Exploring the Anti breast cancer Mechanism of Taro acuminata Based on UPLC-Q-TOF-MS/MS and Network Pharmacology
Globally, breast cancer accounts for 10% of the incidence rate of all cancers and 15% of female tumor related mortality. The latest Global Cancer Report 2020 published by the World Health Organization shows that 2.1 million new breast cancer cases and 27000 people died of breast cancer in 2018, posing a serious threat to human health. Thus, effective treatment of breast cancer is particularly important. According to the theory of syndrome differentiation and treatment, traditional Chinese medicine has obvious curative effect on breast cancer by adjusting the functions of the body, such as yin, yang, qi, blood, viscera and meridians. Traditional Chinese medicine can improve the metabolism of the body, regulate the overall function of the patient’s body, and reduce the recurrence and metastasis of breast cancer to improve the clinical efficacy.
The root and stem of Alocassia cuculata (Lour.) Schott, a plant in the Araceae family, is a traditional folk medicine that has the effects of clearing heat, detoxifying, reducing swelling, relieving pain. Modern pharmacological studies have shown that taro can activate immune factors to inhibit the activity of various tumor cells such as melanoma, improve the body’s immune ability, induce apoptosis of cancer cells, and exert anti-tumor effects. Its related molecular mechanisms involve signaling pathways such as MAPK and PI3K Akt. It is reported in the literature that petroleum ether fraction of Alocasia cucullata (EAC) can inhibit the proliferation of breast cancer MDA-MB-231 cells in vitro cell experiments. The mechanism is related to the inhibition of p-ERK/ERK expression in the MAPK pathway. At the same time, the petroleum ether fraction of Alocasia cucullata is rich in various unsaturated fatty acids such as linoleic acid and various phytosterols such as γ – sitosterol through GC-MS analysis. Modern research shows that unsaturated fatty acids such as linoleic acid and phytosterols can induce apoptosis of breast cancer cells by regulating the level of Bax/Bcl-2. This suggests that the anti breast cancer mechanism of petroleum ether fraction of Taro may also be related to the regulation of Bax/Bcl-2 level. However, at present, the research on chemical composition of petroleum ether part of Taro acuminata is only limited to GC-MS analysis, and there is no relevant report on the mechanism of anti breast cancer action of petroleum ether part of Taro acuminata in vivo. The research on the material basis of its efficacy and the mechanism of anti breast cancer action in vivo are relatively weak. The growth state and metastasis site of 4T1 cell transplantation tumor model can well simulate human advanced breast cancer, and become a good model for evaluating drugs for advanced breast cancer. Therefore, in this paper, the petroleum ether part of Taro acuminata was taken as the research object, and the chemical composition of petroleum ether part of Taro acuminata was determined through UPLC-Q-TOF-MS/MS, while the network pharmacology strategy combined with molecular docking technology was used to clarify the molecular level of the anti breast cancer mechanism of petroleum ether part of Taro acuminata. Finally, the anti breast cancer effect of petroleum ether part of Taro acuminata acuminata in vivo was evaluated through the 4T1 breast cancer tumor bearing mouse model and its anti breast cancer mechanism was verified. In order to provide experimental references for the in-depth research and development of the pointed tail taro.

 

This study used UPLC-Q-TOF-MS/MS technology to analyze 41 chemical components of the petroleum ether fraction of taro. At the same time, 32 anti breast cancer active ingredients and 556 anti breast cancer related targets were analyzed from the petroleum ether fraction of Tara acuminata through network pharmacology strategy. 40 core targets such as MAPK1 and Bcl-2 were predicted by PPI network, and these targets were mainly concentrated in MAPK protein family and PI3K protein family. Furthermore, the GO biological process enrichment analysis showed that the anti breast cancer biological process of petroleum ether fraction of Tara acuminata mainly involved in negative regulation of cell apoptosis and other biological processes. The enrichment analysis of KEGG signal pathway showed that the anti breast cancer target enrichment of petroleum ether fraction of Tara acuminata was mostly in PI3K-Akt and MAPK signal pathways, both of which were related to cell apoptosis. The PPI network predicted that MAPK1, Bcl-2 and other apoptosis related core targets were obtained, and further verified by molecular docking technology that the sterol component digitalis glycoside ligand, the fatty acid component linolenic acid and the coumarin component 7-hydroxycoumarin could bind freely (binding energy<0 kcal/mol) with the related core targets (p-ERK, Bcl-2, Bax) of the petroleum ether part of Amorphophallus senticosus for breast cancer. The above results suggest that the anti breast cancer effect of petroleum ether fraction of taro is related to its multi-component and multi-target characteristics, and its anti breast cancer mechanism is related to apoptosis related signal pathways such as PI3K Akt and MAPK.

