Vol. 39, No. 6 935Biol. Pharm. Bull. 39, 935–945 (2016)

© 2016 The Pharmaceutical Society of Japan

Regular Article

In Vitro Characterization of Derrone as an Aurora Kinase InhibitorNhung Thi My Hoang,*,a Thuong Thien Phuong,b Trang Thi Nhu Nguyen,a,c Yen Thi Hai Tran,a,d Anh Thi Ngoc Nguyen,a,e Thanh Lai Nguyen,a and Khanh Thi Van Buia

a Faculty of Biology, VNU University of Science; 334 Nguyen Trai Street, Hanoi, Vietnam: b National Institute of Medicinal Materials (NIMM); 3B Quang Trung Street, Hanoi, Vietnam: c Assisted Reproductive Technology Center, Vinmec International Hospital; 458 Minh Khai Street, Hanoi, Vietnam: d Korea Advanced Institute of Science and Technology; 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Korea: and e Faculty of Pathological Anatomy, 108 Military Central Hospital; No. 1 Tran Hung Dao Street, Hanoi, Vietnam.Received October 26, 2015; accepted February 19, 2016; advance publication released online March 15, 2016

Among mitotic kinases, Aurora kinases are the most widely studied, since their expression is restricted to mitosis. They play a key role in chromosome segregation and cell polyploidy. Aurora kinases are impor-tant therapeutic targets, and several research groups have directed their efforts toward the identification of kinase inhibitors. The aim of this study is to screen and characterize Aurora kinase inhibitors from natural substances extracted from plants that are used in the Vietnamese pharmacopoeia. We have characterized in vitro Derrone, extracted from Erythrina orientalis L. MURR, as a novel Aurora kinase inhibitor. This com-pound exhibited an ability to inhibit the phosphorylation of histone H3 at ser10 both in kinase assay and at the cellular level. The compound was more effective against Aurora kinase B, with a lower IC50 value as compared to Aurora A. Moreover, it impaired the mitotic spindle checkpoint and led to endoreduplication in cancer cells, a phenomenon caused by an Aurora B inhibitor. Interestingly, using the xCelligence system and real-time cell analysis (RTCA) software, we set up a comparison of cell proliferation profiles between cancer cells treated with Derrone and VX680—a well-known Aurora kinase inhibitor—and we found that these profiles exhibited considerable similarity in cell morphology, growth, and death. Additionally, Derrone significantly inhibited the formation and growth of MCF7 tumor spheroids.

Key words Aurora kinase; Aurora kinase inhibitor; Derrone; H3 phosphorylation; xCelligence system; multicellular tumor spheroid

Aurora kinases are serine/threonine kinases that phosphor-ylate serine and threonine residues in proteins and regulate several essential events in cell division during mitosis. Human Aurora kinase family consists of three members: Aurora A, Aurora B, and Aurora C.1–3) They are patchily distributed throughout the cell cycle. Notably, Aurora B is a member of chromosomal passenger complex (CPC), and thus, its distri-bution is well correlated with chromosome events. Aurora B initially localizes along the length of the chromosomes during prophase, subsequently accumulating in the inner centromere region during prometaphase, leaving for the central region of the mitotic spindle and cell cortex, in which contractile ring is formed later at anaphase, and becoming rarely found at the end of M phase.4,5)

Aurora A regulates several essential events during cell division including centrosome maturation, centrosome separa-tion, mitotic entry, bipolar spindle assembly, and chromosome alignment on the late metaphase. There are a number of tar-gets of Aurora A in centrosome maturation, such as CDK11 and PAK1 lining upstream, and LATS2, NDEL1, and TAC C lining downstream.6–9) Aurora B that is associated with other members of chromosomal passenger complex controls mul-tiple processes during both nuclear and cytoplasmic division: correction of erroneous kinetochore–microtubule attachment, elimination of error in chromosome segregation, promotion of axial shortening of chromosomal arm, and cytokinesis.4,5,10,11) The phosphorylation of histone H3 by Aurora B bears on chromosomes condensation and might serve as “ready produc-tion label,” which is used to mark chromosome in such a way that the cell can progress from metaphase to anaphase.10–13)

