Fig. 1.

Growth inhibitory activity of Nintedanib and Regorafenib toward CRC cells. (A) The growth inhibitory activity of Nintedanib (up right) and Regorafenib (up left) toward CRC cells was determined by the MTT viability assay after 120 h continuous drug exposure. The corresponding IC₅₀ values for each cell line for both drugs are shown in the tables directly below the viability curves. All values are averages of at least 3 independent experiments each done in duplicate. (B) The IC₅₀ values for Nintedanib were plotted against the IC₅₀ values for Regorafenib for each cell line. There was no statistically significant correlation between the 2 parameters (r² = 0.29). (C, D) HT-29 and LoVo cells were exposed to the IC₅₀ dose of Nintedanib or Regorafenib for the indicated times and the cell cycle distribution was determined by flow cytometry analysis. The values indicate the average values for 3 experiments. (E, F) HT-29 and LoVo cells were exposed to the IC₅₀ dose of Nintedanib and Regorafenib for the indicated times, and the protein levels of the cell-cycle regulators p27Kip1 and p21Waf1/Cip1 were determined by Western blot analysis. The protein levels of β-actin are shown as the loading control. The numbers reflect the expression of the indicated cell cycle regulator in drug-exposed cells compared to the corresponding untreated control. (G, H) HT-29 and LoVo cells were exposed to the IC₅₀ dose of Nintedanib and Regorafenib for the indicated times, and the fraction of fragmented DNA present in the sub-G1 fraction was determined by flow cytometry analysis. All data are expressed as a mean ± SD (n = 3). Data were analyzed by two-way ANOVA test and were compared to the corresponding untreated control; ****p < 0.0001; ***p < 0.001; *p < 0.01. ns < 0.05 vs. non treated.