Thujaplicin Enhances TRAIL-Induced Apoptosis Via the Dual Effects of XIAP Inhibition and Degradation in NCI-H460 Human Lung Cancer Cells

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Article β-Thujaplicin Enhances TRAIL-Induced Apoptosis via the Dual Effects of XIAP Inhibition and Degradation in NCI-H460 Human Lung Cancer Cells

Saki Seno 1, Minori Kimura 1, Yuki Yashiro 1, Ryutaro Kimura 1, Kanae Adachi 1, Aoi Terabayashi 1, Mio Takahashi 1, Takahiro Oyama 2, Hideaki Abe 2, Takehiko Abe 2, Sei-ichi Tanuma 3 and Ryoko Takasawa 1,*

1 Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan; [email protected] (S.S.); [email protected] (M.K.); [email protected] (Y.Y.); [email protected] (R.K.); [email protected] (K.A.); [email protected] (A.T.); [email protected] (M.T.) 2 Hinoki Shinyaku Co. Ltd., Chiyoda-ku, Tokyo 102-0084, Japan; [email protected] (T.O.); [email protected] (H.A.); [email protected] (T.A.) 3 Department of Genomic Medicinal Science, Research Institute for Science and Technology, Organization for Research Advancement, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-4-7124-1501

β Abstract: AbstractBackground: -thujaplicin, a natural tropolone derivative, has anticancer effects on various cancer cells via apoptosis. However, the apoptosis regulatory proteins involved in this Citation: Seno, S.; Kimura, M.; process have yet to be revealed. Methods: Trypan blue staining, a WST-8 assay, and a caspase-3/7 Yashiro, Y.; Kimura, R.; Adachi, K.; activity assay were used to investigate whether β-thujaplicin sensitizes cancer cells to TNF-related Terabayashi, A.; Takahashi, M.; apoptosis-inducing ligand (TRAIL)-mediated apoptosis. Additionally, western blotting was per- Oyama, T.; Abe, H.; Abe, T.; et al. formed to clarify the effects of β-thujaplicin on X-linked inhibitor of apoptosis protein (XIAP) in β-Thujaplicin Enhances NCI-H460 cells and a fluorescence polarization binding assay was used to evaluate the binding- TRAIL-Induced Apoptosis via the inhibitory activity of β-thujaplicin against XIAP-BIR3. Results: β- and γ-thujaplicins decreased the Dual Effects of XIAP Inhibition and Degradation in NCI-H460 Human viability of NCI-H460 cells in a dose-dependent manner; they also sensitized the cells to TRAIL- Lung Cancer Cells. Medicines 2021, 8, induced cell growth inhibition and apoptosis. β-thujaplicin significantly potentiated the apoptosis 26. https://doi.org/10.3390/ induction effect of TRAIL on NCI-H460 cells, which was accompanied by enhanced caspase-3/7 medicines8060026 activity. Interestingly, β-thujaplicin treatment in NCI-H460 cells decreased XIAP levels. Furthermore, β-thujaplicin was able to bind XIAP-BIR3 at the Smac binding site. Conclusions: These findings Academic Editor: William Cho indicate that β-thujaplicin could enhance TRAIL-induced apoptosis in NCI-H460 cells via XIAP inhibition and degradation. Thus, the tropolone scaffold may be useful for designing novel nonpeptidic Received: 12 May 2021 small-molecule inhibitors of XIAP and developing new types of anticancer drugs. Accepted: 31 May 2021 Published: 2 June 2021 Keywords: β-thujaplicin; XIAP; TRAIL; apoptosis; cancer; NCI-H460 cells

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- 1. Introduction iations. β-thujaplicin, also known as hinokitiol, is a natural tropolone derivative with a β- diketone structure in its molecule; it forms metal chelates in the presence of various metal ions including ferric ion (Fe2+), copper ion (Cu2+), and zinc ion (Zn2+)[1]. β-thujaplicin is used in various personal care products (e.g., cosmetics, body soaps, and toothpastes) Copyright: © 2021 by the authors. and exhibits a variety of biological properties including antibacterial [2,3], antifungal [4], Licensee MDPI, Basel, Switzerland. This article is an open access article antiviral [5], and anticancer activities [6–13]. It is also reported to induce apoptosis in distributed under the terms and human cancer cells, such as human colon cancer cells [9] and hepatocellular carcinoma conditions of the Creative Commons cells [13]; however, the mechanism and targets of β-thujaplicin’s anticancer activity have Attribution (CC BY) license (https:// yet to be fully elucidated. creativecommons.org/licenses/by/ X-linked inhibitor of apoptosis protein (XIAP), the most potent member of the inhibitor 4.0/). of apoptosis protein (IAP) family of endogenous caspase inhibitors, blocks the initiation

Medicines 2021, 8, 26. https://doi.org/10.3390/medicines8060026 https://www.mdpi.com/journal/medicines Medicines 2021, 8, 26 2 of 11

