Ginsenoside Rh1 inhibits colorectal cancer cell migration and invasion in vitro and tumor growth in vivo

Abstract

Colorectal cancer (CRC) is the third leading cause of cancer-associated mortality worldwide. Ginsenoside Rh1 (Rh1) is a traditional medicine monomer with antitumor activity; however, the effects of Rh1 in CRC remain to be determined. In the present study, SW620 cells were treated with different concentrations of Rh1. Cell Counting Kit-8, wound healing and Transwell assays were performed to measure cell viability and proliferation, migration and invasion, respectively. Subsequently, the mRNA expression levels of matrix metallopeptidase (MMP)1, MMP3 and tissue inhibitor of metalloproteinases 3 (TIMP3) were detected by reverse transcription-quantitative PCR analysis. In addition, the protein expression levels of MMP1, MMP3, TIMP3, and total or phosphorylated (p-)ERK1/2, P38, JNK were detected by western blotting. Furthermore, tumor growth was examined in a nude mouse xenograft model. The results of the present study indicated that Rh1 was not toxic to CRC cells at various concentrations (0, 50 or 100 µM) and treatment durations (24 or 48 h). However, cell proliferation was suppressed by Rh1 in a dose-dependent manner. Rh1 (100 µM) significantly inhibited cell migration and invasion in vitro. Additionally, Rh1 suppressed the mRNA and protein expression of MMP1 and MMP3, and promoted TIMP3 expression. Rh1 decreased the ratios of p-P38/P38, p-ERK1/2/ERK1-2 and p-JNK/JNK in vitro and in vivo, which suggested that Rh1 inactivated the mitogen-activated protein kinase (MAPK) signaling pathway. Notably, Rh1 markedly decreased tumor volume and weight in vivo. In conclusion, the present study demonstrated that Rh1 inhibited the proliferation, migration and invasion of CRC cells in vitro and tumor growth in vivo. This inhibition was at least partially due to the inhibition of MMP1 and MMP3 expression, the increase in TIMP3 expression level and the MAPK signaling pathway inactivation. Therefore, Rh1 may effectively inhibit the development of CRC as an anticancer drug, and may have a supporting effect during CRC treatment.

Keywords: colorectal cancer; ginsenoside Rh1; invasion; migration; tumor growth.

Figure 1.

Rh1 has no toxic effect but inhibits proliferation in colorectal cancer cells. (A) SW620 cells were treated with 0, 50 or 100 µM Rh1 for 24 or 48 h, and cell viability was subsequently measured using a CCK-8 assay. (B) Rh1 (0, 50 or 100 µM) was used to treat SW620 cells for 0, 12, 24 and 48 h, and a CCK-8 assay was performed to detect cell proliferation. Data are presented as the means ± SEM. **P<0.01 and ***P<0.001 vs. control group. CCK-8, Cell Counting kit-8; OD, optical density; Rh1, ginsenoside Rh1.

Figure 2.

Rh1 reduces the migration and invasion capabilities of colorectal cancer cells. (A) Following treatment of SW620 cells with 0 or 100 µM Rh1 for 24 h, cell migration was assessed using a wound healing assay. The percentage of wound width was calculated at 24 h compared with 0 h. (B) SW620 cells were treated with 0 or 100 µM Rh1, and cell invasion was assessed using a Transwell assay. The invasive cells were imaged and counted. Data are presented as the means ± SEM. **P<0.01 vs. control group. Rh1, ginsenoside Rh1.

Figure 3.

Rh1 decreases the mRNA and protein expression levels of MMP1 and MMP3, enhances TIMP3 expression and inhibits the phosphorylation of ERK1/2, P38 and JNK. mRNA expression levels of (A) MMP1, (B) MMP3 and (C) TIMP3 were detected using reverse transcription-quantitative PCR following exposure of SW620 cells to 0 or 100 µM Rh1 for 24 h. (D) Protein expression levels of MMP1 and MMP3 were measured using western blotting. GAPDH was used as an internal control. (E) Fold change of MMP1 and MMP3 protein levels was normalized using GAPDH. (F) Protein expression levels of TIMP3 were detected by western blot analysis. GAPDH was used for normalization. (G) Semi-quantified TIMP3 protein expression. (H) Protein expression levels of P38, p-P38, ERK1/2, p-ERK1/2, JNK and p-JNK were assessed using western blotting. GAPDH was used as an internal control. (I) Relative protein expression levels of p-P38, p-ERK1/2 and p-JNK were compared with their total amount. Data are presented as the means ± SEM. **P<0.01 vs. control group. MMP, matrix metallopeptidase; p-, phosphorylated-; Rh1, ginsenoside Rh1; TIMP3, tissue inhibitor of metalloproteinases 3.

Figure 4.

Rh1 inhibits tumor growth and the mitogen-activated protein kinase signaling pathway in vivo. (A) Tumor volumes were determined every week following Rh1 treatment. (B) Tumor weights were measured when the tumors were dissected. (C) Tumors in control and Rh1 groups were photographed following resection. (D) Protein expression levels of p-P38, P38, p-ERK1/2, ERK1/2, p-JNK and JNK were determined by western blotting. GAPDH was used for normalization. (E) Analysis of western blotting. Data are presented as the means ± SEM. **P<0.01 vs. control group. p-, phosphorylated-; Rh1, ginsenoside Rh1.