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Exploring the Molecular Mechanism of Cinnamic Acid-Mediated Cytotoxicity in Triple Negative MDA-MB-231 Breast Cancer Cells.

BACKGROUND
Cinnamic acid (CA), also known as 3-phenyl-2-propenoic acid, is a naturally occurring aromatic fatty acid found commonly in cinnamon, grapes, tea, cocoa, spinach and celery. Various studies have identified CA to have anti-proliferative action on glioblastoma, melanoma, prostate and lung carcinoma cells.


OBJECTIVE
Our objective was to investigate the molecular mechanism underlying the cytotoxic effect of CA in killing MDA-MB-231 triple negative breast cancer cells.


METHODS
We performed MTT assay and trypan blue assay to determine cell viability and cell death, respectively. Comet analysis was carried out to investigate DNA damage of individual cells. Furthermore, AO/EtBr assay and sub-G1 analysis using flowcytometry was used to study apoptosis. Protein isolation followed by immunoblotting was used to observe protein abundance in treated and untreated cancer cells.


RESULTS
Using MTT assay we have determined CA to reduce cell viability in MDA-MB-231 breast cancer cells and tumorigenic HEK 293 cells but not in normal NIH3T3 fibroblast cells. Subsequently, trypan blue assay and comet assay showed CA to cause cell death and DNA damage, respectively, in the MDA-MB-231 cells. Using AO/EtBr staining and sub-G1 analysis we further established CA to increase apoptosis. Additionally, immunoblotting showed the abundance of TNFA, TNF receptor 1 (TNFR1) and cleaved caspase-8/-3 pro-apoptotic proteins to increase on CA treatment. Subsequently, blocking of TNFA-TNFR1 signalling by small molecule inhibitor, R-7050, reduced the expression of cleaved caspase-8 and caspase-3 at the protein level.


CONCLUSION
Thus, from the above observations we can conclude that CA is an effective anticancer agent that can induce apoptosis in breast cancer cells via TNFA-TNFR1 mediated extrinsic apoptotic pathway.