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Experimental Chemotherapy
Chemotherapy
DOI: 10.1159/000356067
Induction of Apoptosis by the Medium-Chain
Length Fatty Acid Lauric Acid in Colon Cancer
Cellsdue to Induction of Oxidative Stress
J.K.Fauser a,b G.M.Matthews c,d A.G.Cummins b G.S.Howarth a,c
a School of Animal and Veterinary Sciences, Roseworthy Campus, The University of Adelaide, Adelaide,S.A. ,
b Department of Gastroenterology and Hepatology, The Queen Elizabeth Hospital, Woodville, S.A. ,
c Gastroenterology
Department, Children, Youth and Women’s Health Service, North Adelaide, S.A. , and
d Gene Regulation Laboratory,
Cancer Immunology Program, The Peter MacCallum Cancer Institute, Melbourne, Vic. , Australia
acid induced apoptosis in IEC-6 cells compared to butyrate
(p < 0.05). Butyrate protected IEC-6 cells from ROS-induced
damage, whereas lauric acid induced high levels of ROS
compared to butyrate. Conclusion: Compared to butyrate,
lauric acid displayed preferential antineoplastic properties,
including induction of apoptosis in a CRC cell line.
© 2013 S. Karger AG, Basel
Introduction
Scientific evidence describing the potential preventa-
tive and chemotherapeutic properties of fatty acids is in-
creasing for the treatment of intestinal cancers, especially
colorectal cancer (CRC)
[1, 2] . Fatty acids such as butyr-
ate, docosahexaenoic acid (DHA), and conjugated linole-
ic acid (CLA) have demonstrated the capacity to induce
apoptosis in Caco-2, HT-29, and SW620 CRC cell lines
[1, 3] . Fatty acids, therefore, could hold promise as poten-
tial adjunctive chemotherapeutic agents.
Fatty acids are bioactive molecules classified by their
carbon atom chain length as short chain (SCFA; <C8:
0),
medium chain (MCFA; C8:
0–14: 0), or long chain (LCFA;
>C16:ω3–9)
[4–7] . The differing carbon atom chain
length classifications (short, medium, and long) are be-
Key Words
Medium-chain fatty acid · Short-chain fatty acid ·
Colorectalcancer · Caco-2 · IEC-6 · Apoptosis · Redox ·
Cellcycle · Glutathione · Reactive oxygen species
Abstract
Background: Fatty acids are classified as short chain (SCFA),
medium chain (MCFA) or long chain and hold promise as ad-
junctive chemotherapeutic agents for the treatment of
colorectal cancer. The antineoplastic potential of MCFA re-
mains underexplored; accordingly, we compared the MCFA
lauric acid (C12:
0) to the SCFA butyrate (C4: 0) in terms of
their capacity to induce apoptosis, modify glutathione (GSH)
levels, generate reactive oxygen species (ROS), and modify
phases of the cell cycle in Caco-2 and IEC-6 intestinal cell
lines. Methods: Caco-2 and IEC-6 cells were treated with lau-
ric acid, butyrate, or vehicle controls. Apoptosis, ROS, and
cell cycle analysis were determined by flow cytometry. GSH
availability was assessed by enzymology. Results: Lauric acid
induced apoptosis in Caco-2 (p < 0.05) and IEC-6 cells (p <
0.05) compared to butyrate. In Caco-2 cells, lauric acid re-
duced GSH availability and generated ROS compared to bu-
tyrate (p < 0.05). Lauric acid reduced Caco-2 and IEC-6 cells
in G0/G1and arrested cells in the S and G2/M phases. Lauric
Received: January 3, 2013
Accepted after revision: September 30, 2013
Published online: ■ ■ ■
Prof. Gordon S. Howarth
School of Animal and Veterinary Sciences, The University of Adelaide
Roseworthy Campus
Adelaide, SA 5371 (Australia)
E-Mail gordon.howarth @ adelaide.edu.au
© 2013 S. Karger AG, Basel
0009–3157/13/0000–0000$38.00/0
www.karger.com/che
Fauser/Matthews/Cummins/Howarth
Chemotherapy
DOI: 10.1159/000356067
2
lieved to be a critical factor influencing their apoptotic
capacity
[1, 2, 8] . The SCFA butyrate has demonstrated
the capacity to induce apoptosis by: inhibiting histone
deacetylase activity, inducing cell cycle arrest, promoting
differentiation, activating NF-κB, downregulating α
2 β 1 ,
modifying glucose availability, and inducing caspase ac-
tivation in CRC cell lines
[1, 2, 9–12] . LCFA have also
demonstrated apoptotic properties, with palmitic acid,
α-linoleic acid, CLA, and DHA inducing apoptosis in a
range of CRC cell lines, including Caco-2, HTC-116, and
COLO 205 cell lines. This is believed to be a consequence
of lipid peroxidation, modification of cell cycle phases,
induction of p21, an increase in the proapoptotic gene
NAG-1, and altered P13 kinase and p38 MAPK pathways
[13–17] .
Cancer chemotherapy can induce apoptotic cell death
and increased oxidative stress in transformed cells
[18] .
This is often due to reduced levels of the major antioxi-
dant glutathione (reduced form: GSH) or increased levels
of reactive oxygen species (ROS)
[19] . Manipulation of
this system holds promise for cancer treatment
[20, 21] .
The cellular redox state of a cell is a tightly regulated cou-
pled system and a key modulator of intestinal cell homeo-
stasis, regulating the balance between cell proliferation
and apoptosis
[19] . The major regulators of redox balance
in a cell are the redox couplet GSH/GSSH (reduced and
oxidized GSH, respectively)
[19, 22] . Imbalance in this
system can result in oxidative stress, leading not only to
the initiation of diseases including diabetes, cardiovascu-
lar disease, and cancers of the skin, breast, and stomach
but also, paradoxically, to the induction of apoptosis in
neoplastic cells
[19, 23, 24] .
Exogenous manipulation of intracellular GSH levels
has been demonstrated to increase the sensitivity of neo-
plastic cells to chemotherapy and radiotherapy
[25] .
