Cytotoxicity of the coagulant Moringa oleifera lectin (cMoL) to B16-F10 melanoma cells

Accepted Manuscript Cytotoxicity of the coagulant Moringa oleifera lectin (cMoL) to B16-F10 melanoma cells

Luciana de Andrade Luz, Franco Aparecido Rossato, Rute Alves Pereira e Costa, Thiago Henrique Napoleão, Patrícia Maria Guedes Paiva, Luana Cassandra Breitenbach Barroso Coelho PII: DOI: Reference:

S0887-2333(17)30168-6 doi: 10.1016/j.tiv.2017.06.019 TIV 4038

To appear in:

Toxicology in Vitro

Received date: Revised date: Accepted date:

24 February 2017 16 June 2017 19 June 2017

Please cite this article as: Luciana de Andrade Luz, Franco Aparecido Rossato, Rute Alves Pereira e Costa, Thiago Henrique Napoleão, Patrícia Maria Guedes Paiva, Luana Cassandra Breitenbach Barroso Coelho , Cytotoxicity of the coagulant Moringa oleifera lectin (cMoL) to B16-F10 melanoma cells, Toxicology in Vitro (2017), doi: 10.1016/ j.tiv.2017.06.019

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ACCEPTED MANUSCRIPT Cytotoxicity of the coagulant Moringa oleifera lectin (cMoL) to B16-F10 melanoma cells

Luciana de Andrade Luza,*, Franco Aparecido Rossatob, Rute Alves Pereira e Costab, Thiago Henrique Napoleãoa, Patrícia Maria Guedes Paivaa, Luana Cassandra Breitenbach Barroso

Departamento de Bioquímica, Centro de Biociências, Universidade Federal de Pernambuco,

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a

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Coelhoa,*

b

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50670-901 Recife, Pernambuco, Brazil.

Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual

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de Campinas, 13083-887 Campinas, São Paulo, Brazil.

E-mail

addresses:

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*Corresponding author. Tel: +558121268540; fax: +558121268576. [email protected]

Luz),

[email protected]

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(L.C.B.B. Coelho).

(L.A.

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ACCEPTED MANUSCRIPT ABSTRACT

Moringa oleifera seeds are used in alternative medicine to treat inflammation, tumors and bacterial and protozoan infections, for example. The seeds contain lectins, which are carbohydrate-binding proteins with several biological properties including cytotoxicity to

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cancer cells. In this work, we examined the cytotoxicity of the coagulant M. oleifera lectin

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(cMoL) on B16-F10 murine melanoma cells. cMoL cytotoxic effects were evaluated through

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trypan blue assay and flow cytometry analysis. Mitochondrial superoxide levels and activation of caspases 3, 8 and 9 were measured. cMoL (1.5–16 µM) reduced viability and

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caused cell death of B16-F10 cells with an IC50 of 9.72 µM. Flow cytometry analysis indicated induction of necrosis and suggested the presence of cells in late apoptosis.

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Specificity for tumor cells was observed since death of normal human fibroblasts (GN) was not higher than 20% in treatments with cMoL from 1.5 to 16 µM. Microscopy images

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revealed rounded shape and reduction of volume in B16-F10 cells treated with cMoL. cMoL

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increased mitochondrial ROS production and promoted caspases 3, 8 and 9 activation in B16F10 cells, indicating the activation of apoptosis-related pathway. In conclusion, this study

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demonstrates that cMoL is cytotoxic to B16-F10 cells, which stimulates more investigation on

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the anticancer potential of this lectin.

Keywords: Moringa oleifera; lectin; cytotoxicity; melanoma.

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ACCEPTED MANUSCRIPT 1. Introduction

Moringa oleifera Lam. (Moringaceae) is a medium-sized tree autochthonous from northeastern India and widely distributed worldwide throughout the tropics and subtropics [1]. In developing countries, it is a vegetable and medicinal plant, and is source of oil with uses in

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industry and culinary [2]. It has been reported the pharmacological potential of M. oleifera as

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source of anxiolytic, antiepileptic, anticancer, antiulcer, anti-obesity and anti-inflammatory

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agents [3–9].

