Introduction

Colorectal cancer is a common malignancy of the digestive system with a high mortality rate, and its incidence has been increasing globally in recent years. It is estimated that there were approximately 1.9 million new cases of colorectal cancer and over 930,000 reported deaths worldwide in 2020.1–3 The early diagnosis of colorectal cancer is challenging due to the lack of typical symptoms, often leading to missed opportunities for optimal surgical intervention. Therefore, combined surgical and chemotherapy interventions have become the mainstay of treatment for colorectal cancer. Among the chemotherapy regimens, Irinotecan (CPT-11) has served as a cornerstone for the treatment of this disease.4

However, the clinical application of CPT-11 is limited by its significant gastrointestinal toxicity, particularly severe diarrhea, which can severely impact the quality of life for patients and subsequent treatment outcomes.5 Finding strategies to prevent or alleviate the gastrointestinal toxicity of CPT-11 is a critical area of research in oncology.

Oxyresveratrol (OXY) is a natural polyphenolic compound with similar properties to resveratrol, which was first isolated from the core of mulberry called Artocarpus lakoocha,6 and was found to be widely present in the branches, leaves and roots of Moraceae, Myrtaceae, and Tenaceae. OXY exhibiting a wide range of pharmacological activities, including antimicrobial, antioxidant, anticancer, and gastroprotective effects.7,8 Previous studies have demonstrated the ability of OXY to inhibit the growth and metastasis of colorectal cancer cells without significant cytotoxic effects.9,10 Additionally, OXY has been shown to possess potent antioxidant activity, which may contribute to its protective effects on the intestinal mucosa.11–14 These properties make OXY an attractive candidate for potential synergy with CPT-11 in the treatment of colorectal cancer, potentially enhancing antitumor effects while mitigating CPT-11-induced gastrointestinal toxicity.15

In this study, we aimed to investigate whether OXY could alleviate CPT-11-induced diarrhea and enhance the inhibitory effect on colorectal cancer cells when used in combination. Furthermore, we sought to explore the potential mechanisms underlying the synergistic effects of OXY and CPT-11 in colorectal cancer treatment. By elucidating the synergistic mechanisms and assessing the alleviation of CPT-11-induced gastrointestinal toxicity by OXY, we hope to provide valuable insights for the development of more effective and tolerable treatment strategies for colorectal cancer.

Materials and Methods Cell Lines and Reagents

Human colorectal cancer cell lines HCT116 and SW620 were obtained from the American Type Culture Collection (ATCC). Oxaliplatin (OXY, ≥98%) was generously provided by Professor Fengpeng Wang from Sichuan University. Irinotecan hydrochloride (CPT-11, Batch No. HO402A, 5mL:100mg) was purchased from Jiangsu Hengrui Medicine Co., Ltd. Other materials included phosphate-buffered saline (PBS) from Gibco, Gibco DMEM culture medium (Catalog No. 8120494, 2,003,671), fetal bovine serum (FBS) from Biosharp (Catalog No. D02255), CCK-8 assay kit from Beijing Applygen Technologies Inc. (Catalog No. abs70103), dimethyl sulfoxide (DMSO) from MP Biomedicals (Catalog No. YC0907), rabbit polyclonal anti-GAPDH antibody from Wuhan Searview Biotechnology Co., Ltd. (Catalog No.LS20356), and horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody from Beijing Applygen Technologies Inc. (Catalog No. GB23303).

Equipment

Cell cultures were maintained in a CO2 incubator from SANYO (Japan), and observations were made using a fluorescence inverted microscope from Nikon (Japan). Enzyme-linked immunosorbent assay (ELISA) was conducted using an automated plate reader (Model: DNM-9602G) from Beijing Purkinje General New Technology Co., Ltd. Gel electrophoresis was performed using the PowerPacTM Basic electrophoresis power supply from Bio-Rad (USA).