The three members of human Aurora kinases are over-expressed in several human cancers such as breast, bladder, colon, ovarian, and pancreatic cancer. A strong correlation between the overexpression of Aurora kinases A and B and cell transformation and tumorigenesis has been mentioned in several research articles.14–17) Overexpression of the centro-somal protein Aurora A is related to poor prognosis in epi-thelial ovarian cancer patients. Aurora B expression increases in correlation with advanced stages of colorectal cancer, and its overexpression induces metastasis.18,19) Moreover, Aurora A phosphorylates p53 at ser215 and inhibits its DNA bind-ing; and Aurora B interacts with NIR-p53 leads to subsequent functional suppression of this protein. Thus, inhibition of Aurora kinases may rescue the function of tumor suppressor gene and Aurora kinase inhibitors may be the anticancer mol-ecules.20–23)

In this research, we used the bank of natural plant sub-stances used by the Vietnamese pharmacopoeia to search for Aurora kinase inhibitors that may be proposed as new anti-mitotic drugs. First, we screened substances in vitro that showed activity in the inhibition of Aurora kinase domain on kinase assay and then positive extracts was tested on cell culture. We characterized in vitro a natural compound Der-rone extracted from Erythrina orientalis L. MURR as a novel Aurora kinase inhibitor and compared it with the commercial inhibitor, VX680.

MATERIALS AND METHODS

Plant Materials The stem barks of E. variegata L. were

* To whom correspondence should be addressed. e-mail: hoangthimynhung@hus.edu.vn

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collected from Hoa Binh province, Vietnam, in 2010 and were identified by one of the authors (Thuong Thien Phuong). A voucher specimen (No. VDL-0) was deposited in the Depart-ment of Pharmaceutical Analysis and Standardization, Nation-al Institute of Medicinal Materials, Hanoi, Vietnam.

Extraction and Isolation Five kilograms of dried samples were subjected to extraction twice with 96% ethanol at room temperature for 2 weeks, and the combined solvent was evap-orated to obtain the extract (430 g). The extract was suspended in H2O and partitioned twice with CH2Cl2 and the solvents were exhaustively removed to obtain CH2Cl2 fraction (135 g). This fraction (100 g) was subjected to a silica gel column (10×30 cm) and eluted with a gradient of n-hexane–EtOAc (9 : 1, 8 : 2, 7 : 3 … 1 : 1) to yield seven fractions (F. 1, 12 g; F. 2, 21 g; F. 3, 8 g; F. 4, 10 g; F. 5, 11 g; F. 6, 8 g; F. 7, 14 g). The F. 3 (5 g) was separated by a silica gel column (4×30 cm), and eluted with the solvent system n-hexane–EtOAc (9 : 1, 8 : 2, 7 : 3 … 1 : 1) to obtain five fractions (F 3.1, F 3.2, F 3.3, F 3.4, F 3.5). The F. 3.4 (800 mg) was separated by a C18 column (2×25 cm) and eluted with MeOH–H2O (9 : 1), which resulted in the isolation of compound 1 (29 mg). Compound 1 was identified as Derrone (Fig. 1A) by comparison of the MS and NMR data to those in the literature,24) and the purity of com-pound was determined to be 93% by HPLC.

Recombinant Proteins Recombinant Aurora A, Aurora B, Aurora A domain and histone H3 proteins were expressed as N-terminal His6-tagged fusion proteins in Escherichia coli. The proteins were purified by affinity chromatography using nickel–nitrilotriacetic acid (Ni–NTA) agarose to homogeneity. The assay is described in previous studies.25,26)

Protein Kinase Assay This method is already described in our previous studies.25,26) Briefly, the assay was performed in 20 mM Tris–HCl, 20 mM KCl, 20 mM MgCl2, 0.4 mM ATP, 0.4 mM dithiothreitol (DTT), and at pH 7.5. Recombinant his-tone H3 was used as substrate. The reaction was initiated by the addition of the recombinant enzyme. After 1 h of incuba-tion at 37°C, the remaining ATP was monitored by the addi-tion of kinase-GloTM (Promega, U.S.A.) under the conditions suggested by the supplier. Ten minutes later, fluorescence was

recorded with a plate CHAMELEONTMV Multilabel Micro-plate Reader and analyzed by MicroWin® software (Hidex, Turku, Finland). Staurosporine (0.5 mM) was used as positive control.

