Study on the anti colorectal cancer HCT116 cells effect of baicalin based on Hippo signaling pathway
Colorectal cancer (CRC) is a common malignant digestive tract tumor. The latest cancer statistics show that the prevalence and mortality rate of CRC have risen rapidly in the world due to aging, changes in eating habits, obesity, smoking and other factors, ranking third and second respectively. The incidence rate of CRC in developed countries is about four times higher than that in developing countries. Although CRC has made significant breakthroughs in diagnostic techniques and treatment methods in recent years, its five-year survival rate is still less than 10%. Chemotherapy is the cornerstone of anti-tumor treatment. 5-Fluorouracil, oxaliplatin, irinotecan, and other commonly used drugs for the treatment of CRC are often limited in clinical application due to toxic side effects such as mucositis, bone marrow suppression, and dehydration. Therefore, exploring the pathogenesis of CRC and seeking therapeutic drugs with low toxicity and strong targeting remain the focus of current CRC research.
Practice has proven that traditional Chinese medicine has multiple advantages in anti-tumor treatment. It can not only enhance the efficacy of chemotherapy drugs, reduce adverse reactions, reverse drug resistance, but also improve prognosis and enhance patients’ quality of life. Moreover, traditional Chinese medicine contains various active ingredients, and research has shown that many active ingredients can exert anti-tumor effects by affecting cell migration, invasion, and apoptosis. Therefore, traditional Chinese medicine and its active ingredients have good development prospects for the treatment of tumors. Scutellarin (SCU), a flavonoid compound mainly derived from traditional Chinese medicine Scutellaria baicalensis, Scutellaria baicalensis, and Scutellaria baicalensis, has various pharmacological effects such as anti-inflammatory, antioxidant, anti fibrotic, neuroprotective, and cardiovascular and cerebrovascular protection. Recent studies have found that SCU also has extensive anti-tumor activity, which can inhibit the occurrence and development of various malignant tumors such as colorectal cancer, liver cancer, lung cancer, esophageal squamous cell carcinoma, etc. It is a potential anti-tumor drug.
The Hippo signaling pathway has been widely studied in the field of cancer research over the past decade. Research has confirmed that abnormal blockade of the Hippo signaling pathway is closely related to the survival, proliferation, invasion, and development of drug resistance in tumor cells. When the Hippo signaling pathway is inhibited, the main effector factors of the pathway, Yes associated protein (YAP)/Transcription co activator with PDZ binding motif (TAZ) 3, undergo nuclear translocation and aggregate in the nucleus, binding to TEA transcription factors 1-4 and promoting transcription of downstream target genes such as cell cycle regulator cyclin E1 and oncogene c-Myc. The Hippo signaling pathway plays an important role in maintaining normal intestinal homeostasis and repairing and regenerating damaged tissues. High YAP/TAZ activity is often an independent predictor of poor prognosis in CRC, and is positively correlated with tumor staging, lymph node status, and metastasis. In addition, abnormal activation of YAP/TAZ is associated with the absence of upstream regulatory factors in the Hippo signaling pathway. The absence of mammalian sterol 20 like kinase 1/2 (MST1/2) or large tumor suppressor 1/2 (LATS1/2) can lead to varying degrees of activation of YAP/TAZ.
So far, the anti CRC effect of SCU has been confirmed in multiple aspects. Studies have shown that SCU can exert its effects through various signaling pathways, including Hedgehog, Wnt/β – catenin, and others. However, there is currently no research report on the anti CRC effect of SCU by regulating the Hippo signaling pathway. Therefore, this study mainly uses colorectal cancer HCT116 cells as an in vitro model to investigate the effects of SCU on the proliferation, migration, and apoptosis of colorectal tumor cells, and explore its mechanism of action, providing reference and experimental basis for the development of CRC therapeutic drugs.

 

More and more evidence suggests that SCU can inhibit the occurrence and development of tumors through various pathways, and has the potential to be developed into anti-tumor drugs. Sun et al. found that SCU can significantly inhibit the proliferation of non-small cell lung cancer PC-9 and H1975 cells. The research results of Han et al. also indicate that SCU can significantly inhibit the proliferation of human osteosarcoma 143B and U2OS cells in a concentration dependent manner. In this study, SCU significantly reduced the activity of colorectal tumor HCT116 cells, inhibited cell cloning, and hindered their proliferation, consistent with the above conclusion.