However, few studies have investigated the potential role
of fatty acids in modulation of the intracellular redox sys-
tem and regulation of apoptosis in both transformed and
nontransformed intestinal epithelial cells
[19, 23, 26] .
While several studies on the role of SCFA and LCFA in
GSH modulation and regulation of apoptosis have been
undertaken, no such studies have been reported on
MCFA. MCFA could potentially combine the apoptotic
properties of SCFA and LCFA.
We investigated the MCFA lauric acid (C12:
0) for its
capacity to modify the redox state of CRC (Caco-2) and
nontransformed (IEC-6) intestinal cell lines by evaluat-
ing the bioavailability of the antioxidant GSH, the down-
stream effects on phases of the cell cycle, generation of
ROS, and the resultant induction of apoptosis.
Materials and Methods
Materials
Caco-2 and IEC-6 cell lines were obtained from the American
Type Culture Collection (ATCC
® ) (HTB-37 TM ) (CRL-1592 TM )
(Manassas, Va., USA). Dulbecco’s modified Eagle’s medium
(DMEM), Glutamax
TM , penicillin/streptomycin (10,000 units of
penicillin and 10,000 µg of streptomycin), and Ca
2+ - and Mg
2+ -free
phosphate-buffered saline (PBS) were purchased from Gibco
® , In-
vitrogen (Australia). Heat-inactivated fetal bovine serum ( HI-FBS),
sodium lauric acid (NaLa), sodium butyrate (NaB), fatty acid-free
bovine serum albumin (FAF-BSA), propidium iodide, and RNAse
(DNAse free) were all purchased from Sigma-Aldrich (Castle Hill,
N.S.W., Australia). Annexin-V fluorescein isothiocyanate (FTIC)
was obtained from BioSource, Invitrogen (Australia) and 2 ′ ,7 ′ -di-
chlorodihydrofluorescein diacetate (H2-DCF-DA) was obtained
from Molecular Probes
TM (Invitrogen, Australia). Glutathione re-
ductase and L-glutathione (GSH) were obtained from Sigma-Al-
drich. All tissue culture hardware was CELLSTAR products pur-
chased from Greiner Bio-One (Frickenhausen, Germany).
Cell Culture
The Caco-2 and IEC-6 cell lines were determined to be myco-
plasma negative via polymerase chain reaction undertaken at the
Institute of Medical and Veterinary Science, Australia. Both cell
lines were cultured in complete DMEM containing 2 m
M Gluta-
max with 10% HI-FBS and penicillin/streptomycin (73.5 units/ml,
73.5 ug/ml) in 75-cm
2 tissue culture flasks and maintained in a
humidified atmosphere at 37
° C in 5% CO
2 .
E x p e r i m e n t a l P r o t o c o l
For the cytotoxic dose response, the apoptotic index, cell cycle
analysis, and GSH availability, both cell lines were harvested by
enzymatic dissociation and seeded into 24-well tissue culture
plates at 2.5 × 10
5 and 1.5 × 10
5 cells/ml, respectively. After 24 h of
incubation, the medium was replaced with 1 ml of complete
DMEM containing freshly prepared (50 m
M ) NaLa, conjugated
to0.4% FAF-BSA
[27] to final concentrations of 0.1, 0.3, 0.5 , and
1 m
M and compared to NaB (5 m M ) with 50 µl of 0.4% FAF-BSA
as a vehicle control and incubated in a humidified atmosphere at
37
° C in 5% CO
2 and, after enzymatic dissociation, assayed at 24,
48, 72, and 96 h. All experimental studies were undertaken in trip-
licate and measured in duplicate.
Flow Cytometric Analysis of Apoptosis Induced by Lauric Acid
Apoptosis was measured using a standard fluorescence-acti-
vated cell sorting (FACS) assay by annexin V FTIC and propidium
iodide staining as per Matthews et al.
[26] . The Caco-2 and IEC-6
cell lines were incubated with NaLa, NaB, and vehicle controls as
per the experimental design to assess the apoptotic index. Ten
thousand events were collected for all cytometric analyses on a
FACScan
TM flow cytometer (Becton Dickinson Biosciences, San
Jose, Calif., USA) and analyzed using BD CellQuest Pro
TM soft-
ware. Cells were gated into viable, early/late apoptosis, and ne-
crotic cells against standard controls.
Cell Cycle Analysis
Cell cycle analysis was undertaken as previously described by
Matthews et al.
[26] . Caco-2 and IEC-6 cells were cultured in 5%
FCS for 24 h prior to NaLa, NaB, and vehicle control incubation as
Induction of Apoptosis by the MCFA
Lauric Acid
Chemotherapy
DOI: 10.1159/000356067
3
per the experimental protocol. Twenty thousand events were col-
lected on a FACScan (BD). The phases of the cell cycle were catego-
rized into G0/G1, S, and G2/M and analyzed using BD CellQuest
Pro software.
Determination of GSH Availability and ROS Detection
GSH Availability
Assessment of GSH availability was undertaken as previously
described by Matthews et al.
[26] . Caco-2 and IEC-6 cell lines were
incubated with NaLa, NaB, and vehicle controls as per the experi-
mental design.
ROS Detection
A modified assay from Alexandre et al.
[28] and Traver et al.
[29] was employed to detect ROS. Caco-2 and IEC-6 cells were in-
cubated with NaLa, NaB, and vehicle controls as per the experi-
mental design. A final concentration of 5 µ
M H2-DCF-DA was
used. Twenty thousand events were collected on a FACScan (BD).
The resulting difference in fluorescence intensity was compared to
vehicle controls and cell counts adjusted to 5 × 10
5 cells/ml.
S t a t i s t i c s
All results are expressed as means ± SEM of groups and were
compared using Student’s t test or one-way ANOVA with the Bon-
ferroni post hoc test. p < 0.05 was considered statistically significant.
