Integrating network pharmacology and experimental validation to uncover the synergistic effects of Huangqi ()-Ezhu () with 5-fluorouracil in colorectal cancer models

Abstract

Objective: To evaluate the effects of Huangqi (Radix Astragali Mongolici)-Ezhu (Rhizoma Curcumae Phaeocaulis) (HQEZ) on colorectal cancer therapies and to elucidate the potential mechanisms of HQEZ, especially in combination with 5-Fluorouracil (5-FU).

Methods: The anti-tumor effects of HQEZ were evaluated in colorectal cancer models both in vivo and in vitro. The network pharmacological assay was used to investigate potential mechanisms of HQEZ. Potential target genes were selected by Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, protein-protein interaction network (PPI) and molecular docking. Within key targets, potential targets related to drug sensitivity, especially the sensitivity to 5-FU, were evaluated in HCT116 in vitro by immunofluorescence, quantitative real-time polymerase chain reaction (qPCR) and Western-blot. Then, changes in potential targets were assessed in tumors from tumor-bearing mice and the expression of these targets was also evaluated in colorectal cancer (COAD) patients from the Cancer Genome Atlas Program (TCGA) database.

Results: HQEZ significantly enhanced the anti-tumor activity of 5-FU in vivo and inhibit the growth of HCT116 in vitro. By network pharmacological analysis, key targets, such as protein kinase B (AKT1), epidermal growth factor receptor (EGFR), adenosine triphosphate (ATP) binding cassette subfamily B member 1 (ABCB1, also named multidrug resistance protein 1, MDR1), ATP binding cassette subfamily G member 2 (ABCG2), thymidylate synthetase (TYMS, also named TS), prostaglandin-endoperoxide synthase 2 (PTGS2), matrix metallopeptidase 2 (MMP2), MMP9, toll like receptor 4 (TLR4), TLR9 and dihydropyrimidine dehydrogenase (DPYD), were identified. Additionally, 4 potential core active ingredients (Folate, Curcumin, quercetin and kaempferol) were identified to be important for the treatment of colorectal cancer with HQEZ. In key targets, chemoresistance related targets were validated to be affected by HQEZ. Furthermore, 5-FU sensitivity related targets, including MDR1, TS, EGFR, ribonucleotide reductase catalytic subunit M1, Breast and Ovarian Cancer Susceptibility Protein 1 (BRCA1) and mutl homolog 1 were also significantly reduced by HQEZ both in vitro and in vivo. Finally, these validated key targets and 5-FU sensitivity related targets were demonstrated to be up-regulated in COAD patients based on TCGA database.

Conclusion: HQEZ has synergistic effects on the anti-tumor activity of 5-FU in the treatment of colorectal cancer both in vivo and in vitro. The beneficial effect of HQEZ results from the inhibition of the drug sensitivity targets associated with 5-FU. The combination therapy of HQEZ with 5-FU or other chemotherapeutic drugs will also improve the anti-tumor efficacy of chemotherapy.

Keywords: Huangqi ()-Ezhu (); colorectal neoplasms; fluorouracil; network pharmacology; parasitic sensitivity tests.

Figure 1. HQEZ significantly increased sensitivity of colorectal cancer to 5-FU in vivo and in vitro

A: measure of tumor volume; B: increase of tumor volume (n = 5-7); C: at the end of the animal experiment, the tumors were collected for photograph. C1: tumors from mice of control group; C2: tumors from mice of 5-FU group; C3: tumors from HQEZ group, C4: tumors from combined group 5-FU combined with HQEZ group (n = 5-7). And the final weight of tumors from every group (C5). D: in EdU incorporation experiment, HQEZ significantly enhanced the inhibition of 5-FU on the growth of HCT116 by immunofluorescence staining. D1: EdU signaling from control group; D2: DAPI which stains the nucleus of control group; D3: merge image of D1 and D2; D4: EdU signaling from 5-FU (0.18 μg/mL, 48 h) treated group; D5: DAPI which stains the nucleus of 5-FU treated group; D6: merge image of D4 and D5, D7: EdU signaling from 5-FU (0.18 μg/mL, 48 h) combined with HQEZ (30 mg/mL, 48 h) group; D8: DAPI which stains the nucleus of 5-FU (0.18 μg/mL, 48 h) combined with HQEZ (30 mg/mL, 48 h) group; D9: merge image of D7 and D8 (n = 5, repeated 3 times); D10: the rate of EdU positive cells. HQEZ: Huangqi (Radix Astragali Mongolici)-Ezhu (Rhizoma Curcumae Phaeocaulis); 5-FU: 5-Fluorouracil; i.p.: intraperitoneal; p.o.: peros; EdU: 5-Ethynyl-2’- deoxyuridine. Control group (PBS, i.p. every two days and p.o. daily), 5-FU group (30 mg/kg every two days, i.p.), HQEZ group (10 g·kg^-1·d^-1, p.o.), combined group (5-FU, 30 mg/kg every two days, i.p.; HQEZ, 10 g·kg^-1·d^-1, p.o.). Data are presented as mean ± standard deviation. ^aP < 0.05 and ^bP < 0.001, compared with the Control group. ^cP < 0.001, compared with the 5-FU group, differences were evaluated by one-way analysis of variance, bar = 50 μm.

