Synadenium umbellatum Pax. promotes cell cycle arrest and induces apoptosis in K-562 leukemia cells

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disease that shows apoptosis resistance. The introduction of imatinib mesylate has revolutionized the treatment of CML, but imatinib resistance may develop at any time and inevitably leads to disease progression. Synadenium umbellatum Pax. belongs to the Euphorbiaceae family and is popularly used in Brazil for the treatment of cancer. The cytotoxicity of Euphorbiaceae is associated with the ability of these plants and their bioactive compounds to induce apoptotic tumor cell death. Therefore, we aimed to investigate the cytotoxicity and the mechanisms of death induced by S. umbellatum extract in leukemic cells. S. umbellatum cytotoxicity was evaluated by trypan blue exclusion assay and flow cytometric analysis of the cell cycle; the mechanisms involved in K-562 cell death were investigated by light microscopy and flow cytometry. The results demonstrate that S. umbellatum is cytotoxic to leukemic cells in a concentration-dependent manner. Morphological analysis revealed that S. umbellatum treatment induced K-562 cell death by an apoptotic pathway. Furthermore, data indicate ROS overproduction, alterations in mitochondrial membrane potential, phosphatidylserine externalization and activation of caspase 9. Taken together, the results demonstrate that S. umbellatum extract arrested the cell cycle and triggered apoptosis at several levels in K-562 cells.


INTRODUCTION
Chronic myeloid leukemia (CML) is a clonal myeloproliferative disease driven by the BCR-ABL fusion protein that arises from the translocation of chromosomes 9 and 22.This oncoprotein exhibits constitutively active kinase activity that confers growth potential and apoptosis resistance to CML cells (Cortez, Kadlec, Pendergast, 1995).BCR-ABL promotes cell division and blocks apoptosis, leading to unregulated growth of hematopoietic stem cells.Usually, patients with no chemotherapeutic treatment progress after several years from a chronic phase to a blast crisis, which can rapidly lead to death (O 'Hare, Corbin, Druker, 2006).
The introduction of imatinib mesylate (Glivec/ Gleevec; Novartis Pharmaceuticals), a tyrosine kinase inhibitor, has revolutionized the treatment of CML.Imatinib is widely accepted as the standard of care for the first-line treatment of CML, due to its well-documented clinical activity, resulting in durable responses and prolonged survival.Moreover, toxicities associated with this therapy are considered low, and the main toxicities are myelosuppression, edema, and myalgia/arthralgia.On the other hand, imatinib resistance may develop at any time and inevitably leads to disease progression (Fausel, 2007).In this context, new agents are required for the treatment of CML.
Synadenium umbellatum Pax., a species of Euphorbiaceae widely found in South America, is popularly used in Brazil for the treatment of cancer.Studies performed by our group demonstrated scientific evidence for the effectiveness of S. umbellatum extract as an antitumor and antiangiogenic agent in vivo using the experimental model of Ehrlich ascitic tumor (EAT) (Nogueira et al., 2008).Recently, we also demonstrated the mechanisms of EAT cell death induced by S. umbellatum, in vitro.The cytotoxic effects found in EAT cells were mediated by activation of the intrinsic and extrinsic pathways of apoptosis.Furthermore, we determined by HPLC that the main constituents of S. umbellatum crude ethanolic extract are flavonoids and terpenes (Mota et al., 2012).
In the present work, we investigated the cytotoxic activity of S. umbellatum in K-562 and Jurkat leukemia cell lines.Since K-562 is a BCR-ABL-expressing human leukemia cell line, with a drug resistance profile, the mechanisms of death induced by S. umbellatum were investigated in this cell model.

Cell culture
K-562 and Jurkat cell lines were obtained from the American type culture collection (ATCC, Rockville, MD, USA) and maintained in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS), 100 mg/mL streptomycin, 100 U/mL penicillin G, and 2 mM L-glutamine in a humidified atmosphere of 5% CO 2 in air at 37 o C. The cells were passaged twice a week and used in the exponential growth phase.Cell viability was determined by trypan blue exclusion.

