CRC cell lines DLD-1 and LoVo, and the human intestinal epithelial cell line NCM460, were obtained from the Fuheng Cell Center (Shanghai, China). NCM460 and DLD-1 cells were cultured in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Inc.). LoVo cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.). All media contained 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 mg/ml streptomycin. All cells were cultured in a standard humidified incubator at 37°C with a 5% CO₂ atmosphere. Aladdin Industrial Corporation supplied the CVB-D (cat. no. C117989). CVB-D was dissolved in methanol to produce a 71 mmol/l stock solution.

CVB-D cytotoxicity was determined in CRC and NCM460 cells via MTT assay. DLD-1, LoVo and NCM460 cells were seeded into 96-well plates (5×10³ per well) and cultured at 37°C for 24 h until the cells adhered to the plates. Different doses of CVB-D (0, 10, 15, 20, 25, 30, 35, 40, 45 and 50 µM) were added to the plates and cultured at 37°C for 24 or 48 h. MTT solution (5 mg/ml; 20 µl/well; Sigma-Aldrich, Merck KGaA) was then added and the plates were incubated at 37°C for 4 h. DMSO (150 µl/well) was subsequently added to dissolve the formazan crystals. Absorbance at a wavelength of 490 nm was detected using a Microporous Plate automatic regulator (ELx808; BioTek Instruments). The formula used for calculating the cell proliferation inhibition rate was as follows: Inhibition rate (%)=[1-A490 (test)/A490 (blank)] ×100%. Three biologically independent experiments were conducted.

CRC cells were seeded in a 6-cm diameter dish (1,000 cells/dish) and incubated for 24 h. CRC cells were then incubated with different doses of CVB-D (0, 20, 30 and 40 µM) at 37°C for 5 h. The CVB-D was then removed and replaced with normal culture medium. After 14 days, the cells were fixed in methanol at room temperature for 15 min and stained with 0.5% crystal violet at room temperature for 10 min. Three biologically independent experiments were performed.

Adherent CRC cells were treated with CVB-D (30 µM) at 37°C for 48 h, then harvested and fixed in 75% ethanol at 4°C overnight. The cells were digested and stained using RNase and propidium iodide (4A Biotech), respectively. The cells were stained at 37°C for 30 min in the dark. The cell cycle was analyzed using a flow cytometer. ModFit LT V5.0 software (Verity Software House, Inc.) was used for analysis. Three biologically independent experiments were conducted.

CRC cells were attached to a six-well plate (2.5×10⁵ cells/well) and incubated overnight. Cells were then treated with different doses of CVB-D (0, 20, 30 and 40 µM) at 37°C for 48 h, and then harvested and stained using an Annexin V-FITC/PI Apoptosis Detection kit (cat. no. FXP018; 4A Biotech) according to the manufacturer's protocol. A flow cytometer was used to analyze apoptosis followed by analysis with FACS DiVa 6.1.3 software (BD Biosciences). Three biologically independent experiments were conducted.

The 90% confluent monolayer of serum-starved CRC cells was scraped with a 200-µl pipette tip and then treated with CVB-D (0, 10 and 15 µM) at 37°C for 24 h. Subsequently, photographs of the wound widths were obtained at different time periods (0, 24 and 48 h) using a light microscope (magnification, ×100). The migration distances were analyzed by ImageJ V1.8.0 software (National Institutes of Health). Three biologically independent experiments were conducted.

Cell migration and invasion experiments were conducted using Transwell chambers (BD Biosciences). CRC cells were cultured in six-well plates with different doses of CVB-D (0, 10 and 15 µM) at 37°C for 24 h. Culture medium (500 µl) containing 10% FBS was added to the lower chamber. Pretreated CRC cells were resuspended in serum-free medium (10,000 cells/200 µl medium) and injected into the upper chamber. Unlike migration analysis, 50 µl Matrigel (BD Biosciences) was used to cover the upper side of the polycarbonate membrane for the invasion assay. Following incubation for 48 h, migrated or invaded cells were fixed in methanol at room temperature for 15 min. Cells on the polycarbonate membrane were wiped off, and cells under the polycarbonate membrane were stained with 0.5% crystal violet solution at room temperature for 20 min. Under the light microscope, five fields (magnification, ×100) were randomly selected, and the mean number of cells was calculated and used for statistical analysis. Three biologically independent experiments were conducted.