Figure 2. Administration of HQEZ inhibited hub targets and FOXO pathway in HCT116 in vitro

A: in CCK8 assay, HQEZ only significantly inhibited the proliferation of HCT116 in a relatively high concentration (n = 3). B: after 48 h, the influence of different concentration of HQEZ on the mRNA levels of target genes according to network pharmacology were evaluated by qPCR assay. B1: the influence of TYMS; B2: the influence of MDR1; B3: the influence of Bcl-2; B4: the influence of EGFR. C: after 48 hours, the protein levels of Hub targets from network pharmacology were evaluated by Western-blot assay and the bands of were showed. (n = 3). D: after 48 h, the protein levels of Hub targets from network pharmacology were evaluated by western-blot assay which were administrated with HQEZ dose-dependently. D1: the protein levels of BNIP3 were downregulated; D2: the protein levels of PEPCK were not significantly influenced; D3: the protein levels of CCND1 were not significantly influenced; D4: the protein levels of Catalase were significantly downregulated (n = 3). HCT116 cells were treated with different concentrations of HQEZ including 0, 5, 10, 20 and 40 mg/mL for 48 h. TYMS: thymidylate synthetase; MDR1: multidrug resistance protein 1; Bcl-2: B-cell lymphoma-2; EGFR: epidermal growth factor receptor; BNIP3: BCL2/Adenovirus E1B 19 kDa Protein-Interacting Protein 3; PEPCK: phosphoenolpyruvate carboxykinase 2; CCND1: Cyclin D1. Data are presented as mean ± standard deviation. ^aP < 0.05, ^bP < 0.01 and ^cP < 0.001, compared with the control group, differences were evaluated by one-way analysis of variance.

Figure 3. The combination therapy of HQEZ with 5-FU significantly down-regulated 5-FU resistance targets in HCT116 in vitro

A: the influence of 5-FU (0.18 μg/mL) combined with different concentration of HQEZ on the transcription level of target genes in HCT116 cells after 48 h by qPCR assay. A1: BRCA1; A2: MDR1; A3: TYMS; A4: ERCC1; A5: RRM1; A6: POLH; A7: co MLH1; (n = 3). B: HQEZ decreased expression of protein from 5-FU resistance-related genes by western blot assay (n = 3). C: by immunofluorescence staining, combined treatment of 5-FU (0.18 μg/mL) and different concentration of HQEZ significantly increased the inhibition of p-AKT in HCT116 cells after 48 h. (n = 3). C1: p-AKT signaling from control group; C2: p-AKT merged with DAPI signaling from control group; C3: p-AKT signaling from 5-FU group; C4: p-AKT merged with DAPI signaling from 5-FU group; C5: p-AKT signaling from 5-FU combined HQEZ group; C6: p-AKT merged with DAPI signaling from 5-FU combined HQEZ (5 mg/mL) group; C7: p-AKT signaling from 5-FU combined HQEZ (10 mg/mL) group; C8: p-AKT merged with DAPI signaling from 5-FU combined HQEZ (10 mg/mL) group; C9: p-AKT signaling from 5-FU combined HQEZ (20 mg/mL) group; C10: p-AKT merged with DAPI signaling from 5-FU combined HQEZ (20 mg/mL) group; C11: p-AKT signaling from 5-FU combined HQEZ (40 mg/mL) group; C12: p-AKT merged with DAPI signaling from 5-FU combined HQEZ (40 mg/mL) group. D: the immunofluorescence density of p-AKT of C. E: the downregulation of p-AKT was also confirmed by western-blot. The concentration of 5-FU is 0.18 μg/mL and the cells were treated for 48 h. HCT116 cells were treated with 5-FU (0.18 μg/mL) and different concentrations of HQEZ, including 0 mg/mL, 5 mg/mL, 10 mg/mL, 20 mg/mL and 40 mg/mL for 48 h. HQEZ: Huangqi (Radix Astragali Mongolici)-Ezhu (Rhizoma Curcumae Phaeocaulis); 5-FU: 5-Fluorouracil; BRCA1: Breast and Ovarian Cancer Susceptibility Protein 1; MDR1: multidrug resistance protein 1; TYMS: thymidylate synthetase; ERCC1: Excision Repair Cross-Complementation Group 1; RRM1: ribonucleotide reductase catalytic subunit M1; POLH: DNA Polymerase Eta; MLH1: MutL Homolog 1; EGFR: epidermal growth factor receptor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; TS: thymidylate synthetase; p-AKT: phospho- protein kinase B; DAPI: 4',6-Diamidino-2-phenylindole dilactate. Data are presented as mean ± standard deviation. ^aP < 0.05, ^bP < 0.01 and ^cP < 0.001, compared with the Control group, differences were evaluated by one-way analysis of variance, bar = 20 μm.

Figure 4. Combination administration of HQEZ inhibited drug resistance targets relating to 5-FU in vivo and important candidate targets are upregulated in colorectal cancer patients according to TCGA database

A: western-blot analysis of expression of genes in tumors from tumor-bearing mice after treatments; B: results of western-blot analysis of expression of genes in tumors from tumor-bearing mice after treatments; B1: MDR1 was downregulated in the combined group; B2: MLH1 was downregulated in the combined group; B3: EGFR was downregulated in the combined group; B4: RRM1 was downregulated in the combined group; B5: POLH was downregulated in the combined group; B6: p-AKT was downregulated in the combined group; B7: p53 was downregulated in the combined group; B8: Bcl-2 was downregulated in the combined group; B9: TYMS was downregulated in the combined group (n = 3). C: qPCR analysis of expression of genes in tumors from tumor-bearing mice after treatments; C1: Bcl-2 was downregulated in the combined group; C2: MDR1 was downregulated in the combined group; C3: EGFR was downregulated in the combined group; C4: RRM1 was downregulated in the combined group; C5: MLH1 was downregulated in the combined group; C6: POLH was downregulated in the combined group; C7: TYMS was downregulated in the combined group (n = 3). D: gene expressions in colorectal cancer patients according to TCGA database; D1: upregulated expressions of MDR1; D2: upregulated expressions of POLH; D3: upregulated expressions of TYMS; D4: upregulated expressions of RRM1; D5: upregulated expressions of MLH1. Mice were treated with phosphate-buffered saline (PBS, i.p. every two days and p.o. daily), 5-FU alone (30 mg/kg every two days, i.p. HQEZ alone (10 g·kg^-1·d^-1, p.o.) and 5-FU (30 mg/kg every two days, i.p.) combined with HQEZ (10 g·kg^-1·d^-1, p.o.). HQEZ: Huangqi (Radix Astragali Mongolici)-Ezhu (Rhizoma Curcumae Phaeocaulis); 5-FU: 5-Fluorouracil; MDR1: multidrug resistance protein 1; TYMS: thymidylate synthetase; RRM1: ribonucleotide reductase catalytic subunit M1; POLH: DNA Polymerase Eta; MLH1: MutL Homolog 1; p-AKT: phospho- protein kinase B; Bcl-2: B-cell lymphoma-2; p53: Tumor Suppressor P53. Data are presented as mean ± standard deviation. ^aP < 0.05, ^dP < 0.01 and ^eP < 0.001, compared with the control group. ^cP < 0.05 and ^bP < 0.01, compared with the 5-FU group. ^fP < 0.01 and ^gP < 0.001, compared with the normal group, differences were evaluated by one-way analysis of variance.