Isobaric tags for relative and absolute quantitation-based proteomics analysis of the effect of ginger oil on bisphenol A-induced breast cancer cell proliferation

Abstract

Several chemicals in the environment, particularly those with estrogenic activity and small amounts (micromolar or lower) of environmental estrogen can cause changes in cell function and interfere with endocrine functions of animals and humans. These compounds enter the human body and increase the load of estrogen in the body, leading to an increasing incidence of estrogen-related tumors in breast cancer, ovarian cancer and endometrial cancer. Previous studies have demonstrated that ginger can inhibit the expression of estrogen receptors, while the bioactive ingredients of ginger sig-nificantly inhibit proliferation and promote the apoptosis of breast cancer cells. In the present study, a quantitative proteomics technique based on relative and absolute quanti-tative isobaric labeling was used to determine the effect of ginger essential oil (GEO) and BPA combined treatment on the proteomic characteristics of MCF-7 cells. In total, 5,084 proteins were detected. Proteins that were upregulated >1.2-fold and downregu-lated by >0.8-fold were differentially expressed. Overall, 528 differentially expressed proteins were identified. Compared with the control group, MCF-7 cells treated with GEO, BPA and GEO-BPA resulted in 45 (14 up- and 31 downregulated), 481 (141 up- and 340 downregulated) and 34 (13 up- and 21 downregulated) differentially ex-pressed proteins, respectively. Compared with the BPA group, MCF- 7 cells treated with GEO-BPA resulted in 210 (117 up- and 93 downregulated) differentially expressed proteins, among the 210 differentially expressed proteins in the GEO-BPA group, 10 proteins were associated with oxidative phosphorylation pathways, while succinate dehydrogenase (ubiquinone) iron-sulfur subunit (SDHB), succinate dehydrogenase cytochrome b560 subunit, mitochondrial (SDHC), cytochrome c oxidase subunit 2 and superoxide dismutase (Mn), mitochondrial (SOD2) expression was decreased with GEO-BPA combined treatment. Through the analysis of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, the cellular localization, functional annotation and biological pathways of differentially expressed proteins were ex-amined. The results indicated that GEO-BPA may act through the oxidative phosphory-lation pathway, decreased the expression of SDHB and SDHC, affected the tricarbox-ylic acid cycle and decreased the expression of SOD2. This may have led to oxidative stress and the death of breast cancer cells, and the SDH signaling pathway may be an important mediator of the inhibitory effects of GEO in MCF-7 breast cancer cells. GEO can inhibit the proliferation of breast cancer MCF-7 cells induced by BPA, and the underlying mechanism may be associated with oxidative phosphorylation. These results may aid the development of future treatment strategies for breast cancer caused by environmental estrogen exposure.

Keywords: ginger; iTRAQ; oxidative phosphorylation; breast cancer.

Figure 1.

Effect of GEO, BPA, GEO-BPA on the viability of MCF-7 cells. MCF-7 cells were incubated with different concentrations of GEO (0–250 mg/l), BPA (10-5-10-9 mol/l) and GEO (200 mg/l)-BPA (10-5-10-9 mol/l) and the cell viability was measured at different time points. The results are plotted as the means ± SEs (n=3) of the percent-age of viable cells relative to the control. *P<0.05 and **P<0.01 vs. control group. GEO, ginger essential oil; BPA, bisphenol A.

Figure 2.

Experimental process. For quantitative proteomic analysis of design of exper-iment, the experiment was divided into four groups (control, GEO, BPA and GEO-BPA), and each experiment was performed in triplicate. The extracted proteins were prepared by reductive alkylation, digested with trypsin and labeled with Iraqi reagents. Analysis was performed using reversed-phase LC-MS/MS. Bioinformatics tools were further used to analyze the resulting data. GEO, ginger essential oil; BPA, bisphenol A; LC, liquid chromatography; MS, mass spectrometry; KEGG, Kyoto Encyclopedia of Genes and Genomes; PPI, protein-protein interaction.

Figure 3.

Quality control of protein data validation. (A) Protein mass distribution of all identified proteins. (B) Protein length distribution of all identified peptides.

Figure 4.

Cluster analysis of differentially expressed proteins between GEO-BPA and the control. Color changes with the expression of the protein, and blue colors indicate negative correlation, red colors indicates positive correlation, the more intense the color, the strongest the correlation. GEO, ginger essential oil; BPA, bisphenol A.

Figure 5.

MCF-7 cells perform GO analysis of the protein function of GEO. Compared with the control group, there were 17 differentially expressed proteins in the GEO treatment group. GO function analysis of 17 differentially expressed proteins were divided into biological process, molecular function and cellular component. GO, Gene Ontology; GEO, ginger essential oil.

Figure 6.

MCF-7 cells perform GO analysis of the protein function of BPA. Compared with the control group, there were 111 differentially expressed proteins in the BPA treatment group. GO function analysis of 111 differentially expressed proteins were divided into biological process, molecular function and cellular component. GO, Gene Ontology; BPA, bisphenol A.

Figure 7.

MCF-7 cells perform GO analysis of the protein function of GEO-BPA. Compared with the BPA group, there were 128 differentially expressed proteins in the GEO-BPA treatment group. GO function analysis of 128 differentially expressed proteins were divided into biological process, molecular function, and cellular component. Gene Ontology; GEO, ginger essential oil; BPA, bisphenol A.

Figure 8.

KEGG analyses of protein functions in GEO-treated MCF-7 cells. Compared with the control group, 17 differentially expressed proteins in the GEO treatment group were annotated using the KEGG database. KEGG, Kyoto Encyclopedia of Genes and Genomes; GEO, ginger essential oil.

Figure 9.

KEGG analyses of protein functions in BPA-treated MCF-7 cells. Compared with the control group, 111 diffeentially expressed proteins in the BPA treatment group were annotated using the KEGG database. KEGG, Kyoto Encyclopedia of Genes and Genomes; BPA, bisphenol A.

Figure 10.

Compared with the BPA group, 128 differentially expressed proteins in the GEO-BPA combined treatment group was annotated using the KEGG database. KEGG, Kyoto Encyclopedia of Genes and Genomes; BPA, bisphenol A; GEO, ginger essential oil.

Figure 11.

Ginger essential oil and bisphenol A-treated MCF-7 cells protein-protein interaction analysis. The degree value increases from yellow to green, and then to blue. The top ten with degree values in GEO-BPA group are marked yellow: SNRPE, SDHB, SDHC, MRPL13, SOD2, FGB, HNRNPL, MT-CO2, MT-CYB And SFPQ. yellow and green markers are other differential proteins.