрицательных клетках приводила к обращению токсичности исследуемых соединений. При изучении механизмов действия данных веществ селективная цитотоксичность в отношении Nf2-отрицательных клеток была также установлена для ингибитора геранилтрансфераз золедроновой кислоты.
4.Исследование механизмов действия соединений, выбранных по результатам скрининга, показало, что селективная цитотоксичность ловастатина в отношении Nf2-отрицательных клеток обусловлена раъякориванием малой ГТФазы Rac1 и отсоединением её от клеточной мембраны. Под действием
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ловастатина Rac1 транслоцируется в ядро и актвируется в Nf2- отрицательных клетках, вызывая клеточную гибель. Селективная цитотоксичность церуленина обусловлена различиями в уровнях метаболизма жирных кислот в Nf2-отрицательных и нормальных клетках. Было показано, что под влиянием церуленина накопление промежуточных продуктов синтеза насыщенных жирных кислот в Nf2-отрицательных клетках происходит интенсивнее в 1,5 – 2,5 раза по сравнению с контрольными клетками. Также, в Nf2-отрицательных клетках уровень экспрессии ферментов, участвующих в липогенезе, повышен на 50 – 200% по сравнению с контрольными клетками.
5.Ингибитор 3-гидрокси-3-метилглутарил-коА-редуктазы ловастатин, ингибитор геранилтрансферазы I золедроновая кислота и ингибитор синтазы жирных кислот церуленин обладают выраженной противоопухолевой активностью в испытаниях in vivo на мышах с ксенографтной трансплантацией мышиных и крысиных шванном. Все три соединения снижали скорость роста шванном на 80% по сравнению с контрольной группой.
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ПРАКТИЧЕСКИЕ РЕКОМЕНДАЦИИ
Материалы диссертационной работы по изучению метаболического профиля Nf2-отрицательных опухолевых клеток могут быть использованы в разработке методов фармакотерапии для лечения НФ II. Описанные в данной работе изменения уровней синтеза жирных кислот, характерные для опухолей, ассоциированных с НФ II, позволяют применить новый подход – воздействие на участвующие в данных метаболических путях ферменты – к терапии данного заболевания и могут способствовать дальнейшему изучению приложения ингибиторов синтазы жирных кислот в терапии доброкачественных опухолей нервной системы. Препараты золедроновой кислоты могут быть рекомендованы для дальнейших исследований возможности их использования в качестве противоопухолевых средств для лечения Rac-зависимых форм рака (метастазирующий рак молочной железы, немелкоклеточный рак лёгкого и др.)
БЛАГОДАРНОСТИ
Автор благодарит научного руководителя Николая Львовича Шимановского (РНИМУ им. Пирогова) и научного консультанта Джонатана Чернова (ФоксЧейзовский Онкологический Центр) за помощь в организации и планировании работы, сотрудников Фокс-Чейзовского Онкологического Центра: Маргрет Эйнарсон (управление скрининговым роботом), Йан Жу (статистическая обработка результатов скрининга), Йин-Минг Куо (управление хроматографом с масс-спектрометрией) и Галину Семёнову (помощь при измерении опухолей в ксенографтной модели); а также Марко Джованнини (Институт Гематологии, Париж) за предоставленные клетки и клеточные линии.
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