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Micron 59 (2014) 17–23

Contents lists available at ScienceDirect

Micron

j ourna l ho me page: www.elsev ier .com/ locate /micron

ltrastructural aspects of melatonin cytotoxicity on Caco-2ells in vitro

na Paula C. Batistaa, Terezinha G. da Silvab, Álvaro A.C. Teixeiraa,∗,aloma L. de Medeirosc, Valeria W. Teixeiraa, Luiz C. Alvesd,ábio A.B. dos Santosd, Eliete C. Silvac

Department of Animal Morphology and Physiology, University Federal Rural of Pernambuco, Rua Dom Manoel de Medeiros, s/n, 52171-900 Recife, PE,razilDepartment of Antibiotics, University Federal Rural of Pernambuco, Av. Prof. Moraes Rego, 1235, 50670-901 Recife, PE, BrazilDepartment of Histology and Embryology, University Federal Rural of Pernambuco, Av. Prof. Moraes Rego, 1235, 50670-901 Recife, PE, BrazilLaboratory of Immunopathology Keizo Asami (LIKA), and Research Center Aggeu Magalhães, CPqAM/Fiocruz, University Federal of Pernambuco, Av. Prof.oraes Rego, 1235, 50670-901 Recife, PE, Brazil

r t i c l e i n f o

rticle history:eceived 20 April 2013eceived in revised form 4 December 2013ccepted 4 December 2013

eywords:denocarcinomaltrastructureelatonin

ytotoxicityaco-2

a b s t r a c t

Colon adenocarcinoma is a disease expanding worldwide. Cancer of colon and rectum are among thetop ten most insidious types in Brazil. In vitro and in vivo studies have demonstrated the efficacy of thehormone melatonin to prevent and reduce tumor growth. However, there are only few studies addressingthe action of melatonin on Caco-2 cells. Thus, the cytotoxic effect of melatonin on the ultrastructure ofCaco-2 cells was investigated. The MTT colorimetric method was used to assess the cytotoxicity. A totalof 2 × 106 cells/mL were seeded in microplates and incubated at 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78 and0.0 (control) �g/mL of melatonin. For ultrastructural analysis concentrations with low, medium andhigh cytotoxicity plus the control were used for ultrastructural analysis. The concentrations 50, 1.56 and0.78 �g/mL of melatonin showed low, medium and high cytotoxicity, respectively. Ultrastructurally, thecontrol tumor cells were shown to be preserved. Caco-2 cells showed morphological changes at 50 �g/mL

of melatonin, with numerous vacuoles, mitochondrial degeneration and reduced glycogen. However,Caco-2 cells also showed altered morphology in treatments at 1.56 and 0.78 �g/mL of melatonin withcharacteristics of cells in degeneration by the presence of numerous vacuoles, absence of microvilli,mitochondrial degeneration and nuclear fragmentation. Thus, one can infer that concentrations of 1.56and 0.78 �g/mL of melatonin promote cytotoxicity in Caco-2 cells, which can probably be related to the

ygen

generation of reactive ox

. Introduction

Intestinal colon adenocarcinoma is one of the fastest growingiseases in the world (Konishi et al., 2010; Kannen et al., 2011a,b).

n 2009, 106,100 new cases were reported in the USA (Konishi et al.,010). The incidence of colon cancer from 1992 to 2007 led to 842eaths in a population of 2278 men and women diagnosed withon-metastatic colorectal cancer, which accounted for approxi-ately 37% of deaths among patients evaluated (Dehal et al., 2012).In Brazil, cancer has gained more and more importance in the

orbidity and mortality profile with an increasing life expectancyf the population which is becoming a public health issue.

∗ Corresponding author. Tel.: +55 81 33206389.E-mail address: alvaro@dmfa.ufrpe.br (Á.A.C. Teixeira).

968-4328/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.micron.2013.12.003

species (ROS).© 2013 Elsevier Ltd. All rights reserved.

Therefore, cancer of colon and rectum are among the top ten typesof cancer in Brazil affecting both males and females (INCA, 2012).

As an in vitro model, Caco-2, a human colon adenocarcinomacell line, retains many of the characteristics of normal enterocytes.After about five days of culture cells form monolayers and displaymicrovilli, express enzymes, receptors and transporters character-istic for intestinal epithelium, thus being a good model for studieson intestinal transport (Gonc alves et al., 2008).

The treatment of colon adenocarcinoma consists of surgery,chemotherapy and radiation, the latter two being therapies oftencombined with surgery. Surgical resection of the affected site andaccomplishment of a permanent colostomy are the most effec-tive therapies for colon cancer (INCA, 2003), but invasive and withstrong side effects.