and execution phases of the apoptotic cascade through inhibition of caspases-9, -3, and -7 [14]. XIAP contains three baculoviral IAP repeat (BIR) motifs; each BIR domain folds into a functionally independent structure that binds a zinc ion. Additionally, XIAP contains another zinc-binding motif, the RING domain, which has E3 ubiquitin ligase activity [14]. Of all the IAP family members, XIAP is the only one able to directly inhibit both the initiation and execution phases of the caspase cascade [14]. The BIR2 domain of XIAP binds and inhibits caspases-3 and -7, whereas the BIR3 domain binds and inhibits caspase-9 (which is an apical caspase in the mitochondrial pathway of apoptosis). XIAP levels are elevated in many cancer cell lines and suppression of XIAP protein expression sensitizes cancer cells to chemotherapeutic agents [15–17]. Thus, XIAP represents an attractive target for the development of apoptosis-resistant cancer treatments. The inhibition or decrease of XIAP in cancer cells lowers the apoptotic threshold, which in turn induces cell death and/or enhances the cytotoxic action of chemotherapeutic agents. It was previously reported that treatment with the membrane-permeable zinc chelator N,N,N’,N’,-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induced depletion of XIAP at the posttranslational level in human PC-3 prostate cancer cells while sensitizing these cells to TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis [18]. These findings indicate that zinc-chelating agents could be used to sensitize malignant cells to apoptosis-inducing agents via reduction of XIAP. Since β-thujaplicin acts as a chelator of zinc ions [1], it may play an important role in the regulation of XIAP-mediated apoptosis. However, the effect of β-thujaplicin on XIAP has yet to be examined. The BIR3 domain of XIAP, where caspase-9 and Smac proteins bind, is considered an attractive site for the design of small-molecule XIAP inhibitors. Embelin, a natural compound from the Japanese Ardisia herb, is one of the cell-permeable, nonpeptide, small-molecular weight inhibitors that target the XIAP-BIR3 domain as identified through computational structural screening of the herbal medicine compounds database [19]. It binds to the XIAP-BIR3 domain at the position where Smac and caspase-9 also bind and thereby antagonize XIAP-BIR3 (IC50 value = 4.1 µM) [19]. Embelin consists of a di-hydroxyquinone core and long hydrophobic tail, whereas β-thujaplicin has a seven- membered carbon ring and an isopropyl side chain; however, both compounds have similar aromatic rings with α-hydroxy ketone and an alkyl group. Based on the findings detailed above, we hypothesize that β-thujaplicin could act on XIAP both as a zinc chelator and as an inhibitor targeting the XIAP-BIR3 domain to induce and/or enhance apoptosis. To test this hypothesis, we examined the apoptosis inducibility of β-thujaplicin in combination with TRAIL in human non-small cell lung cancer NCI-H460 cells. NCI-H460 cells are considered type II cells, which are dependent on the mitochondrial apoptotic pathway to amplify effector caspase activation during death receptor-induced apoptosis [20]. XIAP is highly expressed in NCI-H460 cells, which show chemoresistance, and binds to the processed form of caspase-9 before suppressing the activation of downstream effector caspases-3 and -7 [21]. Therefore, we investigated whether β- and γ-thujaplicins could sensitize NCI-H460 cells to TRAIL-induced apoptosis. This research is the first to demonstrate that β-thujaplicin enhances TRAIL-induced apoptosis in NCI-H460 cells. Furthermore, we reveal that β-thujaplicin can bind the XIAP-BIR3 domain at the Smac binding site. Moreover, we show that β-thujaplicin treatment decreases the amount of XIAP protein in NCI-H460 cells. Our results validate β-thujaplicin as a potent seed compound that exerts dual effects on XIAP, i.e., XIAP inhibition by binding to the BIR3 domain and XIAP reduction by degrading XIAP protein. Thus, β-thujaplicin represents a new scaffold for the development of novel types of XIAP-targeting anticancer drugs.

2. Materials and Methods 2.1. Materials Tropolone and β-thujaplicin were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). γ-thujaplicin was purchased from Osaka Organic Chemical Industry Ltd. (Osaka, Japan). They were of analytical grade (purity >95%). An antibody against XIAP Medicines 2021, 8, 26 3 of 11

as well as anti-rabbit and anti-mouse immunoglobulin G (IgG) horseradish peroxidase (HRP)-coupled secondary antibodies were purchased from Cell Signaling Technology (Boston, MA, USA). An antibody against β-actin was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).

2.2. Cell Culture Cells of the human non-small cell lung cancer cell line NCI-H460 were maintained in RPMI1640 supplemented with 100 U/mL of penicillin, 100 µg/mL of streptomycin, and 10% fetal bovine serum. The cells were grown at 37 ◦C in a humidiﬁed atmosphere containing 5% CO2.

2.3. Trypan Blue Staining The effects of tropolone derivatives on cell viability were evaluated using trypan blue staining. NCI-H460 cells were seeded in dishes and cultured overnight. The cells were then treated with tropolone derivatives alone or cotreated with SuperKillerTRAIL™, Soluble (human) (rec.) (AdipoGen Life Sciences, San Diego, CA, USA) for 24 h. After treatment, the cells were trypsinised and resuspended in complete medium. Each sample was then mixed with trypan blue solution. Colored (dead) and dye-excluding (viable) cells were counted using a Countess II Cell Counter (Thermo Fisher Scientiﬁc K.K., Tokyo, Japan).

2.4. Measurements of Cell Viability Enhancement of the antiproliferative effect of TRAIL on NCI-H460 cells through a combination treatment with β- or γ-thujaplicin was evaluated by measuring cell growth inhibition. The number of viable cells was estimated via a WST-8 assay using a Cell Counting Kit-8 according to the manufacturer’s instructions (Dojindo Laboratories, Ku- mamoto, Japan).

2.5. Evaluation of the Combinatorial Effects of Drugs The combinatorial effects of TRAIL and β- or γ-thujaplicin were evaluated using an excess-over-Bliss additivism (EOBA) model [22,23] with the following Formula:

EOBA = C − [A + B − (A × B)],

where C is equal to the fractional inhibition of both drugs simultaneously, A is equal to the fractional inhibition of drug A, and B is equal to the fractional inhibition of drug B, while fractional inhibition is equal to 1.0 minus the viability (expressed as a value from 0.0 to 1.0). Positive, negative, and near-zero EOBA values reﬂect synergistic, antagonistic, and additive drug combinations, respectively.

2.6. Caspase-3/7 Activity Assay To analyze caspase activity, NCI-H460 cells were seeded in a 96-well luminometer plate and cultured overnight. The cells were treated with TRAIL or β-thujaplicin alone and cotreated with both reagents for 24 h. A Caspase-Glo® 3/7 Assay was performed according to manufacturer’s protocol (Promega, Madison, WI, USA).

