Morphological and biochemical alterations activated by antitumor clerodane diterpenes

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Morphological and biochemical alterations activated by antitumor

clerodane diterpenes

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, Brazil}

b_{Postgraduate Program in Pharmaceutical Sciences, Federal University of Piauí, Teresina, Brazil} c_{Department of Physiology and Pharmacology, Federal University of Pernambuco, Recife, Brazil}

d_{Department of Biological Sciences, Campus Senador Helvídio Nunes de Barros, Federal University of Piauí, Picos, Brazil} e_{Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Ceará, Fortaleza, Brazil} f_{Faculty of Pharmaceutical Sciences, State University of São Paulo Júlio de Mesquita Filho, Araraquara, Brazil} g_{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 2014

Received in revised form 8 October 2014 Accepted 15 October 2014

Available online 27 October 2014

Keywords: Casearia sylvestris Cytotoxicity

Antiproliferative action Apoptosis

Human cells Murine cells

a b s t r a c t

Casearia sylvestrisSwartz (Salicaceae) is a plant commonly widespread in the Americas. It has oxygenated tricyclic bioactive clerodane diterpenes with antimicrobial, antiulcer, larvicidal, chemopreventive, anti-inflammatory, antioxidant and antiproliferative properties. Due to this requirement for the developing of 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 fromC. sylvestrisleaves 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 were the most active compounds. Cell death analyzes with Cas F (0.5 and 1lM) and Cas X (0.7 and 1.5lM) using the HL-60 leukemia line as experimental model showed DNA synthesis and membrane integrity reduction, DNA fragmentation and mitochondrial depolarization, specially after 24 h exposure, cell cycle arrest in G0/G1phase caused by Cas X, activation of the initiator -8/-9 and effector -3/-7 caspases and phosphatidylserine externalization, all biochemical features of apoptosis corroborated by chromatinic condensation, 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 most functional molecules with more pronounced lethal and discriminating effects on tumor cells and antipro-liferative action predominantly mediated by apoptosis, highlighting clerodane dipertenes as promising lead antineoplastic compounds.

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 mimic or are based on natural compounds. Then, instead of the interest in molecular modeling, combinatorial chemistry and other

chemical synthesis techniques, natural products, and particularly, medicinal plants, remain an important source of new therapeutic agents 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 in tropical and temperate regions around the world and commonly widespread in the Americas. In Brazil, it is present from Amazonas (Tapajós river region) to Rio Grande do Sul states[7,8].

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

http://dx.doi.org/10.1016/j.cbi.2014.10.015

⇑Corresponding author at: Department of Biophysics and Physiology, Campus Ministro Petrônio Portella, Federal University of Piauí, Teresina, Brazil. Tel.: +55 86 32155871.

E-mail addresses:[email protected],[email protected](P.M.P. Ferreira).

Contents lists available atScienceDirect

Chemico-Biological Interactions

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isolated fromC. sylvestrisbelonging to the casearin, caseargrewiin and casearvestrin classes, oxygenated tricyclic bioactive clerodane diterpenes which have presented also excellent cytotoxic potential [4,10,19,20–23]. Thus, we firstly analyzed thein 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 ofC. sylvestriswere collected at the Parque Estadual Car-los Botelho (São Miguel Arcanjo, São Paulo, Brazil). The plant was identified by Dr. Ines Cordeiro (Instituto Botânico do Estado de São Paulo, São Paulo, Brazil). Voucher specimens (numbers AGS04, AGS05, AGS06, AGS13 and AGS19) were deposited at the Herbarium Maria Eneida P. Kaufmann (Instituto Botânico do Esta-do de São Paulo, São Paulo, Brazil). Dried and powdered leaves ofC. sylvestriswere extracted with ethanol in a stainless steel extractor with solvent reflux for ca. 24 h at 40°_{C. The crude extract was}

con-centrated under reduced pressure (rotary evaporator) and dried in desiccators over silica gel under reduced pressure to yield a dry residue. The structures of Cas B, D, X and F were determined by spectrometric data (nuclear magnetic resonance, ultraviolet, infra-red and mass spectrometry) and compainfra-red to the spectral report available in the literature[23,24](Fig. 1).

Fetal calf serum was purchased from Cultilab (Campinas, SP), RPMI 1640 medium, trypsin–EDTA, penicillin and streptomycin were purchased from GIBCOÒ_{(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 (DoxolemÒ

) was pur-chased from Zodiac Produtos Farmacêuticos S/A, Brazil.

2.2. Animals

Adult female Swiss mice (Mus musculusLinnaeus, 1758) were obtained from the animal facilities of the Universidade Federal do Piauí (UFPI), Teresina, Brazil. They were kept in well-ventilated cages under standard conditions of light (12 h with alternate day and night cycles) and temperature (27 ± 2°_{C) and were housed}

with free access to commercial rodent stock diet (Nutrilabor, Cam-pinas, Brazil). All procedures were approved by the Committee on Animal Research at the UFPI (Process no_{102/2011) and followed} the Brazilian (Colégio Brasileiro de Experimentação Animal– COBEA) and International Standards on the care and use of experimental animals (Directive 2010/63/EU of the European Parliament and of the Council).

