Apoptotic Events Induced by Maleimides on Human Acute Leukemia

Article pubs.acs.org/crt

Apoptotic Events Induced by Maleimides on Human Acute Leukemia Cell Lines Karina Elisa Machado,† Kely Navakoski de Oliveira,§ Haíra Maria Slobodianuk Andreossi,‡ Lorena dos Santos Bubniak,‡ Ana Carolina Rabello de Moraes,† Pâmela Cristina Gaspar,† Evilásio da Silva Andrade,§ Ricardo José Nunes,§ and Maria Cláudia Santos-Silva*,†,‡ †

Pós-Graduaçaõ em Farmácia Universidade Federal de Santa Catarina, UFSC, Campus Trindade, CEP, 88040-900 Florianópolis, Santa Catarina, Brazil ‡ Departamento de Análises Clínicas, Universidade Federal de Santa Catarina, UFSC, Campus Trindade, CEP, 88040-900 Florianópolis, Santa Catarina, Brazil § Departamento de Química, Universidade Federal de Santa Catarina, UFSC, CEP, 88040-900 Florianópolis, Santa Catarina, Brazil S Supporting Information *

ABSTRACT: Cyclic imides are known for their antitumor activity, especially the naphthalimide derivatives, such as Mitonafide and Amonafide. Recently, we have demonstrated the cytotoxic effect of a series of naphthalimide derivatives against B16F10 melanoma cells. On the basis of this fact, we have developed a study starting from the synthesis of different cyclic imides and the evaluation of their cytotoxic properties on human acute leukemia cells (K562 and Jurkat). Initially, a screening test was conducted to select the compound with the best cytotoxic effect, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. After this selection, structural modifications were performed in the most active compound to obtain five more derivatives. All compounds presented a good cytotoxic effect. The results of cell cycle analysis, fluorescence microscopy, and Annexin V-FITC assay confirmed that the cells observed in the sub-G0/G1 phase were undergoing apoptosis. From this set of results, cyclic imides 8, 10, and 12 were selected for the evaluation of the mechanisms involved in the apoptotic process. The results demonstrate the involvement of the intrinsic pathway of apoptosis, evidenced by the reduction in mitochondrial potential, an increase in the level of AIF protein expression, a decreased level of expression of anti-apoptotic Bcl-2 protein, and an increased level of expression of pro-apoptotic protein Bax in both K562 and Jurkat cells treated with cyclic imides (8, 10, and 12). Furthermore, cyclic imides 8 and 10 caused an increase in the level of Fas expression in Jurkat cells, indicating the additional involvement of the extrinsic apoptosis pathway. The compounds (8, 10, and 12) also caused a decreased level of expression of anti-apoptotic protein survivin. The biological effects observed with these cyclic imide derivatives in this study suggest promising applications against acute leukemia.

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INTRODUCTION

known to display a wide range of side effects, caused by the low selectivity and high toxicity of the drugs employed.2,3 From this panorama arises an interest in developing new drugs that are more effective, more selective, and less toxic. One of the classes of interest in the search for new chemotherapeutic agents is the group of cyclic imides. These compounds

Leukemias constitute a group of malignancies characterized by uncontrolled hematopoietic cell proliferation in bone marrow and lymphoid tissues. These cells can eventually reach the peripheral circulation and infiltrate other organ systems.1 Various forms of therapy have been used to treat leukemia, such as radiotherapy, chemotherapy, immunotherapy, and transplant of bone marrow, and among them, chemotherapy is the most frequently used. The existing chemotherapeutic treatments are © 2013 American Chemical Society

Received: August 8, 2013 Published: November 25, 2013 1904

dx.doi.org/10.1021/tx400284r | Chem. Res. Toxicol. 2013, 26, 1904−1916

Chemical Research in Toxicology

Article

Figure 2. Cytoxicity of cyclic imides 1−8 on human acute myeloid leukemia cells (K562). Compounds 1−8 (100 μM) were incubated with cells (5 × 104 cells/mL) for 24 h. Cell viability was monitored via the MTT assay. The optical density of control groups was considered as 100% of cell viability. Cell viability was checked at the beginning of the experiment by the Trypan Blue exclusion method. Results are means ± SEM of at least three independent experiments. Asterisks indicate a significant difference compared to the control group (p < 0.001) using ANOVA followed by Bonferroni’s t test.

