Chlorogenic Acid Inhibits Human Glioma U373 Cell Progression via Regulating the SRC/MAPKs Signal Pathway: Based on Network Pharmacology Analysis

Abstract

Introduction: Chlorogenic acid (CGA) is a type of polyphenolic substance that is widely extracted from many traditional Chinese medicines (eg, Lonicera japonica Thunb, Eucommia ulmoides Oliver) and exhibits a wide range of anti-tumor effects. However, the potential molecular mechanisms of CGA in glioma U373 cells remain unclear.

Methods: Network pharmacology analysis was used to explore the potential therapeutic targets of CGA in glioma. Human glioma U373 cells were treated with different concentrations of CGA for 24 h. CCK-8 assays were used to detect the inhibitory rate of cell growth. Annexin V-FITC/PI staining and Hoechst 33342 staining were used to detect apoptosis. PI staining was used to investigate cell-cycle progression. Wound healing assays and transwell assays were used to detect the cell migration and invasion, respectively. Western blotting and immunohistochemistry were used to measure protein levels in vitro and in vivo.

Results: The proliferation of U373 cells was significantly inhibited by CGA in a dose- and time-dependent manner. CGA significantly arrested the cell cycle of U373 cells in the G2/M phase and induced apoptosis. Moreover, CGA significantly suppressed the migration and invasion of U373 cells. Additionally, we found that CGA inhibited the growth of U373 cells in vivo. Furthermore, network pharmacology analysis suggested that the anti-tumor effects of CGA on U373 cells were associated with the down-regulation of the SRC/MAPKs signaling pathway.

Discussion: The present study indicated that CGA had anti-glioma effects on U373 cells by down-regulating SRC/MAPKs signal pathway.

Keywords: SRC/MAPKs signal pathway; apoptosis; chlorogenic acid; migration and invasion; network pharmacology.

Figure 1

The schematic flowchart in the current study combined with a network pharmacology analysis and experimental validation in vitro and in vivo.

Figure 2

Preliminary screening of the candidate targets, PPI network-based cluster analysis and core targets prediction. (A) Venn diagrams of common targets in glioma and chlorogenic acid. As a result, 40 pharmacological potential targets were obtained. (B) Construction of PPI network in CGA in treating glioma by using STRING database. (C) Topological analysis of potential targets in CGA in treating glioma by using Network Analyzer. (D) Cluster analysis of PPI network to extract the core gene modules by using MCODE algorithm. (E) The top 15 core genes visualization obtained by using R software according to the relevance number of nodes.

Figure 3

GO and KEGG pathway analysis. (A) GO analysis of core targets and bubble plot for top 15 biological processes, top 15 cellular components, and top 15 molecular functions. (B) KEGG pathway analysis of core targets and column plot for top 15 pathways. The size of the nodes shows counts of targets, and the gradient of color represents the different adjusted p values.

Figure 4

CGA inhibited the proliferation of U373 cells in a dose- and time-dependent manner. (A–C) The IC50 of CGA on U373 cells for 24 hr, 48 hr, and 72 hr were 139.3 μM, 118.6 μM, and 107.3 μM, respectively. (D) The viability of U373 cells treated with different concentrations of chlorogenic acid for 24 hr, 48 hr, and 72 hr was detected by CCK-8 assays. (E) The inhibitory effect of chlorogenic acid on U373 cells proliferation detected by CCK-8 assays for 24 h, **P<0.01. (F) Cytotoxicity assay of chlorogenic acid on LO2 cells detected by CCK-8 assays for 24 h, ^#P<0.05, compared with 0 group. (G and H) Morphology and Hoechst 33342 staining of U373 cells treated with different concentrations of chlorogenic acid for 24 h, the nuclei of cells treated with CGA showed bright blue color and contained typical apoptotic bodies (red arrows), **P<0.01, compared with 0 group. Data were expressed as mean ± SD (n=6).

Figure 5

CGA inhibited the proliferation of U373 cells by inducing apoptosis and G2/M arrest. (A and C) CGA induced apoptosis in U373 cells. U373 cells were treated with different concentrations of CGA for 24 hr. Cells were stained with Annexin V-FITC/PI staining solution and detected by flow cytometry. *P<0.05, **P<0.01, ^##P<0.01, ^ΔΔP<0.01, both compared with 0 group. (B and D) Chlorogenic acid induces cell cycle arrest in G2/M phase in U373 cells. Cells were treated with different concentrations of chlorogenic acid for 24 h. Samples were stained with PI solution and analyzed by flow cytometry, **P<0.01, ^ΔP<0.05, ^ΔΔP<0.01, both compared with 0 group. Data were expressed as mean ± SD (n=3).

Figure 6

CGA inhibited the migration and invasion of U373 cells. (A and C) Wound healing assay. U373 cells after scratching were treated with different concentrations of chlorogenic acid for 24 hr. Migration ratio of U373 cells were measured by image J software, ^#P<0.05, ^##P<0.01, compared with 0 group. (B and D) Transwell invasion assay. U373 cells were treated as above. The number of invasive cells were measured by image J software, **P<0.01, compared with 0 group. Data were expressed as mean ± SD (n=3).

Figure 7

CGA downregulates the SRC/MAPKs signal pathway in U373 cells. (A and B) Immunofluorescence staining (200×) of SRC in U373 cells treated with or without 200 μM CGA for 24 hr, **P<0.01, compared with 0 group. (C and E) Cells were treated with CGA for 24 hr. The protein levels of MMP2, MMP9, E-cadherin, Vimentin were measured by Western blotting. β-actin was used as the protein loading control, *P<0.05, **P<0.01, both compared with 0 group. (D and G) U373 cells were treated as above. The protein levels of Bcl-2, Bax, cleaved caspase 3 and cleaved caspase 9 were measured by Western blotting. **P<0.01, both compared with 0 group. (F and H) U373 cells were treated as above. The protein levels of SRC/MAPKs signaling were measured by Western blotting. *P<0.05, **P<0.01, both compared with 0 group. The levels of p-SRC/SRC, p-MAPK1/MAPK1, p-MAPK8/MAPK8 were used to compare the levels of phospho-proteins, ^##P<0.01, both compared with 0 group. Data were expressed as mean ± SD (n=6).

Figure 8

CGA suppressed the malignant growth of U373 cells in vivo. (A and B) The mean weight of mice and the mean volume of tumors in the CGA groups were lower significantly than those in the normal saline group, ^##P<0.01, **P<0.01, compared with NS group. (C and E) TUNEL staining assay. With the increase of CGA’s concentrations, the apoptotic cells (green fluorescence) in tumor tissues increased significantly, **P<0.01, compared with NS group. (D and H) Hematoxylin-eosin (H&E) staining and Immunohistochemistry staining. Tumor cells became obvious atypia nuclei, poor differentiation, and apoptosis after treated with CGA. The expressions of Ki-67 and SRC decreased with the increase of CGA’s concentrations, **P<0.01, ^#P<0.05, ^##P<0.01, compared with NS group. (F and G) CGA inhibited the protein levels of SRC/MAPKs signaling in vivo. **P<0.01, ^#P<0.05, ^##P<0.01, both compared with NS group. Data were expressed as mean ± SD (n=3).