2.3. In vitro antiproliferative assays

The cytotoxic potential of the FC, Cas B, D, X and F was assessed after 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 blood mononuclear cells (PBMC). Cell culture was performed in RPMI 1640 medium supplemented with 20% fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin and 100

l

g/mL streptomycin, at 37°_{C with 5% CO}₂_{. Quantification of cell proliferation was}

spectro-photometrically determined using a multiplate reader (DTX 880 Multimode Detector, Beckman Coulter). Control groups (negative and positive) received the same amount of DMSO (0.1%). Doxoru-bicin (Dox, 0.01–8.6

l

M) was used as positive control.

2.3.1. Antiproliferative study on tumor cells evaluated by MTT assay The cytotoxicity against HL-60, MDA-MB/231, Hs578-T, MX-1, PC-3, DU-145, and B16/F-10 cancer cells was determined by MTT assay[25], which analyzes the ability of living cells to reduce the yellow dye 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H -tetrazo-lium bromide (MTT) to a purple formazan product. Briefly, cells

R₁

R₃ R₄ H

O R₂

O

1

3 5 ₇

9

18 19

17 20 11 13

15

2 10

4 6

8 12 16 14

R

1

R

2

R

3

R

4

Casearin B

CH

3

O

CH

3

CO

2

CH

3

CO

2

n-C

3

H

7

CO

2

Casearin D

OH

n-C

3

H

7

CO

2

OH

n-C

3

H

7

CO

2

Casearin X

n-C

3

H

7

CO

2

n-C

3

H

7

CO

2

OH

H

(3)

were plated in 96-well plates (0.3–0.7105cells/well) and incu-bated to allow cell adhesion. Twenty-four hours later, FC and diter-penes were added to each well [(0.04–25

l

g/mL) and (0.01– 10

l

M), respectively]. After 72 h of incubation, the supernatant was replaced by fresh medium containing 10% MTT; the formazan product was dissolved in DMSO and the absorbance was measured at 595 nm wavelength.

2.3.2. Antiproliferative study on Sarcoma 180 cells evaluated by Alamar Blue assay

Ascite-bearing mice between 7 and 9 days postinoculation were sacrificed by cervical dislocation and a suspension of S180 cells was harvested from the intraperitoneal cavity under aseptic condi-tions. The suspension was centrifuged at 500gfor 5 min to obtain a cell pellet and washed three times with RPMI medium. Cell con-centration was adjusted to 0.5106cells/mL in supplemented RPMI 1640 medium, plated in a 96-well plate and incubated with increasing concentrations of the FC and compounds [(0.04–25

l

g/ mL) and (0.01–10

l

M), respectively]. Cell proliferation was deter-mined by the Alamar Blue assay after 72 h[26]. Eight hours befor-e thbefor-e latbefor-e incubation, 10

l

L of stock solution (0.312 mg/mL) of Alamar Blue™ were added to each well. The absorbance was mea-sured at 570 nm and 595 nm and the drug effect was quantified as the control percentage.

2.3.3. Antiproliferative study on peripheral blood mononuclear cells by Alamar Blue assay

Heparinized human blood (from healthy, non-smoker donors who had not taken any drug for at least 15 days prior to sampling, aged between 18 and 35 years-old) was collected and PBMC were isolated by a standard method of density-gradient centrifugation over Ficoll-Hypaque. PBMC were washed and resuspended (3105cells/mL) in supplemented RPMI 1640 medium and phy-tohemagglutinin (4%). Then, PBMC were plated in 96-well plates (3105cells/well). After 24 h, FC and diterpenes were added to each well [(0.04–25

l

g/mL) and (0.01–10

l

M), respectively] and the cells were incubated during 72 h. Twenty-four hours before the late incubation, 10

l

L of stock solution (0.312 mg/mL) of Ala-mar Blue™ were added to each well [26]. The absorbance was measured as described above. All studies were performed in accor-dance with Brazilian research guidelines (Law 466/2012, National Council of Health, Brazil) and with the Declaration of Helsinki.

2.4. Assessment of mechanisms involved in the antiproliferative activity

Since HL-60 cell line was the most sensitive cell line to the sub-stances, it was selected to evaluate their underlying mechanism related to the cytotoxic effects. The substances were added to 24-well tissue culture plates with HL-60 cells (3 105cells/mL) to obtain final concentrations of 1 and 2

l

M (Cas B), 2 and 4

l

M (Cas D), 0.7 and 1.5

l

M (Cas X) and 0.5 and 1

l

M (Cas F). These concentrations were selected based on IC50 values of each com-pound with HL-60 cells after 24 h exposure and corresponds to the IC50/2 and IC50, respectively. Doxorubicin (0.6

l

M) was used as positive control (Dox).

