Arsenic Trioxide ATO exhibits antitumor activities and potent inducer of apoptosis in a variety of cancer types, the exact mechanism of antitumor activity is not clear and its effectiveness in solid cancer remain controversial. Tumor necrosis factor‐alpha (TNF‐alpha) has also been shown to both induce apoptosis and prevent cell proliferation. The present study was designed to further investigate the effect of different concentrations of ATO on the viability of triple negative breast cancer MDA‐MB‐231 cell line, and to investigate apoptosis activated proteins induced by the treatment of ATO, TNF‐alpha, and combination. MDA‐MB‐231 cells exposed to seven different concentrations of ATO from 0 to 12 μM, for 24 hours, cell viability was measured by Alamar blue assay. Results show that ATO treatment effectively caused cell growth inhibition, and induced apoptosis in MDA‐MB‐231 cancer cells in a concentration‐dependent manner. Human apoptosis array is used to evaluate expression of apoptosis‐regulated proteins. Treated cells were labeled as control, ATO (2μM), TNF‐alpha (40 ng/ml) or combinations of ATO and TNF alpha, the arrays were made with dots corresponding to 43 major proteins. The analysis of dot blot intensity indicate that all treatments presented the same 43 proteins with a difference in the intensity of P53, Bcl2, BID, Caspase 3, Caspase 8, Cytochrome C, SMAC, HSP60, HSP70, HTRA2, and surviving proteins. Current results show that arsenic trioxide is useful to treat Breast cancer and apoptosis induced by activation of key regulatory proteins.Support or Funding InformationDepartment of BiologyThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.