Effects of Taraxacum polysaccharide on migration and invasion of triple negative breast cancer cells based on PI3K/Akt/GSK-3 β pathway
Triple negative breast cancer (TNBC) is an estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2), which are all negative subtypes of breast cancer, accounting for only 15%~20% of all breast cancer types. However, due to its high malignancy, strong invasion, and lack of specific targeted drugs, it is more prone to recurrence and metastasis than other types of breast cancer, and its prognosis is worse. At present, surgery, radiotherapy, and chemotherapy are still the main treatment methods for TNBC, but there is still a risk of recurrence and metastasis after surgery. Chemotherapy drugs have serious toxic side effects and are prone to drug resistance. Therefore, searching for new and efficient targeted drugs, screening drug action molecular targets, and elucidating drug targeted anti-tumor molecular mechanisms are undoubtedly effective strategies for the clinical treatment of TNBC.

In recent years, traditional Chinese medicine has become an important source of targeted anti-tumor drugs. Compared with general anti-cancer drugs, traditional Chinese herbal medicines and their active ingredients have the characteristics of multi-target and multi pathway synergistic anti-cancer pharmacological activity, and have the advantages of easy absorption and utilization by the body, low toxicity and side effects. They have gradually become a hot spot in the research and development of anti-cancer drugs. Dandelion, a traditional Chinese medicine, is a dry whole plant of Taraxacum monogolicum Hand. Mazz, a plant in the Asteraceae family, and T. boroisinense Kitam (or other plants of the same species). It has the effects of clearing heat and detoxifying, reducing swelling and dispersing nodules, and promoting diuresis and drainage. It can be used to treat diseases such as breast abscess, scrofula, and edema. Traditional Chinese medicine clinical research has found that dandelion has a significant effect in the treatment of breast cancer, gastric cancer and other malignant tumors. Modern clinical and pharmacological studies have shown that dandelion and its active ingredients not only have pharmacological activities such as anti-inflammatory and antioxidant effects, but also have more significant anti-tumor effects. Dandelion polysaccharide (DP) is a polysaccharide extracted from dandelion roots, consisting of glucose, galactose, arabinose, mannose, rhamnose, and glucuronic acid. It has pharmacological activities such as anti-tumor, antibacterial, anti-inflammatory, antioxidant, hypoglycemic, and immune enhancement. Currently, research has found that DP can exert its anti-tumor pharmacological activity through multiple targets and pathways. DP can inhibit liver cancer angiogenesis in vitro and in vivo by reducing the expression of hypoxia inducible factor-1 alpha (HIF-1 alpha) and vascular endothelial growth factor (VEGF) proteins. DP can also inhibit liver cancer iron metabolism by reducing the expression of iron transporters (Ferroporin) and iron burn, thereby suppressing the growth of HepG2 and Huh7 cells. At the same time, DP can significantly reduce iron deposition in tumor tissues of liver cancer bearing mice. In breast cancer, DP can achieve its inhibitory effect on human breast cancer MCF-7, SK-BR-3 and T47D cells by inhibiting cell proliferation. However, there have been no reports on the anti TNBC effects of DP, and its anti-tumor molecular mechanism is still unclear.
The abnormal activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway has been confirmed to be closely related to the growth, migration, invasion, and epithelial mesenchymal transition (EMT) of TNBC. PI3K is an intracellular phosphoinositol kinase that can be activated by extracellular signal stimulation. Activated PI3K recruits Akt protein to the cell membrane, which in turn activates Akt protein. The activated Akt phosphorylates downstream substrate glycogen synthase kinase-3 β (GSK-3 β), inactivating GSK-3 β. The inactivated GSK-3 β participates in multiple signaling pathways and induces tumor cell proliferation, drug resistance, migration, and invasion, making it a highly promising drug target. So far, a large number of GSK-3 β inhibitors have been synthesized, but due to poor selectivity and side effects of most inhibitors, only a few have entered clinical trials. Therefore, the search for efficient and low toxicity PI3K/Akt/GSK-3 β inhibitors is of great significance for the treatment of TNBC. EMT is a key process in the migration and invasion of tumor cells. During this process, the adhesion between tumor cells weakens, the epithelial cell characteristics decrease, and the stromal cell characteristics increase, allowing them to enter the bloodstream and invade other organs. At the molecular level, a decrease in the expression of epithelial like marker E-cadherin and an increase in the expression of mesenchymal markers N-cadherin and vimentin indicate the occurrence of cellular EMT. Therefore, the search for drugs that inhibit EMT has become a necessary condition for preventing TNBC migration and invasion.
Therefore, based on the PI3K/Akt/GSK-3 β signal pathway, this study took the human triple negative breast cancer MDA-MB-231 cell as the research object. By observing the effects of DP on the proliferation, migration, invasion and EMT of human breast cancer MDA-MB-231 cells, this study analyzed the key molecular targets of DP against TNBC, and then explored the possible molecular regulatory mechanism of DP inhibiting the proliferation, migration and invasion of TNBC cells, aiming to provide pharmacological basis and scientific reference for DP treatment of breast cancer, and lay a scientific theoretical foundation for clinical treatment of breast cancer.