Cck-8

First, the cell suspension was prepared and seeded into a 96-well plate with 100μL per well. Plates were then precultured in an incubator at 37 ° C with 5% CO2 for 2–4 hours to allow cell adherence. Various concentrations of CPT-11 (5, 10, 20, 30, 50, 100 μg/mL) and OXY (10, 30, 60, 90 μg/mL) were selected based on literature16,17 to evaluate their individual inhibitory effects on SW620 cells over 48 hours. Similarly, different concentrations of CPT-11 (10, 20, 30, 50, 100 μg/mL) and OXY (30, 60, 90, 120 μg/mL) were tested on HCT116 cells. To determine the cellular status of HCT116 after 48 hours of combined treatment with OXY at a concentration of 50 μg/mL and different concentrations of CPT-11 (10, 15, 20 μg/mL), as well as the status of SW620 cells after 48 hours of combined treatment with OXY at a concentration of 25 μg/mL and different concentrations of CPT-11 (5, 10, 15 μg/mL). After drug treatment, the cells were incubated for 8 hours. After that, 10μL CCK-8 solution was added to each well. The color reaction was carried out after 4 hours of incubation. Finally, the absorbance at 450 nm was measured using a microplate reader to obtain the results.

The Gold Jun-Quan Q-value method18 was employed for efficacy analysis: Q Q=Ea+Eb/(Ea+Eb-Ea*Eb). Ea + Eb represents the combined cell inhibition rate under dual-drug treatment. The single inhibition rate and Q values for CPT-11 and OXY co-administration were calculated.

Animal Model and Drug Administration

Female SPF-grade Kunming mice, aged 6–7 weeks, were obtained from Sichuan University West China School of Basic Medical Sciences, housed in the animal facility, and provided with standard rodent feed and bedding from Chengdu Dashuo Experimental Animal Co., Ltd. The mice were given ad libitum access to reverse osmosis (RO) water. Ethical approval for the study was obtained (Approval No: 2022ky71). The mice were divided into different groups with 8 mice in each group: control, CPT-11 (50 mg/kg), OXY (20 mg/kg), low-dose OXY + CPT-11 (10, 50 mg/kg), and high-dose OXY + CPT-11 (20, 50 mg/kg). Each repeat was performed as a separate, independent experiment or observation. Drug doses and administration volumes were based on previous studies. CPT-11 was administered intraperitoneally for four consecutive days to induce diarrhea. From the fifth day onwards, mice in the low-dose and high-dose combination groups received OXY or physiological saline intraperitoneally, while mice in the control and OXY groups received OXY or physiological saline for eight consecutive days. On the ninth day, the mice were euthanized and blood was collected, and the spleen was weighed to calculate the spleen coefficient. Colonic tissues were collected for histological examination and Western blot analysis.

Evaluation of Diarrhea and Histological Analysis

Diarrhea was evaluated daily before drug administration based on criteria adapted from Pereira et al.19 Colonic tissues were fixed, dehydrated, embedded in paraffin, and sectioned. Hematoxylin and eosin (HE) staining was performed, and tissue morphology was observed under a light microscope.

Blood Cells Count

Collect the blood sample and treat it with an anticoagulant, mix the sample with a diluent and then drop it into the counting chamber of the hemocytometer, observe and count the number of cells in a specific area using a microscope, calculate the cell concentration per microliter of blood based on the counting results and dilution factor, and finally record the counts of red blood cells (RBC), white blood cells (WBC), and neutrophils (NE).

Target Prediction of OXY and CPT-11

The potential targets of OXY and CPT-11 were predicted using the Swiss Target Prediction (http://www.swisstargetprediction.ch/) and SuperPred (https://prediction.charite.de/) databases. The obtained targets were cross-referenced with the GeneCards (https://www.genecards.org/), OMIM (https://www.omim.org/), and DisGeNET (https://www.disgenet.org/) databases to identify colorectal cancer-related targets. The intersection of drug targets and disease targets was visualized using Venny 2.1.0 online software (https://bioinfogp.cnb.csic.es/tools/venny/), and a drug-target network was constructed using Cytoscape 3.8.2 software.

