Cell Cycle Inhibition and Apoptosis Induction Activities of n-Hexane

Fraction of Cyperus rotundus L. Rhizome

Masfria

, Urip Harahap

, Denny Satria

Department of Pharmaceutical Chemistry,

Department of Pharmacology,

Department of Pharmaceutical Biology

Faculty of Pharmacy, University of Sumatera Utara

Keywords: Cell cycle, Apoptosis, Cyperus rotundus L., rhizome, n-hexane.

Abstract: Background: Breast cancer is one of the leading cause of death and the most common cancer type

amongst women in the world after cervic cancer. Objective: To evaluate the cell cycle inhibition

and apoptosis induction activities on T47D cell lines of n-hexane fraction (nHF) of Cyperus

rotundus L. rhizomes. Methods: Ethanol extract was obtained by maceration method and was

fractionated with n-hexane. Cytotoxic activity was examined with MTT assay, and cell cycle

inhibition, apoptosis induction and cyclin D1 expression were assessed with flow cytometry

method. Results: Cytotoxic activity of nHF was found to have IC

of 71.69 ± 0.34 µg/mL, nHF

at concentration 35 µg/mL caused accumulation in G

-G

and S phase accumulation (56.89% and

19.36%), increased early apoptosis (26.30%) and decreased expression of cyclin D1 (26.30%).

Conclusions: The results reveal that nHF of Cyperus rotundus L. rhizomes has cell cycle

inhibition and apoptosis induction activities. Our further study is to isolation compounds which

responsible for these activities.

1 INTRODUCTION

Breast cancer is one of the leading cause of death

and the most common cancer type amongst women

worldwide in 2012 (WHO, 2015). Breast cancer is

the second cause of cancer death in developed

countries after lung cancer. A recent study has

reported that breast cancer is the first in the

predicted new cancer cases, and the second most

incidence death cause of women suffering from

cancer in the United States (Siegel, et. al., 2015).

Cyperus rotundus L. (Cyperaceae) have been

used as the drug of stomachache, disorders bowel,

menstrual irregularities (Peerzada, et al., 2015).

Bioassay investigations indicated which the extract

of Cyperus rotundus L. exerts antioxidant,

antibacterial, insecticidal activity and its essential oil

have activity as antiradical, antimutagenic and

cytotoxic. Essential oil and steroids/ triterpenoids

could extracted with non polar solvent such as n-

hexane and chloroform (Hemanth, et al., 2014; Hadi,

et al., 2007; Vitaglione, et al., 2004; Lanciotti, et al.,

2004; Kilani, et al., 2008; Tenore, et al., 2011; Nam,

et al., 2016; Sonwa and Konig, 2001; Liu, et al.,

2016; Kilani, et al., 2007; Jirovetz, et al., 2004;

Memariani, et al., 2016). Cyclin D1 has an important

role in cell cycle process in G

-G

phase (Żurynń, et

al., 2016). This study aimed to determine cell cycle

inhibition and apoptosis induction activities of n-

hexane fraction of Cyperus rotundus L. rhizomes.

2 MATERIALS AND METHODS

2.1 Plant and Chemicals Material

Fresh rhizomes of Cyperus rotundus L. was

collected from Paya Tumpi, Aceh Tengah regency,

Nangroe Aceh Darussalam province, Indonesia.

Cyperus rotundus L. was identified in Herbarium

292

Masfria, ., Harahap, U. and Satria, D.

Cell Cycle Inhibition and Apoptosis Induction Activities of n-Hexane Fraction of Cyperus rotundus L. Rhizome.

DOI: 10.5220/0008361602920296

In Proceedings of BROMO Conference (BROMO 2018), pages 292-296

ISBN: 978-989-758-347-6

Medanense, Faculty of Mathematics and Natural

Sciences, University of Sumatera Utara. Chemicals

used were annexin-V (BioLegend), cyclin D1

antibody (Santa Cruz), distilled water, DMSO

(Sigma), [3-(4,5-dimethylthiazole-2-yl)-2,5diphenyl

tetrazolium bromide] (MTT) (Sigma), propidium

iodide reagent (BioLegend).

2.2 Preparation of Extract

Ethanol extract of Cyperus rotundus L. rhizomes

(10g) was repeatedly fractionated with n-hexane

(3x100 mL) at separating funnel. The supernatant

was collected, and then evaporated under reduced

pressure to give a viscous fraction and then dried on

water bath to dry (Satria, et al., 2015; Anggraeni, et

al., 2015; Hasibuan, et al., 2015).

2.3 Cytotoxicity Assay

The cells were treated with nHF. In this test, the

T47D cell line (cancer cells which isolated from a 54

years old woman which mutation on p53) was

grown in RPMI 1640 medium, medium containing

10% Fetal Bovine Serum (Gibco), 1% penicillin-

streptomycin (Gibco), and fungizone 0.5% (Gibco)

in a flask in a humidified atmosphere (5% CO

) at

C. The inoculums seeded at 1x10

cells/mL at an

optimal volume of 0.1 mL per well. After 24 h

incubation, the medium was discharged and treated

by EE. After incubation 24 h, the cells were

incubated with 0.5 mg/mL MTT for 4 h in 37

Viable cells reacted with MTT to produce purple

formazan crystals. After 4 h, SDS 10% as stopper

(Sigma) in 0.01N HCl (Merck) was added to

dissolve the formazan crystals. The cells were

incubated for 24 h in room temperature and

protected from light. After incubation, the cells were

shaken, and absorbance was measured using

microplate reader at λ 595 nm. The data which were

absorbed from each well were converted to

percentage of viable cells

(Harahap, et al., 2018;

Dalimunthe, et al., 2018; Satria, et al., 2017).

2.4 Preparation of Cells for Flow

Cytometry Analysis

T47D cells (5x10

cells/well) were seeded into 6-

well plate and incubated for 24 h. After that, the

cells were treated with nHF and then incubated for

24 h. Both floating and adherent cells were collected

in conical tube using tripsin 0.025%. The cells were

washed thrice with cold PBS and centrifuged 2500

rpm for 5 min. The supernatant was separated, while

the sediment was collected (Satria, et al., 2015;

Anggraeni, et al., 2015).

2.5 Cell Cycle Analysis

Cells were fixed in cold 70% ethanol in PBS at -

C for 2 h. The cells were washed thrice with cold

PBS resuspended and incubated in ethanol 70% for

60 min then centrifuged at 3000 rpm for 3 min,

and PI reagent (containing PI 40 µg/mL and

RNAse 100 µg/mL) added to sediment and

resuspended and incubated at 37

C for 30 min. The

samples were analysed using FACScan flow

cytometer (Dalimunthe, et al., 2017; Nugroho, et al.,

2014).

2.6 Apoptosis Analysis

Annexin V reagent was added to sediment and

suspended and incubated at 37

C for 30 min. The

samples were analyzed using FACScan flow

cytometer (Satria, et al., 2017; Handayani, et al.,

2017).

2.7 Cyclin D1 Expression

Sediment cells were fixed with ethanol 70% stand

for 2 h in -20

C and cyclin D1 antibody was added

and incubated at 37

C for 10 min. The samples were

analyzed using FACScan flow cytometer (Żurynń, et

al., 2016).

2.8 Statistical Analysis

Data was expressed as mean ± SD. All statistics

were analyzed using the SPSS 21 software.

3 RESULTS AND DISCUSSION

3.1 Inhibitory Concentration 50% (IC

)

MTT method was used to determine percentage of

cell viability after incubation for 24 h. In every

treatment nHF was shown to inhibit cells growth.

The IC

value of nHF was 71.69 ± 0.34 µg/mL. The

cytotoxicity estimate of natural product is related to

content of active compound in these plants including

Cyperus rotundus L. This plant contain

monoterpenes, sesquiterpenes, and steroids

estimated as active compounds (Nidugala, et al.,

2016, Yadav, et al., 2012; Nidugala, et al., 2017).

Cell Cycle Inhibition and Apoptosis Induction Activities of n-Hexane Fraction of Cyperus rotundus L. Rhizome

293

3.2 Effect on Cell Cycle and Apoptosis

To evaluate the effect of nHF to increase cell death

by modulating cell cycle, we concentrated on it for

further studies using flow cytometry method. The

effect of nHF is given in Figure 1. Whereas

treatment of nHF in 35 µg/mL caused cell

accumulation at G

-G

and S phase accumulation

(56.89% and 19.36%) and for control cell (52.28%

and 16.80%). This fact was to indicate that nHF can

inhibit cell grow at G

phase. Recent study have

reported which monoterpenes exert anticancer

activities and as chemopreventive agents (Elson and

Yu, 1994; Kellof, et al., 1996; Crowell, et al., 1999).

Figure 1. Cell cycle analysis using flow cytometry. T47D cells were treated by nHF for 24h and stained using propidium

iodide. (a) control cells; (b) nHF 35 µg/mL

As shown in Figure 2, the cells in the upper

and lower right quadrants represent late apoptotic/

necrotic and early apoptotic cells, respectively. The

percentage of nHF at 35 µg/mL and control in early

apoptotic (26.11% and 3.65%), in late

apoptotic/early necrotic (4.26% and 2.96%), and in

late necrotic (4.88% and 2.41).

Figure 2. Apoptosis analysis using flow cytometry. T47D cells were treated by nHF for 24h and stained using Annexin-V.

(a) control cells; (b) nHF 35 µg/mL.

nHF has increased the cells to apoptosis in

early apoptosis if compared to control cell.

Apoptosis is processed in cells which cause

programmed cell death with alters on morphology,

membrane blebbing and chromatine (Ruddin,et al.,

1997).

3.3 Analysis of Cyclin D1 Expression

To evaluate the effect of nHF to decrease cyclin D1

expression, we concentrated on it for further studies

using the flow cytometry method. The effect of nHF

is given in Figure 3. Whereas treatment of nHF in 35

µg/mL caused cell accumulation in M1 area

(26.30%) and for control cell (11.19%). Evaluation

of cyclin D1 expression was performed using flow

cytometry method with cyclin D1 antibody as shown

in Figure 3.

Marker

Events

% Gated

% Total

Mean

Median

All

16415

100.00

82.08

259.35

28.41

223.00

GO-G1

8581

52.28

42.91

196.41

6.51

194.00

S-phase

2757

16.80

13.79

273.57

9.08

274.00

G2-M

4942

30.11

24.71

354.60

5.45

356.00

File: KS T47D CC.001

Total Events: 20000

X Parameter: FL2-A FL2-Area (Linear)

Marker

Events

% Gated

% Total

Mean

Median

All

20000

100.00

295.38

55.50

249.00

845

4.23

15.29

195.18

0.00

GO-G1

8607

43.04

196.42

6.53

194.00

S-phase

2800

14.00

273.64

9.09

274.00

G2-M

5365

26.82

355.52

5.54

357.00

2478

12.39

628.52

29.57

570.00

File: KS T47D CC.001

Total Events: 20000

X Parameter: FL2-A FL2-Area (Linear)

GO-G1

S-phase

G2-M

GO-G1

S-phase

G2-M

Marker

Events

% Gated

% Total

Mean

Median

All

13895

100.00

69.47

224.40

27.77

200.00

GO-G1

7905

56.89

39.52

190.31

7.71

192.00

S-phase

2690

19.36

13.45

253.61

8.51

252.00

G2-M

2164

15.57

10.82

335.50

7.64

334.00

File: HEX RT1/2 T47D CC.005

Total Events: 20000

X Parameter: FL2-A FL2-Area (Linear)

Marker

Events

% Gated

% Total

Mean

Median

All

20000

100.00

240.05

80.35

198.50

4414

22.07

44.48

145.79

0.00

GO-G1

7945

39.73

190.35

7.72

192.00

S-phase

2828

14.14

253.95

8.55

253.00

G2-M

2581

12.90

336.77

7.60

336.00

2403

12.02

642.85

34.60

564.00

File: HEX RT1/2 T47D CC.005

Total Events: 20000

X Parameter: FL2-A FL2-Area (Linear)

GO-G1

S-phase

G2-M

GO-G1

S-phase

G2-M

File: KS T47D APOP.001

Patient ID: 0417.18

Acquisition Date: 17-Apr-18

Gate: No Gate

Total Events: 20000

Quad Location: 64, 79

Quad

% Gated

% Total

1.34

0.06

98.41

0.18

File: KS T47D APOP.001

Patient ID: 0417.18

Acquisition Date: 17-Apr-18

Gate: No Gate

Total Events: 20000

Region

% Gated

% Total

98.37

0.19

0.06

1.38

File: HEX RT1/2 T47D APOP.005

Patient ID: 0417.18

Acquisition Date: 17-Apr-18

Gate: No Gate

Total Events: 20000

Quad Location: 64, 79

Quad

% Gated

% Total

2.54

1.97

69.19

26.30

File: HEX RT1/2 T47D APOP.005

Patient ID: 0417.18

Acquisition Date: 17-Apr-18

Gate: No Gate

Total Events: 20000

Region

% Gated

% Total

65.57

30.23

2.02

2.51

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

294

Figure 3. Cyclin D1 analysis using flow cytometry. T47D cells were treated by nHF for 24h and stained using cyclin D1

antibody. (a) control cells unstaining; (b) control cells; (c) nHF 35 µg/mL

Geraniol is one of monoterpene which has

suppressed the MCF-7 growth through induction cell

cycle arrest in G

phase, reduce the level of cyclin

D1, cyclin dependent kinase 4 (CDK4), cyclin E and

cyclin A (Duncan, et al., 2004).

Based on the results above, we conclude

that n-hexane fraction of Cyperus rotundus L.

rhizome has cytotoxic activity towards T47D breast

cancer cells which have some mechanism such as

induction of apoptosis and inhibition of cell cycle

especially in inhibit cyclin D1 expression.

ACKNOWLEDGEMENTS

We gratefully thank to Research Center University

of Sumatera Utara through “Hibah Penelitian Guru

Besar” Research Grant 2018 “No:2590/

UN5.1.R/PPM/2018” for financial support in the

study.

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Marker

Events

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20000

100.00

5.20

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107

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File: KS UNSTAIN.001

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