Impact and mechanism study of dioscin on biological characteristics of colorectal cancer cells

Figure 1 Effect on dioscin on HCT116 activity. Dioscin at different doses (0, 1.25, 2.5, and 5 μg/mL) was applied to HCT116 cells for 48 hours. The cell counting kit-8 kit was used to measure the dioscin inhibition rate. Three biological repeats were used and reported as mean ± SD. ^aP < 0.01, and ^bP < 0.001 compare to the control group.

Figure 2 Effect of dioscin on HCT116 cell apoptosis. A: Annexin V-FITC and PI staining was utilized to assess apoptosis in various concentrations (0, 1.25, 2.5 and 5 μg/mL) of dioscin-treated HCT116 cells after 48 hours by using flow cytometry; B: Quantification of apoptosis in HCT116 cells. Three biological repeats were reported as mean ± SD. ^aP < 0.01, ^bP < 0.001 compare to the control group.

Figure 3 Effect of dioscin on apoptotic gene and protein expression in HCT116 cells. HCT116 cells were exposed to varying concentrations of dioscin (1.25, 2.5 and 5 μg/mL) for 48 hours. A: RT-qPCR study of Bax/Bcl-2 ratio as well as Caspase-3 mRNA expression; B: Western blot examination of the expression of the proteins Caspase-3, Bcl-2, and Bax; C: Quantification of Caspase-3, Bcl-2, and Bax protein expression levels from the Western blot results. Three biological repeats were reported in mean ± SD format. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 compare to the control group.

Figure 4 Effect of dioscin on HCT116 cells’ invasion (40 ×). A: Transwell method was used to explore the relationship between diosgenin and HCT116 cell invasion and clarify the inhibitory effect of the former on the latter; B: Quantification of HCT116 cell invasion after diosgenin treatment using the Transwell assay. Three biological repeats were used and reported as mean ± SD. ^aP < 0.05, ^bP < 0.01 compare to the control group.

Figure 5 Effect of dioscin on HCT116 cells’ migration (100 ×). A: Wound healing assay was conducted to explore the relationship between diosgenin and HCT116 cell migration and clarify the inhibitory effect of the former on the latter; B: Quantification of HCT116 cell migration following diosgenin treatment using the wound healing assay. Three biological repeats were used and reported as mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 compare to the control group.

Figure 6 Effect of dioscin on HCT116 cells' expression of Notch 1 signaling pathway-related proteins. A: Cells were treated with dioscin for 48 hours. Western blot analysis was conducted to determine protein levels, with GAPDH as control; B: Quantification of protein expression levels from the Western blot results. Three biological repeats were used and reported as mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 compare to the control group.

Figure 7 Notch 1 is involved in the inhibitory effect of dioscin on HCT116. HCT116 cells were treated with dioscin (5 μg/mL), Jagged 1 (5 μg/mL), dioscin (2.5 μg/mL) + Jagged 1 (5 μg/mL) for 48 hours. A: Apoptosis in HCT116 cells was revealed using flow cytometry as well as annexin V-FITC as well as PI staining methods; B: Quantification of apoptosis in HCT116 cells from flow cytometry results; C: RT qPCR analysis was performed using caspase-3 mRNA expression and Bax/Bcl-2 ratio; D: Expression levels of Bax, Bcl-2, as well as other proteins were detected using Western blot analysis; E: Quantification of Bax, Bcl-2, and other protein expression levels from the Western blot analysis; F: Transwell method was used to detect the level of inhibition of HCT116 cell invasion; G: Quantification of HCT116 cell invasion inhibition following dioscin treatment using the Transwell assay; H: Wound healing assay was used to detect the inhibition of HCT116 cell migration; I: Quantification of HCT116 cell migration inhibition following treatment using the wound healing assay. Three biological repeats were used and reported as mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 compare to the control group, ^dP < 0.05, ^eP < 0.01, ^fP < 0.01 compare to the dioscin group.

Figure 8 Notch 1 participates in the inhibitory effect of dioscin on the Notch 1/Hes 1 pathway. A: Western blot analysis was adopted to dig up protein expression levels, with GADPH as the control; B: Quantification of protein expression levels from Western blot analysis. Three biological repeats were used and reported as mean ± SD. ^aP < 0.05, ^bP < 0.01 compare to the control group, ^cP < 0.05 compare to the dioscin group.