The human CRC cell line SW620 was purchased from the Type Culture Collection of the Chinese Academy of Sciences. Cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 1% streptomycin and 1% penicillin (all from Invitrogen; Thermo Fisher Scientific, Inc.). The cultured cells were incubated at 37°C with 5% CO₂.

Ginsenoside Rh1 (purity >97%) was purchased from Chengdu Must Bio-Technology Co., Ltd. (cat. no. A0240). The effects of Rh1 on SW620 cell viability and proliferation were determined by Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.) assays. Briefly, SW620 cells in the logarithmic growth phase were seeded into 96-well plates at a density of 2×10³ cells/well. Next, for examining cell viability, cells were treated with Rh1 at concentrations of 0, 50 or 100 µM at 37°C for 24 or 48 h, and cells were treated with 0, 50 or 100 µM Rh1 at 37°C for 0, 12, 24 and 48 h to examine cell proliferation. Untreated cell served as the control group. Then, 10 µl CCK-8 reagent was added to each well and cells were incubated at 37°C with 5% CO₂ for another 2 h. The absorbance of each well was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, Inc.).

Cell migration was determined by a wound healing test. Cells (5×10⁵ cells/well) were seeded into 6-well plates and incubated at 37°C until >90% confluence was reached. Cell monolayers were carefully scratched using a 10-µl sterile plastic pipette tip to make an artificial wound. The floating cell debris was rinsed twice with PBS. Subsequently, fresh serum-free RPMI-1640 medium containing different concentrations of Rh1 (0 or 100 µM) was added. Images of the wound area were captured at 0 and 24 h under an inverted light microscope (magnification, ×200; Olympus Corporation).

Transwell chambers (pore size, 8-µm; Corning Inc.) were used to examine the cell invasion capacity. The chamber filter was pre-coated with 100 µl diluted Matrigel (BD Biosciences). Cell suspensions (200 µl; 2×10⁵ cells in serum-free RPMI-1640 medium containing 0 or 100 µM Rh1) were added to the upper chamber. The bottom chamber was filled with complete RPMI-1640 medium containing 10% FBS. Following incubation at 37°C with 5% CO₂ for 24 h, the remaining cells on the top surface of the filter were removed. The invasive cells on the lower surface of the filter were fixed in 4% paraformaldehyde for 10 min and stained in 0.1% crystal violet for 15 min both at room temperature. The invasive cells were imaged and counted using a light microscope (Olympus Corporation) at ×200 magnification in five random fields.

SW620 cells (2×10⁵) in the logarithmic growth phase were seeded in 6-well plates until 90% confluence was reached. Subsequently, they were treated with 0 or 100 µM Rh1 at 37°C for 24 h. Total RNA from cells was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA synthesis was performed using a PrimeScript™ RT reagent kit with gDNA Eraser (Takara Biotechnology Co., Ltd.). RT reaction conditions were as follows: 37°C for 15 min followed by 85°C for 5 sec. qPCR was performed using SYBR Premix Ex Taq II (Tli RNaseH Plus; Takara Biotechnology Co., Ltd.), according to manufacturer's protocol, on an ABI PRISM 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The PCR conditions were as follows: Initial denaturation at 95°C for 30 sec, followed by 40 cycles of denaturation at 95°C for 5 sec and annealing/extension at 60°C for 34 sec. The melting curve was analyzed at the end of amplification. The fold change of each gene was calculated using the 2^−ΔΔCq method (23) and normalization to GAPDH. The primers were synthesized by GenScript, and the sequences used were: Matrix metallopeptidase (MMP)1 forward, 5′-AGGACTCCAAGGTAGACACAC-3′ and reverse, 5′-TTGCCGTTCTTGTAGGTGAACGC-3′; MMP3 forward, 5′-CCTGCTTTGTCCTTTGATGC-3′, and reverse, 5′-TGAGTCAATCCCTGGAAAGTC-3′; tissue inhibitor of metalloproteinases 3 (TIMP3) forward, 5′-AGTTACCCAGCCCTATGA-3′ and reverse, 5′-GCAAAGGCTTAAACATCT-3′; and GAPDH forward, 5′-GAAGGTGAAGGTCGGAGTC-3′ and reverse, 5′-GAAGATGGTGATGGGATTTC-3′.

SW620 cells (2×10⁵) treated with 0 or 100 µM Rh1 for 24 h as aforementioned were harvested and lysed in RIPA buffer (Beyotime Institute of Biotechnology). Protein concentration was determined by the bicinchoninic acid (BCA) method using a BCA Protein Assay kit (Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's protocol. A total of 30 µg of protein was separated via 10% SDS-PAGE. Following electrophoresis, proteins were transferred onto PVDF membranes (Thermo Fisher Scientific, Inc.). Membranes were blocked in TBS with 0.05% Tween 20 (Beijing Solarbio Science & Technology Co., Ltd.) containing 5% skimmed milk at room temperature for 1 h, and subsequently incubated with primary antibodies at 4°C overnight. The primary antibodies (all from Abcam) included: Anti-MMP1 (cat. no. ab38929; 1:5,000), anti-MMP3 (cat. no. ab53015; 1:1,000), anti-ERK1+ERK2 (cat. no. ab17942; 1:1,000), anti-phosphorylated (p-)ERK1 + ERK2 (p-T202 + T204; cat. no. ab214362; 1:1,000), anti-P38 (cat. no. ab197348; 1:1,000), anti-p-P38 (p-T180; cat. no. ab178867; 1:1,000 dilution), anti-JNK1+JNK2 (cat. no. ab112501; 1:1,000), anti-p-JNK1 + JNK2 (p-T183 + T185; cat. no. ab4821; 1:1,000), anti-TIMP3 (cat. no. ab39184; 1:1,000) and anti-GAPDH (cat. no. ab181602; 1:10,000). Next, the membranes were incubated with secondary antibody conjugated to horseradish peroxidase (goat anti-rabbit immunoglobulin G; 1:2,000 dilution; cat. no. ab205718; Abcam) for 1 h at room temperature. The immunopositive bands were visualized using a Pierce ECL Western Blotting kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. Band intensity was determined with the ImageQuant^™ LAS 4010 biomolecular imager (GE Healthcare Life Sciences) and normalized to GAPDH.

