Synthesis and characterization of novel n-9 fatty acid conjugates possessing antineoplastic properties

Article abstract of DOI:10.1007/s11745-012-3707-9

The present study enumerates the synthesis, spectroscopic characterization, and evaluation of anticancer potential of esters of two n-9 fatty acids viz., oleic acid (OLA) and ricinoleic acid (RCA) with 2,4- or 2,6-diisopropylphenol. The synthesis strategy involved esterification of the hydroxyl group of diisopropylphenol (propofol) to the terminal carboxyl group of n-9 fatty acid. The synthesized propofol-n-9 conjugates having greater lipophilic character were tested initially for cytotoxicity in-vitro. The conjugates showed specific growth inhibition of cancer cell lines whereas no effect was observed in normal cells. In general, pronounced growth inhibition was found against the human skin malignant melanoma cell line (SK-MEL-1). The anticancer potential was also determined by testing the effect of these conjugates on cell migration, cell adhesion and induction of apoptosis in SK-MEL-1 cancer cells. Propofol-OLA conjugates significantly induced apoptosis in contrast to propofol-RCA conjugates which showed only weak signals for cytochrome c. Conclusively, the synthesized novel ester conjugates showed considerable moderation of anti-tumor activity. This preliminary study places in-house synthesized conjugates into the new class of anticancer agents that possess selectivity toward cancer cells over normal cells.

Full text of DOI:10.1007/s11745-012-3707-9

Lipids
DOI 10.1007/s11745-012-3707-9
ORIGINAL ARTICLE
Synthesis and Characterization of Novel n-9 Fatty Acid
Conjugates Possessing Antineoplastic Properties
•
Azmat A. Khan Ahmad Husain Mumtaz Jabeen
•
•
•
Jamal Mustafa Mohammad Owais
Received: 18 November 2011 / Accepted: 30 July 2012
Ó AOCS 2012
Abstract The present study enumerates the synthesis,
spectroscopic characterization, and evaluation of antican-
cer potential of esters of two n-9 fatty acids viz., oleic acid
(OLA) and ricinoleic acid (RCA) with 2,4- or 2,6-diiso-
propylphenol. The synthesis strategy involved esterifica-
tion of the hydroxyl group of diisopropylphenol (propofol)
to the terminal carboxyl group of n-9 fatty acid. The syn-
thesized propofol-n-9 conjugates having greater lipophilic
character were tested initially for cytotoxicity in-vitro. The
conjugates showed specific growth inhibition of cancer cell
lines whereas no effect was observed in normal cells. In
general, pronounced growth inhibition was found against
the human skin malignant melanoma cell line (SK-MEL-1).
The anticancer potential was also determined by testing the
effect of these conjugates on cell migration, cell adhesion
and induction of apoptosis in SK-MEL-1 cancer cells.
Propofol-OLA conjugates significantly induced apoptosis
in contrast to propofol-RCA conjugates which showed only
weak signals for cytochrome c. Conclusively, the synthe-
sized novel ester conjugates showed considerable modera-
tion of anti-tumor activity. This preliminary study places in-
house synthesized conjugates into the new class of anti-
cancer agents that possess selectivity toward cancer cells
over normal cells.
Keywords Ester conjugates Á Antineoplastic Á Apoptosis Á
Caspase-3 Á Cytochrome c Á Cytotoxicity Á Cancer cell line
Abbreviations
A549
ARA
Human lung carcinoma
Arachidonic acid
DCC
N,N-dicyclohexylcarbodiimide
4-(Dimethyl amino pyridine)
Human liver hepatocellular carcinoma
Human lung fibroblast
DMAP
HepG2
HFL1
HL-60
IC-50
Human leukemia
Electronic supplementary material The online version of this
article (doi:10.1007/s11745-012-3707-9) contains supplementary
material, which is available to authorized users.
Concentration of a test compound that
reduces 50 % of cell viability
Linoleic acid
LNA
MDA-MB-361 Ductal breast carcinoma
A. A. Khan (&) Á M. Owais
Interdisciplinary Biotechnology Unit, Aligarh Muslim
University, Aligarh 202002, India
e-mail: azmatbiotech@gmail.com
MUFA
Monounsaturated fatty acid(s)
Oleic acid
OLA
Propofol
PUFA
Diisopropylphenol
Polyunsaturated fatty acid(s)
Ricinoleic acid
A. Husain
Department of Chemistry, Aligarh Muslim University,
Aligarh 202002, India
RCA
RPMI
Rosewell Park Memorial Institute
Human skin malignant melanoma
Unsaturated fatty acid(s)
SK-MEL-1
UFA
M. Jabeen
Department of Zoology, Aligarh Muslim University,
Aligarh 202002, India
2,4P-OLA
2,6P-OLA
2,4P-RCA
2,6P-RCA
2,4-Diisopropylphenol-oleic acid
2,6-Diisopropylphenol-oleic acid
2,4-Diisopropylphenol-ricinoleic acid
2,6-Diisopropylphenol-ricinoleic acid
J. Mustafa
University Polytechnic, Aligarh Muslim University,
Aligarh 202002, India
123

Lipids
viz., promotes healthy blood lipid profiles, mediates blood
pressure, improves insulin sensitivity and regulates glu-
cose levels [15]. The major MUFA in human diets is oleic
acid (OLA; cis-9-octadecenoic acid). It is found in several
animal and vegetable oils; olive oil being its richest
source. High concentrations of OLA are found to lower
the levels of cholesterol, atherosclerosis and aid in cancer
prevention [16–18]. Curiosity has been stimulated in OLA
since the discovery of its role in protection from breast
cancer [19–23]. Various epidemiological and animal
model studies have correlated the consumption of OLA
rich olive oil with reduction in cancer incidences [24–27].