2.4.1. DNA synthesis immunocytochemistry detection

Leukemia cells were plated (2 mL/well) and incubated with the FC (0.8

l

g/mL) and compounds (Cas B, 2

l

M; Cas D, 4

l

M; Cas F, 1.0

l

M and Cas X 1.5

l

M). After 21 h of incubation, 20

l

L of 5-bromo-20_{-deoxyuridine (BrdU 10 mM) were added and incubated} for additional 3 h at 37°_{C. To determine the amount of BrdU}

incor-porated into the DNA, cells were harvested, transferred to cytospin slides, and allowed to dry for 2 h at room temperature (25°_{C). Cells}

that incorporated BrdU were labeled using direct peroxidase

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

2.4.2. Biological analyzes by flow cytometry

All 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). Five thousand events were evaluated per experiment and cell debris was 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 the highly fluorescent dichlorofluorescein (DCF) in the presence of intracellular 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

l

M during 30 min at 37°_{C. Then, cells were harvested,}

washed, resuspended in PBS and immediately analyzed via flow cytometry[28].b-Lapachone (2

l

M) 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

l

L of treated and untreated cells were incubated with PI (50

l

g/mL). The cells were then incubated for 5 min at 37°_C.

Fluorescence was measured and cell morphology, granularity and membrane integrity were determined[29].

2.4.2.3. Cell cycle and DNA fragmentation.Cell cycle distribution and DNA fragmentation analysis were evaluated after 6, 12 and 24 h of incubation by the incorporation of PI (50

l

g/mL). Briefly, 24 h-trea-ted and untreah-trea-ted cells were incubah-trea-ted at 37°_{C for 30 min in the}

dark, in a lysis solution containing 0.1% citrate, 0.1% Triton X-100 and 50

l

g/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 determined by rhodamine 123, a cell-permeable, cationic, fluorescent dye that is readily sequestered by mitochondria without inducing cytotoxic effects. After 6, 12 and 24 h of treatment with the substances, cells were washed with PBS and incubated with rhodamine 123 at 37°_C

for 15 min in the dark. Cells were incubated again in PBS at 37°_C

for 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™. After 6, 12 and 24 h, leukemia-treated and untreated cells with Cas F and Cas X were washed twice with cold PBS and resuspended in 140

l

L of PBS with 5

l

L of 7-aminoactinomycin D (7-AAD) and 10

l

L of Annexin V-PE. Cells were gently homogenized and incubated for 20 min in the dark at room temperature (22 ± 2°_{C). Afterwards,}

cells were analyzed by flow cytometry. Annexin V is a 35 kDa Ca2+_{phospholipid-binding protein that has high affinity for PS on} the outer layer of the plasma membrane whereas 7-AAD, a cell impermeant dye is used as indicator of membrane structural integ-rity[32]. Fluorescence of annexin V-PE was measured in yellow fluorescence-583 nm and 7-AAD in red fluorescence-680 nm. Then, the percentages of early and late apoptotic cells and necrotic cells were calculated.

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Cas X after 24 h of incubation. HL-60 cells were incubated with Fluorescent Labeled Inhibitor of Caspases (FLICA solution) and maintained for 1 h at 37°_{C and CO}₂_{5%. After incubation, 80}

_l

_{L of}

washing buffer were added and cells were centrifuged at 2000 rpm for 5 min. The pellet was resuspended in 200

l

L of wash-ing buffer and centrifuged once more. Cells were resuspended in the working solution (PI 1:200 in 1washing 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

l

L of cell suspension were transferred to cytospin slides, cytocentrifuged and fixed with 100% methyl alcohol for 30 s. Then, slides were stained with May-Grünwald–Giemsa for 10 s and with Giemsa for additional 10 s. Untreated or Cas F-treated HL-60 cells were examined 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 order to determine differences among treatments, data expressed as mean ± standard error of the mean (S.E.M.) were compared by one-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 in triplicate represented by independent biological evaluations.

3. Results

Clerodane diterpenes were identified at Núcleo de Bioensaios, Biossíntese e Ecofisiologia de Produtos Naturais – NuBBE, Institute of Chemistry, UNESP (Araraquara, São Paulo) using high perfor-mance liquid chromatography (HPLC-DAD) and nuclear magnetic resonance considering literature data [23,33]. In the HPLC-DAD analyses, FC represents 56.5% (mg/g) of the fraction, with case-argrewiin F and casearin X the most present molecules (9.9% and 14.2%, respectively) (Fig. 2).

