Gambogenic acid induces cell growth inhibition, cell cycle arrest and metastasis inhibition in choroidal melanoma in a dose-dependent manner

Abstract

The aim of the present study was to explore the effects of gambogenic acid (GNA) on the malignant behaviors of choroidal melanoma cells, including cell viability, cell cycle, migration and invasion, and to elucidate the underlying regulatory mechanism. The human choroidal melanoma cell line OCM-1 was treated with different concentrations of GNA and cell viability, colony formation ability, cell cycle, migration and invasion were analyzed. Additionally, cells were incubated with or without LY294002, a specific inhibitor of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, for 24 h. Levels of cell cycle-associated proteins (cyclin D1, cyclin E, cyclin-dependent kinase 2 and P21), epithelial-mesenchymal transition (EMT)-associated molecules (epithelial-cadherin, α-smooth muscle actin and vimentin) and phosphorylated (p)-AKT/AKT were determined using reverse transcription-quantitative polymerase chain reaction and western blot analysis. The results demonstrated that GNA significantly inhibited cell viability and induced cell cycle arrest at the G0/G1 phase in a dose-dependent manner (P<0.01). Furthermore, GNA administration significantly suppressed cell migration and invasion in a dose-dependent manner (P<0.01). Treatment with GNA or LY294002 induced a marked decrease in the expression of p-AKT/AKT, a significant downregulation in cell cycle-associated molecules (P<0.01), and a significant decrease in cell viability (P<0.01). Co-treatment with LY294002 and GNA had an additive effect on the growth of OCM-1 cells. In conclusion, the results of the present study suggest that treatment with GNA may inhibit cell viability and induce G0/G1 arrest. Furthermore, GNA may also inhibit cell metastasis via regulating EMT-associated molecules. The PI3K/Akt signaling pathway may be a key mechanism involved in the progression of choroidal melanoma, and GNA may serve as a potential therapeutic reagent for the treatment of this disease.

Keywords: cell cycle arrest; choroidal melanoma; gambogenic acid; growth; metastasis.

Figure 1.

Effects of GNA on (A) cell viability and (B) colony formation ability. Data are presented as the mean ± standard deviation. **P<0.01 vs. control group. GNA, gambogenic acid.

Figure 2.

Effects of GNA on cell cycle. (A) Flow cytometry displayed the effects of GNA on cell cycle. (B) Cell cycle distribution. (C) Western blot analysis of cyclin D1, cyclin E, CDK2 and P21 proteins expressions. (D) Reverse transcription-quantitative polymerase chain reaction results for cyclin D1, cyclin E, CDK2 and P21 mRNA expressions. Data are presented as the mean ± standard deviation. *P<0.05; **P<0.01 vs. control group. GNA, gambogenic acid; CDK, cyclin dependent kinase.

Figure 3.

Effects of GNA and LY294002, a specific inhibitor of the PI3K/Akt signaling pathway, on cell growth. (A) The protein expression of p-AKT/AKT. (B) The expression of cell cycle-associated molecules (cyclin D1, cyclin E, CDK2 and P21) (C) MTT assay of cell viability. Data are presented as the mean ± standard deviation. **P<0.01 vs. control group. GNA, gambogenic acid; LY, LY294002; p, phosphorylated; PI3K, phosphoinositide 3-kinase; AKT, protein kinase B; CDK, cyclin dependent kinase.

Figure 4.

Effects of GNA on cell migration and invasion. (A) Wound healing assay of cell migration following treatment with different concentrations of GNA; (B) Transwell assay of invasive ability. Data are presented as the mean ± standard deviation **P<0.01 vs. control group. GNA, gambogenic acid.

Figure 5.

Effects of GNA on the mRNA expression of (A) E-cadherin, (B) α-SMA and (C) vimentin by reverse transcription-quantitative polymerase chain reaction analysis, and their (D) protein expression by western blot analysis. Data are presented as the mean ± standard deviation. **P<0.01 vs. control group. GNA, gambogenic acid; SMA, smooth muscle actin; E-cadherin, epithelial cadherin.