R e s u l t s
A Cytotoxic Dose of Lauric Acid Significantly Reduced
Caco-2 Cell Viability Compared to Butyrate
After 24 h of treatment with NaLa, Caco-2 cell viabil-
ity was not significantly reduced compared to NaB;
however, NaLa (0.3, 0.5, and 1 m
M
) (70.8 ± 7.4%, 63.4 ±
5.9%, and 55.1 ± 7.5%, respectively) significantly re-
duced cell viability when compared to vehicle controls
(90.1 ± 5.6%) (p < 0.01). NaB (66.5 ± 8.0%) also signifi-
cantly reduced cell viability compared to vehicle con-
trols (p < 0.01) ( fig.1 a). A dose-dependent reduction in
cell viability was demonstrated after 48 h of treatment
with NaLa (0.5 and 1 m
M
) (40.8 ± 6.4% and 7.0 ± 4.3%)
compared to NaB (5 m
M
) (44.5 ± 2.4%) (p < 0.01 and p<
0.0001) and vehicle controls (91.4 ± 3.0%). NaLa (0.1
and 0.3 m
M
) (76.5 ± 3.5% and 50.0 ± 8.8%) and NaB
(44.5 ± 2.4%) reduced cell viability compared to vehicle
controls (91.4 ± 3.0%) (p < 0.01, p < 0.0001, and p <
000.1) ( fig.1 b). NaLa continued to reduce cell viability
at 72 and 96 h postincubation compared to NaB and ve-
0.1 mM NaLa
0.3 mM NaLa
0.5 mM NaLa
1 mM NaLa
NaB
Vehicle
0
20
40
60
80
100
Viable, TA, and
necrotic cells (%)
bb
aa
bbb
bb
bbb
bb
bbb
bb
bbb
bb
bbb
a
0
20
40
60
80
100
Viable, TA, and
necrotic cells (%)
0.1 mM NaLa
0.3 mM NaLa
0.5 mM NaLa
1 mM NaLa
NaB
Vehicle
bbb
bbb
bbb
bbb
bbb
bbb
bbb
bbb
bbb
c
0
20
40
60
80
100
0.1 mM NaLa
0.3 mM NaLa
0.5 mM NaLa
1 mM NaLa
NaB
Vehicle
bbb
bbb
aaa
bbb
aa
bb
bbb
bbb
b
0
20
40
60
80
100
0.1 mM NaLa
0.3 mM NaLa
0.5 mM NaLa
1 mM NaLa
NaB
Vehicle
bbb
bbb
bbb
bbb
bbb
bbb
bbb
bbb
bbb
bb
bbb
d
Necrosis
TA
Viable
Fig. 1. Caco-2 cells treated with increasing concentrations of NaLa
and cell viability, TA, and necrosis compared to NaB (5 m
M ) and
the vehicle control over 24 (
a ), 48 ( b ), 72 ( c ), and 96 h ( d ). Data
are presented as means ± SEM (n = 3). Statistical significance be-
tween treatment groups is denoted as:
aa p < 0.01 (NaLa vs. NaB),
aaa p < 0.0001 (NaLa vs. NaB),
bb p < 0.01 (NaLa or NaB vs. vehicle
control), and
bbb p < 0.0001 (NaLa or NaB vs. vehicle control).
Fauser/Matthews/Cummins/Howarth
Chemotherapy
DOI: 10.1159/000356067
4
hicle controls [(0.5 and 1 m
M
) (44.8 ± 9.8% and 40.1 ±
8.8%) and (18.6 ± 3.9% and 13.6 ± 5.3%) (p < 0.0001)]
( fig.1 c, d).
Lauric Acid Induced Apoptosis in the Caco-2 Cell Line
At 24 h post-NaLa treatment (1 m M ) (41.5 ± 3.5%) the
proportion of Caco-2 cells in total apoptosis (TA) (TA =
early + late apoptosis) was significantly increased com-
pared to NaB (66.5 ± 5.7%) (p < 0.01). All concentrations
of NaLa (0.1, 0.3, 0.5, and 1 m
M ) (16.9 ± 3.3%, 28.1 ± 2.5%,
32.8 ± 3.0%, and 41.5 ± 3.5%) and NaB (66.5 ± 5.7%) in-
duced a significantly higher percentage of cells undergo-
ing TA compared to vehicle controls (p < 0.01, p < 0.0001,
p < 0.0001, p < 0.0001, and p < 0.0001, respectively)
( fig.1 a). After a further 48 h of treatment, NaLa (1 m
M )
(88.0 ± 5.6%) induced a higher percentage of cells in TA
compared to NaB (45.4 ± 3.2%) (p < 0.0001). NaLa, at
concentrations of 0.3 m
M and 0.5 m M (45.0 ± 5.8% and
59.1 ± 6.7%), and NaB (45.4 ± 3.2%) also significantly in-
creased the TA compared to vehicle controls (p < 0.0001)
( fig. 1 b). From 72 to 96 h posttreatment, the rate of TA
induced in Caco-2 cells remained constant and was not
significantly different between NaLa (0.1, 0.3, 0.5, and
1m
M ) and NaB. However, all NaLa and NaB induced sig-
nificant TA compared to vehicle controls (p < 0.0001;
fig.2 c, d). Over the 96-hour assay period, NaLa (1 m
M )
induced an increase in Caco-2 cells (67.6 ± 10.0%) in TA
compared to NaB-treated Caco-2 cells (49.3 ± 7.0%) (p <
0.05). A significant difference in the induction of necrosis
was noted at 96 h postincubation with 5 m
M NaB com-
pared to the vehicle control (p < 0.01).
Lauric Acid Significantly Reduced IEC-6 Cell Viability
To ascertain whether NaLa or NaB altered the viability
of normal intestinal epithelial cells, IEC-6 cells were treat-
ed with NaLa (0.1, 0.3, 0.5, and 1 m
M ) and NaB and com-
pared to the vehicle control. After 24 h, NaLa (0.5 and
1m
M ) (71.5 ± 4.1% and 60.4 ± 5.2%) reduced IEC-6 cell
viability significantly compared to NaB (87.8 ± 6.7%) (p<
0.01) and vehicle controls (91.4 ± 5.5%) (p < 0.05) ( fig.3 a).