2.7. Western Blotting Cell lysates were prepared using Cell Lysis Buffer (Cell Signaling Technology, Boston, MA, USA). Samples containing equal amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk and 0.25% bovine serum albumin in Tris-buffered saline containing 0.1% Tween 20 (TTBS) for 1 h at room temperature and then probed with appropriate primary antibodies overnight at 4 ◦C. Subsequently, washed with TTBS, and then incubated with the appropriate secondary antibody for 1 h at room temperature. After washing, the blotted proteins were visualized with an iBright Western Blot Imaging System (Thermo Fisher Scientiﬁc Medicines 2021, 8, x FOR PEER REVIEW 4 of 11

PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk and 0.25% bovine serum albumin in Tris-buffered saline containing 0.1% Tween 20 (TTBS) for 1 h at room temperature and then probed with appropriate primary Medicines 2021, 8, 26 antibodies overnight at 4 °C. Subsequently, washed with TTBS, and then incubated with4 of 11 the appropriate secondary antibody for 1 h at room temperature. After washing, the blotted proteins were visualized with an iBright Western Blot Imaging System (Thermo Fisher Scientific K.K., Tokyo, Japan) using ImmunoStar LD (FUJIFILM Wako Pure Chemical Cor- poration,K.K., Tokyo, Osaka, Japan) Japan). using ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). 2.8. Expression and Purification of Recombinant GST-Tagged XIAP-BIR3 Domain in an 2.8. Expression and Puriﬁcation of Recombinant GST-Tagged XIAP-BIR3 Domain in an EscherichiaEscherichia coli coli Expression Expression System System A Ahuman human XIAP-BIR3 cDNAcDNA fragment fragment was was subcloned subcloned into pGEX-4T-2into pGEX-4T-2 (GE Healthcare, (GE Healthcare,Chicago, IL,Chicago, USA), IL, and USA), the resulting and the result constructing construct was transformed was transformed into BL21 into to produce BL21 to re- producecombinant recombinant human XIAP-BIR3 human XIAP-BIR3 as a GST-tagged as a GST-tagged protein. The protein. recombinant The recombinant GST-tagged GST- XIAP- taggedBIR3 XIAP-BIR3 domain was domain puriﬁed was with purified Glutathione with Glutathione Sepharose TMSepharose4B (GETM Healthcare, 4B (GE Healthcare, Chicago, IL, Chicago,USA) inIL, native USA) conditionsin native conditions following following the manufacturer’s the manufacturer’s protocol. protocol.

2.9.2.9. Fluorescence Fluorescence Polarization Polarization Binding Binding Assay Assay TheThe interaction interaction between between the theSmac Smac peptide peptide and and the theBIR3 BIR3 domain domain of XIAP of XIAP was was meas- mea- uredsured using using a fluorescence a fluorescence polarization polarization assay, assay, which which was was performed performed at room at room temperature temperature in in384-well 384-well black black polystyrene polystyrene microplates microplates (Thermo (Thermo Fisher Fisher Scientific Scientific K.K., K.K., Tokyo, Japan)Japan) on on anan EnVisionEnVision®® 21042104 multimodemultimode plateplate readerreader(PerkinElmer, (PerkinElmer, Buckinghamshire, Buckinghamshire, UK). UK). PBS PBS ( −) (−)supplemented supplemented withwith 0.05%0.05% TweenTween 20,20, 1 nM of 5-FAM labeled Smac/Diablo Peptide Peptide [AV- [AVPI- PIAQKSE]AQKSE] (AnaSpec, (AnaSpec, Fremont, CA, USA) asas aa fluorescentfluorescent probe,probe,and and 50 50 nM nM of of recombinant recombi- nantGST-tagged GST-tagged XIAP-BIR3 XIAP-BIR3 domain domain were were used used for for all all measurements. measurements.

3. Results3. Results 3.1. β-. and γ-Thujaplicins Decrease the Viability of NCI-H460 Cells 3.1. β-. and γ-Thujaplicins Decrease the Viability of NCI-H460 Cells First, we examined the cytotoxic effect of tropolone, β-thujaplicin, and γ-thujaplicin First, we examined the cytotoxic effect of tropolone, β-thujaplicin, and γ-thujaplicin on NCI-H460 cells using a trypan blue exclusion assay. The chemical structures of these on NCI-H460 cells using a trypan blue exclusion assay. The chemical structures of these compounds are shown in Figure1. As shown in Figure2, β- and γ-thujaplicin treatments compounds are shown in Figure 1. As shown in Figure 2, β- and γ-thujaplicin treatments for 24 h decreased the viability of NCI-H460 cells in a dose-dependent manner. Treatments for 24 h decreased the viability of NCI-H460 cells in a dose-dependent manner. Treat- with >20 µM β- and γ-thujaplicins signiﬁcantly decreased the viability of NCI-H460 cells, mentswhereas with treatments>20 µM β- and at 10 γµ-thujaplicinsM did not affect significantly cell viability. decreased Tropolone the viability did not affect of NCI-H460 NCI-H460 cells,cells whereas at doses treatments up to 50 µ atM. 10 µM did not affect cell viability. Tropolone did not affect NCI-H460 cells at doses up to 50 µM.

Figure 1. The structures of tropolone, β-thujaplicin, and γ-thujaplicin. Figure 1. The structures of tropolone, β-thujaplicin, and γ-thujaplicin. 3.2. β-. and γ-Thujaplicins Enhance the Antiproliferative Effect of TRAIL on NCI-H460 Cells NCI-H460 cells were cotreated with TRAIL and β-thujaplicin (Figure3a) or γ-thujaplicin (Figure3b) for 24 h; cell viability (% of control) was then measured using a WST assay and EOBA scores were calculated. As EOBA values >0.1 denote a synergistic combination [22,23], the data indicated that cotreatment with a relatively low concentration of TRAIL and 10 µM β- or γ-thujaplicin produced a synergistic antiproliferative effect on NCI-H460 cells (Figure3c).

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Figure 2. Quantitation of cell viability by trypan blue staining following tropolone, β-thujaplicin, and γ-thujaplicin treatments on NCI-H460 cells at the indicated concentrations for 24 h. Cell viability (% of control) was measured using a trypan blue exclusion assay. Data are the averages of three independent experiments and error bars show the standard deviation. * p < 0.05 and ** p < 0.01 compared with untreated NCI-H460 cells (Ctrl).