CRC cells were treated with different concentrations of CVB-D (0, 20, 30 and 40 µM) at 37°C for 48 h, and then lysed with RIPA buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) containing protease and phosphatase inhibitors. Following 10 min of centrifugation at 12,000 x g and 4°C, the supernatants were transferred to new test tubes. A Bradford Protein assay kit (cat. no. P0012S) was used to measure the protein concentration. Proteins (25 µg/lane) were separated by 10% SDS-PAGE (Beyotime Institute of Biotechnology) and then eletrotransferred onto polyvinylidene difluoride membranes (EMD Millipore). Membranes were blocked with 5% non-fat milk/TBS-Tween-20 for 1 h at room temperature and then incubated with the corresponding primary antibodies overnight at 4°C. The antibodies used were as follows: CTHRC1 rabbit polyclonal antibody (1:1,000; cat. no. 16534-1-AP; Proteintech Group Inc.), phosphorylated (p)-AKT (Ser473) rabbit polyclonal antibody (1:1,000; cat. no. 4060S; Cell Signaling Technology, Inc.), p-ERK (Thr202/Tyr204) rabbit polyclonal antibody (1:1,000; cat. no. 4370T; Cell Signaling Technology, Inc.), E-cadherin rabbit polyclonal antibody (1:1,000; cat. no. 20874-1-AP; Proteintech Group Inc.), N-cadherin rabbit polyclonal antibody (1:1,000; cat. no. 22018-1-AP; Proteintech Group Inc.), vimentin rabbit polyclonal antibody (1:1,000; cat. no. 10366-1-AP; Proteintech Group Inc.), matrix metalloproteinase (MMP)2 rabbit polyclonal antibody (1:1,000; cat. no. 10373-2-AP; Proteintech Group Inc.), MMP9 rabbit polyclonal antibody (1:1,000; cat. no. 10375-2-AP; Proteintech Group Inc.), CD133 rabbit polyclonal antibody (1:1,000; cat. no. 66666-1-Ig; Proteintech Group Inc.), cyclin D1 rabbit polyclonal antibody (1:1,000; cat. no. 60186-1-Ig; Proteintech Group Inc.), B-cell lymphoma 2 (Bcl-2)-associated X protein (BAX) rabbit polyclonal antibody (1:1,000; cat. no. 50599-2-Ig; Proteintech Group Inc.), Bcl-2 rabbit polyclonal antibody (1:1,000; cat. no. 12789-1-AP; Proteintech Group Inc.), ZEB1 rabbit polyclonal antibody (1:1,000; cat. no. 21544-1-AP; Proteintech Group Inc.), ZEB2 rabbit polyclonal antibody (1:1,000; cat. no. 14026-1-AP; Proteintech Group Inc.), Snail rabbit polyclonal antibody (1:1,000; cat. no. 13099-1-AP; Proteintech Group Inc.), Slug rabbit polyclonal antibody (1:1,000; cat. no. 12129-1-AP; Proteintech Group Inc.), AKT rabbit polyclonal antibody (1:1,000; cat. no. 10176-2-AP; Proteintech Group Inc.), ERK rabbit polyclonal antibody (1:1,000; cat. no. 16443-1-AP; Proteintech Group Inc.), and β-actin mouse polyclonal antibody (1:1,000; cat. no. TA-09; OriGene Technologies, Inc.). The membranes were then incubated with peroxidase-conjugated goat anti-rabbit secondary antibody (1:10,000; cat. no. ZB-2301; ZSGB-BIO) or peroxidase-conjugated goat anti-mouse secondary antibody (1:10,000; cat. no. ZB-2305; ZSGB-BIO) for 1 h at room temperature. Protein signals were visualized using enhanced chemiluminescence substrate (Thermo Fisher Scientific, Inc.) with a Bio-Rad gel imaging system (Bio-Rad Laboratories, Inc.). Image Lab version 2.0.1 (Bio-Rad Laboratories, Inc.) was used for quantification of the western blot data. Three biologically independent experiments were conducted.

