Mechanistic Targets of Diallyl Trisulfide in Human Breast Cancer Cells Identified by RNA-seq Analysis

Abstract

Diallyl trisulfide (DATS), a metabolic by-product of processed garlic, is highly effective in inhibiting growth of human breast cancer cells in vitro and in vivo, but the underlying mechanisms are still not fully understood. In this study, we performed RNA-seq analyses using luminal-type (MCF-7) and basal-like (MDA-MB-231) human breast cancer cells to identify mechanistic targets of DATS. The Reactome Pathway Analysis revealed upregulation of genes associated with SLIT/ROBO tumor suppressor signaling following DATS treatment in both MCF-7 and MDA-MB-231 cells. However, the expression of SLIT2 and ROBO1 proteins or their downstream target C-X-C motif chemokine receptor 4 was not affected by DATS treatment in both cell lines. The Reactome as well as the Gene Ontology Pathways Analyses of the RNA-seq data from DATS-treated cells indicated downregulation of genes associated with G₂/M phase cell cycle arrest in comparison with vehicle-treated control cells. Consistent with the RNA-seq data, DATS treatment caused a significant increase in the fraction of the G₂/M population in both cell lines when compared to corresponding control cells. In addition, Ser10 phosphorylation of histone H3, a mitotic marker, was also increased significantly following DATS treatment in MCF-7 and MDA-MB-231 cells. These results indicate that while SLIT/ROBO signaling is not affected by DATS treatment, cell cycle arrest likely contributes to the antitumor effect of this phytochemical.

Keywords: Allyl compounds; Breast neoplasms; Chemoprevention; Sulfides.

Figure 1. DATS treatment altered breast cancer transcriptomes.

(A) Chemical structure of DATS. (B) Volcano scatter plots showing genes upregulated or downregulated in MCF-7 and MDA-MB-231 cells in response to DATS. (C) Venn diagram showing unique and common genes by DATS treatment in MCF-7 and MDA-MB-231 cells. (D) Heatmap diagram visualizing the differentially expressed genes by DATS treatment in MCF-7 and MDA-MB-231 cells. DATS, diallyl trisulfide; MCF-7, michigan cancer foundation-7; MDA-MB-231, MD anderson-metastatic breast-231.

Figure 2. Reactome enrichment analysis for differentially expressed genes by DATS treatment in human breast cancer cells.

(A, B) Reactome enrichment analysis of (A) upregulated or (B) downregulated genes by DATS exposure in MCF-7 and MDA-MB-231 cells. DATS, diallyl trisulfide; MCF-7, michigan cancer foundation-7; MDA-MB-231, MD anderson-metastatic breast-231; TNFR, tumor necrosis factor receptor; SRP, signal-recognition particle; CENPA, centromere protein A; AURKA, aurora kinase A; TPX2, targeting protein for Xklp2; P_adj, adjusted P-value.

Figure 3. Gene Ontology (GO) enrichment analysis for differentially expressed genes by DATS treatment in human breast cancer cells.

(A, B) GO enrichment analysis of (A) upregulated or (B) downregulated genes by DATS exposure in MCF-7 and MDA-MB-231 cells. DATS, diallyl trisulfide; MCF-7, michigan cancer foundation-7; MDA-MB-231, MD anderson-metastatic breast-231; ER, estrogen receptor; CENP-A, centromere protein A; P_adj, adjusted P-value.

Figure 4. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for differentially expressed genes by DATS treatment in human breast cancer cells.

(A, B) KEGG pathway enrichment analysis of (A) upregulated or (B) downregulated genes by DATS exposure in MCF-7 and MDA-MB-231 cells. DATS, diallyl trisulfide; MCF-7, michigan cancer foundation-7; MDA-MB-231, MD anderson-metastatic breast-231; P_adj, adjusted P-value.

Figure 5. Effect of DATS treatment on the expression of SLIT2/ROBO1 signaling-related proteins.

Immunoblot analysis for SLIT2, ROBO1, CXCR4, and b-Actin proteins using lysates from MCF-7 and MDA-MB-231 cells treated with either DMSO (control) or desired doses of DATS for indicated time points. Numbers above bands are fold change of each protein relative to corresponding DMSO-treated control. Experiments were repeated twice with comparable results. Molecular weights for the observed bands for SLIT2 were indicated. DATS, diallyl trisulfide; MCF-7, michigan cancer foundation-7; MDA-MB-231, MD anderson-metastatic breast-231; CXCR4, C-X-C motif chemokine receptor 4; DMSO, dimethyl sulfoxide.

Figure 6. DATS arrested cells at G₂ as well as mitotic phase in human breast cancer cells.

(A) Representative propidium iodide (PI) histogram displaying 2 N and 4 N DNA content in MCF-7 and MDA-MB-231 cells after 8-hour treatment with DMSO (control) or 20 mmol/L DATS. (B) Quantification of the percentage of cells at G₂ and M phases in MCF-7 and MDA-MB-231 cells treated with DMSO (control) or indicated doses of DATS for 4 or 8 hours. Results shown are mean ± SD (n = 3) and statistical analysis was done by one-way ANOVA followed by Dunnett’s test (*P < 0.05). (C) Representative cell cycle histogram of DNA stained with PI and mitotic marker in MCF-7 and MDA-MB-231 cells after 8-hour treatment with DMSO (control) or 20 µmol/L DATS. (D) Quantification of the percentage of cells undergoing mitosis in MCF-7 and MDA-MB-231 cells treated with DMSO (control) or indicated doses of DATS for 4 or 8 hours. Results shown are mean ± SD (n = 3) and statistical analysis was done by one-way ANOVA followed by Dunnett’s test (*P < 0.05). DATS, diallyl trisulfide; MCF-7, michigan cancer foundation-7; MDA-MB-231, MD anderson-metastatic breast-231; DMSO, dimethyl sulfoxide.