Guanguan Tengzhi G inhibits the proliferation of colorectal cancer cells through the ATM-CH2-p53 signaling pathway
Colorectal cancer (CRC), as one of the common malignant tumors, its incidence rate ranks the third in the world and its mortality ranks the second in the world. Although advances in diagnosis and treatment have extended the lives of some patients, the 5-year survival rate is still only 64%, which is not significantly different from 10 years ago. At present, the main treatment for colorectal cancer is surgery combined with radiotherapy and chemotherapy, and its serious side effects significantly reduce the quality of life of patients. In recent years, relevant studies have found that natural medicines have certain advantages in inhibiting the proliferation of colorectal cancer and reducing the toxic side effects of radiotherapy and chemotherapy, making them an important means of treating colorectal cancer.

Marsdeniatenacissima – (Roxb.) Wight et Arn is a medicinal herb from the family Asclepiadaceae, mainly produced in provinces such as Yunnan and Guizhou. Research has found that Tongguan Vine has a wide range of pharmacological effects, including anti-cancer, asthma relief, immune regulation, blood pressure reduction, pain relief, diuresis, and alleviation of leukopenia in cancer patients caused by radiotherapy and chemotherapy. In addition, it is also used clinically to treat various tumors such as gastric cancer, lung cancer, liver cancer, leukemia, etc., with significant therapeutic effects. Research has shown that the total saponins of Tongguan Vine have anti proliferative effects on liver cancer cells and are its main anti-cancer active ingredients. The chemical structure formula of tenacissoside G (TG) is shown in Figure 1, and it is one of the components in the total saponins of tenacissoside G. There are research reports that TG may be the main active ingredient in the treatment of non-small cell lung cancer in Tongguan Teng, exerting anti lung cancer effects through multiple signaling pathways such as PI3K/Akt. However, there are currently no reports on the role and mechanism of TG in combating colorectal cancer. This experiment takes human colorectal cancer cells RKO and LoVo as the research objects, observes the effect of TG on the proliferation activity of colorectal cancer cells, and explores its possible mechanism of action.

As a traditional Chinese medicine, Tongguan Vine has anti-cancer effects in clinical practice. According to relevant research reports, the anti-cancer agent extracted from Tongguan Vine has a significant inhibitory effect on tumor cell proliferation, and its total saponin extract can induce cell apoptosis and exert its anti-cancer activity. Therefore, we conducted a study on the pharmacological effects and molecular mechanisms of the main active ingredient, Tongguan Vine Glycoside G, on colorectal cancer. The experimental results showed that Tongguan Tengzhi G can significantly inhibit the proliferation of colorectal cancer cells and induce DNA damage, activate the ATM-CHK2-p53 pathway, and cause p53 mediated cell cycle arrest and apoptosis.

Maintaining the integrity of genomic DNA is crucial for the growth and proliferation of cells, however, cells may experience varying degrees of DNA damage due to various physical or chemical factors, both internal and external. Among them, DNA double strand breaks are the most severe type of DNA damage. When DNA undergoes double strand breaks, the cellular DNA damage response mechanism phosphorylates the H2AX histones surrounding the DNA, forming gamma H2AX. Gamma H2AX can cluster at the site of damage within minutes to form gamma H2AX foci, and the number of foci corresponds positively to the level of DNA double strand breaks. Therefore, it is recognized as one of the sensitive molecular markers for DNA double strand breaks. We first used comet assay to detect the intracellular DNA damage after TG administration and found that the tail DNA content of the drug treated group was significantly increased, indicating the induction of DNA damage in colorectal cancer cells by TG. In addition, the results of immunofluorescence staining showed that compared with the control group, the drug treatment group had a significant increase in the number of intracellular γ – H2AX positive foci and a significant increase in the expression level of γ – H2AX, further proving that TG induces DNA double strand breaks to cause DNA damage.

DNA damage response refers to the DNA modification initiated by an organism to protect cells from damage when stimulated; This mainly includes activation of periodic checkpoints, DNA damage repair, and DNA damage induced cell apoptosis. The pathways used to repair DNA damage in cells are complex and diverse. When a DNA double strand breaks, the damage receptor immediately recognizes the damage and adopts homologous recombination repair and non homologous end connected recombination repair. Among them, ATM protein is one of the key points involved in cell damage recognition and repair, and it participates in DNA damage response through the above two pathways. When ATM protein is activated, downstream target CHK2 undergoes phosphorylation and activates p53, causing cell cycle arrest and inducing cell apoptosis. Among them, G1/S cycle progression arrest is mainly caused by the upregulation of p21, a cyclin dependent kinase inhibitor induced by p53. Both flow cytometry and Western blot results showed that TG can induce G0/G1 phase arrest by upregulating p21 levels and inhibiting the expression of cycle related proteins CDK2, CDK4, CDK6, Cyclin D1, and CyclinE.

Research has shown that the ATM-CHK2-p53 signaling pathway plays an important role in DNA damage induced cell apoptosis. Based on its DNA damage induction effect, we conducted molecular mechanism research on the regulatory effect of TG on the ATM-CHK2-p53 signaling pathway. The Western blot experiment results showed that after 48 hours of TG administration, the intracellular phosphorylation levels of ATM and CHK2 significantly increased, and the expression and phosphorylation levels of p53 protein were also significantly upregulated. At the same time, we detected the intracellular apoptosis markers cleanedPARP and cleanedCaspase 3 and found that their expression levels significantly increased with increasing drug concentration, indicating that TG has an apoptosis inducing effect on colorectal cancer cells. It can be inferred that the activated ATM in cells after TG treatment may induce phosphorylation of its downstream protein CHK2 and further activate p53, leading to p53 mediated cell apoptosis.

Cell cycle inhibition plays an important role in the anti proliferation of colorectal cancer cells. Cyclin and cyclin dependent kinase (CDK) regulate the cell cycle by forming CDK Cyclin complexes and phosphorylating target proteins during the cell cycle process. Among them, CDK2 CyclinE and CDK4/6-Cyclin D1 play important roles in the transition of cells from G1 phase to S phase, while p21 has an inhibitory effect on this cycle transformation process. Due to the upregulation of p53 activity by TG through the ATM-CH2-p53 signaling pathway, activated p53 may initiate G1/S phase monitoring points and induce G0/G1 phase cell cycle arrest by activating p21.

In summary, this study found that TG has a proliferative inhibitory effect on colorectal cancer cells and induces G0/G1 phase arrest and apoptosis. In addition, TG can cause cellular DNA damage and activate the ATM-CHK2-p53 signaling pathway. Therefore, it suggests that TG may induce p53 mediated cell cycle arrest and apoptosis, and inhibit colorectal cancer cell proliferation by regulating the ATM-CH2-p53 signaling pathway. This study provides a certain theoretical basis for the treatment of colorectal cancer with Guantongteng G, and other related mechanisms of action need further research.