Chemico-Biological Interactions 222 (2014) 112–125

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Chemico-Biological Interactions

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Morphological and biochemical alterations activated by antitumorclerodane diterpenes

http://dx.doi.org/10.1016/j.cbi.2014.10.0150009-2797/� 2014 Elsevier Ireland Ltd. All rights reserved.

⇑ Corresponding author at: Department of Biophysics and Physiology, CampusMinistro Petrônio Portella, Federal University of Piauí, Teresina, Brazil. Tel.: +55 8632155871.

E-mail addresses: pmpf@ufpi.edu.br, pmifepe@yahoo.com.br (P.M.P. Ferreira).

Paulo Michel Pinheiro Ferreira a,b,⇑, Gardenia Carmen Gadelha Militão c, Daisy Jereissati Barbosa Lima e,Nagilla Daniela de Jesus Costa d, Kátia da Conceição Machado b, André Gonzaga dos Santos f,Alberto José Cavalheiro g, Vanderlan da Silva Bolzani g, Dulce Helena Siqueira Silva g, Cláudia Pessoa e

a Department of Biophysics and Physiology, Campus Ministro Petrônio Portella, Federal University of Piauí, Teresina, Brazilb Postgraduate Program in Pharmaceutical Sciences, Federal University of Piauí, Teresina, Brazilc Department of Physiology and Pharmacology, Federal University of Pernambuco, Recife, Brazild Department of Biological Sciences, Campus Senador Helvídio Nunes de Barros, Federal University of Piauí, Picos, Brazile Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Ceará, Fortaleza, Brazilf Faculty of Pharmaceutical Sciences, State University of São Paulo Júlio de Mesquita Filho, Araraquara, Brazilg Chemistry Institute, State University of São Paulo Júlio de Mesquita Filho, Araraquara, Brazil

a r t i c l e i n f o

Article history:Received 1 September 2014Received in revised form 8 October 2014Accepted 15 October 2014Available online 27 October 2014

Keywords:Casearia sylvestrisCytotoxicityAntiproliferative actionApoptosisHuman cellsMurine cells

a b s t r a c t

Casearia sylvestris Swartz (Salicaceae) is a plant commonly widespread in the Americas. It has oxygenatedtricyclic bioactive clerodane diterpenes with antimicrobial, antiulcer, larvicidal, chemopreventive, anti-inflammatory, antioxidant and antiproliferative properties. Due to this requirement for the developingof new anticancer drugs, it was initially evaluated the cytotoxic activity of a fraction with Casearins(FC) and its clerodane diterpenes Casearin B (Cas B), D (Cas D), X (Cas X) and Caseargrewiin F (Cas F) iso-lated from C. sylvestris leaves against 7 tumor cell lines, Sarcoma 180 cells (S180) and on normal periph-eral blood mononuclear cells (PBMC). All substances tested showed cytotoxic potential. Cas F and X werethe most active compounds. Cell death analyzes with Cas F (0.5 and 1 lM) and Cas X (0.7 and 1.5 lM)using the HL-60 leukemia line as experimental model showed DNA synthesis and membrane integrityreduction, DNA fragmentation and mitochondrial depolarization, specially after 24 h exposure, cell cyclearrest in G0/G1 phase caused by Cas X, activation of the initiator -8/-9 and effector -3/-7 caspases andphosphatidylserine externalization, all biochemical features of apoptosis corroborated by chromatiniccondensation, karyorrhexis, cytoplasmic vacuolation and rarefaction and cellular shrinkage, morpholog-ical findings specially observed after 12 and 24 h of incubation. Therefore, Cas X and F were the mostfunctional molecules with more pronounced lethal and discriminating effects on tumor cells and antipro-liferative action predominantly mediated by apoptosis, highlighting clerodane dipertenes as promisinglead antineoplastic compounds.

� 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Between 1981 and 2010, out of 1073 new chemical entities(NCE) approved as novel medicines by the Food and Drug Admin-istration (FDA), only 36% can be classified as truly synthetic; 64%are unmodified NCEs, derived or synthetic molecules that mimicor are based on natural compounds. Then, instead of the interestin molecular modeling, combinatorial chemistry and other

chemical synthesis techniques, natural products, and particularly,medicinal plants, remain an important source of new therapeuticagents against infectious diseases (bacteria or fungi), insects, can-cer, dyslipidemia and immunomodulation [1–6].

Casearia sylvestris Swartz (Salicaceae), popularly known as‘‘guaçatonga’’, ‘‘café silvestre’’, erva-de-lagarto’’, ‘‘língua-de-tiú’’,‘‘cafezinho-do-mato’’ and ‘‘corta-lengua’’, is a plant distributed intropical and temperate regions around the world and commonlywidespread in the Americas. In Brazil, it is present from Amazonas(Tapajós river region) to Rio Grande do Sul states [7,8].

Different parts of C. sylvestris have shown antimicrobial [9–11],antiulcer [12–14], larvicidal [15], chemopreventive [16], anti-inflammatory and antioxidant properties [14,17,18]. Most of theseproperties are attributed to the different secondary metabolites

P.M.P. Ferreira et al. / Chemico-Biological Interactions 222 (2014) 112–125 113

isolated from C. sylvestris belonging to the casearin, caseargrewiinand casearvestrin classes, oxygenated tricyclic bioactive clerodanediterpenes which have presented also excellent cytotoxic potential[4,10,19,20–23]. Thus, we firstly analyzed the in vitro antiprolifer-ative action of a fraction with casearins (FC) and its isolated com-pounds Casearin B (Cas B), Casearin D (Cas D), Casearin X (Cas X)and Caseargrewiin F (Cas F). Secondly, it was studied the mecha-nism involved in the antiproliferative activity using HL-60 leuke-mia line as experimental model.

2. Methods

2.1. Chemicals, isolation of the compounds and structure identification

Leaves of C. sylvestris were collected at the Parque Estadual Car-los Botelho (São Miguel Arcanjo, São Paulo, Brazil). The plant wasidentified by Dr. Ines Cordeiro (Instituto Botânico do Estado deSão Paulo, São Paulo, Brazil). Voucher specimens (numbersAGS04, AGS05, AGS06, AGS13 and AGS19) were deposited at theHerbarium Maria Eneida P. Kaufmann (Instituto Botânico do Esta-do de São Paulo, São Paulo, Brazil). Dried and powdered leaves of C.sylvestris were extracted with ethanol in a stainless steel extractorwith solvent reflux for ca. 24 h at 40 �C. The crude extract was con-centrated under reduced pressure (rotary evaporator) and dried indesiccators over silica gel under reduced pressure to yield a dryresidue. The structures of Cas B, D, X and F were determined byspectrometric data (nuclear magnetic resonance, ultraviolet, infra-red and mass spectrometry) and compared to the spectral reportavailable in the literature [23,24] (Fig. 1).

Fetal calf serum was purchased from Cultilab (Campinas, SP),RPMI 1640 medium, trypsin–EDTA, penicillin and streptomycinwere purchased from GIBCO� (Invitrogen, Carlsbad, CA, USA). Pro-pidium iodide (PI), acridine orange (AO), ethidium bromide (EB)and Rhodamine 123 (Rho-123) were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). Doxorubicin (Doxolem�) was pur-chased from Zodiac Produtos Farmacêuticos S/A, Brazil.

R1H

OR2

1

3 5

181

2

4

16

R1 RCasearin B CH3O CHCasearin D OH n-CCasearin X n-C3H7CO2 n-CCaseargrewiin F n-C3H7CO2 CH

Fig. 1. Chemical structures of the molecules

2.2. Animals

Adult female Swiss mice (Mus musculus Linnaeus, 1758) wereobtained from the animal facilities of the Universidade Federaldo Piauí (UFPI), Teresina, Brazil. They were kept in well-ventilatedcages under standard conditions of light (12 h with alternate dayand night cycles) and temperature (27 ± 2 �C) and were housedwith free access to commercial rodent stock diet (Nutrilabor, Cam-pinas, Brazil). All procedures were approved by the Committee onAnimal Research at the UFPI (Process no 102/2011) and followedthe Brazilian (Colégio Brasileiro de Experimentação Animal – COBEA)and International Standards on the care and use of experimentalanimals (Directive 2010/63/EU of the European Parliament and ofthe Council).

