Олигонуклеотидные ингибиторы Dnmt1: проникновение и ингибирование роста клеток HeLa и CaSki

The use of Dnmt1 inhibitors allows reactivation of tumor suppressor genes which leads to tumor regression. Currently used drugs have potent toxic and mutagenic effect despite their high effectiveness. Thus, the development of Dnmt1 direct inhibitors with antitumor activity and moderate effect on normal cells remains relevant. Recently we have designed and synthesized competitive oligodeoxyribonucleotide inhibitors of human Dnmt1 demonstrating their ability to inhibit DNA methylation reaction in vitro. The aim of our work was to study the influence of the best of Dnmt1 inhibitors on the cervix carcinoma cells growth. We used cell lines HeLa, CaSki and L-68 from SRC VB "Vector" (Russia) collection. Oligonucleotides were transfected using Lipofectamine 2000 "Invitrogen" (USA) according to the manufacturer''s instructions. The localization of fluorescent labeled oligonucleotides in the cells was evaluated by fluorescence microscopy. Toxic effect was evaluated by spectrophotometry. TC 50values (50% cytotoxic concentration) were calculated from the number of living cells depending on the inhibitor concentration in the medium. We studied fluorescent labeled oligonucleotides localization with oligonucleotides which are similar to inhibitors but have no Dmnt1 inhibition properties. After transfection the oligonucleotides are localized in cell nuclei, and there is no apparent luminescence reduction within 48 hours. This was achieved due to phosphates tophosphothioates replacement that allows a long-term presence of the inhibitor in the cell nucleus. The combination of structural features, such as C: A non-complementary and "pin" had a cumulative effect. Thus, the best TC 50 values were obtained from inhibitors that possess all of these modifications. The presence of unpaired nucleotides in the double-stranded DNA recognition site greatly enhances the ability to inhibit the methylation reaction. Despite the fact that both investigated cervical carcinomas are associated with human papillomavirus (Hela with HPV-18, Caski with HPV-16), the effect of inhibitors occurs with different efficiency: TC 50 values ranged 236-408 nM for HeLa cell line and 118170 nM for CaSki cell line. At the same time, TC 50 value for fibroblast cell line L-68 exceeded 10 ^M for all tested inhibitors which makes them promising for a further study as antitumor drugs.