3.1. Antiproliferative activity of the FC and diterpenes

Firstly,in vitroantiproliferative activity of the substances was performed 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 lmur-ine (B-16/F10)]. As showed in Table 1, all substances tested revealed cytotoxic potential against all tumor lines, being Cas F the most activity molecule, with IC50 values ranging from 0.14

l

M (MDA/MB-231 and B-16/F10) to 0.76

l

M (DU-145). In similar ways, FC and Cas X revealed high antiproliferative action and IC50values lower than 0.5

l

g/mL and 2

l

M, respectively. On the other hand, Cas B and D showed moderate activity on tumor cell lines, ranging from 1.41

l

M (Cas D in PC-3) to 8.53

l

M (Cas D in DU-145). Secondly, primary culture of S180 cells was carried out in order to predict activity of these substances towards an in vivo cancer model. It was noted that Cas F [0.55

l

g/mL (1.0

l

M)] and FC (0.60

l

g/mL) were the most active substances on S180 cells.

Since all substances showedin vitroantitumoral activity, it was also verified the antiproliferative action on primary culture of

0 10 20 30 40 50 60 min

-10 0 10 20 30 40 50 60 70 80 90 100

mAU

235nm,4nm (1.00)

2.709 2.835 5.420 13.223

13.899

15.520

15.959

16.311

21.579

22.895

24.402

25.556

26.814

27.926

28.905

30.272

30.937

32.810

33.739

34.126

36.492

39.659 42.156 45.952

46.414

49.528

Caseargrewiin F (9.9 mg/g)

y = 4E+07x + 536575 R² = 0,9949

0 5000000 10000000 15000000 20000000 25000000

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

Ár

ea

Concentração caseargrewiina F (mg/mL)

Casearin X (14.2 mg/g)

O H

OH O

O

O O

O H

OH O

O

O O

O H

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PBMC after 72 h of incubation. Over again, FC and its diterpenes were cytotoxic on normal proliferating leukocytes, though they have demonstrated different levels of selectivity on HL-60 cells compared to their respective IC50values in PBMC (Table 2). Herein, Cas F showed promising outcomes, whereas it was 53.4-fold more selective against leukemia cells compared to dividing leukocytes with coefficient of selectivity higher than the positive control Dox (44.5).

To confirm the antiproliferative activity of the substances, it was investigated whether the inhibition of cell proliferation is related to DNA synthesis using the BrdU assay. As seen inFig. 3, all clerodanes reduced BrdU incorporation by dividing cells after

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

l

M), D (4

l

M), F (1

l

M), X (1.5

l

M) and FC (0.4

l

g/mL), respectively, in comparison to the negative control (66.4 ± 1.8%) (p< 0.01). Dox treated cells presented 24.5% of BrdU labeling.

3.2. Clerodane diterpenes activated biochemical and morphological changes suggestive of apoptosis

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

Table 1

Cytotoxic activity of a fraction with Casearins (FC) and its clerodane diterpenes [Casearin B (Cas B), Casearin D (Cas D), Casearin X (Cas X) and Caseargrewiin F (Cas F)] isolated fromCasearia sylvestrisleaves on tumor cell lines after 72 h exposure determined by MTT assay.

Substance IC50[lg/mL (lM)]*

HL-60 MDA-MB/231 Hs578-T MX-1 PC-3 DU-145 B-16/F10

FC 0.35 0.26 0.29 0.18 0.29 0.27 0.25

0.28–0.42 0.23–0.29 0.25–0.31 0.15–0.21 0.25–0.34 0.20–0.35 0.22–0.28

Cas B 1.54 (2.67) 2.10 (3.62) 2.10 (3.63) 2.83 (4.89) 1.66 (2.87) 3.11 (5.37) 2.10 (3.63)

1.39–1.51 1.87–2.36 1.87–2.36 2.35–3.40 1.35–2.03 2.48–3.89 1.87–2.36

Cas D 1.91 (3.44) 2.35 (4.23) 2.43 (4.39) 3.60 (6.50) 0.78 (1.41) 4.74 (8.53) 3.61 (6.52)

1.25–2.91 1.52–3.00 1.88–3.14 2.71–4.76 0.59–1.01 3.92–5.74 3.06–4.25

Cas F 0.11 (0.20) 0.08 (0.14) 0.13 (0.26) 0.18 (0.36) 0.17 (0.31) 0.42 (0.76) 0.09 (0.16)

0.10–0.12 0.08–0.09 0.12–0.15 0.16–0.20 0.15–0.19 0.31–0.58 0.08–0.10

Cas X 0.15 (0.28) 0.81 (1.51) 0.61 (1.14) 0.51 (0.95) 0.46 (0.86) 0.64 (1.19) 0.63 (1.15)

0.14–0.16 0.67–0.99 0.48–0.77 0.42–0.63 0.41–0.52 0.58–0.72 0.48–0.81

Dox 0.02 (0.04) 0.03 (0.05) 0.01 (0.02) 0.002 (0.004) 0.24 (0.41) 0.17 (0.29) 0.002 (0.004)

0.01–0.02 0.02–0.03 0.01–0.02 0.001–0.004 0.21–0.27 0.12–0.23 0.001–0.003

*_{Data are presented as IC}

50values and 95% confidence intervals for leukemia (HL-60), breast (MDA-MB/231, Hs578-T, MX-1), prostate (PC-3, DU-145) and skin (B16/F-10) tumor lines. Doxorubicin (Dox) was used as positive control. Experiments were performed in triplicate.