High Throughput Screening The protein kinase assay was performed in a black 96-well plate, and started with the addition of the Aurora A kinase domain. The Z-factor of the assay was estimated to be 0.77.25,27) The primary screening was performed in triplicate at compound concentration of 60 µM (the compounds were dissolved in 0.1% dimethyl sulf-oxide (DMSO)) and the selected hits were tested again at con-centration of 30 µM. To determine the IC50 value of Derrone against whole Aurora kinases A and B, 10 concentrations of the substance were used, ranging from 305 to 0.6 µM. IC50 was defined as the concentration required for 50% of inhibition of the enzyme activity.

Cell Culture HeLa, MCF7, H1299, and KPL4 cells (pur-chased from the American Type Culture Collection) were grown on Dulbecco’s modified Eagle’s medium (DMEM; Gibco, U.S.A.). Media were supplemented with 10% fetal bo-vine serum (Gibco), 100 units/mL of penicillin, and 100 µg/mL of streptomycin (Gibco). The cells were cultured in an incuba-tor at 37°C with 5% CO2.

Cytotoxicity Assay Cell cytotoxicity assays were con-ducted in a 96-well plate and in normal growth conditions. Viable cells were seeded in the growth medium into 96-well plates (104 cells/well) and incubated at 37°C, 5% CO2. The sample was dissolved in DMSO and adjusted to final sample concentrations, ranging from 15 to 238 µM (for Derrone) by diluting with the growth medium. Each sample was prepared in triplicate. The final DMSO concentration was adjusted to 0.1%. The same volume of medium with 0.1% DMSO was added to the control wells. After 72 h of incubation, 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent was added to each well and guidance from the sup-plier (Promega) was followed. The optical density (OD) was measured at 570 nm using a microplate reader (BioRad, U.S.A.). The IC50 value was defined as the concentration of the sample that reduced absorbance by 50% as compared to the

Fig. 1. Derrone Inhibited the Activity in Vitro of Aurora Kinases A and B(A) Structure of Derrone. (B) Semi-logarithm dose of Derrone to Aurora A and Aurora B activity inhibition ratio (%). Curves were fitted and IC50 was determined by

using GraphPad Prism 5 software.

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0.1% DMSO-treated control.Cell Cycle Analysis After incubation with Derrone 30 µM

for 24 h, cells were harvested and washed with phosphate buffered saline (PBS). Cells were fixed for 1 h at 4°C in cold 70% ethanol by adding dropwise to the pellet while vortexing. Subsequently, the cells were washed two times in PBS, and then centrifuged at 250×g in 10 min. Afterwards, the super-natant was discarded, and the cells were treated with 50 µL of a 100 µg/mL stock of RNase (this will ensure that only DNA, not RNA, is stained) and 200 µL propidium iodide (PI) (from 50 µg/mL stock solution). The mixture was incubated for 15 min. Cell population analysis was implemented on FACS Canto II system (BD, U.S.A.).

Immunofluorescence Cells grown on glass coverslips for 24 h were arrested in mitosis by the addition of paclitaxel (0.035 µM, Sigma, U.S.A.), in the presence of either Derrone (30 µM) or VX-680 (0.14 µM, Vertex and Merck, U.S.A.) for 15 h. Cells were fixed with 4% formaldhehyde and 2% sucrose at 37°C then immunofluorescences were performed. Phosphor-ylated histone H3 was detected by a polyclonal rabbit antibody (Abcam, U.S.A.). Aurora B was detected using mouse mono-clonal antibodies (Abcam). DNA was visualized with 0.1 mM Hoechst 33342 (Invitrogen, U.S.A.). Images were collected with a ZEISS 510 Laser Scanning Confocal microscope with 40× or 63× objectives.

