Exploring the mechanism of action of Canmei Formula in treating colorectal cancer based on network pharmacology, molecular docking, and experimental verification
Colorectal cancer (CRC) is considered the third most common cancer in men, the second most common cancer in women, and the second leading cause of cancer death worldwide. The incidence rate and mortality of colorectal cancer are rising rapidly, especially in low-income and middle-income countries. It is estimated that by 2030, the global burden of colorectal cancer will increase by 60%, with more than 2.2 million new cases and 1.1 million cancer deaths. Traditional treatment methods include surgery, medication, chemotherapy, etc., but the overall survival rate is not ideal.
Traditional Chinese medicine, as an important anti-tumor drug, has been clinically proven. Previous studies have shown that combining traditional Chinese medicine with chemotherapy or radiotherapy can significantly improve the survival rate of patients for 1-3 years, reduce adverse reactions, improve quality of life, and prolong survival time. It is a particularly important method for treating colorectal cancer. There is no exact record of the name of colorectal cancer in ancient medical books, and it is now mostly classified as “anal hemorrhoids”, “intestinal wind”, “accumulation” and other categories. The Compendium of Materia Medica records: “When the intestinal wind causes blood to flow, stir fry the silkworms to remove their mouth and feet, roast one or two pieces of black plum meat each, and grind them into powder. Take one hundred pills each time, with white soup before eating, three times a day.” Among them, the silkworms have a mild nature, salty and pungent taste, and are used to dispel wind, stop startle, and disperse phlegm and nodules in the liver, lungs, and stomach meridians; Ume has a flat nature, a sour and astringent taste, and belongs to the liver, spleen, lungs, and large intestine meridians. It can tighten the lungs, astringent the intestines, and eliminate decay. The two drugs are separated to remove yin and yang, and the sour and pungent powder is collected. Therefore, the formula is named Canmei Decoction (CMF). Research has shown that CMF extract has a preventive and therapeutic effect on AOM/DSS induced adenomatous intestinal polyps in mice, and can significantly reduce the incidence of adenomatous polyps in AOM/DSS mice.
Network pharmacology is a new discipline that explores the interaction between drugs and the body from a systemic and holistic perspective. In order to explore the potential active ingredients and molecular mechanisms of traditional Chinese medicine formulas to exert their therapeutic effects, this discipline is based on modern bioinformatics research methods and deeply analyzes the synergistic mechanisms between component component, component target, and target disease networks, reflecting the characteristics of multi-component and multi-target effects in traditional Chinese medicine formulas and highlighting the outstanding advantages of systematicity and holism. Scholars have used network pharmacology methods to explore the potential mechanism of action of CMF against colorectal adenoma (CRA), but there are no research reports on the mechanism of action of CMF against CRC using network pharmacology methods. This study utilizes network pharmacology research methods to explore the signaling pathways through which CMF exerts its effects, providing new ideas for the mechanism of action of CMF multi-component and multi-target therapy.

 

This study systematically analyzed the potential mechanism of action of CMF in the treatment of colorectal cancer based on network pharmacology, molecular docking, and experimental verification. A target network of active ingredients from Bombyx mori and Ume nigra was constructed to predict potential targets and signaling pathways. Through topological data analysis, it was found that quercetin and kaempferol may play a crucial role in the treatment of colorectal cancer with CMF.

Research has shown that quercetin has pharmacological activities such as anti-inflammatory, antioxidant, antiplatelet aggregation, anti-tumor, and immune suppression. It can promote cell apoptosis and autophagy, as well as inhibit angiogenesis and inflammation to suppress the progression of colorectal cancer. Inducing cell apoptosis by inhibiting signaling pathways such as MAPK/Erk, PI3K/Akt, and NF – κ B. Natural flavonol naphthol has various pharmacological activities such as antibacterial, anti-inflammatory, antioxidant, and anti-tumor, and is currently being used in cancer chemotherapy. The above existing research results are basically consistent with the predicted results, indicating that the main active ingredients of CMF have a definite therapeutic effect on the prevention and treatment of colorectal cancer.