Table 2

Antiproliferative activity of a fraction with casearins (FC) and its clerodane diterpenes [Casearin B (Cas B), Casearin D (Cas D), Casearin X (Cas X) and Caseargrewiin F (Cas F)] isolated fromCasearia sylvestrisleaves on primary culture of Sarcoma 180 (S180) cells and peripheral blood mononuclear cells (PBMC) quantified by Alamar Blue assay.

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

FC Cas B Cas D Cas F Cas X Dox

S180 0.60 1.27 (2.20) 3.78 (6.80) 0.55 (1.00) 1.61 (3.00) 1.85 (3.17)

0.41–0.90 1.06–1.52 3.25–4.40 0.43–0.70 1.39–1.86 1.42–2.42

PBMC 0.65 3.63 (6.29) 4.54 (8.17) 5.39 (10.68) 1.10 (2.01) 0.97 (1.78)

0.50–0.86 2.98–4.41 3.90–5.29 4.80–6.06 0.91–1.32 0.52–1.80

HL-60 0.35 2.67 3.44 0.20 0.28 0.04

Selectivity** _1.9 _2.4 _2.4 _53.4 _7.2 _44.5

*_{Data are presented as IC}

50values and 95% confidence interval after 72 h exposure. Doxorubicin (Dox) was used as positive control. ** _{Selectivity coefficient determined by IC50}_{in PBMC/IC50}_{in HL-60 leukemia cells (see}_{Table 1}_{). Experiments were performed in triplicate.}

C Dox 2 4 1 1.5 0.4

0 10 20 30 40 50 60 70

66.4±1.8

25.9±1.4

40.0±4.8

30.8±3.4 32.7±6.1

35.0±1.4

*

(µM)

Cas B Cas D Cas F Cas X

28.5±1.3

*

FC

(µg/mL)

Br

d

U

-p

o

s

it

iv

e

c

e

lls

(%

)

Fig. 3.BrdU (5-bromo-20_{deoxyuridine) incorporation by leukemic HL-60 cells treated by a fraction with casearins (FC) and its clerodane diterpenes [Casearin B (Cas B),}

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3.2.1. Membrane integrity

Analyzes 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

l

M), D (4

l

M), F (1

l

M) and X (1.5

l

M), respectively] (Fig. 4A, p< 0.01). Cas X 1.5

l

M was the exception, whereas it reduced membrane 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

l

M) revealed significant results only after 24 h of incubation (92. 6 ± 0.7%,p< 0.01) (Fig. 4).

3.2.2. DNA fragmentation

Results were obtained by DNA size, and sub-diploid G0/G1 pat-tern was considered fragmented. In these assays, untreated and

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

As demonstrated inFig. 3B, all molecules induced DNA frag-mentation in a concentration- and time-dependent manner. Among the compounds studied, Cas X was the unique that caused DNA fragmentation of HL-60 cells as early as 6 h exposure in the lower concentration (0.7

l

M, 6.6 ± 0.9%) when compared to the negative 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

l

M (24 h, 23.7 ± 1.0%) and 1

l

M (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 6 12 24 6 12 24 6 12 24 6 12 24 6 12 24

0 20 40 60 80 100 C 1 Cas B

Dox 2 2

Cas D

4 0.5 1 0.7

Cas X

1.5 (µM)

*

Cas F

A

Me mb ra n e i n te g ri ty (% )

6 12 24 6 12 24 6 12 24 6 12 24 6 12 24 6 12 24 6 12 24 6 12 24 6 12 24 6 12 24 0 10 20 30 40 50 60

C 1 2 2 4 0.5 1 0.7 1.5 (µM)

*

_*

*

Dox

Cas B Cas D Cas F Cas X

B

D N A f ragm e nt at io n ( % )

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

0 5 10 15 20 25 30 35

C 1 2 2 4 0.5 1 0.7 1.5 (µM)

*

Dox

Cas B Cas D Cas F Cas X

C

Mi to ch o n d ri a l depolar iz a tion ( % )

(7)

at lower concentrations after 24 h (1

l

M, 7.5 ± 0.6%; 2

l

M, 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% and 33.2 ± 1.8%) of incubation at higher doses (2 and 4

l

M, for Cas B and Cas D, respectively). In parallel, Dox also produced fragmenta-tion as early as 6 h (10.9 ± 2.3%) of incubafragmenta-tion. After 12 h and 24 h, fragmentation levels increased significantly (33.4 ± 2.7% and 43.6 ± 4.3%, respectively) (p< 0.01) (Fig. 4)