Functional Enrichment Analysis

The intersectional genes were subjected to functional enrichment analysis using the DAVID database (https://david.ncifcrf.gov/). Gene Ontology (GO) analysis was performed to predict the biological processes (BP), cellular components (CC), and molecular functions (MF) of the targets. Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was conducted to elucidate the potential mechanisms of OXY and CPT-11 in combination therapy for colorectal cancer. The top 10 GO terms and 20 KEGG pathways related to colorectal cancer (P < 0.01) were selected for analysis.

Cell Transfection

Stable low-expression models of PI3K and Akt genes were constructed in SW620 colorectal cancer cell lines using lentiviral plasmid transfection technology. Initially, double-stranded DNA was synthesized based on the siRNA sequences of PI3K and Akt genes obtained from Invitrogen, and then recombined into the lentiviral plasmid pGIPZ. The recombined plasmids were transformed into DH5α competent cells, and recombinant clones were selected. The plasmids were then packaged into 293T cells, cultured for 48 hours, and the virus-containing supernatant was collected. After filtration through a microporous membrane and centrifugation, shRNA lentiviral packaging particles targeting PI3K and Akt genes were obtained. SW620 cells were cultured until reaching 20–30% confluence, then dissociated with trypsin and prepared into single-cell suspensions. The virus-containing supernatant and transfection reagent were added to the target plasmid-transfected cells. After 12–16 hours, the virus-containing supernatant was replaced with complete culture medium, and the cell status was observed. Transfected cells were further cultured for 72 hours, followed by the addition of appropriate concentrations of puromycin to select for transfected positive cells, which were maintained in low concentrations of puromycin to sustain stable transfection. Each constructed stable transfected cell line was amplified and used after successful identification by qPCR or immunoblotting.

Real-Time Fluorescence Quantitative PCR

After cell lysis, RNA was extracted and its concentration and purity were determined. Reverse transcription reactions were performed using a 20μL system, followed by qPCR detection after dilution of the template. PCR reaction parameters were set as follows: 95°C for 30 seconds, followed by 40 cycles of denaturation at 95°C for 5 seconds and annealing/extension at 60°C for 30 seconds, with a final melt curve analysis. The expression levels of target genes were calculated using the 2−ΔΔCt method. The primer sequences were included in Supplementary Table 1.

Transwell Assay

SW620 cells were cultured to logarithmic growth phase, then seeded into pre-coated chambers containing basement membrane collagen. A chemoattractant was added to the lower chamber to create a chemical gradient that induces cell migration. Then the Transwell chamber was cultured for 24 hours. For invasion experiments, add a layer of Matrigel on top of the membrane to simulate the cell invasion process. After incubation, remove the non-migrated cells and stain and count the migrated or invaded cells. Finally, analyze the cell migration and invasion abilities based on the number of migrated or invaded cells.

Scratch Assay

Logarithmic phase cells were seeded into 6-well plates, scratched with a pipette tip, and treated with different concentrations of culture medium. After 24 and 48 hours of incubation, cell migration was observed under a microscope and migration rate was calculated.

Statistical Analysis

Experimental data were processed, analyzed, and graphed using GraphPad Prism 9.0 and SPSS 26.0 software. One-way analysis of variance (ANOVA) was employed for dissecting differences among different groups, with pairwise comparisons conducted between groups. The representation of experimental data followed the format mean± SD, where SD represents the standard deviation. Diarrhea scores were analyzed using the Mann–Whitney U-test. A significance level of P < 0.05 indicated the presence of statistical differences, while P < 0.001 denoted highly significant differences.

