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. 2018 May 9;11:2685-2698.
doi: 10.2147/OTT.S157129. eCollection 2018.

Traditional Chinese Medicine Astragalus Polysaccharide Enhanced Antitumor Effects of the Angiogenesis Inhibitor Apatinib in Pancreatic Cancer Cells on Proliferation, Invasiveness, and Apoptosis

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Free PMC article

Traditional Chinese Medicine Astragalus Polysaccharide Enhanced Antitumor Effects of the Angiogenesis Inhibitor Apatinib in Pancreatic Cancer Cells on Proliferation, Invasiveness, and Apoptosis

Jun Wu et al. Onco Targets Ther. .
Free PMC article

Abstract

Background: Traditional chemotherapy and molecular targeted therapy have shown modest effects on the survival of patients with pancreatic cancer. The current study aimed to investigate the antitumor effects of apatinib, Astragalus polysaccharide (APS), and the combination of both the drugs in pancreatic cancer cells and further explore the molecular mechanisms in vitro.

Materials and methods: Expression of vascular endothelial growth factor receptor-2 (VEGFR-2) in human pancreatic cancer cell lines ASPC-1, PANC-1, and SW1990 was detected by Western blotting. Cell proliferation was measured by MTS, and migration and invasion were detected by wound-healing and Transwell assays, respectively. Cell apoptosis rate was determined by flow cytometry and cellular autophagy level affected by apatinib, and APS was analyzed by Western blotting.

Results: Human pancreatic cancer cell lines ASPC-1 and PANC-1 expressed VEGFR-2, but VEGFR-2 was not detected in SW1990. Either apatinib or APS inhibited cell proliferation in a dose-dependent manner in ASPC-1 and PANC-1. APS in combination with apatinib showed enhanced inhibitory effects on cell migration and invasion compared with apatinib monotherapy in ASPC-1 and PANC-1. Meanwhile, APS combined with apatinib strongly increased cell apoptosis percentage. Western blotting showed that the combination of APS and apatinib significantly enhanced the downregulation of phosphorylated protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) (p-AKT and p-ERK) as well as matrix metalloproteinases-9 (MMP-9) expression. In addition, both apatinib and APS induced cellular autophagy. However, the expression of autophagy-related proteins was not further elevated in the combination group.

Conclusion: The study first demonstrated that apatinib showed potentially inhibitory effects in pancreatic cancer cells and that APS enhanced the antitumor effects of apatinib through further downregulating the expression of phosphorylation of AKT and ERK as well as MMP-9.

Keywords: AKT; ERK; anti-angiogenesis drug; basic medical research; digestive tumor; molecular mechanisms; molecular targeted therapy.

Conflict of interest statement

Disclosure The authors report no conflicts of interest in this work.

Figures

Figure 1
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
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
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
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
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
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.

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