3.2.3. Mitochondrial depolarization

Only Cas F (0.5

l

M, 5.4 ± 0.3%) altered the mitochondrial trans-membrane potential of leukemia cells at lower concentrations in comparison 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

l

M (4.4 ± 0.8%, 5.4 ± 0.6% and 13.0 ± 1.2%), D 4

l

M (16.8 ± 17%, 2.3 ± 0.3% and 9.4 ± 1.0%) and F 1

l

M (8.5 ± 0.7%, 2.9 ± 0.4% and 10.7 ± 0.9%) led to depolarization after 6, 12 and 24 h, respectively. On the other hand, Cas X 1.5

l

M and Dox 0.6

l

M revealed substantial results only after 12 h (5.5 ± 0.4% and 25.7 ± 4.5%) and 24 h (23.2 ± 1.7% and 22.7 ± 1.9%), respectively (p< 0.01) (Fig. 4).

3.2.4. Cell cycle arrest in G0/G1phase

Treated and untreated cells during 6, 12 and 24 h were labelled with PI to measure DNA quantity by flow cytometry. Following analyzes byModFit LT 3.1. software, it was noted that only Cas X (0.7 and 1.5

l

M) after 24 h of incubation was capable to arrest cells in G0/G1phase (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). Dox was 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, were chosen to explore two hallmarks of apoptosis: caspase activation and phosphatidylserine externalization[34].

3.2.5. Caspases-8/-9 and -3/-7 activation

Both the molecules Cas F and X were able to activate the initi-ating caspases-8 and -9. In relation to caspase-8, both compounds led to viable cells’ reduction (55.2 ± 2.5% and 47.4 ± 6.1%, Cas F 0.5 and 1

l

M; 30.7 ± 6.2% and 26.1 ± 6.6%, Cas X 0.7 and 1.5

l

M) and apoptotic cells’ increasing (35.1 ± 5.7% and 42.7 ± 9.2%, Cas F 0.5 and 1

l

M; 63.1 ± 5.0% and 65.9 ± 8.4%, Cas X 0.7 and 1.5

l

M) in Table 3

Effects of clerodane diterpenes [Casearin B (Cas B), Casearin D (Cas D), Casearin X (Cas X) and Caseargrewiin F (Cas F)] isolated fromCasearia sylvestrisleaves on the cell cycle of leukemic 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 (0.6lM) 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 software house).

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 ± 0.7 Dox 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 ± 0.6 2.0 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 ± 0.9 4.0 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 ± 1.0 1.0 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 ± 0.6}

1.5 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}

(8)

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

Caspase-9 assessment showed that Cas F 0.5

l

M (43.2 ± 0.4%, 46.4 ± 0.4% and 10.7 ± 1.2%), F 1

l

M (40.1 ± 1.0%, 69.2 ± 19.0% and 8.3 ± 4.7%), Cas X 0.7

l

M (44.0 ± 4.0%, 41.3 ± 9.0% and 9.6 ± 0.2%) and X 1.5

l

M (21.7 ± 7.0%, 71.8 ± 7.5% and 6.5 ± 5.0%) decreased viability 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 cell number of viable cells and increased the occurrence of early and apoptotic cells (p< 0.01), though these changes have been found only at higher doses of Cas F (1

l

M, 12.6 ± 2.9% and 17.6 ± 3.7%) and in both concentrations of Cas X (0.7

l

M, 12.3 ± 3.3% and 19.1 ± 1.6%; 1.5

l

M, 31.1 ± 9.4% and 42.1 ± 8.9%), indicating caspas-es-3/-7 activation in comparison with control (2.1 ± 0.2% and 1.1 ± 0.3%), respectively (Fig. 6). Cas X was able to augmented necrosis (0.7

l

M, 13.8 ± 4.4%; 1.5

l

M, 8.8 ± 1.6%). Dox (0.6

l

M) 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 externalization Phosphatidylserine externalization was assessed after 6, 12 and 24 h of incubation with Cas F and Cas X.

Dox (0.6

l

M) 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 at higher concentrations (Cas F 1

l

M, 83.5 ± 1.7%; Cas X 1.5

l

M, 54.7 ± 2.5%) compared to the control (97.5 ± 0.6%) (Fig. 7A) and increased the number of Cas F 1

l

M-treated cells in early apoptosis (Fig. 7B) (9.7 ± 1.1%) and both concentrations of Cas X (0.7

l

M, 7.9 ± 0.7%; 1.5

l

M, 17.6 ± 5.6%) (p< 0.01). Substantial levels of late apoptosis were seen after 6 h of incubation with Dox (1.9 ± 0.7%) and in 24 h of treatment with Cas X 1.5

l

M (18.9 ± 2.6%) (Fig. 7C). Low levels of necrosis were detected only after 24 h in the highest doses (Cas F 1

l

M, 2.1 ± 0.2%; Cas X 1.5

l

M, 9.4 ± 0.4%) (Fig. 7D,p< 0.01).

