The Cytotoxicity of Paclitaxel Was Smaller than Doxorubicin

in T47D Breast Cancer Cell

Irma Yanti Rangkuti

Biomedic/Pharmacology Department, Faculty of Medicine, Universitas Islam Sumatera Utara, Indonesia

Keywords: Paclitaxel, doxorubicin, cytotoxicity, T47D breast cancer cell.

Abstract: Breast cancer is a type of cancer commonly found by most women, ranking fifth leading cause of cancer

worldwide cancer, consist of various subtypes. Chemotherapy is one of therapy in breast cancer patients but

the benefit is not equal for all patients and paclitaxel is one of the most important chemotherapeutic drugs.

The descriptive study had been done to paclitaxel and doxorubicin in T47D breast cancer cell. This study

aimed to compare cytotoxic effect of paclitaxel and doxorubicin in T47D breast cancer cell, used paclitaxel

concentrations 1000; 500; 250; 31,25; 15,625 nM and doxorubicin concentrations 500; 250;62,5;31,25;and

15,625 nM for 24 hours. This study was an invitro. The cytotoxic test used MTT method to determine IC

and analized by SPSS. The result showed that IC

paclitaxel was 1577,2 ± 115,3 nM and IC

doxorubicin

202,37 ± 3,99 nM. The cytotoxicity of paclitaxel was smaller than doxorubicin in T47D breast cancer cell.

1 INTRODUCTION

Breast cancer is a disease in which there is excessive

growth or uncontrolled development of breast tissue

cells. Breast cancer is a type of cancer commonly

found by most women, ranking fifth leading cause of

cancer worldwide cancer around 522,000 deaths and

the most common cause of death in women in

developing countries (324,000 deaths). According to

IARC's Globocan data (International Agency for

Research on Cancer) in 2012 there are 14,067,894

new cases of cancer and 8,201,575 deaths from

cancer worldwide with the most cancer types of

breast cancer, prostate cancer and lung cancer.

Estimated 1.67 million new cases breast cancer in

2012. For Indonesia, the incidence of cancer in

women is about 134 per 100,000 population with

most cases of breast cancer of 40 per 100,000

women. Globocan estimates, deaths in Indonesia due

to breast cancer approximately 16.6 deaths per

100,000 population (Kemenkes RI, 2016).

Breast cancer consist of subtypes based on IHC

markers including ER, progesterone receptor (PR)

and human epidermal growth factor receptor 2

(HER2). Breast tumors are grouped into four basic

subgroups according to these markers, i.e.,

[ER+|PR+]HER2 - (tumors with either ER or PR

positivity, and HER2 negativity), [ER+|PR+]HER2+

(tumors with either ER or PR positivity, and HER2

positivity), ER –PR -HER2+ (tumors with ER and

PR negativity, and HER2 positivity, also named

HER2 positive), ER –PR -HER2 - (tumors with ER,

PR, HER2 negativity, also named triple negative)

(Dai et al. 2016).

Chemotherapy is one of therapy in breast cancer

patients and improves survival of patients with stage

I–III breast cancer, but the benefit is not equal for

all patients because there are melocular

characteristics of the cancer affect sensitivity to

chemotherapy (Andre and Pusztai 2006; Hassan et al.

2010). The use of cytotoxic chemotherapy in both

advanced and early stage breast cancer has made

significant progress in the last 10 years with several

landmark studies identifying clear survival benefits

for newer therapies (Hassan et al. 2010).

Paclitaxel is one of the most important

chemotherapeutic drug, isolated from the Pacific

yew, Taxusbrevifolia, was approved for the

treatment of metastatic breast cancer in 1994 (Patt,

Gauthier, and Giordano 2006). This drug causes

abnormal stabilization of the dynamic microtubule

polymerization that alters intracellular signaling,

transport organelle and locomotion, and leading to

the failure of mitosis (Honore, Pasquier, and Braguer

2005). Recent studies showed that paclitaxel is able

to induce reactive oxygen species (ROS) production

in cancer cells and hydrogen peroxide (H2O2) that

Rangkuti, I.

The Cytotoxicity of Paclitaxel Was Smaller than Doxorubicin in T47D Breast Cancer Cell.

DOI: 10.5220/0010061801710176

In Proceedings of the 7th International Conference on Multidisciplinary Research (ICMR 2018) - , pages 171-176

ISBN: 978-989-758-437-4

171

involved in cancer cell death with Paclitaxel

(Alexandre, Batteux, et al. 2006; Ramanathan et al.

2005; Alexandre, Nicco, et al. 2006). The novel

mechanism of paclitaxel by inducing toxic bystander

effect through generation of extracellular H2O2

from the membrane–associated NOX (Alexandre et

al. 2007).