M. oleifera seeds are broadly used in water treatment for human consumption due to

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their coagulant properties [2]. Two lectins deemed cMoL (coagulant M. oleifera lectin) and WSMoL (water-soluble M. oleifera lectin) are among the coagulant proteins present in the

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seeds [10,11]. Lectins are proteins of non-immune origin that interact reversibly and specifically with sugars; this carbohydrate-binding effect endows diverse biological properties

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to lectins, including the ability to alter the functioning of cells [12]. The cMoL is a heat-stable

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and basic lectin (theoretical pI of 11.67) with a native molecular mass of 30 kDa, composed by subunits with 101 amino acids, and belonging to the α/β tertiary structure class. It is able to

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recognize both monosaccharides and oligosaccharide moieties of glycoproteins, with highest affinity for glucose, galactose, asialofetuin and azocasein [10,13]. This lectin demonstrated

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anticoagulant properties on hemostatic parameters of human blood coagulation [13] as well as insecticidal activity against Anagasta kuehniella [14]. However, no study on the anticancer properties of cMoL was performed until now. Mitochondria play an important role in cell death process since they regulate energy production and execution of cell death [15]. Mitochondria are also important in generating reactive oxygen and nitrogen species and are involved in pathways linked to cell proliferation, intracellular death signaling and disease pathogenesis [16]. Lectins have been able to interfere 3

ACCEPTED MANUSCRIPT with mitochondria function causing cell death. The Cratylia mollis seed lectin induced death of Trypanosoma cruzi epimastigote by promoting mitochondrial Ca2+ overload and stimulation of reactive oxygen species (ROS) production followed by necrosis-like cell death [17]. The Canavalia virosa seed lectin was toxic to rat C6 glioma cells by disrupting the mitochondrial membrane potential [18]. The treatment of HT29 human colon adenocarcinoma

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cells with a lectin from Bothrops jararacussu venom lead to mitochondrial respiration

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decrease and increase of cytochrome c release [19].

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In the present work, we evaluated the in vitro cytotoxic effects of cMoL to B16-F10 murine melanoma tumor cells and to normal human fibroblasts (GN) as well as mechanisms

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involved in B16-F10 cell death. In the context of studies searching for positive effects against cancer cells, this cell line was selected since it is very aggressive and presents high capacity

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for metastatic dissemination [20]. Some of the experiments were also conducted with normal fibroblasts obtained from gingival biopsy, since they correspond to untransformed primary

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cells, which brings them very close to a physiological situation. In addition, these cells were

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chosen due to their different origin being appropriate to test the specificity of the lectin

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cytotoxicity.

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2. Materials and methods

2.1. Plant material

The seeds of M. oleifera were collected in Recife City, State of Pernambuco, northeastern Brazil and stored at -20°C. A sample of the collected material is archived as voucher specimen (number 73,345) at the herbarium Dárdano de Andrade Lima from the Instituto Agronômico de Pernambuco (Recife, Brazil). The authors have authorization from 4

ACCEPTED MANUSCRIPT the Instituto Chico Mendes de Conservação da Biodiversidade from Brazilian Ministry of the Environment for plant collection (number 38690).

2.2. Lectin preparation

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cMoL was obtained in accordance with the protocol described by Luz et al. [13]. Seed

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flour proteins were extracted with 0.15 M NaCl at 25°C for 6 h. The extract was treated with

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ammonium sulphate (0-60% saturation) at 25°C for 4 h and a precipitated fraction was obtained, dialyzed and applied (10 mg of protein) on a guar gel column (10 cm × 1.0 cm)

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previously equilibrated (20 mL/h flow rate) with 0.1 M NaCl. After washing step with 0.15 M NaCl, cMoL was eluted with 0.3 M NaCl. UV absorbance was used to monitor protein

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elution. cMoL concentration was evaluated according to Lowry et al. [21] using bovine serum albumin (31.25–500 µg/mL) as standard. Carbohydrate-binding ability was monitored by

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hemagglutinating activity assay performed according to Silva et al. [22].