Western Blotting For the preparation of whole extracts, cells were recovered by trypsinization. Cells were blocked in mitosis for 15 h, in the presence of 0.035 µM paclitaxel and Derrone (30 or 45 µM). Mitotic cells were harvested by mitotic shake-off machine. Cells were lysed in Laemmli sample buffer containing urea (7 M). Lysates were boiled and subjected to sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis (SDS-PAGE), then transferred to nitrocellulose sheets for Western blotting. β-Actin or α-tubulin detected by monoclonal antibodies (Sigma), were used as loading control whereas S10-phospho-histone H3 indicated the percentage of mitotic cells. Blots were developed using the ECL technique (GE-Bioscience, U.S.A.) and signals recovered on radiographic films (BioRad).

Multicellular Tumor Spheroid (MCTS) Culture MCF7 cell line was reported to have a high ability to create spher-oids, when using the hanging drop method,28) with a slight modification. Fifteen microliters medium that contained 5×103 cells was added to each circle on the inverted cover of the 96-well plate for making one spheroid. The cover was then placed upside down on the plate coated with sterile agarose 1.5% (w/v) containing 200 µL complete medium. After 48 h of incubation in a humidified chamber in the presence of 5% CO2 at 37°C, spheroids were transferred from the cover into each well of the agarose-coated plate and further cultivated in fresh growth medium. Images of MCTS in each well were taken by Axiovert 40CFL microscope (Zeiss, Germany) with Powershot G9 camera every two days. These images were analyzed using Axio version 4.5 (Zeiss) to determine the MCTS diameter. The volume of each spheroid was calculated by the formula for the volume of a sphere: V=4/3 πr3. The tumor growth inhi-bition (%TGI) was calculated according to the following equa-tion: % TGI=100−(mean tumor volume of treated group/mean tumor volume of control group)×100.

Real-Time Monitoring of Cell Proliferation Using xCelligence System An in vitro growth curve characteriza-

tion of materials was carried out using xCELLigence system (Roche Inc., U.S.A.). The system measures electrical imped-ance across interdigitated microelectrodes integrated on the bottom of tissue culture E-plates. The impedance measure-ment provides quantitative information on cell number and viability.29,30) The real-time cell assay started with the back-ground reading by adding 50 µL of DMEM (Invitrogen) to each well of the 96-E-plate and then monitored at 15 s inter-vals within 1 min. Next, 130 µL of DMEM media containing 5×103 cells were seeded into each well of the E-plate, and the cells were monitored every 15 min for 24 h to obtain the growth baseline reading. At the time point of treatment, 20 µL of Derrone or VX-680 was added into each well to obtain 5 concentrations in the range of 15 to 238 µM (for Derrone) and 0.07 to 0.7 µM (for VX-680). Dynamic cell proliferation was monitored in 30-min intervals from the time of treatment until the end of the experiment. Medium addition and cell obser-vation were noted during incubation time. Normalized Cell Index values were analyzed by RTCA software (Roche Inc.) to obtain time-dependent IC50 value, doubling time, and other evaluations.

Statistical Analysis Statistical analyses were performed with GraphPad Prism 5. A value of p<0.05 was considered statistically significant.

RESULTS

Identification of Derrone through Kinase Assay We used the kinase assay to screen 100 natural substances from the library of Vietnamese National Institute of Medicinal Materials. Among them, Derrone showed the most effective inhibition of Aurora kinase domain (80%) at the concentration of 60 µM. Derrone exhibited higher activity against Aurora B than against Aurora kinase A, with IC50 values of 6 and 22.3 µM, respectively (Fig. 1B). The results indicated that Der-rone has ability to inhibit both Aurora kinases A and B and more selectivity toward Aurora B.