Doxorubicin is considered to be the most

effective agent in the treatment of breast cancer

patients (Smith et al. 2006). Doxorubicin is an

anthracyline drug first extracted from Streptomyces

peucetius var. caesius in the 1970’s. The

mechanisms of action doxorubicin in cell are

intercalation into DNA and disruption of

topoisomerase-II-mediated DNA repair and

generation of free radicals and their damage to

cellular membranes, DNA and proteins. Doxorubicin

is oxidized to semiquinone, an unstable metabolite,

which is converted back to doxorubicin in a process

that releases reactive oxygen species. Reactive

oxygen species can lead to lipid peroxidation and

membrane damage, DNA damage, oxidative stress,

and triggers apoptotic pathways of cell death (Thorn

et al. 2011).

The T47D cell is one of breast cancer cell that

used for research. The characteristic this cell is a

continuous cell line isolated from a breast ductal

tumor tissue of a 54-year-old woman who expresses

a mutated p53 protein (missense mutation) at a 194

residue (in zinc-binding domain, L2).

2 MATERIALS AND METHODS

This study used paclitaxel and doxorubicin as

materials. Paclitaxel was obtained from PT Dankos

Farma, Indonesia.

This research was an invitro, descriptive study

to compare cytotoxic effect of paclitaxel and

doxorubicin in T47D breast cancer cell, used

paclitaxel concentrations 1000; 500; 250; 31,25;

15,625 nM and doxorubicin concentrations 500;

250;62,5;31,25; and 15,625 nM for 24 hours. The

cytotoxic test used MTT [3-(4,5-dimetiltiazol-2-il)-

2,5-difenil tetrazolium bromida] method to

determine IC

and analized by SPSS.

2.1 Cell Culture

In this study, T47D cells obtained from the

Laboratory of Parasitology, Faculty of Medicine,

Gadjah Mada University were grown in RPMI

medium containing 10% Fetal Bovine Serum (Gibco,

USA), 2% Penicillin-Streptomycin (Gibco, USA),

and Fungizone (Amphotericin B) 0.5% (Gibco, USA)

on the flask in a humidified incubator(5% CO

/95%

air) at 37°C (Doyle and Bryan, 1998).

2.2 Cell Viability Assay

The viability of T47D cells was assessed using the

MTT assay. The cells were cultivated on 96 well

plates (Iwaki, Japan). Each well contains 1x10

cells.

The cells incubated in a humidified incubator

(5% CO

/95% air) for 24 hours. After 24 hours

incubation, the medium culture is discharged and

each well is given paclitaxel with concentration1000;

500; 250; 31,25; 15,625 nM. After 24 hours

incubation, the cells was incubated with 0.5 mg/mL

MTT (Sigma-Aldrich, USA) for 4 hours at 37°C.

The cells that is feasible to react with MTT to

produce of purple crystals formazan. After 4 hours,

10% SDS (Sigma-Aldrich, USA) stopper in 0.01 N

HCl (Merck, USA) was added to dissolve the

formazan crystals. Then, the cells are incubated for

24 hours at room temperature and protected from

light. After incubation, cells were shaken, and cell

absorbance was measured by elisamicroplate reader

(Bio-Rad, USA) at λ 595 nm. The experimental data

were the absorbance of each well, and then

converted to percentage of a cells viable using

equation as indicated below

% of viable cells =

–

A – C

x 100%

(1)

Where A, B, and C (1) respectively are absorbance

of control cells absorbance, treated cells absorbance,

and medium culture absorbance. All data were

expressed as IC

that calculate using probate

regression analysis at SPSS, test were used for

statistical analyses with p values <0.05 were

considered significant (Meiyanto et al. 2008).

3 RESULT AND DISCUSSION

Cytotoxic assay is a preliminary test to determine

the potential toxicity of a compound and IC

as a

mainly parameters. T47D cells were exposed to

paclitaxel using concentration series of 1000; 500;

250; 31,25; 15,625 nM for 24 hours. After analyzed,

paclitaxel = 1577,2 ± 115,3 nM.

The result of cytotoxic test paclitaxel against

T47D cells during 24 hours exposure can be seen in

Figure 1.

ICMR 2018 - International Conference on Multidisciplinary Research

172

Figure 1: Graph the effect of paclitaxel concentration on

T47D cell viability.

In the cytotoxic paclitaxel test, doxorubicin was

used as a positive control, one of the chemotherapy

for breast cancer using concentration series of 500;

250;62,5;31,25 and 15,625 nM. After analysis, IC

doxorubicin = 202,37 ± 3,99 nM. The result of

doxorubicin cytotoxic test can be seen in Figure 2

below.

Figure 2: Graph the effect of doxorubicin concentration on

viability T47D cell.

This study used the MTT assay to test

cytotoxicity of drug, a quantitative method was

measured by elisamicroplate reader (Bio-Rad, USA)

at λ 595 nm. Cytotoxic effects are indicated by IC

values, the concentration that causes death in 50% of

the cell population by calculating living cells.