In-vitro studies conducted by Menendez et al. [19] have
shown the use of OLA in synergistically enhancing the
effectiveness of various anticancer agents. The higher
concentration of OLA has been revealed to induce DNA
fragmentation and loss of membrane structure of various
cancer cell lines [28]. Recently, Brinkmann et al. [29]
suggested that OLA has a great potential to lyse the
membrane of target cells and also induce apoptosis in
cancer cells. The augmentation of the antiproliferative
effect of OLA can be ascribed partly to its unique
structure. The presence of one double bond (monounsat-
urated) at the 9th position in a 18-carbon chain makes it
much less susceptible to oxidation and thereby imparts
higher stability.
Introduction
Dietary constituents are reported to be one of the main
safety factors in the prevention and pathobiology of
cancer [1]. They are now being used effectively as an
alternative to the conventional anticancer drugs. A range
of natural sources like castor oil, olive oil, almonds,
peanuts, red meat, fish oil, etc., contains the n-hydroxy
unsaturated fatty acids (UFA). These fatty acids have
been shown to play an important role in inhibition of
various type of cancer and manifest less side-effects when
compared to standard chemotherapeutic agents [2, 3]. The
UFA are taken up rapidly by tumor cells [4] and their
hydrophobic nature facilitates their rapid incorporation
into the lipid bilayer of cells [5, 6] resulting in disruption
of membrane structure and fluidity [7]. Considering these
characteristics of UFA, they are now being used exoge-
nously to enhance the anticancer activity of various che-
motherapeutic agents [8, 9] and for tumor-targeting drug
delivery.
To achieve effective tumor-specific drug delivery, the
UFA have been chemically modified to enhance their
specificity, potent biological activity and reduction in
toxicity against a range of therapeutic targets [10]. Pre-
viously, we had demonstrated efficient synthesis of n-6
polyunsaturated fatty acid (PUFA)-esters where PUFA
was conjugated with propofol in order to enhance its
activity, lipid-solubility, bioavailability, and decrease its
side-effects [11]. Propofol (diisopropylphenol) is one of
the most popular chemical agents used for induction of
anesthesia and is non-toxic to humans at high concen-
trations (3–8 lg/ml; 20–50 lM) [12]. The presence of
two isopropyl groups at the ortho position with respect to
the hydroxyl group in propofol exerts a steric hindrance
that prevents access of hydrophilic molecules to the
hydroxyl group. This makes the molecule highly hydro-
phobic. Although it is an intravenous sedative-hypnotic
agent, propofol also shows preferential scavenging of
organo-radical species. Clinically relevant concentrations
of propofol are reported to decrease the metastatic
potential of human cancer cells [13] and have been shown
to induce apoptosis involving both extrinsic and intrinsic
pathways [14]. Our preliminary studies showed a notice-
able feature of selective inhibition of cancerous cells by
the novel PUFA-ester conjugates [11]. These results
encouraged the present work, which is directed towards
the synthesis of a new class of n-9 monounsaturated fatty
acid (MUFA)-ester conjugates possessing anticancer
activity.
Ricinoleic acid (RCA; cis-12-hydroxyoctadec-9-enoic
acid), the other MUFA has not yet been investigated for its
role in cancer prevention. In yeast, RCA is a possible
intermediate in the conversion of oleic acid to linoleic acid
[30]. RCA produced through the hydrolysis of castor oil is
a pharmacologically active molecule [31]. Structurally
RCA is similar to OLA, but with a slight difference, where
one hydroxyl group is present at the 12th carbon position.
Unlike other UFA, the presence of an additional hydroxyl
group provides several interesting properties to RCA by
which it can be converted to useful chemical analogs. A
study on the biological action of RCA has shown that it
does not exert any hyper-analgesic effect towards heat and
chemical nociceptive stimuli [32]. These properties render
RCA as a promising analgesic and anti-inflammatory agent
for local application. The surfactant properties of RCA
have been considered to impart its antibacterial activity
[33]. Moreover, its chemical structure has been used as a
basis for the development of novel capsaicin-like com-
pounds [34].
Here, we report the synthesis of four novel n-9 MUFA-
ester derivatives (Scheme 1). The conjugates were spectro-
scopically analyzed to establish their chemical structures,
followed by evaluation of their in-vitro anticancer efficacy.
These conjugates were tested for their ability on apoptosis,
cell migration and cell adhesion.
Amongst UFA, the MUFA, despite being crucial to the
body, have received less attention than n-3 and n-6 PUFA
counterparts. Primarily, MUFA offer major health benefits
123

Lipids
Scheme 1 Schematic
representation of chemical
synthesis of propofol-OLA and
propofol-RCA conjugates
Materials and Methods
EMEM whereas MDA-MB-361, A549, HL-60 and HFL1
were maintained in RPMI medium. To make the complete
growth medium, both types of media were supplemented
with 10 % (v/v) heat-inactivated FCS, 2 mM L-glutamine,
100 U/ml penicillin and 100 lg/ml streptomycin. All cells
were maintained at 37 °C in a 95 % humidified atmosphere
containing 5 % CO2. Cells were screened periodically for
mycoplasma contamination.