Figure 1. Molecular structures of compounds studied.

can exert biological effects that have attracted the attention of researchers for many years. Among the activities assigned to these compounds are the following: antinociceptive,4,5 antiinflammatory,6 antimicrobial,5 and antitumor.6−9 These activities seem to be related to the size and character of the substituent groups in the imide ring, which may change the steric properties of the compounds, potentially altering their activity.10 Although cyclic imides have been the focus of scientific researches for many years, the search for bioactive compounds is still ongoing. In the past four years, new cyclic imides and derivatives have been identified because of their antitumor properties.6,11−18 In a recent work, we investigated the cytotoxic effect of naphthalimides such as 4 and 5 (Figure 1), in which the compound 2-benzyl-1H-benzo[de]isoquinoline-1,3(2H)-dione (5) had an IC50 of 77.75 μM for B16F10 melanoma cells.17 Other researchers have also demonstrated the antitumor activity of this class of compounds, an example being Mitonafide, Amonafide, and their derivatives, which showed significant antitumor activity on various types of tumors, especially leukemias, melanomas, and breast tumors.15,19 An additional characteristic of cyclic imides that makes them a promising class in the search for new lead compounds is their relative ease of preparation, allowing the synthesis of various types of derivatives with planned structural modifications. In view of these data and on our previous results,17 this study proposes the synthesis of different cyclic imide derivatives and the evaluation of their cytotoxic properties and the mechanism of apoptosis involved.

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Cell Cycle Analysis. To assess cell cycle arrest, a PI/RNase solution kit (Immunostep, Salamanca, Spain) was employed. K562 cells (5 × 105 cells/mL) and Jurkat cells (10 × 105 cells/mL) were incubated with vehicle or cyclic imides 8−13, at their respective IC50 values. After being incubated for 24 h, cells were harvested, and cell cycle analysis was conducted according to the kit protocol. Briefly, cells were washed with PBS, fixed with 70% ethanol, washed with PBS supplemented with 2% bovine albumin, and stained with 500 μL of a PI/RNase solution. Analysis was performed by flow cytometry (FACSCantoII, Becton Dickinson Immunocytometry Systems). The data were analyzed with WinMDI 2.8. Analysis of the Apoptotic Effects with Ethidium Bromide and Acridine Orange.35 For the assessment of apoptotic death with ethidium bromide and acridine orange, K562 cells (5 × 105 cells/mL) and Jurkat cells (10 × 105 cells/mL) were incubated with IC50 values of cyclic imides 8−13. After 24 h, the coverslips from the bottom of the plate were removed, washed with PBS, and covered with a solution of ethidium bromide and acridine orange (1:1) at a concentration of 1%. Cells were analyzed under a fluorescence microscope (Olympus BX41). The data were analyzed with NIH ImageJ 1.40 (National Institutes of Health, Bethesda, MD). Analysis of the Apoptotic Effects with Annexin.36 For the assessment of apoptotic death, an Annexin V-FITC apoptosis detection kit was used by following the manufacturer’s instructions. K562 cells (5 × 105 cells/mL) and Jurkat cells (10 × 105 cells/mL) were incubated with cyclic imides 8−13, at their respective IC50 values. After being incubated for 16 h, cells were harvested, washed with PBS, washed with annexin buffer (1:10), and stained with an Annexin V-FITC solution. After incubation, 300 μL of annexin buffer was added and the fluorescence was determined with a flow cytometer. Analyses were performed with a flow cytometer (FACSCanto, Becton Dickinson Immunocytometry Systems). The data were analyzed using WinMDI 2.8. Analysis of Mitochondrial Membrane Potential (MMP). The evaluation of MMP was performed using a MitoView 633 kit (Biotium, Inc., Hayward, CA). K562 cells (5 × 105 cells/mL) and Jurkat cells (10 × 105 cells/mL) were incubated with vehicle or cyclic imides 8, 10, and 12, at their respective IC50 values, for 12 h. Cells were labeled as described by the manufacturer. Briefly, cells treated with cyclic imides were collected by centrifugation and washed twice with PBS. Cells were resuspended in 100 μL of a MitoView 633 solution (1:10000). Following incubation in the dark for 30 min, cells were washed and resuspended with 1 mL of PBS. Analysis was performed by flow

EXPERIMENTAL PROCEDURES

Compounds 1−13 (Figure 1) were synthesized by following reaction procedures reported in the literature.5,12,20−33 The physicochemical data of all synthetized compounds (1−13) are reported in the Supporting Information. Cell Culture and Viability Assay (MTT assay).34 Leukemia cell lines [K562 and Jurkat (ATCC, Manassas, VA)] maintained in RPMI 1640 were cultured in RPMI 1640 (GIBCO, São Paulo, Brazil) supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin, and 10 mM HEPES (pH 7.4) at 37 °C in a 5% CO2 humidified atmosphere in plastic culture flasks. Test compounds 1−14 were added to cells in a maximal volume of 20 μL. For compounds that are soluble in DMSO, the same volume of solvent was added to control wells. Treatments with the imides at the indicated concentrations were conducted for 24−72 h. Cell viability was assessed by using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma Chemical Co., St. Louis, MO)] assay. 1905



