Morphological Changes of Cisplatin-resistant Human Breast Cancer MCF-7 Cell Line | Puspita | International Journal of Integrated Health Sciences 960 4212 2 PB
Original Article
Morphological Changes of Cisplatin-resistant Human Breast Cancer
MCF-7 Cell Line
Nanda Ayu Puspita,1,2 Amy Bedford3
1
Departement of Biochemistry, Faculty Medicine, Syiah Kuala University, Banda Aceh, Indonesia
Biomedical Research Centre, University of Salford, The Crescent, Salford, United Kingdom
3
Faculty of Biology, Medicine and Health, University of Manchester, Oxford Road, Manchester, United Kingdom
2
Abstract
Objective: To evaluate morphological changes of breast cancer cell line
MCF-7 following the development of acquired resistance towards cisplatin.
Resistance towards anticancer agents still plays an important role in the
breast cancer chemotherapy failure.
Methods: The cisplatin resistant subline MCF-7/CisR was developed in-vitro
by cultivating the parental cell line cisplatin-sensitive MCF-7 with raising
concentration of cisplatin (from 0.01 to 2.3 µM) for four months. The cell
morphology was observed by giemsa staining.
Results: The resistance was shown by the increase of IC50 by 9 folds on
MCF-7/CisR compared to the initial IC50 value of MCF-7. The change in
cisplatin cytotoxic potency on the resistant cell line was accompanied by
the morphological modification, including the enlargement of cell size, the
increase of nucleus and cytoplasm ratio, and the increase of the number of
microvesicular and cytoplasmic granules.
Received:
December 14, 2016
Revised:
February 17, 2017
Conclusions: This result has supported the underlying mechanism of
cisplatin resistance, including the ability of the cells to decrease intracellular
cisplatin concentration and repair DNA damage effects.
Keywords: Drug resistance, human breast cancer, cisplatin, MCF-7
Accepted:
February 27, 2017
pISSN: 2302-1381; eISSN: 2338-4506; http://doi.org/10.15850/ijihs.v5n1.960
IJIHS. 2017;5(1):8–14
Introduction
Breast cancer is known as the most common
cancer in women.1,2 In the developed world,
for example, in the United Kingdom, around
150 new cases are diagnosed every day; 1 in
8 women are predicted to be diagnosed with
breast cancer during their lifetime. Meanwhile,
in less developed countries, the breast cancerrelated deaths reach a significant number,
which is more than 50% of the diagnosed
cases.3 In combating breast cancer, the modality
of therapy can include surgery, chemotherapy,
and radiotherapy.1–3 For a systemic approach,
chemotherapy is counted as a key strategy to
treat breast cancer due to eliminate residual
Correspondence:
Nanda Ayu Puspita, Departement of Biochemistry,
Faculty Medicine, Syiah Kuala University
Jl. T. Nyak Arief Darussalam Banda Aceh 23111, Banda
Aceh, Indonesia
e-mail: n.a.puspita@edu.salford.ac.uk
8
cancer after surgery or possible occurrence
of metastatic cells.4 However, chemotherapy
is frequently accompanied by the occurrence
of drug resistance of the cancer cells towards
one or multiple drugs which accounts for the
failure of the breast cancer treatment.1,3,4
Breast cancer cells were notably reported
to develop resistance towards chemotherapy
agents, including platinum derivatives such as
cisplatin.4–6 Until recently, the clear mechanism
of drug resistance of breast cancer cell is still in
debate.4–8 Many studies have reported several
possible mechanisms which are related to
chemo-resistance, including over-expression
of p-glycoprotein, alteration of cell cycle arrest
and apoptosis, the expression of multidrug
resistance-associated protein (MRP).5,6 Overall,
all these molecular mechanisms will result in
the drug-resistant phenotype modification,
which could be accompanied by changes in
proliferation potential and morphological
structure.5 Therefore, in order to observe the
International Journal of Integrated Health Sciences. 2017;5(1):8–14
Nanda Ayu Puspita, Amy Bedford
characteristics in the early stages of acquired
drug-resistance in breast cancer, structure of
the cell cancer might provide an initial insight
of the development of chemo-resistance.
The aim of this study was to observe the
morphological changes on human breast
cancer cell line MCF-7 following the in-house
development of the subline resistant to
cytotoxic effects of cisplatin (MCF-7/CisR).
Methods
In this study, the cell line used was human
breast cancer cell line MCF-7 from ATTC,
United Kingdom (UK). The cell culture and
other laboratory procedures were conducted
in the Biomedical Research Centre (BMC),
University of Salford, UK. Culture media and all
media supplements were obtained from Lonza,
Walkersville, MD. Dimethyl Sulfoxide (DMSO),
cisplatin, and thiazolyl blue tetrazolium
bromide (MTT salt) were purchased from
Sigma-Aldrich, Dorset, UK.