3.2. β-. and γ-Thujaplicins Enhance the Antiproliferative Effect of TRAIL on NCI-H460 Cells Figure 2. 2. QuantitationQuantitation of cell of viab cellility viability by trypan by blue trypan staining blue following staining tropolone, following β-thujaplicin, tropolone, β-thujaplicin, NCI-H460 cells were cotreated with TRAIL and β-thujaplicin (Figure 3a) or γ-thu- and γγ-thujaplicin-thujaplicin treatments treatments on NCI-H460 on NCI-H460 cells at cells the indicated at the indicated concentrations concentrations for 24 h. Cell for via- 24 h. Cell viability bility (% of control) wasjaplicin measured (Figure using 3b) a trypanfor 24 h;blue cell exclusion viability assay. (% of Data control) are the was averages then ofmeasured using a WST (%three of independent control) was experimentsassay measured and and EOBA using error scores bars a trypan show were the bluecalculated. standard exclusion deviation. As EOBA assay. * pvalues < Data 0.05 and are>0.1 ** thedenote p < averages a synergistic of three com- independent0.01 compared with experiments untreatebinationd NCI-H460[22,23], and error the cells data bars (Ctrl). indicated show the that standard cotreatment deviation. with a relatively * p < 0.05 low and concentration ** p < 0.01 of compared with untreatedTRAIL and NCI-H460 10 µM β cells- or (Ctrl).γ-thujaplicin produced a synergistic antiproliferative effect on 3.2. β-. and γ-ThujaplicinsNCI-H460 Enhance cells the (Figure Antiproliferative 3c). Effect of TRAIL on NCI-H460 Cells NCI-H460 cells were cotreated with TRAIL and β-thujaplicin (Figure 3a) or γ-thujaplicin (Figure 3b) for 24 h; cell viability (% of control) was then measured using a WST assay and EOBA scores were calculated. As EOBA values >0.1 denote a synergistic combination [22,23], the data indicated that cotreatment with a relatively low concentration of TRAIL and 10 µM β- or γ-thujaplicin produced a synergistic antiproliferative effect on NCI-H460 cells (Figure 3c).

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(a) (b)

(c)

Figure 3. Enhancement of the antiproliferative effect of TRAIL on NCI-H460 cells by combination treatments with β- or γ- Figure 3. Enhancement of the antiproliferative effect of TRAIL on NCI-H460 cells by combination thujaplicin. NCI-H460 cells were cotreated with the indicated concentrations of TRAIL and β-thujaplicin (a) or γ-thujaplicin (b) for 24 h. Cell viability treatments(% of control) with wasβ measured- or γ-thujaplicin. using a WST NCI-H460 assay and cellsEOBA were scores cotreated were calculated with the (c). indicated concentrations Data are the averages of three independentof TRAIL experi and βments-thujaplicin and error ( abars) or showγ-thujaplicin standard deviations. (b) for 24 h. Cell viability (% of control) was measured using a WST assay and EOBA scores were calculated (c). Data are the averages of three independent 3.3. β-. Thujaplicin Enhances the Cell Death Induction Effect of TRAIL on NCI-H460 Cells experiments and error bars show standard deviations. In NCI-H460 cells, we also investigated the cell death induction effect of combination treatments with β- or γ-thujaplicin and TRAIL using a trypan blue exclusion assay. As shown in Figure 4, 10 µM β-thujaplicin substantially potentiated cell death induction in NCI-H460 cells (to ~60%) in the presence of 0.1 ng/mL TRAIL. On the other hand, γ-thujaplicin only slightly enhanced TRAIL-induced cell death. Both thujaplicins had a small potentiating effect on cell death induced by 0.01 ng/mL TRAIL.

Figure 4. Enhancement of the TRAIL cell death induction effect on NCI-H460 cells through a combination treatment with β-thujaplicin. NCI-H460 cells were cotreated with the indicated concentrations of TRAIL and β- or γ-thujaplicin for 24 h. Cell death (%) was determined by a trypan blue

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(c) MedicinesFigure2021, 83., 26Enhancement of the antiproliferative effect of TRAIL on NCI-H460 cells by combination treatments with β- or γ- 6 of 11 thujaplicin. NCI-H460 cells were cotreated with the indicated concentrations of TRAIL and β-thujaplicin (a) or γ-thujaplicin (b) for 24 h. Cell viability (% of control) was measured using a WST assay and EOBA scores were calculated (c). Data are the averages of three independent experiments and error bars show standard deviations. 3.3. β-. Thujaplicin Enhances the Cell Death Induction Effect of TRAIL on NCI-H460 Cells 3.3. β-. Thujaplicin Enhances the Cell Death Induction Effect of TRAIL on NCI-H460 Cells In NCI-H460 cells, we also investigated the cell death induction effect of combination treatmentsIn NCI-H460 with cells,β- or weγ also-thujaplicin investigated and the TRAIL cell death using induction a trypan effect blue of combination exclusion assay. As treatments with β- or γ-thujaplicin and TRAIL using a trypan blue exclusion assay. As shown in Figure4, 10 µM β-thujaplicin substantially potentiated cell death induction shown in Figure 4, 10 µM β-thujaplicin substantially potentiated cell death induction in in NCI-H460 cells (to ~60%) in the presence of 0.1 ng/mL TRAIL. On the other hand, NCI-H460 cells (to ~60%) in the presence of 0.1 ng/mL TRAIL. On the other hand, γ-thu- γ japlicin-thujaplicin only slightly only enhanced slightly enhanced TRAIL-induced TRAIL-induced cell death. Both cell thujaplicins death. Both had thujaplicins a small had a potentiatingsmall potentiating effect on effectcell deat onh cellinduced death by induced 0.01 ng/mL by TRAIL. 0.01 ng/mL TRAIL.t

FigureFigure 4. 4.EnhancementEnhancement of the of theTRAIL TRAIL cell death cell death inductio inductionn effect on effect NCI-H460 on NCI-H460 cells through cells througha com- a combi- binationnation treatmenttreatment with with ββ-thujaplicin.-thujaplicin. NCI-H460 NCI-H460 cells cells were were cotreated cotreated with withthe indicated the indicated concentra- concentrations tions of TRAIL and β- or γ-thujaplicin for 24 h. Cell death (%) was determined by a trypan blue of TRAIL and β- or γ-thujaplicin for 24 h. Cell death (%) was determined by a trypan blue exclusion assay. Data are the averages of three independent experiments and error bars show standard deviations. Asterisks (*, **, and ***) indicate p value smaller than 0.05 (p < 0.05), 0.01 (p < 0.01), and 0.001 (p < 0.001), respectively.

3.4. Potentiating Effect of β-Thujaplicin on TRAIL-Induced Cell Death in NCI-H460 Cells Is Accompanied by Enhanced Caspase-3/7 Activity Next, we assessed whether cotreatment with β-thujaplicin promotes activation of caspase-3/7, which occurs during TRAIL-induced cell death in NCI-H460 cells. As shown in Figure5, β-thujaplicin signiﬁcantly enhanced caspase-3/7 activity in NCI-H460 cells in the presence of 0.1 ng/mL TRAIL.

