Copyright © 2020 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. Original article 55 0959-4973 Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved. DOI: 10.1097/CAD.0000000000000841 Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's website, www.anti-cancerdrugs.com. Heterocyclic organobismuth(III) compound induces nonapoptotic cell death via lipid peroxidation Katsuya Iuchi, Sayo Shirai, Yuji Tasaki and Hisashi Hisatomi Heterocyclic organobismuth compounds, such as N-tert- butyl-bi-chlorodibenzo[c,f][1,5]azabismocine (compound 1) and bi-chlorodibenzo[c,f ][1,5]thiabismocine (compound 3), exert potent antiproliferative activities in vitro in human cancer cell lines. We showed that compound 3 induced both apoptotic and nonapoptotic cell death via reactive oxygen species production and mitotic arrest in a dose- dependent manner. The mechanisms underlying the dose- dependent effect of these organobismuth compounds were not clear. In the present study, we examined the dose-dependent mechanism underlying cell death induced by compound 1 in a human pancreatic cancer cell line, SUIT-2, and a human colorectal cancer cell line, DLD-1. Compound 1 inhibited cell growth in a dose-dependent manner and induced cell death. Treatment with the pan- caspase inhibitor zVAD-fmk reduced cell death induced by compound 1, whereas the inhibitory effect of zVAD-fmk was limited. Moreover, compound 1 significantly induced lipid peroxidation with concomitant induction of caspase- independent cell death. Our results suggested that eight-membered ring organobismuth compounds induce nonapoptotic cell death via lipid peroxidation. Anti-Cancer Drugs 31:55–59 Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved. Anti-Cancer Drugs 2020, 31:55–59 Keywords: heterocyclic organobismuth compounds, lipid peroxidation, nonapoptotic cell death Department of Materials and Life Science, Faculty of Science and Technology, Seikei University, 3-3-1 Kichijojikitamachi, Musashino-shi, Tokyo, Japan Correspondence to Katsuya Iuchi, Department of Materials and Life Science, Faculty of Science and Technology, Seikei University, 3-3-1 Kichijojikitamachi, Musashino-shi, Tokyo, 180-8633, Japan Tel: +81 0422 37 3523; fax: +81 0422 37 3871; e-mail: iuchi@st.seikei.ac.jp Received 15 June 2019 Revised form accepted 14 August 2019 Introduction A large number of bismuth compounds have been syn- thesized and used for industrial, biological, and med- ical applications [1–4]. Bismuth salts, such as bismuth subsalicylate and bismuth citrate, have been used for the eradication of Helicobacter pylori [5]. Murafuji et al. demonstrated that various types of organobismuth com- pounds exhibit antifungal activity [6–11]. Tris( N, N-dimethylaminomethyl) phenylbismuthane and 2-( N , N -dimethylaminome-thyl)phenylbis (4-methylphenyl) bismuthane exhibit cytotoxicity against vascular endothelial cells [12,13]. 1-[(2Di-p- tolylbismuthanophenyl)diazenyl]pyrrolidine induces apoptosis in the human acute promyelocytic leukemia cell line NB4 by inducing the production of reactive oxy- gen species (ROS), which do not inhibit tubulin polym- erization [14]. Islam et al. showed that other bismuth derivatives inhibit the proliferation of cultured human cells, including chronic myelogenous leukemia (K562), and murine metastatic melanoma (B16F10) [6,15]. Heterocyclic organobismuth(III) compounds exhibit strong antimicrobial activity [16]. In our previous study, we showed that N-tert-butyl-bi-chlorodibenzo[c,f][1,5] azabismocine (compound 1) and bi-chlorodibenzo[c,f] [1,5]thiabismocine (compound 3) induce mitotic arrest and the accumulation of HeLa cells in the G 2 /M phase [17,18]. These studies showed that compounds 1 and 3 disrupt normal spindle pole formation. High concentra- tions of compounds 1 and 3 (>1 µM) induced apoptotic cell death without causing cell cycle arrest at the G 2 /M phase in HeLa cells. In addition, our previous study indicated that these compounds induced apoptotic and nonapoptotic cell death in the human leukemia cell line HL-60 [19]. Compound-3-induced cell death was found to occur in a ROS-dependent manner. However, the mechanisms underlying the induction of nonapoptotic cell death by organobismuth compounds remain unclear. Apoptosis has long been considered as the only regulated type of cell death. However, nonapoptotic and regulated cell death pathways have been reported in the last few decades [20]. Ferroptosis is a regulated form of cell death that is induced by lipid peroxidation [21]. Ferroptosis is biochemically and morphologically distinct from apop- tosis, necroptosis, and autophagy [22,23]. Lipids, such as fatty acids, play roles in the initiation and execution of nonapoptotic cell death [24]. Nonapoptotic cell death was found to be induced by artesunate, a water-soluble sem- isynthetic derivative and a drug that can be used for the treatment of malaria [25]. Metal compounds induce non- apoptotic cell death [26]. Arsenic trioxide (ATO) prefer- entially induces nonapoptotic cell death, autophagy, and mitotic cell death in p53-defcient CHO AA8 cells [27]. In the present study, we examined the effect of com- pound 1 on cell membrane peroxidation and death in the