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Introduction There is increasing evidence that cancer stem cells (CSCs) could mediate resistance to chemo- and radiotherapy, metastasis and recurrence in a wide range of solid tumours. Notch signalling pathway is involved in CSCs maintenance. Notch influences intra- and intercellular communications of various cell types in embryogenesis and adult organs. The oncogenic role of Notch signalling in survival, proliferation, self-renewal, differentiation and migration of cancer cells is demonstrated in most solid tumours and leukaemia. This study aims to explore the effect of Notch1 suppression on CSCs in human lung (A549) and colorectal (HCT116) adenocarcinoma cell lines. Material and methods Knockdown of Notch1 by shRNA was confirmed by PCR and western-blot analyses. PCR was used for estimation of stemness, epithelia-to-mesenchyma transition (EMT) markers and proto-oncogene c-MYC expression. The proportion of CD133+ cells in different subpopulations was identified by Flow cytometry. Cell migration potential was examined by Boyden chamber assay. The effect of Notch1 inhibition on CSCs was established using classical detection methods of CSCs in heterogeneous cancer cell cultures: tumourisphere formation, tumourigenicity in vivo and quantitative assessment of ABC-transporter activity assays. Results and discussions Notch1 inhibition resulted in stemness (SOX2, OCT3/4, NES), EMT (TWIST1, HES1, SNAIL1), CSCs (CD133) markers, c-MYC expression decrease and CDH1 (E-cadherin) expression increase suggesting that Notch pathway is involved in EMT. Loss of Notch1 did not influence cell proliferation in vitro but reduced migration potential. It was confirmed that Notch signalling interruption leads to reduction of stemness features: decreased drug resistance (A549 but not HCT116), ability to form tumourispheres and tumourigenic potential in vivo. ABC-transporter activity in HCT116 could be upregulated by TAZ/YAP signalling pathway and not by Notch. Moreover, reduced Notch1 expression increased the minimum inoculum cell dose for xenograft formation. The xenograft vascularisation assay revealed that Notch1 inhibition is not necessary for neovascularizaton but it is essential for angiogenesis but in tumour. Conclusion The Notch signalling pathway is important for maintaining the CSCs population, stemness and migration so Notch1 could be considered as a potential therapeutic target in lung and colorectal cancer. The study was supported Russian Science Foundation (RSCF), grant No. 14-15-00467. |
