Unnatural enantiomer of chaetocin shows strong apoptosis-inducing activity through caspase-8/caspase-3 activation Yuou Teng a,b, Katsuya Iuchi a,b, Eriko Iwasa a,b,c, Shinya Fujishiro b,d, Yoshitaka Hamashima b, Kosuke Dodo a,b, Mikiko Sodeoka a,b,c,d,* a Sodeoka Live Cell Chemistry Project, ERATO, JST, 2-1, Hirosawa, Wako-shi, Saitama 351-0198, Japan b RIKEN, Advanced Science Institute (ASI), RIKEN, 2-1, Hirosawa, Wako-shi, Saitama 351-0198, Japan c Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan d Graduate School of Biomedical Science, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan a r t i c l e i n f o Article history: Received 27 May 2010 Revised 25 June 2010 Accepted 8 July 2010 Available online 13 July 2010 Keywords: Apoptosis Caspase Dithiodiketopiperazine a b s t r a c t Chaetocin, a natural product isolated from Chaetomium species fungi, was reported to have various bio- logical activities, including antitumor and antifungal activities. Recently, we reported the first total syn- thesis of chaetocin and its derivatives. Here, we examined the cell-death-inducing activity of these compounds in human leukemia HL-60 cells. The unnatural enantiomer of chaetocin (ent-chaetocin) was more potent than chaetocin, and was found to induce apoptosis through the caspase-8/caspase-3 activation pathway. �� 2010 Elsevier Ltd. All rights reserved. Chaetocin (1), a natural product isolated from Chaetomium spe- cies fungi, exhibits antitumor and antifungal activities.1,2 Further, Imhof and co-workers reported that it shows inhibitory activity against lysine-specific histone methyltransferases (HMTs), which are key enzymes in the epigenetic control of gene expression.3 In- spired by the complex chemical structure and interesting biologi- cal activities, we began a synthetic study of chaetocin (1), and recently reported the first total synthesis of chaetocin and its ana- logs (Fig. 1). We then examined the inhibitory activity against H3K9 HMT G9a,4 and found that sulfur-deficient chaetocin (2) and its enantiomer (ent-2) lacked activity, whereas chaetocin (1) and its enantiomer (ent-1) showed comparable activities. These re- sults indicate that the disulfide structure is important for the HMT- inhibitory activity, and also that the chirality of chaeotocin (1) is not strictly recognized by HMT. In addition to HMT inhibition, chaetocin (1) and related epidi- thiodiketopiperazine (ETP) compounds were reported to induce oxidative stress and to be cytotoxic to various cell lines.5���8 The internal disulfide bridge of ETP is reduced to dithiol by cellular reductants, and redox cycling between dithiol and disulfide is thought to mediate the generation of reactive oxygen species (ROS), which induce cell death.9,10 On the other hand, several ETP-binding proteins have been identified and suggested to medi- ate various biological activities.11,12 Several mechanisms, including a ROS-mediated mechanism and a binding-protein-mediated mechanism, have been proposed, but the nature of ETP-induced cytotoxicity has not yet been fully clarified. In this study, in order to clarify the structure���activity relationship (SAR) and the mecha- nism of the cytotoxicity, we examined the cell-death-inducing activity of chaetocin derivatives. Interestingly, the unnatural enantiomer of chaetocin (ent-1) showed more potent activity than 0960-894X/$ - see front matter �� 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2010.07.032 * Corresponding author. Fax: +81 48 467 9373. E-mail address: sodeoka@riken.jp (M. Sodeoka). N N H N O O H N N N H O O H Me Me OH HO S2 S2 N N H N O O H N N N H O O H Me Me OH HO S2 S2 chaetocin (1) ent-chaetocin (ent-1) N N H N O O H N N N H O O H Me Me OH HO H H N N H N O O H N N N H O O H Me Me OH HO H H ent-2 S-deficient chaetocin (2) Figure 1. Structures of chaetocin derivatives. Bioorganic & Medicinal Chemistry Letters 20 (2010) 5085���5088 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl

natural chaetocin (1) at low concentration. Moreover, ent-1 was found to induce apoptosis through the caspase-8/caspase-3 activa- tion pathway. First, we tested the cell-death-inducing activity of chaetocin derivatives using human leukemia HL-60 cells (Fig. 2). Cell viability was determined by alamarBlue assay as described elsewhere.13,14 Chaetocin (1) dose-dependently reduced cell viability, but its sul- fur-deficient derivative 2 was inactive, indicating a correlation be- tween HMT inhibition and anticancer activity. The importance of disulfide structure for anticancer activity was already reported by Isham et al.7 The same structure���activity relationship (SAR) was observed for ent-1 and its disulfide-deficient derivative (ent-2). Interestingly, though, we found that ent-1, but not 1, showed a bell-shaped dose���response curve, that is, recovery of cell viability was seen at moderate concentration. The difference between 1 and ent-1 at low concentration implies the existence of a novel tar- get that recognizes the chirality of chaetocin. The morphology of cells treated at low (0.3 lM) and high (30 lM) concentrations was also distinct (Fig. 3a). Typical apoptotic morphology (cell shrinkage and/or blebbing) was observed in the former case. In contrast, cell swelling (typical necrotic morphology) was observed in the latter case. To examine more precisely the effects of 1 and ent-1 on HL-60 cells, DAPI/PI staining was performed at low (0.3 lM) and high (30 lM) concentrations. DAPI stains nuclear DNA of normal/dead cells, and PI stains nuclear DNA of dead cells whose cellular membrane has become permeabilized. As shown in Figure 3a, 1 and ent-1 induced apoptotic cell death characterized by nuclear fragmentation (a typical marker of apoptosis DAPI stain) at low concentration. On the other hand, at high concentration, 1 and ent-1 induced necrotic cell death characterized by cellular membrane permeabilization (a typical marker of necrosis PI stain). These results indicate that 1 and ent-1 induce apoptosis and necrosis through independent mechanisms. Compared to natural chaetocin (1), ent-1 showed similar necro- sis-inducing activity, but its apoptosis-inducing activity seemed to be stronger. To confirm the difference of apoptosis-inducing activ- ity between 1 and ent-1, we examined DNA ladder formation by DNA electrophoresis.15 As shown in Figure 3b, a typical DNA ladder was detected in the case of ent-1 treatment at low concentration. In contrast, there was little or no cleaved DNA in the case of treat- ment with 1 or with ent-1 at a high concentration. These results suggest that a low concentration of ent-1 induces apoptosis through a mechanism that is able to recognize the chirality of chaetocin. Next, to examine the involvement of oxidative stress, we monitored cellular ROS by flow-cytometric analysis. However, neither 1 nor ent-1 increased cellular ROS (data not shown). These results strongly imply that ROS are not involved in the ent-1-in- duced apoptosis. Moreover, since not much difference was observed in HMT- inhibitory activity between 1 and ent-1, a target protein distinct from HMT is expected to be involved in apoptosis induction by ent-1. In order to investigate the action mechanism of ETP compounds, we planned to investigate the signaling pathway of apoptosis in- duced by ent-1. Typical apoptosis is mediated by a cascade of pro- teases, called caspases,16,17 but recent studies indicate the existence of a caspase-independent pathway for some types of apoptosis.18���20 To examine the involvement of caspases in apopto- sis induced by ent-1, we monitored the activation of caspase-3, an effector caspase. By means of Western blot analysis, cleavage of caspase-3 was detected in ent-1-treated HL-60 cells (Fig. 4a). Moreover, the quantification of caspase activation21 indicated that ent-1 showed much more potent activation of caspase-3 than did 1 (Fig. 4b). Moreover, Z-VAD (a general caspase inhibitor) inhibited apoptosis induced by ent-1 (Fig. 4c). These results taken together indicate that caspases are involved in apoptosis induced by ent-1. To identify the major pathway activated by ent-1, we next examined the effects of subtype-specific caspase inhibitors. Up- 0 20 40 60 80 100 120 Cell viability (%) 0.1 0.3 1 3 10 30 1 (��M) ent-1 2 ent-2 *** *** *** *** NS *** Figure 2. Cell-death-inducing activities of chaetocin derivatives in HL-60 cells. HL- 60 cells were treated with chaetocin derivatives for 4 h, and cell viability was determined by alamarBlue assay. Statistical differences were determined by Student���s t-test (N.S., nonsignificant ***P 0.001). control 0.3 30 0.3 30 1 ent-1 (��M) b a control chaetocin (1) ent-chaetocin (ent-1) 0.3 30 (��M) DAPI stain PI stain 0.3 30 Figure 3. (a) Typical apoptotic and necrotic morphological changes induced by chaetocin (1) or ent-1 in HL-60 cells. HL-60 cells treated with chaetocin derivatives for 4 h were stained with DAPI/PI. Nuclear fragmentation (DAPI, blue) and PI-stained nuclei (PI, red) were observed using confocal laser microscopy. (b) DNA ladder formation induced by chaetocin (1) or ent-1 in HL-60 cells. 5086 Y. Teng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5085���5088