Results Inhibition Effects of CPT-11 and OXY on Colorectal Cancer Cells

Using the CCK-8 assay, we measured the IC50 of the CPT-11 and OXY in both SW620 and HCT116 cells. The IC50 values of OXY for SW620 and HCT116 were 25.93 μg/mL (95% CI=25.12~26.74) and 49.33 μg/mL (95% CI=47.66~50.99), respectively. The IC50 values of CPT-11 were 12.46 μg/mL (95% CI=10.61~14.47) for SW620 and 14.94 μg/mL (95% CI=13.13~16.70) for HCT116. Both CPT-11 and OXY exhibited inhibitory effects on colorectal cancer cells. The dose-response curves are illustrated in Figure 1A. Based on these results, treatment of CRC cells with 15 μg/mL CPT-11 resulted in 36.82±0.73% for SW620 and 48.74±0.40% for HCT116 inhibition of cell viability, respectively. On the basis of administration of fixed concentrations of OXY (25 μg/mL for SW620 and 50 μg/mL for HCT116), CPT-11 inhibited the growth of CRC cells in a dose-dependent manner (Figure 1B). It was observed that when OXY and CPT-11 were used individually, their inhibition rates were lower compared to their combined usage at equivalent doses. Moreover, the efficacy of the combined treatments was superior for SW620 compared to HCT116. In HCT116 cells, the increase in Q values with rising CPT-11 concentrations (15, 20 μg/mL) when combined with OXY administration was 0.86 and 0.92, respectively. For SW620 cells, the Q values for the three concentrations of CPT-11 (5, 10, 15 μg/mL) combined with OXY were 1.01, 1.11, and 1.14, respectively. Notably, HCT116 originates from colorectal adenocarcinoma in situ, while SW620 originates from lymph node metastases of primary rectal adenocarcinoma, indicating the suitability of both drugs for colorectal cancer lymphatic metastasis.

Figure 1 CPT-11 and OXY inhibit CRC cells viability. (A) Inhibition rates of OXY or CPT-11 on HCT116 and SW620 cells after 48 hours of treatment; (B) CPT-11 inhibited the viability of CRC cells in a dose-dependent manner with OXY.

Alleviation of CPT-11-Induced Weight Loss and Diarrhea Symptoms by OXY

Throughout the experiment, mice in the CON group and OXY group showed a gradual increase in body weight. Compared to the CON group, mice in the CPT-11 group experienced continuous weight loss after modeling (Figure 2A, P < 0.001). Different doses of OXY combined with CPT-11 were able to attenuate the rate of weight loss in mice (Figure 2A, P <0.001).Mice in the CON group and OXY group did not experience diarrhea throughout the experiment. Starting from the third day, mice in the CPT-11 group, low OXY+CPT-11 group, and high OXY+CPT-11 group showed varying degrees of diarrhea symptoms, characterized by soft and wet stools. The severity of diarrhea in the CPT-11 group gradually worsened, with some mice developing purulent blood stools. The difference in diarrhea scores between the CPT-11 group and the other four groups was significant (P < 0.001). Mice in the low OXY+CPT-11 group and high OXY+CPT-11 group showed improvement in diarrhea symptoms starting from the sixth day, with significant relief observed on the eighth day, indicating that OXY treatment may help alleviate CPT-11-induced diarrhea and reduce diarrhea scores. The scoring of diarrhea based on stool consistency and perianal erythema is shown in Figure 2B.

Figure 2 OXY alleviated CPT-11-induced weight loss and diarrhea symptoms. (A) Measurement results of mice body weight; (B) Measurement results of mice diarrhea score; (C) Measurement results of mice Blood cell count; (D) Measurement results of mice food intake; (E) Representative photos of mice; *P <0.05, **P <0.01, ***P <0.001, compared with CON group; #P <0.05, ##P <0.01, ###P <0.001, compared with the CPT-11 group.

The blood examination of the mice was performed on the 8th day of the model establishment, and the results showed that the mice in the CPT-11 group had a significant reduction in both white blood cells (WBC, P <0.05) and red blood cells (RBC, P <0.01), and the combination treatment with high concentration of OXY could significantly improve the reduction in both cell levels (Figure 2C, P <0.05 or P <0.01). The trend of neutrophils (NE) in the blood of drug-treated mice in each group was the same as that of the other two cells, but the differences between the groups were not statistically significant.

Besides, the study revealed that mice in the CPT-11 group exhibited poorer conditions compared to the other four groups, manifested by weight loss, reduced activity, decreased food and water intake, lack of gloss in fur, partial development of loose stools into purulent blood stools, and perianal erythema. Figure 2D shows the statistics of the food intake of the mice in each group. CPT-11 treatment significantly inhibited the food intake of the mice (P <0.001), and OXY combination treatment could partially restore the food intake of the mice (P <0.01). Representative photos of each group of mice were shown in Figure 2E, showing the hair changes.