Cytotoxicity assay
The viabilities of K-562 and Jurkat cells were evalu-ated using the trypan blue exclusion method.The cells (1x10 6 cells/mL) were seeded in 96-well flat microtiter plates (Corning, USA) in RPMI 1640 medium supplemented with 10% FBS and incubated with or without eight concentrations (0.007 -1 mg/mL) of S. umbellatum extract in six replicates for 24 h.The cell suspension and a trypan blue solution (0.2% in phosphate buffered saline -PBS) were mixed 1:10 and the viability of cells was estimated using a hemacytometer (Boeco, Germany).Stained cells were scored as dead.IC 50 values (concentration that inhibited cell growth by 50%, compared to untreated controls) were determined.For all subsequent assays, a value near the IC 50 was used.

Light microscopy
The morphological alterations between K-562 cells untreated and treated with S. umbellatum were assessed by light microscopy.Untreated cells and cells treated with 0.06 mg/mL S. umbellatum extract were incubated for 24 h at 37 o C and 5% CO 2 and, after incubation, placed on glass slides by cytospin and stained with Giemsa.After staining, the slides were examined by light microscopy (DM 2000, Leica microsystems, USA) and images were taken using a 40 x objective amplified with 3 x zoom (Canon Powershot S80).

Cell cycle analysis
K-562 cells (1x10 6 /mL) were incubated with or without 0.06 mg/mL S. umbellatum extract for 24 h.After incubation, the cells were washed with ice-cold PBS-EDTA, centrifuged at 296.82 g for 10 min and fixed with 2 mL 70% ice-cold ethanol at 4 o C for 24 h.After fixation, cells were washed again, resuspended in 500 µL PBS solution containing 0.2 mg/mL RNAse and 0.05 mg/mL propidium iodide (PI), and then incubated at 4 o C for 2 h.Fluorescence was measured with a FACSCanto II flow cytometer, using PE x FL2 channels.The assay was carried out in triplicate and 10,000 events were analyzed per experiment using the BDFACSDiva software.

Measurement of intracellular ROS
K-562 cells (1x10 6 /mL) were incubated with or without 0.06 mg/mL S. umbellatum extract for 12 and 24 h.Afterwards, cells were washed with PBS, centrifuged at 296.82 g for 10 min and incubated with 500 µL DCFH-DA (10 µM) for 1 h, and fluorescence determined by flow cytometry using the FL1 channel.

Assessment of mitochondrial membrane potential (Δψm)
K-562 Cells (1x10 6 /mL), untreated and treated with 0.06 mg/mL S. umbellatum extract for 24 h, were washed with PBS-EDTA, centrifuged at 296.82 g for 10 min, and incubated with 1 µg/mL rhodamine 123 at 37 o C for 1 h, and the fluorescence determined by flow cytometry, using the FL1 channel.
Annexin V-FITC/PI double-staining for phosphatidylserine externalization analysis K-562 Cells (1x10 6 /mL), untreated and treated with 0.06 mg/mL S. umbellatum extract for 24 h, were washed with PBS-BSA and centrifuged at 296.82 g for 10 min.The cells were then incubated for 15 min with 100 µL binding buffer (10 mmol/L HEPES/NaOH, 140 mmol/L, NaCl 2.5 mmol/L CaCl 2 ) with 1 µL annexin V-FITC and 10 µL PI solution, and analyzed by flow cytometry.Fluorescence of annexin V-FITC and PI was measured using the FL1 and FL2 channels, respectively.

Caspase activity
The activity of caspase 9 was measured using a colorimetric protease assay kit, following the manufacturer's instructions.After 24 h of incubation with or without 0.015 mg/mL S. umbellatum extract, K-562 cells (1x10 6 /mL) were washed with ice-cold PBS, centrifuged at 296.82 g for 10 min and incubated on ice for 20 min with 25 µL of cell-lysis buffer (provided in the kit).The cell lysate was collected by centrifugation at 29,682.9 g at 4 o C for 15 min.The assay was performed in 96 well plates containing 50 µL cell lysate, 50 µL 2x reaction buffer, 5 µL caspase colorimetric substrate and 10 µL DTT.Duplicate wells were prepared containing blank (no cell extract), negative control (extract from untreated cells), positive controls (no cell lysate and no substrate) and S. umbellatum-treated cells.The plates were incubated at 37 o C for 2 h, and absorbance was read with a microplate reader (Stat Fax 2100, Awareness Technology, Germany) at 405 nm.Caspase 9 activity was determined by measuring p-nitroanaline (pNA) released during the cleavage of LEHD-pNA substrate.