DLD-1 cells were inoculated into a 6-cm culture dish during the exponential growth phase (2.5×10⁵ cells/well) and cultured overnight. After 48 h at 37°C of CVB-D (20 µM) treatment, a cell suspension with <25% of the total cells was selected as the experimental group. The cells were digested with 1.5 ml TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), then collected and transferred to −80°C for storage. This sample collection step was repeated three times. The next steps including RNA isolation, cDNA library preparation and RNA-seq were completed by KangChen Bio-tech Inc. A NanoDrop was used to measure the quantity and quality of RNA. The DNA library was constructed using an Illumina kit, based on the KAPA stranded RNA-Seq Library, and enrichment of RNA was performed with oligo(dT) magnetic beads. The first-strand cDNA was generated by random primer inversion, and dUTP was added for second-strand cDNA synthesis. The final library was obtained by PCR amplification. Agilent 2100 was used to detect the quality of the constructed library, and quantitative PCR was used to quantify the library. After a series of strict quality control steps, the trimmed data were compared to the reference genome.

GEPIA2 (http://gepia2.cancer-pku.cn/#index) analysis included analyses of the differential expression of tumors/normal tissues, the survival of patients and multiple gene comparisons. The human protein atlas (http://www.proteinatlas.org) was employed to evaluate CTHRC1 protein expression in COAD tissues. The Venny online tool (https://bioinfogp.cnb.csic.es/tools/venny/) was used to identify common data in different groups of data. Gene Ontology (GO) (http://www.geneontology.org) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway (http://www.genome.jp/kegg) enrichment analyses were performed based on the downregulated differentially expressed genes (DEGs) obtained from RNA-seq experiments. The GO terms covers three domains: Biological Process (BP), Cellular Component (CC) and Molecular Function (MF). The targets of CVB-D should comply with the following requirements: i) They were oncogenes; ii) they were overexpressed in COAD; and iii) the overexpressed oncogenes were accompanied by a decrease of OS and/or DFS.

A total of six female BALB/c nude mice (5-6-weeks old; weight, 18 g) from Vital River Laboratory Animal Technology Co., Ltd. were selected and reared in a pathogen-free environment. Mice were housed at room temperature in a 12-h light/dark cycle and had access to food and water ad libitum. The Medical Ethics Committee of Harbin Medical University (Harbin, China) approved the animal protocol. Following intraperitoneal injection of 1% pentobarbital sodium into mice (50 mg/kg) (28), DLD-1 cells (0.1 ml; 1×10⁷ cells/ml) were resuspended in culture medium and subcutaneously implanted into the flanks of mice in equal volumes to establish tumor xenografts. Mice were randomly divided into treatment and control groups (three mice in each group) 7 days after xenograft establishment. The experimental group and the negative control group were intraperitoneally injected with 15 mg/kg CVB-D and PBS, respectively, once a day for 4 weeks. Evaluation of tumor growth, angiogenesis, microangiogenesis and tumor hardness was performed using B-ultrasound, color Doppler flow imaging (CDFI), color power angiography (CPA), and ultrasonic elastosonography (USE). Finally, mice were sacrificed and the tumors, hearts, livers, spleens, lungs were collected for pathological examination at the end of the experiment.

For IHC, briefly, formaldehyde-fixed (at room temperature for 48 h) and paraffin-embedded tumour tissues were sliced into 5-µm-thick sections. The sections were deparaffinized by xylene, rehydrated in a graded series of ethanol (100, 95, 85 and 75%), incubated with 3% H₂O₂ for 30 min, and blocked with 3% bovine serum albumin at room temperature for 1 h. Sections were then immunostained with anti-Ki-67 antibody (polyclonal, rabbit anti-human IgG; 1:200; cat. no. WL01384; Wanleibio Co., Ltd.), CTHRC1 rabbit polyclonal antibody (1:200; cat. no. 16534-1-AP; Proteintech Group Inc.), Bcl-2 rabbit polyclonal antibody (1:100; cat. no. WL01556; Wanleibio Co., Ltd.), N-cadherin rabbit polyclonal antibody (1:100; cat. no. WL01047; Wanleibio Co., Ltd.), cyclin D1 rabbit polyclonal antibody (1:200; cat. no. WL01435; Wanleibio Co., Ltd.) and HIF-1α rabbit polyclonal antibody (1:500; cat. no. WL01607; Wanleibio Co., Ltd.) at 4°C overnight. The colour was developed with a 3,3′-diaminobenzidine Substrate kit (cat. no. DA1010; Beijing Solarbio Science and Technology Co., Ltd.), and the sections were counterstained with hematoxylin at room temperature for 3 min. Five visual fields on each slide were randomly selected and photographed under a light microscope (magnification, ×400).