0
5
10
15
GSH availability
(μM/1×106 cells)
a
0.1 mM NaLa
0.3 mM NaLa
0.5 mM NaLa
1 mM NaLa
Butyrate
Vehicle
bb
bbb bbb bbb
0
5
10
15
b
0.1 mM NaLa
0.3 mM NaLa
0.5 mM NaLa
1 mM NaLa
Butyrate
Vehicle
bbb
bb
GSH availability
(μM/1×106 cells)
0
5
10
15
c
0.1 mM NaLa
0.3 mM NaLa
0.5 mM NaLa
1 mM NaLa
Butyrate
Vehicle
bbb
bbb bbb
bbb
bbb
0
5
10
15
d
0.1 mM NaLa
0.3 mM NaLa
0.5 mM NaLa
1 mM NaLa
Butyrate
Vehicle
bb
Fig. 2. GSH availability in Caco-2 cells treated with increasing con-
centrations of NaLa (0.1, 0.3, 0.5, and 1 m
M ) and NaB (5 m M ) and
compared to the vehicle control over 24 (
a ), 48 ( b ), 72 ( c ) and 96h
(
d ). Data are presented as means ± SEM (n = 3). Statistical signifi-
cance between treatment groups is denoted as:
bb p < 0.01 (NaLa/
NaB vs. vehicle control) and
bbb p < 0.0001 (NaLa/NaB vs. vehicle
control).
Induction of Apoptosis by the MCFA
Lauric Acid
Chemotherapy
DOI: 10.1159/000356067
5
At 48 h posttreatment, IEC-6 cell viability was significant-
ly reduced compared to NaLa (0.5 and 1 m
M ) (70.3 ± 7.1%
and 57.4 ± 6.3%), NaB (92.0 ± 4.3%) (p < 0.05 and p <
0.0001, respectively), and vehicle controls (95.0 ± 5.5%)
(p < 0.05 and p < 0.0001, respectively) ( fig.3 b). At 72 h
posttreatment NaLa (0.5 and 1 m
M ) reduced cell viability
significantly compared to NaB (p < 0.0001). NaLa (0.1,
0.3, 0.5, and 1 m
M ) and NaB reduced cell viability signif-
icantly compared to vehicle controls (p < 0.05, p < 0.01,
p < 0.0001, p < 0.0001, and p < 0.0001, respectively)
( fig. 3 c). At 96 h posttreatment, only NaLa (1 m
M ) sig-
nificantly reduced cell viability compared to NaB and ve-
hicle controls (p < 0.0001) ( fig.3 d).
Lauric Acid Induced Apoptotic Cell Death in IEC-6
Cells
To ascertain whether treatment with NaLa or NaB in-
duced apoptosis or necrosis on normal gut epithelia, the
experimental conditions were reproduced in the IEC-6 cell
line evaluating induction of cell death. At 24–96 h post-
treatment with NaLa (0.5 and 1 m
M
), IEC-6 cell viability
was significantly reduced compared to NaB and vehicle
controls due to the preferential induction of apoptosis (p<
0.0001). NaB did not induce apoptosis at any assay time
point compared to vehicle controls ( fig.3 a–d). A signifi-
cantly higher level of necrosis was noted at 48 h posttreat-
ment with NaLa (0.5 and 1 m
M
) and NaB compared to the
vehicle control (p < 0.001, p < 0.0001, and p < 0.05, respec-
tively) and once again at 72 h posttreatment with NaLa (0.5
and 1 m
M
) compared to the vehicle control (p < 0.01 and
p< 0.0001). In the final assay period of 96 h, NaB induced
a significant higher number of cells undergoing necrosis
compared to the vehicle control (p < 0.01) ( fig.3 b–d).
In Caco-2 Cells, Lauric Acid Reduced G0/G1 and
Arrested Cells in the S and M Phases
Over the 72-hour assay period, NaLa at the higher con-
centrations of 0.5 and 1 m
M tended to reduce the mean
percentage of Caco-2 cells (36.3 ± 1.7% and 37.9 ± 5.6%)
in the G0/G1 phase compared to NaB (42.7 ± 0.9%); how-
0.1 mM NaLa
0.3 mM NaLa
0.5 mM NaLa
1 mM NaLa
NaB
Vehicle
0
20
40
60
80
100
Viable, TA, and
necrotic cells (%)
aaa
bbb
aaa
bbb
aaa
bbb
aaa
bbb
a
0
20
40
60
80
100
Viable, TA, and
necrotic cells (%)
0.1 mM NaLa
0.3 mM NaLa
0.5 mM NaLa
1 mM NaLa
NaB
Vehicle
bbb bbb
aaa
bbb
bb
aaa
bbb
aaa
bbb
aaa
bbb
bbb
c
0
20
40
60
80
100
0.1 mM NaLa
0.3 mM NaLa
0.5 mM NaLa
1 mM NaLa
NaB
Vehicle
aaa
bbb
aaa
bbb
bbb
aaa
bbb
aaa
bbb
bb b
b
0
20
40
60
80
100
0.1 mM NaLa
0.3 mM NaLa
0.5 mM NaLa
1 mM NaLa
NaB
Vehicle
aaa
bbb
aaa
bbb
aaa
bbb
aaa
bbb
bb
d
Necrosis
TA
Viable
Fig. 3. IEC-6 cells treated with increasing concentrations of NaLa
and cell viability, TA, and necrosis compared to NaB (5 m
M ) and
the vehicle control over 24 (
a ), 48 ( b ), 72 ( c ), and 96 h ( d ). Data
are presented as means ± SEM (n = 3). Statistical significance be-
tween treatment groups is denoted as:
aaa p < 0.0001 (NaLa vs.
NaB),
b p < 0.05 (NaLa or NaB vs. vehicle control),
bb p < 0.01 (NaLa
or NaB vs. vehicle control), and
bbb p < 0.0001 (NaLa or NaB vs.
vehicle control).