Alleviation of CPT-11 Chemotherapy-Induced Injury of Colon and Spleen

The spleen plays a crucial role in cellular and humoral immunity, and protecting it effectively can ameliorate adverse reactions caused by drugs. The spleen index is also one of the ways to evaluate the immune function status. Compared to the CON group, the spleen weight and index of mice in the CPT-11 group showed significant reduction (Figure 3A and B, P < 0.01 and P < 0.001). OXY combined treatment significantly improved spleen weight and index compared with CPT-11 treatment alone (Figure 3A and B, P < 0.05 and P < 0.01). This indicates that OXY treatment significantly alleviated the decrease in spleen weight and spleen index induced by CPT-11. However, there was no significant difference in spleen weight and spleen index between the low OXY+CPT-11 group and the high OXY+CPT-11 group (P > 0.05), suggesting that even low doses of OXY could alleviate the decrease in spleen weight and spleen index caused by CPT-11, as shown in Figure 3A and B.

Figure 3 OXY alleviates splenic and colonic injury induced by CPT-11 chemotherapy. (A) Representative photos of spleen and measurement results of mouse splenic weight; (B) Measurement results of mouse splenic index; (C) Representative photos of colon and measurement results of mouse colonic lengths; (D) Representative Hematoxylin and Eosin (HE) staining of mouse colonic epithelium (×200 for the first row, ×40 for the second row); (E) Representative photos of TUNEL staining of colon and statistics of apoptosis; *P <0.05, **P <0.01, ***P <0.001, compared with CON group; #P <0.05, ##P <0.01, compared with the CPT-11 group.

Figure 3C showed representative pictures of the colon of each group of mice along with the colon length statistics. Upon dissecting the colons from the anus to the cecum, it was observed that the colonic mucosa of mice in the CON group and OXY group appeared intact and clear, without congestion or ulceration. In contrast, the colonic mucosa of mice in the CPT-11 group was reddish, with evident edema and congestion in the cecum area. The colon appeared thicker, and the length of the colon was significantly shorter compared to the CON group (P < 0.05). The intestines of mice in the low and high OXY+CPT-11 groups were slightly thicker, with a pale red mucosa, occasional congestion, and edema. Although the colon length was slightly shorter compared to the CON group (P > 0.05), the colon length of mice in the high OXY+CPT-11 group was significantly longer than that of the CPT-11 group (P < 0.01).

Hematoxylin and eosin (HE) staining revealed that the colonic epithelium of mice in the CON group and OXY group was intact, with neatly arranged crypts, clear nuclei, and no infiltration of inflammatory cells. In contrast, the colonic epithelium of mice in the CPT-11 group showed structural defects, with atrophied crypts and a large number of inflammatory cells in the basal layer. Most of the colonic epithelium in the low and high OXY+CPT-11 groups remained intact, with relatively dense cell arrangements, and occasional inflammatory cell infiltration. Specific details are depicted in Figure 3D. TUNEL staining (Figure 3E) showed that colon apoptosis was significantly increased in the CPT-11 alone group (P < 0.01), and both high and low concentrations of OXY combined treatment could significantly inhibit colon apoptosis (P < 0.01).

Network Analysis of Regulatory Targets for OXY and CPT-11 in Colorectal Cancer

Swiss Target Prediction database incorporated all genes with a probability greater than 0. SwissTarget Prediction and SuperPred were used to retrieve the target genes regulated by OXY and CPT-11, yielding 201 and 207 targets, respectively. After deduplication, a total of 336 regulatory targets were obtained.

Due to the extensive targets in the GeneCards database, genes with a relevance score greater than 5 were included, and after merging with GeneCards, OMIM, and DisGeNET databases, the regulatory targets were 3501, 496, and 166, respectively. After deduplication, a total of 3938 targets related to colorectal cancer were obtained, which were further validated using the UniProt database. The intersection of these genes revealed 204 common targets for the drugs and colorectal cancer (Figure 4A).