2.3. In vitro antiproliferative assays

The cytotoxic potential of the FC, Cas B, D, X and F was assessedafter 72 h exposure using leukemia (HL-60), breast (MDA-MB/231,Hs578-T, MX-1), prostate (PC-3, DU-145) and skin (B16/F-10)tumor lines, Sarcoma 180 cells (S180) and normal peripheral bloodmononuclear cells (PBMC). Cell culture was performed in RPMI1640 medium supplemented with 20% fetal bovine serum, 2 mMglutamine, 100 U/mL penicillin and 100 lg/mL streptomycin, at37 �C with 5% CO2. Quantification of cell proliferation was spectro-photometrically determined using a multiplate reader (DTX 880Multimode Detector, Beckman Coulter). Control groups (negativeand positive) received the same amount of DMSO (0.1%). Doxoru-bicin (Dox, 0.01–8.6 lM) was used as positive control.

2.3.1. Antiproliferative study on tumor cells evaluated by MTT assayThe cytotoxicity against HL-60, MDA-MB/231, Hs578-T, MX-1,

PC-3, DU-145, and B16/F-10 cancer cells was determined by MTTassay [25], which analyzes the ability of living cells to reduce theyellow dye 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazo-lium bromide (MTT) to a purple formazan product. Briefly, cells

R3

R4

O

O

7

9

9

17

20

11

13

15

10

6

8

12

14

2 R3 R4

3CO2 CH3CO2 n-C3H7CO2

3H7CO2 OH n-C3H7CO2

3H7CO2 OH H 3CO2 OH H

isolated from Casearia sylvestris leaves.

114 P.M.P. Ferreira et al. / Chemico-Biological Interactions 222 (2014) 112–125

were plated in 96-well plates (0.3–0.7 � 105 cells/well) and incu-bated to allow cell adhesion. Twenty-four hours later, FC and diter-penes were added to each well [(0.04–25 lg/mL) and (0.01–10 lM), respectively]. After 72 h of incubation, the supernatantwas replaced by fresh medium containing 10% MTT; the formazanproduct was dissolved in DMSO and the absorbance was measuredat 595 nm wavelength.

2.3.2. Antiproliferative study on Sarcoma 180 cells evaluated byAlamar Blue assay

Ascite-bearing mice between 7 and 9 days postinoculation weresacrificed by cervical dislocation and a suspension of S180 cellswas harvested from the intraperitoneal cavity under aseptic condi-tions. The suspension was centrifuged at 500�g for 5 min to obtaina cell pellet and washed three times with RPMI medium. Cell con-centration was adjusted to 0.5 � 106 cells/mL in supplementedRPMI 1640 medium, plated in a 96-well plate and incubated withincreasing concentrations of the FC and compounds [(0.04–25 lg/mL) and (0.01–10 lM), respectively]. Cell proliferation was deter-mined by the Alamar Blue assay after 72 h [26]. Eight hours befor-e the late incubation, 10 lL of stock solution (0.312 mg/mL) ofAlamar Blue™ were added to each well. The absorbance was mea-sured at 570 nm and 595 nm and the drug effect was quantified asthe control percentage.

2.3.3. Antiproliferative study on peripheral blood mononuclear cells byAlamar Blue assay

Heparinized human blood (from healthy, non-smoker donorswho had not taken any drug for at least 15 days prior to sampling,aged between 18 and 35 years-old) was collected and PBMC wereisolated by a standard method of density-gradient centrifugationover Ficoll-Hypaque. PBMC were washed and resuspended(3 � 105 cells/mL) in supplemented RPMI 1640 medium and phy-tohemagglutinin (4%). Then, PBMC were plated in 96-well plates(3 � 105 cells/well). After 24 h, FC and diterpenes were added toeach well [(0.04–25 lg/mL) and (0.01–10 lM), respectively] andthe cells were incubated during 72 h. Twenty-four hours beforethe late incubation, 10 lL of stock solution (0.312 mg/mL) of Ala-mar Blue™ were added to each well [26]. The absorbance wasmeasured as described above. All studies were performed in accor-dance with Brazilian research guidelines (Law 466/2012, NationalCouncil of Health, Brazil) and with the Declaration of Helsinki.

2.4. Assessment of mechanisms involved in the antiproliferativeactivity

Since HL-60 cell line was the most sensitive cell line to the sub-stances, it was selected to evaluate their underlying mechanismrelated to the cytotoxic effects. The substances were added to24-well tissue culture plates with HL-60 cells (3 � 105 cells/mL)to obtain final concentrations of 1 and 2 lM (Cas B), 2 and 4 lM(Cas D), 0.7 and 1.5 lM (Cas X) and 0.5 and 1 lM (Cas F). Theseconcentrations were selected based on IC50 values of each com-pound with HL-60 cells after 24 h exposure and corresponds tothe IC50/2 and IC50, respectively. Doxorubicin (0.6 lM) was usedas positive control (Dox).

2.4.1. DNA synthesis immunocytochemistry detectionLeukemia cells were plated (2 mL/well) and incubated with the

FC (0.8 lg/mL) and compounds (Cas B, 2 lM; Cas D, 4 lM; Cas F,1.0 lM and Cas X 1.5 lM). After 21 h of incubation, 20 lL of 5-bromo-20-deoxyuridine (BrdU 10 mM) were added and incubatedfor additional 3 h at 37 �C. To determine the amount of BrdU incor-porated into the DNA, cells were harvested, transferred to cytospinslides, and allowed to dry for 2 h at room temperature (25 �C). Cellsthat incorporated BrdU were labeled using direct peroxidase

immunocytochemistry by the chromogen diaminobenzidine(DAB) staining [27]. Slides were counterstained with hematoxylinand coverslipped. The determination of BrdU positivity was per-formed light microscopy (Olympus, Tokyo, Japan). Two hundredcells were counted per sample to determine the percentage ofBrdU-positive cells.

2.4.2. Biological analyzes by flow cytometryAll flow cytometry analyzes were performed in a Guava Easy-

Cyte Mine™ using Guava Express Plus CytoSoft 4.1 software(Guava Technologies Inc. Industrial Blvd. Hayward, CA, USA). Fivethousand events were evaluated per experiment and cell debriswas omitted from the analysis.

2.4.2.1. Reactive oxygen species. Accumulation of intracellular reac-tive oxygen species (ROS) was evaluated using 20,70-dichlorodihy-drofluorescein diacetate (H2-DCF-DA), which is converted to thehighly fluorescent dichlorofluorescein (DCF) in the presence ofintracellular ROS. After the incubation with the substances (FC,Cas B, D, F and X) (1 and 3 h), cells were incubated with H2-DCF-DA 20 lM during 30 min at 37 �C. Then, cells were harvested,washed, resuspended in PBS and immediately analyzed via flowcytometry [28]. b-Lapachone (2 lM) was used as a positive control.

2.4.2.2. Membrane integrity. Cell membrane integrity was evalu-ated by the exclusion of PI after 6, 12 and 24 h of incubation.Briefly, 100 lL of treated and untreated cells were incubated withPI (50 lg/mL). The cells were then incubated for 5 min at 37 �C.Fluorescence was measured and cell morphology, granularity andmembrane integrity were determined [29].

2.4.2.3. Cell cycle and DNA fragmentation. Cell cycle distribution andDNA fragmentation analysis were evaluated after 6, 12 and 24 h ofincubation by the incorporation of PI (50 lg/mL). Briefly, 24 h-trea-ted and untreated cells were incubated at 37 �C for 30 min in thedark, in a lysis solution containing 0.1% citrate, 0.1% Triton X-100and 50 lg/mL PI and fluorescence was measured afterwards [30].Data were analyzed by ModFit LT for Win32 version 3.1.