A total of 6 adult male BALB/c nude mice (4–6 weeks; 20–22 g) were selected for the present study. Nude mice were purchased from Shanghai Experimental Animal Center of the Chinese Academy of Sciences (Shanghai, China). The mice were housed individually in a specific pathogen-free environment with free access to water and food under 26–28°C, 40–60% humidity and 10 h light/14 h dark conditions. The experimental procedures were approved by the Institutional Animal Care and Use Committee of Lanzhou University Second Hospital (Lanzhou, China). Logarithmic growth phase SW620 cells (3×10⁶) were suspended in 250 µl PBS and Matrigel (1:1 ratio; BD Biosciences) and injected subcutaneously into the dorsal right flank of mice. Mice were randomly divided into control and Rh1 groups (3 mice/group). Mice in the control group were orally administered with 2 ml distilled water/day, while mice in Rh1 group were orally administrated with 20 mg/kg/day Rh1, dissolved in 2 ml distilled water, for 35 consecutive days. Tumor volumes were measured once per week. At day 35, mice were sacrificed by decapitation and the tumors were dissected at 12 h after the last drug delivery. The tumor length and width were measured with a caliper, and the volume was calculated using the formula: Volume=0.5 × (length × width)². Images of the tumors in each group were captured and the tumors were weighed.

Statistical analysis was performed using GraphPad Prism version 7 software (GraphPad Software, Inc.). Data are expressed as the means ± SEM of at least three independent experiments. The differences between two groups were analyzed using a Student's t-test, and the differences among multiple groups were assessed using one-way ANOVA followed by a Newman Keuls post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the effects of Rh1 on cell viability and proliferation, SW620 cells were treated with various concentrations (0, 50 or 100 µM) of Rh1, and CCK-8 assays were performed. As shown in Fig. 1A, 50 and 100 µM Rh1 did not influence cell viability in the treatment groups, compared with in the control group. Furthermore, Rh1 treatment for 24 or 48 h did not significantly influence cell viability for the same Rh1 concentrations. These results indicated that Rh1 did not exert significant cytotoxicity on SW620 cells at 0–100 µM for 24 or 48 h. However, 50 and 100 µM Rh1 significantly inhibited cell proliferation (50 µM, P<0.01; 100 µM, P<0.001) in the treatment group compared with in the control group (Fig. 1B). The nontoxic concentration of 100 µM was selected for subsequent experimentation.

To evaluate the effects of Rh1 on cell migration and invasion, SW620 cells were treated with 0 or 100 µM Rh1 for 24 h. The wound healing assay demonstrated that Rh1 significantly inhibited wound closure. The percentage of the wound area in the Rh1 group was significantly greater than that of the control group (P<0.01; Fig. 2A). Similarly, the Transwell invasion assay demonstrated that Rh1 treatment significantly decreased the number of invasive cells, compared with the control group (P<0.01; Fig. 2B). These data suggested that Rh1 effectively inhibited the migration and invasion of CRC cells in vitro.

The expression levels of MMP1, MMP3 and TIMP3 were measured in CRC cells via RT-qPCR and western blot analyses, respectively. The results revealed that Rh1 treatment markedly decreased the mRNA expression levels of MMP1 and MMP3, and increased TIMP3 mRNA expression, compared with the control group (P<0.01; Fig. 3A-C). Consistently, the protein expression levels of MMP1 and MMP3 were significantly decreased, and TIMP3 expression was increased in the Rh1 group (P<0.01; Fig. 3D-G). Additionally, as presented in Fig. 3H and I, it was demonstrated that Rh1 significantly decreased the ratios of p-P38/P38, p-ERK1/2/ERK1-2 and p-JNK/JNK compared with the control group (P<0.01). These results suggested that Rh1 inhibited MAPK signaling.

To further investigate the antitumor effects of Rh1, a xenograft model of nude mice was established with SW620 cells. Rh1 (0 or 20 mg/kg) was orally administered to treat the tumors in mice, and tumor growth was determined. The tumor volume was significantly decreased in the Rh1 group compared with in the control group after 35 days (P<0.01; Fig. 4A). When the tumors were harvested, the tumor weights were measured. Rh1 treatment significantly decreased the tumor weight, compared with the control group (P<0.01; Fig. 4B). Additionally, tumor size was markedly decreased in the Rh1 group compared with that in the control group (Fig. 4C). These results suggested that Rh1 inhibited tumor growth in vivo. Additionally, whether Rh1 regulates the MAPK signaling pathway in vivo remains unclear. The ratios of p-P38/P38, p-ERK1/2/ERK1-2 and p-JNK/JNK were significantly downregulated in Rh1 treated mice tumor tissues compared with in tissues from the control group (P<0.01; Fig. 4D and E).