Saturated Hydroxy Fatty Acids Exhibit a Cell Growth Inhibitory Activity and Suppress the Cytokine-Induced β-Cell Apoptosis

Article abstract of DOI:10.1021/acs.jmedchem.0c00844

The field of bioactive lipids is ever expanding with discoveries of novel lipid molecules that promote human health. Adopting a lipidomic-Assisted approach, two new families of previously unrecognized saturated hydroxy fatty acids (SHFAs), namely, hydroxy

Full text of DOI:10.1021/acs.jmedchem.0c00844

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Article
Saturated Hydroxy Fatty Acids Exhibit a Cell Growth Inhibitory
Activity and Suppress the Cytokine-Induced β‑Cell Apoptosis
Maroula G. Kokotou, Alexandros C. Kokotos,# Dimitrios Gkikas,# Olga G. Mountanea,#
Christiana Mantzourani, Abdulaziz Almutairi, Xiaoyong Lei, Sasanka Ramanadham, Panagiotis K. Politis,
and George Kokotos*
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ABSTRACT: The field of bioactive lipids is ever expanding with discoveries of novel lipid molecules that promote human health.
Adopting a lipidomic-assisted approach, two new families of previously unrecognized saturated hydroxy fatty acids (SHFAs), namely,
hydroxystearic and hydroxypalmitic acids, consisting of isomers with the hydroxyl group at different positions, were identified in
milk. Among the various regio-isomers synthesized, those carrying the hydroxyl at the 7- and 9-positions presented growth inhibitory
activities against various human cancer cell lines, including A549, Caco-2, and SF268 cells. In addition, 7- and 9-hydroxystearic acids
were able to suppress β-cell apoptosis induced by proinflammatory cytokines, increasing the possibility that they can be beneficial in
countering autoimmune diseases, such as type 1 diabetes. 7-(R)-Hydroxystearic acid exhibited the highest potency both in cell
growth inhibition and in suppressing β-cell death. We propose that such naturally occurring SHFAs may play a role in the promotion
and protection of human health.
to activate receptors such as GPR84 more effectively than their
nonhydroxylated counterparts.8,9
INTRODUCTION
■
Lipids are increasingly being recognized as biomolecules that
dynamically affect human physiology and pathophysiology.1
Hydroxylated polyunsaturated fatty acids constitute a class of
bioactive lipids known for their ability to act as potent signaling
mediators. The seminal work of Serhan et al.2 uncovered a
family of protective compounds, termed resolvins (e.g.,
resolvin E2, 1, Figure 1a), which promote the resolution of
inflammation and are generated from the omega-3 fatty acids
docosahexaenoic and eicosapentaenoic. Resolvins together
with protectins and maresins constitute a new genus of
hydroxylated polyunsaturated fatty acid mediators called
specialized proresolving mediators (SPMs).3,4 In contrast,
less is known about the bioactivities of saturated hydroxy fatty
acids (SHFAs) and their ability to act as signaling molecules,
though some of the SHFAs are relatively common. The 2-
hydroxy fatty acids (2HFAs) are abundant in sphingolipids,5
and the 3-hydroxy fatty acids (3HFAs) are intermediates of
fatty acid synthesis and β-oxidation and are constituents of
inflammatory lipopolysaccharides.6 Agonists and antagonists of
free fatty acid and hydroxy carboxylic acid receptors have
received increased interest in recent years,7 and medium-chain
fatty acids with a hydroxyl group at the 2- or 3-position [e.g., 3-
hydroxymyristic acid (3HMA), 2, Figure 1b] have been shown
Recently, fatty acid esters of hydroxy fatty acids (FAHFAs),
mainly composed of saturated fatty acids, have emerged as a
novel class of endogenous lipids with anti-inflammatory and
antidiabetic properties.10 Several branched FAHFAs families
have been identified and each family consists of multiple regio-
isomers, in which the hydroxyl participating in the ester bond
is situated at different positions (5 and higher positions). In
particular, the family of palmitic acid (PA) esters of
hydroxystearic acids is one of the most abundant and has
been recognized as possessing interesting bioactivities.10,11 For
instance, the PA ester of 9-hydroxystearic acid (PAHSA, 3,
Figure 1c) has been reported to activate GPR40, which is
important in maintaining glucose homeostasis, and to regulate
innate and adaptive immune responses preventing mucosal
damage and protecting against colitis.12−15 Although experi-
Received: June 4, 2020
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Figure 1. Classes of hydroxylated fatty acids and their bioactivities.
ments in mice have shown that FAHFAs may be generated
from SHFAs in vivo,10 the origin of SHFAs remains unknown.
SHFAs bearing a hydroxyl group at a position higher than 3
could be either endogenously generated in the body through a
hydroxylation pathway of fatty acids or be obtained
exogenously from food.
Lipidomic-based approaches employing mass spectrometry
(MS) have been recognized as a powerful and rapidly growing
technology that can be used in biomedical research to study
disease mechanisms and identify novel therapeutic targets and
novel bioactive lipids.16 Most recently, Watrous et al. utilizing
the integration of directed nontargeted MS and computational
chemical networking described hundreds of previously
unrecognized inflammatory oxylipin metabolites in human
plasma, thus providing new insight into the role of oxylipins in
human biology.17
In view of this, we adopted a lipidomic approach to identify
previously unrecognized SHFAs existing in natural sources.
Here, we present the asymmetric synthesis of various SHFAs
and the study of their bioactivities. We demonstrate that 7-
hydroxystearic and 7-hydroxypalmitic acids (7HPA) inhibit
cancer cell proliferation and that 7- and 9-hydroxystearic acids
exhibit high potency in suppressing cytokine-induced β-cell
apoptosis. Further, we report that these effects of SHFAs
involve STAT3 signaling pathway, a major player in
proproliferative and proinflammatory cascades.
chromatography (LC)−MS approach with reversed-phase
chromatography and high mass accuracy MS operating in
full-scan negative mode. In an initial experiment, we screened
human plasma and cow milk samples by LC−HRMS and
monitored for peaks corresponding to the exact masses
(deprotonated molecule) of a set of medium- and long-chain
SHFAs, namely, hydroxycaprylic (m/z 159.1027), hydrox-
ycapric (HCA, m/z 187.1340), hydroxy lauric (HLA, m/z
215.1653), hydroxymyristic (HMA, m/z 243.1966), hydrox-
ypalmitic (HPA, m/z 271.2279), hydroxystearic (HSA, m/z
299.2592), hydroxyarachidic (m/z 327.2905), and hydrox-
ybehenic (m/z 355.3218) acids. The accurate mass for each
formula was searched using a 0.01 mass width. Among the
spectra of the above mentioned medium- and long-chain
SHFAs, those of HPAs and HSAs attracted our attention
because only weak peaks were recorded for hydroxy caprylic,
HCA, HLA, HMA, hydroxy arachidic, and hydroxy behenic
acids. Figure 2a,c shows the extracted ion chromatograms
(EICs) for reference synthetic 2- and 3-hydroxypalmitic and
stearic acids. In a representative human plasma sample, 2HFAs
seemed to be the most predominant, with the highest peak
intensities corresponding to 2-hydroxypalmitic acid (2HPA)
(Figure 2b) and 2HSA (Figure 2d) acids. Corresponding
3HFAs were also evident in the human plasma samples, though
they were detected in lower intensities. However, in a
representative cow milk sample, although peaks corresponding
to 2- and 3-hydroxystearic and PAs (Figure 2f,h) appear at the
same retention times with those of reference compounds
(relative tolerance of the retention time lower than 2.5%)
(Figure 2e,g), the most intense peaks were recorded at earlier
times. These findings suggest the presence of other isobaric
regio-isomers (i.e., with the hydroxyl group at positions other
than 2- and 3-) in milk.
RESULTS
■
High-Resolution Mass Spectrometry Screening for
Masses Corresponding to SHFAs. The presence of 2HFAs
and 3HFAs in human plasma18,19 and foods, in particular in
milk,20,21 is well documented. However, only one report refers
to the existence in such sources of two SHFAs possessing the
hydroxyl at higher positions (10-hydroxystearic acid and 8-
hydroxypalmitic acid).22 We utilized a “suspect” liquid
From the subsequent fragmentation studies, we deduced
that the most intense peaks correspond to 7HSA and 10HSA.
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Figure 2. EICs for mass corresponding to hydroxypalmitic acid. (a) Reference compounds; (b) human plasma sample; (e) reference compounds;
(f) milk sample. EICs for mass corresponding to hydroxystearic acid. (c) Reference compounds; (d) human plasma sample; (g) reference
compounds; (h) milk sample.
To address this further, we proceeded to generate the
prerequisite LC−HRMS reference compounds.
sequence of acetylation, hydrogenation, oxidation, and
saponification led to (R)-HFAs 10a−d in high enantiomeric
purity (Scheme 1). The use of the (2R,5S)-2-(tert-butyl)-3,5-
dimethylimidazolidin-4-one trifluoroacetate catalyst enabled
the synthesis of the corresponding (S)-HFAs.
Determination of Various SHFAs in Milk. To facilitate
the identification of the SHFA mixture in milk, we developed a
rapid LC−HRMS method that enabled us to simultaneously
identify a variety of SHFAs in milk within a 10 min sample run.