2.4.2.4. Mitochondrial transmembrane potential. It was determinedby rhodamine 123, a cell-permeable, cationic, fluorescent dye thatis readily sequestered by mitochondria without inducing cytotoxiceffects. After 6, 12 and 24 h of treatment with the substances, cellswere washed with PBS and incubated with rhodamine 123 at 37 �Cfor 15 min in the dark. Cells were incubated again in PBS at 37 �Cfor 30 min in the dark, and fluorescence was measured [30].

2.4.2.5. Phosphatidylserine externalization. Phosphatidylserine (PS)externalization was performed according to Vermes et al. [31],with some modifications, using the Guava Nexin Assay Kit™. After6, 12 and 24 h, leukemia-treated and untreated cells with Cas F andCas X were washed twice with cold PBS and resuspended in 140 lLof PBS with 5 lL of 7-aminoactinomycin D (7-AAD) and 10 lL ofAnnexin V-PE. Cells were gently homogenized and incubated for20 min in the dark at room temperature (22 ± 2 �C). Afterwards,cells were analyzed by flow cytometry. Annexin V is a 35 kDaCa2+ phospholipid-binding protein that has high affinity for PS onthe outer layer of the plasma membrane whereas 7-AAD, a cellimpermeant dye is used as indicator of membrane structural integ-rity [32]. Fluorescence of annexin V-PE was measured in yellowfluorescence-583 nm and 7-AAD in red fluorescence-680 nm. Then,the percentages of early and late apoptotic cells and necrotic cellswere calculated.

2.4.2.6. Caspases-8/-9 and -3/-7. The quantification of the active cat-alytically caspases-8/-9 and -3/-7 was carried out with Cas F and

P.M.P. Ferreira et al. / Chemico-Biological Interactions 222 (2014) 112–125 115

Cas X after 24 h of incubation. HL-60 cells were incubated withFluorescent Labeled Inhibitor of Caspases (FLICA solution) andmaintained for 1 h at 37 �C and CO2 5%. After incubation, 80 lL ofwashing buffer were added and cells were centrifuged at2000 rpm for 5 min. The pellet was resuspended in 200 lL of wash-ing buffer and centrifuged once more. Cells were resuspended inthe working solution (PI 1:200 in 1� washing buffer) and immedi-ately analyzed by flow cytometry.

2.5. Morphological examination by light microscopy

After 6, 12 and 24 h exposure to Cas F and Cas X, 50 lL of cellsuspension were transferred to cytospin slides, cytocentrifugedand fixed with 100% methyl alcohol for 30 s. Then, slides werestained with May-Grünwald–Giemsa for 10 s and with Giemsafor additional 10 s. Untreated or Cas F-treated HL-60 cells wereexamined for morphological changes by light microscopy (Metrim-pex Hungary/PZO-Labimex Modelo Studar Lab™).

2.6. Statistical analysis

For cytotoxicity studies, the IC50 values and their 95% confi-dence intervals were obtained by nonlinear regression. In orderto determine differences among treatments, data expressed asmean ± standard error of the mean (S.E.M.) were compared byone-way analysis of variance (ANOVA) followed by the Newman–Keuls test (p < 0.01) using the Graphpad program (Intuitive Soft-ware for Science, San Diego, CA). All studies were carried out intriplicate represented by independent biological evaluations.

0 10 20 30

-10

0

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mAU235nm,4nm (1.00)

2.70

92.

835

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0

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2313

.899

15.5

2015

.959

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11

21.5

7922

.895

24.4

02 25.5

5626

.814

27.9

2628

.905

30.2

7230

.937

32.8

1033

.739

Caseargrewiin F (9.9 mg/g)

Casearin X(14.2 mg/g)

O

H

OH

O

O

O

OO

O

H

O

H

OH

O

O

O

OO

O

H

Fig. 2. Chromatographic profile of the fraction with casearins (FC) obtained from the Casmost present clerodane diterpenes were quantified based in the calibration curve for ca

3. Results

Clerodane diterpenes were identified at Núcleo de Bioensaios,Biossíntese e Ecofisiologia de Produtos Naturais – NuBBE, Instituteof Chemistry, UNESP (Araraquara, São Paulo) using high perfor-mance liquid chromatography (HPLC-DAD) and nuclear magneticresonance considering literature data [23,33]. In the HPLC-DADanalyses, FC represents 56.5% (mg/g) of the fraction, with case-argrewiin F and casearin X the most present molecules (9.9% and14.2%, respectively) (Fig. 2).

3.1. Antiproliferative activity of the FC and diterpenes

Firstly, in vitro antiproliferative activity of the substances wasperformed on 7 cancer lines by the MTT assay [6 human lines(HL-60, MDA-MB/231, Hs578-T, MX-1, PC-3, DU-145) and 1 mur-ine line (B-16/F10)]. As showed in Table 1, all substances testedrevealed cytotoxic potential against all tumor lines, being Cas Fthe most activity molecule, with IC50 values ranging from0.14 lM (MDA/MB-231 and B-16/F10) to 0.76 lM (DU-145). Insimilar ways, FC and Cas X revealed high antiproliferative actionand IC50 values lower than 0.5 lg/mL and 2 lM, respectively. Onthe other hand, Cas B and D showed moderate activity on tumorcell lines, ranging from 1.41 lM (Cas D in PC-3) to 8.53 lM (CasD in DU-145). Secondly, primary culture of S180 cells was carriedout in order to predict activity of these substances towards anin vivo cancer model. It was noted that Cas F [0.55 lg/mL(1.0 lM)] and FC (0.60 lg/mL) were the most active substanceson S180 cells.

Since all substances showed in vitro antitumoral activity, it wasalso verified the antiproliferative action on primary culture of

40 50 60 min

34.1

2636

.492

39.6

59

42.1

56

45.9

5246

.414

49.5

28

y = 4E+07x + 536575R² = 0,9949

0

5000000

10000000

15000000

20000000

25000000

0 0,1 0,2 0,3 0,4 0,5 0,6

Área

Concentração caseargrewiina F (mg/mL)

earia sylvestris leaves by high performance liquid chromatography (HPLC-DAD). Theseargrewiin F. Detection by UV light at 230 nm.

116 P.M.P. Ferreira et al. / Chemico-Biological Interactions 222 (2014) 112–125

PBMC after 72 h of incubation. Over again, FC and its diterpeneswere cytotoxic on normal proliferating leukocytes, though theyhave demonstrated different levels of selectivity on HL-60 cellscompared to their respective IC50 values in PBMC (Table 2). Herein,Cas F showed promising outcomes, whereas it was 53.4-fold moreselective against leukemia cells compared to dividing leukocyteswith coefficient of selectivity higher than the positive controlDox (44.5).

