INVESTIGATION OF THE EFFECT OF CURCUMIN ON CYTOTOXICITY, GENOTOXICITY, AND APOPTOSIS ON BREAST CANCER CELLS
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WCRJ 2022; 9: e2149
INVESTIGATION OF THE EFFECT
OF CURCUMIN ON CYTOTOXICITY,
GENOTOXICITY, AND APOPTOSIS
ON BREAST CANCER CELLS
K. BOZALI1,2, E. M. GULER2,3, M. CELIKTEN4
1
Avans University of Applied Science, Faculty Life Sciences and Environmental Technology, Breda, the Netherlands
2
Department of Medical Biochemistry, University of Health Sciences Turkey, Faculty of Hamidiye Medicine,
Istanbul, Turkey
3
Department of Medical Biochemistry, Haydarpasa Numune Health Application and Research Center,
Istanbul, Turkey
4
Department of Scientific Research Projects, Bezmialem Vakıf University, Experimental Application and
Research Center, Istanbul, Turkey
Miss Bozali and Dr. Guler contributed equally to this work
Abstract – Objective: In many preclinical studies, curcumin has been anticarcinogenic in several
cancer types. In line with these findings, this research aims to investigate the effects of curcumin
on cytotoxicity, mitochondrial membrane potential (MMP), glutathione (GSH), intracellular reactive
oxygen species generation (iROS), apoptosis, and DNA damage in breast cancer cell lines.
Materials and Methods: In this study, the cytotoxic effect of curcumin concentrations (5-100 µM)
on human mammary gland cancer cell lines (MCF7 and MDA MB231), mammary gland breast/epithe-
lium healthy cell line (184A1) was determined by the colorimetric MTT assay, and the genotoxic effect
was determined by alkaline single gel electrophoresis (Comet Assay) method. Its apoptotic effect was
determined by the Acridine Orange/Ethidium Bromide (AO/EB) method. In addition, intracellular ROS
(iROS) and mitochondrial membrane potential (MMP) levels were measured with the fluorometric
method, and glutathione (GSH) levels were measured with the luminometric method.
Results: Our study demonstrated that treated curcumin concentrations significantly decreased
cell viability, GSH, and MMP levels and increased ROS, apoptosis, and DNA damage in MCF7 and
MDA MB231 and 184A1 cells in a dose-dependent manner (p
when diagnosed at an early stage. In recent years, it ithelium healthy cell line 184A1 (ATCC®
has been stated that more than 3000 plants showing CRL-8798™ ) from human are commercially
anticancer properties are used in many countries as purchased from the American Type Culture
an alternative to clinical treatment in cancer treat- Collection (ATCC, Manassas, VA, USA). The
ment3. One of these herbs, Turmeric, belongs to the cell lines were maintained in a culture medium
group of Curcuma longa plants of the Zingibera- at 37°C. The culture medium contained prima-
ceae family in the Southeast Asian countries4. The ry medium supplemented with 10% fetal bovine
main component of the Curcuma longa plant is cur- serum (FBS), 1% penicillin-streptomycin anti-
cumin. Curcumin (1,7-bis(4-hydroxy-3-methoxy- biotic, and MCF7 as an extra addition 0.01 mg/
phenyl)-1,6-heptadiene-3,5-dione) is a hydrophobic mL human recombinant insulin. The primary
yellow bioactive ingredient of Rhizoma Curcumae medium of MCF7 was Eagle’s Minimum Essen-
and has many biological activities that are anti-in- tial Medium (EMEM), and MDA MB231 was
flammatory, antioxidant, anti-coagulant, and an- RPMI-1640. 184A1 was cultured in a MEGM
ti-bacterial5,6. Commercially available curcumin medium supplemented with 10% FBS, 1 ng/mL
consists of a mixture of three curcuminoids. Cur- cholera toxin, 1% penicillin-streptomycin anti-
cuminoids are the main components of curcumin, biotic, and 0.005 mg/ml transferrin. All three
have been found to prevent lipid accumulation in cell lines were incubated at 37°C with saturated
liver and adipose tissue, ameliorate hyperlipidemia moisture in 5% CO2.
in animals fed a high-fat diet and lower blood sug-
ar in diabetic animals7. In a study, Empagliflozin
(EMPA), a selective inhibitor of sodium-glucose Cell Viability
co-transporter 2 (SGLT-2), has hypoglycemic and
antioxidant effects. EMPA exerted anti-inflamma- By utilizing MTT (3-(4,5 dimethylthiazol-
tory and cardioprotective effects in doxorubicin-in- 2-yl)-2,5-diphenyltetrazolium bromide) assay,
duced cardiotoxicity. This defines a new therapeu- the cell viability of the cell lines on the cytotoxic
tic option8. In addition, a study showed that the effect of curcumin was examined. MCF7, MDA
anti-inflammatory and chemopreventive power of MB231, and 184A1 cell lines were cultured in
curcumin is preserved when curcumin is encapsu- 96-well plates (8×103 cells/plate) for 24 hours.
lated in the systems, based on the selective action After the incubation, the medium was renewed
of curcumin against cancer cells9. and treated with curcumin concentrations (5 to
In many preclinical studies, curcumin is anti- 100 μM) for 24 hours. After the overnight in-
carcinogenic in diverse types of cancer10. Phenolic cubation, 10 μL of 5 mg/mL MTT solution was
compounds show antioxidant and anticancer activi- dissolved in dPBS and added in the wells. The
ty through their free radical scavenging properties11. obtained mixture was incubated for 4 hours at
And besides, curcumin has excellent antioxidant ac- 37°C. 100 μL of dimethylsulphoxide (DMSO)
tivity due to its phenolic and enolic functional groups. was added in wells after the incubation. After
Studies have confirmed that the oxygenated aromatic that, the plate was incubated for 20 minutes at
rings of curcumin show cytostatic activity12. room temperature. The mixture was measured
Last few years, the potential use of curcumin calorimetrically at 540 nm with Varioskan Flash
in the prevention and treatment of cancer has been Multimode Reader (Thermo Fisher Scientific,
confirmed in many in vivo and in vitro studies13. Waltham, MA, USA).
Herbal medicine is believed to bring new hope for
cancer prevention due to the safety of herbs and
the absence of discernible toxicity to normal cells. Intracellular Reactive Oxygen Species
This research aims to investigate the effects of (iROS)
curcumin on viability, glutathione, mitochondri-
al membrane potential, intracellular ROS, DNA The levels of iROS were examined by using the
damage, and apoptosis in breast cancer cells. H2DCF-DA fluorescence dye. First, the cell lines
were incubated with curcumin concentrations in
the range of 5-100 µM for 24 hours. The media
MATERIALS AND METHODS were three-time washed with aspirated 1x dPBS
after the incubation step. Then, the 5 µM H2D-
Cell lines and treatments CF-DA was added and incubated at 37°C for 30
minutes in the dark. And iROS capacity was
Mammary gland breast cancer cell line MCF7 quantified with a fluorimeter using Varioskan
(ATCC® HTB-22™) and MDA MB231 (ATCC® Flash Multimode Reader (Thermo Fisher Scien-
HTB-26™ ), and mammary gland; breast/ep- tific, Waltham, MA, USA).
2
INVESTIGATION OF THE EFFECT OF CURCUMIN ON CYTOTOXICITY, GENOTOXICITY, AND APOPTOSIS ON BREAST CANCER CELLS
Mitochondrial Membrane Potential buffer at 4°C to unbind DNA. After cooling, the
(MMP) slides were electrophoresed for 25 minutes at 4°C
in conditions (26V and 300mA). Subsequently, the
MMP levels of curcumin on breast cancer and electrophoresed slides were three times washed in
the healthy cells were quantified with DiOC6(3) neutralization buffer and fixed with ethanol. After
(3,3’-dihexyloxacarbocyanine iodide) molecu- washing, the dried slides were stained with 2 µg/
lar fluorescence probe in flow cytometry14. First, mL ethidium bromide. The DNA tail percentages
the cells were seeded 24 hours into a 6-well plate in the images were calculated with a fluorescence
in 5% CO2 at 37°C incubator. Subsequently, the microscope using the Comet assay IV software
medium of each plate was refreshed and treated (Leica DM 1000, Solms, Germany).
with curcumin concentrations for 24 hours. After
that, the cells were removed with the aid of 0.25%
trypsin-EDTA and centrifuged at 400 x g at 4°C Apoptosis
with dPBS for 5 minutes. Then, the obtained su-
pernatant was discarded, and the residual pellet After curcumin treatment on cell lines, the changes
was suspended with DiOC6(3). The resulting sus- in nuclear morphology were examined by AO/EB
pension was incubated for 20 minutes at 37°C. (Acridine Orange/Ethidium Bromide) staining, ex-
Eventually, the cell lines were measured by flow plained by McGahon et al16. MCF7, MDA MB231,
cytometry (BD, FACS Canto™II). and 184A1 cells were seeded in the 6-well plates. The
seeded cells were treated with doses of Curcumin
below IC50 (5µM-60 µM) at 37°C for 24 hours. Af-
Glutathione Levels ter the incubation, cells were harvested with 0.25%
trypsin-EDTA, washed with dPBS, and followed
The glutathione levels in MCF7, MDA MB231, AO/EB stain solution (1:1). The inhibition per-
and 184A1 cells were determined and quantified centages were interpreted by counting 100 cells
with GSH/GSSG-Glo (Promega, Madison, WI, with fluorescent microscopy (Leica DM 1000,
USA) assay. All cell lines were seeded into 96 Solms, Germany).
opaque-white plate. After this step, the adhered
cells were treated and incubated with curcumin
concentrations (5 µM-60 µM) for 24 hours. After Statistical Analysis
incubation, 50 µL glutathione reagent for lyse cell
was added to the removed medium. The opaque- The obtained results were given as mean ± stan-
white plate was incubated at room temperature dard deviation (mean ± SD). Data from all exper-
for 5 minutes in a shaker. After that, 100 µL Lu- iments for statistical significance were examined
ciferin Detection Reagent was added in all wells, by one-way ANOVA analysis of variance. The
and luminescence was measured in Thermo Vari- IC50 values of all curcumin-treated cells were
oskan multimode flash reader within 5 minutes. calculated by using nonlinear regression. Rela-
All concentrations were repeated four times. The tionships between all parameters were analyzed
control calculated the obtained results as % rela- with the Pearson correlation coefficient. And
tive luminescence. p-value of
A B Fig. 1. A, Cytotoxicity of human breast cancer cells MCF7, MDA MB231, and healthy cells 184A1. B, Effect of curcumin on intracellular ROS, following incubation of cells with 5-100 µM curcumin for 24 hours. Statistically significant differences in MCF7 cells, xp
INVESTIGATION OF THE EFFECT OF CURCUMIN ON CYTOTOXICITY, GENOTOXICITY, AND APOPTOSIS ON BREAST CANCER CELLS
Effect of Curcumin on apoptosis highest treated concentration of curcumin in the
viability of MCF7 cells decreased, and the apop-
After 24 hours of treatment of curcumin concen- totic cells increased.
tration below IC50 (5-60 µM), the apoptotic cells
increased significantly in a dose-dependent man-
ner (p
Fig. 4. Effect of Curcumin on DNA
damage. A, Percentage DNA in tail
determines in curcumin concen-
trations 5 µM, 10 µM, 20 µM, 40
µM and on MCF7, MDA MB231
and 184A1 cell lines for 24 hours.
B, Representative images of DNA
damage pattern in MCF7 cells.
Statistically significant differences
of relative values in MCF7 cells
x
pINVESTIGATION OF THE EFFECT OF CURCUMIN ON CYTOTOXICITY, GENOTOXICITY, AND APOPTOSIS ON BREAST CANCER CELLS
MDA MB231 cells in a dose-dependent manner. ed cells resulted in an increase of apoptotic cells
Therefore, it can be expected that DNA damage in a dose-dependent manner. The obtained re-
and apoptosis-related cell death due to apoptosis sults of our study indicated that curcumin-treated
induced by the cumulative effect of ROS and bas- mammary gland cancer cells demonstrated more
al ROS, created by the pro-oxidant activity of ac- apoptotic incidents with a decrease in cell viabil-
tive substances in cancer cells. ity compared to healthy cells significantly in a
High ROS levels cause severe damage to dose-dependent manner.
lipids, proteins, and DNA, an essential mecha-
nism for cell death25. This study determined the
genotoxicity of curcumin on mammary gland CONCLUSIONS
breast cancer and mammary gland healthy cell
lines using the comet assay. The obtained results Our study confirmed that curcumin induces cy-
demonstrated that the DNA damage increased totoxicity, genotoxicity, and apoptosis in mam-
significantly after dose-dependent treatment with mary gland cancer cells by increasing iROS and
curcumin. Furthermore, the cancer cell lines were decreasing GSH and MMP. Thus, curcumin has
more sensitive to DNA damage compared to the anticancer activity, and it is highly potential to be
healthy cells. Recent studies have demonstrated an anticancer drug. In vivo and relevant clinical
curcumin-induced DNA damage23-25. Their results trials are required to elucidate the mechanisms by
were consistent with our findings. which curcumin exerts anti-breast cancer proper-
The mitochondrial apoptotic pathways have ties, thereby providing experimental confirmation
been investigated to prove the mechanism of for the treatment of mammary gland cancer and
apoptotic induction in cancer cells. The produc- contributing to new curcumin-related drugs.
tion of iROS causes mitochondrial damage, and
decreased mitochondrial membrane potential ORCIDs:
leads to apoptosis26,27. Our results demonstrate Kubra Bozali: 0000-0003-2416-0773
that curcumin significantly reduces MMP (DΨm) Eray Metin Guler: 0000-0003-4351-1719
in cancer and healthy cell lines in a dose-depen- Mert Celikten: 0000-0002-8215-3638
dent manner. Previous studies have demonstrated
Financial support:
that curcumin decreased MMP in cancer cells and
The authors received no financial support for the research..
caused apoptosis, consistent with our results28,21.
In addition, there was a strong negative correla- Conflict of Interest:
tion between increased intracellular ROS and The authors declare that they have no conflict of interest.
MMP levels.
GSH is a low molecular weight antioxidant. It
plays an essential role in protecting cells from ox-
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