Six regio-isomers of HPA [2HPA, 3-hydroxypalmitic acid
(3HPA), 7HPA, 9-hydroxypalmitic acid (9HPA), 11-hydrox-
ypalmitic acid (11HPA), and 16-hydroxypalmitic acid
(16HPA)], six regio-isomers of HSA [2-hydroxystearic acid
(2HSA), 3-hydroxystearic acid (3HSA), 7-hydroxystearic acid
(7HSA), 9-hydroxystearic acid (9HSA), 10-hydroxystearic acid
(10HSA), and 12-hydroxystearic acid (12HSA)], and three
Synthesis of 7-Hydroxy and 10-Hydroxystearic and
PAs. We have recently reported an enantioselective method-
ology for the synthesis of FAHFAs and 9-hydroxy fatty acids
(stearic and palmitic).23 In the present study, we followed the
same methodology to synthesize saturated 7-hydroxy and 10-
hydroxystearic or PAs. The key step consisted of the
organocatalytic synthesis of asymmetric terminal epoxides
5a,b using monoprotected α,ω-diols 4a,b as starting materials
and MacMillan’s third-generation imidazolidinone [(2S,5R)-2-
(tert-butyl)-3,5-dimethylimidazolidin-4-one trifluoroacetate] as
a catalyst for the induction of chirality (Scheme 1). Treatment
of epoxides 5a,b with the appropriate Grignard reagent
provided either a 16-atom or an 18-atom carbon chain bearing
a hydroxy group at position 7- or 10- (compounds 6a−d). A
C
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Scheme 1. Enantioselective Synthesis of SHFAs; (a) PCC, CH2Cl2; (b) (i) (2S,5R)-2-(tert-Butyl)-3,5-Dimethylimidazolidin-4-
one Trifluoroacetate (20%), 2,3,4,5,6,6-Hexachlorocyclohexa-2,4-dien-1-one, THF; (ii) NaBH4, EtOH; (iii) KOH, EtOH,
H2O; (c) C5H11MgBr or C7H15MgBr or C8H17MgBr or C10H21MgBr, CuI, dry THF; (d) AcCl, Pyridine, CH2Cl2; (e) H2, 10%
Pd/C, EtOH; (f) Jones Reagent, Acetone; (g) LiOH, THF−H2O
Table 1. Contents of Free SHFAs (ng/mL) in Milk Samples
cow milk (ng/mL), 10 samples, triplicates
a
compound
abbreviation
exact mass [M − H]−
tr
minimum value
maximum value
mean value SD
b
c
3-hydroxycapric acid
3-hydroxylauric acid
16-hydroxypalmitic acid
11-hydroxypalmitic acid
9-hydroxypalmitic acid
7-hydroxypalmitic acid
3-hydroxymyristic acid
12-hydroxystearic acid
10-hydroxystearic acid
7-hydroxystearic acid
9-hydroxystearic acid
3-hydroxypalmitic acid
2-hydroxypalmitic acid
3-hydroxystearic acid
3HCA
3HLA
16HPA
11HPA
9HPA
7HPA
3HMA
12HSA
10HSA
7HSA
9HSA
3HPA
2HPA
3HSA
2HSA
187.1340
215.1653
271.2279
271.2279
271.2279
271.2279
243.1966
299.2592
299.2592
299.2592
299.2592
271.2279
271.2279
299.2592
3.06
3.90
4.31
4.44
4.56
4.64
4.67
5.02
5.11
5.17
5.32
5.33
5.57
5.99
6.22
<LOQ
<LOQ
19.3
20.5
16.8
<LOQ
<LOQ
<LOQ
33.3
40.8
8.9
11.0
10.4
10.1
10.0
10.1
36.8
41.5
72.6
45.6
13.1
14.9
21.2
218.5
378.8
42.4
48.1
40.8
54.4
40.9
5.4 1.8
b
c
11.9 6.2
26.9 8.2
45.7 18.6
25.5 9.1
b
c
6.2 4.1
b
c
7.2 3.2
b
c
8.5 6.5
97.5 10.2
167.3 12.5
19.5 10.0
28.3 11.2
21.7 9.3
30.9 18.8
23.6 12.4
2-hydroxystearic acid
299.2592
a
b
c
Retention time. Lower of limit of quantification. The mean value was determined using the medium-bound approach.
3HFAs [3-hydroxycapric acid (3HCA), 3-hydroxylauric acid
(3HLA), and 3HMA] were used in this study. The analytes
and their exact masses lie in the range of 187−300 Da and are
summarized in Table 1. As shown in Figure 3a, the 15
reference compounds were distinctly separable with this
chromatographic technique. Figure 3b shows a representative
chromatogram of a cow milk sample. Ten different cow milk
samples were examined in triplicates, and the contents of free
HFAs are summarized in Table 1. Details of the analytical
method will be published elsewhere.
In an effort to establish the stereochemistry of HPAs and
HSAs present in cow milk, the lipid fraction of milk was
isolated and a chiral column (Chiralpak AD-RH) was
employed for UPLC−HRMS analysis. Using an isocratic
elution system consisting of (A) acetonitrile/1% formic acid
and (B) water (95/5, v/v) and a long run time (120 min), a
nice separation of the peaks corresponding to 3RHPA and
3SHPA was achieved (Figure S3a). In the case of 7HPA and
10HPA, although the peaks corresponding to the enantiomers
of 7HPA (7RHPA and 7SHPA) and 10HPA (10RHPA and
10SHPA) are well separated from each other, overlap of the
peaks of 7SHPA and 10RHPA was observed (Figure S3a). The
EIC of the lipid fraction of a cow milk sample clearly suggests
that both enantiomers of 3HPA are present in milk (Figure
S3b). The simultaneous presence of various positional isomers
and enantiomers of HPAs in a real milk sample complicates the
identification of the various enantiomers, preventing any
conclusive results under these conditions (Figure S3b). Similar
results were obtained for HSAs (data not shown).
Cell Growth Inhibitory Effects of SHFAs on Human
Cancer Cell Lines. In 1991, HSA was identified as a
peroxidation product in Lewis lung carcinoma cells.24
Subsequently, it was reported that 9HSA upregulates
p21WAF1 in HT29 cancer cells,25 inhibits cell growth in
human colon cancer targeting histone deacetylase 1,26
interferes with EGF signaling in a human colon adenocarci-
noma,27 and modulates apoptotic signaling in osteosarcoma
cells.28 We, therefore, sought to determine the effects of our
recently identified milk SHFAs on proliferation of human
cancer cell lines: epithelial lung carcinoma (A549), colorectal
adenocarcinoma (Caco-2), brain glioblastoma (U87-MG), and
astrocytoma (SF268). The MTT assay was utilized to assess
viable cell numbers.29
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Figure 3. EICs of SHFAs. (a) Standard solution (500 ng/mL). (b) Representative cow milk sample.
The growth inhibitory effects of eight enantiomers of HSAs
and five enantiomers of HPAs (10, 25, 50, and 100 μM) on
A549, Caco-2, and SF268 cells are summarized in Figure
4a,c,e, respectively. Interestingly, the position of the hydroxyl
functionality in the long-chain drastically influences the in vitro
potency. In general, 7HSA and 9HSA, as well as the
corresponding PAs, exhibited statistically significant inhibitory
potency at concentrations over 25 μM, while when the
hydroxyl group is at position 2-, 10-, or 12- of the stearic chain
or at position 10- of the palmitic chain, only very weak activity
was observed. All statistical comparisons for inhibitory potency
of the investigated HFAs are included in Table 1S. The IC50
values for the enantiomers 7RHPA, 7SHPA, 7RHSA, and
7SHSA were calculated from the curves depicted in Figure
4b,d,f, respectively, and vary within the range of 27−55 μM. 7-
(R)-Hydroxystearic acid (7RHSA) was found to exhibit the
highest growth inhibitory potency (IC50 values 38, 37, and 27
μM for A549, Caco-2, and SF268 cells, respectively), being
clearly more potent than its regio-isomer, 9RHSA. The (R)-
enantiomer of 7HSA was found to be slightly more potent than
the (S)-enantiomer (IC50 38 vs 50 μM in A549, 37 vs 50 μM
in Caco-2, and 27 vs 47 μM in SF268 cells). In the case of
7HPA, the (R)-enantiomer was found to be almost equipotent
with the (S)-enantiomer (IC50 49 vs 55 μM in A549 and 42 vs
50 μM in Caco-2 cells). The growth inhibitory effect of various
HSAs and HPAs on U87-MG cell line is depicted in the
Supporting Information (Figure S1). None of SHFAs tested
presented inhibitory activity on U87-MG, indicating a cell-
specific activity.
The growth inhibitory effect of PA and stearic acid (SA) was
studied in A549, Caco-2, and SF286 cells for comparison
purposes, and the results are shown in Figure 4a,c,e. A very
weak effect was observed only for stearic acid, proving that the
presence of the hydroxyl group at a particular position
determines the cell growth inhibitory potency. In addition,
the growth inhibitory effect of 7PAHSA was studied in A549,
Caco-2, and SF286 cells. No effect was observed at
concentrations up to 100 μM (Figure 4a,c,e). Similar negative
results were observed for 9PAHSA and 9OAHSA (Figure
4a,c,e). These findings exclude the possibility that the growth
inhibitory effect is caused by FAHFAs that may be generated
in cells after treatment with HSA.
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Figure 4. Cell growth inhibitory potency of various HSAs and HPAs. (a) Human cancer cell line A549. (c) Human cancer cell line Caco-2. (e)
Human cancer cell line SF268. Cells were treated with increasing concentrations (10, 25, 50, and 100 μM) of the test compounds for 72 h, and cell
viability was determined by the MTT assay for a minimum of six experiments (AVG SEM). No effect was observed for any compound at 10 μM
and this concentration is not shown in the graph. (b) IC50 values (μM) of enantiomers 7RHPA, 7SHPA, 7RHSA, and 7SHSA in A549 cells. IC50
values were derived from the dose−response relationship for a minimum of six experiments (95% CI log IC50 −4.49 to −4.36, −4.42 to −4.20,
−4.38 to −4.14, and −4.43 to −4.17, respectively). (d) IC50 values (μM) of enantiomers 7RHPA, 7SHPA, 7RHSA, and 7SHSA in Caco-2 cells.
IC50 values were derived from the dose−response relationship for a minimum of six experiments (95% CI log IC50 −4.46 to −4.40, −4.43 to −4.32,
−4.36 to −4.25, and −4.36 to −4.24, respectively). (f) IC50 values (μM) of enantiomers 7RHSA and 7SHSA in SF268 cells. IC50 values were
derived from the dose−response relationship for a minimum of six experiments (95% CI log IC50 −4.71 to −4.43, −4.47 to −4.19, respectively).
To understand the mode of action of SHFAs, A549 cells
were incubated with 7RHSA, which exhibited the highest
potency, or its regio-isomer 9RHSA (50 μM) for 72 h and then
fixed for immunostaining and cell imaging. Markers of cell
cycle [phosphorylated histone H3 (pH3)30 and apoptosis
[activated caspase 3 (casp3)31 were used to investigate their
antiproliferative mechanism. These analyses revealed that both
7RHSA and 9RHSA induced the arrest of the cell cycle (Figure
5a), as reflected by the decreased phosphorylation of the
histone H3 marker (pH3), but did not promote apoptosis
(Figure 5b), as evidenced by the absence of increased
generation of activated caspase-3.
Subsequently, mRNA analysis was employed to assess which
signaling pathway(s) leading to the cell cycle arrest is affected
by 7RHSA. A variety of different signaling molecules, namely,
cyclin D1, cyclin E1, p21cip1, p27kip1, EGFR, PI3K (PIK3CA),
p53, mTOR, cmyc, RET, and STAT3 (Figure 6a−k), were
examined by real-time RT-qPCR. This approach revealed a
specific reduction of the transcription regulator signal trans-
ducer and activator of transcription 3 (STAT3) mRNA (Figure
6k). Most importantly, the downregulation of STAT3
expression was observed both at mRNA and protein levels of
both total and phosphorylated STAT3 (p-STAT3) (Figure 6l−
n). The latter event is critical for STAT3-mediated
proproliferative and proinflammatory effects, indicating a
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Figure 5. Effect of 7RHSA and 9RSHA on cell death and apoptosis of A549 cells. Cells were treated with 50 μM 7RHSA, 9RSHA, and equivalent
vehicle (DMSO) for 72 h and then immunostained against (a) phosphorylated histone H3 (pH3) and (b) activated caspase 3 (casp3) to further
study their action. Representative images are shown as well as quantification of the percentage of positive-stained cells, as identified by DAPI
staining (n = 8 fields of view, AVG SEM).
possible involvement of 7RHSA compound in suppressing
these functions.
findings suggest differential effects of the four HSAs in
protecting β-cells against cytokine attack.
The inhibitory effect of 7PAHSA, which might be generated
in cells after the treatment with 7HSA, was studied in INS-1
cells at a concentration of 10 μM. As shown in Figure 7e, a
weak effect was observed, leading to the conclusion that the
potent effect observed for 7HSA at a concentration of 10 μM
should be mainly attributed to 7RHSA by itself.
It is well-established that saturated and monounsaturated
FFAs cause apoptosis of β-cells. Numerous studies performed
with insulinoma cell lines, human β-cell lines, murine, and
human islets demonstrate profound β-cell apoptosis following
exposure to either palmitate or stearate,33−38 analogous to
proinflammatory cytokine-induced β-cell apoptosis.
Suppression of Cytokine-Induced β-Cell Apoptosis
by Hydroxystearic Acids. As discussed in Introduction,
FAHFAs have been shown to exhibit attractive antidiabetic
properties.10−12 Recently, it has been reported that PAHSAs
promote β-cell survival and function and protect against type 1
diabetes (T1D).32 However, nothing is known about such an
activity of SHFAs. To examine the potential impact of SHFAs
on β-cell survival, INS-1 cells were treated with proinflamma-
tory cytokines (CTKs) (IL-1β and IFNγ) in the absence or
presence of four different enantiomers of 7- and 9-
hydroxystearic acids (1 or 10 μM), namely, 7RHSA, 7SHSA,
9RHSA, and 9SHSA, and the results are shown in Figure 7.
None of them alone had a significant effect on cell survival, in
comparison with vehicle alone. As expected, CTK alone
promoted cell death (15−23%) relative to vehicle alone. At 1
μM, 9RHSA and 9SHSA, but not 7RΗSA and 7SHSA, reduced
cell death (ca. 40%), relative to CTK alone. At 10 μM, cell
death was further significantly decreased by 9RHSA (73%),
7RΗSA (95%), and 9SHSA (68%); however, 7SHSA did not
promote a significant decrease, relative to CTK alone. These
DISCUSSION
■
Initially, a “suspect” LC−HRMS analysis of human plasma and
cow milk samples revealed the presence of novel SHFAs,
bearing the hydroxyl group at positions other than 2- and 3-, in
milk but not plasma. A variety of SHFAs were synthesized and
a rapid LC−HRMS method for the simultaneous determi-
nation of 15 synthetic SHFAs was developed. Six regio-isomers
of HPA, six regio-isomers of HSA, and three additional 3HFAs
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Figure 6. Effect of 7RHSA on crucial cell growth and proliferation regulators. (a−j) Relative expression levels of cyclinD1, cyclinE1, p21cip1, p27kip1
,
EGFR, PIK3CA (PI3K), p53, mTOR, cmyc, and RET mRNA in DMSO- and 7RHSA-treated A549 cells, measured with quantitative real-time RT-
PCR. (k) Relative expression levels of STAT3 mRNA in DMSO- and 7RHSA-treated A549 cells, measured with quantitative real-time RT-PCR. (l)
Western blots for phospho-STAT3, STAT3, and b-actin protein in DMSO- and 7RHSA-treated A549 cells. (m,n) Relative expression of phospho-
STAT3 and STAT3 proteins in DMSO- and 7RHSA-treated A549 cells.
were well separated by this method, and their concentrations
in samples of cow milk were quantifiable. Our LC−HRMS
study uncovered the presence of previously unrecognized
families of naturally occurring SHFAs in milk. Each family
consists of regio-isomers of a particular fatty acid (e.g., stearic
acid) hydroxylated at various positions (e.g., 7, 9, 10, etc.). In
cow milk, 7HSA and 10HSA are the predominant HFAs with
an 18-carbon saturated chain, and they were present in
considerably higher concentrations (167.3 12.5 and 97.5
10.2 ng/mL, respectively) than other detected SHFAs. In the
seminal work of Kahn et al.,10 it was demonstrated that gavage
of mice with 9-hydroxyheptadecanoic acid resulted in the
synthesis of FAHFAs containing this fatty acid, indicating that
FAHFAs can be synthesized in vivo. Thus, SHFAs (e.g., 7-, 9-,
10-) present in foods and taken by humans in daily dietary
intake may serve as the precursors of the physiologically
relevant corresponding FAHFAs, although the direct hydrox-
ylation (enzymatic or nonenzymatic) of SHFAs within the
body cannot be excluded.
The in vitro growth inhibition study using the MTT viability
assay demonstrated that the (R)-enantiomer of 7HSA
exhibited the highest potency (IC50 values of 38, 37, and 27
μM against human cancer cells A549, Caco-2, and SF268,
respectively) among the HFAs tested, being more potent than
the corresponding 9HSA and the other regio-isomers (2-, 10-,
12-). The (R)-enantiomer of 7HSA exhibited slightly higher
potency than the (S)-enantiomer (IC50 38 vs 50 μM in A549
and 37 vs 50 μM in Caco-2 cells). In the case of HPAs, again
H
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Figure 7. Comparison of the effects of SHFA enantiomers on β-cell apoptosis. INS-1 insulinoma cells were cultured in six-well plates to 70%
confluency at 37 °C under an atmosphere of 5% CO2/95% air. The cells were then pretreated with vehicle (DMSO) or the enantiomers (1 or 10
μM) for 1 h. The cells were then exposed to vehicle alone or CTK (20 ng/mL IL-1β + 200 ng/mL IFNγ) for 16 h. (a) 9RHSA; (b) 9SHSA; (c)
7RHSA; (d) 7SHSA; (e) 7PAHSA. The cells were then processed for TUNEL analyses to determine the extent of apoptotic cell death. The data
are the mean SEM of apoptotic cells, relative to total cell number, and were generated from three to five independent experiments.
7HPA exhibited higher potency than 9HPA and 10HPA, while
the (R)-enantiomer was found almost equipotent with the (S)-
enantiomer. The observation that none of SHFAs exhibited
growth inhibition on U87-MG cells indicated a cell-specific
effect. PA and SA did not cause any effect in A549, Caco-2,
and SF268 cells, highlighting the importance of a hydroxyl
functionality at a particular distance from the carboxyl group of
the fatty chain. No effect was also caused by 7PAHSA,
9PAHSA, and 9OAHSA, indicating that acylation of the
hydroxyl functionality by a fatty chain destroys the growth
inhibitory potency.
The mechanism CORRECT of cell proliferation inhibition
was attributed to the ability to arrest the cell cycle while not
interfering with cell apoptosis. This phenotype was further
attributed to reduction in STAT3, a transcription factor that
regulates the expression of genes related to cell cycle, cell
survival, and immune response.39 STAT3 is activated by CTK
and growth factors through kinase-mediated tyrosine phos-
phorylation and dimerization,40 leading to a nuclear trans-
location and transcription activation. Recently, it has been
demonstrated that STAT3 is post-translationally S-palmitoy-
lated at the SRC homology 2 domain, which also promotes its
dimerization and transcriptional activation.41 Although in
normal cells, activation of STAT3 signaling is transient and
controlled, STAT3 remains constitutively active in approx-
imately 70% of human solid and hematological tumors.42
Prolonged STAT3 activation has been heavily implicated in
tumorigenesis, while increased STAT3 levels correlate with
poor patient prognosis,42 and as a consequence, the STAT3
signaling pathway represents an attractive drug target for
cancer therapy. Although a variety of small-molecule STAT3
inhibitors have been developed,43,44 the traditional orthosteric
inhibitors of STAT3 have shown to exhibit limited clinical
efficacy, with low selectivity and numerous side effects.
Therefore, allosteric inhibitors targeting STAT3 or its
upstream molecules have emerged as a more promising
approach.44 The STAT3 mRNA reduction uncovered here
provides a very promising mechanism for cell cycle arrest and
suggests that the post-translation modification levels of STAT3
can also be affected. Overall, our cell biology analyses render
this new class of naturally existing substances as promising
novel antiproliferative and anti-inflammatory agents.
