Target Analysis and Mechanism of Podophyllotoxin in the Treatment of Triple-Negative Breast Cancer.

Wenfeng Zhang,Cun Liu,Yan Yao,Changgang Sun,Ruijuan Liu,Jing Zhuang,Fubin Feng,Jie Li

doi:10.3389/fphar.2020.01211

Abstract

BackgroundAs the original compound of many podophyllotoxin derivatives, podophyllotoxin has a beneficial antitumor effect. The mechanism of podophyllotoxin activity in triple-negative breast cancer still needs to be explored.MethodsWe used cell proliferation assay, scratch and transwell experiments, and cell cycle and apoptosis analyses to observe the intervention effect of podophyllotoxin on breast cancer. Furthermore, we analyzed the differences between GSE31448, GSE65194, and GSE45827 in the Gene Expression Omnibus database (GEO) and explored the differential genes using a STRING database. Centiscape2.2, MCODE cluster analysis and KEGG pathway analysis were used to identify the most significant gene differences. Next, we utilized BATMAN-TCM and TCMSP databases for further screening to identify key genes. Finally, quantitative RT-PCR (qRT-PCR) and Western blotting were performed to detect the expression of key targets.ResultsOur research confirmed that podophyllotoxin could not only inhibit the migration and invasion of triple-negative breast cancer but also affect the cell cycle and induce apoptosis. In total, 566 differential genes were obtained by using the GEO database. After topological network analysis, cluster analysis, and molecular docking screening, we finally identified PLK1, CCDC20, and CDK1 as key target genes. The results of the qRT-PCR assay showed that the mRNA levels of PLK1, CDC20, and CDK1 decreased, while the expression of upstream P53 increased, after drug induction. The Gene Set Enrichment Analysis (GSEA) and conetwork analysis showed that PLK1 is a more critical regulatory factor. Further Western blotting analysis revealed that there was a negative regulatory relationship between the key gene PLK1 and P53 on the protein level. The results were presented as the mean ± standard deviation of triplicate experiments and P<0.05 was considered to indicate a statistically significant difference.ConclusionPodophyllotoxin has an intervention effect on the development of triple-negative breast cancer. The expression of PLK1, CDC20, and CDK1 in the cell cycle pathway is inhibited by regulating P53. Our research shows that natural drugs inhibit tumor activity by regulating the expression of cyclins, and the combination of natural drugs and modern extensive database analysis has a wide range of potential applications in the development of antitumor therapies.

Highlights

Breast cancer is a malignant tumor that seriously threatens women’s health around the world
First we investigated whether podophyllotoxin has an effect on breast cancer cell lines MDA-MB-231 and MDA-MB-468 were treated with 0 mmol/L, 0.375 mmol/L, 0.75 mmol/L, 1.5 mmol/L, 3 mmol/L, 6 mmol/L, 12 mmol/L, and 24 mmol/L podophyllotoxin for 12, 24, and 36 h, respectively
Our research innovatively proposed that podophyllotoxin effectively regulates the triple-negative breast cancer (TNBC) cell cycle and promotes cell apoptosis

Summary

Introduction

Breast cancer is a malignant tumor that seriously threatens women’s health around the world. TNBC is a type of breast cancer with negative estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) genes (Brown et al, 2008). At present, targeted and hormone types of therapy have no beneficial therapeutic effect on TNBC. More attention has been paid to some natural plant drugs that can interfere with critical molecules in cell growth and development signaling pathways. Natural plant drugs are widely used in inhibiting tumor activity (Muniraj et al, 2019). Podophyllotoxin is a kind of natural aryl lignin, which is the leading compound of many derivatives (Li et al, 2013). Podophyllotoxin inhibits microtubule polymerization, leading to mitotic failure and cell cycle arrest (Chen et al, 2013). The mechanism of podophyllotoxin activity in triple-negative breast cancer still needs to be explored

Methods

Results

Discussion

Conclusion