To confirm the antiproliferative activity of the substances, itwas investigated whether the inhibition of cell proliferation isrelated to DNA synthesis using the BrdU assay. As seen in Fig. 3,all clerodanes reduced BrdU incorporation by dividing cells after

Table 1Cytotoxic activity of a fraction with Casearins (FC) and its clerodane diterpenes [Casearinfrom Casearia sylvestris leaves on tumor cell lines after 72 h exposure determined by MTT

Substance IC50 [lg/mL (lM)]*

HL-60 MDA-MB/231 Hs578-T

FC 0.35 0.26 0.290.28–0.42 0.23–0.29 0.25–0.31

Cas B 1.54 (2.67) 2.10 (3.62) 2.10 (3.63)1.39–1.51 1.87–2.36 1.87–2.36

Cas D 1.91 (3.44) 2.35 (4.23) 2.43 (4.39)1.25–2.91 1.52–3.00 1.88–3.14

Cas F 0.11 (0.20) 0.08 (0.14) 0.13 (0.26)0.10–0.12 0.08–0.09 0.12–0.15

Cas X 0.15 (0.28) 0.81 (1.51) 0.61 (1.14)0.14–0.16 0.67–0.99 0.48–0.77

Dox 0.02 (0.04) 0.03 (0.05) 0.01 (0.02)0.01–0.02 0.02–0.03 0.01–0.02

* Data are presented as IC50 values and 95% confidence intervals for leukemia (HL-60),tumor lines. Doxorubicin (Dox) was used as positive control. Experiments were perform

Table 2Antiproliferative activity of a fraction with casearins (FC) and its clerodane diterpenes [Caisolated from Casearia sylvestris leaves on primary culture of Sarcoma 180 (S180) cells and

Cell culture IC50 [lg/mL (lM)]*

FC Cas B Cas D

S180 0.60 1.27 (2.20) 3.78 (6.80.41–0.90 1.06–1.52 3.25–4.4

PBMC 0.65 3.63 (6.29) 4.54 (8.10.50–0.86 2.98–4.41 3.90–5.2

HL-60 0.35 2.67 3.44Selectivity** 1.9 2.4 2.4

* Data are presented as IC50 values and 95% confidence interval after 72 h exposure. D** Selectivity coefficient determined by IC50 in PBMC/IC50 in HL-60 leukemia cells (see

C Dox 2 40

10

20

30

40

50

60

7066.4 ± 1.8

25.9 ± 1.4

40.0 ± 4.835.0 ±*

**

Cas B Cas

BrdU

-pos

itive

cells

(%)

Fig. 3. BrdU (5-bromo-20deoxyuridine) incorporation by leukemic HL-60 cells treatedCasearin D (Cas D), Casearin X (Cas X) and Caseargrewiin F (Cas F)] isolated from Caseavehicle used for diluting the tested substances. Doxorubicin (0.6 lM) was used as posit(S.E.M) from three independent experiments. ⁄p < 0.01 compared to control by ANOVA f

24 h exposure (40.0 ± 4.8%, 35.0 ± 1.4%, 32.7 ± 6.1%, 30.8 ± 3.4%and 28.5 ± 1.3% for Cas B (2 lM), D (4 lM), F (1 lM), X (1.5 lM)and FC (0.4 lg/mL), respectively, in comparison to the negativecontrol (66.4 ± 1.8%) (p < 0.01). Dox treated cells presented 24.5%of BrdU labeling.

3.2. Clerodane diterpenes activated biochemical and morphologicalchanges suggestive of apoptosis

Flow cytometry analyzes were performed to delineate themechanism responsible for compounds’ antiproliferative action.

B (Cas B), Casearin D (Cas D), Casearin X (Cas X) and Caseargrewiin F (Cas F)] isolatedassay.

MX-1 PC-3 DU-145 B-16/F10

0.18 0.29 0.27 0.250.15–0.21 0.25–0.34 0.20–0.35 0.22–0.282.83 (4.89) 1.66 (2.87) 3.11 (5.37) 2.10 (3.63)2.35–3.40 1.35–2.03 2.48–3.89 1.87–2.363.60 (6.50) 0.78 (1.41) 4.74 (8.53) 3.61 (6.52)2.71–4.76 0.59–1.01 3.92–5.74 3.06–4.250.18 (0.36) 0.17 (0.31) 0.42 (0.76) 0.09 (0.16)0.16–0.20 0.15–0.19 0.31–0.58 0.08–0.100.51 (0.95) 0.46 (0.86) 0.64 (1.19) 0.63 (1.15)0.42–0.63 0.41–0.52 0.58–0.72 0.48–0.810.002 (0.004) 0.24 (0.41) 0.17 (0.29) 0.002 (0.004)0.001–0.004 0.21–0.27 0.12–0.23 0.001–0.003

breast (MDA-MB/231, Hs578-T, MX-1), prostate (PC-3, DU-145) and skin (B16/F-10)ed in triplicate.

searin B (Cas B), Casearin D (Cas D), Casearin X (Cas X) and Caseargrewiin F (Cas F)]peripheral blood mononuclear cells (PBMC) quantified by Alamar Blue assay.

Cas F Cas X Dox

0) 0.55 (1.00) 1.61 (3.00) 1.85 (3.17)0 0.43–0.70 1.39–1.86 1.42–2.427) 5.39 (10.68) 1.10 (2.01) 0.97 (1.78)9 4.80–6.06 0.91–1.32 0.52–1.80

0.20 0.28 0.0453.4 7.2 44.5

oxorubicin (Dox) was used as positive control.Table 1). Experiments were performed in triplicate.

1 1.5 0.4

30.8 ± 3.432.7 ± 6.11.4 **

(µM)

D Cas F Cas X

28.5 ± 1.3*

FC

(µg/mL)

by a fraction with casearins (FC) and its clerodane diterpenes [Casearin B (Cas B),ria sylvestris leaves after 24 h exposure. Negative control (C) was treated with theive control (Dox). Results are expressed as mean ± standard error of measurementollowed by Student Newman–Keuls test.

P.M.P. Ferreira et al. / Chemico-Biological Interactions 222 (2014) 112–125 117

3.2.1. Membrane integrityAnalyzes of membrane integrity showed that all compounds

caused membrane disruption only in higher concentrations (IC50

values) following 12 h (98.8 ± 0.1%, 95.7 ± 0.4%, 94.4 ± 1.0%,95.7 ± 0.6% and 94.9 ± 0.5%) and 24 h [96.1 ± 0.8%, 89.1 ± 1.6%,71.8 ± 3.4%, 74.9 ± 2.1% and 94.1 ± 0.4% for negative control, Cas B(2 lM), D (4 lM), F (1 lM) and X (1.5 lM), respectively] (Fig. 4A,p < 0.01). Cas X 1.5 lM was the exception, whereas it reducedmembrane integrity as early as 6 h of treatment (92.7 ± 1.0%) com-pared to control (98.7 ± 0.2%). On the other hand, Dox (0.6 lM)revealed significant results only after 24 h of incubation (92.6 ± 0.7%, p < 0.01) (Fig. 4).

3.2.2. DNA fragmentationResults were obtained by DNA size, and sub-diploid G0/G1 pat-

tern was considered fragmented. In these assays, untreated and

6 12 24 6 12 24 6 12 24 6 12 24 6 12 240

20

40

60

80

100

C 1Cas B

Dox 2 2C

* * *

A

Mem

bran

e in

tegr

ity (%

)

6 12 24 6 12 24 6 12 24 6 12 24 6 12 240

10

20

30

40

50

60

C 1 2 2

*

*

*

*

**

*

*

DoxCas B Ca

B

DNA

frag

men

tatio

n (%

)

6 12 24 6 12 24 6 12 24 6 12 24 6 12 240

5

10

15

20

25

30

35

C 1 2 2

* *

**

*

DoxCas B Ca

C

Mito

chon

dria

l de

pola

rizat

ion

(%)

Fig. 4. Flow cytometry analyzes of leukemic HL-60 cells treated with clerodane diterpeneF)] isolated from Casearia sylvestris leaves after 6, 12 and 24 h exposure performed witcitrate (DNA fragmentation – B) and rhodamine 123 (mitochondrial depolarization –substances. Doxorubicin (0.6 lM) was used as positive control (Dox). Results are expexperiments. ⁄p < 0.01 compared to control by ANOVA followed by Student Newman–K

treated cells were incubated in a solution with PI and triton X-100. Triton permeabilizes cell membrane, allowing that PI entriesinto cells and matches the DNA. Cells containing intact nuclei ema-nate high fluorescence and cells with condensed chromatin andfragmented DNA emit low fluorescence [32].