Effect of Derrone on Cell Growth and Mitotic Spindle Check Point The compound was toxic to the 4 cancer cell lines, with the most sensitive being MCF7 and H1299 cells. The IC50 values of the compound against these cell lines are shown in Table 1. During the cytotoxicity experiment, we observed that in cells treated with lower dose of Derrone, the size of cells increased with the duration of incubation. Since Aurora kinases play an important role in cell division, especially in the mitotic cell checkpoint, this directed us to evaluate the effect of the substance on the cell cycle by using flow cytometry technique. DNA histogram indicated that Der-rone caused treated cells to endoreduplicate with multiplica-tion in chromosomes number. In the untreated sample, most cells accumulated on the region of G1/G0 phase which had chromosome set 2N, and there were few cells in area which

Table 1. IC50 Values of C14 on Cancer Cell Lines

Cancer cell line IC50 (µM)

H1299 23.8±2.4MCF7 24.4±3.9HeLa 31.2±8.3KPL4 45.8±5.7

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possessed chromosomes set>4N. In comparison, percentage of chromosome set 2N was two-fold lower and 5-fold higher than percentage of chromosome set>4N in cells exposed to 30 µM Derrone (Fig. 2).

To examine if endoreduplication caused by the escape of cells from the mitotic spindle checkpoint without completing cytokinesis that lead to the mitotic slippage phenomenon, we collected the paclitaxel-arrested mitotic cells that remained after exposure to Derrone. The percentage of mitotic cells in the treated sample at 45 µM decreased 1.6 and 2.9 fold in HeLa and MCF7, respectively, compared to the results of the pacli-taxel-treated only cells. Moreover, we observed many cells remained spread out in the cell culture flask after incubation with Derrone. These phenomena were observed in both HeLa (Fig. 3) and MCF7 cells.

Derrone Inhibited the Phosphorylation of H3 at ser10 but Did Not Effect on the Expression of Aurora B Immu-nofluorescence-based methods quantifying phospho-histone H3 levels have been utilized to characterize Aurora kinase inhibitors. The fluorescent images showed a decrease in the level of phospho-histone H3 (ser10) in samples treated with

Derrone. Control sample was distinguished by a clear and strong fluorescent signal; dividing cells were easily identified because they were abundant, with distinctive round shapes, chromosomes aligning at the metaphase plate, and histone H3 heavily phosphorylated. In contrast, cells exposed to the tested compound demonstrated a dramatic decrease in the level of phospho-histone H3 at ser10, shown by their extremely weak fluorescent signals. In some cells, fluorescent signal could not be detected (Figs. 4A, C). Similar results were obtained from Western blot analysis of the HeLa sample (Fig. 4B). There was no difference in Aurora B expression between the treated and untreated cells (Figs. 4C, D). This indicates the down-regulation of phospho-H3 (ser10) was due to the inactivation of Aurora B kinase activity rather than the down-regulation of this enzyme. Furthermore, these results demonstrated that Derrone not only had effect on Aurora kinase assay but also on cellular endogenous protein.

Characterization of Derrone as an Aurora Kinase In-hibitor Using xCelligence System We chose MCF7 and H1299 cells to characterize Derrone as an Aurora B inhibitor by using xCelligence system. These two cell lines were most

Fig. 2. Distribution of Distinct HeLa Cell Populations in the Cell Cycle Based on FSC and SSC Values(A) In the untreated sample (control), most cells accumulated on the G1 phase, which has chromosome set 2N, and least cells possessed chromosomes set >4N; however,

the opposite was observed in Derrone treated samples (30 µM, 24 h), with the most cells contained 4N and >4N DNA contents. (B) % of cells in each phase of the cell cycle in control and Derrone treated samples.

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sensitive to Derrone and had different morphology.A normalized cell index (NCI) is introduced to reduce the

influence of inter-experimental variations.31,32) Cell indexes were normalized with the last time point before the addition of

the compound. The biological status of MCF7 and H1299 cells varied dependently on the time and concentration of Derrone throughout the course of experiment.

The behavior of cells varied in different treated doses of

Fig. 3. Effect of Derrone on the Mitotic Spindle Checkpoint(I) HeLa cells were arrested at M phase by incubating with paclitaxel (0.035 µM) for 15 h then treated with 0.1% DMSO (I-C) or Derrone (30 µM) (I-E); Derrone (45 µM)

(I-F), or VX680 (0.14 µM) (I-D) for 8 h. (I-A): control without paclitaxel; (I-B): cells before the addition of substances. (II) HeLa cell nuclear staining with Hoescht in the control or treated with Derrone or VX680. Note that these cells were remained attaching to the flask after washing with PBS. (III) Mitotic cells were obtained after 1 h shaking on the machine. Cells were analyzed the DNA content relative percentages of mitotic cells were shown. * p<0.05; ** p<0.01.

