A Synthetic Pan-Aurora Kinase Inhibitor, 5-Methoxy-2-(2-methoxynaphthalen-1-yl)-4H-chromen-4-one, Triggers Reactive Oxygen Species-Mediated Apoptosis in HCT116 Colon Cancer Cells

Aurora kinases are Ser/%r kinases that function as mitotic regulators. 5-Methoxy-2-(2-methoxynaphthalen-1-yl)-4H-chromen-4-one (DK1913) is a synthetic pan-Aurora kinase inhibitor. However, the mode of action of DK1913 concerning the induction of apoptosis is unclear. Here, we report that DK1913 triggered apoptosis, as revealed by flow cytometry and Annexin V staining. DK1913 enhanced the intracellular levels of reactive oxygen species (ROS) and stimulated the endoplasmic reticulum (ER) and genotoxic stress responses.We also found that DK1913 induced the loss of mitochondrial membrane potential, leading to the activation of caspase-9, caspase-7, and caspase-3. In addition, the antioxidant, butylated hydroxyanisole (BHA), abrogated DK1913-induced loss of mitochondrial membrane potential and activation of the caspase cascade. %ese findings demonstrate that pan-Aurora kinase inhibitor DK1913 triggers apoptosis through ROSmediated ER and genotoxic stress responses.


Introduction
Cell division is an essential event for normal growth and development. One of the primary purposes of mitosis is the correct segregation of the duplicated chromosomes to two daughter cells. Mitotic progression is regulated by a complex network of mitotic kinases [1]. e aurora kinase family members (Aurora-A, Aurora-B, and Aurora-C) are Ser/ r protein kinases that play critical roles in the regulation of mitotic progression [2]. e subcellular distribution and functions of the aurora kinases are largely distinct; Aurora-A is a mitotic centrosomal kinase required for centrosomal maturation, centrosomal microtubule organization, and bipolar spindle assembly during mitosis [3], Aurora-B is present on the mitotic spindle and plays a role in chromosome condensation, sister chromatid cohesion, mitotic spindle assembly, and chromosomal alignment [2], and Aurora-C is localized to the centrosome and plays a role in centrosome function at a later stage of mitosis [4]. Aurora kinases are overexpressed in many human cancers, including colon cancer [5], and deregulation of their kinase activity can lead to chromosomal instability, which results in tumorigenesis [6]. Inhibition of aurora kinases induces mitotic cell cycle arrest and eventually triggers apoptotic cell death [7]. erefore, targeting Aurora kinases is a valuable strategy when designing novel drugs in cancer therapy.
In a previous study, we investigated the structural features of flavone derivatives that inhibit the aurora kinase activity [8]. In that study, we found that 5-methoxy-2-(2methoxynaphthalen-1-yl)-4H-chromen-4-one (DK1913) directly binds to Aurora-A and Aurora-B and induces cell cycle arrest at the G2/M phase, eventually leading to apoptotic cell death. As the aurora kinases are associated with mitotic signaling, it seems clear that inhibition of the aurora kinase could be linked to the cell cycle arrest at the G2/M phase. However, the molecular mechanisms responsible for the induction of apoptosis remain elusive. e mitotic arrest can increase the intracellular level of reactive oxygen species (ROS), thereby inducing oxidative stress [9]. ROS comprises oxygen-containing reactive molecules, such as superoxide anion radical ( · O 2 − ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical ( · OH), and singlet oxygen ( 1 O 2 ). Aberrant ROS production can lead to cellular damages known as oxidative stress. Although oxidative stress has been implicated in various human diseases, there is increasing evidence regarding the beneficial effects of ROS with respect to the selective killing of cancer cells [10,11]. Aberrant accumulation of ROS can activate the unfolded protein response (UPR) in the endoplasmic reticulum (ER). Prolonged UPR has been implicated in the induction of ER stress, resulting in apoptotic cell death [12,13].

