Traditional Chinese medicine Astragalus polysaccharide enhanced antitumor effects of the angiogenesis inhibitor apatinib in pancreatic cancer cells on proliferation, invasiveness, and apoptosis

Figures & data

Figure 1 Expression of VEGFR-2 in ASPC-1 and PANC-1 cells and APS enhanced the inhibitory effects of apatinib on cell proliferation.

Notes: Western blotting showed that ASPC-1 and PANC-1 expressed VEGFR-2 and that HUVEC cell line was used as the positive control for VEGFR-2 (A). MTS assay showed that either apatinib or APS reduced A490 value in a dose-dependent manner after 24 hours of treatment in ASPC-1 (B and C) and PANC-1 (E and F). A combination of 40 μM apatinib and 200 μg/mL APS showed stronger inhibition on cell proliferation compared with the single use of apatinib group in ASPC-1 (D) and PANC-1 (G). **P<0.01, ***P<0.001 versus control; ^##P<0.01, ^###P<0.001 versus 40 μM apatinib.
Abbreviations: APS, Astragalus polysaccharide; VEGFR-2, vascular endothelial growth factor receptor-2; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; HUVEC, human umbilical vein endothelial cell.

Figure 2 APS enhanced the inhibition of cell migration suppressed by apatinib.

Notes: Wound healing analysis of ASPC-1 (A and B) and PANC-1 (C and D) cell migration for 0 and 24 hours; cells were treated with control, 40 μM apatinib, 200 μg/mL APS, and 40 μM apatinib + 200 μg/mL APS. *P<0.05, ***P<0.001 versus control; ^###P<0.001versus apatinib. Original magnification ×40.
Abbreviation: APS, Astragalus polysaccharide.

Figure 3 APS enhanced the inhibition of cell invasion suppressed by apatinib.

Notes: Transwell analysis of ASPC-1 (A and B) and PANC-1 (C and D) cell invasion for 24 hours; cells were treated with control, 40 μM apatinib, 200 μg/mL APS, and 40 μM apatinib + 200 μg/mL APS. Original magnification ×100. (E) MMP-9 protein expression treated with different groups after 24 hours were determined by Western blotting. β-Actin was used as the internal control. **P<0.01, ***P<0.001 versus control; ^#P<0.05 versus apatinib.
Abbreviations: AP, apatinib; APS, Astragalus polysaccharide; MMP-9, matrix metalloproteinases-9.

Figure 4 APS increased apoptosis induced by apatinib in pancreatic cancer cells.

Notes: ASPC-1 (A and B) and PANC-1 (C and D) exposed to control, 40 μM apatinib, 200 μg/mL APS, and 40 μM apatinib + 200 μg/mL APS after 24 hours followed by Annexin V-FITC and PI staining, and apoptosis percentage was detected by flow cytometry. (E) Proapoptotic (Bax) and antiapoptotic (Bcl-2) proteins expression treated with 0 (control), AP (40 μM apatinib), APS (200 μg/mL) and AP + APS (40 μM apatinib + 200 μg/mL APS) after 24 hours were determined by Western blotting. β-Actin was used as the internal control. ***P<0.001 versus control; ^#P<0.05, ^###P<0.001 versus apatinib.
Abbreviations: AP, apatinib; APS, Astragalus polysaccharide; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Figure 5 APS increased the inhibition of p-AKT and p-ERK expression in AKT and ERK signaling pathway.

Notes: ASPC-1 and PANC-1 were treated with 0, AP (40 μM apatinib), APS (200 μg/mL), and AP + APS (40 μM apatinib + 200 μg/mL APS) for 24 hours. The proteins were assessed by Western blotting.
Abbreviations: AP, apatinib; APS, Astragalus polysaccharide; p-AKT, phosphorylated AKT; p-ERK, phosphorylated ERK; AKT, protein kinase B; ERK, extracellular signal-regulated kinase.

Figure 6 Apatinib and APS induced cellular autophagy.

Notes: ASPC-1 (A) and PANC-1 (B) were incubated with 0, 20, 40, and 60 μM apatinib and 0, 200, 400, and 600 μg/mL APS for 24 hours. (C) ASPC-1 and PANC-1 cells were treated with 0 (control), AP (40 μM apatinib), APS (200 μg/mL), and AP + APS (40 μM apatinib + 200 μg/mL APS) for 24 hours. The autophagy-related protein (LC3) from different groups was determined by Western blotting.
Abbreviations: AP, apatinib; APS, Astragalus polysaccharide; LC3, light chain 3.

Figure S1 Expression of VEGFR-2 in SW1990 cell line and the influence of apatinib and APS on cell proliferation.

Notes: Western blotting showed that VEGFR-2 was not expressed in SW1990, and HUVEC was used as the positive control for VEGFR-2 (A). Apatinib and APS inhibited cell proliferation in SW1990. MTS assay showed that apatinib (B) and APS (C) reduced A490 value at 60 μM and 600 μg/mL, respectively. The combination of 40 μM apatinib and 400 μg/mL APS showed inhibition on cell proliferation significantly compared with the single use of apatinib group in SW1990 (D). *P<0.05, ^#P<0.05 versus 40 μM apatinib.
Abbreviations: APS, Astragalus polysaccharide; VEGFR-2, vascular endothelial growth factor receptor-2; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt.

Figure S2 Apatinib inhibited cell proliferation more significantly after adding rhVEGF-165 in ASPC-1 and PANC-1.

Notes: MTS assay showed that apatinib reduced A490 value at a lower concentration after adding rhVEGF-165 (20 ng/mL) after 24 hours of treatment in ASPC-1 (A) and PANC-1 (B). APS showed that the same concentration compared with the treatment without rhVEGF-165 in ASPC-1 (C) and PANC-1 (D). A combination of 20 μM Apatinib and 200 μg/mL APS showed stronger inhibition on cell proliferation compared with the single use of apatinib group in ASPC-1 (E) and PANC-1 (F). *P<0.05, **P<0.01, ***P<0.001 versus control; ^#P<0.05, ^##P<0.01 versus 20 μM apatinib.
Abbreviations: APS, Astragalus polysaccharide; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt.