Dual Effects of Omega -6, and -9 Fatty Acids on Ovarian Cancer Cell Viability and Their Ability to Induce Apoptosis
Saeideh Hajighasemi1, Mehdi Azad1, Morteza Karimipoor2, Hamzeh Rahimi2, Farshad Foroughi1, Nematollah Gheibi3*
1Department of Medical Biotechnology, School of Paramedicine, Qazvin
University of Medical Sciences, Qazvin, Iran
2Molecular Medicine Department, Biotechnology Research Center, Pasteur
Institute of Tehran, Tehran, Iran
3Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran
*Corresponding author: Nematollah Gheibi, Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran. Tel: +982833324970; Email: Nasr_bio@yahoo.com
Received Date: 01 March, 2018; Accepted Date: 22 May, 2018;
Published Date: 29 May, 2018
Citation: Hajighasemi S,
Azad M, Karimipoor M, Rahimi H, Foroughi F, et al. (2018) Dual Effects of Omega
-6, and -9 Fatty Acids on Ovarian Cancer Cell Viability and Their Ability to
Induce Apoptosis. Adv Proteomics Bioinform: APBI-106. DOI: 10.29011/APBI -106.100006
1. Abstract
It has been proposed that unsaturated fatty acids (UFAs) have cytotoxic effects on different cancer cell lines mostly colorectal, breast and prostate cancer cells. Unsaturated fatty acids with more than one double band are called polyunsaturated fatty acids and consist of omega-3 and omega-6 fatty acids. The omega-3 series of fatty acids seem to possess anti- cancer actions and have the ability to inhibit cell division and induce apoptosis, whereas the products of omega-6 fatty acids are believed to enhance cancer cell proliferation. The cytotoxic effects of some UFAs including Linoleic Acid (LA), Arachidonic Acid (AA), Α-Linolenic Acid (ALA), and Oleic Acid (OA) was investigated in SKOV-3 human ovarian adenocarcinoma cells using MTT assay. The apoptosis induction of LA and OA was further characterized using an annexin-V-FLUOS staining kit. Linoleic acid (LA) and oleic acid (OA), as omega-6 and omega-9 fatty acids respectively, were abletoinhibitSKOV-3cell growth at concentrations above 500µmol, while at low concentrations (300–500 µmol) they promoted the proliferation of cells. However, ALA and AA, omega-3 and - 6 fatty acids respectively, showed no remarkable effect on viability of SKOV-3 cells. LA and OA had significant apoptotic effect on SKOV3 ovarian cancer cell line. It seems that there is a critical concentration for some UF as in confronting with some cancer cells and this critical concentration depends on the type of cell and also unsaturated fatty acid itself. UFAs put cytotoxic effect on cancer cells and this cytotoxicity is resulted from apoptosis induction.
2. Keywords: Apoptosis; Cytotoxicity; Unsaturated Fatty Acids
1.
Introduction
Despite recent progress in cancer
treatment, cancer still remains as a great challenge for medicine. In
gynecologic oncology, ovarian cancer is a major problem because of its
therapeutic resistance [1]. Epithelial ovarian cancer (EOC) is at the first
place among other gynecologic malignancies to drive fatality [2] and in women
suffering from cancer, is at the fourth place to cause morbidity and mortality
[3,4]. Although there are moderate advances in chemotherapy for epithelial
ovarian cancer, it still remains as one of the most aggressive cancer types
which have a survival rate of 5years and an overall cure rate of
approximately30% [5]. So, finding new drugs and approaches for epithelial ovarian
cancer treatment seems to be very crucial.
There is considerable evidence that
unsaturated fatty acids, despite their role as an energy source, might affect
both cancer development and progression [6-8]. Unsaturated fatty acids are
categorized into two groups of polyunsaturated fatty acids (PUFAs) and
monounsaturated fatty acids (MUFAs) based on the number of carbon-carbon double
bonds in their carbon chain [9,10] which both have specific effects on cancer
[11,12]. It should be noted that most investigations in this field have been
focused on the effects of PUFAs and there are less studies about the effects of
MUFAs, such as Oleic acid (OA; n-918:1), on cancer. PUFAs including omega-3 and
omega-6 families are able to exert anti-cancer activities both in vitro and in
vivo [13-16]. The omega-6 Linoleic acid (LA; n-618:2) andomega-3α-linolenic
acid (ALA; n-318:3) are essential fatty acids (EFAs) and used to synthesis of
long chain PUFAs of their family. The omega-6 long chain polyunsaturated fatty
acids (LCPUFAs) include γ-linolenic acid (GLA; n-6 18:3), dihomo-GLA (DGLA; n-6
20:3) and arachidonic acid (AA; n-6 20:4), and LCPUFAs of omega-3 family are
eicosapentaenoic acid (EPA; n-3 20:5) and docosahexaenoic acid (DHA; n-3 22:6)
[17]. These long chain PUFAs can give rise to the action of their precursors
(EFAs), and hence areal so called functional EFAs [18]. It has been
demonstrated that EFAs and their products can remarkably inhibit the cancer
cell growth both in vitro and in vivo [15]. Previously, it has been clarified
that some PUFAs exhibit selective cytotoxic effect on various cancer cells in
vitro and in vivo, meaning that when the appropriate concentration is used, the
tumor cells are killed without any harm or damage to the normal cells. But the
mechanisms behind this tumoricidal function of PUFAs are not fully understood.
Oleic acid (OA; 18:1) is an omega-9
monounsaturated fatty acid abundantly found in olive oil. Traditionally it has
been demonstrated that olive oil consumption links to an anti-cancer effect due
to the existence of OA [19,20]. A broad range of epidemiological and animal
researches have shown a protective effect for OA against several cancers such
as breast, colorectal, prostate and ovarian [21,27]. OA can act selectively on
cell growth as it has a promoting effect on growth of non-malignant cells and
an inhibitory effect on malignant ones [28,29]. Several studies clarified that
OA can inhibit proliferation of various cancer cell lines [11,30,31]. Yet, in
this context some controversial results have been also reported, representing
different effects (from non-promoting to completely promoting) on tumor cell
growth [32,35]. But all these investigations have been focused on breast cancer
cell lines.
In a large population-based case–control
study, there was no evidence that the more consumption
oftotalomega-3polyunsaturatedfattyacidsoreachomega-3fatty acid (ALA, EPA, DPA or
DHA) individually helps to the lowered incidence of ovarian cancer. But
consumption of higher a mounts of total omega-6 fatty acids inversely related
to ovarian cancer development, and this appeared to be primarily driven by
linoleic acid [36].
According to our knowledge, the cytotoxic
and apoptotic effect of unsaturated fatty acids such as ALA(n-3;18:3), LA(n-6;18:2),
AA(n-6;20:4), and OA(n-9;18:1) on SKOV-3cellshasnotyetbeen examined. Thus, we
conducted present study to evaluate such effects on mentioned cell line.
2.
Materials and Methods
The unsaturated fatty acids including
Alpha-linolenic acid (n-3; 18:3), Linoleic acid (n-6; 18:2), Arachidonic acid
(n-6;20:4), and Oleic acid (n-9;18:1) were purchased from Sigma-Aldrich. The
unsaturated fatty acids were dissolved in pure ethanol, filter-sterilized and
stored at -70°C. The stock solutions were diluted with cell culture medium for
use. The SKOV-3, a human epithelial ovarian cancer cell line, was obtained from
NCBI (National Cell Bank of Iran). The MTT powder (Sigma-Aldrich, M2128) was
kindly provided by Dr. Elham Tafsiri, Molecular Medicine Department,
Biotechnology Research Center, Pasteur Institute of Iran. The annexin-V-FLUOS
staining kit (Roche Life Science) was kindly provided by Dr.Mehdi Edalati
Fathabad, Hematology Department, Tehran University of Medical Sciences.
2.1.
Cell Culture
The SKOV-3 cell line was cultured in RPMI-1640
medium (Biosera, LM-R1637) supplemented with 10% heat-inactivated FBS (fetal
bovine serum, Gibco, 26140-079) and 1% penicillin- streptomycin (Gibco,
15140-122). The cells were incubated at 37°C in a humidified incubator
containing 5% CO2, and sub-cultured beyond 80% confluency.
2.2.
Cell Viability
Assessment
The cell viability was evaluated by usage
of MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5- di phenyl tetrazolium bromide] dye.
The SKOV-3 cells were seeded in 96-well plates at 1.0×104 cells/well density
and incubated overnight to attach. Then the medium was gently removed and fresh
medium (RPMI-1640, 10% FBS) containing different concentrations of ALA, LA, AA
and OA was added and incubated for 24, 48, and 72 hours without replacement of
the medium. In all the experiments, untreated cells which didn't receive any
concentration of unsaturated fatty acids but were treated with equal content of
ethanol served as controls. The doses for LA and OA fatty acids ranged from 300
to 700µM, and from 300 to 1000µM for ALA and AA. After spending proper
incubation time, the medium was gently discarded, and each well was treated
with 10µl of MTT solution [5 mg/ml in phosphate buffered saline (PBS)] and 90µl
medium at 37°C for 2½ hours. The formed formazan crystals were then dissolved
in 100 µl/ well dimethyl sulfoxide (DMSO) and the absorbance was measured at
570 nm by a 96-well micro plate ELISA reader (Synergy4, nBioTek). The viability
of cells in each well was presented as percentage of the control. Triplicate
tests were set up for each treatment and for control. The IC50 was calculated
from mean±SD values.
2.3.
Quantification of
Apoptosis Using Flow Cytometry
In order to detect the apoptotic and
necrotic cells, the annexin-V-FLUOS staining kit (RocheLife Science, 11 858 777
001) was utilized. According to its dual-staining protocol, the Annexin-V-
FLUOS (green fluorescence) was used to stain the apoptotic cells, and the
propidium iodide (PI; red fluorescence) was used to stain the necrotic ones.
The SKOV-3 cells were seeded in 6-well plates at 3×105cells/well density and
incubated overnight. Then, the medium was discarded and freshRPMI-1640medium
with LA and OA each at 600µM concentration was added and the cells were treated
for 24,48, and 72h. After the treatment, the cells were trypsinized, washed
with PBS, and labeled according to the manufacturer’s instruction. The labeled
cells were finally analyzed using a flow cytometry (Cyflow, Partec) and the
percentages of apoptotic cells were determined using Cyflogic software.
2.4.
Statistical Analysis
All the experiments were performed in
triplicate. The statistical analysis was accomplished by repeated measures
ANOVA and Tukey’s post hoc tests (α = 0.05). Values are means (n=3) ± SD.
(P-value ˂0.05). Data were analyzed using SPSS19.
3. Results
3.1.
SKOV-3 Cell
Viability After UFA Treatment
To evaluate the cytotoxicity of omega-3,
-6 and -9 UFAs on SKOV-3 cells, the cells were treated with different
concentrations (300, 400, 500, 600, and 700 µM) of LA and OA, and (300,
400,500, 600, 700, and 1000 µM) of ALA and AA for 24, 48, and 72 hours. Then,
the viability of treated cells was measured by MTT assay. The LA
(shortchainomega-6PUFA) and OA (omega- 9 MUFA) induced cytotoxic effects on
human ovarian adenocarcinoma SKOV-3 cells. On the other hand, ALA (shortchainomega-3PUFA)
and AA (longchainomega-6PUFA) were notable to affect the viability of SKOV-3
cells at used concentrations after 72 hours. The LA and OA performed dual
effects on SKOV-3 cells at concentrations of 300-700 µM as they promoted the
cell proliferation at concentrations below 500 µM, while reduced the cell
viability at concentrations above 500 µM (Figure 1). The half maximal
inhibitory concentrations (IC50) of LA and OA were calculated and demonstrated
in Table 1.
3.2.
Flow Cytometric
Analysis of Apoptosis
To determine whether the cytotoxicity of
LA and OA was caused by apoptosis induction, the LA- and OA-treated SKOV-3
cells were stained with Annexin-V-FLUOS staining kit. According to the flow
cytometry results, it was revealed that the cytotoxic action of LA and OA on
SKOV-3 cells is caused by the ability of these FAs to induce apoptosis. It was
also indicated that the apoptosis induction ability of mentioned fatty acids is
promoted through time (Figure 2 and3).
4.
Discussion
In this study, it has been demonstrated
that LA and OA unsaturated fatty acids have cytotoxic and apoptotic effects
onSKOV-3 cancer cell line. In contrast, we didn't observe significant changes
in cell viability of SKOV-3 cells by ALA and AA. The LA and OA cytotoxic
effects on SKOV-3 were obtained in concentrations higher than 500 µM which were
enhanced in a time-dependent manner. On the other hand, there was a minor
increase in cell proliferation through the concentrations under500µM.Previous
studies have shown that unsaturated fatty acids can induce cell death in tumor
cells [37] but the sensitivity of these cells seems to be diverse based on
their nature, fatty acid type, and the used concentration [38,40]. Also, it was
mentioned that the concentration at which unsaturated fatty acids can reduce
cell viability is dependent on the cell density [41]. Similar to the current
study, the dual effects of some types of unsaturated fatty acids have been
referred in other studies i.e., they promote cell proliferation at low
concentrations and inversely induce the cell death at higher concentrations. It
was noted that when cancer cells at a density of 1×104 were exposed to LA at
the concentration of 40 μg/ml, the growth was inhibited,
whileatconcentrationsof5-10μg/ml,the cell proliferation was enhanced in some,
if not all, types of cancer cells tested [42]. In another study performed on
colorectal cancer cells, LA was growth- promoting at concentrations below 300
µM/L, but induced the inhibition at higher concentrations [43].
InMOLT-4leukemia cell line, the inhibitory and stimulatory effects of LA were
obtained at 400µM and 200µM, respectively [44]. There is also evidence
supporting such effect for OA [45]. The stimulatory effect LA and the
inhibitory and/or cytotoxic effect of OA are reported in other studies [46,47].
In line with this investigation, it has been shown that AA is not able to
influence the viability of some cell lines of breast cancer, but has
cytotoxicity on others [39]. The in vitro studies have revealed that the
cytotoxic potency is different among PUFAs and it seems that ALA and AA have
the weakest [48]. In addition, according to some reports on tumor cells, ALA
seems to need more time than other PUFAs to have an influence on cancer cells
[49].
Also, it has been clarified that the
cytotoxic effect of LA and OA on SKOV-3 cells is caused by apoptosis induction.
Apoptosis maintains the balance between cell birth and cell death in our body
and as a result, many biological circumstances are regulated [50]. Cancerous
cells can block apoptotic pathways, so that they continue to growth and
proliferation [51]. There are many efforts to find the elements for apoptosis
induction in different cancer cells. The results of the present study are
clearly compatible with recent reports that demonstrate the apoptosis induction
of unsaturated fatty acids in various cell lines at high concentrations
[15,52]. According to other studies it seems that fatty acids can cause cell
death through apoptosis or necrosis (at higher concentrations) [53]. As
observed in recent study and also previous studies, LA is more potent to induce
apoptosis than OA. Unsaturated fatty acids induce apoptosis through the
activation of caspases- 3, 6, 7, 8, and 9 [40,54,55]. They can disrupt redox
state of cells by generating free radicals and lipid peroxides through lipid
peroxidation, compelling cells to be a rapoptosis [38,56]. Lipid peroxidatin
increases after treatment with PUFAs. The lipid derived metabolites may
activate caspases and induce apoptosis [57]. Moreover, unsaturated fatty acids
can cause loss of mitochondrial potential which may lead to the elevated levels
of reactive oxygen species (ROS) [58]. In addition, PUFAs can cause the
cleavage of Bid and the cytochrome c leakage from mitochondria [40,59].
5.
Conclusions
OurresultsindicatedthatLAandOAhavedualeffectsonSKOV-3ovariancancercellline.These
effects include cell death induction at high concentrations (≥500 µM) and the
promotion of cell proliferation at low concentrations (<500µM). In general,
different cells have different sensitivity against unsaturated fatty acids.
Also, the fatty acid concentration and the exposure time of cells affect the
degree of cell death and it seems that there is difference in the potency of
unsaturated fatty acids to induce apoptosis.
Given our results and according to the
previous studies in this content, a critical concentration seems to exist for
some unsaturated fatty acids in confronting with some cancer cells and it
relies on the cell type and also the unsaturated fatty acid itself. LA and OA
have cytotoxic effect on SKOV-3 cells through their apoptosis induction
ability. However, the distinct molecular mechanisms behind the apoptosis
induction of LA and OA need more investigations.
6.
Acknowledgement
We gratefully appreciate the Department of
Molecular Medicine, Biotechnology Research Center, Pasteur Institute of Tehran,
Iran, for providing the possibility to use the experimental equipment. We would
like to thank Dr. Elham Tafsiri, Molecular Medicine Department, Biotechnology
Research Center, Pasteur Institute of Iran, and Dr. Mehdi Edalati Fathabad,
Hematology Department, Tehran University of Medical Sciences for their kind
assistance through this study. We are also grateful to Reyhaneh Sadeghinejad,
Department of Biostatistics, Tarbiat Modares University for providing SPSS
measurements.
Figure 1: MTT assay results: The viability of SKOV-3 cells after 24, 48, and 72 hours of treatment with A) ALA; B) OA; C) LA; and D) AA. The dual effect of LA and OA on SKOV-3 cell viability is evident.
Figure
2: Flow cytometric assessment of
apoptosis after treatment with LA and OA.
A) Control; B) LA-treated cells after 24h; C) LA-treated cells after 48h;
D) LA-treated cells after 72h; E) OA- treated cells after 24h; F) OA- treated
cells after 48h; and G) OA- treated cells after 72h.
Figure
3: Apoptosis
in SKOV-3 cells after treatment with LA and OA. A) 24 hours after the
treatments, B)48 hours after the treatments, and C) 72 hours after the
treatments with LA and OA. LA and OA are able to induce apoptosis in SKOV-3
cell line. The percentage of apoptotic cells after treatment with LA and OA is
significant. Q1: Debries, Q2: Necrotic, Q3: Living, and Q4: Apoptotic cells.
Cell line |
Unsaturated fatty
acid |
IC50 (µM) |
|||
24 h |
48 h |
72 h |
|||
SKOV-3 |
Linoleic acid (LA) |
541.92 |
522.5 |
513.21 |
|
|
Oleic acid (OA) |
669.52 |
589 |
532.3 |
|
Table 1: The IC50 of cytotoxic unsaturated fatty acids.
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