Human CRC SW480, HT-29, Lovo, and NCM460 cells were purchased from American Type Culture Collection (ATCC), and cells were cultured in DMEM medium (06-1055-57-1ACS, BI, Israel), which added 10% Fetal Bovine Serum (FBS, A3160801, Hyclone, United States) and 1% Pecinillin-Streptomycin (P/S, UB89609, GBICO, United States), cultured in 37°C incubation containing 5% CO₂.

Nodosin was purchased from Huzhou Zhanshu Biotechnology Co., Ltd. (10391-09-0, molecular formula: C₂₀H₂₆O₆, molecular weight: 362.42, purity: 99.98%). DMSO (dimethyl sulfoxide, D8371) was obtained from Solarbio Technology Ltd.

Cells with a density of 25% were seeded in 96-well plates, and treated with 0–16 μM nodosin when the total cell density was about 40%. The relative cell activities of SW480, HT-29, LoVo, and NCM460 after nodosin treatment were then detected following the manufacturer’s instruction of CCK-8 kit (CK04, DOJINDO, Japan).

SW480 cells were seeded in the 12-well plates at the confluence of 1 × 10⁵ cells/well. After the cells adhered and returned to normal, the cell morphology at T0 moment was recorded, then treated with 0–12 μM of nodosin for 48 h, and recorded the morphological changes after different times of treatment with an inverted microscope (Nikon, Japan).

Before the cells were seeded into 12-well plates, the inserts (80469, IBIDI, Germany) were stuck onto plate bottom following the manufacturer’s instruction. When the cell confluence reached 80%, the insert was removed with forceps to produce a scratch of 500 μm width. The T0 moment was recorded with an inverted microscope (Nikon, Japan). Then added 0–12 μM nodosin and incubated with cells for 24 h, observed and recorded the width of cell scratches with a microscope (Nikon, Japan).

An appropriate amount of cells were seeded in 12-well plates, treated with nodosin for 24 h. Nodosin-treated cells were washed using PBS and fixed with 4% paraformaldehyde for 15 min. After washed 2 to 3 times with PBS, the fixed cells were stained with double dyes (Hoechst 33342 1:1000, C1022, Beyotime; Dio 1:100, C1038, Beyotime) for 20 min, washed twice with PBS, and then photographed with a fluorescence inverted microscope (Nikon, Japan).

The cells were seeded in a 12-well plate at a confluence of 500 cells/well. 48 h later, different concentration of nodosin was added. The medium with nodosin was changed every 3 days. After incubation with/without nodosin, the cells were fixed with 4% paraformaldehyde for 20 min at ambient temperature and then stained with crystal violet staining solution (KGA229, KeyGEN BioTECH, China) for 5–10 min. The stained colonies were recorded under an inverted microscope (Nikon, Japan).

SW480 cells were seeded in 12-well plates at a number of 1 × 10⁵ cells/well. After the cell density was about 50%, nodosin was added for treatment. The cell viability was detected using a Calcein AM Cell Viability Assay Kit (C2013FT, Beyotime, China), and the stained cells were recorded microscopically (Nikon, Japan).

DNA synthesis in nodosin-treated SW480 cells was detected using EdU-594 Cell Proliferation Assay Reagent (C0078S, Beyotime, China) in the light of the manufacturer’s instruction. The staining results were recorded microscopically (Nikon, Japan), followed by quantitative analysis using ImageJ software.

The nodosin-treated SW480 cells were centrifuged and collected. The cells were resuspended with 70% cold ethanol, fixed on ice for 60 min. After centrifugated at 2,000 rpm, the supernatant was discarded and the pellet cells were resuspended with a mixed solution containing PI (KGA214, KeGEN BioTECH, China), RNase (ST579, Beyotime, China) and Triton X-100 (P0096, Beyotime, China). The stained cells were detected with a flow cytometer (Beckman, cytolfex, United States).

The cultured cells were fixed with 4% paraformaldehyde at room temperature for 20 min, then permeabilized with 0.3% Triton X-100 for 15 min, blocked with 3% BSA for 30 min. After discarding the block solution, the samples were incubated with Ki67 (1:300, ab15580, abcam, United Kingdom) overnight, and then incubated with CoraLite 488-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (SA00013-2, proteintech, China) for 1.5 h. The nucleus was stained with DAPI (C1002, Beyotime, China) and the cytoskeleton was stained with Actin-Tracker Red 594 (F-Actin, C2205S, Beyotime, China). The number of the cell proliferation marker Ki67 was observed and recorded with a fluorescence inverted microscope (Nikon, Japan) at a wavelength of 550 nm.