Immunoblot
Analysis. HCT116 cells were extracted in lysis buffer containing 20 mM HEPES (pH 7.2), 1% Triton X-100, 10% glycerol, 150 mM NaCl, 10 μg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride (PMSF). e protein extracts (20 μg each) were electrophoresed on a 10% SDSpolyacrylamide gel and transferred onto a nitrocellulose membrane. e blots were incubated with the appropriate primary and secondary antibodies, and antibody-reactive proteins were visualized using an enhanced chemiluminescence detection system (GE Healthcare, Piscataway, NJ, USA). Relative band intensities were analyzed using the ImageJ software version 1.52a (National Institute of Health, Bethesda, MD, USA) and expressed relative to the basal control band (set to 1) after normalizing with GAPDH levels.
2.6. Apoptosis Assay by Annexin V Staining. Apoptosis assay was performed using a FITC-conjugated Annexin V kit according to the manufacturer's instructions (BD Pharmingen). Fluorescence was measured using a Nucleo-Counter NC-3000 image cytometer (ChemoMetec), as described previously [17].

Detection of Intracellular Reactive Oxygen Species (ROS).
Intracellular levels of ROS were determined using oxidantsensing fluorescent probe DCF-DA. After treatment with DK1913, the cells were incubated with 10 μM DCF-DA for an additional 30 min. DCF fluorescence resulting from ROS production was measured using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Fluorescent intracellular ROS were also visualized by an EVOSf1 fluorescence microscope (Advance Microscopy Group, Bothell, WA, USA).

Measurement of the Mitochondrial Membrane Potential (ΔΨm).
Loss of the mitochondrial membrane potential was monitored by measuring the fluorescence of the lipophilic cationic probe JC-1 (5,5′,6,6′-tetrachloro1,1′,3,3′-tetraethylbenzimi-dazolylcarbocyanine chloride), according to the manufacturer's instruction (Cayman Chemical). HCT116 cells were seeded into a 24-well plate and treated with 5 μM DK1913. After 18 h, the cells were treated with JC-1 dye and incubated for an additional 30 min at 37°C, according to the manufacturer's instructions (Cayman Chemical). Fluorescent images were captured under an EVOSf1 ® fluorescence microscope. e aggregated JC-1 in mitochondria indicates high membrane potential and emits red fluorescence, while monomeric JC-1 indicates loss of membrane potential and emits green fluorescence.

Statistical Analysis.
Statistical significance was analyzed using GraphPad PRISM software version 8.2.1 (GraphPad Software Inc., La Jolla, CA). A P value of less than 0.05 was considered significant. All experiments were performed in triplicate.

DK1913 Inhibits the Aurora Kinases in HCT116 Colon
Cancer Cells. To investigate the mode of action of DK1913 (Figure 1(a)) concerning the induction of apoptosis, we first confirmed the inhibitory effect of DK1913 on the phosphorylation of Aurora kinases by immunoblot analysis. Treatment with DK1913 significantly (P < 0.001, n � 3) prevented phosphorylation of Aurora-A, Aurora-B, and Aurora-C at a concentration of less than 5 μM (Figure 1(b)).

DK1913 Induces G2/M Cell Cycle Arrest and Apoptosis.
To determine the cytotoxicity of DK1913, we performed a cell viability assay using a CCK-8. e cell viability was 95.7 ± 4.16%, 82.7 ± 8.62%, 73.3 ± 7.09%, 61.7 ± 4.51%, and 36.7 ± 11.2% at 1, 5, 10, 20, and 40 μM, respectively, as compared with the vehicle-treated control (0 μM). ese results suggest that DK1913 reduced cell viability in a dosedependent fashion (Figure 2(a)). We next tested the effect of DK1913 on cell cycle and apoptosis by flow cytometry. As expected, DK1913 increased the population of G2/M phase cells from 11.9% to 58.3% with a concomitant decrease in G1 and an increase in sub-G1 cell population (Figure 2(b)). Translocation of phosphatidylserine (PS) from the inner layer to the outer leaflet is a typical marker of apoptosis [18]. We thus examined the effect of DK1913 on the localization of PS on the membrane surface using fluorescein-5-isothiocyanate (FITC)-conjugated Annexin V (AV). Propidium iodide (PI) was used to detect necrotic cells. Treatment with DK1913 increased the population of AV-positive cells from 9% to 97% (Figure 2(c)).
ese results demonstrate that DK1913 induces cell cycle arrest at the G2/M phase and triggers apoptotic cell death in HCT116 colon cancer cells. (ROS). Several reports have demonstrated that inhibition of the aurora kinase can induce G2/M arrest and apoptosis in various cancer cells [19,20]. However, very little is understood about how DK1913 causes apoptosis. Many polyphenols, such as quercetin and resveratrol, produce intracellular ROS to induce apoptosis in various cancer cells Total cell lysates were immunoblotted with a phospho-specific antibody, which can detect phospho-Aurora-A (T288)/Aurora-B (T232)/Aurora-C (T198). Band intensities of phosphorylated Aurora-A, Aurora-B, and Aurora-C were measured using ImageJ software and expressed relative to the basal control bands (set to 100%) after normalizing with total proteins (Aur-A and Aur-B) or GAPDH (Aur-C) levels. Aur, Aurora; bars, means ± SD (n � 3). * * * P < 0.001 by Sidak's multiple comparisons test. [21,22]. As aberrant ROS production in cancer cells may increase the efficacy of chemotherapeutic anticancer agents [23,24], we investigated whether DK1913 elevated the levels of intracellular ROS. We detected ROS using the redoxsensitive 2′,7′-dichlorofluorescein diacetate (DCF-DA) dye. Flow cytometry showed that DK1913 increased DCF fluorescence within 6 h, which remained elevated relative to the control for more than 12 h (Figure 3(a)). To further assess ROS generation by DK1913, we visualized intracellular ROS using DCF-DA as a fluorescent probe in cultured cells with fluorescence microscopy. ere was no noticeable fluorescence in vehicle-treated cells; however, after 12 h of   DK1913 treatment, the number of fluorescent cells increased dose-dependently (Figure 3(b)). ese data suggest that DK1913 generates intracellular ROS.