The PPI protein interaction network analysis results show that the potential target genes for CMF prevention and treatment of colorectal cancer include TP53, AKT1, MAPK8, MAPK1, JUN, and IL6. TP53 is a tumor suppressor protein that strictly regulates cell growth by promoting cell apoptosis and DNA repair. Research has shown that mutated TP53 can lead to abnormal proliferation of cancer cells and tumor progression in colorectal cancer patients. AKT1 (AKT) is a core target in the PI3K/AKT signaling pathway, involved in the growth, proliferation, apoptosis, metabolism, tumor drug resistance, and immune escape of tumor cells. MAPK1 and MAPK8 are both members of the MAP kinase family. MAP kinase, as an integration point of various biochemical signals, participates in various cellular processes such as differentiation, proliferation, transcriptional regulation, etc. Experimental results have shown that miR-422A inhibits colon cancer cell proliferation by targeting MAPK1 and AKT1. C-jun is a protein of JUN gene, and colorectal cancer patients show susceptibility to c-jun, which is closely related to survival rate. IL-6 is a key cytokine in acute inflammatory response, involved in the pathogenesis of various chronic inflammatory diseases including cancer. IL-6 family cytokines have been identified as biomarkers for detecting cancer. In summary, the effective ingredient targets of CMF interact with the disease targets of colorectal cancer, fully leveraging the unique advantages of traditional Chinese medicine in treating colorectal cancer with multiple components and targets.

GO functional and KEGG pathway enrichment analysis revealed that CMF is involved in biological processes such as cell proliferation, cell cycle, apoptosis, signal transduction, oxidative stress, and regulation of protein serine/threonine kinase activity; The number of genes enriched in cancer pathways, IL-17 signaling pathway, PI3K-AKT signaling pathway, HIF-1 signaling pathway and other pathways is the highest, indicating that these pathways may play an important role in the mechanism of CMF therapy for colorectal cancer.
Cytokines are important inflammatory mediators for cancer growth and invasion. IL-17 is the main cytokine produced by Th17 cells, which can induce neutrophils and macrophages to produce inflammatory cytokines and chemokines. It participates in various processes including host defense, tissue repair, pathogenesis of inflammatory diseases, and cancer progression, and plays a key role in human malignant tumors. In the intestine, IL-17 signaling promotes adenoma formation by enhancing the proliferation and survival of intestinal cells with Apc mutations, impairing intestinal barrier function, activating IL-17 response within tumors, and promoting tumor growth. In addition, IL-17-induced cytokine and chemokine granulocyte mobilization inhibits cells, promoting angiogenesis and suppressing anti-tumor immunity. The classic autophagy signaling pathway PI3K/AKT/mTOR is involved in the growth, proliferation, survival, apoptosis, metabolism, as well as tumor drug resistance and immune escape of tumor cells. The activation of this signaling pathway is a hallmark of cancer development. PI3K signaling pathway activation mutations are common in advanced breast cancer, gastric cancer, colorectal cancer and other solid cancers, and the mutation rate can increase by 30%~60%. The active ingredients of CMF can affect the occurrence and development of colorectal cancer by regulating multiple pathways, providing new ideas for clinical treatment.
The molecular docking results of “active ingredient target” showed that the core active ingredients of CMF, quercetin and kaempferol, exhibited good affinity with key targets, and the binding site conformation was stable with strong binding ability, further verifying the prediction of network pharmacology in this study.
Based on the above research results, the main active ingredient quercetin was selected as the subsequent cell experiment to explore its potential mechanism in depth. The experimental results showed that intervention of different concentrations of quercetin in HCT116 cells can significantly inhibit cell proliferation and promote apoptosis, reduce the expression of anti apoptotic protein Bcl-2, and increase the expression of pro apoptotic protein BAX. The differences were statistically significant (P<0.05).
The analysis of the potential mechanism of CMF in the prevention and treatment of colorectal cancer from the perspective of systems biology is in line with the characteristics of traditional Chinese medicine using compound Chinese medicine with multiple targets, pathways, and systems to regulate the body and prevent diseases as a whole. It also helps to reveal the scientific connotation of traditional Chinese medicine compound and guide the development of new Chinese medicine drugs. Traditional Chinese medicine has broad prospects in the prevention and treatment of tumors, but there are certain limitations in network pharmacology research methods, and multidimensional experiments are still needed for further verification in the later stage.