Melatonin, N-acetyl-5-methoxy-tryptamine, is a smalllipophilic molecule secreted in a circadian fashion by the pinealgland (Macchi and Bruce, 2004; Claustrat et al., 2005). The liter-ature reports this hormone to inhibit tumor cell proliferation by

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Fig. 1. Percentage of viable Caco-2-cells after treatment with melatonin at different

8 A.P.C. Batista et al. /

ifferent mechanisms. In the case of hepatoma and lung cancer,elatonin acts on the specific membrane and nuclear receptors

s well as by restricting the transport of linoleic acid which is aumor growth factor (Blask et al., 2004).

In vitro and in vivo studies on the efficacy of physiological con-entrations of melatonin as well as exogenous administration of theormone have shown excellent results in prevention and reductionf certain types of tumor growth, in animal experiments (Sauert al., 2001; Blask et al., 2002; Medeiros et al., 2003). Studies onumans (Lissoni et al., 2003) with a variety of different cancersave also suggested the oncostatic action of melatonin (Reiter et al.,003; Bartsch and Bartsch, 2006; Jung and Ahmad, 2006).

There have been only a few clinical trials conducted using mela-onin in relation to colorectal cancer. These studies have not yieldedata to confirm the efficacy of melatonin for colorectal cancerGarcía-Navarro et al., 2007).

It was also found that increased colonic crypt cell proliferationad occurred in rats after pinealectomy (Callaghan, 1995), indicat-

ng that melatonin pathways were involved in tumorigenesis inhe colon (Dalio et al., 2006). Another study suggested that smallowel crypt cell hyperplasia occurred after the pinealectomy, buthe mechanism by which this occurred was not evaluated (Chent al., 2011). In a recent study, it has been shown that melatoninegulates preneoplastic patterns caused by constant light exposuren the colon (Callaghan, 1995).

Controlled clinical trials are still needed in order to establishelatonin’s role in the treatment of colon adenocarcinoma. Thus,

he present study examined the in vitro effects of exogenous mela-onin on the cytotoxicity and ultrastructure of Caco-2, a humanolon adenocarcinoma cell line.

. Materials and methods

.1. Site where experiments were performed

The research was conducted at the Department of Histologynd Embryology, Federal University of Pernambuco and Labora-ory of Immunopathology Keiso Asami (LIKA), Federal Universityf Pernambuco. The experimental protocol was approved by thenstitutional ethics committee under the no. 012/2012

.2. Cell culture

Cells of the human colonic lineage Caco-2 representing an ade-ocarcinoma were obtained from the INCA (Instituto Nacionalo Câncer). The DMEM culture medium (Sigma) supplementedith 1% (v/v) fetal bovine serum (GIBCO), 1% (w/v) 2.0 mM

lutamine (Sigma) and 1% (w/v) antibiotic solution (1000 IU peni-illin/mL + streptomycin 250 mg/mL) was used for maintenance ofhe cells.

.3. Melatonin

Melatonin was obtained from Sigma (St. Louis, MO, USA), dis-olved in ethanol (0.2 mL) and diluted in 0.9% saline solution0.8 mL) to obtain the following concentrations: 50, 25, 12,5, 6.25,.125, 1.56, 0.78 and 0.0 �g/mL (control).

.4. Cytotoxic activity of melatonin on Caco-2 cells

The cytotoxicity was assessed by the colorimetric method MTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium – SIGMA],

hich is based on the conversion of the tetrazolium salt

nto the colored formazan product, the concentration of whichan be determined spectrophotometrically. Cells were seeded2 × 106 cells/mL) with DMEM medium in 96-well microplates and

concentrations. Means followed by different letters indicating statistically signifi-cant differences according to the Wilcoxon–Mann–Whitney test (a: p = 0.0102, b:p = 0.0079; c: p = 0.0231, compared to controls).

