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. 2021 Apr 21;11:8626. doi: 10.1038/s41598-021-88133-7

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Tiliroside targeted CAXII enzyme as U-104 did and reduced its quantity and activity. Tiliroside also modulated the expression of E2F1, E2F3 whose relative expressions were higher in Hep3B and SNU-449 than in THLE-3. CAXII was predicted to be the potential targets of Tiliroside by Swiss target prediction tool (A). In both 2D and 3D culture systems, Tiliroside has significantly decreased the relative concentration of CAXII compared to NC group in both Hep3B, SNU-449 and THLE-3 at 24 h, 48 h and 72 h respectively (B). Dose-dependent inhibition of CAXII esterase activity by Tiliroside showed the IC50s were 47.54 ± 3.6 μM and 34.67 ± 2.7 μM for Hep3B and SNU-449, respectively (C), and that for U-104 were 6.3 ± 3.6 μM in Hep3B and 4.78 ± 3.6 μM in SNU-449 (D) (The concentration showed x axis stands for 0.5, 1.5, 5,10,20,80 μM, respectively). The relative expression levels of E2F1 and E2F3 were significantly higher in Hep3B and SNU-449 compared to that in THLE-3 (E). The relative expression levels of E2F1 (F) and E2F3 (G) in Hep3B, SNU-449 and THLE-3 cells were significantly downregulated by 40 μM Tiliroside in both 2D and 3D culture systems. Data are presented as the mean ± standard deviation (SD); NS not significant, NC negative control.