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2.3. Cell culture

B16-F10 cells were obtained from American Type Culture Collection (Virginia, USA)

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and cultured in 75-cm2 plastic bottles (TPP, Trasadingen, Switzerland) in RPMI 1640 (Vitrocell, São Paulo, Brazil) supplemented with 10% fetal bovine serum (FBS), 100 µg/mL gentamicin, 100 IU/mL penicillin and 100 µg/mL streptomycin (Vitrocell, São Paulo, Brazil). Primary GN fibroblasts were obtained from normal gingival biopsy and kindly provided by Dr. Ricardo Della Coletta from the Universidade Estadual de Campinas. GN cells were grown in DMEM with high glucose supplemented with 10% FBS (Vitrocell, São Paulo,

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ACCEPTED MANUSCRIPT Brazil), 2 mM glutamine, 100 µg/mL gentamicin, 100 IU/mL penicillin, and 100 µg/mL streptomycin. The cells were kept at 37°C in a humidified atmosphere with 5% CO2.

2.4. cMoL effects on B16-F10 cell viability

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The effect of cMoL on cell viability was evaluated as previously described [23].

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Briefly, B16-F10 cells were plated in 6-well culture plates (3.5 x 104 cells per well) and after

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24 h, the medium was replaced and cells were treated with the lectin (1.5–16 µM). The concentrations were calculated considering a native molecular mass of 30 kDa for cMoL [10].

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After further 48 h, cells were washed in PBS, treated with a trypsin-EDTA solution (Vitrocell, São Paulo, Brazil), centrifuged at 1500 g for 4 min and resuspended in 300 µL of medium

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containing FBS. Cell viability was assessed using trypan blue dye (0.1%, w/v) added to aliquots of cell suspensions, and the percentages of stained (unviable) cells were determined

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microscopically. Cell viability in lectin treatments was expressed as the percentage of the

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control was above 95%.

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number of viable cells in control. The assay was considered valid only when cell viability in

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2.5. Flow cytometry analysis of cell death

Samples were analyzed in a FACSCalibur flow cytometer (BD Biosciences, USA) equipped with an argon laser and Cell-Quest software (version 4.1). For the cell death analysis, B16-F10 cells (106) from control or treated with cMoL (1.5–16 µM; 48 h) were incubated in labeling buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2 and 1.8 mM CaCl2) containing annexin V (Anx V) conjugated to FITC (1:500) and propidium iodide (PI; 20 µg/mL) at room temperature for 20 min in the dark [23]. Ten 6

ACCEPTED MANUSCRIPT thousand events were acquired for each sample and apoptosis was quantified as the percentage of Anx Vpos/PIneg while necrosis was quantified as the number of Anx Vneg/PIpos cells. GN cells treated or not with cMoL (1.5–16 µM; 48 h) were also evaluated for cell death.

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2.6. Microscopy

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B16-F10 melanoma cells (3.5 × 104 cells/mL) were treated with cMoL at 8 or 16 µM

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for 48 h. Then, the cells were photographed under a microscope Leica DFC360 FX with an

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increase of 20 x, using LAS AF software (Leica Microsystems, Germany).

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2.7. Determination of mitochondrial superoxide generation

B16-F10 cells (106) were treated with cMoL (8 µM) for 6 h, harvested, and stained

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with 5 µM MitoSOX (Molecular Probes, USA) for 10 min at 37°C in order to detect

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mitochondrial superoxide levels. MitoSOX fluorescence intensity was analyzed by flow

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cytometry (FACSCalibur, BD, San Jose, USA).

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2.8. Detection of caspases 3, 8 and 9 activation

To determine caspases 3, 8 and 9 activation, B16-F10 cells were treated with cMoL at 8 µM and, after 48 h, 106 cells were stained with the fluorescent markers FITC-DEVD-FMK, FITC-IETD-FMK or FITC-LEHD-FMK (1:300, Calbiochem, USA) in serum-free medium for 40 min at 37°C in a humidified atmosphere with 5% CO2. Then, cells were washed, resuspended in the same medium, and analyzed by flow cytometry according to the manufacturer’s instructions. B16-F10 cells treated with 1.25 µg/mL cycloheximide (Sigma7

ACCEPTED MANUSCRIPT Aldrich, USA) and 10 nM tumor necrosis factor α (TNFα; Peprotech, USA) for 24 h were used as a positive controls.

2.9. Statistical analysis

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Data were expressed as the mean ± the standard error of the mean (S.E.M.) of at least

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five independent experiments. Differences between means values were analyzed using one-

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way ANOVA followed by Tukey’s multi-comparison test. A p value < 0.05 was considered significant. The concentration of cMoL that causes 50% death of melanoma cells (IC 50) was

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calculated by probit analysis using the software Statplus LE (AnalystSoft, Canada).