Fauser/Matthews/Cummins/Howarth
Chemotherapy
DOI: 10.1159/000356067
6
ever, this was not significant (p > 0.05). Caco-2 cells were
arrested in the S phase following NaLa treatment. Once
again, higher doses of NaLa (0.5 and 1 m
M ) arrested a sig-
nificantly higher percentage of Caco-2 cells (26.2 ± 0.7%
and 26.8 ± 1.6%) compared to NaB (21.5 ± 0.8%) (p <
0.05). Cells were also arrested in the M phase after treat-
ment with NaLa (1 m
M ) (39.4 ± 2.3%) compared to NaB
(29.6 ± 0.7%) (p < 0.0001). All fatty acids reduced Caco-2
cells in G0/G1 and arrested cells in the S and M phases
compared to vehicle controls (G0/G1, 62.7 ± 0.6; S, 14.9±
0.7, and M, 18.7 ± 1.3%), NaLa (0.3, 0.5, and 1 m
M ), and
NaB for all phases (p < 0.0001).
Lauric Acid Reduced G0/G1 and Arrested the S and M
Phases in IEC-6 Cells
IEC-6 cells treated with NaLa had a reduced mean per-
centage of cells in the G0/G1 phase with treatment of 0.5
and 1 m
M (60.4 ± 0.06% and 53.6 ± 1.8%) compared to
NaB (75.4 ± 0.2%; p < 0.001 and p < 0.0001) and the ve-
hicle control (81.5 ± 0.3%) (p < 0.0001) over the 72-hour
assay period. NaLa at doses of 0.3, 0.5, and 1 m
M (8.8 ±
0.2%, 11.5 ± 0.2%, and 11.9 ± 0.3%) arrested IEC-6 cells
in the S phase compared to NaB (7.3 ± 0.1%; p < 0.05).
The same NaLa doses arrested a mean percentage of cells
compared to vehicle controls (6.4 ± 0.3%; p < 0.05). Once
again, NaLa at does of 0.5 and 1 m
M arrested a mean per-
centage of IEC-6 cells (24.5 ± 0.4% and 29.8 ± 0.1%) in
the M phase compared to NaB (15.2 ± 0.2%) (p < 0.05)
and vehicle controls (14.0 ± 0.2%) (p < 0.05). NaB did not
significantly reduce the G0/G1, S, or M phase at any time
point in IEC-6 cells ( table2 ).
Reduction of GSH Availability and Generation of ROS
GSH Availability
After 24 h, the GSH concentration was significantly
reduced in NaLa-treated cells (0.3, 0.5, and 1 m
M ) (p <
0.01, p < 0.0001, and p < 0.0001) and NaB-treated Caco-2
cells (p < 0.0001) compared to the vehicle control. At 48h
Table 2. Cell cycle results for IEC-6 cells following treatment with
NaLa compared to NaB
Cell cycle
phase
Fatty acid
(mM)
Duration of treatment (h)
24 48 72
G0/G1 0.1 NaLa 68.0±0.7 80.9±0.1b83.2±0.3
0.3 NaLa 59.0±0.6e71.8±0.1 64.7±0.4f
0.5 NaLa 56.4±0.1a, e 68.6±0.05 56.3±0.1c, f
1 NaLa 56.9±0.1a, e 53.4±0.1c, e 50.5±0.1c, f
5.0 NaB 68.0±0.3 72.1±0.01 86.0±0.3
Vehicle control 78.6±0.4 78.3±0.3 87.5±0.05
S 0.1 NaLa 9.5±0.7 6.2±0.03 6.7±0.1
0.3 NaLa 14.5±0.2b, e 5.8±0.1 6.1±0.2a
0.5 NaLa 14.2±0.1b, e 7.5±0.5a, d 12.7±0.2b, e
1 NaLa 13.5±0.4a, d 8.8±0.1b, e 13.5±0.3b, e
5.0 NaB 8.1±0.2 5.2±0.1 8.6±0.3
Vehicle control 6.4±0.2 5.4±0.2 7.4±0.5
M 0.1 NaLa 13.3±1.3 13.6±0.06 8.4±0.03d
0.3 NaLa 18.2±0.5b19.5±0.2 17.8±0.4b, d
0.5 NaLa 20.7±0.1c, d 21.3±0.4b, d 31.7±0.1c, f
1 NaLa 22.3±0.1c, e 31.1±0.1c, e 36.2±0.1c, f
5.0 NaB 14.5±0.2 18.2±0.2 13.0±0.3g
Vehicle control 15.6±0.5 16.5±0.3 10.0±0.06
Data are presented as mean percentages ± SEM (n = 3).
Cell cycle phases are compared between NaLa, NaB, and the
vehicle control. Statistical significance between treatment
groups is denoted as:
a
p < 0.05 (NaLa vs. NaB),
b
p < 0.01
(NaLavs. NaB),
c
p < 0.0001 (NaLa vs. NaB),
d
p < 0.05 (NaLa
vs.vehicle control),
e
p < 0.01 (NaLa vs. vehicle control),
f
p <
0.0001 (NaLa vs. vehicle control),
g
p < 0.05 (NaB vs. vehicle
control).