The changes of ROS in nodosin-induced SW480 cells were detected with the cell-permeable ROS probe H₂DCFDA (HY-D0940, MCE, United States). SW480 cells in the nodosin-treated and control group were incubated with H₂DCFDA in a CO₂ incubator at 37°C for 30 min in the dark. The changes of ROS in the cells were detected by flow cytometry (Beckman, cytolfex, United States). To detect whether ROS was involved nodosin-inhibited cell activity, SW480 cells were pretreated with the antioxidant NAC (ST1546, Beyotime, China) for 1 h, and co-incubated with 12 μM nodosin for 48 h. Finally, the cell activity was detected using the CCK-8 kit.

Nodosin-treated SW480 cells were digested with EDTA-free trypsin (T1350, Solarbio, China). The cells were incubated with Annexin V-FITC (C1062M, Beyotime, China) and PI (KGA214, KeyGEN BioTECH, China) for 20 min in the dark. The rate of apoptosis was determined and recorded by flow cytometry (Beckman, cytolfex, United States).

Lipofectamine 3000 Transfection Kit (L3000015, Invitrogen, United States) was used to transfer lamp1 OFP, ER, and mito plasmids (gifts from Camilla Raiborg, University of Oslo, Norway) into SW480 cells. After the target plasmids expressed, different concentrations of nodosin were added. Then the confocal laser microscopy (Leica TCS SP8, Leica, Germany) was used to examine the changes of intracellular lysosomes, endoplasmic reticulum, and mitochondria.

SW480 cells were cultured in a 96-well plate at 37°C with 5% CO₂ for 12 h. Next, they were treated with 12 μM of nodosin for 48 h. SW480 cells were then incubated with 6.25–250 nM of the autophagy blocker chloroquine (CQ, HY-17589A, MCE, United States) for 2 h, and cell viability was detected using the CCK-8 kit.

In transcriptome sequencing, three nodosin-treated samples and three control samples were lysed with Trizol reagent (15596026, ambition, Japan) respectively (5 × 10⁶ cells in each sample). Library construction, mRNA sequencing, and bioinformatics analysis were performed at BIOMARKER (Beijing, China).

Total RNA in cells was extracted with Trizol reagent (15596026, ambition, Japan), then RNA was reversed into cDNA with FastKing RT Kit (With gDNase) (KR116, TIANGEN, China), validated with agarose gel, and then qPCR was conducted using SuperReal PreMix Color (SYBR Green, FP215, TIANGEN, China), relative mRNA levels were normalized to β-actin levels, and relative gene expression changes were calculated. The primers of PCR and q-PCR are shown in Tables 1, 2.

The nodosin-treated SW480 cells were lysed and proteins were extracted from them using lysis buffer (P0013, Beyotime, China). The protein extracts were processed by SDS polyacrylamide gel electrophoresis and blotted on PVDF membranes. Western blotting (WB) was performed using the following primary antibodies: HMOX1 (Rabbit, 1:3,000, 10701-1-AP, proteintech, China), LC3 (Rabbit, 1:2,000, 14600-1-AP, proteintech, China), β-Actin (Rabbit, 1:2,000, GB11001, Servicebio, China). After the primary antibody was incubated and washed, the immunohy bridization signal was captured using HRP-conjugated secondary antibody. The secondary antibodies were listed as follows: HRP Goat Anti-Rabbit IgG (H + L) (1:4,000, AS014, ABclonal, China). The development was then performed using ECL solution (P0018S, Beyotime, China).

BALB/c-nude nude mice were provided by Jinan Pengyue Laboratory Animal Breeding Co.,Ltd., eight females, 6 weeks old, about 20 g, SPF grade, the laboratory animal certificate number was No. 370726211100958867. This assay was performed with the approval of the Ethics Committee of Jining Medical University. BALB/c-nude female mice were subcutaneously injected with CRC SW480 cells. The mice were randomly divided into two groups (4 nude mice/group) when the tumor grew to 100 mm³. In the nodosin-treated group, the mice were treated by nodosin with a dose of 3 mg/kg, once every 3 days. The control group was treated with DMSO at the same injection volume and frequency. After 10 injections, the CRC xenograft tumors of mice were removed, weighed and photographed. Paraffin blocks were made after the tumors were fixed with 10% neutral formalin and dehydrated for subsequent studies.

The CRC xenograft tumors paraffin blocks were cut into slices (the thickness was 4 μm). The tumor sections were stained in the light of the instructions of the HE staining kit (G1120, Solarbio, China). The changes of the pathological sections were observed under a Pannoramic Desk (3DHISTECH, Hungary).