Materials
Oleic acid, ricinoleic acid, 2,6-diisopropylphenol, 2,4-diiso-
propylphenol,
N,N-dicyclohexylcarbodiimide
(DCC),
4-dimethylaminopyridine (DMAP), Rosewell Park Memo-
rial Institute (RPMI)-1640 medium, Eagle’s Minimum
Essential Medium (EMEM); 2-[4-(-2 hydroxyethyl)pipera-
zine-1-yl]ethane sulfonic acid (HEPES); phenylmethylsul-
fonyl fluoride (PMSF); EDTA; dithiothreitol (DTT); 3-4,
5-dimethylthiazol-2-yl-2, 5-diphenyl-tetrazolium bromide
(MTT), propidium iodide, sodium dodecyl sulphate (SDS)
were acquired from Sigma-Aldrich. Fetal calf serum (FCS)
was procured from Bio-Whittaker. Thin-layer chromatogra-
phy plates (60A, 0.2 mm thick) and silica gel (60–120 mesh)
were purchased from Fisher Scientific. Dichloromethane
(DCM), methanol, ethanol, chloroform, n-hexane, diethyl
ether, sucrose, KCl, MgCl2, dimethyl sulfoxide (DMSO) and
acetic acid were procured from Merck. Cell migration and
adhesion kits were obtained from Cell Biolabs, Inc.
Synthesis and Purification of Propofol-n-9 Conjugates
A synthesis approach was conceived using the protocol of
Siddiqui et al. [35]. However, several modifications were
incorporated in our lab for synthesis of the final product
[11]. A 1 mmol amount of the respective MUFA was
dissolved in 5 ml dichloromethane (DCM). To this mix-
ture, 0.45 mmol of coupling reagent N,N-dicyclohexyl-
carbodiimide (DCC) was added and the reaction mixture
was stirred for 10 min at room temperature (23–25 °C).
Finally, 1 mmol of one of the propofol isomers (2,6-pro-
pofol/2,4-propofol) was added and the reaction mixture
was esterified in the presence of a catalyst, 0.152 mmol
4-dimethylaminopyridine (DMAP). The reaction mixture
was stirred for a period of 10 h in the dark. The reaction
was stopped by filtration and the filtrate was concentrated
under reduced pressure to yield the product. The progress
of the reaction was monitored on thin layer chromatogra-
phy (TLC) in iodine vapour. Finally, the synthesized
product was purified by silica gel column chromatography
with solvent system n-hexane and diethyl ether (1:1).
Monoclonal antibodies such as anti-caspase-3, anti-
cytochrome c and anti-b-actin were purchased from BD
Biosciences (San Diego, CA). Secondary anti-mouse per-
oxidase-conjugated antibody was from Amersham Phar-
macia Biotech. A chemiluminescence detection kit was
purchased from GE healthcare.
Cell Lines and Culture Conditions
Cell lines SK-MEL-1 (ATCC# HTB-67), HepG2 (ATCC#
HB-8065), MDA-MB-361 (ATCC# HTB-27), A549
(ATCC# CCL-185), HL 60 (ATCC# CCL-240) and non-
cancerous HFL1 (ATCC# CCL-153) were obtained from
the American Type Culture Collection (Rockville, MD).
SK-MEL-1 and HepG2 cell lines were maintained in
2,4-Diisopropylphenol-Oleic Acid (2,4P-OLA)
1
Yield 82 %; H NMR (CDCl3, dH): 0.88 (t, J = 3.76 Hz,
3H), 1.20 (d, J = 6.92 Hz, 12H), 1.25–1.46 (m, 22H), 1.79
123

Lipids
(quin, J = 7.32 Hz, 2H), 2.0 (q, 2H), 2.6 (t, J = 7.64 Hz,
2H), 2.8 (t, J = 6.68 Hz, 1H), 2.9 (m, 1H), 5.3 (m, 2H),
7.14 (d, J = 2.2 Hz, 1H), 7.16 (s, 1H), 7.20 (d, J =
2.88 Hz, 1H); 13C NMR (CDCl3, dC): 14.1, 22.6, 25.06,
25.66, 27.23, 27.53, 29.18–29.63, 31.55, 34.22, 123.87,
126.42, 127.91, 128.12, 130.02, 130.25, 140.32, 145.62,
172.38. FAB-MS: [M]? 442. Anal. calc. for C30H50O2: C,
81.39; H, 11.38. Found: C, 80.93; H, 11.43. IR (KBr,
cm-1): 2926, 2860, 1758, 1460, 1137.
assessed by UV spectroscopy on a UV Mini-1240 spec-
trophotometer. The absorption spectra were measured
between 200 and 600 nm. The infrared spectra of the
conjugates were recorded on a Nikolet-6700 FT-IR
instrument. An aliquot (ten microliter of 1 lg/ll) of the
conjugate was deposited exactly within the cell limit and
scanned as a thin film after evaporation of solvent.