For H&E, briefly, tissues were fixed at room temperature for 48 h and embedded in paraffin, sliced into 5-µm-thick sections, stained with hematoxylin at room temperature for 5 min, treated with 1% acid ethanol for 3 sec, washed with distilled water, dyed with eosin solution at room temperature for 3 min, dehydrated with graded alcohol and cleaned with xylene. After drying, five visual fields were randomly selected on each slide and photographed using a light microscope (magnification, ×200).

Apoptosis in pathological tissues was detected using a TUNEL Apoptosis Detection kit (Wanlei Co., Ltd.; www.wanleibio.cn), according to the manufacturer's protocol. Five visual fields were randomly selected on each slide and photographed using a light microscope (magnification, ×400).

CTHRC1 expression was silenced by siRNA. CTHRC1 siRNA and negative control siRNA was synthesized by General Biosystems, Inc. and 100 nM was transfected into DLD-1 and LoVo cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The cells were incubated for 48 h and then used in subsequent experiments. The siRNA sequences are presented in Table SI.

Statistical analysis was conducted using SPSS 22.0 software (IBM Corp.). Data are presented as the mean ± standard deviation. The difference of means between two groups was assessed using Student's t-test. The difference of means between multiple groups was assessed using one-way analysis of variance, and a comparison between two groups was assessed using Bonferroni's correction. P<0.05 was considered a statistically significant difference.

To evaluate the inhibitory effects of CVB-D on cell viability, CRC cells were exposed to increasing concentrations (0-50 µM) of CVB-D for 24 and 48 h. The results demonstrated that CVB-D significantly inhibited the CRC cell viability in a dose-dependent manner (Fig. 1A). According to probit regression analysis, the IC₅₀ of CVB-D in DLD-1 and LoVo cells at 48 h was ~23.20 and ~26.12 µM, respectively. Significant dose-dependent reductions in colony formation rate were also observed in DLD-1 and LoVo cells treated with CVB-D, as determined by colony formation assay (Fig. 1B), possibly due to a decrease in stemness. CD133 is a transmembrane glycoprotein that is considered to be a surface marker of tumor stem cells and is overexpressed in CRC (29). Patients with CRC with high CD133 expression are prone to metastasis and recurrence (30,31). The present results demonstrated that CD133 expression significantly decreased with an increase in drug concentration (Figs. 1F and S2), indicating that stemness may be suppressed by CVB-D. In addition, CVB-D had an insignificant effect on the viability of NCM460 cells (Fig. S1).

In cancer, control of the cell cycle is altered, leading to uncontrolled cell proliferation, thereby making the cell cycle an important target of cancer treatment (32). The overexpression of cyclin D1 in patients with CRC indicates a poor prognosis (33). Therefore, enhancing the expression of cyclin D1 and/or inhibiting its degradation is another important mechanism by which oncogenic signaling pathways, including the PI3K/AKT signaling pathway, regulate the proliferation and growth of tumor cells (34,35). The regulation of cyclin D1 expression is a dynamic process in the cell. The expression levels of cyclin D1 in the G1-S-G2 phases are high, low, and low, respectively. The expression of cyclin D1 is low in the S phase; however, it is not the decrease of cyclin D1 expression that leads to the stagnation of S phase cells (36). The cell cycle experiments demonstrated that CRC cells were arrested at the S phase following CVB-D treatment, with a significantly higher percentage of cells in this phase (Fig. 1D and E). The expression of cyclin D1 in CRC cells was decreased following CVB-D treatment, as shown by western blot analysis (Figs. 1F and S2).

Apoptosis is the key steady-state mechanism that balances cell division and cell death to restrain the uncontrolled proliferation of cancer cells; thus, induction of apoptosis is an important mechanism of anticancer drugs (37). The present study found that CVB-D significantly induced apoptosis of CRC cells in a dose-dependent manner (Fig. 2A and B). Western blot analysis demonstrated that Bcl-2 expression significantly decreased and Bax expression significantly increased in CRC cells following treatment, indicating that CVB-D induced apoptosis (Figs. 2C and S3).