As demonstrated in Fig. 3B, all molecules induced DNA frag-mentation in a concentration- and time-dependent manner.Among the compounds studied, Cas X was the unique that causedDNA fragmentation of HL-60 cells as early as 6 h exposure in thelower concentration (0.7 lM, 6.6 ± 0.9%) when compared to thenegative control (1.5 ± 0.3%) (Fig. 4B). Maximum levels of fragmen-tation with Cas X were seen after 24 h (25.4 ± 0.9%). Highest mea-sures of sub-diploid DNA were detected with Cas F 0.5 lM (24 h,23.7 ± 1.0%) and 1 lM (12 h, 44.5 ± 5.7%; 24 h, 44.2 ± 1.1%)(p < 0.01) in comparison with the control (12 h, 3.2 ± 0.4%; 24 h,0.9 ± 0.1%) (p < 0.01). Casearins B and D caused DNA disintegration

6 12 24 6 12 24 6 12 24 6 12 24 6 12 24

as D4 0.5 1 0.7

Cas X1.5 (µM)

**

**

** *

Cas F

6 12 24 6 12 24 6 12 24 6 12 24 6 12 244 0.5 1 0.7 1.5 (µM)

*

*

**

*

*

**

*

*

*

*

s D Cas XCas F

6 12 24 6 12 24 6 12 24 6 12 24 6 12 244 0.5 1 0.7 1.5 (µM)

*

*

*

**

*

**

*

s D Cas XCas F

s [Casearin B (Cas B), Casearin D (Cas D), Casearin X (Cas X) and Caseargrewiin F (Cash propidium iodide (membrane integrity – A), propidium iodide, triton X-100 andC). Negative control (C) was treated with the vehicle used for diluting the testedressed as mean ± standard error of measurement (S.E.M) from three independenteuls test.

Table 3Effects of clerodane diterpenes [Casearin B (Cas B), Casearin D (Cas D), Casearin X (Cas X) and Caseargrewiin F (Cas F)] isolated from Casearia sylvestris leaves on the cell cycle ofleukemic HL-60 cells after 6, 12 and 24 h exposure determined by flow cytometry. Negative control (C) was treated with the vehicle used for diluting the tested substances.Doxorubicin (0.6 lM) was used as positive control (Dox). On the left, Cas X cell cycle effects are represented in graphics produced by the ModFit LT 3.1 software (Verity softwarehouse).

Compound Concentration (lM) Cell cycle phases (%)

6 h 12 h 24 h

G0/G1 S G2/M G0/G1 S G2/M G0/G1 S G2/M

C – 13.1 ± 0.9 78.6 ± 1.5 6.3 ± 0.8 21.5 ± 0.6 71.4 ± 1.2 7.1 ± 1.2 17.1 ± 1.0 76.5 ± 1.4 6.5 ± 0.7Dox 0.6 14.1 ± 2.7 77.2 ± 4.0 7.5 ± 1.2 17.5 ± 2.0 79.3 ± 1.8* 1.3 ± 0.5 21.5 ± 3.3 77.3 ± 3.6 1.1 ± 0.6*

Cas B 1.0 12.1 ± 0.3 83.0 ± 0.4 4.6 ± 0.7 20.7 ± 2.0 71.3 ± 2.3 8.0 ± 0.7 18.1 ± 0.4 74.5 ± 0.8 7.3 ± 0.62.0 16.1 ± 2.3 77.2 ± 3.1 6.0 ± 0.8 22.4 ± 1.9 70.4 ± 1.0 5.9 ± 0.7 17.9 ± 1.6 76.7 ± 2.1 5.4 ± 1.1

Cas D 2.0 12.4 ± 0.7 78.3 ± 2.0 7.0 ± 0.8 26.3 ± 1.5 68.3 ± 1.2 5.0 ± 1.0 17.3 ± 2.1 77.3 ± 2.4 6.4 ± 0.94.0 7.6 ± 1.5 82.8 ± 0.8 9.6 ± 2.0 22.9 ± 1.1 72.0 ± 1.5 5.2 ± 0.9 20.3 ± 1.4 74.4 ± 0.8 5.1 ± 1.3

Cas F 0.5 14.3 ± 0.6 79.9 ± 0.7 6.6 ± 0.6 24.3 ± 1.6 72.3 ± 2.9 5.7 ± 0.8 19.4 ± 2.9 73.5 ± 2.5 7.2 ± 1.01.0 10.9 ± 0.3 82.9 ± 0.7 6.1 ± 0.8 18.1 ± 1.8 75.4 ± 1.4 6.4 ± 0.6 17.6 ± 1.8 81.4 ± 2.5 4.7 ± 1.9

Cas X 0.7 21.0 ± 3.4 72.3 ± 1.9 7.3 ± 0.9 20.7 ± 0.9 74.3 ± 0.7 5.1 ± 0.4 63.1 ± 2.5* 30.2 ± 2.8* 6.6 ± 0.61.5 22.5 ± 3.9 71.8 ± 2.1 5.6 ± 0.4 26.9 ± 1.3 67.3 ± 1.0 5.7 ± 0.6 63.1 ± 10.6* 29.2 ± 10.5* 5.7 ± 1.9

Results are expressed as mean ± standard error of measurement (S.E.M) from three independent experiments.* p < 0.01 compared to control by ANOVA followed by Student Newman–Keuls test.

118 P.M.P. Ferreira et al. / Chemico-Biological Interactions 222 (2014) 112–125

at lower concentrations after 24 h (1 lM, 7.5 ± 0.6%; 2 lM,13.0 ± 0.8%) and after 6 h (7.1 ± 0.3% and 26.3 ± 1.9%), 12 h(20.0 ± 0.9% and 30.1 ± 1.2%) and 24 h (22.3 ± 2.2% and33.2 ± 1.8%) of incubation at higher doses (2 and 4 lM, for Cas Band Cas D, respectively). In parallel, Dox also produced fragmenta-tion as early as 6 h (10.9 ± 2.3%) of incubation. After 12 h and 24 h,fragmentation levels increased significantly (33.4 ± 2.7% and43.6 ± 4.3%, respectively) (p < 0.01) (Fig. 4)

3.2.3. Mitochondrial depolarizationOnly Cas F (0.5 lM, 5.4 ± 0.3%) altered the mitochondrial trans-

membrane potential of leukemia cells at lower concentrations incomparison with the negative control (1.1 ± 0.1%) after 24 h expo-sure. Meanwhile, the other molecules showed statistically signifi-cant results only at higher concentrations (p < 0.01, Fig. 4C).Thus, casearins B 2 lM (4.4 ± 0.8%, 5.4 ± 0.6% and 13.0 ± 1.2%), D4 lM (16.8 ± 17%, 2.3 ± 0.3% and 9.4 ± 1.0%) and F 1 lM(8.5 ± 0.7%, 2.9 ± 0.4% and 10.7 ± 0.9%) led to depolarization after6, 12 and 24 h, respectively. On the other hand, Cas X 1.5 lM andDox 0.6 lM revealed substantial results only after 12 h(5.5 ± 0.4% and 25.7 ± 4.5%) and 24 h (23.2 ± 1.7% and22.7 ± 1.9%), respectively (p < 0.01) (Fig. 4).

3.2.4. Cell cycle arrest in G0/G1 phaseTreated and untreated cells during 6, 12 and 24 h were labelled

with PI to measure DNA quantity by flow cytometry. Followinganalyzes by ModFit LT 3.1. software, it was noted that only Cas X(0.7 and 1.5 lM) after 24 h of incubation was capable to arrest cellsin G0/G1 phase (63.1 ± 2.5% and 63.1 ± 10.6%) and to reduce S phase(30.2 ± 2.8% and 29.2 ± 10.5%) compared to the control (G0/G1,17.1 ± 1.0%; S, 76.5 ± 1.4%), respectively (Table 3) (p < 0.01). Doxwas active in 12 h (arrest in S stage, 79.3 ± 1.8%) and 24 h (reduc-tion of G2/M cells, 1.1 ± 0.6%).