The MCF-7 cell line was cultivated in
Dulbecco’s Modified Eagle (DMEM) medium
supplemented with 10% (v/v) fetal bovine
serum (FBS), 2 mM L-glutamine, 50 IU/ml
penicillin, and 50 µg/ml streptomycin, at 37
⁰C in a humidified atmosphere containing 5%
CO2. The cells were subcultured twice a week
or after reaching 70–80% confluence.
The cisplatin resistant variant (MCF-7/
CisR) was developed from the parental line
MCF-7 by cultivating the parental cell line
with raising concentration of cisplatin (from
0.01 to 2.3 µM). Cisplatin was added twice a
week following subculturing. The cell viability
and confluence were checked regularly before
reseeding. The drug was only given when the
viability >90% and the confluence >50%.
Otherwise, the cell line was grown in drug-free
media until the cells returned to confluence
and the viability has recovered. Sensitivity to
cisplatin was measured every 8 weeks using
MTT-colorimetric test.
Healthy cells, which had been sub-cultured
at least once and subsequently reached 70–
80% confluency, were used for the MTT assay.
Briefly, 100 µl cell suspensions (105 cells/ml)
were plated in 96-wells microplate. Cisplatin
with raising concentration was added into
the corresponding well; the control wells
were left untreated. After 5 days incubation
in 37 ⁰C with 5% CO2, 30 µl of MTT solution
(5 mg/mL) was added into each well and
the incubation was continued for a further 4
hours. Subsequently, 100 µl DMSO was added
into each well to dissolve the formazan crystal.
The optical density (OD) was measured using
Multiskan FC® (test wavelength of 590 nm and
reference wavelength at 690nm). Cell viability
was calculated using the following equation:
Cell viability (%) = OD0/ODn x 100%
Where OD0 is the OD from the control wells
and ODn is the OD of the treated cells. The IC50
values were calculated as the concentrations
that show 50% inhibition of cell.
The cell slides were prepared from cell
suspension (105 cells/mL) and stained with
hematoxylin-eosin staining. The morphological
changes were observed to evaluate the signs
of apoptosis, such as plasma membrane
blebbing, condensation of chromatins, nuclear
condensation, and formation of apoptotic
bodies.7
The statistical analyses were performed
using Graphpad prism software version 5.0
(GraphPad Software, San Diego California
USA). Data was presented as mean±standard
mean error (SEM). The comparison of means
was performed by using Student’s t-test and
ANOVA with Tukey multiple comparison as a
post hoc analysis. P-value smaller than 0.05
was considered statistically significant.
Results
In this study, the MCF-7 resistance to cisplatin
was developed in-vitro by introducing an
increasing dose of cisplation, using a low startup dose (0.01 µM). Cisplatin is known as one of
several platinum based anticancer drugs which
are widely used in cancer therapy and have
demonstrated a successful outcome in breast
cancer therapy.4,5,7 In this study, the parental
cell line MCF-7 exerted sensitivity to cisplatin,
demonstrated by a low IC50 value (2.72±0.51
µM) (Fig. 1). The sensitivity towards cisplatin
and high successful rate of cisplatin therapy is
attributed to multiple molecular mechanisms
in combating cancer cells, which is mediated
by its ability to interact with deoxyribonucleic
acid (DNA) strands causing DNA adduct.6,11,12
The DNA damage is recognised by several
proteins as a signal to activate further
pathways, including p53, ATR, and MAPK. The
crosstalk between these pathways ultimately
leads to cell cycle arrest, apoptosis and cell
death.8
Despite the effectiveness of cisplatin as
chemotherapy agent, cancer cells might have
a natural ability to turn this cytotoxic effect,
which might be a result from continuous drug
exposure.4,5,9 In the establishment of the drug
International Journal of Integrated Health Sciences. 2017;5(1):8–14
9
Morphological Changes of Cisplatin-resistant Human Breast Cancer MCF-7 Cell Line
b
a
Fig. 1 Cell Growth Curve Showing the Percentage of MCF-7 Cell Growth againts a
Serial Dose of Cisplatin (1a). The IC50 of Parental Cell Line before Exposure to
Cisplatin (Control), after 3 Months, and after 4 Months ((1b). For the Calculation
of IC50 Value, the Largest Value in the Data Set Corresponded to 100% and the
Smallest Value Corresponded to 0%. Log-Transformed Drug Concentrations
were Plotted against the Dose Response and the IC50 Values were Determined
Using Non-Linear Regression Analysis (*P
Morphological Changes of Cisplatin-resistant Human Breast Cancer
MCF-7 Cell Line
Nanda Ayu Puspita,1,2 Amy Bedford3
1
Departement of Biochemistry, Faculty Medicine, Syiah Kuala University, Banda Aceh, Indonesia
Biomedical Research Centre, University of Salford, The Crescent, Salford, United Kingdom
3
Faculty of Biology, Medicine and Health, University of Manchester, Oxford Road, Manchester, United Kingdom
2
Abstract
Objective: To evaluate morphological changes of breast cancer cell line
MCF-7 following the development of acquired resistance towards cisplatin.
