Canthin-6-one Displays Antiproliferative Activity and Causes

Article pubs.acs.org/jnp

Canthin-6-one Displays Antiproliferative Activity and Causes Accumulation of Cancer Cells in the G2/M Phase Camille Dejos, Pierre Voisin, Marianne Bernard, Matthieu Régnacq, and Thierry Bergès* Signalisation & Transports Ioniques Membranaires, CNRS ERL 7368, University of Poitiers, Poitiers, France

ABSTRACT: Canthinones are natural substances with a wide range of biological activities, including antipyretic, antiparasitic, and antimicrobial. Antiproliferative and/or cytotoxic effects of canthinones on cancer cells have also been described, although their mechanism of action remains ill defined. To gain better insight into this mechanism, the antiproliferative effect of a commercially available canthin-6-one (1) was examined dose-dependently on six cancer cell lines (human prostate, PC-3; human colon, HT-29; human lymphocyte, Jurkat; human cervix, HeLa; rat glioma, C6; and mouse embryonic fibroblasts, NIH-3T3). Cytotoxic effects of 1 were investigated on the same cancer cell lines by procaspase-3 cleavage and on normal human skin fibroblasts. Strong antiproliferative effects of the compound were observed in all cell lines, whereas cytotoxic effects were very dependent on cell type. A better definition of the mechanism of action of 1 was obtained on PC-3 cells, by showing that it decreases BrdU incorporation into DNA by 60% to 80% and mitotic spindle formation by 70% and that it causes a 2-fold accumulation of cells in the G2/M phase of the cell cycle. Together, the data suggest that the primary effect of canthin-6-one (1) is antiproliferative, possibly by interfering with the G2/M transition. Proapoptotic effects might result from this disturbance of the cell cycle.

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HCT-8 (colon adenocarcinoma), CAKI-1 (kidney carcinoma), MCF-7 (breast carcinoma), and SK-MEL-2 (melanoma).12 No attempt was made at defining the mechanism of action of canthinones on cancer cell proliferation, and further studies were mostly focused on their cytotoxic activities. Uncharacterized cell death was reported in A-549 (lung) and MCF-7 human cancer cell lines treated with 9-methoxycanthin-6-one and canthin-6-one (1) isolated from the roots of Eurycoma longifolia Jack.13 The proapoptotic activity of canthinones was first reported on HeLa, SAOS-2, U87MG, and U-937 (osteosarcoma) cancer cell lines treated with an extract of Ailanthus altissima (Mill.) Swingle.15 It was confirmed in a detailed study of the apoptotic symptoms evoked by 1methoxycanthin-6-one in Jurkat (leukemia), ARO, NPA (thyroid), and HuH7 (liver) cancer cell lines.16 Considering the wide range of biological activities of canthinones, their apparently safe use in traditional medicine, and the relative ease of high-yield synthesis in vitro,18 these molecules may represent a valuable complement to current chemotherapies. As a first step toward a possible use of canthinones in evidence-based

atural products have long been a source of anticancer substances until the emergence of targeted therapies and rational drug design in the 1990s but regained interest in the past decade.1 Among these compounds, antimalarials represent an underexplored source of novel anticancer drugs.2 First described in 1952 by Haynes et al.,3 canthinones, isolated from the Australian rainforest tree Pentaceras australis (F. Muell.) Benth. (Rutaceae family), belong to the class of β-carbolin molecules.4 Canthinones are also produced by plants belonging to the Simaroubaceae and Zygophyllaceae families.5,6 Traditional medicine makes use of plants producing canthinones for their antipyretic and antiparasitic properties (against malaria and leishmaniasis) and for the treatment of gastric ulcers and diarrhea.7 During the past decade, the biological activities of canthinones have been evaluated, and several studies described antimicrobial,8 antifungal,9−11 and antiproliferative and/or cytotoxic activities on various human cancer cell lines.12−17 Some simaroubaceous species have been used in Chinese Traditional Medecine for their antitumoral properties, e.g., Ailanthus altissima (Mill.) Swingle.15 Among the first studies undertaken, Xu et al. reported that 1-methoxycanthinone and 5-methoxycanthinone extracted from Leitneria f loridana Chapm. suppressed the growth of a panel of human cell lines: HeLa (cervix adenocarcinoma), A-549 (lung carcinoma), © 2014 American Chemical Society and American Society of Pharmacognosy