The biomarkers in nude mice transplanted tumors were detected by IHC methods. Cut the wax block into 4-μm wax slices, and use an oven at 60°C for 1–2 h. The sections were removed for dewaxing, hydration, and then antigen repair with improved sodium citrate antigen repair solution (P0083, Beyotime, China). Incubated for 30 min with inactivated endogenous peroxidase 3% H₂O₂ (SV0004, BOSTER, China) to avoid light. Afterwards, they were blocked with ready-to-use normal goat serum (AR0009, BOSTER, China) for 30 min. After shaking off the blocking solution, Ki67 (1:300, ab15580, abcam, United Kingdom), HMOX1 (Rabbit, 1:200, 10701-1-AP, proteintech, China), LC3 (Rabbit, 1:500, 14600-1-AP, proteintech, China), CTSL (Rabbit, 1:500, proteintech, China) were added and placed in a refrigerator at 4°C overnight for incubation. Polymeric HRP-labeled anti-rabbit/mouse IgG (SV0004, BOSTER, China) was added after washing with PBS and incubated for 30 min at room temperature in the dark. The color was developed with DAB chromogen solution (C02-12, ORIGENE, America). After color development, counterstain with hematoxylin (AR0005, BOSTER, China). Afterwards, it was blue with alkaline PBS. Finally, dehydration was performed and the slides were mounted. Scanning observations were performed using a Pannoramic Desk (3DHISTECH, Hungary).

The difference between the two groups was assessed by two-tailed t-test. The three groups and above were evaluated with one-way ANOVA analysis followed by LSD Post Hoc multiple comparison to assess its significance, and p-value < 0.05 was considered significant.

The chemical structure of nodosin is shown in Figure 1A. According to the CCK-8 assay results (Figures 1B–D), nodosin significantly repressed CRC cells proliferation in a dose-dependent manner. The IC₅₀ of nodosin in SW480, HT-29, and LoVo cells were 7.4, 7.7, and 6.6 μM, respectively. However, the toxicity to human normal colonic mucosal epithelial cells NCM460 was lower, with an IC₅₀ of 10.2 μM (Figure 1E). In addition, the morphological characteristics of CRC SW480 cells were significantly changed after nodosin treatment (Figure 1F). At low concentrations, the cells underwent significant shrinkage and elongation; at high concentrations, the cells were unable to adhere and became rounded. Figure 1G shows that nodosin dose-dependently restrained the migration ability of SW480 cells. Cell mobility decreased gradually with increased nodosin concentration, and almost no cell migration occurred at 12 μM. Therefore, the relative activity of cells gradually decreased with increased nodosin concentration and prolonged treatment (Figures 1H,I), indicating that nodosin could inhibit the proliferation of CRC cells in a dose- and time-dependent manner.

Proliferation and death are important processes in the development of tumor cells. As nodosin concentration increased, the number of cells gradually decreased, and the cell membrane became larger and damaged (Figure 2A). Additionally, nodosin inhibited the clonogenic ability of SW480 cells. When nodosin concentration was greater than 4 μM, the cells could not form clonal colonies (Figure 2B). Interestingly, nodosin only inhibited 10% of cell activity at a concentration of 4 μM, whereas nodosin at the same concentration inhibited majority of the clonogenic ability of SW480 cells. Moreover, the number of live cells decreased, and the number of dead cells gradually became elevated with increased nodosin concentrations in the live/dead cell staining assay (Figure 2C).

DNA replication speed is an important indicator for evaluating the cell proliferation rate, activity, and physiological status. This study found that nodosin inhibited DNA replication ability of SW480 cells by EdU assay (Figure 3A). After nodosin treatment, the total number of cells decreased to a certain extent. The relative ratio of EdU/H33342 gradually decreased as the nodosin concentration increased, which indicated that nodosin effectively inhibited the DNA replication ability of SW480 cells. Moreover, the cell cycle of SW480 cells was detected after nodosin treatment, and nodosin affected the synthesis (S) phase of the cell cycle; the S phase gradually decreased with increased nodosin concentration (Figure 3B). Ki67 is an antigen related to cell proliferation and exerts an important role in mitosis. In Ki67 immunofluorescence staining (Figure 3C), the Ki67-labeled antigen gradually decreased with an increase in nodosin treatment concentrations, which is consistent with the experimental results shown in Figures 3A,B. These results demonstrate that nodosin can inhibit DNA synthesis from multiple perspectives.