The principle of MTT assay is colorimetry

(measurement of color intensity) based on the

formation of formazan crystals (purple and

filamentous) in living cells, penetrating the

membrane and accumulating in them (Figure 3 - 8).

Color formation in living cells as a result of

metabolizing a substrate by living cells into colored

products. The tetrazolium succinate reductase

system found in the living cell mitochondria

included in the respiratory chain will reduce the

MTT yellow to form purple formazan crystals (van

Meerloo, Kaspers, and Cloos, 2011).

Dead cells cannot form formazan crystals

because dead cells are unable to aspire so that

tetrazolium succinate enzymes which can reduce

MTT salt to formazan products are not produced, so

the color of dead cells is not purple but will remain

yellow. The more cells that live, the purple will

become thicker (Freshney, 2015). These figures

below were T47D breast cancer cell looked by

microscope.

A. T47D breast cancer cell before MTT

These figures below showed T47D cell before

MTT, consist of control cell, T47D with

doxorubicin exposure and paclitaxel (Figure 3

– 5).

Figure 3: Control cell (T47D without drug).

Figure 4: T47D cell after giving doxorubicin 500 nM for

24 hours.

The Cytotoxicity of Paclitaxel Was Smaller than Doxorubicin in T47D Breast Cancer Cell

173

Figure 5: T47D cell after giving paclitaxel 1000 nM for 24

hours.

B. T47D breast cancel after MTT

These figures below showed T47D cell after

MTT, consist of control cell, T47D with

doxorubicin exposure and paclitaxel (Figure 6

– 8).

Figure 6: Control cell (T47D without drug).

Figure 7: T47D cell after giving doxorubicin 500 nM for

24 hours (= formazan crystal).

Figure 8: T47D cell after giving paclitaxel 1000 nM for 24

hours (= formazan crystals).

Based on IC

50,

from cytotoxic test, it showed that

cytotoxicity of paclitaxel was smaller than

doxorubicin. Study in 2012 found that paclitaxel was

resistant in T47D breast cancer cell associated with

highly expresses Lin28 in T47D cells than the

MCF7, Bcap-37 or SK-BR-3 cancer cell lines, which

had low-level expression of Lin28. This study

knocked down of Lin28 in Lin28 high expression

T47D cells, the result showed increasing the

sensitivity to paclitaxel treatment, while stable

expression of Lin28 in breast cancer cells effectively

attenuated the sensitivity to paclitaxel treatment,

resulting in a significant increase of IC

values of

paclitaxel. Transfection with Lin28 also significantly

inhibited paclitaxel-induced apoptosis. This study

showed that Lin28 expression was dramatically

increased in tumor tissues after neoadjuvant

chemotherapy or in local relapse or metastatic breast

cancer tissues. Moreover, further studies showed

that p21, Rb and Let-7 miRNA were the molecular

targets of Lin28. Overexpression of Lin28 in breast

cancer cells considerably induced p21 and Rb

expression and inhibited Let-7 miRNA levels (Lv et

al. 2012).

p21, universal inhibitor of cyclin kinases inhibits

the activity of each member of the cyclin/CDK

family (Xiong et al. 1993), promote cell cycle arrest

in cell cycle (Karimian, Ahmadi, and Yousefi, 2016).

Cell cycle is a cell proliferation process that

mediates the growth and development of living

things (Nurse, 2000). The Rb protein is a tumor

suppressor, which plays a pivotal role in the negative

control of the cell cycle and in tumor progression. It

Formazan crystal

Formazan

cristals

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174

has been shown that Rb protein (pRb )is responsible

for a major G1 checkpoint, blocking S-phase entry

and cell growth (Giacinti and Giordano, 2006;

Foster et al. 2001).

Doxorubicin is an anthracycline breast cancer

drug that is still used in combination regimens and

also for other types of cancer such as leukemia

(Wattanapitayakul et al. 2005). Doxorubicin was

used as a positive control in this study, because this

drug is still used, but also doxorubicin showed an

anticancer effect on T47D cells (Barzegar et al.

2015). Cytotoxic activity of doxorubicin through

topoisomerase II inhibition, DNA intercalation, cell

membrane binding and semiquinone free radical

formation and oxygen free radicals (Bruton et al,

2005). Doxorubicin causes the activation of various

molecular signals from AMPK (AMP‐activated

protein kinase inducing apoptosis) to influence the

Bcl‐2/Bax apoptosis pathway. By altering the Bcl‐

2/Bax ratio, downstream activation of different

caspases can occur resulting in apoptosis (Tacar,

Sriamornsak, and Dass, 2013)

4 CONCLUSION

paclitaxel was 1577,2 ± 115,3 nM and IC

doxorubicin 202,37 ± 3,99 nM. The cytotoxicity of

paclitaxel was smaller than doxorubicin in T47D

breast cancer cell.

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