incubated for 24 h at 37 ◦C in a humidified incubator with atmo-sphere of 5% CO2. After 24 h, the culture medium was removedand cells were incubated with 1.0 mL of different concentrationsof melatonin (50, 25, 12.5, 6.25, 3.125, 1.56, 0.78 and 0.0 �g/mL) (inquadruplicate). DMEM medium (1.0 mL) + ethanol (0.2 mL) + 0.9%saline solution (0.8 mL) (positive control) and containing pureDMEM (1.0 mL) (negative control) were also added. After 72 h ofincubation with melatonin, solutions from wells were removed byaspiration and 25 �L of MTT solution (5 mg/mL) were added. After3 h of incubation at 37 ◦C, MTT solution was aspirated and 100 �LDMSO were added for the solubilization of the formazan crystals.After solubilization, the absorbance was determined in a spec-trophotometer at 595 nm wavelength. The results were expressedas percentage of relative viability of cells to the negative controlgroup (Mosman, 1983). Cell preparations with a viability higherthan 95% were used for testing the cytotoxicity of Caco-2 cells inresponse to melatonin. Based on the cytotoxic analyses, melatoninconcentrations that showed low, medium and high cytotoxicity aswell as the control were used. Data were submitted to the nonpara-metric Kruskal–Wallis test, and means were compared by using theWilcoxon–Mann–Whitney test (P < 0.05).

2.5. Transmission electron microscopy

Cells were trypsinized and plated (1 mL of DMEM culture) for24 h at a concentration of 1 × 105. After forming a monolayer,cells were washed twice with sterile PBS and 1 mL of the con-centrations of melatonin which showed that high, medium andlow cytotoxicity was added to each well. The control was treatedwith ethanol (0.2 mL) and saline solution (0.9 mL). The culture platewas trypsinized and cells were centrifuged to form pellets. Pelletswere washed in PBS, pH 7.2, fixed in 2% glutaraldehyde and 4%paraformaldehyde in PHEM buffer, pH 7.2. After 1 h, the material

was washed in 0.1 M sodium cacodylate buffer, pH 7.2 and post-fixed in 1% osmium tetroxide, 0.8% potassium ferrocyanide and5 mM calcium chloride for 60 min at room temperature in the dark.Cells were then washed in the same buffer solution, dehydrated in

A.P.C. Batista et al. / Micron 59 (2014) 17–23 19

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ig. 2. Electron micrographs of Caco-2 cells of control. (A) Well-preserved cells wicrovilli and numerous ribosomes. (C) Mitochondria in the apical region and some

, mitochondria; arrows, ribosomes; asterisk, glycogen. Bars 1 �m.

cetone (ascending series) and embedded in epoxy resin. Ultra-thinections were obtained and collected on copper grids, contrasted in% uranyl acetate for 40 min and lead citrate for 5 min. The obser-ation was performed under the Transmission Electron Microscopearl Zeiss 900 (Oberkochen, Baden-Württemberg, Germany).

. Results

The cytotoxicity was inversely proportional to the melatoninoncentrations, the concentration 0.78 �g/mL caused the highestoxicity in Caco-2 cells followed by 1.56 �g/mL of melatonin.he concentration of 50 �g/mL melatonin showed the lowest

rge nuclei with euchromatic and heterochromatic areas. (B) Note the presence ofles. (D) Large reserves of glycogen granules. N, nucleus; n, nucleolus; mv, microvilli;

cytotoxicity and did not differ from the other concentrations orthe control (Fig. 1).

The ultrastructural appearance of tumor cells in the controlgroup showed well preserved cells forming a monolayer. Therewas the presence of microvilli, large nuclei with euchromatic andheterochromatic areas, well-organized cytoplasms with numerousribosomes, mitochondria in the apical region, some vacuoles andlarge reserves of glycogen granules (Fig. 2a–d).

In groups treated with melatonin there were no monolayers.In the group where Caco-2 cells showed less cytotoxicity towardmelatonin (50 �g/mL) there were changes in cell morphology, pres-ence of microvilli, large nuclei with peripheral heterochromatin

20 A.P.C. Batista et al. / Micron 59 (2014) 17–23

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ig. 3. Electron micrographs of Caco-2 cells treated with melatonin at a concentraeterochromatin areas. (B) Presence of microvilli. (C and D) Cytoplasmic vacuoles,ucleus; mv, microvilli; dm, degenerated mitochondria; v, vacuoles; asterisk, glyco

reas, numerous cytoplasmic vacuoles, mitochondrial degener-tion, absence ribosomes and reduction of glycogen reservesFigs. 3a–d). However, Caco-2 cells also showed altered morphol-gy by treatments with melatonin leading to medium and highytotoxicity (1.56 and 0.78 �g/mL melatonin, respectively), buthowing characteristics of cells in degeneration due to the pres-nce of numerous vacuoles, sometimes containing electron-lucentubstance, no microvilli, mitochondrial degeneration and nuclearragmentation (Fig. 4a–d).

