The Inhibitory Effects of Organosulfide Diallyl Trisulfide on TNF‐α induced CCL2 release through MAPK/MAPK8 and NFқB/IKBKE signaling in TNBC cell lines

Abstract

Inflammation and immune response play a key role in triple-negative breast cancer (TNBC) progression and metastasis. Previous studies have shown that CCL2 plays a significant role in breast cancer cell signaling and can increase cell proliferation and differentiation, immune suppression, epithelial-to-mesenchymal transition, and angiogenesis. CCL2 expression is modulated via mitogen-activated protein kinase (MAPK) and nuclear factor-kappa beta (NFĶB) signaling pathways. Compounds that can inhibit the release of CCL2 have been shown to prevent cancer-associated inflammatory and pro-oncogenic processes. Diallyl Trisulfide (DATS), an organosulfide found in garlic, has been previously reported to be effective in chemoprevention, apoptosis, and cell cycle arrest in DNA damaged normal breast epithelial cells, breast cancer cells, as well as other cancers. The current study explored the effect of DATS on the genetically different TNBC MDA-MB-231 and MDA-468 cells, stimulated by TNF-α. Cytokine arrays, ELISA, and RT-PCR using individual primers were used. DATS downregulated CCL2 protein expression in MDA-MB-231, but not in MDA-MB-468 cells. These results were confirmed with ELISA analysis that showed a reduction in the expression of CCL2 in the TNF-α stimulated MDA-MB-231 cells treated with DATS. The data also showed that DATS treatment reduced by 50% the mRNA expression of CCL2 in MDA-MB-231cells after a 24-h period. To study a possible mechanism for DATS inhibitory effect over CCL2 expression, this compound's effect on the mRNA expression of IKBKE and MAPK8, which belong to the NFĶB and MAPK signaling pathways, respectively, was also investigated. DATS downregulated both genes in the TNF-α stimulated MDA-MB-231 and MDA-MB-468 cells. In conclusion, this study showed that DATS could downregulate CCL2 expression in the protein and transcription level in MDA-MB-231 cells. A possible mechanism for DATS inhibitory effect may involve the downregulation of IKBKE and MAPK8 mRNA expression. IKBKE gene was reported to be overexpressed in approximately 30% of human breast tumors and promotes cytokine release and pro-survival signaling through the activation of NFĶB and JAK-STAT pathways. Our findings demonstrate that DATS may have potential in breast cancer treatment, slowing down cancer progression by the downregulation of CCL2 expression and that genetically different TNBC cells may respond differently to DATS treatment.