3.5. Tropolone, β-Thujaplicin, and γ-Thujaplicin Treatments Decrease the Amount of XIAP Protein in NCI-H460 Cells To examine whether tropolone derivatives (which are zinc chelators) decrease XIAP protein levels in cells, NCI-H460 cells were treated with 10 µM of tropolone, β-thujaplicin, and γ-thujaplicin for 24 h, and then XIAP protein levels were measured in cell extracts via western blot analysis. Interestingly, the amount of XIAP protein in cells was reduced by 10 µM of tropolone derivatives, especially by β- and γ-thujaplicins (Figure6a). Furthermore, the β-thujaplicin-mediated decrease in XIAP was dose-dependent (Figure6b). As shown in Figure6c, treatment with TRAIL alone at 0.1 ng/mL did not affect the amount of XIAP in cells; however, at 1 ng/mL, TRAIL treatment markedly decreased XIAP protein levels. When cotreated with 0.1 ng/mL TRAIL and 10 µM β- or γ-thujaplicin, the decrease in XIAP levels was considerably enhanced. Medicines 2021, 8, x FOR PEER REVIEW 7 of 11

exclusion assay. Data are the averages of three independent experiments and error bars show standard deviations. Asterisks (*, **, and ***) indicate p value smaller than 0.05 (p < 0.05), 0.01 (p < 0.01), and 0.001 (p < 0.001), respectively.

3.4. Potentiating Effect of β-Thujaplicin on TRAIL-Induced Cell Death in NCI-H460 Cells Is Accompanied by Enhanced Caspase-3/7 Activity Next, we assessed whether cotreatment with β-thujaplicin promotes activation of Medicines 2021, 8, 26 caspase-3/7, which occurs during TRAIL-induced cell death in NCI-H460 cells. As shown7 of 11 in Figure 5, β-thujaplicin significantly enhanced caspase-3/7 activity in NCI-H460 cells in the presence of 0.1 ng/mL TRAIL.

FigureFigure 5. 5. EnhancementEnhancement of of caspase-3/7 caspase-3/7 activity activity in inTR TRAIL-treatedAIL-treated NCI-H460 NCI-H460 cells cells through through combina- combination tiontreatment treatment with withβ-thujaplicin. β-thujaplicin. NCI-H460NCI-H460 cells cells were were cotrea cotreatedted with with 0.1 0.1 ng/mL ng/mL TRAIL TRAIL and 10 and µM 10 µM β-thujaplicin for 24 h. Caspase-3/7 activity was then measured by a Caspase-Glo® 3/7 Assay System. β-thujaplicin for 24 h. Caspase-3/7 activity was then measured by a Caspase-Glo® 3/7 Assay System. Data are the averages of three independent experiments and error bars show standard deviations. * Medicines 2021, 8, x FOR PEER REVIEWData are the averages of three independent experiments and error bars show standard deviations.8 of 11 p < 0.05. * p < 0.05. 3.5. Tropolone, β-Thujaplicin, and γ-Thujaplicin Treatments Decrease the Amount of XIAP Protein in NCI-H460 Cells To examine whether tropolone derivatives (which are zinc chelators) decrease XIAP protein levels in cells, NCI-H460 cells were treated with 10 µM of tropolone, β-thujaplicin, and γ-thujaplicin for 24 h, and then XIAP protein levels were measured in cell extracts via western blot analysis. Interestingly, the amount of XIAP protein in cells was reduced by 10 µM of tropolone derivatives, especially by β- and γ-thujaplicins (Figure 6a). Further- more, the β-thujaplicin-mediated decrease in XIAP was dose-dependent (Figure 6b). As shown in Figure 6c, treatment with TRAIL alone at 0.1 ng/mL did not affect the amount of XIAP in cells; however, at 1 ng/mL, TRAIL treatment markedly decreased XIAP protein levels.( Whena) cotreated with 0.1 ng/mL TRAIL and 10 µM( bβ)- or γ-thujaplicin, the decrease in XIAP levels was considerably enhanced.

(c)

FigureFigure 6. 6.Decrease Decrease in in XIAP XIAP protein protein levels levels in NCI-H460 in NCI-H460 cells cells following following tropolone, tropolone,β-thujaplicin, β-thujaplicin, and γ -thujaplicinand γ-thujaplicin treatments. treatments. (a) NCI-H460 cells were treated with 10 µM of tropolone, β-thujaplicin, and γ-thujaplicin for 24 h. (b) NCI-H460 (a) NCI-H460 cells were treated with 10 µM of tropolone, β-thujaplicin, and γ-thujaplicin for 24 h. (b) NCI-H460 cells cells were treated with 0, 10, 20, and 40 µM of β-thujaplicin for 24 h. (c) NCI-H460 cells were cotreated with the indicated were treated with 0, 10, 20, and 40 µM of β-thujaplicin for 24 h. (c) NCI-H460 cells were cotreated with the indicated concentrations of TRAIL and β-thujaplicin or γ-thujaplicin for 24 h. In (a–c), cell lysates were subjected to western blot β γ a c concentrationsanalysis using ofXIAP TRAIL antibody. and -thujaplicin or -thujaplicin for 24 h. In ( – ), cell lysates were subjected to western blot analysis using XIAP antibody. 3.6. Tropolone, β-Thujaplicin, and γ-Thujaplicin Inhibit the Binding of the Smac N-Terminal 3.6.Peptide Tropolone, to the XIAP-BIR3β-Thujaplicin, Domain and γ -Thujaplicin Inhibit the Binding of the Smac N-Terminal Peptide to the XIAP-BIR3 Domain Since the core structure of embelin is similar to that of tropolone, we evaluated the Since the core structure of embelin is similar to that of tropolone, we evaluated the inhibitory effect of tropolone derivatives on the binding of the Smac N-terminal peptide inhibitory effect of tropolone derivatives on the binding of the Smac N-terminal peptide to to the XIAP-BIR3 domain using a fluorescence polarization assay. Under our experimental the XIAP-BIR3 domain using a ﬂuorescence polarization assay. Under our experimental conditions, the IC50 value for embelin was 3.1 ± 0.6 µM (Table 1), similar to the previously conditions, the IC50 value for embelin was 3.1 ± 0.6 µM (Table1), similar to the previously reported IC50 value [19]. As shown in Table 1, tropolone, β-thujaplicin, and γ-thujaplicin reported IC value [19]. As shown in Table1, tropolone, β-thujaplicin, and γ-thujaplicin inhibited the50 binding of the Smac N-terminal peptide to the XIAP-BIR3 domain in a dose- dependent manner. The IC50 values of tropolone, β-thujaplicin, and γ-thujaplicin were 83.1 ± 6.9 µM, 58.5 ± 2.8 µM, and 56.2 ± 2.9 µM, respectively. The inhibitory activities of β- and γ-thujaplicins were higher than that of tropolone.