Statistical analysis
Statistical analysis was performed using Graph Pad Prism 5 (version 5.00 for Windows 98, Graph Pad Software, San Diego, CA) software.The evaluation of cytotox-icity was carried out in three separate experiments.Results were converted to percentages of the control and IC 50 values were obtained by non-linear regression analysis.Results are expressed as the mean ± SD of six replicates.All other assays were analysed by Student's t-test.Results were considered statistically significant when P < 0.05.

Cytotoxic effects of S. umbellatum in leukemic cells
S. umbellatum cytotoxicity was evaluated in the leukemic cell lines, K-562 and Jurkat, by the trypan blue exclusion method, after 24 h of exposure to different concentrations (0.007 -1 mg/mL).As shown in Figure 1, both cell lines studied were highly sensitive to S. umbellatum extract, in a concentration-dependent manner.Differences in the susceptibility of the two leukemic cell lines to S. umbellatum were observed in the IC 50 values (IC 50 Jurkat = 0.041 mg/mL and IC 50 K-562 = 0.073 mg/mL).Additionally, the effects of S. umbellatum on normal lymphocytes from primary culture were studied; these experiments revealed a high cytotoxic activity (data not shown).Based on these findings, the K-562 cell line was chosen for subsequent mechanistic studies.

K-562 cell morphology after S. umbellatum treatment
Morphological analysis of K-562 cells stained with Giemsa revealed alterations after treatment with S. umbellatum extract.As can be seen in Figure 2A, control cells exhibited nuclei with dispersed chromatin and organized plasma membrane.In contrast, cells treated with S. umbellatum for 24 h (Figure 2B) showed membrane blebbing and chromatin condensation.These microscopic changes, caused by S. umbellatum treatment, suggested an activation of apoptosis.

Effects of S. umbellatum in cell cycle distribution
Flow cytometric analysis revealed changes in cell cycle progression between untreated and treated K-562 cells (Figures 3A, B). S. umbellatum extract (0.06 mg/mL) increased the number of cells in G 0 /G 1 phases by 19.7 ± 7.9% (P <0.05), followed by a reduction of 74.0 ± 2.4% in the S phase (P <0.001) and an increase of 51.2 ± 5.7% FIGURE 1 -Cytotoxic effects of Synadenium umbellatum extract on K-562 and Jurkat cells after 24 h of treatment (0.007-1 mg/mL).The cytotoxic activity was determined by trypan blue exclusion assay.Experiments were performed in sextuplicate.Each bar presents the mean ± SD of three independent experiments.The IC 50 was 0.041 mg/mL and 0.073 mg/mL in Jurkat and K-562 cells, respectively.in G 2 (P <0.01).These results suggest that S. umbellatum arrested K-562 cells in the G phases.

S. umbellatum-induced oxidative stress
In addition to cell cycle arrest and morphological changes induced by S. umbellatum treatment, a timedependent increase in intracellular ROS was found after 12 and 24 h of incubation with 0.06 mg/mL S. umbellatum extract.As shown in Figure 4, the generation of ROS dramatically increased with incubation time.Treatment with S. umbellatum for 12 h increased intracellular ROS levels by 36.2 ± 13.7%, reaching 111.4% at 24 h (P <0.05).

S. umbellatum-induced alterations in mitochondrial membrane potential
The most common alteration in Δψm after oxidative stress is cellular depolarization, but an early hyperpolarization can occur.As shown in Figure 5, treatment with 0.06 mg/mL S. umbellatum extract for 24 h increased Δψm in K-562 cells by 117.6 ± 1.9%.exposure or necrosis by rupture of plasma membrane in S. umbellatum-mediated cell death, K-562 cells were labeled with annexin V-FITC (A) and PI.As shown in Figure 6, after 24 h of treatment with 0.06 mg/mL S. umbellatum extract, the number of live cells (A-/PI-) decreased by 16.0 ± 0.8% (P < 0.01), while the number of apoptotic cells (A+/PI+) increased by 286.1 ± 30.8% (P <0.001).