. Discussion

Evaluating the cytotoxicity of drugs is important in the phar-aceutical industry to establish their efficiency and safety for

f 50 �g/mL. (A) Note changes in cell morphology and large nuclei with peripheralhondrial degeneration, absence ribosomes and reduction of glycogen reserves. N,ars 1 �m.

administration. In this context, Caco-2 cells are used as a modelto develop drugs which can be administered orally due to theirabsorption via enterocytes (Leonard et al., 2000; Rodriguez-Juanet al., 2001; Miret et al., 2004; Artursson et al., 2012).

In this work the cytotoxicity in Caco-2 cells was inverselyproportional to the melatonin concentrations tested, with 50and 0.78 �g/mL of the hormone causing the lowest respectivelyhighest cytotoxicity. The melatonin action in neoplastic diseases isoften attributed to its often dose-dependent antioxidant as well asendocrine effects (Cos and Sanchez-Barcelo, 2000; Bartsch et al.,2001).

The dosages used in this study are well above the amount pro-duced by the body (nanomolar concentrations), which accordingto Hevia et al. (2010) would be necessary to inhibit the growth ofmany tumor types. A stronger suppressive effect of melatonin on

A.P.C. Batista et al. / Micron 59 (2014) 17–23 21

Fig. 4. Electron micrographs of Caco-2 cells treated with melatonin at a concentration of 1.56 mg/mL (A and B) and 0.78 mg/mL (C and D). Note characteristics of cells ind -lucenN hout c

cpmscHtP(2

at2t

egeneration by the presence of numerous vacuoles, sometimes containing electron, nucleus; vl, vacuoles electron lucent; dm, degenerated mitochondria; arrow, wit

ell proliferation in slower growing melanoma cells from earlierassages had been previously noted in both micromolar andillimolar concentrations (Bartsch et al., 1986). Some cancer cells,

uch as androgen-independent prostate cancer cells or gliomaells, are insensitive to nanomolar concentrations of melatonin.owever, their growth is inhibited by millimolar concentrations of

he indole. LNCaP androgen-dependent and androgen-insensitiveC3 human prostate cancer cells show decreased proliferation60%) after six days of treatment with melatonin 1 mM (Sainz et al.,005; Martín et al., 2006).

In a very similar way studies showed that 2 �g/L melatonin

dministered in rodents via the drinking water were more effec-ive to inhibit the growth of breast and lung tumors compared to0 �g/L (Anisimov et al., 2003; Vesnushkin et al., 2006). Moreover,he melatonin absorption by the Caco-2 cells at 400 �g/mL showed

t substance, no microvilli, mitochondrial degeneration and nuclear fragmentation.ell surface microvilli; asterisk, nuclear fragmentation. Bars 1 �m.

that they result in no damage of the plasma membrane or leads todecreased viability (Hafner et al., 2009). This seemingly puzzlingobservation may be related to the ability of melatonin to promotethe generation of intracellular reactive oxygen species (ROS) whichare highly toxic to some types of tumors even at low concentrations(Bejarano et al., 2010).

Recent findings indicate that ROS are involved in the induction ofapoptosis in some cell types (Albertini et al., 2006; D’Agostino et al.,2007; Morgado et al., 2008; Radogna et al., 2009; Bejarano et al.,2009; Gonzolez et al., 2010). The ultrastructural analysis of Caco-2cells revealed changes characteristic of apoptosis by treatment with

1.56 and 0.78 �g/mL of melatonin mainly exemplified nuclear frag-mentation and mitochondrial degeneration. This means that ROSmay exert dual effects on cell growth and apoptosis of cancer cells:ROS may initiate the transformation of cells leading to mutations

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uring DNA replication (Gackowski et al., 2002), whereas in thoseells which already have undergone changes, ROS could play anmportant role in the initiation and execution of apoptosis (Wenzelt al., 2005; Kroemer et al., 2007).

The balance between ROS and antioxidant levels criticallyetermines apoptosis in cancer cells and exceeds the capacityf antioxidant defense system, due to an accelerated productionf mitochondrial ROS (Kroemer et al., 2007). According to Zhangt al. (2011), melatonin at micromolar concentrations induceshe mitochondrial ROS generation, leading to the activation ofaspase-9 and 3 and induction of cell death (Shankar et al.,007).

Therefore, one can infer that doses of 1.56 and 0.78 �g/mL ofelatonin promote cytotoxicity in Caco-2 cells, which can probably

e related to the ROS generation. This suggests that there is a corre-ation between supra-physiological determined concentrations of

elatonin and cytotoxic effect on certain types of tumors.

onflict of interest statement

The authors declare that there is no conflict of interest that couldffect the impartiality of the research reported.

cknowledgment

The authors like to thank the Coordination of Improvement ofigher Education Personnel – CAPES for the scholarship doctorate.

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