Pro-apoptotic chemotherapeutic agents decrease the mobility of tumor cells (38). Thus, the effects of CVB-D on cell migration and invasion were evaluated by wound healing and Transwell assays. As presented in Fig. 3A and B, the wound in the untreated cells healed rapidly, while the wound of the CVB-D-treated cells healed more slowly in a dose-dependent manner. Consistent with these observations, Transwell assays demonstrated a significantly dose-dependent decrease in the number of migrating and invading cells following CVB-D treatment (Fig. 3C and D). These results confirmed that CVB-D inhibited the mobility of CRC cells in a dose-dependent manner.

E-cadherin, vimentin, and N-cadherin are main biomarkers of the EMT (39). Western blot analysis revealed that CVB-D treatment significantly increased the expression of E-cadherin and significantly decreased the expression of vimentin and N-cadherin (Fig. 4A and B). Degradation of the extracellular matrix (ECM) promotes the ability of malignant tumor cells to invade adjacent tissues, blood and lymphatic vessels in an MMP-dependent manner (40). In particular, MMP2 and MMP9 have been shown to promote cancer cell invasion and lymph node metastasis in gastrointestinal tumors (41). In the present study, MMP2 and MMP9 expression levels were decreased following treatment with CVB-D (Fig. 4A and B). In addition, western blotting demonstrated that CVB-D significantly decreased the expression of Snail in CRC cells in a dose-dependent manner. In DLD-1 cells, the expression of Slug was significantly inhibited when the concentration reached 40 µM, and the expression levels of ZEB1 and ZEB2 were reduced by CVB-D in a dose dependent manner. In LoVo cells, the expression levels of Slug and ZEB1 were decreased in a dose-dependent manner, and the expression of ZEB2 was significantly decreased at 40 µM. Therefore, compared with Slug, ZEB1 and ZEB2, only the expression of Snail was significantly decreased both in DLD-1 and LoVo cells in a dose-dependent manner (Fig. 4C and D). In view of these results, the present study mainly focused on Snail. The GEPIA database confirmed that Snail expression was significantly higher in COAD compared with adjacent tissue (Fig. S4A). Furthermore, a higher expression of Snail was significantly associated with a reduced OS rate of patients with COAD (Fig. S4B). These data suggested that inhibition of the EMT by CVB-D was regulated by Snail.

RNA-seq was used to investigate the mechanisms underlying the effects of CVB-D on CRC. The experimental flowchart is presented in Fig. 5A. Hierarchical clustering and volcano plots demonstrated that 1,932 DEGs were upregulated and 2,559 DEGs were downregulated, and 7,255 DEGs were unchanged in RNA-seq data (Fig. 5B and C). According to the functional annotation in the GO database, the downregulated DEGs were mostly enriched in the following BP terms 'metabolic process' and 'heterocycle metabolic process'; the CC terms 'intracellular' and 'mitochondrion'; and the MF terms 'catalytic activity' and 'RNA binding' (Fig. 5D). As cellular metabolism is the foundation of all biological activities, studying the mechanism of the metabolic process may enhance cancer treatment methods (42). The classical MAPK, PI3K-AKT and cell cycle pathway pathways were associated with the group of downregulated DEGs in the RNA-seq dataset (Fig. 5E). Western blot analysis verified that the protein expression levels of p-AKT, p-ERK1/2 and PI3K were inhibited by CVB-D in a dose-dependent manner, while, the levels of total AKT and ERK1/2 demonstrated no difference, indicating that CVB-D exerts anticancer effects on CRC by inhibiting the AKT/ERK signaling pathway (Figs. 5F and S5).

By mining the GEPIA2 database, a total of 5,331 DEGs related to COAD and 926 most differentially expressed survival genes associated with the OS and/or disease-free survival (DFS) rates of patients with COAD were identified. Using the Venny online tool, 24 overlapping genes were found between the two aforementioned groups of data and the downregulated genes revealed by RNA-seq (Fig. 6A). Then, potential targets were selected according to three conditions: i) They were oncogenes; ii) they were overexpressed in COAD (Fig. S6); and iii) the overexpressed oncogenes were accompanied by a decrease in OS and/or DFS (Figs. S7 and S8).