In cancer, cancer cells often acquire the ability to proliferate and invade through epithelial mesenchymal transition. In hepatocellular carcinoma, Liu et al. found that SCU can inhibit epithelial mesenchymal transition of liver cancer cells and reduce their ability to invade and metastasize by downregulating the activity of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Li et al. found that in gastric cancer, SCU can regulate the activity of the PTEN/PI3K signaling pathway, downregulate the expression levels of E-cadherin in gastric cancer MGC-803 and AGS cells, upregulate the expression levels of vimentin and N-cadherin, and inhibit the epithelial mesenchymal transition of gastric cancer cells. In this study, the results of cell scratch assay showed that compared with the blank control group, the migration rate of HCT116 cells in the SCU treated group was significantly reduced in a concentration dependent manner, indicating that SCU has an inhibitory effect on the migration ability of colorectal tumor cells, which is consistent with previous research results.
The currently widely accepted view is that insufficient cell apoptosis is an important cause of tumor occurrence and development. Therefore, inducing tumor cell apoptosis is considered an effective means of tumor treatment. Cao et al.’s study confirmed that SCU can block cisplatin resistant lung adenocarcinoma A549 cells in the G0/G1 phase and induce apoptosis by inhibiting the AKT/mTOR/4EBP1 and STAT3 pathways. In liver cancer, Xu et al. found that SCU can reduce the activity of the STAT3 signaling pathway by inhibiting the production of reactive oxygen species (ROS) in HepG2 liver cancer cells, thereby downregulating the expression levels of downstream target proteins BCL-XL and MCL-1 and inducing cell apoptosis. Similarly, in this study, we found through Hoechst 33342/PI double staining and flow cytometry detection that SCU can induce cell apoptosis and significantly increase the apoptosis rate of HCT116 cells. However, the specific regulatory mechanism of SCU is still unclear, so further exploration has been conducted in subsequent studies.
Studies have shown that the Hippo signaling pathway can participate in the regulation of cell proliferation, and excessive activation or expression of YAP can lead to excessive cell proliferation in multiple tissues such as the liver, gastrointestinal tract, skin, and heart. In addition, YAP and TAZ are active inducers of epithelial mesenchymal transition in cells. High activity YAP and TAZ can induce the expression of the key transcription regulator ZEB1/2 in epithelial mesenchymal transition, thereby promoting epithelial mesenchymal transition. The Hippo signaling pathway also plays an important regulatory role in cell apoptosis. YAP can promote cell apoptosis by regulating the transcription of downstream anti apoptotic genes such as COX-2, Survivor, and Glut1; Moreover, overexpressed YAP can also block tumor necrosis factor, FAS, and chemotherapy induced apoptosis; The study by He et al. also confirmed that YAP can drive AKT dependent phosphorylation of NR4A1 through Ser351, isolating it in the nucleus and weakening its role in promoting cell apoptosis.
The results of this study indicate that SCU can upregulate the mRNA and protein expression levels of MST1 and LATS1 in HCT116 cells, downregulate the mRNA and protein expression levels of YAP1 and TAZ, promote YAP phosphorylation, and increase the protein expression level of p-YAP (Ser127). Therefore, it is speculated that the role of SCU may be related to the activation of the Hippo signaling pathway. The qRT PCR detection results showed that SCU treatment for 24 hours effectively increased the mRNA expression levels of Bax, caspase-3, and casepase-9 in HCT116 cells, while reducing the mRNA expression level of Bcl-2; Western blot analysis also showed that SCU significantly increased the expression level of pro apoptotic protein Bax and decreased the expression level of anti apoptotic protein Bcl-2 in HCT116 cells. In addition, the mRNA and protein expression levels of c-Myc were significantly reduced after 24 hours of SCU treatment. The above results suggest that SCU may activate the Hippo YAP/TAZ cascade, promote YAP/TAZ phosphorylation, inhibit its transcriptional activity, suppress the expression of downstream target gene c-Myc, reduce the expression level of apoptosis protein family member Bcl-2, increase the expression level of Bax, and migrate into mitochondria to promote the release of cytochrome C and the formation of apoptosis complex, thereby activating the apoptosis initiating factor caspase-9 in the caspase family and downstream apoptosis executing factor caspase-3, ultimately inducing cell apoptosis through the mitochondrial pathway, hindering the growth of colorectal cancer cells, and exerting anti colorectal cancer effects.
In summary, this study found that SCU can significantly inhibit the proliferation and migration of colorectal cancer HCT116 cells, and induce cell apoptosis. Its mechanism of action may be related to the activation of the Hippo signaling pathway. However, considering the complex causes of colorectal cancer formation and the differences between tumor cell lines from different sources, the anti colorectal cancer effect of SCU still needs to be validated on multiple other colorectal cancer cell lines and further comprehensively explored through in vivo animal experiments. In addition, it has been reported that Wnt, Notch, Hedgehog, and BMP signaling pathways can all participate in the regulation of intestinal homeostasis and regeneration, and studies have found that the Hippo signaling pathway can interact with these pathways, leading to crosstalk. Therefore, it is necessary to further explore whether SCU exerts its anti colorectal cancer effect by comprehensively regulating the activity of Hippo signaling pathway and its crosstalk pathway.