Figure 4 Network, GO and KEGG analysis of common targets of OXY and CPT-11 with colorectal cancer. (A) Venn diagram of drugs (OXY, CPT-11) and colorectal cancer targets; (B) Network diagram of common targets between drugs (OXY, CPT-11) and colorectal cancer; (C) Protein-protein interaction network diagram of common targets between drugs (OXY, CPT-11) and colorectal cancer; (D) GO analysis of drug-disease co-regulated target genes; (E) KEGG analysis of drug-disease co-regulated target genes.

Utilizing drug-target data, “network.xlsx” and “type.xlsx” files were constructed and imported into Cytoscape 3.8.2 for network visualization. The network contained 206 nodes and 252 edges (Figure 4B). After intersecting the regulatory targets of both drugs with colorectal cancer targets, 204 common targets for colorectal cancer and the combination of both drugs were identified. This network was imported into the String database for protein-protein interaction prediction, with Homo sapiens set as the species and confidence set to 0.7. The network file was saved in TSV format and imported into Cytoscape 3.8.2 software for protein-protein interaction network construction. Nodes with a degree greater than 5 were included, resulting in a network containing 111 nodes and 1686 edges. Topological analysis was performed on the network, with node size and color reflecting the degree value of the targets, and edge thickness reflecting the combined score, thereby constructing the protein-protein interaction network, as depicted in Figure 4C. Core targets included HSP90AA1, SRC, AKT1, EGFR, PIK3CA, PTPN11, JAK2, FYN, ESR1, and LCK.

GO Enrichment Analysis

Taking the jointly regulated genes of drugs and diseases, GO functional enrichment analysis was conducted using the DAVID database, resulting in a total of 559 GO entries. A significance threshold of P < 0.01 was used to filter significant enrichments for OXY and CPT-11 combination therapy in colorectal cancer. Among these, 380 entries were related to biological processes (BP), mainly involving positive regulation of protein phosphorylation, signal transduction, positive regulation of cell proliferation, peptidyl-tyrosine phosphorylation, autophosphorylation of proteins, negative regulation of apoptosis, inflammatory response, response to drugs, response to exogenous stimuli, and positive regulation of gene expression. Cellular components (CC) related to 64 entries included cytoplasm, cytosol, nucleus, plasma membrane, nucleoplasm, membrane, extracellular vesicle, integral components of membrane, extracellular region, and cytoplasmic side of the nucleus. Molecular function (MF) related to 115 entries involved protein binding, ATP binding, identical protein binding, protein kinase activity, protein serine/threonine kinase activity, protein tyrosine kinase activity, enzyme binding, kinase binding, protein homodimerization activity, and receptor binding. The top 10 entries for each aspect suggested that OXY and CPT-11 combination therapy might inhibit colorectal cancer cell proliferation by affecting cytoplasm, protein phosphorylation, and ATP binding at the cellular level, as shown in Figure 4D.

KEGG Pathway Enrichment Analysis

Using the DAVID database for pathway enrichment analysis, a total of 150 pathways related to OXY and CPT-11 combination therapy in colorectal cancer were enriched. After filtering based on P < 0.01, 130 pathways related to colorectal cancer were selected, including the PI3K-Akt signaling pathway, microRNAs in cancer, MAPK signaling pathway, Ras signaling pathway, chemokine signaling pathway, HIF-1 signaling pathway, EGFR tyrosine kinase inhibitor resistance, thyroid hormone signaling pathway, Rap1 signaling pathway, PD-L1 expression and PD-1 checkpoint pathway in cancer, regulation of actin cytoskeleton, phospholipase D signaling pathway, JAK-STAT signaling pathway, chronic myeloid leukemia, apoptosis, NOD-like receptor signaling pathway, T cell receptor signaling pathway, sphingolipid signaling pathway, platelet activation, and FoxO signaling pathway. Among these, OXY and CPT-11 combination therapy most likely inhibits colorectal cancer cell proliferation by suppressing the phosphorylation process of the PI3K-Akt signaling pathway, as illustrated in Figure 4E.

Comparison of PI3K/AKT Signaling Pathway-Related Gene Expression

The expression of PI3K and AKT in cells of all groups was significantly downregulated compared to the control group (P < 0.01 or P < 0.001). When comparing with the low PI3K/Akt expression group, the PI3K/Akt low expression + OXY combined with CPT-11 group showed a further reduction in the expression levels of PI3K and AKT (P < 0.05, Figure 5A). These findings suggest that both low expression of PI3K/Akt and OXY combined with CPT-11 intervention can interfere with the PI3K/Akt signaling pathway. However, the inhibitory effect of OXY combined with CPT-11 on the migration and invasion of colorectal cancer cells was significantly weakened after inhibition of the PI3K/Akt signaling pathway.

Figure 5 Effects of PI3K/Akt downregulation and combined OXY with CPT-11 on the migration and invasion ability of colon cancer cells. (A) Changes in gene expression levels of the PI3K/AKT signaling pathway; (B) Measurement results of cell migration; (C) Measurement results of cell invasion; (D) Photographs of scratch assays at 24 and 48 hours and statistical results. (A) **P <0.01, ***P <0.001, compared with Control group; #P <0.05, compared with PI3K/Akt siRNA group. (B-D): *P <0.05, **P <0.01, compared with CON group; #P <0.05, ##P <0.01, compared with the CPT-11 group; andP <0.05, andandP <0.01 compared with OXY + CPT-11 group; $P <0.05, compared with si-PI3K-Akt group.

Transwell Migration and Invasion Experiment Results

Compared to the CON group, the numbers of migrated and invaded cells across the membrane were significantly reduced in the other groups (P < 0.05 or P < 0.01). Cell migration and invasion levels were significantly reduced in the OXY + CPT-11 group compared to the CPT-11 alone treatment group (P < 0.01). Additionally, when compared to the PI3K/Akt low expression group, the numbers of migrated and invaded cells were also decreased in the PI3K/Akt low expression + OXY combined with CPT-11 group (P < 0.05). Conversely, compared to the OXY combined with CPT-11 group, the numbers of migrated and invaded cells were increased in the PI3K/Akt low expression + OXY combined with CPT-11 group (P < 0.05) (Figure 5B and C). These results indicate that both OXY combined with CPT-11 and low expression of PI3K/Akt can inhibit the migration and invasion of colorectal cancer cells. However, the inhibitory effect of OXY combined with CPT-11 on the migration and invasion of colorectal cancer cells is weakened after inhibition of the PI3K/Akt signaling pathway.

Scratch Test

Compared to the CON group, the migration distances of cells in the remaining groups at 12h and 24h were significantly shortened (P < 0.05 or P < 0.01). The migration distances were significantly reduced in the OXY + CPT-11 group compared to the CPT-11 alone treatment group at both 12h and 24h (P < 0.05 or P < 0.01). Moreover, when compared to the PI3K/Akt low expression group, the migration distances of cells in the PI3K/Akt low expression + OXY combined with CPT-11 group were also shortened at 12h and 24h (P < 0.05). Conversely, compared to the OXY combined with CPT-11 group, the migration distances of cells in the PI3K/Akt low expression + OXY combined with CPT-11 group were prolonged at 12h and 24h (P < 0.05) (Figure 5D). These results indicate that both low expression of PI3K/Akt and OXY combined with CPT-11 can inhibit cell migration. However, the inhibitory effect of OXY combined with CPT-11 on cell migration is weakened after inhibition of the PI3K/Akt signaling pathway.

