Ginsenoside Rg3 enhances the anticancer effect of 5‑FU in colon cancer cells via the PI3K/AKT pathway

Abstract

Chemotherapy is one of the most commonly used treatments for patients with advanced colon cancer, yet the toxicity of chemotherapy agents, such as 5‑fluorouracil (5‑FU), limits the effectiveness of chemotherapy. Ginsenoside Rg3 (Rg3) is an active ingredient isolated from ginseng. Rg3 has been shown to display anticancer effects on a variety of malignancies. Yet, whether Rg3 synergizes the effect of 5‑FU to inhibit the growth of human colon cancer remains unknown. The present study was designed to ascertain whether Rg3 is able to enhance the anti‑colon cancer effect of 5‑FU. The results revealed that combined treatment of Rg3 and 5‑FU significantly enhanced the inhibition of the proliferation, colony formation, invasion and migration of human colon cancer cells (SW620 and LOVO) in vitro. We also found that combined treatment of Rg3 and 5‑FU significantly enhanced the apoptosis of colon cancer cells by activating the Apaf1/caspase 9/caspase 3 pathway and arrested the cell cycle of the colon cancer cells in G0/G1 by promoting the expression of Cyclin D1, CDK2 and CDK4. In addition, the PI3K/AKT signaling pathway in colon cancer cells was suppressed by Rg3 and 5‑FU. In vivo, Rg3 synergized the effect of 5‑FU to inhibit the growth of human colon cancer xenografts in nude mice. Similarly, combined treatment of Rg3 and 5‑FU altered the expression of colon cancer protein in vivo and in vitro. Collectively, the present study demonstrated that ginsenoside Rg3 enhances the anticancer effect of 5‑FU in colon cancer cells via the PI3K/AKT pathway.

Keywords: ginsenoside Rg3; colon cancer; 5-fluorouracil; PI3K/AKT.

Figure 1.

Effect of the combined treatment of Rg3 and 5-FU on proliferation of colon cancer cells in vitro. SW620 and LOVO cells were treated with different doses of (A) Rg3 (mmol/l) or (B) 5-FU (µmol/l), and then the MTT assay was used to detect cell viability. (C and D) After treatment with Rg3 (1 mmol/l) or 5-FU (50 µmol/l) or their combination, the colony formation of the colon cancer cells was photographed. WT group was used as a baseline for cell viability and cell colony formation. Three independent repetitions were performed for each experiment. *P<0.05, **P<0.01 and ***P<0.001, compared with the WT group. 5-FU, 5-fluorouracil; Rg3, ginsenoside Rg3.

Figure 2.

Effect of the combined treatment of Rg3 and 5-FU on migration and invasion of colon cancer cells in vitro. Treatment of colon cancer cells with combined treatment of Rg3 (1 mmol/l) and 5-FU (50 µmol/l) inhibited the migration (A) and invasion (B) abilities of the SW620 and LOVO cells. Scale bar, 100 µm. (C) Western blot analysis was used to detect the expression of EMT-related protein (N-cadherin, E-cadherin and MMP-9). The Solvent group was used as a baseline for the migration and invasion of cells. Three independent repetitions for each experiment were performed. *P<0.05, **P<0.01 and ***P<0.001 compared with the Rg3+5-FU group. 5-FU, 5-fluorouracil; Rg3, ginsenoside Rg3; EMT, epithelial-mesenchymal transition; MMP, matrix metalloproteinase.

Figure 3.

Effect of the combined treatment of Rg3 and 5-FU on the apoptosis of colon cancer cells in vitro. (A) The percentage of apoptotic SW620 and LOVO cells in the different groups. (B) Apoptosis-related proteins [(cleaved (Cl)-caspase 9, Cl-caspase 3 and Apaf-1] were assessed by western blot analysis in SW620 and LOVO cells. Three independent repetitions for each experiment were carried out. ***P<0.001, compared with the Rg3+5-FU group. 5-FU, 5-fluorouracil; Rg3, ginsenoside Rg3; Apaf-1, Apoptotic protease activating factor 1.

Figure 4.

Effect of the combined treatment of Rg3 and 5-FU on cell cycle progression of colon cancer cells in vitro. (A) Flow cytometry was used to analysis the cell cycle in colon cancer cells after treatment with Rg3 (1 mmol/l) or 5-FU (50 µmol/l) or the combination. (B) Cell cycle-associated protein (cyclin D1, CDK2 and CDK4) were assessed by western blot analysis. Three independent repetitions were performed for each experiment. *P<0.05, **P<0.01 and ***P<0.001, compared with the Rg3+5-FU group. 5-FU, 5-fluorouracil; Rg3, ginsenoside Rg3; CDK, cyclin-dependent kinase.

Figure 5.

Effect of the combined treatment of Rg3 and 5-FU on PI3K/AKT signaling in colon cancer cells in vitro. (A and B) Western blot analysis was used to detect the expression of key proteins in the PI3K/AKT signaling pathway after treatment with Rg3 (1 mmol/l) or 5-FU (50 µmol/l) or the combination. Three independent repetitions were performed for each experiment. *P<0.05, **P<0.01 and ***P<0.001, compared with the Rg3+5-FU group. 5-FU, 5-fluorouracil; Rg3, ginsenoside Rg3.

Figure 6.

Effects of the combined treatment of Rg3 and 5-FU on tumor growth and protein expression of colon cancer cells in vivo. After 3 weeks of treatment, the mice were sacrificed, tumor tissues were excised, and the weight (A) and volume (B) of tumor tissues were measured. (C-E) Total protein was extracted from the colon cancer tumor tissues, and the expression of proteins was detected by western blot analysis. Five nude mice in each group, and at least 3 tumor tissues were used to evaluate protein expression., *P<0.05, **P<0.01 and ***P<0.001, compared with the Rg3+5-FU group. 5-FU, 5-fluorouracil; Rg3, ginsenoside Rg3.