The remaining four genes, CTHRC1, C2orf70, NIFK and COMT, were considered the most likely potential targets for CVB-D. GEPIA2 was used to analyze the correlation between the four genes (CTHRC1, COMT, C2orf70 and NIFK) and transcription factors. The correlations were low between Snail and COMT (r=0.35, P=1.1×10^-10), Snail and C2orf70 (r=0.26, P=2.4×10^-6), and Snail and NIFK (r=0.38, P=1.6×10 ^-12). However, there was a significantly high correlation between Snail and CTHRC1 (r=0.69; P=3.1×10^-45) (Fig. S9A). In addition, the correlation between other transcription factors and the four potential targets was also analyzed. COMT, C2orf70 and NIFK had no or low correlation with Slug, ZEB1 and ZEB2. Whereas, CTHRC1 had a certain degree of correlation with other three transcription factors (CTHRC1 and Slug (r=0.57, P=4.5×10^-29); CTHRC1 and ZEB1 (r=0.43, P=1×10^-15); CTHRC1 and ZEB2 (r=0.37, P=8.7×10 ^-12) (Fig. S9). The protein and mRNA expression analysis using the online databases demonstrated that the expression of CTHRC1 was significantly higher in COAD tissues compared with adjacent normal tissues (Fig. S10A and B). The CTHRC1 mRNA expression in COAD ranked 13th highest among 31 types of tumors (Fig. S10C). Patient survival analysis revealed that overexpression of CTHRC1 predicted poor OS and DFS rates in COAD (Fig. S10D). Western blotting reveled that the expression of CTHRC1 in DLD-1 and LoVo cells decreased in a dose-dependent manner following treatment with CVB-D (Fig. 6B and C). IHC results demonstrated that CTHRC1 expression in the CVB-D group was lower compared with the control group (Fig. 6D). RNAi2 and RNAi3 effectively downregulated the expression of CTHRC1 (Figs. 6E and S11). Western blot analysis demonstrated that the ratios of p-AKT/AKT and p-ERK/ERK significantly decreased after siRNA-mediated CTHRC1-knockdown; the expression level of Snail was also decreased after siRNA-mediated CTHRC1-knockdown (Figs. 6F and S12), indicating that CTHRC1 may be located upstream of p-AKT, p-ERK and Snail. In the siRNA3 rescue experiment, siRNA3-mediated suppression of CTHRC1 in CRC cells resulted in a partial reversal of CVB-D-induced growth inhibition (Fig. 6G). These results suggest that the growth of CRC cells inhibited by CVB-D is mediated by a CTHRC1-dependent mechanism.

The DLD-1 xenograft model was established in BALB/C nude mice to evaluate the antitumor ability of CVB-D. After 4 weeks, tumor weight and size in the CVB-D treatment group were significantly smaller compared with the control group (Fig. 7A-C). The expression levels of Ki67, cyclin D1, Bcl-2, HIF1A and N-cadherin were decreased in the CVB-D treatment compared with the control group, as detected by IHC (Fig. 7E).

In addition to dynamically observing the growth of the transplanted tumor and comparing the size of the excised tumor after execution, ultrasonic imaging (USI) was employed to dynamically detect the tumor indicators. USI can continuously and dynamically scan the lesions, and can show stereoscopic changes, which are not limited by image stratification, and does not damage the nude mice.

USI can also be evaluated systematically with multi-parameters such as Doppler and elastography (43). The ultrasound scan mode in this experiment included B-mode, CDFI, CPA and USE. The results demonstrated that tumor growth slowed after CVB-D treatment; angiogenesis decreased, especially microvascular; the elasticity score, which is highly related to tumor malignancy, decreased; and hardness of tumor decreased (Fig. 7D). The H&E staining result revealed that the number of blood vessels in the experimental group was decreased after CVB-D treatment (Fig. S13). H&E staining demonstrated that there were no obvious morphological changes in the heart, liver, spleen and lung (Fig. S14). The apoptosis rate of xenograft tumor cells, as detected by the TUNEL assay, was 15.91% in the CVB-D treatment group and 3.96% in the control group, demonstrating the antitumor effects of CVB-D (Fig. 7F).

The chemical and 3D structure of CVB-D is presented in Fig. 8A. A schematic presentation of the mechanism underlying CVB-D-induced inhibition of CRC and the relevant molecular mechanism associated with the AKT/ERK-Snail signaling pathway is presented in Fig. 8B.