Author's Accepted Manuscript

Antiproliferative mechanism of the methano-

lic extract of Enterolobium cyclocarpum (Jacq.)
Griseb. (Fabaceae)
Abimbola Sowemimo, Luanne Venables, Mod-
eola Odedeji, Trevor Koekemoer, Maryna van
de Venter, Liu Hongbin

www.elsevier.com/locate/jep

PII: S0378-8741(14)00807-1
DOI: http://dx.doi.org/10.1016/j.jep.2014.11.023
Reference: JEP9143

To appear in: Journal of Ethnopharmacology

Received date: 2 September 2014

Revised date: 28 October 2014
Accepted date: 14 November 2014

Cite this article as: Abimbola Sowemimo, Luanne Venables, Modeola Odedeji,
Trevor Koekemoer, Maryna van de Venter, Liu Hongbin, Antiproliferative
mechanism of the methanolic extract of Enterolobium cyclocarpum (Jacq.)
Griseb. (Fabaceae), Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.
jep.2014.11.023

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Antiproliferative mechanism of the methanolic extract of Enterolobium cyclocarpum

(Jacq.) Griseb. (Fabaceae)

a*
Abimbola Sowemimo , Luanne Venables b, Modeola Odedeji a, Trevor Koekemoer b,

Maryna van de Venter b, Liu Hongbin c

a
Department of Pharmacognosy, Faculty of Pharmacy, University of Lagos, Nigeria;
b
Department of Biochemistry and Microbiology, Nelson Mandela Metropolitan University,
Port Elizabeth 6031, South Africa.
c
Key Laboratory of Marine Drugs, School of Medicine and Pharmacy, Ocean University of
China, 266003 Qingdao, People’s Republic of China.

*Correspondence

Dr Abimbola Sowemimo

Department of Pharmacognosy,

Faculty of Pharmacy

University of Lagos

Telephone: +234 803 936 3361

E-mail: asowemimo@unilag.edu.ng; bimsowemi@yahoo.com




 ABSTRACT

Ethnopharmacological relevance: Enterolobium cyclocarpum (Jacq.) Griseb. is a tropical

tree that has folkloric implications against many ailments and diseases including cancer.

Materials and methods: To explore the ethnopharmacological claims against cancer, the

cytotoxicity of the methanolic extract of the leaves, was investigated using the brine shrimp

lethality assay, MTT assay using cervical (HeLa) and breast (MCF7) cancer cell lines, cell

cycle analysis and Annexin V-FITC/PI assay.

Results: In the brine shrimp lethality assay, the extract showed cytotoxic activity with LC50

value of 31.63 µg/ml. Significant growth inhibition was observed in both cell lines with IC50

values of 2.07 ± 1.30µg/mL and 11.84 ± 1.18µg/mL for HeLa and MCF7 respectively. Cell

cycle analysis indicated that HeLa cells were arrested in the G2/M phase while MCF7 cells

arrested in the G1/G0 phase. The Annexin V-FITC/PI assay revealed phosphatidylserine

translocation in both cell lines and thus apoptosis induction upon treatment with the extract.

Conclusion: The study demonstrated the potential antiproliferative activity of E. cyclocarpum

thereby supporting the traditional claim and provides basis for further mechanistic studies and

isolation of active constituents.

Keywords: Cancer; Enterolobium cyclocarpum; Cell cycle analysis; Apoptosis




 1. Introduction

Enterolobium cyclocarpum (Jacq.) Griseb. (Fabaceae) is a tree of up to 20-25m tall

native to Mexico, Central America, West Indies and introduced to many tropical countries

including West Africa. In traditional medicine, a leaf poultice of the plant is used in the

treatment of inflammatory tumours while the syrup from the bark is used for the treatment of

cold and the gum as a bronchitis remedy (Burkill, 1995). Pharmacological investigations

revealed that the aqueous extract of the heartwood of the plant exhibited a lethal effect

against Incisitermes marginipennis (Latreille) (Raya-González et al., 2013) while the

ethanolic extract showed antifungal activity against Trametes versicolor, Coniophora

puteana, Trichoderma viride and Chaetomium globosum (Rutiaga Quinones et al., 1995).

Saponins from E. cyclocarpum have also been reported to have spermicidal activity as well as

an effect on the fermentation of Pennisetum purpureum in vitro (Primorac at al., 1985;

Rodríguez and Fondevila, 2012). D-limonene, terpineol, eugenol and d-(+)-pinitol have been

isolated from the heartwood (Raya-Gonzalez et al., 2008; Raya-González et al., 2013) while

the gum has been reported to contain ȕ-(1-->3)-galactan (León de Pinto et al., 1994).

The nutritive value of the leaf, seed, pod and fruit has been reported as well as the physico-

chemical characteristics of the seed oil (Babayemi, 2006; Folarin and Igbon, 2009).

Although the plant material has been long used to treat tumours, no scientific work has

been carried out to ascertain the claimed property hence the aim of this study is to investigate

the apoptotic effect of Enterolobium cyclocarpum on cervical and breast cancer cells.

2. Materials and methods

2.1. Plant material

The leaves of Enterolobium cyclocarpum (Jacq.) Griseb. (Fabaceae) were collected in

Ibadan, Oyo State, Nigeria. The plant specimen was identified and authenticated at the



Herbarium of the Department of Botany and Microbiology, University of Lagos, Akoka,

Lagos, Nigeria where a voucher specimen (FHI 2941) was deposited. The collected leaves

were oven dried at 40ºC and powdered. The powder (250 g) was extracted by maceration

with methanol at room temperature for 3 days with occasional shaking. The extract was

filtered and concentrated in vacuo at 40ºC using the rotary evaporator (Buchi, Switzerland).

2.2. Brine shrimp lethality assay

For this assay, the method of Meyer et al. (1982) was employed. The eggs of the brine

shrimp were purchased from a local pet shop and hatched in filtered sea water for 48 hr. 1 mL

stock solution of the extract (50, 500, 5000 µg/mL) was put into a test tube already calibrated

into 5 mL and made up to 5 mL with the filtered sea water to give final concentrations of

10,100 and 1000 µg/mL. Vincristine sulphate was used as the positive control and sea water

as negative control. Ten shrimp nauplii were added to each of the test tubes. The number of

shrimps which survived were counted using a magnifying lens and recorded after 24 h. All

experimental assays were done in triplicates. The LC50 was calculated using probit analysis

(Wardlaw, 1985).

2.3. Cytotoxicity assay

Cervical (HeLa) and breast (MCF-7) cancer cells cultured in nutrient medium (RPMI

1640 medium supplemented with 10% heat inactivated foetal bovine serum (FBS),

streptomycin (100 Ig/mL) and penicillin (100 IU/mL) were seeded at 30,000 cells/mL in 96-

well plates and left to attach overnight in a humidified 5% CO2 incubator at 37°C. Stock

solutions of the extract and melphalan were made in dimethylsulfoxide (DMSO) and further

diluted with culture medium to 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91 µg/ml and 100, 50

and 25 µM, respectively. These concentrations were applied to the cells. The final




concentration of DMSO never exceeded 0.25% (v/v). The cells were treated for 48 h after

which the medium was replaced with 200 ȝL MTT (Sigma) (0.4 mg/mL in RPMI 1640:10%

FBS). After a further 3 hr incubation at 37°C, the MTT was removed and the purple

formazan product dissolved in DMSO and absorbance measured at 540 nm using a BioTek®

PowerWave XS spectrophotometer (Winooski, VT, USA).

Kidney cells (Vero) established from normal adult African green monkey (Cercopithecus

aethiops) were used as a representative of normal cells (Yasumara and Kawakita, 1963). The

cells were cultured in DMEM medium supplemented with 10% foetal bovine serum and

antibiotics (100 IU/mL penicillin and 100 ȝg/mL streptomycin). Cells were seeded at a

density of 6,000 cells per well and incubated in a humidified 5% CO2 incubator at 37 °C. All

experiments were done in triplicate. IC50 values were calculated using GraphPad Prism 4.0

software.

2.4. Cell cycle analysis

HeLa and MCF-7 cells were seeded in 10 cm cell culture dishes (Nunc) at 1.15x105

cells cells/mL and incubated for 24 h at 37°C in a humidified incubator as previously

described to allow attachment of cells. Cells were then treated for 24 h using IC50 of the plant

extract. Melphalan was used as a positive control for this assay. DNA cell cycle analysis was

performed using the Coulter® DNA PrepTM Reagents Kit (Beckman Coulter). The assay was

performed as per kit instructions and the results analyzed on a Beckman Coulter Cytomics

FC500 flow cytometer. A minimum of 10,000 events were recorded for each sample.

MultiCycle software for cell cycle analysis was used to calculate the percentage of cells in

each of the cell cycle phases.

2.5. Apoptosis assay




 MCF-7 and HeLa cells were seeded at 100,000 cells/mL in 24-well plates separately

and treated with E. cyclocarpum ethanolic extracts (11.84 ȝg/mL and 2.07 ȝg/mL for MCF7

and HeLa cells, respectively). Cells were incubated for 24 and 48 h at 37 ºC. After the

incubation periods, cells were transferred to polypropylene tubes and cells collected by

centrifuging at 500 x g for 5 min at room temperature. Cells were washed in ice-cold

Dulbecco’s Modified Eagle’s Medium (DMEM), treated and stained according to the

protocol of the Annexin V-FITC Apoptosis Detection kit (Miltenyi Biotec). In brief, cells

were washed with cold DMEM and centrifuged at 500 x g for five min at 4 °C. Supernatant

was discarded and cell pellets resuspended in ice-cold 1X binding buffer. Annexin V-FITC (1

ȝL; 25 ȝg/mL) and PI (5 ȝL; 250 ȝg/mL) were added to each tube. Compensation control

tubes contained cells with Annexin V-FITC only, PI only and combination of Annexin V-

FITC and PI. Tubes were gently mixed and incubated on ice for 15 min in the dark. Samples

were read within 30 min on a Beckman Coulter Cytomics FC500 and a minimum of 20,000

events were recorded for each sample.

2.6. Statistical analysis

All experiments were carried out in triplicate. Data were analysed by two-tailed

student’s t-test and expressed as mean ± SEM. Results were considered statistically

significant if p < 0.05. IC50 values were determined using GraphPad Prism Version 4.0

(GraphPad Software, San Diego, USA). Cell cycle analysis results were analysed by using a

Multicycle version 4.0 software.

3. Results

3.1. Brine shrimp lethality assay




 The methanolic extract of Enterolobium cyclocarpum showed significant lethality

against brine shrimp. The LC50 value was 31.63 µg/ml (Table 1).

3.2. Cytotoxicity assay

Based on the significant activity observed against the shrimp larvae, the in vitro

cytotoxicity of the extract was evaluated at different concentrations against malignant cell

lines (human cervix carcinoma HeLa cells and human breast carcinoma MCF-7 cells) and

normal kidney cells (Vero) after 48 h of treatment. Melphalan was used as positive control.

Significant cytotoxic effect of the extract was observed with IC50 values for the HeLa and

MCF-7 cancer cell lines as 2.07 ± 1.30 ȝg/mL and 11.84 ± 1.18 ȝg/mL respectively (Table 1)

while that of the Vero cells was > 250 ȝg/mL and hence considered to be non-toxic.

3.3. Cell cycle analysis

Cell cycle progression and the possibility of induction of apoptosis in the cells were

analysed by flow cytometry. After 24 h of exposure, an arrest of the cell cycle in the G2/M

phase was noticed in HeLa cells (Fig. 1) and G1/ G0 phase in MCF-7 cancer cells (Fig. 2)

respectively.

3.4. Apoptosis assay

In order to determine whether the cytotoxicity induced by E. cyclocarpum is due to

apoptosis or necrosis, HeLa and MCF-7 cells were incubated with the extract for 24 h and the

percentage of cells undergoing apoptosis or necrosis was determined by dual staining with

Annexin-V FITC and PI (Table 2). An increase of apoptotic cells from 1.13 ± 0.21% to 12.27

± 3.16% and 1.6 ± 0.17% to 23.63 ± 8.03% was recorded in HeLa and MCF-7 cancer cells

respectively.



4. Discussion and conclusion

The brine shrimp lethality assay is considered as a reliable assay for preliminary

assessment of toxicity and it can be extrapolated for cell line toxicity (Meyer et al., 1982;

Anderson et al., 1991). It has been used as a preliminary tool for the detection and isolation of

bioactive compounds from plant extracts (Almeida et al., 2002; Ogunnusi and Dosumu,

2008) and from a pharmacological perspective there exists a good correlation between the

brine shrimp lethality assay and the detection of anticancer compounds from plant extracts

(Solis et al., 1993; Meyer et al., 1982; Mackeen et al., 2000). According to Meyer et al.

(1982), crude extracts and pure substances with LC50 values less than 1000 µg/ml in the brine

shrimp assay are considered to be cytotoxic. Therefore, based on the significant activity of

the extract of E. cyclocarpum against the shrimp larvae, further in vitro cytotoxicity of the

extract was evaluated against human MCF-7 breast and HeLa cervical adenocarcinoma cells

after 48 h of treatment using the MTT assay.

The MTT assay quantifies metabolically viable cells through their ability to reduce a

soluble yellow tetrazolium salt to blue-purple formazan crystals by the cleavage of the

tetrazolium ring by succinate dehydrogenase within the mitochondria. This formazan product

is impermeable to cell membranes and thus accumulates in viable cells (Mossman, 1983).

In this assay, significant inhibition of cell viability was observed in both cell lines with IC50

values of 2.07±1.30 µg/ml and 11.84±1.18 µg/ml for HeLa and MCF-7 respectively. In

contrast to these two cancer cell lines, an IC50 value in excess of 250
g/ml was obtained for

Vero cells, a non-tumour forming cell line. The high IC50 on the normal cells and low IC50

on cancer cells may be due to a lack of cytotoxicity on the normal cells and inhibition of

proliferation in the cancer cells respectively.

Cytotoxic activity of the aqueous extract of E. contortisiliquum against BAC1.2F5 murine

macrophage cell line as well as that of the alcoholic extract of E. timbouva against human



breast (MCF-7), colon (HCT116), larynx (Hep2), cervical (HeLa), prostate (PC-3) and liver

(Huh-7) carcinoma cell lines have been reported (Mimaki et al., 2003; Mimaki et al., 2004;

Gamal El-Din et al., 2014) and the results compare favourably with those obtained in this

study. This is however, the first report of the cytotoxic activity of E. cyclocarpum.

Apoptosis is said to be an important mechanism that balances cell division and cell

death for controlling the tissue kinetics (Kerr et al., 1972; Hengartner, 2000). Impairments in

apoptosis are related to cell immortality, carcinogenesis, and the induction of apoptosis in

neoplastic cells (Shinomiya et al., 1994). Most anti-cancer agents are reported to exert their

cytotoxic effects by inducing apoptosis in tumour cells (Kaufman, 1989).

Annexin V staining allows the identification of apoptosis

through binding to the exposed phospholipid phosphatidylserine

which occurs at the early stages of apoptosis. The addition of

PI to the assay allows one to distinguish between apoptosis

and necrosis by virtue of the integrity of the cell membrane.

The necrotic cells with increased permeability will be stained

by the vital dye PI (Dedoussis et al., 2005) whereas early

apoptotic cells will resist PI uptake.

In this study, treatment with the extract of E. cyclocarpum

gave a significant increase in the Annexin V stained positive

cells (A4 of Table 2) implying the fact that apoptosis was

triggered by the treatment with the extract in both HeLa and

MCF-7 cells.

DNA cell cycle analysis was performed in order to determine if the cytotoxic mechanism

involves cell cycle arrest and to determine which phase of the cell cycle the cells arrest in.

Cell cycle analysis via flow cytometry distinguishes between different cell cycle phases and


detects apoptotic DNA fragmentation, simultaneously, so that the proliferative (S + G2/M) as

well as the apoptotic (degraded DNA) effects of the crude plant extracts can be measured

(Nunez, 2001). In this study, the HeLa cells were arrested at the G2/M phase while in the

MCF7 cells, growth was arrested in the G1/G0 phase after treatment.

The presence of cells with a DNA content less than 2N (i.e. a sub-G1 peak which appears to

the left of the G1/G0 peak of the histogram) in HeLa cells treated with extract (Figure 1) but

not in MCF7 (Figure 2) cells, indicates an increase in DNA fragmentation in the HeLa cells

but not in MCF7 cells treated with E. cyclocarpum.

Although these findings appear to contradict the Annexin V data described above where it is

shown that E. cyclocarpum treatment produced a greater apoptotic

fraction in the MCF-7 cells, it should be emphasised that DNA

fragmentation is a late event in the apoptotic cascade. Therefore it would appear that while

E. cyclocarpum treatment induces apoptosis in both cell lines, the MCF-7 cells are more

resistant to DNA degradation. As caspases trigger the activation of endonucleases

responsible for DNA cleavage, it is tempting to speculate that the reported absence of

caspase-3 expression in MCF-7 cells may be related to the lack of DNA fragmentation

despite the initiation of apoptosis. The greater resistance of the MCF-7 cells is also reflected

in the fivefold difference in the respective IC50 values (Table 1). Apart from these

differences in DNA fragmentation, the striking dissimilarity in the cell cycle analysis profile

between E. cyclocarpum treated HeLa and MCF-7 cells further

indicate that the cytotoxic response to E. cyclocarpum

treatment is cell type dependent. These differential effects may be due to

other differences between the cell lines such as the presence of different surface receptors and

varying apoptotic sensitivity other than caspase 3.




 In conclusion, our results demonstrate that E.

cyclocarpum has cytotoxic effect and cell death is induced by

cell cycle arrest and apoptosis. Further studies are in

progress to isolate and identify the compounds responsible for

the observed activity.

Acknowledgement

This work has been supported by the African Laser Centre (Project number LHEAB01) and

the South African National Research Foundation (Grant number 81780).

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 LIST OF FIGURES

Figure 1: Cell cycle analysis of HeLa cells treated with E. cyclocarpum extract for 24 h

HeLa cells were treated with 40 µM Melphalan as positive control, 2.07 µg/mL of

E. cyclocarpum

Figure 2: Cell cycle analysis of MCF-7 cells treated with E. cyclocarpum extract for 24 h

MCF-7 cells were treated with 40 µM Melphalan as positive control, 11.84 µg/mL

of E. cyclocarpum




Table 1: Cytotoxic activity of Enterolobium cyclocarpum

Treatment Brine shrimp

HeLa cell MCF-7 cell Vero cells
lethality test
line line

LC50 (µg/ml) IC50 (µg/ml) IC50 (µg/ml) IC50 (µg/ml)

E. cyclocarpum (Jacq.) Griseb. 31.63 2.07 ± 1.30 11.84 ± 1.18 > 250

Vincristine sulphate 0.52 ND ND ND

Melphalan ND 40 µM 370 µM 172.8 µM

ND- Not determined




Table 2

HeLa cells MCF-7 cells

Treatment
A1 A2 A3 A4 A1 A2 A3 A4
% Necrosis % late apoptosis % Live % Apoptosis % Necrosis % late apoptosis % Live % Apoptosis

PI +/Annexin - PI +/Annexin + PI -/Annexin - PI -/Annexin + PI +/Annexin - PI +/Annexin + PI -/Annexin - PI -/Annexin +

Control 2.40 ± 0.1 1.13 ± 0.12 95.33 ± 0.23 1.13 ± 0.21 0.73 ± 0.25 1.07 ± 0.21 96.6 ± 0.17 1.6 ± 0.17

Melphalan 4.43 ± 1.19 3.53 ± 0.65 82.97 ± 1.90 9.1 ± 0.95 2.27 ± 1.5 1.53 ± 0.42 81.6 ± 2.19 14.67 ± 3.42

E. cyclocarpum 11.0 ± 3.8 5.7 ± 1.0 71.07 ± 2.48 12.27 ± 3.16 10.0 ± 4.59 2.4 ± 0.61 64.03 ± 3.13 23.63 ± 8.03

Table 2. Results obtained from dot plots of Annexin V-FITC/PI stained HeLa and MCF-7 cells after 24 h exposure to medium only (control),

Enterolobium cyclocarpum or 40 μM melphalan. Four columns represent necrotic cells (A1: Annexin V-negative; PI-positive), late apoptotic

cells (A2: Annexin V-positive; PI-positive), unstained/live cells (A3: Annexin V-negative; PI-negative) and early apoptotic cells (A4: Annexin

V-positive; PI-negative). A minimum of 20,000 events were read for each sample (n=3).
Figure 1
Figure 2