Absorption peaks of synthesized conjugates were observed
at 1757.40/1758.73 (2,6P-OLA/2,4P-OLA) and 1740.07/
1735.00 (2,6P-RCA/2,4P-RCA). The presence of m(C=O),
m(C–O), aromatic C–H (propofol) and aliphatic C–H bond
and absence of hydroxyl (–OH) absorption peak corre-
sponds to the data published previously [11]. The forma-
tion of new conjugates was elucidated with 1H- and
13C-NMR spectra on a BRUKER AVANCE II 400 NMR
spectrometer. The FAB-MS spectra were recorded on a
JEOL SX 102 Mass Spectrometer/Data System using
Argon/Xenon (6 kV, 10 mA) as the FAB gas.
2,6-Diisopropylphenol-Oleic Acid (2,6P-OLA)
1
Yield 85 %; H NMR (CDCl3, dH): 0.88 (t, J = 3.92 Hz,
3H), 1.19–1.43 (m, 34H), 1.76 (m, 2H), 2.0 (m, 2H), 2.57
(t, J = 7.48 Hz, 2H), 2.9 (m, 2H), 5.35 (m, 2H), 6.89
(d, J = 5.96 Hz, 2H), 7.0 (t, J = 6.08 Hz, 1H); 13C NMR
(CDCl3, dC) 14.23, 22.71, 24.31, 24.97, 27.22, 27.50,
29.15–29.79, 33.56, 34.13, 121.90, 124.08, 124.37, 130.06,
130.25, 140.32, 146.52, 172.68. FAB-MS: [M]? 442. Anal.
calc. for C30H50O2: C, 81.39; H, 11.38. Found: C, 80.99; H,
11.47. IR (KBr, cm-1): 2929, 2863, 1757, 1453, 1142, 786,
720.
Analysis of Growth Inhibition of Cancer Cells
The conjugates were examined for their cytotoxicity
against five different types of cancer cell lines viz., SK-
MEL-1 (human skin malignant melanoma), HepG2 (human
liver hepatocellular carcinoma), MDA-MB-361 (human
ductal carcinoma, breast), A549 (human lung carcinoma),
HL-60 (human leukemia, acute promyelocytic) as well as
one non-cancerous HFL1 (human lung fibroblast) using a
standard MTT reduction assay. Cells in exponential growth
were seeded into 96-well plates at a concentration of
5 9 105 cells/200 ll/well and allowed to grow in their
respective medium containing 5 % FCS. After 24 h, cells
were treated with different concentrations of test conjugate
or parent controls (OLA and RCA only/propofol only) at a
concentration range of 0–15 lM. Vehicle control (ethanol
only) and positive control (doxorubicin) cells were cultured
using the same conditions. Following 94 h incubation, the
medium was removed and replaced with fresh medium.
MTT reagent (5 mg/ml in PBS) was added to each well at a
volume of 1:10 and incubated for 2–3 h at 37 °C. After
treatment, 100 ll of DMSO was added to each well after
carefully aspirating the supernatants. Absorbance was
measured at 620 nm in a multi-well plate reader. Triplicate
wells were prepared for each individual concentration.
Dose–response curves were plotted as percentages of the
cell absorbance. Drug sensitivity was expressed in terms of
the concentration of drug required for a 50 % reduction in
cell viability (IC50).
2,4-Diisopropylphenol-Ricinoleic Acid (2,4P-RCA)
1
Yield 84 %; H NMR (CDCl3, dH): 0.88 (t, J = 9.88 Hz,
3H), 1.19–1.48 (m, 30H), 1.77 (m, 2H), 2.07 (q, 2H), 2.23
(t, J = 13.72 Hz, 2H), 2.56 (t, J = 15.0 Hz, 2H), 2.8 (m,
1H), 2.9 (m, 1H), 3.6 (m, 1H), 5.4 (m, 1H), 5.5 (m, 1H),
6.88–6.90 (d, J = 5.76 Hz, 1H), 7.04 (d, J = 8.0 Hz, 1H),
7.12 (s, 1H); 13C NMR (CDCl3, dC): 71.58, 125.24, 133.42,
134.1, 141.02, 150.93, 172.73. FAB-MS: [M]? 458,
[M-17]? 441. Anal. calc. for C30H50O3: C, 78.55; H, 10.99.
Found: C, 78.93; H, 11.23. IR (KBr, cm-1): 2955, 2874,
1735, 1499, 1447, 1252, 1174, 817, 740, 634, 544.
2,6-Diisopropylphenol-Ricinoleic Acid (2,6P-RCA)
1
Yield 86 %; H NMR (CDCl3, dH): 0.80 (t, J = 6.96 Hz,
3H), 1.11–1.40 (m, 30H), 1.68-1.72 (m, 2H), 1.98 (q, 2H), 2.1
(t, J = 13.6 Hz, 2H), 2.54 (t, J = 15.08 Hz, 2H), 2.8
(m, 2H), 3.5 (m, 1H), 5.3 (m, 1H), 5.5 (m, 1H), 6.80–6.82
(d, J = 15.44 Hz, 1H), 6.9 (t, J = 7.6 Hz, 1H); 13C NMR
(CDCl3, dC) 71.52, 120.6, 123.87, 126.42, 133.67, 139.54,
145.61, 172.40. FAB-MS: [M-17]? 441. Anal. calc. for
C30H50O3: C, 78.55; H, 10.99. Found: C, 78.98; H, 11.13. IR
(KBr, cm-1): 2934, 2869, 1749, 1452, 1364, 1171, 820, 733.