Discussion

OXY has been found to inhibit various types of tumors. For instance, OXY has been shown to significantly inhibit the proliferation and migration of hepatocellular carcinoma cell lines QGY-7701 and SMMC-772113. Additionally, OXY can suppress the proliferation of NCI-H520 non-small cell lung cancer cells and induce apoptosis through the activation of the intrinsic pathway.20 In colorectal cancer, OXY is able to significantly inhibit the migration of HCT116 cells and TGF-β-induced HT-29 cells, thereby hindering tumor progression. CPT-11 is a commonly used drug in the treatment of colon cancer.21–23 Although there have been studies on the combined use of CPT-11 with various drugs for the treatment of colorectal cancer, research on the combination of OXY and CPT-11 is lacking. For example, CPT-11 and 5-azacytidine synergistically inhibit cancer cell growth.24 Additionally, CPT-11 inhibits cancer cell growth by modulating HIF-1α, and its combination with rapamycin effectively suppresses cancer cell proliferation.25 Furthermore, CPT-11 combined with flavonoids synergistically inhibits cancer cell growth, showing lower tumor volumes compared to monotherapy26. However, there are currently no reports on the combined treatment of colorectal cancer with OXY and CPT-11.

In our study, both OXY and Irinotecan CPT-11 demonstrated dose-dependent inhibition of colon cancer cell growth, supporting the potential efficacy of their combined application as reported in the literature. Furthermore, the co-administration of OXY and CPT-11 exhibited a synergistic additive effect, resulting in a stronger inhibitory effect on the proliferation of colon cancer cells. This may be attributed to OXY’s independent action on inhibiting cell growth and proliferation, and its synergistic effect when combined with CPT-11, resulting in a more effective suppression of tumor cell proliferation.

Several studies have found that the use of CPT-11 leads to diarrhea and weight loss. For instance, CPT-11 can cause diarrhea in up to 87% of patients27. It can also result in diarrhea, weight loss, and increased fecal water content28. Additionally, CPT-11 is associated with severe diarrhea, neutropenia, nausea, and vomiting, with weight loss considered a notable side effect.29 In our experiment, CPT-11-induced intestinal toxicity manifested as diarrhea and weight loss. However, in the group of mice treated with the combination of OXY, the symptoms of diarrhea were significantly alleviated, and the rate of weight loss was reduced. This suggests that OXY can to some extent mitigate the intestinal toxicity of CPT-11 and alleviate its impact on the overall health of the mice.

Since the spleen plays a crucial role in the body’s immune system, we examined the spleen index. The results showed a significant increase in the spleen index in the CPT-11 group of mice, while the spleen index in the group of mice treated with the combination of OXY and CPT-11 was relatively lower. This indicates that OXY may attenuate the negative impact of CPT-11 on the immune system of mice by modulating immune function.

Drug target prediction and enrichment analysis revealed that OXY may exert its alleviating effect on CPT-11-induced intestinal toxicity by influencing multiple signaling pathways and genes related to cellular immunity. This provides important clues for further exploration of the molecular mechanisms and pathways of OXY’s action.

Cell experiments further validated the inhibitory effect of OXY on the proliferation and invasion of colon cancer cells induced by CPT-11. Additionally, through cell transfection experiments, it was found that OXY may modulate the PI3K/Akt signaling pathway to affect the growth and invasion capacity of colon cancer cells. This is consistent with previous findings that OXY specifically inhibits the AKT/GSK-3β/MCL-1 axis, thereby reducing the viability, proliferation, and migration of cervical cancer cells.30

In conclusion, the combined application of Oxyresveratrol (OXY) and Irinotecan (CPT-11) holds potential clinical value in alleviating CPT-11-induced intestinal toxicity and enhancing the inhibition of colon cancer cell proliferation. However, further research is warranted to validate the mechanism of action and the safety and efficacy of OXY in clinical applications. This study provides important experimental groundwork for the clinical translation of the combined application of OXY and CPT-11 in the treatment of colon cancer.

Data Sharing Statement

The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

Ethics Approval and Consent to Participate

The study protocol was approved by the Ethics Committee of Sichuan University. Procedures involving animals and their care were conducted in conformity with NIH guidelines (NIH Pub. No. 85-23, revised 1996) and was approved by Animal Care and Use Committee of the Sichuan University. Informed consent to participate was waived because this study does not involve any human participants.

Consent for Publication

Informed consent for publication was waived because this study does not involve any human participants.

Funding

No funding was received for this study.

Disclosure

The authors declare that they have no competing interests.

References

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