Activation of Autophagy and Nrf2 Signaling in Human Breast Adenocarcinoma MCF-7 Cells by Novel Monophenolic Antioxidants Abstract The effect of novel water-soluble structurally related monophenolic compounds on the activity of two most important mechanisms of maintaining intracellular homeostasis, autophagy and the redox-sensitive signal system Keap1/Nrf2/ARE, has been studied in human breast adenocarcinoma cell line MCF-7 using confocal microscopy. Autophagy processes were analyzed on the basis of the amount of intracellular vesicles that were positive for the autophagy marker (LC3B). The activation of the Keap1/Nrf2/ARE system was determined by the translocation of the transcription factor Nrf2 into the nucleus. It was found that the effect of the tested compounds depended on their structure and concentration. When the inhibitor of autophagosome–lysosome fusion chloroquine was added to the culture medium (20 μM), the asymmetrically hindered by the tert-butyl group phenols with thiosulfonate (TS-13) and sulfonate group in the para-propyl substituent increased the rate of autophagosome elimination in MCF-7 cells. Shortening of the para-alkyl substituent by one methylene unit abolished the effect. The addition of the second ortho-tert-butyl substituent had the reverse result. Both tested compounds enhanced the translocation of the transcription factor Nrf2 into the nucleus of MCF-7 cells (which is a critical step in Keap1/Nrf2/ARE activation). It was observed after incubation with asymmetrically hindered by the tert-butyl group phenol with selenosulfonate group in para-propyl substituent (5−100 μM) for 4 h and with TS-13 (5−100 μM) for 24 h. Taking into account our previous findings on the toxicity of this group of compounds for MCF-7 cells we can conclude that these compounds exert different effect on autophagy and activation of the antioxidant response element signaling system Keap1/Nrf2/ARE.