Table 1. Cell cycle results for Caco-2 cells following treatment with
NaLa compared to NaB
Cell cycle
phase
Fatty acid
(mM)
Duration of treatment (h)
24 48 72
G0/G1 0.1 NaLa 48.7±0.1a, e 41.7±0.1e40.1±0.5
0.3 NaLa 37.6±0.8d48.0±0.4d48.1±0.4
0.5 NaLa 30.0±0.4b, f 40.3±0.1e38.2±0.2b
1 NaLa 32.0±0.6a, e 49.3±0.4d32.6±0.4c
5.0 NaB 40.9±0.2h43.9±1.0h42.4±0.1i
Vehicle control 62.1±0.3 63.7±0.3 64.1±0.1
S 0.1 NaLa 21.0±0.1d23.7±0.3d20.8±0.1a, f
0.3 NaLa 24.4±0.1d23.6±0.3d22.8±0.2a, e
0.5 NaLa 29.0±0.2e24.4±0.1d25.0±0.■c, e
1 NaLa 35.9±0.4a, i 21.9±0.1d32.6±0.4c, e
5.0 NaB 24.1±0.3 21.8±0.9g18.6±1.0h
Vehicle control 15.6±0.2 12.7±0.9 13.8±0.4
M 0.1 NaLa 32.3±0.1e32.9±0.1a24.2±0.1b
0.3 NaLa 34.1±0.1b, e 22.4±0.8 29.5±0.5d
0.5 NaLa 31.1±0.1b, e 26.2±0.3b34.2±0.2b, e
1 NaLa 32.2±0.1d24.7±0.3 43.0±0.9b, e
5.0 NaB 28.4±0.5g26.2±0.2g30.1±0.3g
Vehicle control 18.3±0.4 15.7±0.2 19.6±0.1
Data are presented as mean percentages ± SEM (n = 3). Cell
cycle phases are compared between NaLa, NaB, and the vehicle
control. Statistical significance between treatment groups is de-
noted as: ap < 0.05 (NaLa vs. NaB), bp < 0.01 (NaLa vs. NaB), cp<
0.0001 (NaLa vs. NaB), dp < 0.05 (NaLa vs. vehicle control), ep <
0.01 (NaLa vs. vehicle control), fp < 0.0001 (NaLa vs. vehicle con-
trol), gp < 0.05 (NaB vs. vehicle control), hp < 0.01 (NaB vs. ve-
hicle control), and ip < 0.0001 (NaB vs. vehicle control).
Induction of Apoptosis by the MCFA
Lauric Acid
Chemotherapy
DOI: 10.1159/000356067
7
posttreatment, only 1 m M NaLa (p < 0.0001) and NaB
(p< 0.001) significantly reduced GSH levels compared to
the vehicle control. Interestingly, after 72 h of incubation
with NaLa (0.1, 0.3, 0.5, and 1 m
M ) (all p < 0.0001) and
NaB (p < 0.0001) compared to the vehicle control. At the
last time point of measurement, only NaLa at 1 m
M (p <
0.001) led to significantly reduced levels of GSH com-
pared to the vehicle control. GSH levels of the vehicle
control at 96 h (7.4 ± 0.6
M /1 × 10
6 cells) were less than
those of vehicle controls at 72 h (10.2 ± 0.9
M /1 × 10
6
cells), which may have had an impact on the significance
of the results at 96 h ( fig.2 a–d). GSH availably in the IEC-
6 cell line did not significantly change following treat-
ment with either NaLa or NaB at 24 h postincubation.
However, at the time points of 48–96 h the NaLa dose of
1 m
M significantly reduced GSH levels compared to 0.5
m
M NaLa and NaB (p < 0.0001; fig. 4 a–d).
ROS Generation
In Caco-2 cells, at 24 h posttreatment NaLa (0.5 and
1m
M ) induced a significant increase in ROS compared to
NaB (p < 0.05 and p < 0.05) and vehicle controls (p < 0.01
and p < 0.01). However, no significant increase was de-
tected between NaB and vehicle controls (p > 0.05). ROS
generation at 48 h was significantly increased for NaLa
(0.5 and 1 m
M ) compared to NaB (p < 0.01 and p < 0.0001)
and vehicle controls (p < 0.01 and p < 0.0001). NaB in-
duced a significant increase in ROS compared to vehicle
controls (p < 0.01). In the final 72 h of analysis, the in-
crease in ROS generation continued significantly between
NaLa (0.5 and 1 m
M ) and NaB and vehicle controls (p <
0.0001, p < 0.0001, and p < 0.0001). NaB also induced a
significant difference in ROS generation compared to the
vehicle control (p < 0.01) ( fig.5 a).
In IEC-6 cells, at 24 h posttreatment NaLa (0.5 and
1m
M ) induced a significant increase in ROS (p < 0.0001
and p < 0.01, respectively) compared to NaB and vehicle
controls (p < 0.05). Interestingly, NaB generated lower
levels of ROS than the vehicle controls at all time points
(p < 0.0001, p < 0.001, and p < 0.05). ROS levels at 48 h
after treatment with NaLa (0.5 and 1 m
M ) were signifi-
cantly increased compared to NaB and vehicle controls
(p< 0.0001, p < 0.0001, and p < 0.0001). At the final assay
point of 72 h, once again, NaLa (0.5 and 1 m
M ) increased
GSH availability
(μM/1×106 cells)
a
0.1 mM NaLa
0.3 mM NaLa
0.5 mM NaLa
1 mM NaLa
Butyrate
Vehicle
0
2
4
6
8
10
0
10
b
0.1 mM NaLa
0.3 mM NaLa
0.5 mM NaLa
1 mM NaLa
Butyrate
Vehicle
8
6
4
2aaa
bbb
GSH availability
(μM/1×106 cells)
0
10
c
0.1 mM NaLa
0.3 mM NaLa
0.5 mM NaLa
1 mM NaLa
Butyrate
Vehicle
8
6
4
2aaa
bbb
0
10
d
0.1 mM NaLa
0.3 mM NaLa
0.5 mM NaLa
1 mM NaLa
Butyrate
Vehicle
8
6
4
2aaa
bbb
Fig. 4. GSH availability in IEC-6 cells treated with increasing con-
centrations of NaLa (0.1, 0.3, 0.5 , and 1 m
M ) and NaB (5 m M ) and
compared to the vehicle control over 24 (
a ), 48 (b), 72 ( c ), and
96h (
d ). Data are presented as means ± SEM (n = 3). Statistical
significance between treatment groups is denoted as:
aaa p < 0.0001
(NaLa vs. NaB) and
bbb p < 0.0001 (NaLa/NaB vs. vehicle control).