To further study the antitumor mechanism of nodosin, H₂DCFDA fluorescent probe was used to test intracellular ROS levels after nodosin treatment. Figure 4A shows that after the cells were stimulated by nodosin, oxidative stress occurred, and the ROS level in the cells increased continuously with an increase in nodosin treatment concentration. In this regard, after pretreatment with the antioxidant N-acetylcysteine (NAC), the relative cell activity reached saturation when the NAC concentration was 0.5 mM, and the relative cell activity could be increased to 80%, indicating that nodosin induced oxidative stress-mediated cell death. The evidence that pretreatment with NAC reversed oxidative stress-induced changes in SW480 cell death validated the dependence on oxidative stress (Figure 4B). Oxidative stress may lead to cell apoptosis and autophagy; therefore, an apoptosis assay using flow cytometry was performed. These results revealed that nodosin induced cell apoptosis. The apoptosis rate gradually elevated with increased nodosin concentration. When the concentration of nodosin was 12 μM, the apoptosis rate of the cells was 16.11% (Figures 4C,D). Therefore, we speculated that there are other modes of cell death in addition to apoptosis. Elevated levels of ROS usually lead to stress in mitochondria, endoplasmic reticulum, and lysosomes; therefore, the changes in these cell organelles after nodosin treatment were observed by plasmid transfection assay. This study’s findings showed that mitochondria and endoplasmic reticulum gradually disappeared with increased nodosin concentration, while lysosomes gradually increased. The endoplasmic reticulum and mitochondria almost completely disappeared at a nodosin concentration of 6 μM (Figure 4E). Based on this phenomenon, we hypothesized that these cells may have undergone autophagy. To verify whether nodosin can induce autophagy in cells, the autophagy blocker chloroquine was used to rescue nodosin-treated SW480 cells. Chloroquine rescued nodosin-induced SW480 cells cell death to a certain extent. At a chloroquine concentration of 6.25 nM, the relative cell activity increased by approximately 10%. These results illustrate that nodosin induced autophagy in SW480 cells (Figure 4F).

To investigate the anticancer mechanism of nodosin, transcriptomic sequencing was performed. Figure 5A shows a volcano plot of differentially expressed genes (DEGs) between the nodosin treatment and control groups. With nodosin treatment, 205 genes were upregulated, and 376 genes were downregulated. Among them, aldo-keto reductase family 1 member C1 (AKR1C1), aldo-keto reductase family 1 member B10 (AKRIB10), OSGIN1, HMOX1, and CTSL were significantly upregulated, whereas transcription factor 4 (TCF4), TRIB3, syntrophin beta 1 (SNTB1), kinase family member 21B (KIF21B), mitogen-activated protein kinase 6 (MAP2K6), cadherin 11 (CDH11), amphiregulin (AREG), and epiregulin (EREG) were significantly downregulated. Most of these genes were involved in signaling pathways related to cell proliferation inhibition and cell death induction. Figure 5B shows that gene ontology (GO) analysis was enriched in biological processes with more significant DEGs changes. We drew a heatmap of the DEGs involved in apoptosis and death. HMOX1 was also upregulated (Figure 5C). These changes induced oxidative stress in the cells, which in turn induced apoptosis. To verify the high-throughput sequencing results, specific primers were designed for verification at the messenger RNA (mRNA) level. The results of PCR and qPCR showed that the HMOX1 and CTSL levels, which are related to apoptosis and autophagy, were significantly increased (Figures 5D,E). Furthermore, WB was performed to verify whether nodosin affected the above pathways at the protein level. The results showed that the apoptosis-and autophagy-positive relative molecules HMOX1 and LC3 were significantly elevated (Figure 5F). Since the transcriptomic results indicated that nodosin affected the expression of genes related to oxidative stress, apoptosis, and autophagy in CRC cells, we validated it using the commonly used gene expression profiling interactive analysis (GEPIA) database in clinical practice. The results revealed that the gene TRIB3 negatively associated with oxidative stress was significantly downregulated, the genes PHLDA3, LC3, and CTSL related to apoptosis and autophagy were significantly upregulated in normal colorectal tissues compared to colorectal tumors (Figure 5G), while nodosin also reversed the expression of these genes, indicating that nodosin is beneficial for the treatment of CRC.

To investigate the antitumor effect of nodosin on CRC in vivo, nude mouse subcutaneous tumorigenesis models were used for administration. Figure 6A shows the growth process of xenografted tumors in nude mice. Tumors in the nodosin-treated group grew slower than those in the control group. After the mice were terminated, tumors were isolated and photographed (Figure 6B). When the tumors were weighed, there was a significant difference in tumors weight between the treatment and control group (Figure 6C). There was no significant change in the bodyweight of nude mice in the two groups (Figure 6D), indicating that nodosin had no obvious side effects. H&E and IHC stainings of the pathological tumor sections were performed. H&E staining showed that compared with the control group, the cell structure of the tumor was destroyed, and the number of nuclei was also reduced (Figure 6E). The results of IHC staining revealed that the level of proliferation marker Ki67 was significantly lower in the nodosin-treated group than in the control group, indicating that treatment with nodosin could supress the proliferation of colorectal xenografted tumors. In addition, the expression of the apoptosis marker HMOX1 and the autophagy markers LC3 and CTSL were significantly increased in the nodosin-treated group, which indicated that nodosin could induce apoptosis and autophagy in colorectal tumor cells (Figure 6F).