Pancreatic β-cells are central players in the pathogenesis of
both T1D and T2D and a decrease in β-cell number and/or
deficiency in the function of existing β-cells contribute to the
development of the disease. Therefore, there is an increasing
effort to identify novel methods of improving the number and
function of β-cells.45 Dysregulated cytokine signaling resulting
to β-cell apoptosis has been targeted by either genetic or small-
molecule approaches.45 Along this line, we have previously
identified small-molecule inhibitors of calcium-independent
phospholipase A2 able to suppress the cytokine-induced β-cell
apoptosis.46,47 In view of their effects in cancer cells described
above, we sought to assess the effects of SHFAs on β-cell
apoptosis. INS-1 insulinoma cells were treated with proin-
flammatory CTK, recognized as mediators of β-cell death
leading to T1D. As expected, CTK exposure promoted ca. 20−
25% apoptotic cell death. Cotreatment with four HSAs
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produced differential effects: the 9HSAs, but not the 7HSAs,
significantly inhibited apoptosis at 1 μM. At 10 μM, both of
the (R)-enantiomers promoted further inhibition of apoptosis,
with the 7RHSA nearly completely inhibiting apoptosis. In
contrast, 9SHSA did not promote such a marked decrease in
cell death, while 7SHSA exhibited the weakest effects. These
finding suggest that the (R)-enantiomer of 7HSA is a more
potent inhibitor of β-cell death than the (S)-enantiomer.
Importantly, these HFAs warrant consideration as small-
molecule inhibitors that may be effective in preventing
autoimmune destruction of β-cells and counter development
of T1D. The ability of 7HSA to suppress the apoptosis of β-
cells is in direct contrast to the effect reported for PA and
SA,33−38 which cause apoptosis of β-cells.
Kahn et al. have recently reported that PAHSAs promote β-
cell survival and function and protect against T1D.32 In the
present study, we demonstrate that simple, not esterified,
HSAs (either 7- or 9-) are able to protect β-cells from
cytokine-induced apoptosis. It seems that only the hydroxy
fatty acid part of PAHSAs is enough to produce the beneficial
effect in β-cells. If we consider potential exogenous
administration as a therapeutic strategy for β-cell protection,
it is advantageous and more attractive to use HFAs instead of
FAHFAs because HFAs may be synthesized more easily in
comparison to the more complex structured FAHFAs, avoiding
additional steps for synthesizing the esterified FAHFAs from
HFAs. Furthermore, as recently demonstrated in mice,
FAHFAs may be metabolized in vivo by the endogenous
hydrolases AIG1 and ADTRP.48
The potential health benefits from the consumption of milk
fatty acids have been recently reviewed discussing that whole
dairy products could promote human health because of the
presence of certain bioactive fatty acids.49 There is
considerable evidence, although not conclusive, that an
increased consumption of milk or dairy products is associated
with a reduction in colon cancer.50−52 However, the role of
milk fat content in cancer prevention remains unclear. In the
present study, we demonstrate that milk contains previously
unrecognized lipid components, such as 7-hydroxy and 9-
hydroxy stearic acids and PAs, which inhibit in vitro growth of
human cancer cell lines, including the epithelial colorectal
adenocarcinoma line Caco-2, with IC50 values between 40 and
75 μM. It has to be noted that a number of SHFAs are present
in milk, contributing to a cumulative effect. Thus, the newly
uncovered naturally occurring milk components SHFAs may
constitute a novel class of chemopreventive agents analogous
to the plant food components flavonoids, which are
polyphenols with a recognized protective role in tumor
development. A detailed comparative analysis has shown that
flavonoids possess growth inhibitory effects in different cancer
cell lines with IC50 values ranging from 40 to 200 μM.53 The
results of the present study could suggest that SHFAs might
play a cancer preventive role, especially in the intestinal tract
because of direct exposure of intestinal epithelia to these
dietary milk ingredients.
associated with a lower risk of T2D.55 Another study in the
Dutch population has shown that total SFAs do not relate to
T2D risk, but the association may depend on the type and food
sources of the SFAs. For example, individual SFAs that are
commonly found in cheese were significantly related to lower
T2D risks.56 The previously unrecognized SHFAs described in
the present study may contribute to lower the risk of T2D by
milk consumption.
CONCLUSIONS
■
In summary, we identified two previously unrecognized
families of fatty acids in milk consisting of regio-isomers
possessing the hydroxyl functional group at different positions
and we demonstrated that the 7- and 9-hydroxystearic and PAs
manifest cell growth inhibitory properties as well as
suppression of cytokine-induced β-cell apoptosis. 7-(R)-
Hydroxystearic acid exhibited the highest potency both in
cell growth inhibition and in suppressing the β-cell death in
INS-1 cells. The research workflow we followed in the present
work, that is, “suspect” LC−HRMS analysis in a food matrix
organic synthesistargeted LC−HRMS analysis using refer-
ence compounds in this food matrixstudy of the in vitro
bioactivities, may be employed in searching other food
matrices, providing a foundation for the discovery of previously
unrecognized naturally occurring lipids that may play a role in
the protection and promotion of human health.
EXPERIMENTAL SECTION
■
General. All commercially available products and solvents were
purchased from Sigma-Aldrich, Fluka, Merck, and Alfa Aesar. Solvents
were used as received or dried over molecular sieves (4 Å). All water-
or air-sensitive reactions have been performed under an argon
atmosphere with dry solvents and anhydrous conditions. All reactions
were monitored by thin-layer chromatography performed on
aluminum-backed silica plates (0.2 mm, 60 F254). Purification by
flash chromatography was performed on Merck silica gel 60 (230−
400 mesh). Melting points were determined using a Buchi 530
apparatus and were uncorrected. Optical rotations were measured
using a PerkinElmer 343 polarimeter in a 10 cm cell at room
1
temperature. H and 13C NMR spectra were recorded on a Varian
Mercury (200 and 50 MHz, respectively) or a Bruker AVANCE NEO
(400 and 100 MHz, respectively) in CDCl3. Chemical shifts δ are
given in parts per million, and coupling constants (J) are given in Hz.
Peak multiplicities are described as follows: s, singlet, d, doublet, t,
triplet, and m, multiplet. High-resolution mass spectra were obtained
on a Bruker Maxis Impact QTOF spectrometer or an AB Sciex 4600
Triple TOF mass spectrometer. The purity of all compounds
subjected to biological tests was determined by analytical high-
performance liquid chromatography (HPLC) and was found to be
≥95%. For the measurement of enantiomeric excess (ee) of 5a,b,
HPLC experiments were performed on an Agilent 1100 Series with a
DAD UV detector, and the peak intensities were measured in the UV
range between 206 and 280 nm. A Daicel Chiralpak OD-H
chromatography column (250 × 4.6 mm ID) was used with HPLC
i
grade hexane and PrOH as solvents.
2HPA, 2HSA, and 16HPA were commercially available. 3HCA,
3HLA, 3HMA, 3HPA, and 3HSA were synthesized as previously
described, and their analytical data are in accordance with the
literature.57 9HPA, 9HSA, 11HPA, and 12HSA were synthesized as
previously described, and their analytical data are in accordance with
the literature.23
General Procedure for the Synthesis of Epoxides (S)-5a,b
and (R)-5a,b. To a solution of (2S,5R)-2-(tert-butyl)-3,5-dimethy-
limidazolidin-4-one trifluoroacetate (for S-epoxide) or (2R,5S)-2-
(tert-butyl)-3,5-dimethylimidazolidin-4-one trifluoroacetate (for R-
epoxide) (57 mg, 0.20 mmol) in THF (0.5 mL), 2,3,4,5,6,6-
The association between milk fatty acids intake and T2D is
not well established.54 Saturated fatty acids such as PA could
increase the risk of T2D. However, milk also contains several
other fatty acids that are beneficial for human health. A recent
large meta-analysis, which pooled the findings from 16
prospective cohort studies, discusses the potential health
effects of selected dairy products, highlighting that higher levels
of odd-chain saturated fatty acids C15:0 and C17:0 are
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hexachlorocyclohexa-2,4-dien-1-one (381 mg, 1.10 mmol) was added,
followed, after 5 min, by the corresponding aldehyde (1.00 mmol).
After stirring for 20 min at room temperature, the resulting mixture
was cooled to 0 °C before NaBH4 (95 mg, 2.50 mmol) and EtOH (1
mL) were added. After 10 min, the mixture was warmed to room
temperature for 5 min before a freshly prepared solution of aqueous
KOH (1.70 g KOH diluted in 2.7 mL of water) and EtOH (1.3 mL)
were added. The resulting mixture was stirred vigorously for 30 min,
and then, water (20 mL) was added. The mixture was extracted with
Et2O (3 × 20 mL), washed with brine (1 × 20 mL), dried over
Na2SO4, filtered, and concentrated under reduced pressure. The
epoxide was purified by silica gel flash chromatography eluting with
petroleum ether (bp 40−60 °C)/ethyl acetate 8/2 or 7/3.
(S)-2-(6-(Benzyloxy)hexyl)oxirane (S)-5a. Colorless oil; yield 68%;
1H NMR (200 MHz, CDCl3): δ 7.45−7.14 (5H, m, ArH), 4.50 (2H,
s, ArCH2), 3.46 (2H, t, J = 6.5 Hz, OCH2), 2.89−2.80 (1H, m,
OCH), 2.73 (1H, t, J = 4.5 Hz, OCH), 2.45 (1H, dd, J = 5.0, 2.7 Hz,
OCH), 1.65−1.22 (10H, m, 10 × CHH); 13C NMR (50 MHz,
CDCl3): δ 138.5, 128.3, 127.5, 127.4, 72.8, 70.3, 52.3, 47.0, 32.3, 29.6,
29.2, 26.0, 25.9; [α]D20 −5.0 (c 1.0, CH2Cl2); HRMS (ESI+) m/z:
calcd for C15H22O2Na+, 257.1512; [M + Na]+ found, 257.1512;
HPLC analysis: 93% ee.
31.9, 29.8, 29.7, 29.6, 29.5, 29.4, 29.3, 26.2, 25.6, 22.7, 14.1; [α]D20
+1.5 (c 1.0, CH2Cl2); HRMS (ESI+) m/z: calcd for C25H44O2Na+,
399.3234; [M + Na]+ found, 399.3236.
(S)-1-(Benzyloxy)octadecan-10-ol (S)-6b. White solid; mp 47−49
°C; yield 82%; [α]2D0 −1.4 (c 1.0, CH2Cl2).