DK1913 Induces the Endoplasmic Reticulum (ER) Stress.
ROS can induce apoptosis through unfolded protein response-(UPR-) mediated ER stress response [25]. A typical feature of the UPR is the accumulation of ER-resident UPR sensor proteins. To determine whether DK1913 stimulates the UPR pathway, we investigated the effect of DK1913 on the expression of ER-resident UPR sensor proteins. As shown in Figure 4, DK1913 increased the amounts of ER stress sensor (GRP78), ER stress effectors (PERK and IRE1α), and transcription factors ATF4, and spliced form of XBP1 (sXBP1) in a time-dependent manner, suggesting the activation of the ER stress response by DK1913. Under sustained ER stress conditions, ATF4 and sXBP1 contributed to the induction of apoptosis by upregulating the proapoptotic transcription factor C/EBP homologous protein (CHOP), also known as growth arrest-and DNA damage-inducible gene 153 (GADD153) [26]. We also found that DK1913 increased the amounts of CHOP and proapoptotic BCL2-interacting protein (BIM), a downstream target of CHOP [27], 12 h after treatment. ese data suggest that DK1913 causes the activation of the UPR-mediated ER stress.

DK1913 Induces the Genotoxic Response.
Tumor suppressor p53 plays a central role in cellular genotoxic stressinduced apoptosis [28]. Prolonged ER stress can upregulate p53 expression. To test whether p53 plays a role in DK1913induced apoptosis, we examined the expression levels of p53. As shown in Figure 5, the amounts of phosphorylated p53 at Ser-15 were significantly increased after 6 h of DK1913 treatment. BCL2-associated X protein (BAX), a downstream target of p53 [29], was significantly increased after 12 h. Phosphorylation of histone variant H2AX at Ser-139 (termed c-H2AX), a hallmark of DNA damage [30], was significantly increased 24 h after DK1913 treatment. ese results suggest the induction of genotoxic stress by DK1913. Caspase-2, a cysteine protease that is a member of the interleukin-1β-converting enzyme (ICE) family, mediates DNA damage-induced apoptosis by producing a truncated form of BID (t-BID) [31,32]. DK1913 also increased the proteolytic cleavage of pro-caspase-2 and t-BID in a timedependent manner. ese data suggest that DK1913 induces a genotoxic stress response.

DK1913 Induces Activation of the Caspase Cascade.
Mitochondria play a pivotal role in triggering the intrinsic apoptosis pathway. Caspases are cysteine-dependent aspartate-specific proteases that mediate apoptotic signaling [33].
Oligomerization of proapoptotic BAX results in the formation of a membrane channel and facilitates the release of Cyt c from the mitochondria into the cytosol [34]. BCL2 homology domain 3-(BH3-) containing proteins, including BIM and t-BID, stimulate the release of Cyt c through inhibition of BCL2 [35]. e Cyt c released in the cytosol binds to APAF1 and pro-caspase-9 to form a complex called an apoptosome. Within the apoptosome, pro-caspase-9 becomes activated, leading to activation of the executioner caspases (caspase-3 and caspase-7), which triggers apoptosis [36].