Received: July 11, 2014 Published: November 7, 2014 2481

dx.doi.org/10.1021/np500516v | J. Nat. Prod. 2014, 77, 2481−2487

Journal of Natural Products

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increase in procaspase-3 cleavage after 48 h treatment with 10 μM or 30 μM 1, respectively (Figure 1L). HeLa cells could not be analyzed at 96 h of treatment, due to severe cell death and inability to harvest sufficient protein material. Jurkat, C6, and NIH-3T3 cells showed no increase in procaspase-3 cleavage at 40 μM 1 (Figure 1I, O, and R), in agreement with the absence of cell count reduction by the drug (Figure 1G, H, M, N, P, and Q). These data indicate that the proapoptotic effect of 1 is cellline-dependent, whereas a strong antiproliferative effect of this compound was consistently observed in all cell lines. Canthin6-one (1)’s cytostatic feature is thus not automatically associated with cytotoxic effects. Cell death could be induced by several mechanisms including cytotoxicity but also cell cycle arrest.23 The rest of the study was focused on the prostate cancer PC-3 cell line,24 with the aim of better characterizing the effect of 1 on cell proliferation. Effect of Canthin-6-one (1) on DNA Synthesis. Because cell counts reflect a balance between proliferation and cell death, we sought to obtain a more direct assessment of the effects of canthin-6-one (1) on cell proliferation by measuring BrdU incorporation into DNA. PC-3 cells were precultured for 30 h in the presence or absence of 1 and then labeled with BrdU for 18 h. BrdU incorporation into genomic DNA was quantified by immunoblotting (Figure 2A), and the proportion of postmitotic nuclei, characterized by a strong, uniform labeling of the chromatin, was estimated by immunofluorescence microscopy (Figure 2B). Southern blot analysis revealed an 80% drop in BrdU incorporation into DNA after treatment with 1 (Figure 2A). Further information was obtained with immunofluorescence microscopy, as it indicated that 85% of the cells were postmitotic in control cultures, after 18 h of BrdU labeling (Figure 2C). This high-efficiency labeling is in agreement with a doubling time of PC-3 cells around 24 h, as observed above (Figure 1A). In contrast, only 35% of the cells treated with 1 had a postmitotic nucleus (Figure 2C). This represents a 60% drop in the frequency of postmitotic nuclei (Figure 2C). Decrease of DNA synthesis in the presence of 1 brings additional evidence that its primary action consists of antiproliferative activity rather than a cytotoxic effect. Altogether, the data clearly show that canthin-6-one (1) lowers the occurrence of cells replicating their DNA and raise the question of which phase of the cell cycle is affected. Canthin-6-one (1) Causes Cell Accumulation in G2/M. To gain deeper insight into the mechanism of action of canthin6-one (1), its effect on cell cycle distribution of PC-3 cells was assessed by flow cytometry with detection of PI-stained DNA. In controls, distribution of PC-3 cells in the cell cycle was consistent with previous studies: 58% of cells were in G1 phase, 22% in S phase, and 20% in G2 or mitosis.25 After 48 h incubation with 1 (40 μM) PC-3 cells showed a clear accumulation in G2/M, characterized by 4N DNA content (Figure 3). In response to treatment with 1, the proportion of G2/M cells increased from 20% to 48%, the fraction of cells in G1 phase decreased from 58% to 38%, and those in S phase decreased from 22% to 13% (Figure 3). These observations suggest that 1 interferes with processes occurring in the late S phase and/or in G2, leading to G2/M checkpoint induction. Since this checkpoint prevents cells from entering mitosis when DNA is damaged, leaving the cells enough time and giving them the opportunity to repair their DNA, these results raise the possibility that canthin-6-one (1) treatment may result in DNA lesions, directly or indirectly. The putative genotoxicity of this drug or its derivatives has not been examined thus far and

therapies, a better understanding of the mechanisms of action of these molecules is required. Specifically, the mechanism of action of canthinones on cancer cell proliferation remains illdefined as compared to well-documented effects on apoptosis.16 However, because apoptosis may be the consequence of cell cycle arrest in established cancer cell lines,19−22 previous studies have not ruled out the possibility that the primary effect of canthinones may be to interfere with cell cycle progression. Therefore, this study sought to re-examine the antiproliferative effects of a commercially available canthin-6-one (1) in order to obtain a better definition of its impact on the cell cycle and possibly to identify its mode of action.

