Toxicology and Applied Pharmacology 437 (2022) 115887 Available online 19 January 2022 0041-008X/© 2022 Published by Elsevier Inc. Deregulation of the CD44-NANOG-MDR1 associated chemoresistance pathways of breast cancer stem cells potentiates the anti-cancer effect of Kaempferol in synergism with Verapamil Sourav Kumar Nandi a , Tanaya Roychowdhury b , Samit Chattopadhyay b, c , Sudarshana Basu a , Krishti Chatterjee d , Pritha Choudhury e , Nirmalya Banerjee d , Prosenjit Saha e , Soma Mukhopadhyay a, * , Ashis Mukhopadhyay f , Rittwika Bhattacharya a, * a Department of Molecular Biology, Netaji Subhas Chandra Bose Cancer Research Institute, 3081 Nayabad, Kolkata 700094, India b Cancer Biology and Infammatory Disorder Division, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata 700032, India c Department of Biological Sciences, BITS Pilani, K K Birla Goa Campus, India d Department of Pathology, Netaji Subhas Chandra Bose Cancer Research Institute, 3081 Nayabad, Kolkata 700094, India e Department of Cancer Chemoprevention, Chittaranjan National Cancer Institute, 37, Shyamaprasad Mukherjee Rd, Kolkata, West Bengal 700026, India f Department of Haematooncology, Netaji Subhas Chandra Bose Cancer Research Institute, 3081 Nayabad, Kolkata 700094, India A R T I C L E INFO Keywords: Neoadjuvant therapy Breast cancer stem cells Induced pluripotent stem cell Kaempferol Anti-cancer Multidrug resistance Synergism ABSTRACT Chemoresistance is an imminent therapeutic challenge for breast cancer. Previous evidence suggests that breast cancer stem cells (BCSC) develop resistance through upregulation of stemness and chemo-evasion markers viz. SOX2, OCT4, NANOG, MDR1 and CD44, following anticancer chemotherapeutic treatments. Early studies sug- gest an inhibitory role of Kaempferol in BCSC propagation through downregulation of epithelial to mesenchymal transition. We hypothesized that the pathway involved in chemoresistance could be effectively addressed through Kaempferol (K), alone or in combination with Verapamil (V), which is an inhibitor of MDR1. We used K in combination with V, in multiple assays to determine if there was an inhibitory effect on BCSC. Both K and KV attenuated pH-dependent mammosphere formation in primary BCSC and MDA-MB-231 cells. RNA and protein (immunocytochemistry, western blot) expression of candidate markers viz. SOX2, OCT4, NANOG, MDR1 and CD44 were carried out in the presence or absence of candidate drugs in ex-vivo grown primary BCSC and MDA- MB-231 cell line. Immunoprecipitation assay, cell cycle analysis was carried out in MDA-MB-231. Our candidate drugs were not only anti-proliferative, but also downregulated candidate genes expression at RNA and protein level in both settings, with more robust effcacy in KV treatment than K; induced G2/M dependent cell cycle arrest, and interrupted physical association of CD44 with NANOG as well as MDR1 in MDA-MB-231. In primary tumor explant but not in adjacent normal tissue, our candidate drugs K and KV induced robust γH2AX expression. Thus, our candidate drugs are effective in attenuating BCSC survival. 1. Introduction Acquisition of chemoresistance has a close association with stem cell renewal, the complex mechanism of which is not fully unravelled. The phenomena of the early development of chemoresistance are charac- teristically observed in triple negative breast cancers (TNBCs), the most heterogeneous subtype of breast cancer, with characteristic lack of expression of estrogen receptor (ER), the progesterone receptor (PgR) and defciency of overexpression/gene amplifcation of human epidermal growth factor receptor 2 (HER2). Therefore, the therapeutic options targeting these hormone receptors are not available for TNBC patients (Anders and Carey, 2009). However, chemoresistance is not only the prerogative of TNBC, as hormone receptor-positive breast tu- mors also acquire chemoresistance with time (Brand˜ ao et al., 2019). Abbreviations: K, Kaempferol; V, Verapamil; G, Gemcitabine; KV, Kaempferol with Verapamil; TNBC, triple negative breast cancer; NACT, neo adjuvant chemotherapy; iPSC, induced pluripotent stem cell. * Corresponding authors at: Department of Molecular Biology, Department of Molecular Biology and Gynaecological Oncology, Netaji Subhas Chandra Bose Cancer Research Institute, 3081 Nayabad, Kolkata 700094, India. E-mail addresses: admin@nscri.in (S. Mukhopadhyay), rittwika@nscri.in (R. Bhattacharya). Contents lists available at ScienceDirect Toxicology and Applied Pharmacology journal homepage: www.elsevier.com/locate/taap https://doi.org/10.1016/j.taap.2022.115887 Received 13 September 2021; Received in revised form 11 January 2022; Accepted 12 January 2022