(R)-1-(Benzyloxy)hexadecan-7-ol (R)-6c. White solid; mp 50−53
1
°C; yield 71%; H NMR (200 MHz, CDCl3): δ 7.39−7.20 (5H, m,
ArH), 4.50 (2H, s, ArCH2), 3.63−3.52 (1H, m, CHOH), 3.46 (2H, t,
J = 6.5 Hz, OCH2), 1.73−1.51 (3H, m, 2 × CHH, OH), 1.51−1.03
(24H, m, 24 × CHH), 0.88 (3H, t, J = 5.3 Hz, CH3); 13C NMR (50
MHz, CDCl3): δ 138.6, 128.3, 127.6, 127.4, 72.8, 71.9, 70.4, 37.5,
37.4, 31.9, 29.7, 29.6, 29.5, 29.3, 26.2, 25.6, 22.7, 14.1; [α]2D0 +1.3 (c
1.0, CH2Cl2); HRMS (ESI+) m/z: calcd for C23H40O2Na+, 371.2921;
[M + Na]+ found, 371.2911.
(S)-1-(Benzyloxy)hexadecan-7-ol (S)-6c. White solid, mp 50−53
°C; yield 70%; [α]2D0 −1.8 (c 1.0, CH2Cl2).
(R)-1-(Benzyloxy)hexadecan-10-ol (R)-6d. White solid; mp 46−48
1
°C; yield 86%; H NMR (200 MHz, CDCl3): δ 7.42−7.18 (5H, m,
ArH), 4.50 (2H, s, ArCH2), 3.63−3.51 (1H, m, CHOH), 3.46 (2H, t,
J = 6.6 Hz, OCH2), 1.74−1.54 (3H, m, 2 × CHH, OH), 1.53−1.09
(24H, m, 24 × CHH), 0.89 (3H, t, J = 6.3 Hz, CH3); 13C NMR (50
MHz, CDCl3): δ 138.6, 128.3, 127.5, 127.4, 72.8, 71.9, 70.4, 37.4,
31.8, 29.7, 29.6, 29.5, 29.4, 29.3, 26.1, 25.6, 22.6, 14.1; [α]2D0 +2.1 (c
1.0, CH2Cl2); HRMS (ESI+) m/z: calcd for C23H40O2Na+, 371.2921;
[M + Na]+ found, 371.2909.
(R)-2-(6-(Benzyloxy)hexyl)oxirane (R)-5a. Colorless oil; yield 78%;
[α]D20 +6.0 (c 1.0, CH2Cl2); lit.58 for (R)-epoxide [α]2D5 +5.6 (c 2.0,
CHCl3).
(S)-2-(9-(Benzyloxy)nonyl)oxirane (S)-5b. Colorless oil; yield 75%;
(S)-1-(Benzyloxy)hexadecan-10-ol (S)-6d. White solid; mp 46−48
1H NMR (200 MHz, CDCl3): δ 7.44−7.16 (5H, m, ArH), 4.50 (2H,
°C; yield 87%; [α]2D0 −2.8 (c 1.0, CH2Cl2).
s, ArCH2), 3.46 (2H, t, J = 6.6 Hz, OCH2), 2.97−2.84 (1H, m,
OCH), 2.74 (1H, t, J = 4.5 Hz, OCH), 2.46 (1H, dd, J = 5.0, 2.8 Hz,
OCH), 1.73−1.15 (16H, m, 16 × CHH); 13C NMR (50 MHz,
CDCl3): δ 138.7, 128.3, 127.6, 127.4, 72.8, 70.5, 52.4, 47.1, 32.5, 29.7,
29.5, 29.4, 26.1, 25.9; [α]2D0 −4.0 (c 1.0, CH2Cl2); lit.59 for (S)-
epoxide [α]2D0 −4.2 (c 1.0, CHCl3); HRMS (ESI+) m/z: calcd for
C18H28O2Na+, 299.1982; [M + Na]+ found, 299.1977; HPLC
analysis: 92% ee.
General Procedure for the Synthesis of Compounds (R)-7a−
d and (S)-7a−d. To a flame-dried flask containing a solution of the
secondary alcohol 6a−d (1.00 mmol) in dry dichloromethane (10
mL) at 0 °C, dry pyridine (0.12 mL, 1.50 mmol) was added, followed
by acetyl chloride (0.14 mL, 2.00 mmol). The mixture was then left to
reach room temperature, and stirring was continued for 16 h. Then, a
saturated aqueous solution of NH4Cl (10 mL) was added, and the
product was extracted with Et2O (3 × 20 mL), dried over Na2SO4,
filtered, and concentrated under reduced pressure. The acetyl
protected product was purified by flash silica chromatography eluting
with petroleum ether (bp 40−60 °C)/ethyl acetate 9/1.
(R)-2-(9-(Benzyloxy)nonyl)oxirane (R)-5b. Colorless oil; yield
75%; [α]2D0 +3.6 (c 1.0, CH2Cl2); lit.59 for (R)-epoxide [α]2D3 +3.9
(c 1.0, CHCl3).
General Procedure for the Synthesis of Compounds (R)-6a−
d and (S)-6a−d. To a flame-dried flask which contained copper(I)
iodide (40 mg, 0.20 mmol) under an inert atmosphere,
pentylmagnesium bromide (or heptylmagnesium bromide or
octylmagnesium bromide or decylmagnesium bromide) (2 M solution
in diethyl ether, 2 mL, 2.00 mmol) was added. After cooling to −40
°C and stirring for 10 min, epoxide 5a,b (1.00 mmol) in dry THF (10
mL) was added dropwise. The mixture was left stirring at −40 °C for
1 h and then warmed to room temperature. Then, a saturated aqueous
solution of NH4Cl (10 mL) was added, and the aqueous layer was
extracted with Et2O (3 × 20 mL). The organic phase was washed with
brine (1 × 50 mL), dried over Na2SO4, filtered, and concentrated
under reduced pressure. The alcohol was obtained by flash silica
chromatography eluting with petroleum ether (bp 40−60 °C)/ethyl
acetate 8/2 or 7/3.
(R)-1-(Benzyloxy)octadecan-7-yl Acetate (R)-7a. Yellowish oil;
yield 89%; 1H NMR (200 MHz, CDCl3): δ 7.39−7.20 (5H, m, ArH),
4.92−4.78 (1H, m, CHOCO), 4.50 (2H, s, ArCH2), 3.45 (2H, t, J
= 6.5 Hz, OCH2), 2.03 (3H, s, CH3CO), 1.70−1.40 (8H, m, 8 ×
CHH), 1.40−1.14 (22H, m, 22 × CHH), 0.87 (3H, t, J = 6.0 Hz,
CH3); 13C NMR (50 MHz, CDCl3): δ 171.0, 138.6, 128.3, 127.6,
127.5, 74.4, 72.8, 70.4, 34.1, 34.0, 31.9, 29.6, 29.5, 29.3, 27.6, 26.1,
25.3, 25.2, 22.7, 21.3, 14.1; [α]2D0 +1.4 (c 1.0, CHCl3); HRMS (ESI+)
m/z: calcd for C27H46O3Na+, 441.3339; [M + Na]+ found, 441.3339.
(S)-1-(Benzyloxy)octadecan-7-yl Acetate (S)-7a. Yellowish oil;
yield 98%; [α]2D0 −0.9 (c 1.0, CHCl3).
(R)-18-(Benzyloxy)octadecan-9-yl Acetate (R)-7b. Yellowish oil;
yield 94%; 1H NMR (200 MHz, CDCl3): δ 7.38−7.21 (5H, m, ArH),
4.91−4.79 (1H, m, CHOCO), 4.50 (2H, s, ArCH2), 3.46 (2H, t, J
= 6.6 Hz, OCH2), 2.04 (3H, s, CH3CO), 1.67−1.36 (8H, m, 8 ×
CHH), 1.36−1.13 (22H, m, 22 × CHH), 0.87 (3H, t, J = 6.1 Hz,
CH3); 13C NMR (50 MHz, CDCl3): δ 170.9, 138.7, 128.3, 127.6,
127.4, 74.4, 72.8, 70.5, 34.1, 31.8, 29.8, 29.5, 29.2, 26.2, 25.3, 22.6,
21.3, 14.1; [α]2D0 +1.1 (c 1.0, CHCl3); HRMS (ESI+) m/z: calcd for
C27H46O3Na+, 441.3339; [M + Na]+ found, 441.3340.
(R)-1-(Benzyloxy)octadecan-7-ol (R)-6a. White solid; mp 56−58
1
°C; yield 80%; H NMR (200 MHz, CDCl3): δ 7.44−7.16 (5H, m,
ArH), 4.50 (2H, s, ArCH2), 3.66−3.52 (1H, m, CHOH), 3.47 (2H, t,
J = 6.5 Hz, OCH2), 1.75 (1H, s, OH), 1.69−1.54 (2H, m, 2 × CHH),
1.53−1.08 (28H, m, 28 × CHH), 0.89 (3H, t, J = 6.3 Hz, CH3); 13
C
NMR (50 MHz, CDCl3): δ 138.5, 128.2, 127.5, 127.4, 72.8, 71.8,
70.3, 37.4, 37.3, 32.7, 31.9, 29.7, 29.6, 29.5, 29.4, 29.3, 26.1, 25.6,
25.5, 22.6, 14.1; [α]2D0 +1.2 (c 1.0, CH2Cl2); HRMS (ESI+) m/z: calcd
for C25H44O2Na+, 399.3234; [M + Na]+ found, 399.3238.
(S)-1-(Benzyloxy)octadecan-7-ol (S)-6a. White solid; mp 56−58
°C; yield 83%; [α]2D0 −0.9 (c 1.0, CH2Cl2).
(S)-18-(Benzyloxy)octadecan-9-yl Acetate (S)-7b. Yellowish oil;
yield 87%; [α]2D0 −0.9 (c 1.0, CHCl3).
(R)-1-(Benzyloxy)hexadecan-7-yl Acetate (R)-7c. Yellowish oil;
yield 96%; 1H NMR (200 MHz, CDCl3): δ 7.42−7.19 (5H, m, ArH),
4.92−4.79 (1H, m, CHOCO), 4.50 (2H, s, ArCH2), 3.46 (2H, t, J
= 6.5 Hz, OCH2), 2.03 (3H, s, CH3CO), 1.67−1.43 (6H, m, 6 ×
CHH), 1.43−1.04 (20H, m, 20 × CHH), 0.88 (3H, t, J = 6.3 Hz,
CH3); 13C NMR (50 MHz, CDCl3): δ 170.9, 138.6, 128.3, 127.6,
127.4, 74.4, 72.8, 70.4, 34.1, 34.0, 31.9, 29.7, 29.5, 29.3, 26.1, 25.3,
25.2, 22.7, 21.3, 14.1; [α]D20 +1.3 (c 1.0, CHCl3); HRMS (ESI+) m/z:
calcd for C25H42O3Na+, 413.3026; [M + Na]+ found, 413.3022.
(R)-1-(Benzyloxy)octadecan-10-ol (R)-6b. White solid; mp 47−49
1
°C; yield 83%; H NMR (200 MHz, CDCl3): δ 7.38−7.20 (5H, m,
ArH), 4.50 (2H, s, ArCH2), 3.64−3.52 (1H, m, CHOH), 3.46 (2H, t,
J = 6.6 Hz, OCH2), 1.72−1.52 (3H, m, 2 × CHH, OH), 1.52−1.04
(28H, m, 28 × CHH), 0.88 (3H, t, J = 6.3 Hz, CH3); 13C NMR (50
MHz, CDCl3): δ 138.7, 128.3, 127.6, 127.4, 72.8, 72.0, 70.5, 37.5,
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(S)-1-(Benzyloxy)hexadecan-7-yl Acetate (S)-7c. Yellowish oil;
yield 95%; [α]2D0 −1.0 (c 1.0, CHCl3).
(R)-7-Acetoxyoctadecanoic Acid (R)-9a. Colorless oil; yield 82%;
1H NMR (200 MHz, CDCl3): δ 4.91−4.78 (1H, m, CHOCO),
2.34 (2H, t, J = 7.3 Hz, CH2COOH), 2.03 (3H, s, CH3CO), 1.74−
1.43 (6H, m, 6 × CHH), 1.43−0.99 (22H, m, 22 × CHH) 0.87 (3H,
t, J = 6.1 Hz, CH3); 13C NMR (50 MHz, CDCl3): δ 179.5, 171.0,
74.3, 34.1, 33.9, 31.9, 29.6, 29.5, 29.3, 28.9, 25.3, 24.9, 24.5, 22.7,
21.3, 14.1; [α]2D0 +1.4 (c 0.5, CHCl3); HRMS (ESI−) m/z: calcd for
(R)-16-(Benzyloxy)hexadecan-7-yl Acetate (R)-7d. Yellowish oil;
yield 92%; 1H NMR (200 MHz, CDCl3): δ 7.41−7.21 (5H, m, ArH),
4.91−4.79 (1H, m, CHOCO), 4.50 (2H, s, ArCH2), 3.46 (2H, t, J
= 6.5 Hz, OCH2), 2.03 (3H, s, CH3CO), 1.63−1.39 (6H, m, 6 ×
CHH), 1.39−1.11 (20H, m, 20 × CHH), 0.87 (3H, t, J = 6.2 Hz,
CH3); 13C NMR (50 MHz, CDCl3): δ 170.9, 138.7, 128.3, 127.6,
127.4, 74.4, 72.8, 70.5, 34.1, 31.7, 29.7, 29.5, 29.4, 29.2, 26.6, 26.1,
25.3, 22.6, 21.3, 14.0; [α]D20 +1.8 (c 1.0, CHCl3); HRMS (ESI+) m/z:
calcd for C25H42O3Na+, 413.3026; [M + Na]+ found, 413.3016.
(S)-16-(Benzyloxy)hexadecan-7-yl Acetate (S)-7d. Yellowish oil;
yield 89%; [α]2D0 −2.0 (c 1.0, CHCl3).
−
C20H37O4 , 341.2697; [M − H]− found, 341.2691.
(S)-7-Acetoxyoctadecanoic Acid (S)-9a. Colorless oil; yield 77%;
[α]2D0 −1.0 (c 0.5, CHCl3).
(R)-10-Acetoxyoctadecanoic Acid (R)-9b. Colorless oil; yield 92%;
1H NMR (200 MHz, CDCl3): δ 4.92−4.78 (1H, m, CHOCO),
2.34 (2H, t, J = 7.4 Hz, CH2COOH), 2.03 (3H, s, CH3CO), 1.72−
1.42 (6H, m, 6 × CHH), 1.42−1.09 (22H, m, 22 × CHH) 0.87 (3H,
t, J = 5.5 Hz, CH3); 13C NMR (50 MHz, CDCl3): δ 179.7, 171.0,
74.4, 34.1, 34.0, 31.8, 29.5, 29.4, 29.3, 29.2, 29.1, 29.0, 25.3, 24.6,
22.6, 21.3, 14.1; [α]2D0 +1.4 (c 0.5, CHCl3); HRMS (ESI−) m/z: calcd
General Procedure for the Removal of Benzyl Group. A
mixture of (R)- or (S)- 7a−d (1.00 mmol) in MeOH (10 mL) and
10% palladium on activated charcoal was left stirring under a
hydrogen atmosphere for 16 h. After filtration through a Celite pad,
the solvent was evaporated to afford the desired alcohol without
further purification.
−
for C20H37O4 , 341.2697; [M − H]− found, 341.2690.
(S)-10-Acetoxyoctadecanoic Acid (S)-9b. Colorless oil; yield 93%;
[α]2D0 −1.2 (c 0.5, CHCl3).
(R)-1-Hydroxyoctadecan-7-yl Acetate (R)-8a. Colorless oil; yield
1
88%; H NMR (200 MHz, CDCl3): δ 4.90−4.78 (1H, m, CHOC
(R)-7-Acetoxyhexadecanoic Acid (R)-9c. Colorless oil; yield 96%;
1H NMR (200 MHz, CDCl3): δ 4.90−4.78 (1H, m, CHOCO),
2.34 (2H, t, J = 7.4 Hz, CH2COOH), 2.03 (3H, s, CH3CO), 1.70−
1.42 (6H, m, 6 × CHH), 1.42−1.04 (18H, m, 18 × CHH) 0.87 (3H,
t, J = 6.4 Hz, CH3); 13C NMR (50 MHz, CDCl3): δ 179.6, 171.0,
74.3, 34.1, 33.9, 31.9, 29.5, 29.3, 28.9, 25.3, 24.9, 24.5, 22.7, 21.3,
14.1; [α]2D0 +3.0 (c 0.5, CHCl3); HRMS (ESI+) m/z: calcd for
C18H34O4Na+, 337.2349; [M + Na]+ found, 337.2351.
O), 3.62 (2H, t, J = 6.5 Hz, CH2OH), 2.03 (3H, s, CH3CO), 1.76
(1H, s, OH), 1.66−1.38 (8H, m, 8 × CHH), 1.38−1.16 (22H, m, 22
× CHH), 0.86 (3H, t, J = 6.4 Hz, CH3); 13C NMR (50 MHz,
CDCl3): δ 171.0, 74.3, 62.9, 34.1, 34.0, 32.6, 31.9, 29.6, 29.5, 29.3,
29.2, 25.6, 25.3, 25.2, 22.7, 21.3, 14.1; [α]2D0 +1.4 (c 1.0, CHCl3);
HRMS (ESI+) m/z: calcd for C20H40O3Na+, 351.2870; [M + Na]+
found, 351.2862.
(S)-1-Hydroxyoctadecan-7-yl Acetate (S)-8a. Colorless oil; yield
(S)-7-Acetoxyhexadecanoic Acid (S)-9c. Colorless oil; yield 92%;
72%; [α]2D0 −1.2 (c 1.0, CHCl3).
[α]2D0 −2.5 (c 0.5, CHCl3).
(R)-18-Hydroxyoctadecan-9-yl Acetate (R)-8b. Colorless oil; yield
(R)-10-Acetoxyhexadecanoic Acid (R)-9d. Colorless oil; yield
1
1
87%; H NMR (200 MHz, CDCl3): δ 4.91−4.78 (1H, m, CHOC
83%; H NMR (200 MHz, CDCl3): δ 4.90−4.78 (1H, m, CHOC
O), 3.63 (2H, t, J = 6.5 Hz, CH2OH), 2.03 (3H, s, CH3CO),1.69−
1.40 (8H, m, 8 × CHH), 1.40−1.03 (23H, m, 22 × CHH, OH), 0.87
(3H, t, J = 6.4 Hz, CH3); 13C NMR (50 MHz, CDCl3): δ 171.0, 74.4,
63.0, 34.1, 32.8, 31.8, 29.5, 29.4, 29.3, 29.2, 25.7, 25.3, 22.6, 21.3,
14.1; [α]2D0 +1.3 (c 1.0, CHCl3); HRMS (ESI+) m/z: calcd for
C20H40O3Na+, 351.2870; [M + Na]+ found, 351.2875.
O), 2.33 (2H, t, J = 7.4 Hz, CH2COOH), 2.03 (3H, s, CH3CO),
1.69−1.42 (6H, m, 6 × CHH), 1.42−1.11 (18 H, m, 18 × CHH) 0.86
(3H, t, J = 6.0 Hz, CH3); 13C NMR (50 MHz, CDCl3): δ 180.0,
171.0, 74.4, 34.1, 34.0, 31.7, 29.4, 29.3, 29.2, 29.1, 29.0, 25.2, 24.6,
22.6, 21.3, 14.0; [α]2D0 +1.1 (c 1.0, CHCl3); HRMS (ESI+) m/z: calcd
for C18H34O4Na+, 337.2349; [M + Na]+ found, 337.2343.
(S)-18-Hydroxyoctadecan-9-yl Acetate (S)-8b. Colorless oil; yield
(S)-10-Acetoxyhexadecanoic Acid (S)-9d. Colorless oil; yield 82%;
78%; [α]2D0 −1.6 (c 1.0, CHCl3).
[α]2D0 −1.3 (c 1.0, CHCl3)
(R)-1-Hydroxyhexadecan-7-yl Acetate (R)-8c. Colorless oil; yield
General Procedure for the Removal of the Acetyl Group.