Fauser/Matthews/Cummins/Howarth
Chemotherapy
DOI: 10.1159/000356067
8
ROS compared to NaB and vehicle controls (p < 0.0001,
p < 0.0001, and p < 0.0001) ( fig.5 b). The reduced levels
of ROS induced by NaB compared to vehicle controls
suggest a protective effect of this SCFA.
D i s c u s s i o n
The current study primarily investigated the antineo-
plastic potential of the MCFA lauric acid on a CRC cell
line in vitro. It was hypothesized that this (C12:
0) MCFA
acid, with a carbon atom chain length positioned between
the SCFA (<C8:
0) and the LCFA (>C16:ω3–9) would
augment its antineoplastic properties. It was shown that
lauric acid induced apoptosis in the Caco-2 cell line, pro-
posed to be due to a reduction in the levels of antioxidant
glutathione (reduced) (GSH), concomitant with a reduc-
tion of G0/G1 and arrest of the S and M cell cycle phases,
together with increased generation of ROS.
In the evaluation of new chemotherapeutic bioactive
agents, few in vitro investigations have evaluated the del-
eterious side effects of the target agents in nontrans-
formed cell lines. This study used the rat small intestinal
cell line IEC-6 as a model of normal intestinal epithelial
cells. It was demonstrated that lauric acid at concentra-
tions of 0.5 and 1 m
M induced considerable cell death due
to apoptosis in IEC-6 cells. This was associated with a re-
duction of GSH, a reduction of cells in the G0/G1 phase,
an increase in cells in the S and M phases, and an increase
in generation of ROS. Once again, in this cell line higher
doses of LA induced significant cell death which did not
correspond with a reduced availability of GSH. There-
fore, this high level of apoptosis may have been associated
with reductions in phases of the cell cycle and increased
generation of ROS independent of GSH availability. Bu-
tyrate did not induce apoptosis in the IEC-6 cell line, nor
did it decrease GSH availability, although cells accumu-
lated in the G0/G1 phase at the initial time points. Inter-
estingly, butyrate generated lower levels of ROS than PBS
control values, which is consistent with previous studies
[44] .
Previous studies have alluded to the cytotoxic capa-
bilities of lauric acid over other MCFA
[8, 30] . Little is
known about the cytotoxic actions of lauric acid on any
in vitro neoplastic cell type. However, Lima et al.
[8] treat-
ed the leukemic cell lines Jurkat and Raji with increasing
concentrations of lauric acid (0.005–1 m
M ) and deter-
mined that the optimal cytotoxic dose was greater than
0.2, although the underlying mechanism of cell death was
not determined
[8] . Differential rates of cell death in-
duced by fatty acids have been proposed to be influenced
by cell lineage, and the study of Lima et al.
[8] did not de-
tect differences in fatty acid-induced cytotoxicity be-
tween T and B lymphocytes. However, it has been sug-
gested that blood-borne cancers are more sensitive to cy-
totoxic agents than are solid malignancies
[8, 26, 31] .
In the current study, levels of apoptosis induced by the
cytotoxic doses of lauric acid (0.5 and 1 m
M ) were evalu-
ated against the SCFA butyrate, and rates of apoptosis
comparable to those published previously were induced
in butyrate-treated Caco-2 cells
[12, 32] . Litvak et al. [32]
employed flow cytometric methods similar to those of the
0
2
4
6
8
10
12
DCF relative fluroescence
intensity (5 × 105 cells × 102)
24
a
bb
a
bb
48
aa
bb
aaa
bbb
bb
72
aaa
bbb
aaa
bbb
bb
0.5 mM NaLa
1 mM NaLa
5 mM NaB
Vehicle
Time (h)
a
0
2
4
6
8
10
DCF relative fluroescence
intensity (5 × 105 cells × 102)
24
aa
bb
aaa
bbb
48
aa
bb aaa
bbb
bb
72
aaa
bbb
aaa
bbb
bb
0.5 mM NaLa
1 mM NaLa
5 mM NaB
Vehicle
b
bbb
Time (h)
Fig. 5. a , b ROS detected in Caco-2 cells ( a ) and IEC-6 cells ( b )
treated with increasing concentrations of NaLa (0.1, 0.3, 0.5, and
1m
M ) as detected by flow cytometric analysis of oxidation of the
probe dichlorodihydrofluorescein diacetate to DCF compared to
NaB (5 m
M ) and the vehicle control over 24, 48, and 72 h. Data are
presented as means ± SEM (n = 3). Statistical significance between
treatment groups is denoted as:
a p < 0.05 (NaLa vs. NaB),
aa p <
0.01 (NaLa vs. NaB),
aaa p < 0.0001 (NaLa vs. NaB),
bb p < 0.01
(NaLa/NaB vs. vehicle control), and
bbb p < 0.0001 (NaLa/NaB vs.
vehicle control).
Induction of Apoptosis by the MCFA
Lauric Acid
Chemotherapy
DOI: 10.1159/000356067
9
current study and reported a significant increase in apop-
tosis in butyrate-treated Caco-2 cells
[32] . This is further
supported by the findings of Roy et al.
[33] , who demon-
strated that Caco-2 cells treated with butyrate induced a
65.7% increase in apoptosis compared to negative con-
trols, as detected by flow cytometry and further con-
firmed by ELISA
[32, 33] . In the current study, lower con-
centrations of lauric acid (0.3 m
M ) also induced cell death
compared to controls but not to butyrate. Moreover, low-
er lauric acid concentrations ( ≤ 0.1 m
M ) did not display
any cytotoxic activity.
In the current study, lauric acid induced a significant-
ly higher mean rate of cell death (TA) compared to butyr-
ate and, more importantly, the mechanism of cell death
was determined to be due to apoptosis as opposed to ne-
crosis. This result was surprising considering that lauric
acid has previously demonstrated inflammatory respons-
es in vivo and in vitro
[34] . To date, few studies have in-
vestigated the apoptotic properties of lauric acid. How-
ever, published literature has reported that lauric acid ex-
erts antimicrobial effects by inhibiting growth or reducing
cell viability in bacterial and viral species
[35–37] .