Spectral Analysis of Propofol-n-9 Conjugates
Toxicity Analysis of Propofol-n-9 Conjugates
The formation of propofol-n-9 conjugates was confirmed
by various spectroscopic studies. The presence of diiso-
propyl-phenolate in the synthesized conjugates was
Preliminary acute toxicity of conjugates was tested through
erythrocyte lysis test wherein hemoglobin, released as a
123

Lipids
result of membrane leakage or disruption caused by
exposure to high doses of the drug, was measured spec-
trophotometrically [36]. Fresh human blood in heparin was
subjected to centrifugation at 1,000g for 15 min at 4 °C.
Buffy coat as well as plasma was discarded. The washed
erythrocytes were diluted with isotonic buffer (10 mM
phosphate buffer, 150 mM NaCl) and 50 % hematocrit was
prepared. To study the extent of haemolysis, the suspension
of RBCs was incubated with 150 lM (dose that induces
cytotoxicity concentrated ten times) of conjugates, propo-
fol only as well as OLA and RCA only at 37 °C for 1 h.
Ethanol was taken as the vehicle control. After 1 h, the
reaction mixture was centrifuged at 1,500g and the super-
natant was collected and analyzed by UV–Visible spec-
troscopy (kmax = 576 nm) for released hemoglobin.
4–5 times with distilled water. After air drying the wells,
200 ll of extraction solution per well was added and then
incubated for 10 min on an orbital shaker. Then 150 ll
from each extracted sample was transferred to a 96-well
microtiter plate and the absorbance at 560 nm was read in a
plate reader. Absorbance of dye in the control (vehicle-
treated) cells was regarded as 100 % adherence and the
percentage adherence of treated cells was calculated in
comparison with that of the control cells.
Apoptosis Assay
Cancer cells (1 9 107 cells per well) were grown in 6-well
plates in a serum free culture medium (respective to the
cell line used) in a humidified CO2 incubator at 37 °C.
After 24 h, confluent cells were treated with control
(ethanol only) or parent control (OLA/RCA only/propofol
only) or test conjugates and incubated for 24 h again.
Following incubation, cells were harvested and washed
twice in PBS. The cells were suspended in 50 ll of ice-cold
TNN buffer containing 50 mM Tris–HCl pH 7.4, 100 mm
NaCl, 5 mM EDTA, 0.5 % Nonidet P-40, 1 lg/ml pep-
statin, 0.5 mM EGTA, 200 lM PMSF, 0.5 mM DTT and
1 lg/ml of leupeptin and homogenized in a Teflon
homogenizer. A post-nuclear fraction was prepared by
centrifugation for 5 min at 2,000 rpm at 4 °C. The super-
natant was further centrifuged for 20 min at 10,000 rpm at
4 °C and the resultant cytosolic fraction was used for
detecting the expression of two apoptotic factors; cyto-
chrome c and caspase-3.
Analysis of Cancer Cell Migration
Cell migration was performed with CytoSelect 24-Well
Cell Migration Assay (Cell Biolabs, Inc.). Under sterile
conditions, the 24-well migration plate was allowed to
warm up at room temperature for 10 min. The lower well
of the migration plate was supplemented with 500 ll of
EMEM containing 10 % fetal bovine serum with or with-
out (control) test conjugates. To the inside of each insert
100 ll of SK-MEL-1 (0.5–1.0 9 106 cells/ml) cell sus-
pension was added. The plates were incubated for 8 h at
37 °C in a humidified CO2 incubator. After incubation, the
media from inside the inserts was carefully aspirated and
the non-migratory cells were removed using cotton-tipped
swabs. The inserts were transferred to a clean well con-
taining 400 ll of cell stain solution and incubated for
10 min at room temperature. The stained inserts were
gently washed several times and then transferred again to
an empty well. Finally, 200 ll of extraction solution per
well was added and incubated for 10 min on an orbital
shaker. From each sample, 100 ll was taken in a 96-well
microtiter plate and the absorbance at 560 nm was read in a
plate reader.
Western Blotting
Equal amounts of protein in SDS sample buffer were
subjected to 10 % SDS-polyacrylamide gel electrophoresis
[37]. Immunoblotting of resolved proteins was done on a
nitrocellulose membrane. Nonspecific binding on the
nitrocellulose membrane was minimized by blocking for
1 h at room temperature with PBS-T [PBS (pH 7.5) and
0.05 % Tween-20] containing 5 % (w/v) non-fat dry milk.
The treated membrane was washed in PBS-T and then
incubated overnight at 4 °C with specific primary anti-
bodies (monoclonal anti-cytochrome-c or monoclonal anti-
caspase-3) in PBS-T containing 5 % (w/v) BSA. The
membranes were again washed in PBS-T, anti-mouse
peroxidase-conjugated secondary antibodies in PBS-T
were added for 2 h, and immunoreactive bands were
detected by enhanced chemiluminescence detection kit.
Blots were re-probed with an antibody for b-actin to con-
trol for equal protein loading and transfer. Densitometric
values of protein bands were quantified using Alpha Image
Analysis software on Alpha Image Gel Documentation
System.