Table 1. Effects of tropolone derivatives on the binding of the Smac N-terminal peptide to the XIAP-BIR3 domain.

Compound Structure IC50 (μM) a

OH Tropolone 83.1 ± 6.9

HO β- 58.5 ± 2.8 thujaplicin

OH γ- 56.2 ± 2.9 thujaplicin

MedicinesMedicinesMedicines 2021 2021 2021, ,8 ,8 ,8 ,x ,x xFOR FOR FOR PEER PEER PEER REVIEW REVIEW REVIEW 88 8of of of 11 11 11

((a(aa)) ) ((b(bb)) )

((c(c)c) ) FigureFigureFigure 6. 6.6. Decrease DecreaseDecrease in inin XIAP XIAPXIAP protein proteinprotein levels levelslevels in inin NCI-H460 NCI-H460NCI-H460 cells cellscells following followingfollowing tropolone, tropolone,tropolone, β ββ-thujaplicin,-thujaplicin,-thujaplicin, and andand γ γγ-thujaplicin-thujaplicin-thujaplicin treat- treat-treatments.ments.ments. ( (a(a)a) ) NCI-H460 NCI-H460NCI-H460 cells cellscells were werewere treated treatedtreated with withwith 10 1010 µM µMµM of ofof tropolone, tropolone,tropolone, β ββ-thujaplicin,-thujaplicin,-thujaplicin, and andand γ γγ-thujaplicin-thujaplicin-thujaplicin for forfor 24 2424 h. h.h. ( (b(bb)) ) NCI-H460 NCI-H460NCI-H460 cellscellscells were were were treated treated treated with with with 0, 0, 0, 10, 10, 10, 20, 20, 20, and and and 40 40 40 µM µM µM of of of β β β-thujaplicin-thujaplicin-thujaplicin for for for 24 24 24 h. h. h. ( ( c(c)c) )NCI-H460 NCI-H460 NCI-H460 cells cells cells were were were cotr cotr cotreatedeatedeated with with with the the the indicated indicated indicated concentrationsconcentrationsconcentrations of ofof TRAIL TRAILTRAIL and andand β ββ-thujaplicin-thujaplicin-thujaplicin or oror γ γγ-thujaplicin-thujaplicin-thujaplicin for forfor 24 2424 h. h.h. In InIn ( (a(a–a–c–c),c),), cell cellcell lysates lysateslysates were werewere subjected subjectedsubjected to toto western westernwestern blot blotblot analysisanalysisanalysis using using using XIAP XIAP XIAP antibody. antibody. antibody.

3.6.3.6.3.6. Tropolone, Tropolone,Tropolone, β ββ-Thujaplicin,-Thujaplicin,-Thujaplicin, and andand γ γγ-Thujaplicin-Thujaplicin-Thujaplicin Inhibit InhibitInhibit the thethe Bind BindBindinginging of ofof the thethe Smac SmacSmac N-Terminal N-TerminalN-Terminal PeptidePeptidePeptide to toto the thethe XIAP-BIR3 XIAP-BIR3XIAP-BIR3 Domain DomainDomain SinceSinceSince the thethe core corecore structure structurestructure of ofof embelin embelinembelin is isis simila similasimilarr r to toto that thatthat of ofof tropolone, tropolone,tropolone, we wewe evaluated evaluatedevaluated the thethe inhibitoryinhibitoryinhibitory effect effecteffect of ofof tropolone tropolonetropolone derivatives derivativesderivatives on onon the thethe binding bindingbinding of ofof the thethe Smac SmacSmac N-terminal N-terminalN-terminal peptide peptidepeptide tototo the the the XIAP-BIR3 XIAP-BIR3 XIAP-BIR3 domain domain domain using using using a a a fluorescence fluorescence fluorescence polarization polarization polarization assay. assay. assay. Un Un Underderder our our our experimental experimental experimental Medicines 2021, 8, 26 8 of 11 conditions,conditions,conditions, the thethe IC ICIC505050 value valuevalue for forfor embelin embelinembelin was waswas 3.1 3.13.1 ± ±± 0.6 0.60.6 µM µMµM (Table (Table(Table 1), 1),1), similar similarsimilar to toto the thethe previously previouslypreviously reportedreportedreported IC ICIC505050 value valuevalue [19]. [19].[19]. As AsAs shown shownshown in inin Table TableTable 1, 1,1, tropolone, tropolone,tropolone, β ββ-thujaplicin,-thujaplicin,-thujaplicin, and andand γ γγ-thujaplicin-thujaplicin-thujaplicin inhibitedinhibitedinhibited the thethe binding binding binding of ofof the thethe Smac SmacSmac N-terminal N-terminal N-terminal peptide peptidepeptide to toto the thethe XIAP-BIR3 XIAP-BIR3XIAP-BIR3 domain domain domain in inin a a a dose- dose- dose- inhibited thedependentdependentdependent binding manner. ofmanner.manner. the Smac The TheThe N-terminal IC ICIC505050 values valuesvalues peptide of ofof tropolone, tropolone,tropolone, to the XIAP-BIR3 β ββ-thujaplicin,-thujaplicin,-thujaplicin, domain and andand in γ γγ-thujaplicin a-thujaplicin-thujaplicin dose- were werewere dependent83.183.183.1 manner. ± ±± 6.9 6.96.9 µM, µM,µM, The 58.5 58.558.5 IC50 ± ± ± values2.8 2.8 2.8 µM, µM,µM, of and andand tropolone, 56.2 56.2 56.2 ± ±± 2.9 2.92.9β µM,-thujaplicin, µM,µM, respectively. respectively. respectively. and γ The TheThe-thujaplicin inhibitory inhibitory inhibitory were activities activities activities of ofof β β β-- - 83.1 ± 6.9andµandandM, γ γ58.5γ-thujaplicins-thujaplicins-thujaplicins± 2.8 µM, were werewere and higher56.2 higherhigher± 2.9than thanthanµ M, that thatthat respectively. of ofof tropolone. tropolone.tropolone. The inhibitory activities of β- and γ-thujaplicins were higher than that of tropolone. TableTableTable 1. 1. 1. Effects Effects Effects of of of tropolone tropolone tropolone derivatives derivatives derivatives on on on the the the binding binding binding of of of the the the Smac Smac Smac N-terminal N-terminal N-terminal peptide peptide peptide to to to the the the Table 1. EffectsXIAP-BIR3XIAP-BIR3XIAP-BIR3 of tropolone domain. domain. domain. derivatives on the binding of the Smac N-terminal peptide to the XIAP-BIR3 domain.