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Derrone. Among the 5 concentrations of substance used in this experiment, the two highest concentrations exhibited the strongest cytotoxicity to H1299 cells. Soon after addition of the compound (3 h), all cells in these two concentrations died as shown by the growth curve reached the base line corre-sponding to NCI=0. In the middle concentration (60 µM), cell growth was relatively slower than the DMSO control. Surpris-ingly, in the H1299 cell population, at the two lowest concen-trations, the NCI increased and was higher than the control after 2.5 d of incubation. The curves continued to increase until the NCI doubled the control value on day 4 of treatment; however, a sudden decrease was observed afterwards. Similar

behavior was also observed in MCF7 cells, except only the lowest concentration showed the highest NCI value when compared to those of the control. All treated growth curves eventually decreased and reached 0 value far before the con-trol curve did. The comparison of cellular profiling between H1299 cells treated with Derrone and VX680 also showed similar results: the NCI values of the two lowest doses were higher than the control and quickly dropped, meanwhile the highest dose inhibited the growth of cells at the early stage of treatment (Fig. 5A).

We further evaluated the effects of Derrone on cell growth by analyzing the doubling time (DT) of the cell population

Fig. 4. Effect of Derrone on the Cellular Phospho-H3 (ser10)(A) The level of phosphorylated ser10 in Histone H3 in HeLa cells detected by immunofluorescence staining, observed under fluorescent microscopy. The fluorescent

images showed a decrease in the expression level of histone H3 phosphorylation at ser10 in samples treated with Derrone at concentration of 30 µM for 15 h. Scale bar: 5 µm; arrows—cells expressed phosphor-H3 (ser10) when treating with Derrone. (B) Western blotting results of phosphorylation of H3 at ser10 in the presence of Derrone; β-Actin was used as sample loading control. (C) Western blotting results of Aurora B expression in the presence of Derrone; α-tubulin was used as sample loading control. (D) Derrone (30 µM for 15 h) inhibited phospho-H3 (ser10) in MCF7 cells and showed no effect on the expression of Aurora B kinase at the indicated concentrations.

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following incubation. The DT in control cells increased gradu-ally following the time of culture, reflecting the decrease in proliferation along with the increase in confluence. At the two highest concentrations (119, 238 µM), the DT values were all negative, indicating no cell growth rather than death. The middle concentration (60 µM) demonstrated DT value relatively 2.8-fold (on day 2 of incubation) and 1.6-fold (on day 3 of

incubation) higher than that of control. Notably the DT values of the two lowest concentrations were lower than the control from day 2 to day 4 of treatment. After day 4 of treatment, all DT values (except the two highest) were more than the control (Fig. 5B).

The growth curve slope demonstrated stable cell population and growth/death rates of cells.29,33,34) We compared the slope

Fig. 5. xCelligence RTCA Biosensor Technology Revealed the Profile of Cells Treated with Compounds(A) Real-time cell analyzer (RTCA) profiles generated with Derrone on H1299 and MCF7 cells, and VX680 on H1299 cells. These cells were exposed to 5 concentra-

tions of each compound as indicated for 8 d. The cell index profiles reflected the initial cell attachment, logarithmic growth phase, and responded to the compound treat-ment. Cell indexes were normalized with the last time point before addition of the compound. The normalized time point is indicated by the vertical line. Each data point was calculated from triplicate values. Data represent the average±S.D. (B) Doubling time of H1299 cells in real-time analyzer from the normalized time point to day 6 of incubation with Derrone at 5 concentrations. Note that the two highest concentrations showed minus values indicating that the cells died when exposed to the compound at an earlier stage and no growth was counted.