Since all substances revealed cytotoxic properties with equiva-lent standard, Cas F and Cas X, the most active molecules, werechosen to explore two hallmarks of apoptosis: caspase activationand phosphatidylserine externalization [34].

3.2.5. Caspases-8/-9 and -3/-7 activationBoth the molecules Cas F and X were able to activate the initi-

ating caspases-8 and -9. In relation to caspase-8, both compoundsled to viable cells’ reduction (55.2 ± 2.5% and 47.4 ± 6.1%, Cas F 0.5and 1 lM; 30.7 ± 6.2% and 26.1 ± 6.6%, Cas X 0.7 and 1.5 lM) andapoptotic cells’ increasing (35.1 ± 5.7% and 42.7 ± 9.2%, Cas F 0.5and 1 lM; 63.1 ± 5.0% and 65.9 ± 8.4%, Cas X 0.7 and 1.5 lM) in

C Dox 0.5 1 0.7 1.50

20

40

60

80

100

(µM)

Cas X Cas F

** *

* ** *

** *

A

Viable cells Apoptotic cells Necrotic cells

Cas

pase

-8ac

tivat

ion

(%)

C Dox 0.5 1 0.7 1.50

20

40

60

80

100

(µM)

Cas X Cas F

*

*

* **

*

*

*

*

*

** * * *

B

Necrotic cellsViable cells Apoptotic cells

Cas

pase

-9 a

ctiv

atio

n (%

)

Fig. 5. Activation of the initiator caspases-8 (A) and -9 (B) in leukemic HL-60 cells treated with Caseargrewiin F (Cas F) and Casearin X (Cas X) isolated from Casearia sylvestrisleaves after 24 h exposure determined by flow cytometry with propidium iodide and FLICA™ solution. Negative control (C) was treated with the vehicle used for diluting thetested substances. Doxorubicin (0.6 lM) was used as positive control (Dox). Results are expressed as mean ± standard error of measurement (S.E.M) from three independentexperiments. ⁄p < 0.01 compared to control by ANOVA followed by Student Newman–Keuls test.

P.M.P. Ferreira et al. / Chemico-Biological Interactions 222 (2014) 112–125 119

comparison with the control (9.3 ± 0.7 e 3.1 ± 0.6%, viables andapoptotic cells, respectively) (Fig. 5A, p < 0.01).

Caspase-9 assessment showed that Cas F 0.5 lM (43.2 ± 0.4%,46.4 ± 0.4% and 10.7 ± 1.2%), F 1 lM (40.1 ± 1.0%, 69.2 ± 19.0% and8.3 ± 4.7%), Cas X 0.7 lM (44.0 ± 4.0%, 41.3 ± 9.0% and 9.6 ± 0.2%)and X 1.5 lM (21.7 ± 7.0%, 71.8 ± 7.5% and 6.5 ± 5.0%) decreasedviability and augmented cell number in apoptosis and necrosis,respectively (Fig. 5B, p < 0.01).

In a similar way to those results found with caspases-8 and -9,both compounds triggered effecting caspases-3/-7, reduced cellnumber of viable cells and increased the occurrence of early andapoptotic cells (p < 0.01), though these changes have been foundonly at higher doses of Cas F (1 lM, 12.6 ± 2.9% and 17.6 ± 3.7%)and in both concentrations of Cas X (0.7 lM, 12.3 ± 3.3% and19.1 ± 1.6%; 1.5 lM, 31.1 ± 9.4% and 42.1 ± 8.9%), indicating caspas-es-3/-7 activation in comparison with control (2.1 ± 0.2% and1.1 ± 0.3%), respectively (Fig. 6). Cas X was able to augmentednecrosis (0.7 lM, 13.8 ± 4.4%; 1.5 lM, 8.8 ± 1.6%). Dox (0.6 lM)diminished the viability (38.6 ± 2.5%) and increased the early apop-tosis (38.4 ± 6.0%) (p < 0.01).

3.2.6. Cas F and Cas X induces phosphatidylserine externalizationPhosphatidylserine externalization was assessed after 6, 12 and

24 h of incubation with Cas F and Cas X.Dox (0.6 lM) reduced the number of viable leukemic cells in all

exposure times (6 h, 91.4 ± 2.4%; 12 h, 47.7 ± 6.8%; 24 h,55.6 ± 0.7%) and increased early apoptotic cells in 12 h(50.8 ± 6.3%) and 24 h (43.6 ± 1.4%). Meanwhile, Cas F and X

showed activity after 24 h only. Then, they decreased viability athigher concentrations (Cas F 1 lM, 83.5 ± 1.7%; Cas X 1.5 lM,54.7 ± 2.5%) compared to the control (97.5 ± 0.6%) (Fig. 7A) andincreased the number of Cas F 1 lM-treated cells in early apoptosis(Fig. 7B) (9.7 ± 1.1%) and both concentrations of Cas X (0.7 lM,7.9 ± 0.7%; 1.5 lM, 17.6 ± 5.6%) (p < 0.01). Substantial levels of lateapoptosis were seen after 6 h of incubation with Dox (1.9 ± 0.7%)and in 24 h of treatment with Cas X 1.5 lM (18.9 ± 2.6%)(Fig. 7C). Low levels of necrosis were detected only after 24 h inthe highest doses (Cas F 1 lM, 2.1 ± 0.2%; Cas X 1.5 lM,9.4 ± 0.4%) (Fig. 7D, p < 0.01).

3.2.7. Reactive oxygen speciesAll substances (FC, Cas B, D, F and X) were studied regarding the

ROS generation after 1 and 3 h of incubation. None of them wascapable to induce ROS production in comparison with negativecontrol (1 h: 0.1 ± 0.0%; 3 h: 0.4 ± 0.1%). The positive control b-lapachone caused significant ROS production (1 h: 45.6 ± 3.4%;3 h: 12.2 ± 2.3%) (p < 0.01).

3.3. Morphological changes

Under light microscopy, control cells displayed a typical non-adherent and round morphology, homogeneous cytoplasm, pres-ence of mitotic figures and visualization of the cellular plasmamembrane bound (Figs. 7A, 8A and 9A). Cells treated with Cas F(0.5 and 1 lM) and Cas X (0.7 and 1.5 lM) presented morphologi-cal features of death by apoptosis after 6 and 12 h of incubation.

C Dox 0.5 1 0.7 1.50

20

40

60

80

100

(µM)

Viable cells Early apoptosis Late apoptosis Necrosis

*

**

**

*

Cas X Cas F

*

*

** **

* *

Casp

ases

-3/-7

activ

atio

n(%

)

Fig. 6. Activation of the executing caspases-3/-7 in leukemic HL-60 cells treated with Caseargrewiin F (Cas F) and Casearin X (Cas X) isolated from Casearia sylvestris leavesafter 24 h exposure determined by flow cytometry with propidium iodide and FLICA™ solution. Negative control (C) was treated with the vehicle used for diluting the testedsubstances. Doxorubicin (0.6 lM) was used as positive control (Dox). Results are expressed as mean ± standard error of measurement (S.E.M) from three independentexperiments. ⁄p < 0.01 compared to control by ANOVA followed by Student Newman–Keuls test.