a aaa CompoundCompoundCompoundCompound StructureStructure Structure IC50IC(ICICµM)505050 ( (μ(μμM)M)M)

O OO OH OHOH TropoloneTropoloneTropoloneTropolone 83.1 83.1±83.183.16.9 ± ±± 6.9 6.96.9

O OO HO HOHO βββ--- β-thujaplicin 58.5 58.5±58.558.52.8 ± ±± 2.8 2.82.8 thujaplicinthujaplicinthujaplicin

O OO OH OHOH γγ-- γ- 56.2 ± 2.9 Medicines 2021, 8, x FOR PEER REVIEWγ-thujaplicin 56.2 56.2±56.22.9 ±± 2.92.9 9 of 11 thujaplicinthujaplicinthujaplicin

OH EmbelinEmbelin 3.1 ± 0.63.1 ± 0.6 HO

a a 50 The IC50, i.e., the The inhibitor IC , i.e., concentration the inhibitor at which concentration 50% of bound at which Smac/Diablo 50% of peptide bound probe Smac/Diablo is displaced, peptide was probe is determined fromdisplaced, the dose–response was determined curve. Data from represent the dose–respons the mean ± standarde curve. deviation Data represent of three independentthe mean ± standard experiments. deviation of three independent experiments.

4. Discussion4. Discussion β-Thujaplicinβ-Thujaplicin is reported tois exhibitreported anticancer to exhibit activity anticanc iner various activity human in various cancer human cell cancer cell lines [6–13].lines In relation [6–13]. to In this relation anticancer to this activity, anticancerβ-thujaplicin activity, β has-thujaplicin been shown has been to induce shown to induce apoptosis inapoptosis human colonin human cancer colon cells cancer through cells the through activation the activation of caspases-9 of caspases-9 and -3 [ 9and] -3 [9] and and in humanin hepatocellularhuman hepatocellular carcinoma carcinoma cells through cells a mitochondria-dependentthrough a mitochondria-dependent intrinsic intrinsic pathway [13pathway]. Recently, [13]. it Recently, was reported it was that re nontoxicported that doses nontoxic of β-thujaplicin doses of β-thujaplicin inhibit the inhibit the migration ofmigration A549 lung of A549 cancer lung cells cancer through cells suppression through suppression of matrix of metalloproteinases matrix metalloproteinases and and inductioninduction of apoptosis of apoptosis via caspases-9 via caspases-9 and -3 and activation -3 activation [12]. To [12]. date, To however,date, however, the the mech- mechanism andanism targets and targets of β-thujaplicin’s of β-thujaplicin’s anticancer anticancer activity activity have not have been not fully been elucidated. fully elucidated. β In the presentIn study,the present we hypothesized study, we hypothesized that -thujaplicin that β acts-thujaplicin on XIAP acts both on as XIAP a zinc both as a zinc chelator andchelator an inhibitor and an targeting inhibitor thetargeting XIAP-BIR3 the XIAP-BIR3 domain domain to induce to induce and/or and/or enhance enhance apop- apoptosis. Wetosis. used We NCI-H460 used NCI-H460 cells to cells test to this test hypothesis this hypothesis because because they arethey considered are considered type II type II cellscells that that depend depend on on the the mitochondrial mitochondrial apoptotic apoptotic pathway pathway to to amplify amplify effectoreffector caspase ac- caspase activation during death receptor-induced apoptosis [20]. We assumed that, if tivation during death receptor-induced apoptosis [20]. We assumed that, if β-thujaplicin β-thujaplicin decreases and/or inhibits XIAP, it would enhance TRAIL-induced apoptosis decreases and/or inhibits XIAP, it would enhance TRAIL-induced apoptosis via the liber- via the liberation of caspases-9, -3, and -7 from XIAP-mediated inhibition. ation of caspases-9, -3, and -7 from XIAP-mediated inhibition. We found that β- and γ-thujaplicins, but not tropolone, treatments decreased the We found that β- and γ-thujaplicins, but not tropolone, treatments decreased the vi- viability of NCI-H460 cells in a dose-dependent manner. These results suggest that an alkyl ability of NCI-H460 cells in a dose-dependent manner. These results suggest that an alkyl side chain, in this case an isopropyl group, attached to tropolone contributes to the cell side chain, in this case an isopropyl group, attached to tropolone contributes to the cell death induction abilities of the tropolone derivatives. We therefore investigated whether β- death induction abilities of the tropolone derivatives. We therefore investigated whether and γ-thujaplicins could sensitize NCI-H460 cells to TRAIL-induced apoptosis. We found β- and γ-thujaplicins could sensitize NCI-H460 cells to TRAIL-induced apoptosis. We that 10 µM of β- and γ-thujaplicins alone did not induce a signiﬁcant degree of cytotoxicity found that 10 µM of β- and γ-thujaplicins alone did not induce a significant degree of and cell death, whereas a combined treatment of TRAIL and β- or γ-thujaplicin resulted in a synergisticcytotoxicity antiproliferative and effectcell death, on NCI-H460 whereas cells. a combined Furthermore, treatment 10 µM of βTRAIL-thujaplicin and β- or γ-thujaplicin resulted in a synergistic antiproliferative effect on NCI-H460 cells. Furthermore, 10 µM of β-thujaplicin substantially potentiated the cell death induction effect of 0.1 ng/mL TRAIL on NCI-H460 cells. Thus, we further investigated β-thujaplicin as a cotreatment, particularly whether it could promote activation of caspase-3/7, which occurs during TRAIL-induced apoptosis in NCI-H460 cells. β-Thujaplicin cotreatment with TRAIL was shown to significantly enhance the increase in caspase-3/7 activity in NCI-H460 cells. To our knowledge, this is the first report of β-thujaplicin promoting TRAIL-induced apoptosis by enhancement of caspase-3/7 activity in cancer cells. XIAP is probably the only bona fide inhibitor that selectively binds and inhibits caspases-9, -3, and -7, with zinc-binding BIR domains being responsible for this inhibitory function [14]. We found that a β-thujaplicin-mediated reduction in XIAP levels coincides with enhanced cell death and caspase-3/-7 activity in NCI-H460 cells treated with TRAIL. When NCI-H460 cells were treated with β-thujaplicin alone, the level of caspases-3/-7 activity remained consistently low throughout 24-h incubation. Although treatment of NCI- H460 cells with TRAIL alone did induce noticeable levels of caspase-3/-7 activity, cotreatment with β-thujaplicin significantly enhanced this activity. It seems that the β-thujaplicin-mediated decrease in XIAP protein levels contributes to the enhancement of caspase-3/-7 activity in TRAIL-treated NCI-H460 cells. The mechanism underlying the β- thujaplicin-induced decrease in XIAP levels remains to be elucidated. Interestingly, Ma- khov et al. [18] analyzed the action of TPEN on XIAP protein and their results implied that the addition of zinc chelators triggers a serine protease-dependent reduction of XIAP. It should be noted, however, that further experiments will be necessary to determine how β-thujaplicin decreases XIAP protein levels.