3.2.7. Reactive oxygen species

All substances (FC, Cas B, D, F and X) were studied regarding the ROS generation after 1 and 3 h of incubation. None of them was capable to induce ROS production in comparison with negative control (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 plasma membrane bound (Figs.7A,8A and9A). Cells treated with Cas F (0.5 and 1

l

M) and Cas X (0.7 and 1.5

l

M) presented morphologi-cal features of death by apoptosis after 6 and 12 h of incubation.

C Dox 0.5 1 0.7 1.5

0 20 40 60 80 100

(µM) Cas X

Cas F

*

_*

*

A

Viable cells Apoptotic cells Necrotic cells

C

a

sp

as

e

-8

ac

tiv

a

tio

n

(%

)

C Dox 0.5 1 0.7 1.5

0 20 40 60 80 100

(µM)

Cas X Cas F

*

_*

*

_*

*

B

Necrotic cells

Viable cells Apoptotic cells

Ca

sp

ase-9 act

iv

a

tio

n

(%

)

(9)

C Dox 0.5 1 0.7 1.5 0

20 40 60 80 100

(µM)

Viable cells Early apoptosis Late apoptosis Necrosis

*

Cas X

Cas F

*

* *

*

* *

C

asp

ase

s-

3/-7

act

ivat

io

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 fromCasearia 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 the tested substances. Doxorubicin (0.6lM) was used as positive control (Dox). 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.}

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

0 20 40 60 80 100

*

* *

*

C

Cas X Cas F

0.5 1 0.7 1.5

*

Dox

A

(µM)

V

iab

le ce

lls

(%

)

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

0 2 4 6 8 10

*

D

C

Cas X Cas F

0.5 1 0.7 1.5

Dox (µM)

N

ecr

o

ti

c

cel

ls

(%

)

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

0 10 20 30 40 50 60

C

Cas X Cas F

0.5 1 0.7 1.5

Dox (µM)

*

* _*

*

B

Ea

rl

y

ap

o

p

to

ti

c

ce

lls

(%

)

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

0 5 10 15 20 25

C

Cas X Cas F

0.5 1 0.7 1.5

Dox (µM)

*

C

La

te

ap

opt

o

ti

c

ce

lls

(%

)

(10)

Chromatin condensation, nuclear fragmentation, karyolysis, cellu-lar shrinking and rarefaction in presence of membrane integrity were 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 showed occurrence of cytoplasmic vacuoles (Fig. 9C–F, respectively). On the other hand, a typical necrosis characteristic – membrane disin-tegration – was found after 12 h exposure in presence of Cas F 1

l

M (Fig. 9F) and after 24 h of treatment with Cas F and Cas X (Fig. 10C–F).

Dox-treated cells exhibited morphological alterations only after 24 h (chromatin condensation, nuclear fragmentation, cellular shrinking and rarefaction) (Fig. 10B). Shorter exposures with Dox revealed parallel cell morphology to the negative control, though rare mitotic cells had been found (Figs.8B and9B).

4. Discussion

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

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

Knowing the pharmacological and popular importance ofC. syl-vestris [38]and that mammal cells are commonly used tools to evaluate the cytotoxic properties of new compounds[5,30,39,40], we firstly detailed the antiproliferative activity of a fraction rich in casearins (FC) and its major components (Cas B, D, F and X), all obtained from leaf ethanol extracts. FC showed encouraging outcomes and IC50values lower than 0.5

l

g/mL against all tumor lines. According to the American National Cancer Institute (NCI, USA), the IC50limit to consider an active crude extract for further purification is a value lower than 30

l

g/mL [41]. Subsequently, among the isolated clerodanes, Cas F was the most active molecule, showing IC50values lower than 1

l

M, followed by Cas X.

In last years, bioguided phytochemical investigations led to expand of new clerodane diterpenes fromCaseariaspecies, 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 cruzi amastigotes [42,43], Plasmodium falciparum strains resistant to chloroquine[44], Chinese hamster V-79 cells, fibroblasts (L-929) Dividing cells _{Chromatin condensation}

Cell shrinking

A B

D

C

E

_F

(11)

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 uponin vivo assess-ments, the activity on Sarcoma 180 cells was determined by the Alamar Blue assay. Sarcoma 180 tumor is an useful model extre-mely utilized in research of natural products with antineoplastic action[5,26,33,40,48]. Herein, once more, it was noted that Cas F and FC were the most effective substances against S180 cells, con-firming the antitumoral potential of all studied substances. Previ-ously, ethanolic extract from C. sylvestrisleaves reportedin vivo antitumor 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 fromC. sylvestrisleaves – isobutyl gal-late-3,5-dimethyl ether (IGDE) and methyl galgal-late-3,5-dimethyl ether (MGDE) – also showed significant chemotherapeutic poten-tial against Ehrlich and Lewis lung cancer ascite tumor cells[22].