Fig. 6. Effect of Derrone on the Death Rate of MCF7Slope values of MCF7 death rates in those treated with Derrone at two concentrations: 15 µM (sky blue) and 30 µM (pink) compared to the DMSO control (purple). Note:

the death of MCF7 was induced after 5 d incubation with Derrone at two indicated concentrations. The slope values were calculated within 24 h from the time point the curve decreased (148-172 h for Derrone and 166–190 h for DMSO control) indicated by the vertical lines (purple for Derrone-treated cells and red for DMSO control cells). The minus values exhibit the death rate of cells. ** p<0.01.

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of the growth curve between 30 µM concentration treated cells and the control on MCF7 cells at the point of decrease (Fig. 6). We examined this concentration because its growth curve was the most similar to that of the control in the maximum NCI value. As shown in Fig. 6, the absolute value of the slope in the treated curve was 4-fold higher than that of the con-trol. It appears that as the concentrations increase, the higher

the absolute slope value. This indicates cell death in treated samples was occurring faster than the untreated cells.

Derrone Inhibited the Formation and Growth of MCF7 Multicellular Tumor Spheroid To evaluate the effect of Derrone on the formation of MCF7 tumor spheroid, we in-cubated cells with 2 concentrations of the compound: 15 and 30 µM. After 2d in hanging drop, no spheroid was formed at

Fig. 7. MCF7 Tumor Spheroid Formation and Growth Were Inhibited by Derrone(A) In the presence of Derrone at concentration of 30 µM no spheroid was established. In the meantime, spheroids were successfully formed at a dose of 15 µM, but the

growth was less than the control. Images were taken at the magnification of 80×. (B) The spheroids in each group were imaged and the volume was measured every two days following the addition of the compound. Scale bar represents 200 µm. (C) Dose–response curves of MCF7 tumor spheroids under Derrone treatment at two concentra-tions as indicated. The bars represent the mean±S.D. values (n=4). * p<0.05; ** p<0.01.

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the higher concentration tested. Cells died and separated in the medium. At concentration of 15 µM, spheroids were suc-cessfully formed but the growth was slower than that of the control, with the necrotic core appeared on day 3 of culture instead of day 5 as in the control. On day 7 of treatment, cells at the outer layer detached from the spheroids and eventually died (Fig. 7A).

To assess the effect on tumor growth inhibition, the com-pound was added on to MCF7 spheroids on day 3 of culture, on agarose-coated plate with two concentrations: 30 and 60 µM. We observed that all treated-spheroids decreased in size compared to those of the control. On day 3 of treatment, Derrone showed an effect on the morphology of spheroids. Some cells of the outer layer—the highest proliferated part in spheroid structure, became more loosely bound together. This was clearer on day 7 of treatment when the cells in the outer layer began to die and the spheroids lost their tightly-linked border (Fig. 7B). Moreover, from day 5 of the treatment, the size of spheroids at all treatment doses was smaller than that in the control at the same stage. Especially at the highest dose of 60 µM, there was only a slight increase in the size of spher-oids from day 5 of treatment (Fig. 7C) with the average size of spheroids was around 5.3±0.4 mm3. On day 15 of treatment, the tumor spheroid growth inhibition (% TGI) was 17.5% and 65.4% for 30 and 60 µM Derrone, respectively. After day 15 of treatment, some spheroids at dose of 60 µM were loosen or broken apart and the size could not be measured.

These results indicated that Derrone inhibited the formation and suppressed the growth of MCF7 tumor spheroids.

DISCUSSION

To date, more than 30 Aurora inhibitors have been identi-fied, and are under different stages of preclinical and clini-cal studies. They are either pan-Aurora kinase inhibitors or specific inhibitors of Aurora A and Aurora B. Some clinical studies have shown that Aurora kinase inhibitors were well tolerated, and no thrombocytopenia was observed following treatment with these molecules. Even though Aurora kinases have shown to be promising drug targets in preclinical models and have also stabilized disease in some clinical trials, the responses reported in phase I studies in patients with solid tu-mors are rather poor. Combination of Aurora kinase inhibitors with other anticancer agents is considered a promising solu-tion to increase their antitumor activity.33–36) Moreover, new Aurora kinase inhibitors, especially natural inhibitors, will contribute to the development of efficient and safe anticancer drugs.37,38)

