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. 2020 Aug 7:11:1211.
doi: 10.3389/fphar.2020.01211. eCollection 2020.

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

Wenfeng Zhang  1 , Cun Liu  2 , Jie Li  3 , Ruijuan Liu  4 , Jing Zhuang  4 , Fubin Feng  4 , Yan Yao  1 , Changgang Sun  5
Affiliations

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

Wenfeng Zhang et al. Front Pharmacol. .

Abstract

Background: As 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.

Methods: We 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.

Results: Our 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.

Conclusion: Podophyllotoxin 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.

Keywords: breast cancer; cell cycle; inhibition; network pharmacology; podophyllotoxin.

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Figures

Figure 1
Figure 1
Cell viability assay (MTT) of (A) MDA‐MB‐231 and (B) MDA‐MB‐468 cells treated with 0–24 μM of podophyllotoxin 12–36 h. The inhibition rate of the two kinds of cells treated by drugs was the highest at 24 h. Each data represents the mean ± SD from three independent experiments *p < 0.05, **p < 0.01.
Figure 2
Figure 2
Antimigration effect of podophyllotoxin. After 24 h of drug intervention, the migration distance of the (A) MDA‐MB‐231 and (B) MDA‐MB‐468 in the control group was significantly narrower than that in the treatment group. Podophyllotoxin intervention could significantly inhibit tumor migration. The moving distance was measured by inverted microscope and photographed. (C) The proportion of migration between the cells treated with podophyllotoxin and the control group. Each data represents the mean ± SD from three independent experiments. **p < 0.01 was considered to indicate a statistically significant difference.
Figure 3
Figure 3
Antiinvasive effect of podophyllotoxin. In vitro invasion assay was used to observe the invasion of drugs on triple negative breast cancer cells (MDA-MB-231, MDA-MB-468). (A) After 24 h of intervention with podophyllotoxin, we found that the number of cells passing through the chamber decreased significantly, and podophyllotoxin could inhibit the invasion of triple negative breast cancer. (B) Bar graphs show the quantitative expression of by the number of invasive cells in transwell experiment. *p < 0.05 is considered statistically significant.
Figure 4
Figure 4
Effect of podophyllotoxin on the cycle and apoptosis of triple negative breast cancer cells. (A) After 0.75 μM podophyllotoxin treatment, the proportion of G1 phase decreased and the proportion of G2/M phase increased. Podophyllotoxin blocked triple-negative breast cancer cells in G2/M phase. (B) Statistics of cell cycle changes. P<0.05, the difference was statistically significant. (C) After 24 h of Podophyllotoxin (0 and 0.75 μM) intervention, and cell apoptosis was analyzed by flow cytometry. (D) Bar graphs show the quantification of apoptosis in MDA-MB-231and MDA-MB-468 cells. The data are expressed as the mean value ± SD.*p < 0.05, **p < 0.01, ***p < 0.001.
Figure 5
Figure 5
Identification of differential genes and key genes. (A) Volcanic maps of all the genes and identification of DEGs. (B) Venn diagram of GSE31448, GSE45827, and GSE65194. (C) protein-protein interaction (PPI) network of DEGs and screening of key genes. The color degree of the node is proportional to the degree of the node. The darker the color, the better the connectivity between genes. (D) Using MCODE to cluster in the huge gene (protein) network to construct functional modules, with a total of 14 nodes and 82 edges. The best protein module is selected through the score value of the node by MCODE, and its score value is 12.615. (E) Functional analysis of genes, including functional categories or cellular localization. The abscissa is the logarithm of the p-values with a base of 10 and a negative value; the vertical is different enrichment pathways. The higher the -log10 value ranked above, the smaller the p-values and the more significant the enrichment (the darker the color). (F) Enrichment analysis of GO and KEGG. Enrichment of key genes in KEGG pathway. The higher the proportion, the more enrichment in the pathway. (G) Protein-protein interaction network of DEGs. Selecting more valuable nodes according to degree UnDir and betweenness UnDir. CDK1, NDC80, PLK1, CDC20, AURKB, BUB1 were identified as key genes.
Figure 6
Figure 6
Expression of three hub genes. The expression of PLK1 (A), CDK1 (B), CDC20 (C) in triple negative breast cancer is higher than that in other breast cancer tissues.
Figure 7
Figure 7
Molecular docking diagram of key genes and mRNA expression. (A) 3D model of podophyllotoxin. Schematic diagram of binding sites of (B) CDK1, (C) PLK1, and (D) CDC20. Small molecular proteins are connected to the corresponding sites of the protein through hydrogen bonding. The mRNA expression is regulated by podophyllotoxin in (E) MDA-MB-231and (F) MDA-MB-468. Cells were treated with 0.75-μM podophyllotoxin, qRT-PCR detection promoted the expression of p53 at the mRNA level and inhibited the expression of CDK1, CDC20, Polo-like kinase1 (PLK1). *p < 0.05, **p < 0.01.
Figure 8
Figure 8
Further screening of key targets. (A) Gene Set Enrichment Analysis (GSEA) enrichment of Polo-like kinase1 (PLK1) in cell cycle signaling pathway. NES=2.229, p-value=0, FDR-q-value=0.00213. (B) The Venn diagram is the intersection of key disease targets and drug targets. Purple: key target for triple-negative breast cancer (TNBC) disease, yellow: drug target. We end up with a common gene: PLK1. (C) The protein expression levels of p53 were assayed by Western blot. The expression of PLK1 protein decreased and the expression of p53 increased with the increase of podophyllotoxin in both cell lines.
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