Analysis of Cancer Cell Adhesion
The cell adhesion assay was performed with a CytoSelect
24-Well Cell Adhesion Assay kit (Cell Biolabs, Inc.).
Under sterile conditions, the ECM adhesion plate was
allowed to warm up at room temperature for 10 min. Then
150 ll of the SK-MEL-1 cell suspension (0.1–1.0 9 106
cells/ml) in serum free media with control or test conju-
gates was added to each well. The plates were incubated
for 30–90 min in a CO2 incubator. The wells were washed
three times with PBS and the adhered cells were stained
with 200 ll of cell stain solution for 10 min at room
temperature. The excess stain was removed by washing
123

Lipids
Protein Determination
respectively. These peaks were shifted to 278 and
279 nm, for 2,4P-RCA and 2,6P-RCA conjugates,
respectively.
Protein content was determined by the BCA method [38].
The mixture of solutions A and B (1:49) of BCA reagent
was added to the protein sample and further incubated at
37 °C for 45 min. The absorbance was measured at
562 nm and the protein concentration was calculated using
a standard curve of BSA.
Nuclear Magnetic Resonance Spectral Studies
Propofol-OLA and propofol-RCA conjugates The forma-
tion of propofol-OLA and propofol-RCA conjugates was
further confirmed with the 1H- and 13C-NMR spectra.
Assignments of the signals are based on the chemical shift
and intensity pattern. The absence of a signal of the
hydroxyl group confirms the synthesis of 2,4P-OLA and
2,6P-OLA (Supplementary Fig. 2a, b, respectively). The
two olefinic protons of 2,4P-OLA and 2,6P-OLA (k and
i) were observed as a multiplet at dH 5.36 while that of
2,4P-RCA and 2,6P-RCA (l and k) were resonating as
multiplet at dH 5.4 ppm and 5.5 ppm respectively (Sup-
plementary Fig. 2c, d) [39]. Due to the proximity of the
hydroxy group to the double bond, the signal of the ole-
finic protons of 2,4P-RCA and 2,6P-RCA were split, with
the downfield signal being assigned to the k proton at
C-10 and the upfield signal assigned to the l proton at C-9
[40]. The –OH proton is exchangeable with deuterium and
might not always be visible as in our case. A triplet was
observed at 0.88 ppm corresponding to the terminal
methyl protons of the fatty acid chain. A conspicuous
multiplet corresponding to the methylene protons of the
fatty acid chain appeared in the range 1.25–1.46 ppm. The
methyl protons of the isopropyl chain of the ring of 2,4P-
OLA were observed as a doublet at 1.20 ppm while the
signal corresponding to methyl protons of a isopropyl
chain of 2,6P-OLA, 2,4P-RCA and 2,6P-RCA was
merged with the methylene protons appearing as a strong
multiplet at 1.19–1.48 ppm range. The methylene groups
(–CH2–CH=CH–CH2–) attached to the olefinic carbons
were resonating as a quartet at 2.00–2.07 ppm (relatively
at lower field) as a consequence of olefinic bond. The
methylene protons –CH = CH–CH2–CH(OH)– of 2,4P-
RCA and 2,6P-RCA were observed at 2.23 ppm as triplet
with a coupling of 13.72 Hz. The –CH2–CH(OH)–CH2–
protons of 2,4P-RCA and 2,6P-RCA resonate at 3.62 ppm
as multiplet, since it is attached to the –OH group,
therefore is shifted downfield from the others. The
–COCH2– protons adjacent to a carbonyl group resonate
in the range 2.50–2.60 ppm, while the –CO–CH2–CH2–
protons were resonating in the range 1.75–1.85 ppm as
a multiplet. In the case of 2,4P-OLA and 2,4P-RCA,
Ar–CH\ gives two signals in the range 2.9–2.6 ppm [12]
while for 2,6P-OLA and 2,6P-RCA, it gives a single
signal at 2.9 ppm [41–43]. The aromatic protons appear in
the usual range 6.80–7.30 ppm.
Statistical Analysis
Results are expressed as the means SD of three experi-
ments for each treatment and were plotted accordingly.
Individual treatments were tested against the control by
using the Student t tests. Significant differences from
controls were considered at P \ 0.05. Analyses were
conducted with SPSS version 13.0.
Results
Chemistry
The two isomers of propofol viz. 2,4-propofol and 2,6-
propofol were conjugated with OLA and RCA separately.
The four novel propofol-n-9 conjugates were obtained
from the esterification of hydroxyl (1C–OH) group of
propofol with the terminal carboxylic group of MUFA in
the presence of DCC and DMAP. The reaction sequences
are outlined in Scheme 1. The newly synthesized ester
derivatives of propofol-OLA and propofol-RCA were
located at specific RF values when subjected to thin layer
chromatography analysis (Supplementary Fig. 1). The RF
values of the products significantly differ from those of
the parent controls viz. OLA/RCA and propofol. The
conjugates were synthesized in good yield and had a
colorless viscous liquid (oily) consistency at room
temperature.
Spectral Characterization of Propofol-n-9 Conjugates
UV Absorbance
Propofol-OLA conjugates The spectra of parent OLA
(Fig. 1a, b) showed an absorption peak at 268 nm whereas,
absorption peaks of 2,4-propofol and 2,6-propofol were
observed at 282.8 and 286 nm, respectively. In 2,4P-OLA
and 2,6P-OLA conjugates, the characteristic peaks
appeared at 263 and 266 nm, respectively.