Antioxidant Butylated Hydroxyanisole (BHA) Abrogates DK1913-Induced Activation of the Caspase Cascade.
Besides BAX-dependent channel formation, loss of mitochondria membrane potential (ΔΨm) contributes to the release of Cyt c as well as that of other mitochondrial apoptogenic factors, such as apoptosis-inducing factor (AIF) and Smac/Diablo [38]. To further evaluate whether DK1913 alters the ΔΨm, we used a membrane potentialsensitive fluorochrome, JC-1, which emits red fluorescence in intact mitochondria with high ΔΨm; in contrast, it emits green fluorescence upon loss of ΔΨm. As shown in Figure 7(a), DK1913 treatment shifted the emission from red to green fluorescence, indicating the loss of ∆Ψm in DK1913-induced apoptosis. It has been reported that ROS generation is associated with loss of ΔΨm and mitochondria-mediated apoptosis [39]. To determine the causal role of ROS generation in DK1913-mediated apoptosis, we tested the effect of butylated hydroxyanisole (BHA), a synthetic antioxidant, on DK1913-mediated loss of ΔΨm. Pretreatment with BHA switched the green fluorescence induced by DK1913 to red fluorescence (Figure 7(a)). To further clarify the role of ROS in DK1913-induced activation of the caspase cascade, we tested the effect of BHA on DK1913-induced caspase activation by immunoblotting. We found that the DK1913 treatment-induced cleavage of caspase-7, caspase-3, and PARP was decreased in the presence of BHA (Figure 7(b)). ese results suggest that DK1913-induced elevation in ROS levels is closely associated with the loss of ΔΨm of the mitochondria, which in turn facilitates the activation of the mitochondrial caspase cascade.
In this study, we have ascertained the effect of aurora kinase inhibitor on ROS-induced apoptosis in HCT116 colon cancer cells. Similar findings were observed in previous studies demonstrating that Aurora-A inhibitor TCS10 [15] and Aurora-B inhibitor barasertib [40] trigger apoptotic cell death through the excess generation of ROS. Currently, it remains unclear how the aurora kinases inhibition by DK1913 can lead to ROS generation. One possible explanation is that mitotic arrest induced by aurora kinase inhibition could promote oxidative metabolism, increasing intracellular ROS levels. is hypothesis is supported by the previous observation that ROS levels are elevated in mitotic cancer cells [9] and that 200 μM) for 30 min, followed by treatment with 5 μM DK1913. After 24 h, cell lysates were prepared and immunoblotting was performed using antibodies against cleaved caspase-7, caspase-3, and PARP. Arrow: proform; arrowhead: cleaved form; GAPDH: internal control. Band intensities of each blot were measured using ImageJ software and expressed relative to the basal band intensity (ser to 1) after adjusting to GAPDH levels. e data are presented as means ± SD (n � 3). NS, not significant; * P < 0.05; * * P < 0.01; * * * P < 0.001; compared with the 0time control according to Dunnett's multiple comparisons test. 8 Journal of Chemistry accumulated cyclinB/Cdk, a cell cycle regulator involved in driving mitotic entry and progression, during mitotic arrest enhances mitochondrial oxidative metabolism [41]. Indeed, microtubule stabilizer paclitaxel, an antimitotic drug resulting in mitotic arrest, causes oxidative stress [42]. Additional studies are needed to provide mechanistic insight into the mode of action of DK1913 in the generation of ROS.

Conclusion
e synthetic Aurora kinase inhibitor, DK1913, triggers apoptosis through a ROS-mediated mitochondrial caspase pathway activated via genotoxic and ER stress responses in HCT116 colon cancer cells ( Figure 8). As most cancer cells are more sensitive to ROS than normal cells, ROS-producing DK1913 could be used for the selective targeting of cancer cells [43][44][45][46]. Further studies are needed to evaluate the in vivo anticancer efficacy of DK1913.

Data Availability
e data used to support the findings of this study are included within the article and are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.