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RESULTS AND DISCUSSION Differential Effects of Canthin-6-one (1) on Cancer Cell Proliferation and Apoptosis. The in vitro antiproliferative effects of canthin-6-one (1) were studied on six cell lines: human prostate adenocarcinoma (PC-3), human colon adenocarcinoma (HT-29), human cervix epitheloid carcinoma cells (HeLa), human leukemic T cell lymphoblasts (Jurkat), rat glioma (C6), and mouse embryonic fibroblasts (NIH-3T3). Growth curves were established by cell counts over 4 days of culture, in the absence or presence of 40 μM canthin-6-one (1), a concentration previously shown to affect yeast growth.11 For most cell lines, this treatment completely arrested cell growth, while causing only minor decreases in cell populations relative to the initial plating density (Figure 1A, D, G, M, and P). In contrast, 1 was highly toxic to HeLa cells at this concentration, with more than a 90% decrease in cell counts after 2 days of culture (Figure 1J). In HeLa cells, 10 μM canthin-6-one (1) was sufficient for complete inhibition of cell growth without reducing the initial cell density (Figure 1J). The dosedependent effect of 1 was further examined on all cell lines. For most cell lines, half-maximal inhibition of cell growth was observed at 15−20 μM, maximal inhibition at 30 μM, and up to 30% cell death at 40−60 μM (Figure 1B, E, H, K, N, and Q). The dose−response curve of HeLa cells confirmed their higher sensitivity, with half-maximal inhibition of cell growth at 5 μM, complete inhibition at 10 μM, and cell death above 15 μM (Figure 1K). Altogether these results suggest that 1’s primary effect was to block the growth of cancer cell lines. Given that proapoptotic effects of 1-methoxycanthin-6-one have been reported,15,16 procaspase-3 cleavage was monitored by Western blot analysis at different treatment times with 1. PC-3 cells cultured in the presence of 40 μM 1 showed a 2-fold increase in caspase-3 fragments at 48 h and a 7-fold increase at 96 h (Figure 1C), in agreement with the 20−30% decrease in cell viability observed on the time-course and dose−response curves (Figure 1A and B). Weaker signs of procaspase-3 cleavage (maximum 2-fold increase at 48 h) could be observed in HT-29 cells (Figure 1F), in agreement with a less reproducible decrease in cell counts (Figure 1D and E). HeLa cells, which appeared most sensitive to the cytotoxic effect of the drug (Figure 1J and K), showed 1.5- and 3.5-fold 2482



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Figure 1. Antiproliferative and proapoptotic effects of canthin-6-one (1) on several cell lines. Growth curves, dose−response curves, and Western blots of procaspase-3 cleavage were obtained for a series of cancer cell lines: (A, B, and C) PC-3, (D, E, and F) HT-29, (G, H, and I) Jurkat, (J, K, and L) HeLa, (M, N, and O) C6, and (P, Q, and R) NIH-3T3. Time-course and dose−response curves were obtained by counting dissociated cells in a Malassez chamber. Results are expressed as mean ± SEM (standard error of the mean), n = 4. Procaspase-3 cleavage was detected by immunofluorescence on Western blots after 2−4 days’ treatment with the indicated dose of 1. Immunofluorescence of β-actin was used as loading control.

Figure 2. Canthin-6-one (1) reduces the proportion of postmitotic cells. PC-3 cells were treated for 30 h with either 40 μM 1 or 0.2% DMSO (control), before adding BrdU for 18 h. (A) BrdU incorporation into DNA was measured by immunofluorescence on a Southern blot of genomic DNA and normalized to ethidium bromide (EtBr) fluorescence. (B) Example of postmitotic nuclei labeling. (C) Percentage of postmitotic nuclei observed in 28 (control) or 46 (1) random microscopic fields, representing a total of 1500 and 1800 cells, respectively. Results are expressed as mean ± SEM, and similar results were obtained in a second experiment (control: 70% postmitotic nuclei, 1: 31% postmitotic nuclei).

Figure 3. Canthin-6-one (1) induces accumulation of PC-3 cells in G2/M. PC-3 cells were treated for 2 days with either 1 (40 μM) or 0.2% DMSO (control). Cells were fixed and stained with propidium iodide before analyzing the cell cycle distribution by flow cytometry. Single cells (at least 20 000 per condition) were sorted according to the intensity of PI-labeling: 2N DNA content (PI = 100) was interpreted as cells in G1, 4N DNA content (PI = 200) was interpreted as cells in G2 and mitosis (M), cells in the intermediate state (100 < PI < 200) were interpreted as replicating their DNA (S). Percentage of cells in G1, S, and G2/M phases are reported in the tables. Results are expressed as mean ± SEM of 3 independent cultures superimposed on the graph.

mitotic spindle, thereby leading to G2/M arrest. The putative effects of this compound on mitosis were thus investigated. Effect of Canthin-6-one (1) on Mitosis. To analyze the effect of canthin-6-one (1) on the M phase of the cell cycle, mitotic spindles were visualized by tubulin immunolabeling (Figure 4A). The proportion of mitotic cells in a control culture

deserves further investigation. However, given that this compound is only poorly proapoptotic, its effect on the cell cycle could be heightened with the use of molecules that abrogate the G2/M checkpoint (e.g., caffeine), an attractive therapeutic strategy that has emerged in the past decade.26 Alternatively, canthin-6-one (1) may also interfere with the 2484



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Figure 4. Mitotic spindles are less frequent in the presence of canthin-6-one (1). PC-3 cells were treated for 2 days with either 1 (40 μM) or 0.2% DMSO (control). (A) Two representative images of normal-looking mitotic spindles in both control and 1-treated cells, as revealed by β-tubulin immunofluorescence (scale bar: 20 μm). (B) Percentage of mitotic cells in random microscope fields of control and 1-treated cultures (at least 6000 cells were counted in each group). (C) Tubulin polymerization in vitro was measured by absorbance at 350 nm in the presence of 1 (25 μM) or DMSO.

The highest concentration tested (160 μM) induced a sharp drop of cell viability (33%) accompanied by morphological evidence of toxicity (Figure 5). Half-maximal inhibition of NHDF viability by 1 was estimated at 115 μM, whereas it was 5−20 μM on cancer cell lines. A previous study showed that proapoptotic and toxic activity of 1-methoxycanthin-6-one on the Jurkat leukemia cell line was not detected on peripheral blood mononuclear cells from normal donors.16 The very low cytotoxicity of canthin-6-one (1) on NHDF at concentrations that efficiently inhibit cancer cell proliferation is encouraging for further examination of its potential as an anticancer drug. Canthin-6-one (1) is a natural substance produced by plants used in traditional medicine.29 Indeed, this compound already proved its therapeutic potential in antimalarial treatments, and its low toxicity in murine models suggests that it may be considered for long-term therapies.30,31,20 The recently formulated hope to discover new anticancer agents among antimalarial substances1,2 has received further support from the present study, as it describes a strong antiproliferative effect of this compound on cancer cells and narrows down its mechanism of action on a delayed transition from G2 to mitosis. This warrants further studies aimed at elucidating the origin of this modification of the cell cycle, whether it is due to DNA damage or to an inhibition of G2 to M transition enzymes. The effect of 1 on yeast growth may help solve this issue because of the genetic amenability of the yeast model system. 11 Previous studies have argued for a strong pharmacological potential of drugs that stop cells in the G2/ M phase of the cell cycle.32 However, the damage caused by aristolochic acid, another natural substance that stops cancer cells in G2/M, calls for caution.33,34 Further studies on the mechanism of action of canthin-6-one (1) should include thorough studies of its tissue toxicity before it can be considered useful for clinical purposes.

was 2.5% (Figure 4B), in agreement with previous reports on PC-3 cells and consonant with the notion that mitosis contributes 30−60 min of the 24 h cell cycle.27,28 After 48 h treatment with 1, mitotic spindles were observed in 0.4% of the cells, corresponding to an 80% decrease in frequency (Figure 4B). No evident distortion of the mitotic spindles could be observed in the presence of 1 (Figure 4A). In addition, 1 had no effect on tubulin polymerization in vitro (Figure 4C). The data suggest that canthin-6-one (1) delayed the entry into mitosis, thereby causing the G2/M cell accumulation described above. Evaluation of Canthin-6-one (1) Cytotoxicity on Normal Human Dermal Fibroblasts. If canthin-6-one (1) is to become of therapeutic value, its antiproliferative effect on cancer cells should not be sullied by cytotoxic effects on normal cells. To examine this point, the effect of increasing doses of 1 on normal human dermal fibroblasts (NHDF) was monitored by MTT assay and morphological observations. With up to 20 μM canthin-6-one (1), NHDF were not drastically affected by the treatment, with more than 80% viability according to MTT assay and normal morphology of the cell population (Figure 5). When treated with 40 μM 1, a concentration that completely blocked cell growth of most proliferative cancer cell lines, NHDF viability remained at 80% (Figure 5). At 80 μM 1, NHDF viability was significantly decreased (64%, Figure 5).

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EXPERIMENTAL SECTION

Chemicals and Reagents. Canthin-6-one (1) was purchased at Alpha Chimica (Châtenay-Malabry, France).35 The purity of 1 (99%) was determined by GC (supplier’s product specification). 1 was dissolved in DMSO to a concentration of 80 mM and stored at −20 °C. 1 was added to the cell cultures from a 50-fold concentrated solution prepared in 10% DMSO. Culture media were from Life Sciences Technologies (Cergy Pontoise, France). All laboratory chemicals were from Sigma-Aldrich (Saint-Quentin Fallavier, France). Cell Proliferation Assay. A panel of six cell lines (human prostate adenocarcinoma PC-3, human colon adenocarcinoma HT-29, human cervix epitheloid carcinoma cells HeLa, human leukemic T cell

Figure 5. Canthin-6-one (1) cytotoxic effects on normal human dermal fibroblast cells (NHDF). Viability of NHDF was investigated after 3 days in the presence of 1 at indicated concentrations or DMSO in controls. MTT test results are expressed as percentage of viability (mean ± SEM, n = 3). Morphologic observations were reported as (+) for normal cell population, (+/−) for growth inhibition, (−) for toxicity, and (0) for mortality. 2485



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lymphoblast Jurkat, rat glioma C6, and mouse embryonic fibroblasts NIH-3T3 (all from ECACC-Sigma, Saint-Quentin Fallavier, France)) was used to study antiproliferative activity. Cells were cultured at 37 °C, 5% CO2, in appropriate medium supplemented with 100 U/mL penicillin−streptomycin, 2 mM glutamine, and 10% fetal calf serum (FCS) (all from Sigma-Aldrich, Saint-Quentin-Fallavier, France): Ham’s F12 for PC-3 cells, RPMI for Jurkat cells, and DMEM for HT-29, HeLa, C6, and NIH-3T3 cells. Growth curves and dose− response curves were established by cell counts in 24-wells plates. Cells were seeded at 40 000 per well and cultured for the indicated time, with the indicated concentration of 1 or with 0.2% DMSO in controls. At each experimental point, quadruplicate wells were trypsinized (except for Jurkat cells) and cells were counted in a Malassez chamber. Results are expressed as mean ± SEM, n = 4. Western Blot Analysis of Procaspase-3 Cleavage. The influence of canthin-6-one (1) on procaspase-3 cleavage was investigated by Western blot. Cells were seeded in 24-wells plates (40 000 cells per well) and treated with 1 at 40 μM (10 and 30 μM for HeLa cells) or with 0.2% DMSO in control conditions for 2−4 days. After rinsing in phosphate-buffered saline (PBS), cells were scratched in 1 mL of PBS and recovered by centrifugation (6000g for 30 s). Cell pellets were resuspended in Laemmli buffer,36 and then proteins were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12.5% gels) and electrotransferred on nitrocellulose membranes. Nonspecific sites were blocked in PBS containing 0.05% Tween-20 and 5% bovine serum albumin (PBS-Tween-BSA). Membranes were incubated overnight at 4 °C with a rabbit monoclonal antibody anticaspase-3 (#9665 from Cell Signaling Technology, Ozyme Saint-Quentin Yvelines France) diluted 1/1000 in PBS-Tween-BSA. After rinsing in PBS-Tween-BSA, antigen− antibody complexes were detected by Alexa-488-labeled goat antirabbit antibody (#A-11029 from Molecular Probes, Life Technologies, Saint-Aubin, France) diluted 1/500 in PBS-Tween-BSA (2 h, room temperature). β-Actin was used as loading control in Western blot analysis. Nitrocellulose membranes were blocked in PBS-Tween-BSA and then in normal goat serum 1/50 in PBS-Tween-BSA before incubation overnight at 4 °C with a mouse monoclonal antibody antiβ-actin (clone AC-15, #A5441, Sigma-Aldrich) diluted 1/1000 in PBSTween-BSA. Antigen−antibody complexes were detected by Alexa555-labeled goat anti-mouse antibody (#A-21422 Molecular Probes) diluted 1/500 in PBS-Tween-BSA. Fluorescence was measured on a laser scanner (Typhoon Trio, GE Healthcare, Europe, VelizyVillacoublay, France) coupled to ImageQuant software (GE Healthcare). Flow Cytometric Analysis of Cell Cycle Arrest. Cell cycle distribution after treatment with 1 was determined by flow cytometry DNA analysis after cells were stained with propidium iodide (PI). PC3 cells were seeded in six-well plates (100 000 cells in control groups and 200 000 cells in 1-treated groups) and cultured for 2 days with either 40 μM 1 or 0.2% DMSO control. Cells were collected after incubation with 0.25% trypsin and 1 mM EDTA for 5 min at 37 °C and resuspended in 150 μL of PBS. Cells were immediately fixed by the addition of 850 μL of cold 70% ethanol. After standing for 30 min at 4 °C, cells were centrifuged, resuspended in 1 mL of DNA-staining solution (5 μg/mL PI, 0.1% Triton X-100, 200 μg/mL RNase A in PBS) and incubated for 2 h at room temperature in the dark. Cells were analyzed using a BD FACSVerse flow cytometer (Becton Dickinson, BD Biosciences, Le Pont-de-Claix, France). Fluorescence emitted from the PI−DNA complex was captured at 488 nm using 20 000 single cells per sample and analyzed using BD FACSuite software (BD Biosciences). BrdU Incorporation. BrdU incorporation into DNA was monitored in PC-3 cells using a commercially available immunodetection kit (#11 296 736 001 from Roche Applied Bioscience, Mannheim, Germany) according to the manufacturer’s instructions. For immunofluorescence microscopy, PC-3 cells were seeded in fourwell chamber slides (10 000 cells per well) and cultured for 30 h with 1 (40 μM) or 0.2% DMSO in controls, before addition of BrdU and further incubation for 18 h. Cells were briefly rinsed in PBS and fixed in 500 μL of 70% ethanol and 15 mM glycine (pH 2) for 20 min at

−20 °C. DNA denaturation was achieved for 1 h at 37 °C in 500 μL of 2 N HCl, followed by neutralization with three rinses in 0.1 M borate (pH 9.5). After washing in PBS, nonspecific sites were blocked for 2 h in PBS-Tween-BSA. Mouse monoclonal anti-BrdU antibody diluted 1/ 20 in PBS-Tween-BSA was added for 1 h at 37 °C, and after rinsing in PBS-tween-BSA, Alexa 488-labeled goat anti-mouse antibody (#A11029 from Molecular Probes, 1/400 in PBS-Tween-BSA) was added and incubated for 1 h at room temperature. After a final rinse in PBS, preparations were mounted in Mowiol 4.88 (Calbiochem, St-Quentinen-Yvelines, France) and examined by fluorescence microscopy (Olympus BX41, Olympus France, Rungis, France). For Southern blot analysis of BrdU incorporation into DNA, 106 PC-3 cells were seeded in 25 cm2 flasks, treated for 30 h, and labeled for 18 h as described above. Cells were then washed with 10 mL of PBS and incubated for 3 h at 50 °C in 600 μL of proteinase K buffer (10 mM Tris pH 7, 150 mM NaCl, 0.5% SDS) and proteinase K (60 μg). The lysate was transferred into an Eppendorf tube, and 30 μL of RNase A (1 mg/mL) was added and incubated for 1 h at 37 °C. After two washes in 1 volume of phenol/chloroform (1:1) and 1 volume of chloroform, DNA was precipitated in 0.15 M sodium acetate (pH 6) and 50% 2-propanol (overnight, −20 °C). The DNA pellet was recovered by centrifugation (13000g, 20 min), washed in 1 mL of 70% ethanol, and resuspended in TE buffer (10 mM Tris pH 7.4, 1 mM EDTA). A 400 ng aliquot of DNA was electrophoresed on a 1% agarose gel containing ethidium bromide, denatured, and transferred by capillarity on a nitrocellulose membrane. After cross-linking at 72 °C for 2 h, ethidium bromide DNA fluorescence was photographed on a UV light station coupled to Biocapt software (Vilber Lourmat, Marne La Vallée, France). Nitrocellulose-immobilized DNA was denatured with 2 N HCl for 1 h at 37 °C and washed twice with 0.1 M borate (pH 9.5). Nonspecific sites were blocked in PBS-Tween-BSA, and the membrane was probed with mouse monoclonal anti-BrdU antibody and Alexa 647-labeled donkey anti-mouse antibody (#A31571 from Molecular Probes) as described above. Immunofluorescence was recorded on a Typhoon scanner, quantified with ImageQuant TL, and normalized to BrEt-induced DNA fluorescence. Tubulin Immunofluorescence. Mitotic spindles were labeled with anti-β-tubulin antibody (#T4026 from Sigma-Aldrich). PC-3 cells were seeded in four-well chamber slides (10 000 cells per well) and cultured for 2 days with either 40 μM of 1 or 0.2% DMSO in controls. Cells were fixed in 500 μL of 70% ethanol and 15 mM glycine pH 2 (20 min, −20 °C) and washed in PBS, and nonspecific sites were blocked in PBS-Tween-BSA. Incubation with a mouse anti-β-tubulin antibody 1/200 was performed for 2 h at 37 °C, and antigen−antibody complexes were detected with Alexa 488-labeled goat anti-mouse antibody (#A-11029 from Molecular Probes, 1/400 in PBS-TweenBSA, 1 h at 20 °C). After a final wash with PBS, preparations were mounted in Mowiol 4.88 (Calbiochem) and examined on a fluorescence microscope (Olympus BX41). At least 10 000 cells in each group were counted to determine the percentage of cells in mitosis. Viability Assay on Normal Human Skin Fibroblasts. Viability assay was performed by BIOalternatives (Gencay, France). Normal human dermal fibroblasts were cultured for 3 days in DMEM medium supplemented with 10% FCS in the presence of increasing concentrations of 1 (up to 160 μM) or equivalent concentrations of DMSO in controls. Methyl thiazolyl tetrazolium (MTT) reduction was measured by absorbance at 540 nm (results expressed as mean ± SEM, n = 3), and microscopic observation of cell morphology was performed. Tubulin Polymerization in Vitro. The tubulin polymerization assay in vitro was performed by Ecrins Therapeutics (La Tronche, France). The assay was achieved in the presence of 50 μM tubulin and 1 mM GTP in polymerization buffer at 37 °C. Tubulin polymerization was measured (every 30 s for 40 min) by absorbance at 350 nm with a spectrometer (FLUOstar Omega, BMG LABTECH, Champigny-surMarne, France) coupled to OMEGA software, in the presence of 1 (25 μM) or DMSO. Results are expressed as mean ± SEM, n = 3. 2486



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(19) Kelkel, M.; Cerella, C.; Mack, F.; Schneider, T.; Jacob, C.; Schumacher, M.; Dicato, M.; Diederich, M. Carcinogenesis 2012, 33, 2162−2171. (20) Ferreira, J. R.; Cavalcanti, B. C.; Da Costa, P. M.; De Arantes, F. F.; De Alvarenga, E. S.; Maltha, C. R.; De Almeida Barbosa, L. C.; Militao, G. C.; Pessoa, C.; Ferreira, P. M. Toxicol. in Vitro 2013, 27, 1458−1466. (21) Han, C. R.; Jun Do, Y.; Kim, Y. H.; Lee, J. Y.; Kim, Y. H. Biochim. Biophys. Acta 2013, 1833, 2220−2232. (22) Czepukojc, B.; Baltes, A. K.; Cerella, C.; Kelkel, M.; Viswanathan, U. M.; Salm, F.; Burkholz, T.; Schneider, C.; Dicato, M.; Montenarh, M.; Jacob, C.; Diederich, M. Food Chem. Toxicol. 2014, 64, 249−257. (23) Vermeulen, K.; Berneman, Z. N.; Van Bockstaele, D. R. Cell Prolif. 2003, 36, 165−175. (24) Fizazi, K.; Navone, N. M. Bull. Cancer 2005, 92, 129−141. (25) Sarkar, S.; Brautigan, D. L.; Parsons, S. J.; Larner, J. M. Oncogene 2014, 33, 26−33. (26) Bucher, N.; Britten, C. D. Br. J. Cancer 2008, 98, 523−528. (27) Camphausen, K.; Brady, K. J.; Burgan, W. E.; Cerra, M. A.; Russell, J. S.; Bull, E. E.; Tofilon, P. J. Mol. Cancer Ther. 2004, 3, 409− 416. (28) Schorl, C.; Sedivy, J. M. Methods 2007, 41, 143−150. (29) Silva, J. R.; Ramos Ade, S.; Machado, M.; De Moura, D. F.; Neto, Z.; Canto-Cavalheiro, M. M.; Figueiredo, P.; Do Rosario, V. E.; Amaral, A. C.; Lopes, D. Mem. Inst. Oswaldo Cruz 2011, 1, 142−158. (30) Milliken, W. Plants for Malaria for Fever, Medicinal Species in Latin America; The Royal Botanical Garden: Kew, London, 1987. (31) Ferreira, M. E.; Nakayama, H.; de Arias, A. R.; Schinini, A.; de Bilbao, N. V.; Serna, E.; Lagoutte, D.; Soriano-Agatón, F.; Poupon, E.; Hocquemiller, R.; Fournet, A. J. Ethnopharmacol. 2007, 109, 258−263. (32) Jenkins, R. H.; Davies, L. C.; Taylor, P. R.; Akiyama, H.; Cumbes, B.; Beltrami, C.; Carrington, C. P.; Phillips, A. O.; Bowen, T.; Fraser, D. J. Am. J. Pathol. 2014, 184, 996−1009. (33) Xue, X.; Gong, L. K.; Maeda, K.; Luan, Y.; Qi, X. M.; Sugiyama, Y.; Ren, J. Mol. Pharmaceutics 2011, 8, 2183−2192. (34) Hoang, M. L.; Chen, C. H.; Sidorenko, V. S.; He, J.; Dickman, K. G.; Yun, B. H.; Moriya, M.; Niknafs, N.; Douville, C.; Karchin, R.; Turesky, R. J.; Pu, Y. S.; Vogelstein, B.; Papadopoulos, N.; Grollman, A. P.; Kinzler, K. W.; Rosenquist, T. A. Sci. Transl. Med. 2013, 5, 197ra102. (35) Soriano-Agaton, F.; Lagoutte, D.; Poupon, E.; Roblot, F.; Fournet, A.; Gantier, J. C.; Hocquemiller, R. J. Nat. Prod. 2005, 68, 1581−1587. (36) Laemmli, U. K. Nature 1970, 227, 680−685.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +33 5 4945 3735. Fax: +33 5 4945 4014. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