Resistance towards anticancer agents still plays an important role in the
breast cancer chemotherapy failure.
Methods: The cisplatin resistant subline MCF-7/CisR was developed in-vitro
by cultivating the parental cell line cisplatin-sensitive MCF-7 with raising
concentration of cisplatin (from 0.01 to 2.3 µM) for four months. The cell
morphology was observed by giemsa staining.
Results: The resistance was shown by the increase of IC50 by 9 folds on
MCF-7/CisR compared to the initial IC50 value of MCF-7. The change in
cisplatin cytotoxic potency on the resistant cell line was accompanied by
the morphological modification, including the enlargement of cell size, the
increase of nucleus and cytoplasm ratio, and the increase of the number of
microvesicular and cytoplasmic granules.
Received:
December 14, 2016
Revised:
February 17, 2017
Conclusions: This result has supported the underlying mechanism of
cisplatin resistance, including the ability of the cells to decrease intracellular
cisplatin concentration and repair DNA damage effects.
Keywords: Drug resistance, human breast cancer, cisplatin, MCF-7
Accepted:
February 27, 2017
pISSN: 2302-1381; eISSN: 2338-4506; http://doi.org/10.15850/ijihs.v5n1.960
IJIHS. 2017;5(1):8–14
Introduction
Breast cancer is known as the most common
cancer in women.1,2 In the developed world,
for example, in the United Kingdom, around
150 new cases are diagnosed every day; 1 in
8 women are predicted to be diagnosed with
breast cancer during their lifetime. Meanwhile,
in less developed countries, the breast cancerrelated deaths reach a significant number,
which is more than 50% of the diagnosed
cases.3 In combating breast cancer, the modality
of therapy can include surgery, chemotherapy,
and radiotherapy.1–3 For a systemic approach,
chemotherapy is counted as a key strategy to
treat breast cancer due to eliminate residual
Correspondence:
Nanda Ayu Puspita, Departement of Biochemistry,
Faculty Medicine, Syiah Kuala University
Jl. T. Nyak Arief Darussalam Banda Aceh 23111, Banda
Aceh, Indonesia
e-mail: n.a.puspita@edu.salford.ac.uk
8
cancer after surgery or possible occurrence
of metastatic cells.4 However, chemotherapy
is frequently accompanied by the occurrence
of drug resistance of the cancer cells towards
one or multiple drugs which accounts for the
failure of the breast cancer treatment.1,3,4
Breast cancer cells were notably reported
to develop resistance towards chemotherapy
agents, including platinum derivatives such as
cisplatin.4–6 Until recently, the clear mechanism
of drug resistance of breast cancer cell is still in
debate.4–8 Many studies have reported several
possible mechanisms which are related to
chemo-resistance, including over-expression
of p-glycoprotein, alteration of cell cycle arrest
and apoptosis, the expression of multidrug
resistance-associated protein (MRP).5,6 Overall,
all these molecular mechanisms will result in
the drug-resistant phenotype modification,
which could be accompanied by changes in
proliferation potential and morphological
structure.5 Therefore, in order to observe the
International Journal of Integrated Health Sciences. 2017;5(1):8–14
Nanda Ayu Puspita, Amy Bedford
characteristics in the early stages of acquired
drug-resistance in breast cancer, structure of
the cell cancer might provide an initial insight
of the development of chemo-resistance.
The aim of this study was to observe the
morphological changes on human breast
cancer cell line MCF-7 following the in-house
development of the subline resistant to
cytotoxic effects of cisplatin (MCF-7/CisR).
Methods
In this study, the cell line used was human
breast cancer cell line MCF-7 from ATTC,
United Kingdom (UK). The cell culture and
other laboratory procedures were conducted
in the Biomedical Research Centre (BMC),
University of Salford, UK. Culture media and all
media supplements were obtained from Lonza,
Walkersville, MD. Dimethyl Sulfoxide (DMSO),
cisplatin, and thiazolyl blue tetrazolium
bromide (MTT salt) were purchased from
Sigma-Aldrich, Dorset, UK.