Medicines 2021, 8, 26 9 of 11

substantially potentiated the cell death induction effect of 0.1 ng/mL TRAIL on NCI-H460 cells. Thus, we further investigated β-thujaplicin as a cotreatment, particularly whether it could promote activation of caspase-3/7, which occurs during TRAIL-induced apoptosis in NCI-H460 cells. β-Thujaplicin cotreatment with TRAIL was shown to significantly enhance the increase in caspase-3/7 activity in NCI-H460 cells. To our knowledge, this is the first report of β-thujaplicin promoting TRAIL-induced apoptosis by enhancement of caspase-3/7 activity in cancer cells. XIAP is probably the only bona fide inhibitor that selectively binds and inhibits caspases- 9, -3, and -7, with zinc-binding BIR domains being responsible for this inhibitory function [14]. We found that a β-thujaplicin-mediated reduction in XIAP levels coincides with enhanced cell death and caspase-3/-7 activity in NCI-H460 cells treated with TRAIL. When NCI-H460 cells were treated with β-thujaplicin alone, the level of caspases-3/-7 activity remained consistently low throughout 24-h incubation. Although treatment of NCI-H460 cells with TRAIL alone did induce noticeable levels of caspase-3/-7 activity, cotreatment with β-thujaplicin significantly enhanced this activity. It seems that the β-thujaplicin-mediated decrease in XIAP protein levels contributes to the enhancement of caspase-3/-7 activity in TRAIL-treated NCI-H460 cells. The mechanism underlying the β-thujaplicin-induced decrease in XIAP levels remains to be elucidated. Interestingly, Makhov et al. [18] analyzed the action of TPEN on XIAP protein and their results implied that the addition of zinc chelators triggers a serine protease-dependent reduction of XIAP. It should be noted, however, that further experiments will be necessary to determine how β-thujaplicin decreases XIAP protein levels. The BIR3 domain of XIAP, i.e., where caspase-9 and Smac proteins bind, is a promising site for the design of small-molecule XIAP inhibitors. Embelin, a small-molecule inhibitor that targets the XIAP-BIR3 domain [19], has a similar structure to β-thujaplicin, i.e., an aromatic ring with an α-hydroxy ketone and an alkyl group. Therefore, using a fluorescence polarization assay, we evaluated the inhibitory effect of tropolone derivatives on the binding of the Smac N-terminal peptide to the XIAP-BIR3 domain; both β- and γ-thujaplicins inhibited this binding in a dose-dependent manner and their inhibitory activities were greater than that of tropolone. These results suggest that the attachment of an isopropyl group to tropolone contributes to the binding activity to the XIAP-BIR3 domain. Some synthetic β-thujaplicin derivatives have been reported to have higher cytotoxic effects than β-thujaplicin itself in cancer cell lines including NCI-H460 cells [3]. Thus, more potent XIAP-depleting and XIAP-BIR3-binding tropolone derivatives may exist. A trial was recently conducted in which dual action compounds, consisting of a Smac mimetic moiety and zinc-chelating moiety, were constructed [24]. The results of the present study reveal that β-thujaplicin might enhance TRAIL-induced apoptosis in NCI-H460 cells through the dual effects on XIAP, i.e., through XIAP depletion and binding to the XIAP-BIR3 domain. Thus, a tropolone scaffold may be useful for designing novel nonpeptidic small-molecular XIAP inhibitors that exert dual effects on XIAP in a single structure. Indeed, the screening and design of novel XIAP-inhibiting tropolone derivatives is currently in progress with tropolone being used as a starting compound. In future studies, the mechanism by which XIAP levels are reduced by tropolone derivatives should be investigated using more potent XIAP inhibitors derived from the tropolone structure.

Author Contributions: Conceptualization, R.T.; methodology, R.T.; investigation, S.S., M.K., Y.Y., R.K., K.A., A.T., and M.T.; resources, T.O., H.A., and T.A.; data curation, R.T.; writing—original draft preparation, R.T. and S.S.; writing—review and editing, R.T., S.S., and S.-i.T.; supervision, R.T. and S.-i.T. project administration, R.T. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: The authors would like to thank staff members from Laboratory for Medical Molecular Biology, Department of Pharmacy, Faculty of Pharmaceutical Sciences, Tokyo University of Science, for their assistance. Conﬂicts of Interest: The authors declare no conﬂict of interest. Medicines 2021, 8, 26 10 of 11

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