Cell Cycle Analysis of Plectranthus amboinicus, (Lour.) Spreng.

Leaves Ethanol Extract Nanoparticles on T47D Cell Lines

Poppy Anjelisa

, Zaitun Hasibuan

and Sumaiyah

Nanomedicine Center of Innovation,Universitas Sumatera Utara,Medan, Indonesia

Department of Pharmacology, Faculty of Pharmacy, Universitas Sumatera Utara,Medan, Indonesia

Department of Pharmaceutical Technology, Faculty of Pharmacy, Universitas Sumatera Utara,Medan, Indonesia

Keywords: Cell Cycle, Plectranthus Amboinicus, Nanoparticle, T47D Cell Line.

Abstract: The development of molecular targeted therapy in cancer is necessary to reduce the occurrence of cell

resistance and toxicity towards normal cell. The aim of this study is to evaluate the effect of Plectranthus

amboinicus (Lour.) Spreng. leaves ethanol extract nanoparticle on T47D cell cycle. The dried leaves powder

of Plectranthus amboinicus (Lour.) Spreng. was extracted with ethanol by maceration method. Ionic

gelation method was implemented for the preparation of Plectranthus amboinicus leaves ethanolic extract

nanoparticles. The cell cycle of T47D treated with ethanol extract nanoparticle of Plectranthus amboinicus

(Lour.) Spreng. leaves were analyzed using FACScan flow cytometer. Treatment of PAEEN with IC

concentration, ½ IC

(44.582 µg/mL, and ¼ IC

(22.291 µg/mL) caused cell accumulation at G

– G

phase. At S phase, the percentage of accumulation at ¼ IC

(22.291 µg/mL) higher than control. The study

showed that cells underwent apoptosis indicated by occurrence of inhibition of cell cycle on G0-G1 phase

and S phase.

1 INTRODUCTION

Several advantages have been achieved for breast

cancer treatment, including combination treatment

of chemotherapy, antibody therapy and endocrine

therapy. However, the resistance of cancer cells

being one of a major problem in breast cancer

treatment (Lifiani, 2018).

The development of molecular targeted therapy

in cancer is necessary to reduce the occurrence of

cell resistance and toxicity towards normal cell.

Therapeutic targets may involve many proteins and

mechanisms, including the inhibition of protein in

the signaling process which regulates the growth and

the development of cancer cells and of proteins

which cause the resistance of cancer treatment

(Hasibuan, 2016).

The previous studies have demonstrated the

activity of Plectranthus amboinicus (Lour.) Spreng.

leaves extracts on cancer cell could be due to the

inhibition of cell cycle. The study showed that

ethylacetate extracts of Plectranthus amboinicus

(Lour.) Spreng. leaves changed the accumulation of

cell cycle phase from G0-G1 phase (54.61%) to sub

G1 phase (69.73%) (Hasibuan, 2014). In this study,

we aimed to investigate the effect of Plectranthus

amboinicus (Lour.) Spreng. leaves ethanol extract

nanoparticle on T47D cell cycle.

2 METHODS

The extraction was conducted by maceration

method. Dried leaves powder of Plectranthus

amboinicus (Lour.) Spreng. was extracted with

ethanol for 3 days at room temperature. The extract

then concentrated using rotary evaporator, and was

dried by freeze-dryer.

2.1 The Preparation of Nanoparticles

of Plectranthus amboinicus (Lour.)

Spreng. Leaves Ethanolic Extract

(PAEEN)

Ionic gelation method was implemented for the

preparation of Plectranthus amboinicus leaves

ethanolic extract nanoparticles. 0.3% PAEEN

(Plectranthus amboinicus (Lour.) Spreng. leaves

ethanolic extract nanoparticles) was diluted in 1.5%

Anjelisa, P., Hasibuan, Z. and Sumaiyah, .

Cell Cycle Analysis of Plectranthus amboinicus, (Lour.) Spreng. Leaves Ethanol Extract Nanoparticles on T47D Cell Lines.

DOI: 10.5220/0010072904590461

In Proceedings of the International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches (ICOSTEERR 2018) - Research in Industry 4.0, pages

459-461

ISBN: 978-989-758-449-7

459

acetic acid. The sodium tripolyphosphate (1mg/ml)

was added to the extract with dropwise under

magnetic stirring for an hour. The mixture result of

PAEEN nanoparticles was separated by centrifuge at

the speed of 15000 rpm for 20 minutes. The pellet

was collected and used for characterization (Raj,

2015).

1 gram of the ethanolic extract of Plectranthus

amboinicus leaves was diluted into 35 mL ethanol

p.a, added by 15 mL distilled water, chitosan in 100

mL glacial acetic acid 1% and 350 mL NaTPP

solution. Stirred by using magnetic stirrer for ± 2

hours. Then the colloid of nanoparticle chitosan-

NaTPP of Plectranthus amboinicus leaves ethanolic

extract were separated by centrifugation. The result

was put in to the freezer (± -4

C) for ± 2 days. Then,

it was moved to refrigerator (± 3°C) to dry. The

resulting nanoparticles were characterized using

PSA (Particle Size Analyzer). The formed solids are

characterized using TEM (Transmission Electron

Microscope) to determine the morphological form in

its solid form.

2.2 Cell Line and Culture Condition

T47D cell lines were obtained from Parasitology

Laboratory, Faculty of Medicine, Gadjah Mada

University, Indonesia. The cell line was maintained

in RPMI 1,640 suplemented with 10% Foetal

Bovine Serum v/v (Gibco). Cells were cultured in

the presence of 1% penicillin-streptomycine

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

at 37

C in humidified atmosphere containing 5%

2.3 Cell Cycle Analysis

The cell cycle analysis was carried out according to

our previous study (Hasibuan, 2014). T47D cells

(5×10

cells/well) were treated with 89.166 µg/mL

(IC

concentration), 44.582 µg/mL (½ IC

), and

22.291 µg/mL (¼ IC

) PAEEN for 24 hrs. Cells

then washed, harvest and fixed with 70% ice-cold

ethanol. Cells were washed 3 times with ice-cold

PBS, resuspended and centrifuged at 3000 rpm for 3

minutes. Cells were treated with RNAse 100 μg/mL

containing PI 40 μg/mL incubated at 37°C for 30

minutes. Cell cycle distribution was then were

analyzed using FACScan flow cytometer. Data were

calculated using ModFit Lt. 3.0.s; (Satria, 2017).

3 RESULTS AND DISCUSSIONS

The cell cycle involves mainly four steps which lead

to cell growth and cell division in order to produce 2

daughter cells. The phases are G1, S, G2 and M

(Dalimunthe, 2017). Recent study has showed that

of PAEEN was 89.166 µg/mL which is

potential as anticancer. Next, we explore the

mechanism of PAEEN by analyzing cell cycle

distribution using propidium iodide staining and

flowcytometry. Propidium iodide is a fluorogenic

dye wich binds to DNA, allowing the DNA content

of the stained cells to be anayzed by flow cytometry.

Cells can be classified according to phases of the

cell cycle ((G0/G1, S, and G2/M) based on DNA

content (Xuereb and Blundell, 2008). Treatment of

PAEEN with IC

concentration, ½ IC

(44.582

µg/mL, and ¼ IC

(22.291 µg/mL) caused cell

accumulation at G

– G

phase. At S phase, the

percentage of accumulation at ¼ IC

(22.291

µg/mL) higher than control which mean that

PAEEN could inhibit on S phase as well. During the

G1/S phase checkpoint, DNA damage is sensed and

the cell cycle is paused until the DNA is thoroughly

repaired. This ensure that the S phase is embarked

only when the DNA damage accumulated

throughout the entire cycle has been eliminated. If

the damage is so severe, apoptosis can be induced.

The cell cycle accumulations were showed on Figure

1-4 as follow:

Figure 1: Control cells T47D.

Figure 2: cell cycle inhibition by Plectranthus amboinicus

ethnolic extract nanoparticles with IC

GO-G1

S-phase

G2-M

ICOSTEERR 2018 - International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches

460

Figure 3: cell cycle inhibition by Plectranthus amboinicus

ethnolic extract nanoparticles with ½ IC

Figure 4: cell cycle inhibition by Plectranthus amboinicus

ethnolic extract nanoparticles with ¼ IC

The percentage of cell accumulation on every

phase showed that there is a cell cycle arrest on G0-

G1 phase because the percentage was higher than

control as shown on table 1.

Table 1: The percentage of cell accumulation.

We have previously reported that PAEEN can

prevent proliferation and induce apoptosis on T47D

breast cancer cell lines. On this study, we showed

that cells underwent apoptosis indicated by

occurrence of inhibition of cell cycle on G0-G1

phase and S phase. Cell cycle control is deregulated

in cancer cells in spite of defects in their genome,

they can easily pass cell cycle protein such as cyclin

D1 and drive the cycle to proliferation without any

interruption (Xuereb and Blundell, 2008).

4 CONCLUSIONS

PAEEN could inhibit the cell cycle T47D cell lines

at G0-G1 phase and S phase. The data provide the

promising candidate for further studies.

ACKNOWLEDGEMENTS

This research is funded by Universitas Sumatera

Utara, Indonesia. The support is under the “Hibah

Talenta” Research Grant 2018 No.

233/UN5.2.3.1/PPM/KP-TALENTA USU/2018.

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GO-G1

S-phase

G2-M

GO-G1

S-phase

G2-M

Treatments

concentr

ations

Phase (%)

–

S G

Control 59.07 18.93 21.36

PAEEN IC

63.92 17.30 18.32

½ IC

66.89 16.19 16.74

¼ IC

60.94 20.46 19.05

Cell Cycle Analysis of Plectranthus amboinicus, (Lour.) Spreng. Leaves Ethanol Extract Nanoparticles on T47D Cell Lines

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