The MCF-7 cell line was cultivated in
Dulbecco’s Modified Eagle (DMEM) medium
supplemented with 10% (v/v) fetal bovine
serum (FBS), 2 mM L-glutamine, 50 IU/ml
penicillin, and 50 µg/ml streptomycin, at 37
⁰C in a humidified atmosphere containing 5%
CO2. The cells were subcultured twice a week
or after reaching 70–80% confluence.
The cisplatin resistant variant (MCF-7/
CisR) was developed from the parental line
MCF-7 by cultivating the parental cell line
with raising concentration of cisplatin (from
0.01 to 2.3 µM). Cisplatin was added twice a
week following subculturing. The cell viability
and confluence were checked regularly before
reseeding. The drug was only given when the
viability >90% and the confluence >50%.
Otherwise, the cell line was grown in drug-free
media until the cells returned to confluence
and the viability has recovered. Sensitivity to
cisplatin was measured every 8 weeks using
MTT-colorimetric test.
Healthy cells, which had been sub-cultured
at least once and subsequently reached 70–
80% confluency, were used for the MTT assay.
Briefly, 100 µl cell suspensions (105 cells/ml)
were plated in 96-wells microplate. Cisplatin
with raising concentration was added into
the corresponding well; the control wells
were left untreated. After 5 days incubation
in 37 ⁰C with 5% CO2, 30 µl of MTT solution
(5 mg/mL) was added into each well and
the incubation was continued for a further 4
hours. Subsequently, 100 µl DMSO was added
into each well to dissolve the formazan crystal.
The optical density (OD) was measured using
Multiskan FC® (test wavelength of 590 nm and
reference wavelength at 690nm). Cell viability
was calculated using the following equation:
Cell viability (%) = OD0/ODn x 100%
Where OD0 is the OD from the control wells
and ODn is the OD of the treated cells. The IC50
values were calculated as the concentrations
that show 50% inhibition of cell.
The cell slides were prepared from cell
suspension (105 cells/mL) and stained with
hematoxylin-eosin staining. The morphological
changes were observed to evaluate the signs
of apoptosis, such as plasma membrane
blebbing, condensation of chromatins, nuclear
condensation, and formation of apoptotic
bodies.7
The statistical analyses were performed
using Graphpad prism software version 5.0
(GraphPad Software, San Diego California
USA). Data was presented as mean±standard
mean error (SEM). The comparison of means
was performed by using Student’s t-test and
ANOVA with Tukey multiple comparison as a
post hoc analysis. P-value smaller than 0.05
was considered statistically significant.
Results
In this study, the MCF-7 resistance to cisplatin
was developed in-vitro by introducing an
increasing dose of cisplation, using a low startup dose (0.01 µM). Cisplatin is known as one of
several platinum based anticancer drugs which
are widely used in cancer therapy and have
demonstrated a successful outcome in breast
cancer therapy.4,5,7 In this study, the parental
cell line MCF-7 exerted sensitivity to cisplatin,
demonstrated by a low IC50 value (2.72±0.51
µM) (Fig. 1). The sensitivity towards cisplatin
and high successful rate of cisplatin therapy is
attributed to multiple molecular mechanisms
in combating cancer cells, which is mediated
by its ability to interact with deoxyribonucleic
acid (DNA) strands causing DNA adduct.6,11,12
The DNA damage is recognised by several
proteins as a signal to activate further
pathways, including p53, ATR, and MAPK. The
crosstalk between these pathways ultimately
leads to cell cycle arrest, apoptosis and cell
death.8
Despite the effectiveness of cisplatin as
chemotherapy agent, cancer cells might have
a natural ability to turn this cytotoxic effect,
which might be a result from continuous drug
exposure.4,5,9 In the establishment of the drug
International Journal of Integrated Health Sciences. 2017;5(1):8–14
9
Morphological Changes of Cisplatin-resistant Human Breast Cancer MCF-7 Cell Line
b
a
Fig. 1 Cell Growth Curve Showing the Percentage of MCF-7 Cell Growth againts a
Serial Dose of Cisplatin (1a). The IC50 of Parental Cell Line before Exposure to
Cisplatin (Control), after 3 Months, and after 4 Months ((1b). For the Calculation
of IC50 Value, the Largest Value in the Data Set Corresponded to 100% and the
Smallest Value Corresponded to 0%. Log-Transformed Drug Concentrations
were Plotted against the Dose Response and the IC50 Values were Determined
Using Non-Linear Regression Analysis (*P