Network Pharmacology Analysis and Experimental Pharmacology Study Explore the Mechanism of Gambogic Acid against Endometrial Cancer

Abstract

Endometrial cancer (EC) is one of the three most common gynecological cancers in female groups. Gambogic acid (GA), a natural caged xanthone, exerts significantly antitumor effects on many cancers. However, its efficacy on EC and pharmacological mechanism of action remain marginal up to now. This study suggested that GA had significant inhibitory effects on EC in vitro and in vivo, and no toxicity to normal cells or mice. In detail, GA suppressed cell proliferation, induced cell apoptosis, and cell cycle arrest at G₀/G₁ stage, complied with the network pharmacology analysis, showed that the PI3K/Akt pathways were the most important signaling, and their protein and mRNA expression levels were confirmed by qRT-PCR and Western blot experiments. In all, our study first proved that GA could inhibit cell proliferation, induce cell apoptosis, and cell cycle arrest at G₀/G₁ stage via the PI3K/Akt pathways, so GA would be a good therapy for EC.

Figure 1

GA effectively suppresses cell viability in endometrial cells. (A) Chemical structure of gambogic acid. (B, C) Inhibitory action of GA on Ishikawa cells. (D) Inhibitory action of GA on HEC-1B cells. Data are presented as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, vs the control group.

Figure 2

GA induced the morphological changes of human EC cells Ishikawa and ECC-1 (100×, scale bar = 50 μm).

Figure 3

Network pharmacology of the inhibition of EC by GA. (A) Venn map of endometrial cancer-related genes and GA-target genes. (B) “Endometrial cancer targets–GA” network.

Figure 4

KEGG enrichment analysis of potential targets of GA. The top 20 with lower p-value are shown.

Figure 5

GA induced apoptosis in ECC-1 cells. (A) Apoptosis of ECC-1 cells treated with GA for 24 h, which was examined by Annexin V-FITC/PI staining. (B) Percentage of cell apoptosis. (C) Relative mRNA level of apoptosis-related genes treated with GA for 24 h, which was examined by qRT-PCR. Error bars represent mean ± SD. *p < 0.05, **p < 0.01, vs control (no GA).

Figure 6

GA mediated the cell cycle arrest in G₀/G₁ of ECC-1 cells. (A) Results showed the percent of cell population in G₀/G₁, S, and G₂/M phases of the cell cycle analyzed using FACScan. (B) Rates of the cell cycle in different stages. (C) mRNA expression levels of the cell cycle of the involved genes were measured by RT-PCR. Error bars represent mean ± SD. *p < 0.05, **p < 0.01, vs control (no GA).

Figure 7

Relative expression level of PI3K/AKT pathway-related genes in GA-treated ECC-1 cells. (A) Protein expression profile and (B) expression levels of related proteins. (C) mRNA expression profile. (D) Effects of AKT inhibitor (inhibitor III) and activator (SC79) on GA-induced apoptosis in ECC-1 cells. ± SD is the mean value for the data (n = 3). *p < 0.05, **p < 0.01, vs control (no GA).

Figure 8

GA inhibited the growth of ECC-1 cell xenografts in nude mice. (A) Images of tumors at the end of experiments. (B) Average tumor weight in mice. (C) Average tumor volume in mice. (D) Bodyweight changes of mice. Error bars represent mean ± SD. * p < 0.05, **p < 0.01 vs control. n.s., no significant.