Identification of bioactive anti-angiogenic components targeting tumor endothelial cells in Shenmai injection using multidimensional pharmacokinetics

Figures

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  Figure 1. Effect of SMI on tumor vessels in LoVo colon cancer xenograft mice. (A) Schematic plans of animal experiments for synergic anti-tumor effect study. Mice were intraperitoneally injected with SMI (10 mL/kg/day) and 5FU (15 mg/kg/3 days) for 14 days. (B) Images and volumes of tumors dissected from mice with 14-day treatment (n = 6). (C) Schematic plans of animal experiments for anti-angiogenic effect study. Mice were intraperitoneally injected with SMI for 15 days. (D) Morphologies of tumor vessels captured using multi-photon laser scanning microscopy (Zeiss) after 13 days of SMI treatment. Disordered vessels and branch points are indicated by white and yellow arrows, respectively. The color represents the depth of the vessel (depth = 180–250 μm). Statistical quantification of microvessels (<10 μm in vessel diameter) and branches were in the right side of the graph (n = 3–4). (E) Maximum intensity projection of the Z-positions with CD31 staining of tumor vascular endothelial cells. Disordered vessels and branch points are indicated by white and yellow arrows, respectively. Statistical quantification of CD31+ microvessels (<10 μm in vessel diameter) and vessel branches were shown in…

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  Figure 2. Concentration–time profiles of ginsenosides in plasma after single or multiple administration of SMI to Balb/c nude mice harboring LoVo xenografts. (A) and (B) Mice were intraperitoneally injected with SMI (10 mL/kg/day) once a day for one day (A) or for eight days (B). Ginsenosides in plasma were detected at 5, 10, 20 40 min, 1, 2, 4, 8, 12, 24, 48 and 96 h after the last SMI administration (n = 5 mice at each time point). (C) AUC0–∞ of ginsenosides in plasma after single or multiple SMI injection. The ratio between multiple and single administration was calculated to evaluate the accumulation of different ginsenosides in plasma after multiple SMI administration. Data were expressed as mean ± SEM. AUC: area under curve.

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  Figure 3. Concentration–time profiles of ginsenosides in tumors after single or multiple administration of SMI to Balb/c nude mice harboring LoVo xenograft. (A) and (B) Mice were intraperitoneally injected with SMI (10 mL/kg/day) once a day for one day (A) or for eight days (B). Ginsenosides in tumors were detected at 20 min, 2, 12, 24 and 96 h after the last SMI administration (n = 5 mice at each time point). (C) AUC0–∞ of ginsenosides in tumors after single or multiple SMI injection. The ratio between multiple and single administration was calculated to evaluate the accumulation of different ginsenosides in tumors after multiple SMI injections. Data were expressed as mean ± SEM.

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  Figure 4. The concentration of ginsenosides in tumor endothelial and non-endothelial cells. (A) Identification of TECs sorted by magnetic bead using Flow-cytometry. (B) Concentration of ginsenosides in tumor endothelial or non-endothelial cells at 20 min after 15-day SMI treatment (n = 3). Data were expressed as mean ± SEM. TECs: tumor endothelial cells; THCs: tumor hematopoietic cells; OCs: other cells; ND, not detected.

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  Figure 5. The accumulation of ginsenosides in plasma, tumor tissues, and endothelial cells after multiple administration of SMI to Balb/c nude mice harboring LoVo xenograft. (A) The constituent ratio–time profiles of ginsenosides in plasma and tumors after single or multiple (8-day) administration of SMI (10 mL/kg/day). (B) The constituent ratio of ginsenosides in SMI, plasma, tumor, and endothelial cell measured 20 min after 15-day SMI treatment. The ratio changes of different types of ginsenosides are separated by red dotted lines in the graph. Data were expressed as mean ± SEM. PPD: protopanaxadiol-type ginsenosides; PPT: protopanaxatriol-type ginsenosides.

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  Figure 6. Effect of Rd on tumor vessels in Balb/c mice harboring LoVo xenografts. Mice were intraperitoneally injected with SMI, Rd, Rb1, Rg1, or vehicle for 15 days. Maximum intensity projection of the Z-positions with CD31 staining of tumor vascular endothelial cells (n = 3–4; thickness = 100 μm; scale bar = 100 μm). (B) and (C) Statistical quantification of CD31+ microvessels (<10 μm in vessel diameter, B) and vessel branches (C) in (A). (D) Double staining of CD31 (red) and α-SMA (green) in tumors revealing pericyte coverage around tumor vessels (n = 4; thickness = 12 μm; scale bar = 100 μm). (E) Statistical quantification of pericyte coverage based on calculations of the α-SMA+ fractions in (D). (F) and (G) The secretion level of VEGF (F) and FGF (G) in tumors (n = 6–7). Data were expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle group.

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  Figure 7. Effect of Rd on angiogenesis in vitro. (A) HUVECs were treated with SMI Rd for 24 h under normoxia or hypoxia conditions. Cell viability was tested by MTT assays (n = 6). (B) and (C) Rd has no significant effect on HUVEC proliferation in hypoxia condition. HUVECs were treated with SMI or Rd for 24 h followed by EDU/HOECHST staining. (B) Representative images of EDU and HOECHST. Scale bar, 200 μm. (C) Ratio of EDU/HOECHST (n = 5). (D) and (E) Rd represses the migration of HUVECs under hypoxia condition. HUVECs were scratched by pipette tips and treated with SMI or Rd for 24 h. (D) Representative images of wound healing at 0 and 24 h. Scale bar = 1 mm. (E) Quantification of migration length (n = 6). (F) and (G) Rd suppresses tube formation of HUVECs under hypoxia condition. HUVECs were pre-treated with SMI or Rd for 24 h and seeded on Matrigel for 5.5 h followed by 0.5 h of calcein staining. (F) Representative images of tubes. Scale bar = 1 mm. (G) Quantification of tubes in (D) (n = 4). Data were expressed as mean ± SEM. **P < 0.01, ***P < 0.001 vs. normoxia + DMSO group, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. hypoxia + DMSO group.

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  Figure 8. Influence of Rd on the anti-tumor effect of chemotherapy in vivo. Schematic plans of animal experiments. Mice were intraperitoneally injected with SMI (10 mL/kg/day), Rd (5 mg/kg/day) or 5FU (15 mg/kg/3 days) for 20 days. (B) and (C) SMI and Rd enhance the inhibition effect of 5FU on tumor growth. (B) Images of tumors resected from mice after 20-day treatment. (C) Tumor volumes after 20-day treatment. (D) Changes in mice body weight after 17-day treatment. (E) Concentration of 5FU in tumor tissues after 20-day treatment. Data were expressed as mean ± SEM. *P < 0.05, **P < 0.01 vs. vehicle + saline group, #P < 0.05, ##P < 0.01 vs. vehicle+5FU group, n = 6–7.