Acetyl Habaside induces apoptosis in colon cancer HCT116 cells through Wnt signaling pathway
Colon cancer is the fourth deadliest cancer in the world and a common malignant tumor of the digestive system. Genetics, obesity, lack of exercise, and smoking are risk factors for developing colon cancer. At present, surgical treatment is mainly relied on in clinical practice for colon cancer. Due to the difficulty in detecting colon cancer in its early stages, the best surgical opportunity has been missed when diagnosed. At this time, patients often need radiotherapy and chemotherapy to improve their prognosis. However, traditional chemotherapy drugs have more adverse reactions, while natural drugs have fewer adverse reactions and are more affordable.

Ajuga ciliata Bunge is a perennial herbaceous plant in the family Lamiaceae, mainly distributed in Hebei, Shandong, Shaanxi, Gansu, Sichuan and other regions of China. The whole plant of Gynostemma pentaphyllum can be used as medicine and is widely used in Chinese folk medicine to treat diseases such as pulmonary heat hemoptysis, traumatic injury, tonsillitis, pharyngitis, etc. The content of 8-O-acetylharpagide (8-OA) in Gynostemma pentaphyllum is the highest. According to previous studies by You et al., acetyl rhamnoside has strong anti-inflammatory effects and can alleviate early inflammatory exudate in capillaries. As inflammatory mediators themselves can play a regulatory role in tumor progression, acetyl rhamnoside may exert anti-tumor effects through the inflammatory pathway. In addition, Konoshima et al. found that acetyl rhamnoside has a significant inhibitory effect on nitric oxide induced skin cancer in mice and can inhibit the proliferation of liver cancer cells induced by N-nitrosodiethylamine and phenobarbital, indicating that acetyl rhamnoside may be a potential anti-tumor drug.

The aim of this study is to explore the role of acetylhababine in anti-tumor effects by investigating a series of biological behavioral changes after acetylhababine acts on colon cancer HCT116 cells.

 

Colon cancer accounts for approximately 10% of cancer-related deaths worldwide each year. At present, surgery is the main treatment for colon cancer, supplemented by chemotherapy and radiation therapy. For advanced chemotherapy patients, the efficacy of chemotherapy is low and the side effects are significant, with a survival period of only about 18 months. In recent years, searching for anti-tumor drugs in natural plants has become a major research hotspot, as some traditional chemotherapy drugs, including paclitaxel and vincristine, are derived from natural plants.

Anti proliferation is an important entry point for the development of anticancer drugs. The CCK8 method showed that the inhibitory effect of acetyl rhamnoside on HCT116 cells is concentration time dependent, with IC50 values of 0.91, 0.39, and 0.39 mmol/L at three time points, indicating that acetyl rhamnoside mainly exerts its effect within the first 48 hours. In addition, it was demonstrated through plate cloning experiments that acetyl rhamnoside can significantly reduce the colony forming ability of HCT116 cells.

The proliferation and survival ability of tumor cells are much stronger than normal cells, and most anticancer drugs can inhibit tumor cell proliferation by inducing cell cycle arrest and apoptosis. During the process of cell death, there are often phenomena such as increased levels of reactive oxygen species, activation of cysteine protease, and chromatin condensation. Among them, the loss of mitochondrial transmembrane potential induces the release of cytochrome c from mitochondria to cytoplasm, which is a key step in inducing apoptosis. Bax and Bcl2 are crucial for the release of cytochrome c and the activation of downstream caspase proteins. We found through flow cytometry analysis that acetyl rhamnoside can dose dependently affect the cell cycle of colon cancer HCT116 cells, causing cell cycle arrest in the G1 phase and significantly increasing cell apoptosis rate.

Research has confirmed that the Wnt signaling pathway is highly correlated with the development of colon cancer. Among them, β – catenin is a key molecule in the classic Wnt pathway, and the E-cadherin/β – catenin complex helps stabilize intercellular adhesion, thereby reducing cell invasion and metastasis. In tumor cells, β – catenin can activate gene transcription related to cancer cell proliferation, cycle, apoptosis, migration, invasion, and drug resistance, leading to malignant proliferation of tumors. In addition, β – catenin has been proven to be a promising target for cancer prevention and treatment. Many natural products can act as inhibitors of β – catenin signaling, mainly through phosphorylation, ubiquitination, and inhibition of its nuclear translocation. Natural product inhibitors have shown good preventive and therapeutic effects in various tumor models in vitro and in vivo. β – catenin can promote the expression of downstream molecule Myc in the nucleus. Myc belongs to the oncogene family and encodes essential nuclear transcription factors in the nucleus, mainly involved in regulating cell growth, proliferation, differentiation, cycle, metabolism, apoptosis, etc. In mammals, the Myc family of proteins includes three categories: c-Myc, n-Myc, and l-Myc. c-Myc is associated with genomic instability and tumorigenesis. In quiescent cells, the expression level of c-Myc is low, and once the cell enters the cell cycle, the expression of c-Myc rapidly increases several times. In addition, significant upregulation of c-Myc protein was found in approximately 70% of colon cancers, indicating a positive correlation between c-Myc and the malignancy of colon cancer.

In this experiment, high-throughput transcriptome sequencing was used to identify 4129 differentially expressed genes in HCT116 cells induced by acetyl rhamnoside, including 1921 upregulated genes and 2208 downregulated genes. The differentially expressed genes were mainly related to cell growth, cell cycle, cell invasion, cell immunity, oxidative stress, cell metabolism, molecular transport, and cell death pathways. After analyzing the differentially expressed genes in combination with KEGG database, it was found that acetyl rhamnoside significantly affected the Wnt signaling pathway in HCT116 cells. The significant downregulation of β – catenin and c-Myc was confirmed through gene expression and protein detection of acetyl rhamnoside and the control group. The combination of acetyl rhamnoside and Wnt small molecule inhibitor XAV939 further inhibited the growth of HCT116 colon cancer cells, indicating that the Wnt/β – catenin pathway also mediates the drug resistance of HCT116. Inhibiting the Wnt/β – catenin pathway can significantly enhance the anti-tumor activity of acetyl rhamnoside.

This study showed that acetyl rhamnoside can effectively inhibit the proliferation activity of colon cancer cell line HCT116. Genomic testing revealed significant differences in the Wnt/β – catenin signaling pathway of cells, suggesting that acetyl rhamnoside may inhibit normal cell growth by regulating the Wnt/β – catenin signaling pathway, further inducing cell apoptosis and cycle arrest, thereby inhibiting tumor cell growth. However, this speculation requires further evidence, and its specific mechanism of action requires further in-depth research.