Artesunate, as a HSP70 ATPase activity inhibitor, induces apoptosis in breast cancer cells Masoud Pirali a , Maryam Taheri b , Sevda Zarei b , Mashad Majidi b , Hossein Ghafouri b, ⁎ a Department of Microbiology, Faculty of Basic Sciences, Islamic Azad University, Lahijan, Iran b Department of Biology, Faculty of Basic Sciences, University of Guilan, Rasht, Iran abstract article info Article history: Received 9 April 2020 Received in revised form 24 August 2020 Accepted 25 August 2020 Available online 27 August 2020 Keywords: Artesunate Breast cancer HSP70 Apoptosis The present study aims to evaluate the inhibitory effects of artesunate (a semi-synthetic derivative of artemisinin) on HSP70 and Bcl-2 expression in two breast cancer cell lines, 4T1 and MCF-7. In addition, to deter- mine in vitro inhibitory effect of artesunate against the ATPase activity of purified recombinant HSP70, it was tested in a carbonic anhydrase refolding assay with purified HSP70. Our results demonstrated that the artesunate not only induced apoptosis but also lead to the inhibition of HSP70 ATPase activity the in vitro (P < 0.001). The extent of HSP70 refolding inhibition increased with increasing μM concentrations of artesunate. Incubation of HSP70 with 50 μM artesunate showed significant inhibition of refolding activity by 38%. The IC 50 values of artesunate for 4T1 cells, were lower than MCF-7 cells, indicating the higher sensitivity of the triple-negative phenotype. Furthermore, artesunate significantly down-regulated the expression of Bcl-2 and HSP70 while en- hancing the expression of cleaved caspase-9 in MCF-7 and 4T1 cells. It also induced caspase-9 activity at 18 h in a dose-dependent manner in two breast cancer cell lines. Generally, our results show that the artesunate in- duces caspase-dependent apoptosis through the inhibition of HSP70 expression. © 2020 Elsevier B.V. All rights reserved. 1. Introduction Mammalian heat shock proteins (HSPs) are classified by their rela- tive molecular weight and function into two groups: high molecular weight HSPs and low molecular weight HSPs [1]. High molecular weight HSPs include the HSP110, HSP90, HSP70, and HSP60 families, which act in an ATP-dependent manner, whereas low molecular weight HSPs (molecular weight of 34 kDa or less) are ATP-independent chaperones [2]. HSP70 is a member of the molecular chaperone family and found in nearly all living organisms. HSP70 proteins are expressed constitu- tively at low levels under normal physiological conditions, thereby fur- ther induced in response to environmental, physical and chemical stresses [3]. The main function of HSP70, as well as other HSPs, is to protect cells from injury by promoting the refolding of denatured substrates or preventing the aggregation of misfolded proteins. The HSP70 proteins have two major functional domains; N-terminal ATPase domain (45-kDa) and C-terminal substrate-binding domain (25-kDa) [4–6]. The domain in HSP40 co-chaperone that is responsible for substrate binding and HSP70 ATPase activity is the J-domain, and it is present in all DnaJ/Hsp40 family members [7]. Binding and releasing protein substrate to HSP70 are facilitated by binding ATP [8]. Not sur- prisingly, HSP70 has therefore been linked to numerous diseases, in neurodegeneration diseases such as Alzheimer's disease (AD) and many types of cancers [9]. Overproduction of HSP70 is important for tumor cell survival and leads to increased resistance against apoptosis-inducing drugs such as staurosporine and doxorubicin [10]. On the other hand, down-regulation of HSP70 expression leads to an increase in sensitivity towards these drugs [11]. The anti-apoptotic function of the HSP70 family of heat shock proteins and their role in cancer is well documented [12]. HSP70 is a powerful anti-apoptotic protein that can block both the extrinsic and intrinsic pathways of apoptosis [13]. Over the last decade, the small molecule inhibitors' effects of apoptosis-inducing compounds on HSP70 activity have attracted the at- tention of researchers as potent anticancer agents [14,15]. Based on the structure of HSP70 proteins, there are two potential inhibitor-binding sites: the ATP-binding site and the substrate-binding cleft [16]. The HSP70 inhibitors, VER-155008 and PFT-μ, which are proposed to target different sites in HSP70s, VER-155008 is an ATP-competitive inhibitor of HSP70 and it binds in the ATP-binding pocket, and PFT-μ is proposed to interact with the substrate-binding domain [17,18]. In apoptotic signal- ing, highly expressed HSP70 interferes at three levels: up-stream to mi- tochondria, at the mitochondria, and post-mitochondria. The intrinsic pathway is largely centered around and/or regulated by the mitochon- dria [19]. Release of cytochrome c from intermembrane space of mito- chondria by apoptotic signals induces ATP-dependent formation of the oligomeric Apaf-1-caspase-9 apoptosome [20]. Cytosolic cytochrome c International Journal of Biological Macromolecules 164 (2020) 3369–3375 ⁎ Corresponding author. E-mail address: h.ghafoori@guilan.ac.ir (H. Ghafouri). https://doi.org/10.1016/j.ijbiomac.2020.08.198 0141-8130/© 2020 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac