Canthin-6-one induces cell death, cell cycle arrest ...

Biochimica et Biophysica Acta 1861 (2017) 958–967

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Canthin-6-one induces cell death, cell cycle arrest and differentiation in human myeloid leukemia cells Heron F. Vieira Torquato a,b, Antonio C. Ribeiro-Filho c, Marcus V. Buri b, Roberto T. Araújo Júnior b, Renata Pimenta d, José Salvador R. de Oliveira d, Valdir C. Filho e, Antonio Macho a, Edgar J. Paredes-Gamero b,c,⁎, Domingos T. de Oliveira Martins a,⁎⁎ a Department of Basic Sciences in Health, Faculty of Medicine, Federal University of Mato Grosso (UFMT), Av. Fernando Correa da Costa, no. 2367, Boa Esperança, Cuiabá, Mato Grosso 78060-900, Brazil b Department of Biochemistry, Federal University of São Paulo (UNIFESP), Av. Pedro de Toledo, no. 669, São Paulo, São Paulo 04039-401, Brazil c Centro Interdisciplinar de Investigação Bioquı́mica, Universidade de Mogi das Cruzes, Av. Dr. Cândido Xavier de Almeida Souza, 200, Mogi das Cruzes, São Paulo, Brazil d Department of Medicine (Hematology), Federal University of São Paulo (UNIFESP), Av. Diogo de Faria, 824, São Paulo, São Paulo 04037-002, Brazil e Chemical-Pharmaceutical Research Center, University of Vale of Itajaí (UNIVALI), Rua Uruguai, no. 458, Centro, Itajaí, Santa Catarina 88302-202, Brazil

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Article history: Received 25 October 2016 Received in revised form 18 January 2017 Accepted 30 January 2017 Available online 2 February 2017 Keywords: Canthin-6-one Cell death Cell differentiation Cell cycle arrest Leukemia Leukemic stem cells

a b s t r a c t Background: Canthin-6-one is a natural product isolated from various plant genera and from fungi with potential antitumor activity. In the present study, we evaluate the antitumor effects of canthin-6-one in human myeloid leukemia lineages. Methods: Kasumi-1 lineage was used as a model for acute myeloid leukemia. Cells were treated with canthin-6one and cell death, cell cycle and differentiation were evaluated in both total cells (Lin+) and leukemia stem cell population (CD34+ CD38− Lin−/low). Results: Among the human lineages tested, Kasumi-1 was the most sensitive to canthin-6-one. Canthin-6-one induced cell death with apoptotic (caspase activation, decrease of mitochondrial potential) and necrotic (lysosomal permeabilization, double labeling of annexin V/propidium iodide) characteristics. Moreover, canthin-6-one induced cell cycle arrest at G0/G1 (7 μM) and G2 (45 μM) evidenced by DNA content, BrdU incorporation and cyclin B1/histone 3 quantification. Canthin-6-one also promoted differentiation of Kasumi-1, evidenced by an increase in the expression of myeloid markers (CD11b and CD15) and the transcription factor PU.1. Furthermore, a reduction of the leukemic stem cell population and clonogenic capability of stem cells were observed. Conclusions: These results show that canthin-6-one can affect Kasumi-1 cells by promoting cell death, cell cycle arrest and cell differentiation depending on concentration used. General significance: Canthin-6-one presents an interesting cytotoxic activity against leukemic cells and represents a promising scaffold for the development of molecules for anti-leukemic applications, especially by its anti-leukemic stem cell activity. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Acute myeloid leukemia (AML) is a biologically complex and clinically heterogeneous disease, characterized by clonal expansion of blasts, in incomplete stages of cell differentiation, in the bone marrow and peripheral blood [1]. In this group, AML has a particularly dismal outcome with b10% of older adults being long-term survivors. The poor outcome is both patient and disease-related, especially co-comorbidities that

⁎ Corresponding author at: Centro Interdisciplinar de Investigação Bioquímica, Universidade de Mogi das Cruzes, Mogi das Cruzes, São Paulo, 08780 - 911, Brazil. ⁎⁎ Corresponding author. E-mail addresses: [email protected] (E.J. Paredes-Gamero), [email protected] (D.T. de Oliveira Martins).

http://dx.doi.org/10.1016/j.bbagen.2017.01.033 0304-4165/© 2017 Elsevier B.V. All rights reserved.

limit their dose-intensity of applied therapies [2–4]. In turn, a reduction in the number of healthy blood cells leads to severe cytopenias in the patient, requiring recurrent blood product transfusion support and long hospitalization [5,6]. Recently, myeloid cancers are considered to be clonal diseases arising from genetic defects occurring in hematopoietic stem cells (HSCs), leading to the rise of leukemic stem cells (LSCs) [7,8]. LSCs are a subset of cancer stem cells, which have been postulated to represent rare populations of malignant cells responsible for maintaining the bulk tumor and represent a therapy refractory reservoir for relapse. These cells have distinctive features common to normal HSCs, such as self-renewal, quiescence and an undifferentiated state, which make them more resistant to conventional chemotherapy [9]. Targeting these cells among actively dividing cancer cells may significantly contribute to solving the problem of resistance and relapse.

H.F. Vieira Torquato et al. / Biochimica et Biophysica Acta 1861 (2017) 958–967

For many years, the combination of anthracycline plus cytarabine has been a common intensive induction regime for patients with AML [10]. However, there has been little improvement in this combination since it was developed. Furthermore, these treatments are highly toxic and often do not lead to long-term complete remission [11,12]. Several trials showed that further enhancing the dose of anthracycline and cytarabine, augments complete remission rates and overall survival in both younger and older patients, but, an enhanced toxicity was observed [13–15]. Although, despite the development of new techniques for the discovery of new drugs, such as chemical synthesis, combinatorial chemistry, and molecular modeling, the use of natural sources, especially plants, remains one of the most importance sources for the discovery and development of new anti-cancer drugs. In this regard, natural products are considered as important sources for the discovery of new antitumor drugs [16–21], and various herbal compounds have also demonstrated activity against leukemic cells by way of induced cell death or differentiation [22–25]. In this study, we report canthin-6-one inducing cell death, cell cycle arrest and cell differentiation in leukemic cells and on an LSC population. Canthin-6-one is a beta-carboline alkaloid isolated from various plants sources, principally from the Rutaceae and Simaroubaceae families, and recently from fungi [26]. This compound and congeners showed different pharmacological activities such as antifungal, antibacterial and anticancer activities [27–30]. 2. Material and methods 2.1. Canthin-6-one Canthin-6-one (IUPAC name: 6H-indolo[3,2,1-de][1,5]naphthyridin6-one; CAS no: 479-43-6) was purchased from Alpha Chimica (Châtenay-Malabry, France), with purity certified as N 96%. It was dissolved in DMSO, stored at −20 °C and diluted in culture medium before use. The final concentration of DMSO in the culture medium at any time was not higher than 0.25%. 2.2. Cell cultures Human leukemia cell lines (Kasumi-1, KG-1, K562, Jurkat and ARH77) were purchased from ATCC (Virginia, USA); MS-5, a murine stroma cell line, was kindly provided by Dr. Giselle Zenker Justo (Universidade Federal de São Paulo). The cells were maintained in RPMI 1640 (SigmaAldrich, Germany) culture medium, supplemented with 10% fetal bovine serum, 100 U/mL penicillin (Sigma-Aldrich, Germany) and 100 μg/mL streptomycin (Sigma-Aldrich, Germany). Cells were cultured in a humidified incubator containing 5% CO2 at 37 °C. 2.3. Normal human hematopoietic cells Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors after informed patient consent, as approved by the local Ethical Committee of the Federal University of São Paulo (document number 0225/10). To separate the fraction of mononuclear cells, Ficoll-Histopaque (density 1.077 g/mL, Sigma-Aldrich) centrifugation at 400g for 30 min was used. 2.4. Cell differentiation by immunophenotyping Cells (105/mL) were treated with 14 μM canthin-6-one for three consecutive days. Then, the cells were collected and stained to identify mature cells and leukemic stem cells [25]. The LSC markers used were CD34-APC, CD38-PE and lineage (Lin) PE markers (CD2, CD3, CD4, CD7, CD8, CD14, CD19, CD20, CD235a). CD15-FITC and CD11b-Cy7/PE were also used in some experiments. All antibodies were purchased from Becton Dickinson (USA). The measurements were performed

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using an Accuri C6 flow cytometer (Becton Dickinson, USA); 300,000 events were acquired.

2.5. Bone marrow cultures MS-5 murine cells were grown (105 cells) on 6-well plates in RPMI 1640 medium supplemented with 10% fetal serum bovine, penicillin and streptomycin. Half of the culture medium was removed and PBMCs (2 × 106 cells) obtained from three normal donors were added to MS-5 cells. Normal healthy donors were previously treated with granulocyte colony-stimulating factor for mobilization of hematopoietic stem cells. Samples were treated with 14 μM canthin-6-one for three consecutive days. Then, the cells were collected and stained to identify mature cells (lineage markers) and hematopoietic stem cells (CD34APC and CD38-PE), as mentioned above. SYTOX green and CD45 PECy7 were used to exclude dead cells and to identify human hematopoietic cells, respectively. The measurements were performed using an Accuri C6 flow cytometer and 300,000 events were acquired.

2.6. Annexin V/propidium iodide assay Leukemic cells or PBMCs were seeded (105 cells/mL) in 96-well microplates and treated with canthin-6-one (28, 56, 113 and 227 μM). Likewise, Kasumi-1 cells were treated with cisplatin (Sigma-Aldrich, Germany) for 24 h. Then, the cells were centrifuged and resuspended in binding buffer (0.14 M NaCl, 2.5 mM CaCl2, 0.01 M HEPES, pH 7.4) and incubated at room temperature with Annexin V-FITC (BD Biosciences, USA) and 5 μg/mL PI (Sigma-Aldrich, Germany) for 20 min. To investigate the mechanisms of canthin-6-one-induced cell death, Kasumi1 cells were pretreated with E64 (20 μM), z-VAD-fmk (20 μM), SB203580 (20 μM) and SP600125 (20 μM) inhibitors for 1 h before the treatment with canthin-6-one (45 μM). All inhibitors were purchased from Tocris (United Kingdom). Sample analysis was performed using an Accuri C6 flow cytometer with the acquisition of 10,000 events

2.7. Determination of lysosomal membrane permeabilization For the lysosomal leakage assays, Kasumi-1 cells were seeded at 105 cells/mL, treated with 45 μM canthin-6-one for 4 h, and then labeled with 5 μg/mL AO (Sigma-Aldrich) and examined using an Accuri C6 flow cytometer with the acquisition of 10,000 events, and an SP8 confocal microscope (Leica, Germany) with a 63× objective (numerical aperture 1.43) [31]. The AO was excited using an argon laser (λEx = 488 nm) and emission collected at λEmgreen = 505–530 nm/λEmred = 560–600 nm by confocal microscopy, or FL-2 channel (585/40) by flow cytometry.

2.8. Analysis of mitochondrial membrane potential (Ψm) Changes in Ψm were measured using Rhodamine 123 (Rho 123) (Sigma-Aldrich, Germany) dye. Kasumi-1 cells (105/mL) were stimulated with canthin-6-one (45 μM) for 24 h. Then, the cells were incubated with 6 μM Rho123 for 1 h and washed. A total of 10,000 events were acquired using an Accuri C6 flow cytometer.

2.9. Reactive oxygen species measurement To measure ROS, Kasumi-1 cells (105/mL) were treated with 45 μM canthin-6-one for 24 h. Then, the cells were incubated with 10 μM 5(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (Life Technologies, USA) for 30 min. A total of 10,000 events were acquired using an Accuri C6 flow cytometer.

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2.10. Activation of caspases 3, 8 and 9 Kasumi-1 cells (105/mL) were treated with 45 μM canthin-6-one for 24 h. Then the cells were fixed with 2% paraformaldehyde in PBS for 30 min and permeabilized with 0.01% saponin for 15 min at room temperature. Afterward, the cells were incubated for 1 h at 37°C with anticleaved-caspase 3, 8, and 9 monoclonal antibodies (Cell Signaling, USA). As secondary antibody, an anti-IgG rabbit Alexa Fluor 488 conjugate (Life Technologies) was used. After incubation for 40 min, the fluorescence was analyzed using an Accuri C6 flow cytometer. A total of 10,000 events were acquired. 2.11. Cell cycle analysis Distribution of cell cycle phases was determined by PI staining and flow cytometry analysis. Kasumi-1 cells (105/mL) were treated with canthin-6-one (7 and 45 μM) for 24 h. Then, the cells were fixed and permeabilized as previously described and treated with 4 μg/mL RNase (Sigma-Aldrich, Germany) for 45 min at 37°C. For DNA labeling, cells were incubated with 5 μg/mL of PI (Sigma-Aldrich, Germany). Percentages of cells within cell cycle compartments (G1, S and G2/M) were performed using an Accuri C6 flow cytometer (Becton Dickinson, USA). A total of 10,000 events were acquired. 2.12. BrdU assay Kasumi-1 cells (105 cells/mL) were treated with canthin-6-one (7 μM and 45 μM) in the presence of 10 μM BrdU (Sigma-Aldrich, Germany) for 24 h. BrdU labeling was performed according to the manufacturer's instructions (BrdU-FITC Flow Kit, BD Biosciences, USA). DNA content was labeled using 7-aminoactinomycin D (7-AAD, BD Biosciences, USA). Data acquisition was performed using an Accuri C6 flow cytometer; 100,000 events were acquired. 2.13. Clonogenic assay After treatment for three consecutive days with canthin-6-one, 103 cells were mixed with a methylcellulose-based medium (Methocult H4100, Stem Cell Technologies, USA). The mixture was placed in 35 mm dishes and cultured in a humidified incubator for 21 days. At the end of this period, colonies consisting of N50 cells were counted using an inverted microscope at 40× magnification. 2.14. Intracellular protein labeling Kasumi-1 cells (105/mL) were treated with canthin-6-one and were fixed with 4% paraformaldehyde for 10 min, washed with BD Perm/ Wash buffer and permeabilized with BD Perm Buffer III for 30 min. Then, the cells were labeled with 5 μL Ki-67-FITC antibody (BD Biosciences). To label intracellular proteins, cells were incubated for 1 h with the following antibodies: p-p38 (Thr182-180/Tyr) PE/CF594, pJNK (pT183/pY185) Alexa Fluor 647, p-Histone H3 (Ser10) Alexa Fluor 488 and Cyclin B1 Alexa Fluor 647, all purchased from BD Biosciences (USA). p-Rb (Ser780) Alexa Fluor 488, PU.1 Alexa Fluor 488, phospho checkpoint kinase 1 (p-Chk1) Ser 345, phospho checkpoint kinases 2 (p-Chk2) Thr68, p-p53 (Ser15), p-H2A.X (Ser139), p16 INK4A and p27 Kip1 were purchased from Cell Signaling (USA). Anti-rabbit or mouse

IgG secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 647 (Invitrogen, USA) were used for at least 40 min. Protein analyses were performed by quantification of the fluorescence geometric mean (Gm) [32,33]. 2.15. Statistical analyses All data represent at least three independent experiments and were expressed as mean ± standard error of the mean (SEM). Statistical analyses were performed using Student's t-test for comparison between two groups, and analysis of variance (ANOVA) and Dunnett's post hoc test for multiple comparisons among groups. A probability value of p b 0.05 was considered significant. GraphPad Prism 5 software version 5.01 was used for data analyses. 3. Results 3.1. Canthin-6-one produced cell death of an AML lineage Cell death features were investigated to determine the cytotoxic potential of canthin-6-one in some leukemic lineages. Canthin-6-one reduced cell viability in a concentration-dependent manner after 24 h in different human leukemic lineages (supplementary information Fig. S1). KG-1 (AML), K562 (chronic myeloid leukemia), Jurkat (acute T cell leukemia) and ARH-77 (plasma cell leukemia) lineages were tested. Among them, KG-1 was the most sensitive lineage to canthin-6-one (EC50 = 39.1 ± 1 μM) (supplementary information Fig. S1, Fig. 1a). Cisplatin, a chemotherapeutic drug, was used as a positive control (EC50 = 16.2 ± 1.2 μM) (Fig. 1a). Additionally, canthin-6-one was less cytotoxic to PBMCs (EC50 = 79.5 ± 1 μM) (Fig. 1b) than Kasumi-1. Cell death was investigated by double staining with Annexin V-FITC and PI. Kasumi-1 cells showed predominant double staining (Fig. 1c), whereas PBMCs preferentially showed a stain of Annexin V-FITC. Moreover, canthin-6one (45 μM) also altered lysosomal integrity, verified by the extravasation of AO. A decrease in red fluorescence in lysosomes was observed in Kasumi-1 cells treated for 4 h (Fig. 1e[i]) indicating extravasation of AO from lysosomes to the cytosol increasing red fluorescence in the cytosol (Fig. 1e[ii]). The protease inhibitor E64 was used to corroborate participation of lysosomal proteases in cell death. E64 partially reduced canthin-6-one-dependent cell death (Fig. 1f). Other features investigated were Ψm and ROS production. A decrease in Ψm was observed by the reduction intensity of Rho 123 in Kasumi-1 cells (Fig. 1g). ROS production was quantified by H2-DCFDA and it was also able to induce an increase of ROS after 24 h (Fig. 1h). Afterward, activation of caspases was observed by quantification of the active form of caspases (cleaved caspase) after 24 h of stimulation. Canthin-6-one induced the activation of capase-8, capase-9 and caspase-3 in a stimulated group (Fig. 1i). The pan caspase inhibitor z-VAD-fmk partially reduced cell death induced by canthin-6-one (Fig. 1j). Subsequently, participation of proteins associated with cellular stress such as ASK-1, p38 and JNK was investigated (Fig. 2a–c). Canthin-6-one induced an increase of the active forms of ASK-1, p38 and JNK (solid black histograms) as observed in cytometry histograms; quantification of histograms is shown on the right. Stimulation with phorbol 12-myristate 13 acetate (PMA) and ionomycin (IO) was used as positive controls of cellular stress. Participation of p38 (SB203580) and JNK (SP600125) was confirmed by the use of specific inhibitors.

Fig. 1. Canthin-6-one (Cant) promoted cell death by different mechanisms in Kasumi-1 cells. Cells were stimulated with 45 μM canthin-6-one for 24 h. (a–b) The cytotoxic activity of canthin-6-one and cisplatin was assessed by analysis of annexin V/PI staining on Kasumi-1 and PBMCs. (c–d) Representative flow cytometry dot plots of annexin V-FITC and PI; quantitative measurements are shown at the right. (e) Lysosomal disruption was monitored using 5 μg/mL AO by flow cytometry and confocal microscopy. (f) Cell death was partially reduced by E64 inhibitor. (g) Mitochondrial depolarization, (h) increase of ROS production and (i–j) caspase activation. These results are the means ± SEM of three independent experiments performed in triplicate. ANOVA test followed by Dunnett's post hoc test or Student's t-test * p b 0.05, ** p b 0.01, and *** p b 0.001 vs. untreated. # p b 0.001 vs. Cant.


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Fig. 2. Cell death induced by Canthin-6-one (Cant), involves cellular stress response. Kasumi-1 cells were stimulated with 45 μM canthin-6-one for 24 h and activation of proteins was evaluated by flow cytometry. Expression analyses of (a) p-ASK-1, (b) p-p38 and (c) p-JNK activation are shown by cytometry histograms. Quantification of intensity is shown at the right. PMA and IO were used as positive control of cellular stress. These results are the means ± SEM of three independent experiments. ANOVA test followed by Dunnett's post hoc test. ** p b 0.01 and *** p b 0.001 vs. untreated. # p b 0.001 vs. Cant.

SB203580 and SP600125, partially inhibited the cytotoxic effect of canthin-6-one (Fig. 2d and e). 3.2. Canthin-6-one induces cell cycle arrest Change in cell proliferation of Kasumi-1 was observed during cytotoxicity experiments. Thus, the ability of canthin-6-one to produce cell cycle arrest in Kasumi-1 was evaluated. We decided to test two concentrations: 7 μM, which does not induce cell death; and 45 μM, close to the EC50. The cell cycle showed an increase of G0/G1 phase when stimulated with 7 μM canthin-6-one (Fig. 3a), whereas 45 μM induced an increase of G2/M phase with a reduction of G0/G1 (Fig. 3a) after 24 h. Cell cycle arrest was confirmed by BrdU assay, where 7 μM canthin-6-one induced an increase of G1 phase and 45 μM canthin-6-one induced G2/M arrest after 24 h (Fig. 3b). Additionally, Ki-67 protein, which is strictly expressed during cell proliferation in all active cell cycle phases G1, S, G2/M, was observed to have a reduced expression (Ki-67 positive cells) and an increase in Ki-67 negative population after canthin-6one treatment at both concentrations (Fig. 3c). Cell cycle arrest was also confirmed by quantification of phosphorylation of RB protein, which arrests the cell cycle when it is not phosphorylated. A decrease of phospho-RB was observed by treatment with canthin-6-one at both concentrations (Fig. 3d). In order to differentiate G2 phase and mitosis, phosphorylation of histone 3 (p-H3), a marker of mitosis, and cyclin B, a cyclin expressed in G2/M phase, was quantified. Canthin-6-one at 7 μM decreased G2 phase (H3−/Cyclin B1+), whereas at 45 μM canthin-6-one there was an increase in G2 (H3−/Cyclin B1+) (Fig. 3e).

Furthermore, we evaluated the activation of proteins associated with checkpoint/cell cycle arrest such as Chk1, Chk2, p53 and H2.A.X. In Kasumi-1 cells stimulation with canthin-6-one (45 μM) activated Chk2, but not Chk1, only at a concentration of 45 μM (Fig. 4a and b). Evaluations of proteins activated by Chk2 such as p53, a regulator of DNA repair, and p-H2A.X, a histone family member associated with checkpoint-mediated cell cycle arrest, were then carried out. Canthin6-one at a concentration of 45 μM induced activation of Chk2, p53 and H2A.X (Fig. 4b–d). These results indicate that canthin-6-one, in a concentration-dependent manner, affects cell cycle of Kasumi-1 cells. The cell cycle arrest in the G2/M phase rise along with active forms of Chk2, p53 and p-H2A.X suggesting DNA damage response.

3.3. Canthin-6-one induced differentiation of total Kasumi-1 cells and leukemic stem cells An important activity evaluated in antitumor compounds is the ability to induce differentiation of cancer cells. Thus, we evaluated myeloid differentiation of Kasumi-1 cells treated with 14 μM canthin-6-one (a concentration that produces low cell death and sufficient to induce cell differentiation) once a day during three consecutive days. Differentiation status was determined by expression of mature myeloid markers CD15 and CD11b. The expression was significantly increased (2.5-fold) by canthin-6-one (Fig. 5a). Similar results were observed in KG-1 and K562 (supplementary information Fig. S2). Additionally, canthin-6one was able to increase expression of PU.1 (Fig. 5b).


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Fig. 3. Canthin-6-one (Cant) induces cell cycle arrest in Kasumi-1 cells. Kasumi-1 cells were treated with canthin-6-one at indicated concentrations for 24 h. (a) Cell cycle analysis was performed by flow cytometry using PI. Percentages of cell cycle distributions are shown. (b) Cell cycle arrest was confirmed by BrdU assay. Expression of (c) Ki-67 and (d) phospho-RB was also quantified. (e) Analysis of cyclin B1 and phospho-Histone H3. Quantifications of cytometry histograms are shown at the right. These results are the means ± SEM of three independent experiments. ANOVA test followed by Dunnett's post hoc test. * p b 0.05, ** p b 0.01 and *** p b 0.001 vs. untreated.

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Fig. 4. Canthin-6-one (Cant) promoted activation of proteins involved in checkpoints. Kasumi-1 cells were stimulated by canthin-6-one at indicated concentrations for 24 h and activation of proteins was evaluated by flow cytometry. Expression of (a) pChk1, (b) pChk2, (c) pp53 and (d) pH2A.X is shown in cytometry histograms. Quantifications of cytometry histograms are shown at the right. These results are the means ± SEM of three independent experiments performed. ANOVA test followed by Dunnett's post hoc test. *** p b 0.001 vs. untreated.

Currently, the search for compounds that affect the stem cell population in cancer is increasing. Thus, we also investigated the effects of canthin-6-one in an LSC (CD34+ CD38− Lin−; Fig. 5c: dot plots) subset present in leukemia lineages [25] such as Kasumi-1. Canthin-6-one reduced the percentage of CD34+ CD38− Lin− cells of Kasumi-1 (Fig. 5c) and K562 lineages (supplementary information Fig. S2b) and also reduced the ability of Kasumi-1 cells to form colonies (Fig. 5d), indicating the loss of clonal ability of LSCs. On the other hand, the percentage of

CD34+ CD38− Lin− obtained from healthy donors increased after canthin-6-one treatment (Fig. 5h). Since canthin-6-one induced cell cycle arrest we investigated if the cell cycle of LSCs was affected. Treatment with canthin-6-one induced an increase of a quiescent population as determined by the decrease of Ki-67 expression in an LSC population (supplementary information Fig. S3). Moreover, quantification of the expression of proteins associated with cell cycle arrest, such as p16INK4A and p27-Kip1, showed that canthin-6-one produced an increase

Fig. 5. Effect of Canthin-6-one (Cant) in differentiation. The cells were treated for 3 consecutive days with 14 μM canthin-6-one and immunophenotyping was evaluated by flow cytometry. (a) Expression of mature myeloid marker CD11b and CD15. (b) Expression of transcription factor PU.1. (c) Quantification of percentage of LSCs (CD34+ CD38− Lin−). (d) CFU assay (methylcellulose assay) quantification. (e) Expression of Ki-67 and DNA content of LSCs. Quantification of cell cycle regulatory proteins (f) p16INK4 and (g) p27 Kip1, and (h) quantification of HSCs from healthy donors (CD34+ CD38− Lin−). Each column represents the mean of 3 independent experiments performed in duplicate. Quantifications of cytometry histograms are shown at the right. These results are the means ± SEM of three independent experiments performed in duplicate. * Students t-test p b 0.05 * p b 0.05, ** p b 0.01, and *** p b 0.001 vs. untreated.


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of expression of both proteins (Fig. 5f and g). However, the DNA content did not show clearly the specific phase of cell cycle arrest by canthin-6one (Fig. 5f and g). 4. Discussion Canthinones and beta-carboline derivates are natural substances with a wide range of biological activities, including cytotoxic effects [30,34–36]. In this report, in order to evaluate the antileukemic action of canthin-6-one, in vitro models of leukemia were employed. Among the cells tested Kasumi-1 exhibited the highest sensibility. Kasumi-1 is a human AML cell line with t(8;21) translocation, which is one of the most common cytogenetic alterations found in this disease [37]. A plant-based compound, canthin-6-one treatment revealed a predominance of double labeling with Annexin V-FITC/PI, AO release from lysosomes, loss of Ψm, ROS increase, and activation of intrinsic and extrinsic caspase-dependent pathways, which are common features of apoptosis [38]. Additionally, the MAPK family, p38, JNK and ASK-1, recognized kinases that regulate a variety of cellular actions, including apoptosis in response to stress [39,40], were activated. These facts suggested that different intracellular pathways contribute to promote cell death in Kasumi-1 cells, a common characteristic of several compounds, especially other beta-carboline derivatives [19,31]. Apoptotic stimulation occurs through distinct signaling cascades. Especially, intrinsic pathway through release of apoptogenic factors from mitochondria, a key event in the triggering of cell death and integrates signals generated by different stressors, including DNA damage and ROS [41]. ROS have been reported to activate p38, JNK and ASK-1 proteins [42–44]. In this scenario, the mitochondrial pathway seems important for canthin-6-one to induce cell death as Harmine, a beta carboline alkaloid isolated from the seeds of Peganum harmala, trigger mitochondrial pathway-mediated cellular apoptosis in SW620 cells [45]. Additionally, we further explored the antitumor properties of canthin-6-one, particularly antiproliferative effects. A low concentration of canthin-6-one induced cell arrest in G1/G0 phase, whereas a concentration close to the EC50 induced cell arrest in G2 phase. It is known that the planar structured of beta-carbolines can bind to DNA and induce DNA damage [46,47]. Likewise, canthin-6-one, at a concentration close to EC50, is likely aggressive enough to produce DNA damage activating Chk2, p53 and H2A.X proteins. A similar result of the arrest of cell cycle in G2/M by canthin-6-one was observed in other tumoral lineages [48]. These observations showed that canthin-6-one induced cell death and cell cycle arrest in G2 phase with DNA damage using concentration close to EC50. Besides cytotoxic treatment, therapies based on cell differentiation have shown beneficial effects in AML [19,49]. For example, different cytotoxic compounds used at lower concentrations can elicit cell differentiation [50]. We demonstrated that expression of the mature myeloid markers CD11b and CD15, and transcription factor PU.1, associated with myeloid differentiation [25], was increased after treatment with canthin-6-one. Moreover, canthin-6-one also affected LSCs of Kasumi1 leukemia. Cancer stem cell is associated with relapse of several cancers such as leukemias [1,8,19]. Canthin-6-one was able to reduce this population and produce cell arrest. Further investigations are still necessary to understand the actions of canthin-6-one on cell differentiation and in the reduction of LSCs, because this particular characteristic is important against leukemias. This point is of great interest as it indicates potential applications in the development of new antileukemic drugs, where the eradication of LSCs could help in reducing the frequency of relapse of the disease, which commonly occurs in leukemic patients [9,19,51]. Various studies with natural products have been successful and some are currently in use in medicine, such as paclitaxel, vincristine, vinblastine [52], among others [53]. Natural product classes such as terpenes, flavonoids, isoflavones, lignans, neolignans, glycosides, coumarin, chromones, quinones, phytosterols and alkaloids, among others,

attest to the exceptional structural diversity present in plants, thereby making natural products an inexhaustible source of drug templates. Canthin-6-one is a molecule obtained from different plants and fungi and this study showed the diverse biological activities that it possesses against leukemic cells. Thus, canthin-6-one is emerging as a natural product with potential antileukemic activity. 5. Conclusion In conclusion, this study demonstrates the ability of canthin-6-one to promote cell death, cell cycle arrest and cell differentiation in an AML lineage. These properties of canthin-6-one in inducing concomitant effects could be of help in the fight against leukemia. Together, these findings may be explored for the development of more effective treatments for AML. Notes The authors declare no competing financial interest. Transparency document The Transparency document associated with this article can be found, in the online version. Acknowledgments The authors wish to thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq n. 551737/2010-7), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES/Pró-Amazônia 23038.000731/2013-56), Fundação de Amparo e Pesquisa do Estado de São Paulo (FAPESP: 2015/16799-3), and the Instituto Nacional de Ciência e Tecnologia em Áreas Úmidas (INAU/CNPq n. 4.02), for financially supporting this work. We thank the INFAR/UNIFESP Confocal and Flow Cytometry Facility. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbagen.2017.01.033. References [1] D.S. Krause, R.A. Van Etten, Right on target: eradicating leukemic stem cells, Trends Mol. Med. 13 (2007) 470–481. [2] R.B. Walter, E.H. Estey, Management of older or unfit patients with acute myeloid leukemia, Leukemia 29 (2015) 770–775. [3] G. Ossenkoppele, B. Lowenberg, How I treat the older patient with acute myeloid leukemia, Blood 125 (2015) 767–774. [4] L.D. Eleni, Z.C. Nicholas, S. Alexandros, Challenges in treating older patients with acute myeloid leukemia, J. Oncol. 2010 (2010) 943823. [5] G.P. Tuszynski, V.L. Rothman, Angiocidin induces differentiation of acute myeloid leukemia cells, Exp. Mol. Pathol. 95 (2013) 249–254. [6] F. Miraki-Moud, F. Anjos-Afonso, K.A. Hodby, E. Griessinger, G. Rosignoli, D. Lillington, L. Jia, J.K. Davies, J. Cavenagh, M. Smith, H. Oakervee, S. Agrawal, J.G. Gribben, D. Bonnet, D.C. Taussig, Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation, Proc. Natl. Acad. Sci. U.S.A. 110 (2013) 13576–13581. [7] F. Ferrara, C.A. Schiffer, Acute myeloid leukaemia in adults, Lancet 381 (2013) 484–495. [8] T. Reya, S.J. Morrison, M.F. Clarke, I.L. Weissman, Stem cells, cancer, and cancer stem cells, Nature 414 (2001) 105–111. [9] H. Zhang, J.Q. Mi, H. Fang, Z. Wang, C. Wang, L. Wu, B. Zhang, M. Minden, W.T. Yang, H.W. Wang, J.M. Li, X.D. Xi, S.J. Chen, J. Zhang, Z. Chen, K.K. Wang, Preferential eradication of acute myelogenous leukemia stem cells by fenretinide, Proc. Natl. Acad. Sci. U.S.A. 110 (2013) 5606–5611. [10] M. Medinger, C. Lengerke, J. Passweg, Novel therapeutic options in acute myeloid leukemia, Leuk. Res. Rep. 6 (2016) 39–49. [11] A. Burnett, M. Wetzler, B. Lowenberg, Therapeutic advances in acute myeloid leukemia, J. Clin. Oncol. 29 (2011) 487–494. [12] A. Mims, R.K. Stuart, Developmental therapeutics in acute myelogenous leukemia: are there any new effective cytotoxic chemotherapeutic agents out there? Curr. Hematol. Malig. Rep. 8 (2013) 156–162.

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