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3. Results

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3.1. cMoL reduces cell viability and causes cell death on B16-F10 melanoma

cMoL was successfully isolated following the previous published protocol and showed

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hemagglutinating activity (256-1), revealing that its ability to bind carbohydrates was preserved. In the present study, the effect of cMoL on the viability and death of B16-F10 cells

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was followed using trypan blue exclusion assay and flow cytometry, respectively, after 48 h of exposure in culture. The treatment with increasing concentrations of cMoL progressively reduced cell viability (Figure 1) and caused cell death (Figure 2A). IC50 value was estimated to be 9.72 µM. To characterize the cell death, the rates of apoptosis and necrosis were analyzed by annexin V and PI staining. Figure 2A shows that majority of damaged cells in treatments with cMoL was stained with PI but a considerable subpopulation of PIpos cells was also positive for 8

ACCEPTED MANUSCRIPT annexin V staining. Since cMoL at 8 µM induced 47.6% ± S.E.M. cell death in B16-F10 cells, the treatment at this concentration was established for the following experiments. The effect of cMoL on normal fibroblasts (GN) was also analyzed. In contrast to results obtained with B16-F10 melanoma cells, cMoL did not cause death of more than 20% of GN cells after 48 h (Figure 2B). Both apoptotic and necrotic cells were detected.

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Marked morphologic alterations were observed by optical microscopy in B16-F10

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cells cultured in the presence of cMoL at 8 and 16 µM for 48 h (Figure 2C). Progressive

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decrease in cell number and morphological changes as rounded cells and reduction of cellular

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volume were observed.

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3.2. cMoL induces caspase activation and ROS production by B16-F10 cell line

Activation of caspases 3, 8 and 9 was detected in B16-F10 cells treated with cMoL

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(Figure 3), suggesting that this lectin induces apoptotic cell death in a caspase-dependent

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manner. Positive controls cycloheximide (1.25 µg/mL) and TNF-α (10 nM) promoted caspase 8 activation as expected. The evaluation of ROS production revealed that, after 6 h, cMoL

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4. Discussion

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promoted a two-fold increase of mitochondrial ROS level (Figure 4).

The results obtained in the present work show that cMoL causes cell death on B16F10 melanoma and was less cytotoxic to GN cells. This finding suggests that this lectin bears a greater recognition for malignant cells. Other lectins have demonstrated to be active on cancer cells with low toxicity to normal cells. For example, the Microgramma vacciniifolia rhizome lectin was toxic to lung carcinoma cell NCI-H292 cells (IC50 = 25.23 µg/mL) but did 9

ACCEPTED MANUSCRIPT not reduce the viability of human peripheral blood mononuclear cells (PBMCs) even when tested at 100 µg/mL [24]. Similarly, the lectin from Calliandra surinamensis leaf pinnulae reduced the viability of leukemic K562 cells (IC50 = 67.04 µg/mL) and breast cancer T47D cells (IC50 = 58.75 µg/ mL) without any cytotoxic effect on PBMCs [25]. This behavior can be explained by the binding specificity of the lectin regarding the differential expression of

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carbohydrates at the plasma membranes of normal and malignant cells. When the lectin is

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able to bind plasma membrane receptors and/or is internalized by endocytosis, it may trigger a

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variety of cellular events such as apoptosis, autophagy, necrosis or mitotic catastrophe [26]. Raz et al. [27] studied the composition of surfaces of melanoma cells, including B16-

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F10. They reported evidences of the presence of galactose and sialic acid and investigation through lectin binding showed that the B16-F10 surface was recognized by concanavalin A

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(glucose/mannose binding protein), soybean agglutinin (that recognizes galactose) and wheat germ agglutinin (N-acetylglucosamine-binding lectin). Santos et al. [10] showed that cMoL

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has higher affinity for the monosaccharides glucose and galactose. In addition, they proved

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that this lectin is able to recognize not only monosaccharides but also sugars present in the oligosaccharide moieties of glycoproteins. In this sense, cMoL may be recognizing glucose

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and galactose residues present in glycoconjugates found at B16-F10 cells surface and thus trigger intracellular events leading to cell death.