Compounds 9a−d (1 mmol) were dissolved in a mixture of THF and
H2O (1:1, 5 mL), followed by the addition of LiOH (96 mg, 4
mmol), and the reaction mixture was left under stirring at room
temperature for 16 h. After the addition of a solution of HCl 1 N (10
mL) to pH 1, the mixture was extracted with Et2O (3 × 10 mL),
washed with brine (1 × 30 mL), dried over Na2SO4, filtered, and
evaporated under reduced pressure. The hydroxy fatty acids were
purified by silica gel flash chromatography eluting with petroleum
ether (bp 40−60 °C)/ethyl acetate 2/8.
1
99%; H NMR (200 MHz, CDCl3): δ 4.93−4.76 (1H, m, CHOC
O), 3.63 (2H, t, J = 6.4 Hz, CH2OH), 2.03 (3H, s, CH3CO), 1.65−
1.43 (7H, m, 6 × CHH, OH), 1.43−1.11 (20H, m, 20 × CHH), 0.87
(3H, t, J = 6.0 Hz, CH3); 13C NMR (50 MHz, CDCl3): δ 171.0, 74.3,
62.9, 34.1, 34.0, 32.6, 31.9, 29.5, 29.3, 29.2, 25.6, 25.3, 25.2, 22.7,
21.3, 14.1; [α]2D0 +1.1 (c 0.5, CHCl3); HRMS (ESI+) m/z: calcd for
C18H36O3Na+, 323.2557; [M + Na]+ found, 323.2550.
(S)-1-Hydroxyhexadecan-7-yl Acetate (S)-8c. Colorless oil; yield
97%; [α]2D0 −1.2, (c 0.5, CHCl3).
(R)-16-Hydroxyhexadecan-7-yl Acetate (R)-8d. Colorless oil;
yield 99%; 1H NMR (200 MHz, CDCl3): δ 4.90−4.78 (1H, m,
CHOCO), 3.62 (2H, t, J = 6.5 Hz, CH2OH), 2.02 (3H, s, CH3C
O), 1.63−1.41 (7H, m, 6 × CHH, OH), 1.41−1.05 (20H, m, 20 ×
CHH), 0.86 (3H, t, J = 6.4 Hz, CH3); 13C NMR (50 MHz, CDCl3): δ
171.0, 74.4, 63.0, 34.1, 32.7, 31.7, 29.4, 29.3, 29.2, 25.7, 25.2, 22.6,
21.3, 14.0; [α]2D0 +2.7 (c 1.0, CHCl3); HRMS (ESI+) m/z: calcd for
C18H36O3Na+, 323.2557; [M + Na]+ found, 323.2552.
(R)-7-Hydroxyoctadecanoic Acid (R)-10a. White solid; mp 72−75
°C; yield 72%; 1H NMR (400 MHz, CDCl3): δ 6.22 (1H, br s,
COOH), 3.68−3.50 (1H, m, CHOH), 2.33 (2H, t, J = 7.4 Hz,
CH2COOH), 1.81−1.52 (3H, m, 2 × CHH, OH), 1.52−1.10 (26H,
m, 26 × CHH), 0.87 (3H, t, J = 6.4 Hz, CH3); 13C NMR (100 MHz,
CDCl3): δ 179.2, 72.0, 37.4, 37.0, 33.9, 31.9, 29.7, 29.6, 29.3, 29.1,
25.6, 25.2, 24.6, 22.7, 14.1; [α]2D0 −3.6 (c 1.0, CHCl3); HRMS (ESI−)
−
m/z: calcd for C18H35O3 , 299.2592; [M − H]− found, 299.2592.
(S)-7-Hydroxyoctadecanoic Acid (S)-10a. White solid; mp 72−75
(S)-16-Hydroxyhexadecan-7-yl Acetate (S)-8d. Colorless oil; yield
°C; yield 73%; [α]2D0 +4.5 (c 1.0, CHCl3).
86%; [α]2D0 −2.2 (c 1.0, CHCl3).
(R)-10-Hydroxyoctadecanoic Acid (R)-10b. White solid; mp 82−
84 °C; yield 69%; 1H NMR (400 MHz, CDCl3): δ 3.63−3.54 (1H, m,
CHOH), 2.34 (2H, t, J = 7.5 Hz, CH2COOH), 1.70−1.52 (3H, m, 2
× CHH, OH), 1.52−1.15 (26H, m, 26 × CHH), 0.88 (3H, t, J = 6.7
Hz, CH3); 13C NMR (100 MHz, CDCl3): δ 179.0, 72.1, 37.5, 37.4,
33.9, 31.9, 29.7, 29.6, 29.3, 29.1, 29.0, 25.6, 24.6, 22.7, 14.1; [α]D20
General Procedure for the Oxidation of Alcohols to
Compounds (R)-9a−d and (S)-9a−d. To a solution of alcohol
8a−d (1 mmol) in acetone (10 mL) at 0 °C, the Jones reagent (2 M,
1.5 mL, 3 mmol) was added dropwise at 0 °C. The reaction was left
under stirring at 0 °C for 1 h. Then, a saturated solution of NaHSO3
(10 mL) was added at room temperature. The mixture was extracted
with Et2O (3 × 20 mL), washed with brine (1 × 50 mL), dried over
Na2SO4, filtered, and concentrated under reduced pressure. The acids
were purified by flash chromatography eluting with petroleum ether
(bp 40−60 °C)/ethyl acetate 6/4.
−
−2.4 (c 1.0, CHCl3); HRMS (ESI−) m/z: calcd for C18H35O3 ,
299.2592; [M − H]− found, 299.2591.
(S)-10-Hydroxyoctadecanoic Acid (S)-10b. White solid; mp 82−
84 °C; yield 65%; [α]2D0 +2.8 (c 1.0, CHCl3).
L
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(R)-7-Hydroxyhexadecanoic Acid (R)-10c. White solid; mp 60−62
ANOVA statistical analysis was performed for each separate
immunostaining set comparing all conditions to control.
1
°C; yield 67%; H NMR (400 MHz, CDCl3): δ 5.29, (1H, br s,
COOH), 3.66−3.53 (1H, m, CHOH), 2.34 (2H, t, J = 7.4 Hz,
CH2COOH), 1.72−1.56 (2H, m, 2 × CHH), 1.55−1.09 (23H, m, 22
× CHH, OH), 0.88 (3H, t, J = 6.7 Hz, CH3); 13C NMR (100 MHz,
CDCl3): δ 179.1, 72.0, 37.5, 37.1, 33.9, 31.9, 29.7, 29.6, 29.3, 29.1,
25.6, 25.2, 24.6, 22.7, 14.1; [α]2D0 −2.6 (c 1.0, CHCl3); HRMS (ESI−)
RNA Extraction and Real-Time RT-qPCR Analysis. Total RNA
was isolated by TRI reagent solution (AM9738, Ambion/RNA, Life
Technologies), according to the manufacturer’s instructions, followed
by treatment with RQ1 DNase (Promega, Madison, WI, USA). RNA
concentration and purity were measured by Nanodrop 2000c
(Thermo), and 1.5 μg was used for cDNA synthesis using the
SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad,
USA) together with random hexamer primers. Quantitative real-time
RT-qPCR analysis was performed in a LightCycler 96 Instrument
(Roche). Measured values were normalized using beta actin mRNA
levels as internal references. Primers used for real-time RT-qPCR are
shown in Table 1S.
Immunoblotting. Total protein was isolated from treated and
untreated A549 cell with lysis buffer RIPA. The homogenates were
centrifuged at 17,000g for 15 min at 4 °C. The supernatants were
collected, and protein concentration was measured with Bradford
protein assay (Bio-Rad protein assay). Protein samples (35 μg) were
loaded each time into sodium dodecyl sulfate-polyacrylamide gel
electrophoresis gels and transferred to nitrocellulose membranes
(Amersham) using the semidry transfer system (Bio-Rad). The
membranes were blocked with BSA (AppliChem, A1391) 5%
dissolved in Tris-buffered saline (1×) containing 0.1% Tween 20
for 1 h at r.t. The membranes were incubated with primary antibodies
at 4 °C overnight, followed by secondary antibodies for 1.30 h at r.t.
The primary antibodies in the Western blot were rabbit anti-phospho-
STAT3 (Cell Signaling, 9145) (1:1000 dilution), rabbit anti-STAT3
(Santa Cruz, sc-482) (1:1000 dilution), and mouse anti-beta actin
(Sigma, A5441) (1:20,000 dilution). The secondary antibodies were
rabbit anti-mouse IgG (Sigma, A9044) (1:20000 dilution) and goat
anti-rabbit IgG (Sigma, A6154) (1:10000 dilution) IgGs.
Cell Culture and Immunostaining. A549 cells were cultured in
a 37 °C humidified incubator with 5% CO2 cells in DMEM (4.5 g/L
glucose, Biosera) supplemented with 10% (v/v) heat-inactivated FBS
(Biosera) and pen-strep (100 and 100 μg/mL, respectively;
Invitrogen). For the immunostaining experiments, A549 cells were
cultured onto poly-L-lysine (Sigma)-coated coverslips in 24-well
plates. After treatment, the cells were fixed on the coverslips with 4%
PFA (paraformaldehyde) and prepared for immunostaining experi-
ments. The coverslips were blocked with 5% FBS dissolved in
phosphate-buffered saline (PBS) (1×) containing 0.1% Triton X-100
for 1 h at r.t. Next, they were incubated with primary antibodies at 4
°C overnight, followed by secondary antibodies for 1 h at r.t. Finally,
they were incubated with DAPI diluted in 1× PBS for 10 min at r.t.,
followed by mounting with MOWIOL. The primary antibodies in the
immunofluorescence were rabbit anti-phospho-Histone 3 (Abcam,
Ab5176) (1:600 dilution) and rabbit anticleaved caspase 3 (Cell
Signaling, 9661) (1:800 dilution). The secondary antibody was
donkey anti-rabbit 488 (AlexaFluor). Immunostained cells were
imaged on an upright microscope (Leica).