The bioactivities displayed by caseargrewiins, casearins and other clerodane diterpenes have a structure–activity relationship basically attributed to the diterpene skeleton substitutions at positions C-2 (R1_{), C-6 (R}4_{), C-7 (R}5_{), C-18 (R}2_{) and C-19 (R}3_{) or}

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

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

A B

D

C

E

_F

Dividing cells Chromatin condensation

Cell shrinking

Destabilization of the plasma membrane

Vacuolization of the cytoplasm

(12)

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

To determine the mechanism responsible for the cytotoxic effects, we firstly evaluated the ability of substances to inhibit DNA 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 with antiproliferative activity, since it is anin vitroreliably nonradioac-tive and immunocytochemical method widely used to determine the percentage of cell division [30,50]. All substances inhibited BrdU incorporation after 24 h of incubation, revealing high anti-proliferation activity in perceptual values similar to the positive control Dox (Cas F and X), confirming the results obtained with the MTT and Alamar Blue assays.

Biochemical and morphological alterations were also investi-gated using flow cytometry and May-Grünwald–Giemsa staining to determine the cellular death pattern. In general, when death is elicited by apoptosis pathways, different factors can act as activa-tors, including chemotherapeutic agents, ionizing radiation, DNA damage, heat shock proteins, unfolded proteins, deprivation of growth 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 leads to 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-G1cells)[51]. All clerodanes caused DNA disintegration in a concentration-dependent manner, while membrane integrity has been significantly altered only at highest concentrations and following 12 or 24 h exposure. This membrane integrity maintenance validates the results obtained by Santos et al.[23], emphasizing that cytotoxic action of the molecules is not related to direct cell membrane injury.

DNA internucleosomal fragmentation is regularly attributed to caspases, enzymes belonging to the family of Ca2+_{- and Mg}2+ -dependent cysteine proteases, which recognize and cleave sub-strates exhibiting aspartate residues. There are 14 human caspases and six of them (executing caspases3, 6, 7; initiator caspases8, -9, -10) are involved in the apoptotic process[34,52]. Herein, it was found 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 caspase activation was confirmed by mitochondrial potential decrease noted after exposure to the diterpenes, proposing mitochondrial

A

D

C

E

F

μ

B

Dividing cells Chromatin condensation

Cell shrinking

Destabilization of the plasma membrane

Vacuolization of the cytoplasm

(13)

membrane permeabilization and cytochrome c release. Cyto-chromecbinds 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 as effective DNase to slice the genomic DNA into nucleosomes, pro-ducing fragments of 180–200 base pairs, nuclear and cellular reduction 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 from Casearia membranacea also presented cytotoxicity on PC-3 cells and induced caspases-8, -9 and -3 activation and Bid cleavage[45]. To better comprehend the kind of cell death, we evaluated the PS externalization, a gold standard test used to confirm apoptosis triggering[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 plasma membrane, 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-AAD while both events overlapped in necrotic cells, findings detected in dotplot 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 cell death by apoptosis (karyorrhexis, nuclear rarefaction, cell shrink-age and extensive cytoplasmic vacuoles) were expressly detected in leukemia-treated cells after 12 h. In higher concentrations and longer exposure periods, plasma membrane disintegration, unbal-anced caspase activation and higher red fluorescence levels expressed in flow cytometry investigations exhibited after PI or 7-AAD labeling were seen, supposing that dose-dependent effects are also consistent with late apoptosis or secondary necrosis [31,55]. Previously studies with Cas X also reported characteristics of 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 determines how cell death occurs. If the necrosis prevails, early lesions on the plasma membrane occur instead of cell shrinking[32]. Therefore, depending on the concentration used, many different processes may be influenced and/or altered, suggesting that dose-dependent regulation of cellular process reflects signalization triggered by bioactive compounds as stated here and by others[4,30].

Anticancer drugs that kill tumor cells by apoptosis possess physiological advantages due to externalized PS specific recogni-tion and cellular removal by macrophages, preventing tissue dam-age resulting fromin situcell lysis[31,56]. However, most of the chemotherapeutic agents interfered with replication and mitotic spindle formation[37,57,58]. Among all substances studied, only Cas X caused cell cycle arrest in G0/G1phase after 24 h of incuba-tion. Kauranic diterpenes promote arrest in G1phase and, conse-quently, reduction of cells in S and G2 phases [59]. Comparable results were found with Cas X, indicating that these diterpenes might 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 strains causing DNA acetylation and cell death[9].

5. Conclusions

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

effective antiproliferative activity predominantly mediated by apoptosis, highlighting clerodane dipertenes as promising lead antineoplastic compounds.

Conflict of Interest

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

Transparency Document

TheTransparency documentassociated with this article can be found in the online version.

Acknowledgements

We wish to thank the Brazilian agencies Conselho Nacional de Desenvolvimento 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 França dos Santos and Maria de Fátima Teixeira for technical assistance and Priscila Murolo, a skilled author of English language papers, for her help with editing of the manuscript.

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