The authors thank Dr. T. Janet for providing the BrdU incorporation kit and anti-caspase3 antibody. The authors acknowledge Dr. A. C. Balandre, Dr. S. Bensalma, and Dr. V. Coronas for their valuable expertise in culturing primary cell lines and cancer cell lines. The authors wish to thank Ms. L. ̈ Cousin and Ms. J. Colas for their technical assistance. O. Haida and M. Gestin are acknowledged for their participation during their undergraduate internship. This work has benefited from the facilities and expertise of A. Delwail of ImageUP platform (University of Poitiers). The authors are grateful to F. X. Bernard (BIOalternatives, Gençay) for the cytotoxicity assays. C.D. was supported by a grant from the Région PoitouCharentes. This work was supported by an Action Concertée Incitative (Chimie-Biologie) of the University of Poitiers.

(1) Basmadjian, C.; Zhao, Q.; Bentouhami, E.; Djehal, A.; Nebigil, C. G.; Johnson, R. A.; Serova, M.; de Gramont, A.; Faivre, S.; Raymond, E.; Désaubry, L. G. Front. Chem. 2014, 2, 20. (2) Duffy, R.; Wade, C.; Chang, R. Drug Discovery Today 2012, 17, 942−953. (3) Haynes, H. F.; Nelson, E. R.; Price, J. R. Aust. J. Sci. Res. 1952, 5, 387−400. (4) Showalter, H. D. J. Nat. Prod. 2013, 76, 455−467. (5) Anderson, L. A.; Harris, A.; Phillipson, J. D. J. Nat. Prod. 1983, 46, 374−378. (6) Ma, Z. Z.; Hano, Y.; Nomura, T.; Chen, Y. J. Phytochemistry 2000, 53, 1075−1078. (7) De Souza Almeida, E. S.; Filho, V. C.; Niero, R.; Clasen, B. K.; Balogun, S. O.; De Oliveira Martins, D. T. J. Ethnopharmacol. 2011, 134, 630−636. (8) O’Donnell, G.; Gibbons, S. Phytother. Res. 2007, 21, 653−657. (9) He, W.; Van Puyvelde, L.; De Kimpe, N.; Verbruggen, L.; Anthonissen, K.; Van Der Flaas, M.; Bosselaers, J.; Mathenge, S. G.; Mudida, F. P. Phytother. Res. 2002, 16, 66−70. (10) Thouvenel, C.; Gantier, J. C.; Duret, P.; Fourneau, C.; Hocquemiller, R.; Ferreira, M. E.; Rojas De Arias, A.; Fournet, A. Phytother. Res. 2003, 17, 678−680. (11) Dejos, C.; Regnacq, M.; Bernard, M.; Voisin, P.; Berges, T. FEBS Lett. 2013, 587, 3045−3051. (12) Xu, Z.; Chang, F. R.; Wang, H. K.; Kashiwada, Y.; Mcphaila, T.; Bastow, K. F.; Tachibana, Y.; Cosentino, M.; Lee, K. H. J. Nat. Prod. 2000, 63, 1712−1715. (13) Kuo, P. C.; Shi, L. S.; Damu, A. G.; Su, C. R.; Huang, C. H.; Ke, C. H.; Wu, J. B.; Lin, A. J.; Bastow, K. F.; Lee, K. H.; Wu, T. S. J. Nat. Prod. 2003, 66, 1324−1327. (14) Miyake, K.; Tezuka, Y.; Awale, S.; Li, F.; Kadota, S. Nat. Prod. Commun. 2010, 5, 17−22. (15) De Feo, V.; De Martino, L.; Santoro, A.; Leone, A.; Pizza, C.; Franceschelli, S.; Pascale, M. Phytother. Res. 2005, 19, 226−230. (16) Ammirante, M.; Di Giacomo, R.; De Martino, L.; Rosati, A.; Festa, M.; Gentilella, A.; Pascale, M. C.; Belisario, M. A.; Leone, A.; Turco, M. C.; De Feo, V. Cancer Res. 2006, 66, 4385−4393. (17) Peduto, A.; More, V.; De Caprariis, P.; Festa, M.; Capasso, A.; Piacente, S.; De Martino, L.; De Feo, V.; Filosa, R. Mini Rev. Med. Chem. 2011, 11, 486−491. (18) Gollner, A.; Koutentis, P. A. Org. Lett. 2010, 12, 1352−1355. 2487


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