Activation of caspase 9 by S. umbellatum
To investigate whether caspases were activated during S. umbellatum-induced cell death, a colorimetric assay was performed.As shown in Figure 7, after 24 h of exposure to 0.015 mg/mL S. umbellatum, there was a significant increase in caspase 9 activity (15.9± 0.4%), as detected by cleavage of LEHD-pNA substrate.Thus, induction of caspase activity may be involved in S. umbellatuminduced apoptosis.

DISCUSSION AND CONCLUSION
In the present study, a BCR-ABL-expressing human leukemic cell line, K562, was used as a model system of resistance to apoptosis induced by conventional chemo-   therapeutics to investigate the mechanisms of the antitumor activity found in S. umbellatum.The results presented herein showed that S. umbellatum was cytotoxic to human leukemia cells.The mechanistic investigation using K-562 showed that S. umbellatum promoted cell growth arrest and induced death via apoptosis by increasing ROS production, alterations in mitochondrial functions, PS externalization and activation of caspases.
Plant families possessing bioactive compounds, particularly terpenes, have shown antitumor properties (Mota et al., 2012).The cytotoxic activity of the Euphorbiaceae has been associated with induction of apoptosis in tumor cells.The extract of Croton malambo H. Karst.was reported to induce tumor cell apoptosis (Morales et al., 2005).In this study, we demonstrated morphological and biochemical changes in K-562 cells after S. umbellatum treatment, corroborating the reported induction of apoptosis by Euphorbia cheiradenia Boiss.& Hohen. in K-562 cells (Amirghofran et al., 2006).
For the apoptotic process, a disruption of mitochondrial membrane integrity is decisive.An increase in ROS production and consequent loss of Δψm are reported as typical phenomena in the process of mitochondrialmediated apoptosis (Fang et al., 2007).Mitochondrial membrane depolarization is a universal event in apoptosis, but it has been suggested that the inner membrane of mitochondria may undergo a transient hyperpolarization during apoptosis (Liu et al., 2005;Matsuyama, Reed, 2000;Poot, Pierce, Kavanagh, 2002).Our data revealed an increase in ROS levels, followed by an early increase in Δψm (hyperpolarization) after S. umbellatum treatment, suggesting that apoptosis, induced by S. umbellatum, is mitochondriadependent.S. umbellatum-induced apoptosis in K-562

FIGURE 2 -
FIGURE 2 -Morphological changes of Synadenium umbellatum-induced apoptosis in K-562 cells.Cells were treated with 0.06 mg/mL Synadenium umbellatum extract for 24 h and images were taken using a 40 x objective amplified with 3 x zoom.(A) Control cells stained with Giemsa, showing nuclei with dispersed chromatin and organized plasma membrane (arrow).(B) Synadenium umbellatum-treated cells displaying membrane blebbing and chromatin condensation (arrows).Bars: 20 µm.

FIGURE 3 -
FIGURE 3 -Flow cytometry analysis of K-562 cell cycle with and without treatment with Synadenium umbellatum extract (0.06 mg/mL) for 24 h.(A) Histograms showing cells untreated and treated with 0.06 mg/mL S. umbellatum for 24 h.(B) Percentage of K-562 cells in G 1, S and G 2 phases with and without S. umbellatum treatment.Each bar represents the mean ± SD of two independent experiments (*P < 0.05; **P < 0.01;***P < 0.001).

FIGURE 7 -
FIGURE 7 -Activation of caspase 9 during Synadenium umbellatum-induced apoptosis.K-562 cells were treated with 0.015 mg/mL S. umbellatum extract for 24 h.Kinetics of the enzymatic activity of caspase 9 was evaluated.Each bar represents the mean ± SD of two independent experiments (* P < 0.05 compared to control).