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Necrosis can be attributed as important cell death type induced by cMoL on B16-F10 cells due to the remarkable staining with PI. In addition, the presence of double staining for both annexin V and PI suggests cells in late apoptosis or necrotic cells already dead [28]. Annexin V can stain cells in advanced necrotic stage because it may enter the cell through the ruptured membranes and then access phosphatidylserine at the inner layer of the plasma membrane [29]. To shed some light on the death mechanisms promoted by cMoL, we

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ACCEPTED MANUSCRIPT investigated a hypothesis that part of the B16-F10 cells treated with the lectin entered in an apoptosis-related pathway by assessing the activation of caspases. Indeed, the activation of caspase-3 confirms apoptotic cell death induced by cMoL in B16-F10 cells and the activation of caspases 8 and 9 reflects the involvement of both extrinsic and intrinsic signaling pathways, respectively. The cascades involved in extrinsic and intrinsic

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pathways are different but both can be merged into caspase activation. To initiate the extrinsic

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pathway, it is necessary an extracellular stimuli such as UV, X-ray or a toxin. Then, the

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combination between Fas-L (Fas ligand) and Fas (membrane receptor) forms a death complex and results in the intracellular activation of initiator caspase-8 and executioner caspases 3 and

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6 [30]. cMoL should act as a potent stimulus for activation of extrinsic pathway. The activation of caspase-3 is involved in the cell death mechanism described for

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other lectins that induce apoptosis. The Polygonatum odoratum lectin is able to induce human lung adenocarcinoma A549 cells apoptosis with activation of caspase-3 mediated by the

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microRNA miR-15a-3p [31]. A lectin from the hemolymph of the millipede Gluttonous

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beauts induced apoptosis in MCF-7 cells through up-regulation of Bad and caspase-3 expression [32]. Similarly to cMoL, the lectin from Rhizoctonia bataticola activated extrinsic

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apoptotic pathway mediated by caspase-8 in leukemic cells Molt-4 and Jurkat; the authors also reported that this lectin was not active on normal lymphocytes [33].

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Numerous pro-apoptotic signals are stimulated by several factors, among them, increased ROS production, which in turn characterizes the condition of oxidative stress and can precede cell death [16,34]. Thus, we also evaluated whether cMoL stimulated the ROS production by mitochondria of B16-F10 cells, which was confirmed by the results obtained. The increased levels of ROS may result in oxidation of amino acids, polydesaturated fatty acids as well as cause DNA damage, leading cell to death [35]. A pro-oxidant status induced by Aronia melanocarpa juice is involved in the apoptosis induced in Jurkat cells [36] and the 11

ACCEPTED MANUSCRIPT Polygonatum cyrtonema lectin induces apoptosis and autophagy of melanoma A375 cells inducing the massive ROS accumulation in mitochondria [37]. Apoptosis is accompanied by cell rounding-up, retraction of pseudopodes, reduction of cellular volume (pyknosis), chromatin condensation, nuclear fragmentation (karyorrhexis), little or no ultrastructural modifications of cytoplasmic organelles, and plasma membrane

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cMoL also induced apoptotic death of B16-F10 melanoma cells.

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blebbing [38]. Some of the results obtained in microscopy analysis support the possibility that

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Although apoptosis and necrosis have often been treated as excluding events, it is now known that they have common sub-cellular sites and organelles, may share initiator and

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effector molecules and can co-operate in a balanced interplay [39]. Interesting, our results show that part of B16-F10 cells entered in a necrotic process when incubated with cMoL but

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we also found evidences that some cells also underwent apoptotic process. These findings stimulate future studies focusing on the evaluation of deeper molecular mechanisms aiming at

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understanding in detail the action of cMoL on B16-F10 cell death.