 

TNBC is a highly invasive breast cancer, which is prone to recurrence and metastasis, has strong heterogeneity, and lacks effective therapeutic targets. Therefore, in-depth analysis of the potential molecular mechanisms driving TNBC migration and invasion, and the development of efficient and low toxicity anti-tumor drugs are effective strategies for clinical treatment of TNBC. It has been found that many traditional Chinese medicine extracts and active ingredients have multiple pathways, targets, high efficiency and low toxicity anti-tumor activities. Research has shown that DP can inhibit liver cancer angiogenesis by reducing the expression of HIF-1 α and VEGF; In addition, DP can not only inhibit the proliferation of human breast cancer SK-BR-3 and T47D cells, but also inhibit the proliferation of human breast cancer MCF-7 cells and induce cell apoptosis by down regulating the expression of apoptosis related proteins P53 and Bcl-2, and up regulating the expression of Bcl-2 associated x protein (Bax). This study revealed for the first time that DP has pharmacological activity against TNBC, and found that DP can inhibit the proliferation, migration, and invasion of MDA-MB-231 cells, while showing no significant toxicity to normal breast epithelial cells MCF-10A.

The PI3K/Akt signaling pathway plays an important regulatory role in the proliferation, apoptosis, migration, invasion, and angiogenesis of malignant tumor cells. The abnormal activation of PI3K/Akt signaling pathway is one of the most common carcinogenic factors. In triple negative breast cancer, about 25%~30% of patients have PI3K/Akt activation mutations, which means that PI3K/Akt signaling pathway may be an effective target for TNBC treatment. It has been shown that activating PI3K/Akt signaling pathway can promote cell proliferation, migration and invasion of breast cancer cells MCF-7 and BT-20, while inhibiting PI3K/Akt signaling pathway can induce autophagy and apoptosis of breast cancer cells. In this study, we found that DP can downregulate the expression levels of p-PI3K and p-Akt proteins in MDA-MB-231 cells, suggesting that DP can inhibit the activation of the PI3K/Akt signaling pathway in this cell. In addition, GSK-3 is the downstream substrate of PI3K/Akt. GSK-3 is a serine/threonine protein kinase, which has two main subtypes, GSK-3 α and GSK-3 β. GSK-3 α is mainly involved in the glycogen metabolism process, and GSK-3 β is closely related to the cancer process. It is overexpressed in many cancers, including breast cancer. P-Akt can phosphorylate GSK3 β, thereby promoting tumor cell proliferation, migration, and invasion. Therefore, inhibiting the activity of the PI3K/Akt/GSK-3 β signaling pathway is crucial for suppressing tumor cell proliferation, migration, and invasion. This study observed the effect of DP on the expression of GSK-3 β protein in MDA-MB-231 cells, and the results showed that DP significantly reduced the expression level of p-GSK-3 β protein in MDA-MB-231 cells. It can be inferred that the molecular mechanism by which DP inhibits the proliferation, migration, and invasion of MDA-MB-231 cells may be related to blocking the PI3K/Akt/GSK-3 β signaling pathway.

The EMT process is one of the important mechanisms for tumor cell migration and invasion, which is related to structural changes in intercellular connections. At the molecular level, a decrease in the expression of epithelial like marker E-cadherin and an increase in the expression of mesenchymal markers N-cadherin and Vimentin indicate the occurrence of cellular EMT process. During the EMT process, tumor cells transition from a non fixed epithelial phenotype to an active and invasive mesenchymal phenotype. Once the EMT process is activated, tumor cells undergo a series of changes, including dissolution of tight junctions, disruption of apical basal polarity, and remodeling of the cytoskeleton structure, all of which are beneficial for tumor cell migration and invasion. TNBC in vitro and in vivo studies have shown that inhibiting the Akt/GSK-3 β signaling pathway can effectively increase the expression of E-cadherin and decrease the expression levels of N-cadherin and Vimentin, thereby reversing the EMT process. The results of this study showed that in MDA-MB-231 cells, DP upregulated the expression of E-cadherin and downregulated the expression levels of N-cadherin and Vimentin in a dose-dependent manner, indicating that DP can prevent the EMT process in this cell.

In conclusion, DP can significantly inhibit the proliferation, migration, invasion and EMT of MDA-MB-231 cells, which may be achieved by inhibiting the PI3K/Akt/GSK-3 β signaling pathway, which provides a new targeting basis for DP to become a potential new anti breast cancer drug. Although current studies have shown that DP exerts its anti breast cancer effect through multiple channels and targets, the molecular regulation mechanism of breast cancer proliferation, migration and invasion is complex, and the specific mechanism of its anti breast cancer effect needs further exploration. In addition, this study only provides in vitro experimental evidence for DP to inhibit TNBC cell proliferation, migration, invasion, and EMT by suppressing the PI3K/Akt/GSK-3 β signaling pathway. To further reveal the molecular mechanism of DP’s anti TNBC effect, further animal in vivo experiments are needed.