Vol.:(0123456789) 1 3 Apoptosis https://doi.org/10.1007/s10495-020-01594-5 Exploitation of a novel phenothiazine derivative for its anti‑cancer activities in malignant glioblastoma S. I. Omoruyi 1,2  · O. E. Ekpo 1  · D. M. Semenya 3  · A. Jardine 3  · S. Prince 2 © Springer Science+Business Media, LLC, part of Springer Nature 2020 Abstract Glioblastoma remains the most malignant of all primary adult brain tumours with poor patient survival and limited treatment options. This study adopts a drug repurposing approach by investigating the anti-cancer activity of a derivative of the antipsy- chotic drug phenothiazine (DS00329) in malignant U251 and U87 glioblastoma cells. Results from MTT (3-(4,5-dimethylth- iazol-2-yl)-2,5-diphenyltetrazolium bromide) and clonogenic assays showed that DS00329 inhibited short-term glioblastoma cell viability and long-term survival while sparing non-cancerous cells. Western blot analysis with an antibody to γH2AX showed that DS00329 induced DNA damage and fow cytometry and western blotting confrmed that it triggered a G1 cell cycle arrest which correlated with decreased levels in Cyclin A, Cyclin B, Cyclin D1 and cyclin dependent kinase 2 and an increase in levels of the cyclin dependent kinase inhibitor p21. DS00329 treated glioblastoma cells exhibited morpho- logical and molecular markers typical of apoptotic cells such as membrane blebbing and cell shrinkage and an increase in levels of cleaved PARP. Flow cytometry with annexin V-FITC/propidium iodide staining confrmed that DS00329 induced apoptotic cell death in glioblastoma cells. We also show that DS00329 treatment of glioblastoma cells led to an increase in the autophagosome marker LC3-II and autophagy inhibition studies using baflomycin A1 and wortmannin, showed that DS00329-induced-autophagy was a pro-death mechanism. Furthermore, DS00329 treatment of glioblastoma cells inhibited the phosphatidylinositol 3′-kinase/Akt cell survival pathway. Our fndings suggest that DS00329 may be an efective treat- ment for glioblastoma and provide a rationale for further exploration and validation of the use of phenothiazines and their derivatives in the treatment of glioblastoma. Keywords Drug repurposing · Phenothiazines · Glioblastoma · Apoptosis · Autophagy Abbreviations PTZ Phenothiazine MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphe- nyltetrazolium bromide γH2AX Phosphorylated H2A histone family member X CDK2 Cyclin-dependent kinase 2 PARP Poly (ADP-ribose) polymerase FITC Fluorescein isothiocyanate (FITC) LC3 Microtubule-associated protein light chain 3 FACS Fluorescence-activated cell sorting AVO Acidic vesicular organelles PI3K/Akt Phosphatidylinositol 3′ kinase/Akt MAPK Mitogen activated protein kinase ATM Ataxia-telangiectasia mutated Introduction Malignant brain tumours have continued to draw attention not only because of their poor prognosis, but also for their direct impact on neurological function, psychological health, and quality of life [1]. Of these, glioblastoma multiforme (World Health Organization [WHO] grade IV), which ema- nate from the glial cells of the brain, is the most frequent and aggressive and has a very poor prognosis. Indeed, they are highly vascular and invasive neoplasms and the median * S. Prince sharon.prince@uct.ac.za 1 Department of Medical Bioscience, Faculty of Natural Sciences, University of the Western Cape, Bellville, Cape Town 7530, South Africa 2 Division of Cell Biology, Department of Human Biology, Faculty of Health Sciences, University of Cape Town, Observatory, Cape Town 7925, South Africa 3 Department of Chemistry, Faculty of Science, University of Cape Town, Rondebosch, Cape Town 7701, South Africa