6 12 24 6 12 24 6 12 24 6 12 24 6 12 24 6 12 240

20

40

60

80

100 *

*

*

*

C Cas X Cas F

0.5 1.50.71

*

Dox

A

(µM)

Viab

le c

ells

(%)

6 12 24 6 12 24 6 12 24 6 12 24 6 12 24 6 12 240

2

4

6

8

10

*

*D

C Cas X Cas F

0.5 1.50.71Dox (µM)

Nec

rotic

cells

(%)

6 12 24 6 12 24 6 12 24 6 12 24 6 12 24 6 12 240

10

20

30

40

50

60

C Cas X Cas F

0.5 1.50.71Dox (µM)

*

*

* *

*

B

Early

apop

totic

cells

(%)

6 12 24 6 12 24 6 12 24 6 12 24 6 12 24 6 12 240

5

10

15

20

25

C Cas X Cas F

0.5 1.50.71Dox (µM)

*

*

C

Late

apop

totic

cells

(%)

Fig. 7. Phosphatidylserine externalization in leukemic HL-60 cells treated with Caseargrewiin F (Cas F) and Casearin X (Cas X) isolated from Casearia sylvestris leaves after 6,12 and 24 h on incubation determined by flow cytometry with Annexin-V and 7-amino-actinomycin-D (7-AAD). Negative control (C) was treated with the vehicle used fordiluting the tested substances. Doxorubicin (0.6 lM) was used as positive control (Dox). Results are expressed as mean ± standard error of measurement (S.E.M) from threeindependent experiments. ⁄p < 0.01 compared to control by ANOVA followed by Student Newman–Keuls test.

120 P.M.P. Ferreira et al. / Chemico-Biological Interactions 222 (2014) 112–125

Dividing cells Chromatin condensation

Cell shrinking

A B

D C

E F

Fig. 8. Morphology of leukemia HL-60 cells after 6 h of treatment with Caseargrewiin F [0.5 lM (C), 1 lM (D)] and Casearin X [0.7 lM (E), 1.5 lM (F)] isolated from Caseariasylvestris leaves. Negative control (A) was treated with the vehicle used for diluting the tested substances. Doxorubicin [B, (0.6 lM)] was used as positive control. May-Grünwald–Giemsa staining. Magnification, 400�. Scale bar = 20 lm.

P.M.P. Ferreira et al. / Chemico-Biological Interactions 222 (2014) 112–125 121

Chromatin condensation, nuclear fragmentation, karyolysis, cellu-lar shrinking and rarefaction in presence of membrane integritywere seen after 6 h (Fig. 8C–F) and 12 h (Fig. 9C–F). Interestingly,Cas F and Cas X-treated leukemia cells during 12 h also showedoccurrence of cytoplasmic vacuoles (Fig. 9C–F, respectively). Onthe other hand, a typical necrosis characteristic – membrane disin-tegration – was found after 12 h exposure in presence of Cas F1 lM (Fig. 9F) and after 24 h of treatment with Cas F and Cas X(Fig. 10C–F).

Dox-treated cells exhibited morphological alterations only after24 h (chromatin condensation, nuclear fragmentation, cellularshrinking and rarefaction) (Fig. 10B). Shorter exposures with Doxrevealed parallel cell morphology to the negative control, thoughrare mitotic cells had been found (Figs. 8B and 9B).

4. Discussion

The development of novel cytotoxic entities has revolutionizedthe anticancer therapy, since their use as adjunctive treatment hasdemonstrated an undeniable advantage compared to the tradi-tional trials based on surgery and monochemotherapy, making itpossible to cure tumors such as acute childhood leukemia, Hodg-kin’s disease, non-Hodgkin and Hodgkin’s lymphoma and germ cellneoplasms [35,36]. Therefore, chemotherapy remains the most

important line of defense against hematological malignancies andaggressive forms of solid tumors. However, cancer remains the sec-ond cause of death worldwide. In this scenario, plant compoundshave importantly contributed to the discovery of new naturallyoccurring anticancer agents [6,37].

Knowing the pharmacological and popular importance of C. syl-vestris [38] and that mammal cells are commonly used tools toevaluate the cytotoxic properties of new compounds [5,30,39,40],we firstly detailed the antiproliferative activity of a fraction richin casearins (FC) and its major components (Cas B, D, F and X),all obtained from leaf ethanol extracts. FC showed encouragingoutcomes and IC50 values lower than 0.5 lg/mL against all tumorlines. According to the American National Cancer Institute (NCI,USA), the IC50 limit to consider an active crude extract for furtherpurification is a value lower than 30 lg/mL [41]. Subsequently,among the isolated clerodanes, Cas F was the most active molecule,showing IC50 values lower than 1 lM, followed by Cas X.

In last years, bioguided phytochemical investigations led toexpand of new clerodane diterpenes from Casearia species, enlight-ening that most of them, particularly those rich in oxygen radicals,have activity on innumerous cell types, such as bacteria, fungi [9–11], Leishmania donovani promastigotes [42], Trypanosoma cruziamastigotes [42,43], Plasmodium falciparum strains resistant tochloroquine [44], Chinese hamster V-79 cells, fibroblasts (L-929)

A B

D C

E F Dividing cells Chromatin condensation

Cell shrinking

Destabilization of the plasma membrane

Vacuolization of the cytoplasm

Fig. 9. Morphology of leukemia HL-60 cells after 12 h of treatment with Caseargrewiin F [0.5 lM (C), 1 lM (D)] and Casearin X [0.7 lM (E), 1.5 lM (F)] isolated from Caseariasylvestris leaves. Negative control (A) was treated with the vehicle used for diluting the tested substances. Doxorubicin [B, (0.6 lM)] was used as positive control. May-Grünwald–Giemsa staining. Magnification, 400�. Scale bar = 20 lm.

122 P.M.P. Ferreira et al. / Chemico-Biological Interactions 222 (2014) 112–125

and transformed lines of colon (HCT-116), leukemias (HL-60,MOLT-4, CEM, K-562), ovarian (A2780), prostate (PC-3), skin(MDA/MB-435) and glioblastoma (SF-295) [4,10,19–21,23,39,45–47].

In attempt to envisage an antitumor action upon in vivo assess-ments, the activity on Sarcoma 180 cells was determined by theAlamar Blue assay. Sarcoma 180 tumor is an useful model extre-mely utilized in research of natural products with antineoplasticaction [5,26,33,40,48]. Herein, once more, it was noted that Cas Fand FC were the most effective substances against S180 cells, con-firming the antitumoral potential of all studied substances. Previ-ously, ethanolic extract from C. sylvestris leaves reported in vivoantitumor activity on Sarcoma 180 transplanted mice of the casea-rins A, B, C, D, E and F [33,48,49]. More recently, two gallic acid-derived compounds isolated from C. sylvestris leaves – isobutyl gal-late-3,5-dimethyl ether (IGDE) and methyl gallate-3,5-dimethylether (MGDE) – also showed significant chemotherapeutic poten-tial against Ehrlich and Lewis lung cancer ascite tumor cells [22].

The bioactivities displayed by caseargrewiins, casearins andother clerodane diterpenes have a structure–activity relationshipbasically attributed to the diterpene skeleton substitutions atpositions C-2 (R1), C-6 (R4), C-7 (R5), C-18 (R2) and C-19 (R3) or

hydroxylation or O-methylation at C-2 (Fig. 1) and their bioactivitydepend on the oxygenated diacetal ring structure formed by car-bons C-18 and C-19, which displays a rare functional assemblingin natural molecules and can be considered a protected dialdehyde[24]. Acid hydrolysis and other causes that open this ring leads tomolecular instability and loss of antiproliferative effects, as seenwith Cas X dialdehyde [23]. In addition, (�)-hardwickiic acid, aclerodane without diacetal ring and with low cytotoxic properties,was one of the first diterpenes found in C. sylvestris without a typ-ical oxygenated structure [4,23,24], suggesting that oxygenationalso has influence on the cytotoxic activity.

Cell type antiproliferative specificity is observed in some plantextracts and this is probably due to the presence of different clas-ses of compounds [39]. Hence, the use of more than one cell line isconsidered necessary for detection of cytotoxic compounds. SinceHL-60 cells were very sensible to all substances, we chose this lineto study underlying mechanisms involved in the cytotoxicity. Leu-kemias are the fifth leading cause of death for men and sixth forwomen, being the most deadly type of neoplasm in people agedup to 20 years. Although many current systems of treatment havebeen relatively effective in achieving cure, the majority of drugsused to treat leukemias have severe side effects. The human

A

D C

E F

μB

Dividing cells Chromatin condensation

Cell shrinking

Destabilization of the plasma membrane

Vacuolization of the cytoplasm

Fig. 10. Morphology of leukemia HL-60 cells after 24 h of treatment with Caseargrewiin F [0.5 lM (C), 1 lM (D)] and Casearin X [0.7 lM (E), 1.5 lM (F)] isolated from Caseariasylvestris leaves. Negative control (A) was treated with the vehicle used for diluting the tested substances. Doxorubicin [B, (0.6 lM)] was used as positive control. May-Grünwald–Giemsa staining. Magnification, 400�. Scale bar = 20 lm.

