Xiaotan Sanjie decoction attenuates tumor angiogenesis by manipulating Notch-1-regulated proliferation of gastric cancer stem-like cells

Figure 1 High-performance liquid chromatography-DAD of the samples. The samples were randomly tested ten times, and the absorbance was read at 280 nm.

Figure 2 Results of Western blot 48 h after FACS sorting, indicating significant differences in Notch-1, Hes1, VEGF and Ki-67 expression between CD44⁺ and CD44^- cells.

Figure 3 Xiaotan Sanjie decoction inhibits cell viability in a dose-dependent manner. A, B: The effect of Xiaotan Sanjie (XTSJ) decoction on the viability of CD44⁺ and CD44^- cells. The cells were treated with XTSJ decoction for 24, 48 or 72 h. Cell viability was determined by MTT assay. Dots: Mean of six independent experiments; bars: SD; C, D: Results of the Western blot for the expression of Notch-1, Hes1, VEGF, and Ki-67 in the cells incubated with XTSJ decoction for 48 h; E, F: The statistical analysis of the expression of Notch-1, Hes1, VEGF and Ki-67. The densitometry analysis results of the displayed bands were normalized to β-actin; the data are expressed as arbitrary units (one-way ANOVA, followed by the Bonferroni test). ^aP < 0.05 vs CD44⁺ group.

Figure 4 Xiaotan Sanjie decoction inhibits tumor weight and angiogenesis in a dose-dependent manner. A: Tumor weight after eight consecutive weeks of treatment (n = 6 in each group). Control CD44⁺ group had larger tumors than control CD44- group (Student’s t-test). Treatment with Xiaotan Sanjie (XTSJ) decoction inhibited tumor growth in a dose-dependent manner in gastric cancer stem-like cell transplanted mice (one-way ANOVA, followed by the Bonferroni test); B: Comparison of the microvessel density (MVD) in different groups. The control CD44⁺ mice showed higher MVD than the control CD44- mice (n = 6, P < 0.05), and XTSJ decoction reduced the MVD in a dose-dependent manner. Statistically significant differences in MVD could be detected in the medium- and high-dose CD44⁺ groups (Nonparametric tests); C: Immunohistochemical (IHC) results of the MVD. The MVD was defined as a discrete CD34-positive endothelial cell aggregate, with or without definable lumina. (original magnification × 400, positive areas are indicated by black arrows). ^aP < 0.05 vs CD44⁺ group; ^cP < 0.05 vs CD44^- group.

Figure 5 Xiaotan Sanjie decoction inhibits CD44 expression in a dose-dependent manner. A: Immunohistochemical (IHC) staining for CD44. CD44 was highly expressed in the cancer cell membrane (original magnification × 400, positive areas are indicated by black arrows); B: The comparison of CD44 in different groups. CD44 was highly expressed in the control CD44⁺ group compared with the rest of the groups and was rarely detected in the control CD44- group. The results also showed that not all of the cells in the CD44⁺ group were CD44 positive. Xiaotan Sanjie decoction inhibited the expression of CD44 in a dose-dependent manner. Statistically significant differences in the expression of CD44 were also detected in the medium- and high-dose groups in CD44⁺ cells (nonparametric tests).

Figure 6 Western blot and real-time quantitative polymerase chain reaction analysis of Notch-1, Hes1, VEGF and Ki-67 expression in vivo. A, B: Western blot revealed that Notch-1 and Hes1 were highly expressed in the control CD44⁺ group compared with the control CD44- group. Xiaotan Sanjie decoction inhibited the expression of Notch-1 and Hes1 in a dose-dependent manner, and significant differences could be detected in the medium- and high-dose groups (P < 0.05); C: The variation in Notch-1, Hes1, VEGF and Ki-67 expression in the experimental groups was confirmed by quantitative polymerase chain reaction (one-way ANOVA, followed by the Bonferroni test). ^aP < 0.05 vs CD44⁺ group.