Propofol-RCA conjugates The spectra of parent RCA
(Fig. 1c, d) showed two absorption peaks at 255 and
270 nm whereas, absorption peaks of 2,4-propofol and
2,6-propofol were observed at 281.2 and 285.3 nm,
In the 13C-NMR spectra, the assignments of the 13C
signal for[C=O group of an ester is quite straightforward
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Lipids
Fig. 1 UV absorbance spectra
of propofol-OLA and propofol-
RCA conjugates. a 2,4P-OLA,
b 2,6P-OLA, c 2,4P-RCA,
d 2,6P-RCA. Absorbance of
parent controls is also shown
and were observed in the range of 172.0–173.0 ppm [41,
43, 44]. The terminal methyl groups gave signals in
14–15 ppm range. Olefinic carbons were recorded at
133–125 ppm [39, 40, 45]. The –CH(OH)– carbon of 2,4P-
RCA and 2,6P-RCA was recorded at 71.58 ppm. Assign-
ment of other significant carbon signals has been shown in
the Supplementary Fig. 2a–d.
of the terminal carbon as a methylene group from the base
peak of propofol [46].
Analysis of Growth Inhibition
Consistent with a previous report [11], the newly synthesized
conjugates significantly inhibited cancer cell growth in a
dose-dependent manner. All tested cells after treatment with
the conjugates showed variable growth inhibitions till a
maximum tested concentration of 15 lM was reached,
making them potent, less potent or equipotent (IC50 values:
Table 1). However, SK-MEL-1 was observed to be the most
sensitive to all the four conjugates (Figs. 2, 3). The conju-
gates showed statistically significant (P \ 0.05) anticancer
activity in comparison with the vehicle control. The in-vitro
evaluation revealed that structural modifications of propofol
and MUFA resulted in conjugates that were non-toxic to
non-cancerous HFL1 cells. This feature imparts extra edge
to the synthesized products and put them into the class
of anticancer agents in terms of selectivity and specific-
ity. Although conjugates of propofol-linoleic acid and
FAB-MS Studies
Propofol-OLA and propofol-RCA conjugates The mass
spectrum and fragmentation pattern of the 2,4P-OLA, 2,6P-
OLA, 2,4P-RCA and 2,6P-RCA are illustrated in Supple-
mentary Fig. 3a–d, respectively. They are typical in that
they have a prominent ion peak at m/z = 163, 177, 264,
265 and 281, which are all fragments about the ester group.
The molecular ion [M ? 3H]? (m/z = 445), [M ? H]?
(m/z = 443) and [M]? (m/z = 458) are easily distin-
guished. The base peak was observed at m/z = 177 with its
isotopic peak at m/z = 178 and represents a cleavage in the
ester group. There is an ion at m/z = 163 representing loss
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HFL1
Table 1 Analysis of cytotoxic effect of propofol-n-9 conjugates in several cancer cell lines
Conjugates
Cell lines (IC50, lM)
HepG2
MDA-MB-361
SK-MEL-1
A549
HL-60
2,4P-OLA
2,6P-OLA
2,4P-RCA
2,6P-RCA
2,4P-LNA
2,6P-LNA
2,4P-ARA
2,6P-ARA
Doxorubicin
3.9
2.8
4.8
4.2
4.1
3.3
3.7
3.1
2.2
3.1
2.6
LA
LA
3.2
2.7
7.5
7.2
2.0
2.5
2.3
4.1
3.9
4.9
4.1
LA
LA
1.9
3.7
3.7
LA
LA
LA
LA
5.3
4.6
3.6
3.4
3.2
7.4
5.4
LA
LA
4.4
3.9
3.1
NA
NA
NA
NA
NA
NA
LA
LA
NA
The highest concentration tested was 15 lM. Values are the means of three observations
NA not active, LA less active, SK-MEL-1 human skin malignant melanoma, HepG2 human liver hepatocellular carcinoma, MDA-MB-361 ductal
breast carcinoma, A549 human lung carcinoma, HL-60 human leukemia, HFL1 human lung fibroblast
Effect of Propofol-n-9 Conjugates on SK-MEL-1
Cancer Cell Adhesion
propofol-arachidonic acid, (previously synthesized in our
laboratory) [11] were also reported to be significantly cyto-
toxic to cancer cell lines (Table 1), the propofol-oleic acid
conjugates synthesized in the present study were also found
to be more potent anticancer molecules.
We also tested the effect of the propofol-OLA and pro-
pofol-RCA conjugates on the adhesion of the skin cancer
cells to a fibronectin surface. While, OLA and RCA or
propofol alone did not significantly affect skin cancer cell
adhesion (Fig. 6), in contrast, propofol-OLA and propofol-
RCA inhibited cell adhesion by *40 % (significant at
P \ 0.05) and 35 %, respectively.