Little is known about the mechanisms promoting
these growth inhibitory properties and there are even
fewer reports of apoptotic effects, either in vivo or in vi-
tro, in any prokaryote or eukaryote cell type. However,
two separate studies demonstrated that lauric acid treat-
ment of Jurkat and Raji cells induced morphological
changes characteristic of apoptosis which included cell
shrinkage, loss of membrane integrity, increased granu-
larity and reduction of cell viability
[8] . Isolated rat co-
lonic cells treated with LA (10 m
M ) induced activated cy-
tochrome c, reduced expression of Bcl-2, activated cas-
pases 9 and 3, and induced DNA fragmentation, indicative
of apoptosis
[34] . These few studies support the postulate
that lauric acid can induce apoptosis both in vitro and in
vivo. Due to increasing carbon atom chain lengths, this
action could be comparable to the apoptotic actions of
LCFA including palmitic acid (C14:
0) and linoleic acid
(18:
2), which have previously demonstrated morpholog-
ical changes characteristic of apoptosis in Caco-2 cells,
although definitive rates of apoptosis were not measured
[13, 14] .
Modulation of the redox system underpins the induc-
tion of apoptosis, and previous studies have reported that
butyrate-treated colorectal (HT-29) and gastric cancer
cells (Kato III) reduced GSH levels corresponding to an
increase in alkaline phosphatase activity and increased
levels of apoptosis
[26, 38] . Longer-chain fatty acids
which may be closer in carbon atom chain length to
MCFA also reduce GSH levels in CRC cell lines [13] . To
date, there are no cited reports of the effects of lauric acid
on GSH levels, either in vivo or in vitro. To this end, the
current study demonstrated that lauric acid (1 m M ) re-
duced GSH levels in Caco-2 cells. However, GSH avail-
ability to the cell was not significantly reduced compared
to butyrate. This is an interesting finding, as lauric acid
induced a higher percentage of cells in TA compared to
butyrate. Furthermore, the lower concentrations of lauric
acid also reduced GSH to levels comparable to the cyto-
toxic dose of lauric acid without inducing high levels of
apoptosis. This suggests that reduction of the intracellu-
lar availability of GSH in lauric acid-treated Caco-2 cells
may not be the only contributing factor in the induction
of apoptosis. GSH availability was reduced in IEC-6 cells
treated with 1 m
M lauric acid at 48–96 h posttreatment.
This corresponds with increased levels of apoptosis. In-
terestingly, low doses of lauric acid initially elevated GSH
levels, which then returned to control levels over the
study period . Lima et al.
[8] demonstrated that the fatty
acid concentration influenced cytotoxicity in leukemic
cell lines treated with a wide range of carbon chain atom
length fatty acids which may have influenced the GSH
levels in IEC-6 cells
[8] . However, the redox couplet of
GSH/GSSH was not determined; thus it could be argued
that this was not a true measure of the redox potential.
Nevertheless, in the current study, the generation of ROS
was indicative of an increased oxidative state of the cell
[39] .
In further defining the underlying mechanism driving
lauric acid-induced apoptosis, the current study demon-
strated that both lauric acid and butyrate reduced G0/G1
and arrested the S and M phases. Previous studies have
determined that apoptosis can also be induced in Caco-2
cells due to modification of phases of the cell cycle by re-
dox modulation, specifically G1 to the S phase
[40] . In a
study by Xiang et al.
[41] , butyrate increased the number
of cells in the M phase and decreased cells in the S phase
[41] . This inconsistency with the current study was pos-
sibly due to a lower dose of butyrate (1 m
M ) and a short-
er assay period
[41] . However, Matthews et al. [1] dem-
onstrated that butyrate (5 m
M ) treatment of Caco-2 cells
reduced cells in G0/G1 and arrested cells in the S and
G2/M phases
[1] . Treatment of Caco-2 cells with longer-
chain fatty acids (fish oil-based emulsions) demonstrated
G2/M cycle arrest and, interestingly, cotreatment with
fish oil and 5-fluorouracil further arrested cells in the S
phase
[42] .
Increased generation of ROS is presumed to be a final
consequence of imbalance of the redox system and a shift
Fauser/Matthews/Cummins/Howarth
Chemotherapy
DOI: 10.1159/000356067
10
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generated ROS over the entire time course of the assay,
whereas butyrate only increased during the final 96-hour
assay period. In the evaluation of new chemotherapeutic
bioactive agents, few in vitro investigations have evalu-
ated deleterious side effects on nonneoplastic cell lines.
The current study determined, not unexceptionally, that
lauric acid at concentrations of 0.5 and 1 m
M induced
considerable apoptosis in IEC-6 cells. This was associated
with a reduction in GSH, a reduction in the number of
cells in G0/G1 and arrested cells in the S and M phases,
and an increase in ROS generation. Once again, in this
cell line, higher doses of lauric acid induced significant
cell death which did not correspond with increased GSH
availability. Therefore, this high level of apoptosis may
have been associated with modification of phases of the
cell cycle and increased generation of ROS independent
of GSH availability. Butyrate did not induce apoptosis in
the IEC-6 cell line, nor did it decrease GSH availability or
modify any phase of the cell cycle. Interestingly, butyrate
generated lower levels of ROS than vehicle control values,
consistent with previous studies
[44] .
While lauric acid at higher concentrations reduced vi-
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antineoplastic properties in a CRC cell line. Advances in
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muco-
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more intensive investigations into the specific apoptotic
mechanisms of fatty acid action together with the possible
augmentative and protective effects of MCFA and SCFA.
A c k n o w l e d g m e n t s
Prof. Gordon Howarth is supported by the Sally Birch Cancer
Council Australia Senior Research Fellowship in Cancer Control.
The authors would like to thank the Renal Transplantation Immu-
nology Laboratory at the Basil Hetezl Institute of The Queen Eliz-
abeth Hospital, Australia, for assistance with flow cytometry, in
particular Mrs. Svjetlana Kireta.
Induction of Apoptosis by the MCFA
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