Jianwei taro is commonly used in folk medicine to treat various cancers, and the compounds in its tubers can inhibit the growth of various cancer cells. The previous research of our research group found that the water extract of taro can inhibit the growth of breast cancer and prolong the survival period of breast cancer mice by enhancing the activity of mouse spleen cells. The cell experiment found that taro can significantly catalyze the differentiation of immune monocytes into macrophages, stimulate the function of megaphagocytes, and activate the immune system to achieve anti-tumor effect. The petroleum ether fraction of taro can inhibit the growth of human MDA-MB-231 breast cancer cells and promote the apoptosis of cancer cells. The results of this experiment showed that the petroleum ether fraction of taro can inhibit the growth rate of breast cancer in tumor bearing mice, reduce the tumor weight, and the tumor inhibition rate showed a concentration dependent trend. The spleen is an important immune organ in animals, which can reflect the impact of drugs on the immune system. The mice in the medium and high-dose groups of EAC administered orally had higher spleen index and significant differences compared to the model group mice, indicating that the petroleum ether fraction of taro can improve the immune function of tumor bearing mice. The HE staining results of tumor tissue showed that the tumor cells in the model group of mice grew densely, while the 5-fluorouracil group and the group treated with taro had loose cell arrangement, blurred cell contours, and obvious necrotic areas of tumor cells, indicating membrane rupture of tumor cells. The petroleum ether site of taro could induce apoptosis of tumor cells.

A large number of studies have shown that inflammation can mediate a variety of diseases, including cardiovascular disease, cancer, diabetes, arthritis, Alzheimer’s disease, etc. The persistent presence of causative factors can lead to inflammation that cannot be eliminated, and inflammation can become chronic, resulting in cell mutations and proliferation, which is more conducive to the occurrence and development of cancer. Tumor necrosis factor (TNF) is one of the important proinflammatory cytokines. The microenvironment related to breast cancer is characterized by chronic inflammation. TNF – α can promote the occurrence and development of tumors by stimulating estrogen metabolism in tumor cells and surrounding stromal cells and accumulating estrogen. Studies have found that exogenous TNF – α cannot induce tumor cell apoptosis in MDAMB-468, SK-BR3, and MDA-MB-231 cells, while endogenous TNF – α has protective anti apoptotic activity in HS578T cells. The main sources of pro-inflammatory cytokine interleukin-1 β (IL-1 β) include myeloid cells such as MDSCs, macrophages, and DCs, which play a role in promoting tumor growth or enhancing anti-tumor immune detection in the tumor microenvironment. IL-1 β has been shown to promote the accumulation of MDSCs in immature myeloid cells in the tumor microenvironment, thereby promoting tumor progression. In this study, it was found through ELISA kit detection that compared with the model group, the levels of TNF – α and IL-1 β in the serum of the positive drug group and the groups treated with different concentrations of taro were reduced, and the high-dose group had the lowest levels. Therefore, it is speculated that after tumor inoculation in mice, the petroleum ether fraction of taro may inhibit tumor development by reducing pro-inflammatory cytokine levels. In conclusion, the petroleum ether fraction of Taro acuminata has significant anti breast cancer effect in vivo.

MAPK is a serine protein kinase that belongs to the endogenous apoptotic pathway. ERK, JNK, and p38 are the three most important signaling pathways discovered in mammals, with different activation states. They participate in the signaling effects of various growth factors, cytokines, and hormone receptors after activation, regulating cell growth and differentiation. In this experimental study, there was no significant difference in the levels of MAPKs signaling pathway related proteins, ERK, JNK, p38, p-JNK, and p-p38 proteins, and the expression of p-ERK protein was reduced, suggesting that the petroleum ether part of Taro may play an anti breast cancer role by inhibiting the expression of p-ERK/ERK, which is consistent with the results of previous studies in vitro and network pharmacology prediction of related pathways. In addition, EAC can increase the expression of Bax/Bcl-2 protein and the level of apoptosis gene Bax/Bcl-2 mRNA, suggesting that EAC plays an anti breast cancer role by promoting the apoptosis of tumor cells.
To sum up, the petroleum ether fraction of taro can inhibit the tumor growth of 4T1 breast cancer bearing mice. Its anti breast cancer effect is related to its multi-component and multi-target characteristics. Its mechanism may be related to the down-regulation of p-ERK/ERK expression, the increase of Bax/Bcl-2 expression, the increase of Bax/Bcl-2 mRNA level, and the promotion of tumor cell apoptosis. This study provides a theoretical reference for the clinical application of Taro acuminata in the treatment of breast cancer, but its more in-depth material basis of efficacy, target and mechanism need to be further explored.