TUNEL Analyses. HFAs and PAHSA were dissolved in DMSO to
prepare a stock solution. Additional dilutions were made, if needed, in
DMSO and the final added volume (to achieve any final
concentration) was 1 μL in 1000 μL media. Thus, the final
concentration of DMSO was always 1:1000 (v/v) or 0.10%. INS-1
cells were cultured in RPMI (containing 10% fetal calf serum, 2 mmo/
L glutamine, 1% of 100× penicillin/streptomycin, 10 mM HEPES, 1
mM sodium pyruvate) at 37 °C under an atmosphere of 5% CO2/
95% air for up to 24 h until ca 70% confluency was achieved. The cells
were treated with either 1 or 10 μM of each SHFAs for 1 h, prior to
exposures to vehicle (DMSO, 1 μL/mL) alone or CTK IL-1β (20 ng/
mL, R&D systems, 501-RL) + IFNγ (200 ng/mL, R&D systems, 585-
IF) for 16 h. The cells were then harvested and washed twice with ice-
cold PBS, immobilized on slides by cytospin, and fixed with 4%
paraformaldehyde (in PBS, pH 7.4, for 1 h at r.t.). The INS-1 cells
were then washed with PBS and incubated in permeabilization
solution (0.1% Triton X-100 in 0.1% sodium citrate in PBS for 30 min
at r.t.). The permeabilization solution was then removed and
processed for TUNEL analyses using the In Situ Cell Death
−
m/z: calcd for C16H31O3 , 271.2279; [M − H]− found, 271.2266.
(S)-7-Hydroxyhexadecanoic Acid (S)-10c. White solid; mp 60−62
°C; yield 65%; [α]2D0 +3.0 (c 1.0, CHCl3).
(R)-10-Hydroxyhexadecanoic Acid (R)-10d. White solid; mp 64−
1
67 °C; yield 77%; H NMR (400 MHz, CDCl3): δ 6.61 (1H, br s,
COOH), 3.67−3.52 (1H, m, CHOH), 2.31 (2H, t, J = 7.5 Hz,
CH2COOH), 1.69−1.53 (2H, m, 2 × CHH), 1.53−1.09 (23H, m, 22
× CHH, OH), 0.87 (3H, t, J = 6.7 Hz, CH3); 13C NMR (100 MHz,
CDCl3): δ 179.4, 72.1, 37.3, 37.2, 34.0, 31.8, 29.5, 29.3, 29.1, 29.0,
25.5, 24.6, 22.6, 14.0; [α]D20 −3.0 (c 1.0, CHCl3); HRMS (ESI−) m/z:
−
calcd for C16H31O3 , 271.2279; [M − H]− found, 271.2269.
(S)-10-Hydroxyhexadecanoic Acid (S)-10d. White solid; mp 64−
67 °C; yield 83%; [α]2D0 +3.2 (c 1.0, CHCl3).
Suspect Screening Analysis and Determination of SHFAs.
LC−MS/MS measurements were performed with an ABSciex Triple
TOF 4600 combined with a micro-LC Eksigent and an autosampler
set at 5 °C and a thermostated column compartment. Electrospray
ionization (ESI) in negative mode was used for all the MS
experiments. The data acquisition method consisted of a TOF-MS
full scan m/z 50−850 and several information dependent acquisition-
TOF-MS/MS product ion scans using 40 V collision energy with 15 V
collision energy spread used for each candidate ion in each data
acquisition cycle (1091). A Halo C18 2.7 μm, 90 Å, 0.5 × 50 mm
column from Eksigent was used, and the mobile phase consisted of a
gradient (A: acetonitrile/0.01% formic acid/isopropanol 80/20 v/v;
B: H2O/0.01% formic acid). The data acquisition was carried out with
MultiQuant 3.0.2 and PeakView 2.1 from AB SCIEX. EICs were
obtained with the use of MultiQuant 3.0.2, which created the base
peak chromatograms for the masses that achieve a 0.01 mass accuracy
width. The relative tolerance of the retention time was set within a
margin of 2.5%. Ten samples of human plasma and 10 samples of
cow milk were analyzed by LC−HRMS. Ten brand products of fresh
(pasteurized) whole cow milk (fat content 3.5−3.7%) were collected
from the local market in Athens, Greece.
MTT Assay. All four cells lines used were cultured as described.
After 24 h, the medium was changed to one containing the desired
compounds at selected concentrations, including control samples with
the equivalent vehicle amount. HFAs, FAs, and FAHFAs were
dissolved in DMSO to prepare a stock solution of 10 mM. The final
solutions were prepared by appropriate dilution of the stock solution
in media maintaining a final 0.5% DMSO content. A triplicate for
each condition was always executed in the same plate. Following 72 h,
MTT was added at a final concentration of 0.25 mg/mL and
incubated for 3 h. The medium was removed, and DMSO was added
to dissolve the formazan crystals. The formazan absorbance was
measured at 570 nm using an ELISA Plate reader (ELX800,
BIOTEC), while the background absorbance was measured at 690
nm.
Cell Growth Inhibition Analysis. Data analysis was performed
using Microsoft Excel (Office Professional Plus 2016). The 690 nm
background absorbance was subtracted from the 570 nm values, and
the average of the three repeats per condition was calculated. The data
set was normalized to DMSO control cells as 100% survival value.
The curves were generated using GraphPad Prism version 6.01, and
the IC50 values were calculated from the resultant plots. Two-way
ANOVA statistical analysis with multiple comparisons was performed
on this data set comparing the various concentrations of the
compounds investigated to DMSO control.
Image Data Analysis. Cell nuclei were identified in all samples
using DAPI staining. pH3 and casp3 positive nuclei were overlaid
onto the DAPI images and quantified using ImageJ (NIH). Values
were normalized to DMSO control conditions using Microsoft Excel,
while graphs were created using GraphPad Prism 6.01. One-way
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Detection Kit, Fluorescein (catalog number 11684795910; Roche
Applied Science, Mannheim, Germany), as described.47 The cells
were washed with PBS and counterstained with 1 μg/mL DAPI in
PBS for 10 min to identify cellular nuclei. Incidence of apoptosis was
assessed under a fluorescence microscope using a FITC filter. Cells
with TUNEL-positive nuclei were considered apoptotic. DAPI
staining was used to determine the total number of INS-1 cells in a
field. A minimum of three fields per slide was used to calculate the
percentage of apoptotic islet cells.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c00844
Author Contributions
#A.C.K., D.G., O.G.M. authors contributed equally.
Notes
The authors declare no competing financial interest.
Statistics. Comparisons between two data groups (with n ≥ 3
independent experiments) were analyzed using a Student’s t-test.
Values of <0.05 were considered significant and are indicated in the
graphs as *p < 0.05, **p < 0.01, or ***p < 0.001. All data represent
the mean SEM (n ≥ 3).
ACKNOWLEDGMENTS
■
The research presented was carried out within the framework
of a Stavros Niarchos Foundation grant to the National and
Kapodistrian University of Athens (G.K.). This work was
partially supported by a Fondation Sante grant and the
Hellenic Foundation for Research and Innovation (H.F.R.I.)
under the “First Call for H.F.R.I. Research Projects to support
Faculty members and Researchers and the procurement of
high-cost research equipment grant” (Project Number: 1782)
(P.K.P.) and by NIH Grants R01DK-69455 and R01
DK110292 (S.R.). The research work was supported by the
Hellenic Foundation for Research and Innovation (HFRI)
under the HFRI PhD Fellowship grant (Fellowship Number:
1338) (O.M).
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c00844.
Cell growth inhibitory potency of HSAs and HPAs on
human cancer cell line U87-MG, 2-way ANOVA
statistical analysis, effect of 7RHSA and 9RSHA on
cell death and apoptosis of SF268 cells, primers used for
real-time RT-qPCR, and 1H NMR and 13C NMR
spectra of the compounds synthesized (PDF)
ABBREVIATIONS
■
Molecular formula strings and inhibition data (CSV)
Casp3, activated caspase 3; CTK, cytokines; DMSO, dimethyl
sulfoxide; ee, enantiomeric excess; EICs, extracted ion
chromatograms; FAHFAs, fatty acid esters of hydroxy fatty
acids; HCA, hydroxycapric acid; 2HFAs, 2-hydroxy fatty acids;
3HFAs, 3-hydroxy fatty acids; HLA, hydroxylauric acid; HMA,
hydroxymyristic acid; HPA, hydroxypalmitic acid; HRMS,
high-resolution mass spectrometry; HSA, hydroxystearic acid;
PAHSA, palmitic acid ester of 9-hydroxystearic acid; PCC,
pyridinium chlorochromate; pH3, phosphorylated histone H3;
p-STAT3, phosphorylated STAT3; SHFAs, saturated hydroxy
fatty acids; SPM, specialized proresolving mediators; STAT3,
transcription regulator signal transducer and activator of
transcription 3; THF, tetrahydrofuran
AUTHOR INFORMATION
Corresponding Author
■
George Kokotos − Department of Chemistry, National and
Kapodistrian University of Athens, Athens 15771, Greece;
orcid.org/0000-0003-3753-7082; Phone: +30 210
7274462; Email: gkokotos@chem.uoa.gr; Fax: +30 210
7274761
Authors
Maroula G. Kokotou − Department of Chemistry, National and
Kapodistrian University of Athens, Athens 15771, Greece
Alexandros C. Kokotos − Center of Basic Research, Biomedical
Research Foundation of the Academy of Athens, Athens 11527,
Greece
Dimitrios Gkikas − Center of Basic Research, Biomedical
Research Foundation of the Academy of Athens, Athens 11527,
Greece
Olga G. Mountanea − Department of Chemistry, National and
Kapodistrian University of Athens, Athens 15771, Greece
Christiana Mantzourani − Department of Chemistry, National
and Kapodistrian University of Athens, Athens 15771, Greece
Abdulaziz Almutairi − Department of Cell, Developmental, and
Integrative Biology and Comprehensive Diabetes Center,
University of Alabama at Birmingham, Birmingham, Alabama
35294, United States
Xiaoyong Lei − Department of Cell, Developmental, and
Integrative Biology and Comprehensive Diabetes Center,
University of Alabama at Birmingham, Birmingham, Alabama
35294, United States
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