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Exogenous glycan-binding proteins (e.g. plant lectins) may establish multivalent interactions with transmembrane glycoproteins and transduce intracellular signals leading to

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cell death. The interaction of lectin with glycosylated cell death receptors may modulate positively or negatively the apoptotic machinery. These interactions may also affect signaling

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thresholds and receptor clustering as well as lead to lectin endocytosis [40]. The lectin from Artocarpus heterophyllus seeds was cytotoxic to NB4 (human myelocytic leukemia) cells strongly inducing reactive oxygen species generation and cell death was attributed to recognition by the lectin of a trimannosyl core of N-glycans containing a β1,6-GlcNAc branch linked to α1,6-mannose [41]. The cMoL may exert its cytotoxic effects on B16-F10 cells leading to drastic deregulation of cell homeostasis (resulting in necrosis) and triggering caspase activation and ROS production pathways. The initiation of these processes may 12

ACCEPTED MANUSCRIPT include internalization by the cell or interaction with glycosylated receptors, which remain to be defined in the future.

5. Conclusions

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The lectin cMoL showed cytotoxic effects on B16-F10 melanoma cells, with

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specificity for these cancer cells compared to normal human fibroblasts (GN). The study

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stimulates more investigation on the anticancer potential and possible therapeutic uses of

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cMoL.

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Conflict of Interest statement

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Acknowledgements

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The authors declare that there are no conflicts of interest.

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We are thankful to Dr. Anibal Eugênio Vercesi from the Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP)

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for support and scientific assistance. We are also grateful to Dr. Carlos Amilcar Parada and Raffaela Silvestre Ignarro, from UNICAMP, for assistance in optical microscopy. The authors express their gratitude to the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; 11/50400-0), to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for research grants (446902/2014-4) and fellowships (THN, PMGP and LCBBC). The Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco

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ACCEPTED MANUSCRIPT (FACEPE; APQ-0661-2.08/15) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for research grants and financial support are also acknowledged.

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Zhivotovsky B, Melino G. Classification of cell death: recommendations of the Nomenclature Committee on Cell Death. Cell Death Differ 2009;16:3-11. [39] Nikoletopoulou V, Markaki M, Palikaras K, Tavernarakis N. Crosstalk between apoptosis, necrosis and autophagy. Biochim Biophys Acta 2013;1833:3448–3459.

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Figure captions

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Figure 1. cMoL reduces B16-F10 melanoma cell viability. B16-F10 cells were treated with

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increasing concentrations of the lectin for 48 h. Cell viability was expressed as the percentage of viable cells in regard to control. Cell viability under control condition was above 95%.

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Values are mean ± S.E.M. of at least five independent experiments. A p-value < 0.05 was

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considered to indicate significance; p-value < 0.05 (*) and p-value < 0.001 (**).

Figure 2. cMoL induces cell death in B16-F10 melanoma cells (A) but not on GN normal fibroblasts (B). Cells were treated with increasing concentrations of cMoL for 48 h, stained with annexin V and propidium iodide. The percentages of necrotic (PI+) or apoptotic (AnxV+) cells were determined by flow cytometry. Values are mean ± S.E.M. of at least five independent experiments. *Significantly different from control at p < 0.05, Student’s t-test.

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ACCEPTED MANUSCRIPT (C) Representative images of control and cMoL-treated cells provided by a Leica DFC360 FX microscope. Bars represent 100 μm.

Figure 3. cMoL treatment induces caspase 3 (A), 8 (B) and 9 (C) activation. Caspase activation was determined by flow cytometry using FITC-DEVD-FMK after 48 h of treatment

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with the lectin. Values are mean ± S.E.M of at least 5 independent experiments; p-value <

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0.05 (*) and p-value < 0.001 (**).

Figure 4. Treatment of B16-F10 melanoma cells with cMoL increases mitochondrial

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superoxide levels. Cells (106/mL) were treated with cMoL for 6 h, then washed and probed with 5 µM MitoSOX at 37 °C for 10 min. Superoxide levels were analyzed by flow

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cytometry. Values are the means ± S.E.M. of at least five independent experiments.

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*Significantly different from control at p < 0.05 (*) and p-value < 0.001 (**) level.

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ACCEPTED MANUSCRIPT Highlights

 The cytotoxicity of the lectin cMoL on B16-F10 murine melanoma cells was evaluated.  cMoL reduced viability of B16-F10 cells and was low toxic to normal human

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fibroblasts.

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 Flow cytometry analysis revealed necrotic and late apoptotic cells

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 Rounded shape and volume reduction were observed in B16-F10 cells treated with cMoL.

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 cMoL increased mitochondrial ROS and activated caspases 3, 8 and 9 in B16-F10.

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