P.M.P. Ferreira et al. / Chemico-Biological Interactions 222 (2014) 112–125 123

HL-60 cell line, acute promyelocytic leukemia with prevailing ofneutrophilic promyelocytes, is commonly used in the research fornovel cytotoxic agents [4,5,30,40].

To determine the mechanism responsible for the cytotoxiceffects, we firstly evaluated the ability of substances to inhibitDNA synthesis by BrdU assay. The cellular uptake of BrdU, a thymi-dine analogue during the S phase of the cell cycle, have been exten-sively applied in biomedical research to identify drugs withantiproliferative activity, since it is an in vitro reliably nonradioac-tive and immunocytochemical method widely used to determinethe percentage of cell division [30,50]. All substances inhibitedBrdU incorporation after 24 h of incubation, revealing high anti-proliferation activity in perceptual values similar to the positivecontrol Dox (Cas F and X), confirming the results obtained withthe MTT and Alamar Blue assays.

Biochemical and morphological alterations were also investi-gated using flow cytometry and May-Grünwald–Giemsa stainingto determine the cellular death pattern. In general, when death iselicited by apoptosis pathways, different factors can act as activa-tors, including chemotherapeutic agents, ionizing radiation, DNAdamage, heat shock proteins, unfolded proteins, deprivation ofgrowth factors, low quantities of nutrients and increased levels

of ROS, which usually trigger the intrinsic pathway. On the con-trary, binding of molecules to the membrane receptors often leadsto the activation of the extrinsic route [32]. In the intrinsic path-way, cells concomitantly exhibit membrane integrity and high lev-els of DNA fragmentation (sub-G1 cells) [51]. All clerodanes causedDNA disintegration in a concentration-dependent manner, whilemembrane integrity has been significantly altered only at highestconcentrations and following 12 or 24 h exposure. This membraneintegrity maintenance validates the results obtained by Santoset al. [23], emphasizing that cytotoxic action of the molecules isnot related to direct cell membrane injury.

DNA internucleosomal fragmentation is regularly attributed tocaspases, enzymes belonging to the family of Ca2+- and Mg2+-dependent cysteine proteases, which recognize and cleave sub-strates exhibiting aspartate residues. There are 14 human caspasesand six of them (executing caspases-3, -6, -7; initiator caspases-8, -9, -10) are involved in the apoptotic process [34,52]. Herein, it wasfound that both molecules Cas F and X, as well as the positive con-trol Dox, stimulate intense enzymatic activity of the initiator casp-ases-8 and -9 and effector caspases-3 and -7. This caspaseactivation was confirmed by mitochondrial potential decreasenoted after exposure to the diterpenes, proposing mitochondrial

124 P.M.P. Ferreira et al. / Chemico-Biological Interactions 222 (2014) 112–125

membrane permeabilization and cytochrome c release. Cyto-chrome c binds to Apaf-1 (Apoptotic protease-activating factor 1)and generate the catalytically active form of caspase-9, which acti-vates caspase-3, the most important effector caspase that acts aseffective DNase to slice the genomic DNA into nucleosomes, pro-ducing fragments of 180–200 base pairs, nuclear and cellularreduction and pyknosis [53], all morphological alterations fre-quently seen in HL-60 cells after Cas F and X exposure. Analo-gously, investigations with caseamembrin C isolated fromCasearia membranacea also presented cytotoxicity on PC-3 cellsand induced caspases-8, -9 and -3 activation and Bid cleavage [45].

To better comprehend the kind of cell death, we evaluated thePS externalization, a gold standard test used to confirm apoptosistriggering [31,32]. Cells exhibit asymmetrical distribution of phos-pholipids in the bilayer membrane, with prevalence of phosphati-dylcholine and sphingomyelin in the outer leaflet of the plasmamembrane, whereas phosphatidylethanolamine, phosphatidylino-sitol and phosphatidylserine predominate in the inner leaflet[54]. Apoptotic cells display loss of this asymmetry and PS exter-nalization, as seen in HL-60-treated cells with Cas F and X.

Our studies revealed a gap dividing positivity for PS and 7-AADwhile both events overlapped in necrotic cells, findings detected indotplot graphics of HL-60 cells incubated com Dox, Cas F and Cas X,likely explained by the fact that 7-AAD (and propidium iodide)penetrate in cells only after the plasma membrane becomes per-meable [29,51]. Besides, morphological changes indicative of celldeath by apoptosis (karyorrhexis, nuclear rarefaction, cell shrink-age and extensive cytoplasmic vacuoles) were expressly detectedin leukemia-treated cells after 12 h. In higher concentrations andlonger exposure periods, plasma membrane disintegration, unbal-anced caspase activation and higher red fluorescence levelsexpressed in flow cytometry investigations exhibited after PI or7-AAD labeling were seen, supposing that dose-dependent effectsare also consistent with late apoptosis or secondary necrosis[31,55]. Previously studies with Cas X also reported characteristicsof apoptosis or secondary necrosis [4].

In toxic stimulus analyzes, higher doses regularly develops con-comitant features of apoptosis and necrosis. Under such conditions,severity and not specificity of the stimulus selectively determineshow cell death occurs. If the necrosis prevails, early lesions on theplasma membrane occur instead of cell shrinking [32]. Therefore,depending on the concentration used, many different processesmay be influenced and/or altered, suggesting that dose-dependentregulation of cellular process reflects signalization triggered bybioactive compounds as stated here and by others [4,30].

Anticancer drugs that kill tumor cells by apoptosis possessphysiological advantages due to externalized PS specific recogni-tion and cellular removal by macrophages, preventing tissue dam-age resulting from in situ cell lysis [31,56]. However, most of thechemotherapeutic agents interfered with replication and mitoticspindle formation [37,57,58]. Among all substances studied, onlyCas X caused cell cycle arrest in G0/G1 phase after 24 h of incuba-tion. Kauranic diterpenes promote arrest in G1 phase and, conse-quently, reduction of cells in S and G2 phases [59]. Comparableresults were found with Cas X, indicating that these diterpenesmight inhibit DNA duplication during the transition G1/S. Possibly,this cell cycle arrest is related to the DNA break and repair machin-ery triggering. It was also observed that casearins G, S and T dam-aged DNA molecules of Saccharomyces cerevisiae mutant strainscausing DNA acetylation and cell death [9].

5. Conclusions

Cas X and F were the most active molecules with morepronounced lethal and discriminating effects on tumor cells and

effective antiproliferative activity predominantly mediated byapoptosis, highlighting clerodane dipertenes as promising leadantineoplastic compounds.

Conflict of Interest

The authors have declared that there is no conflict of interest.

Transparency Document

The Transparency document associated with this article can befound in the online version.

Acknowledgements

We wish to thank the Brazilian agencies Conselho Nacional deDesenvolvimento Científico e Tecnológico (CNPq), Fundação Cea-rense de Apoio ao Desenvolvimento Científico e Tecnológico (FUN-CAP), Fundação de Amparo à Pesquisa do Estado de São Paulo(FAPESP) and Fundação de Amparo à Pesquisa do Estado do Piauí(FAPEPI) for financial support. We are grateful to Silvana Françados Santos and Maria de Fátima Teixeira for technical assistanceand Priscila Murolo, a skilled author of English language papers,for her help with editing of the manuscript.

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