From the library of natural medicinal materials, we screened more than 100 natural substances, (which were used in Vietnamese folk remedies) on Aurora kinase assay which are well developed.25,26) Through this assay, we selected Der-rone that inhibited both Aurora A and B kinases, but showed more selective activity against Aurora B kinase. Based on the fact that some molecules are Aurora kinase inhibitors in in vitro assay but were not suitable for cell-based assays, we characterized Derrone as an Aurora kinase inhibitor in cancer cells. As expected, Derrone reduced the phosphorylation of histone H3 on ser10, a natural substrate of Aurora B that is used as an indicator of Aurora B inhibition.13) This compound prevented the proliferation of 4 cancer cell lines, impaired

the mitotic spindle checkpoint, and induced formation of hyperploid cells called endoreduplication. These phenomena were based on a process termed “mitotic slippage,” which can be induced by anti-mitotic drugs that inhibit microtubule organization and Aurora kinases.22,23,26) Moreover Derrone showed efficient inhibition of spheroid growth on MCF7 tumor spheroids. Multicellular tumor spheroid culture systems demonstrated intermediate complexity reflecting particular as-pects of tumor tissues including multilayer cell systems. Any compound that has the ability to prevent the growth of tumor spheroids would be a candidate of anti-cancer drug.39,40)

The xCelligence real-time cell analysis high-throughput (RTCA HT) system is now considered as an alternative to the traditional single time-point assay, and it has the potential to become routine setting for evaluating cell-based in vitro assay. Cell index (CI) values were relative to cellular changes includ-ing viability, morphology, and adhesion degree, because they probably alternated electrode impedance.29,31,32) We compared the cytotoxicity response profile of living cells between Der-rone and VX680 via this system on H1299 cells. Cells that were exposed to both of these compounds demonstrated simi-lar dose- and time-dependent response. Cells either died or delayed growth at high and medium doses. However, at lower doses, both cell profiles showed an increase in NCI value due to the increase in the size but not the number of cells. The expansion of site attachment probably led to elevation of the cell index of treated cells, whereas the increase in cell num-ber, cell viability, or attach degree is accountable for increas-ing CI values of the untreated sample.41,42) VX-680 is a potent inhibitor of Aurora kinases that induces the accumulation of cells with ˃ 4N DNA content, followed by cell death. In cells lacking p53, endoreduplication and apoptosis in response to VX680 are markedly enhanced.43,44) H1299 is reported to have homozygous partial deletion of the p53 protein and lacked expression of p53.45,46) Treatment with VX680 and Derrone at low doses showed that these cells were more likely to undergo endoreduplication, and resulted in high CI values. VX-680 caused progressive endoreduplication until after 96 h in which apoptosis occurred. Similar cell profile was also found in cells treated with Derrone. All of these results present strong evidence that Derrone exhibits characteristics of an Aurora kinase inhibitor.

CONCLUSION

Natural dietary agents are now being increasingly studied for their potential in the treatment of cancer because of their safety, low toxicity, and antitumor properties. The aim of the current study was to identify a novel natural substance as an Aurora kinase inhibitor. Further studies will be required, such as testing on different cancer cell lines and more ki-nases, evaluating the effect of Derrone on in vivo model, and structure modification in order to improve the potency and efficiency of the compound. However, our results provide the evidence that Derrone inhibited Aurora kinases in vitro from assay level to cellular level. Additionally, Derrone inhibited the growth of tumor spheroids. Thus, this compound could be a potential anticancer drug.

Acknowledgments We deeply thank Dr. Annie Molla for helpful discussions and advices regarding the kinase assays

944 Vol. 39, No. 6 (2016)Biol. Pharm. Bull.

and high throughput screening. We are thankful to Dr. Stefan Dimitrov and Veronique Gerson for preparing and giving us the vectors. This study was supported by Nafosted Grant, project Code: 106.02-2010.55. Flow cytometry and xCelligence system were performed in the Key Laboratory of Enzyme and Protein Technology facilities (HUS).

Conflict of Interest The authors declare no conflict of interest.

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