Figure 3.

Effect of OPE and polymethoxylated flavones (PMFs) in HT29 cell spheroid proliferation. (A) Antiproliferative effect of OPE (24 and 72 h) and the mixture of four PMFs (tested in the same concentration as in the extract—72 h). Data are mean ± SD of four independent experiments performed with six replicates. (B) Relative mRNA expression of the apoptosis marker BIRC5 in HT29 cell spheroids treated with OPE (1.00 mg/mL, ~EC₅₀), the mixture of four PMFs and the isolated compounds (nobiletin—49.11 μM, sinensetin—46.62 μM, tangeretin—10.41 μM, and scutellarein tetramethylether—31.55 μM) for 72 h. Results were normalized relative to the control (untreated spheroids) and expressed in mean ± SD of two independent experiments. (C) Relative mRNA expression of cell cycle markers (CDKN1A, CCNA2) in HT29 cell spheroids treated with OPE (1.00 mg/mL), the mixture of four PMFs and the isolated compounds (nobiletin—49.11 μM, sinensetin—46.62 μM, tangeretin—10.41 μM, and scutellarein tetramethylether—31.55 μM) for 72 h. Results were normalized relative to the control (untreated spheroids) and expressed in mean ± SD of at least two independent experiments. Statistically significant differences were calculated according to one-way ANOVA for multiple comparisons by Tukey’s method (a P ≤ 0.05, b P ≤ 0.01, c P ≤ 0.001, d P ≤ 0.0001). Legend: N—nobiletin; S—sinensetin; T—tangeretin; Sc—scutellarein tetramethylether.