Mercier, Anne ElisabethPrudent, RenaudPepper, Michael SeanDe Koning, LeanneNolte, ElsiePeronne, LauralieNel, MarcelLafanechere, LaurenceJoubert, Annie M.2022-06-072022-06-072021-01-29Mercier, A.E.; Prudent, R.; Pepper, M.S.; De Koning, L.; Nolte, E.; Peronne, L.; Nel, M.; Lafanechère, L.; Joubert, A.M. Characterization of Signalling Pathways That Link Apoptosis and Autophagy to Cell Death Induced by Estrone Analogues Which Reversibly Depolymerize Microtubules. Molecules 2021, 26, 706. https://DOI.org/10.3390/molecules26030706.1420-3049 (online)10.3390/molecules26030706https://repository.up.ac.za/handle/2263/85702SUPPLEMENTARY DATA: DOCUMENT: Mercier et al. VIDEO S1: Time-lapse imaging.The search for novel anti-cancer compounds which can circumvent chemotherapeutic drug resistance and limit systemic toxicity remains a priority. 2-Ethyl-3-O-sulphamoyl-estra-1,3,5(10)15- tetraene-3-ol-17one (ESE-15-one) and 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16) are sulphamoylated 2-methoxyestradiol (2-ME) analogues designed by our research team. Although their cytotoxicity has been demonstrated in vitro, the temporal and mechanistic responses of the initiated intracellular events are yet to be determined. In order to do so, assays investigating the compounds’ effects on microtubules, cell cycle progression, signalling cascades, autophagy and apoptosis were conducted using HeLa cervical- and MDA-MB-231 metastatic breast cancer cells. Both compounds reversibly disrupted microtubule dynamics as an early event by binding to the microtubule colchicine site, which blocked progression through the cell cycle at the G1/S- and G2/M transitions. This was supported by increased pRB and p27Kip1 phosphorylation. Induction of apoptosis with time-dependent signalling involving the p-JNK, Erk1/2 and Akt/mTOR pathways and loss of mitochondrial membrane potential was demonstrated. Inhibition of autophagy attenuated the apoptotic response. In conclusion, the 2-ME analogues induced a time-dependent cross-talk between cell cycle checkpoints, apoptotic signalling and autophagic processes, with an increased reactive oxygen species formation and perturbated microtubule functioning appearing to connect the processes. Subtle differences in the responses were observed between the two compounds and the different cell lines.en© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license.MicrotubulesReactive oxygen speciesApoptosisAutophagy2-Methoxyestradiol analoguesAnti-cancerMitochondrial membrane potentialCell cycle arrestp27Kip1JNKArticle