Article

Figure 3. Cytotoxicity of cyclic imides 8−13 on human acute myeloid leukemia cells (K562). Cyclic imides 8 (A), 9 (B), 10 (C), 11 (D), 12 (E), and 13 (F) were incubated with K562 cells (5 × 104, 2.5 × 104, and 1.25 × 104 cells/mL) at different concentrations (0.1−100 μM) and for different periods of time (24, 48, and 72 h). Cell viability was monitored via the MTT assay. Cell viability was checked at the beginning of the experiment by the Trypan Blue exclusion method. IC50 values were calculated for each incubation time (G). Results are means ± SEM of at least three independent experiments. Asterisks indicate statistically significant differences compared to the control group (**p < 0.01, and ***p < 0.001) using ANOVA followed by Bonferroni’s t test. cytometry (FACSCantoII, Becton Dickinson Immunocytometry Systems). A total of 10000 events were acquired, and duplicates were removed using the characteristics of FSC high and SSC area. The data were analyzed using Infinicyt version 1.7.0 (Cytognos S.L., Salamanca, Spain). Analysis of the Expression of the AIF, Bax, Bcl-2, Caspase 3, and FasR Proteins with a Flow Cytometry. For the analysis of AIF, Bax, Bcl-2, FasR, and active caspase 3 proteins, K562 cells (5 × 105 cells/mL) and Jurkat cells (10 × 105 cells/mL) were incubated with vehicle or cyclic imides 8, 10, and 12, at their respective IC50 values, for 3, 6, 9, 12, or 24 h. At the end of the incubation, cells were fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences, San Diego, CA), with the exception of cells used in Fas protein analysis. Subsequently, cells were incubated in the dark at room temperature with anti-AIF-FITC, anti-Bax-PerCP, anti-Bcl-2-FITC, anti-caspase 3-PE, and anti-FasR-PE antibodies for the detection of AIF, Bax, Bcl-2, caspase 3, and Fas, respectively. After being incubated, cells were washed with

Perm/Wash Buffer and resuspended in PBS for cytometric analysis. Analysis was performed by flow cytometry (FACSCantoII, Becton Dickinson Immunocytometry Systems). The data were analyzed using Infinicyt version 1.7.0 (Cytognos S.L.). Analysis of the Expression of Survivin Protein. Immunocytochemistry assays for the evaluation of survivin expression were performed on cytospins from cell suspensions using the peroxidaselabeled streptavidin−biotin method. K562 cells (5 × 105 cells/mL) and Jurkat cells (10 × 105 cells/mL) were incubated with vehicle or cyclic imides 8, 10, and 12, at their respective IC50 values, for 24 h. At the end of the incubation, the cells were centrifuged in a cytocentrifuge (Cytopro − Wescor Inc. An Elitech Company) on pretreated slides with a silane solution. Cytospins from cell suspensions were fixed in 95% ethanol for 1 h at room temperature and incubated for 20 min with a 3% hydrogen peroxide solution to prevent nonspecific false positive reactions. Subsequently, the cells were permeabilized with Triton X-100-PBS 0.2 and incubated for 12 h at 8 °C with primary 1906



Article

Figure 4. Cytotoxicity of cyclic imides 8−13 on human acute lymphocytic leukemia cells (Jurkat). Cyclic imides 8 (A), 9 (B), 10 (C), 11 (D), 12 (E), and 13 (F) were incubated with Jurkat cells (10 × 105, 5 × 105, and 2.5 × 105 cells/mL) at different concentrations (0.1−100 μM) and for different periods of time (24, 48, and 72 h). Cell viability was monitored via the MTT assay. Cell viability was checked at the beginning of the experiment by the Trypan Blue exclusion method. IC50 values were calculated for each incubation time (G). Results are means ± SEM of at least three independent experiments. Asterisks indicate a statistically significant difference compared to the control group (*p < 0.5, **p < 0.01, and ***p < 0.001) using ANOVA followed by Bonferroni’s t test. Statistical Analysis. Results are presented as means ± the standard error of the mean (SEM). Statistical significance between groups was determined by analysis of variance (ANOVA) followed by Bonferroni’s multiple-comparison test. P values of

Apoptotic Events Induced by Maleimides on Human Acute Leukemia

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