Toxicity Level of Propofol-n-9 Conjugates
The toxicity levels of conjugates were determined by in-vitro
hemolysis testing. Figure 4 displays lysis of erythrocytes by
various conjugates expressed as a percentage of total lysis
(100 % lysis) induced with Triton X-100. The propofol-
OLA conjugates caused negligible RBC lysis as compared to
propofol-RCA conjugates. Among various conjugates 2,6P-
OLA (*8 % hemolysis) and 2,4P-OLA (*10 % hemoly-
sis) imparted less toxicity to erythrocytes than does
2,6P-RCA (*18 % hemolysis) and 2,4P-RCA (*19 %
hemolysis). The intrinsic lytic ability of the propofol only,
OLA and RCA only was also tested. RCA (*21 % hemo-
lysis) caused a higher toxicity than OLA (*6 % hemolysis),
propofol (*17 % hemolysis) and ethanol (*4 % hemoly-
sis) thereby resulting in significant hemolysis of RBCs.
Note: Further in-vitro evaluations of anticancer efficacy
of propofol-n-9 conjugates were done on the SK-MEL-1
skin cancer cell line since it was observed to be more prone
to the cytotoxic effect of conjugates.
Effect of Propofol-n-9 Conjugates on Induction
of Apoptotic Factors
Protein fractions of SK-MEL-1 cancer cells after incuba-
tion with n-9 or propofol alone or with test conjugates
individually were screened by Western blotting after 24 h
of treatment. Propofol-OLA conjugates were able to induce
apoptosis in SK-MEL-1 cells at 15 lM concentration
whereby a significant increase in the expression of cyto-
chrome c and caspase-3 was distinctly visualized (Fig. 7a,
lanes 4 and 5). Whereas, weak signals of cytochrome c and
no signals of caspase-3 were recorded with propofol-RCA
conjugates (data not shown).
Mean densitometric values of individual bands of
cytochrome c and caspase-3 were quantified using image
analysis gel documentation system software. Each value
was mean of three experiments. At 15 lM tested concen-
tration, increased expression of both apoptotic factors was
recorded with slight variations in the activity of 2,4P-OLA
and 2,6P-OLA conjugates. Release of 32.8 and 36.6 %
cytochrome c and expression of 28.7 and 34.1 % caspase-3
was observed after treatment with 2,4P-OLA and
2,6P-OLA, respectively (Fig. 7b, c).
Effect of Propofol-n-9 Conjugates on SK-MEL-1
Cancer Cell Migration
The results shown in Fig. 5 demonstrate that OLA, RCA
and propofol alone had a minimal effect on skin cancer cell
migration. On the other hand, propofol-OLA and propofol-
RCA inhibited cell migration by about 50 % (P \ 0.05)
and 30 % respectively to that of the control group.
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Lipids
Fig. 2 Viability assessment of various cell lines after treatment with propofol-OLA conjugates. The effects of novel esters along with parent
OLA and 2,4-/2,6-propofol are expressed as the means SD of three experiments
Discussion
molecules possess more specific and potent biological
activity, in addition to possibly affecting a wider range of
therapeutic targets [10]. Over the last decade several
attempts have been made to synthesize novel anticancer
compounds by conjugating fatty acids with various anti-
cancer drugs [35, 45, 47–49]. Many of these agents have
Efforts made in medicinal and combinatorial chemistry
continue to give rise to a wide range of anticancer agents
with remarkable therapeutic potential. A number of studies
have concluded that chemically modified fatty acid
123

Lipids
Fig. 3 Viability assessment of various cell lines after treatment with propofol-RCA conjugates. The effects of novel esters along with parent
RCA and 2,4-/2,6-propofol are expressed as the means SD of three experiments
solubility, stability or toxicological issues, which hamper
their development into viable treatment strategies. To
overcome these issues, the administration of antioxidants
such as propofol is speculated to enhance the impact of
chemotherapy. The co-administration of propofol with
chemotherapeutic UFA, is supposed to enhance the
cytotoxicity and inhibit the oxidative damage, thereby
inhibiting the growth of tumors. The cytotoxicity of dif-
ferent propofol-fatty acid esters have been investigated
previously [11, 35, 50]. The present work is based on the
chemical conjugation of MUFA with propofol isomers.
The presence of DMAP (catalyst) and DCC (coupling
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Lipids
Fig. 4 The extent of damage caused to blood erythrocytes by
propofol-n-9 conjugates was measured as the percentage lysis of the
total red blood cells added. The data are the means SD of three sets
of different experiments
Fig. 6 Effect of propofol-n-9 conjugates on adhesion of SK-MEL-1
cancer cells. Results are the means SD for three experiments. The
level of significance from the control (P \ 0.05) is indicated with an
asterisk
When the NMR signals of the conjugates were com-
pared with the parent propofol [35], the hydroxyl proton
signal at about 4.82 ppm in propofol was not visualized in
the spectra of conjugates. The absence of hydroxyl group
1
signal in H-NMR spectra of the conjugates confirms the
synthesis of new product, distinct from parent controls. The
overall shift in signals indicates that the ester bond was
formed at C-19 hydroxyl group of propofol. The molecular
mass of the novel MUFA-ester derivatives determined
by mass spectrometry was very close to the theoretically
calculated molecular mass values viz., 442.47 Da for pro-
pofol-OLA conjugates and 458 Da for propofol-RCA
conjugates.
After establishing the structures of the conjugates, their
inherent toxicity in terms of cell membrane lysis was
determined. The in-vitro erythrocyte lysis test showed that
the newly synthesized esters cause only negligible damage
to RBCs (Fig. 5). The four novel MUFA-esters were also
screened for potential anticancer activities against a range
of human cancer cell lines (Figs. 2, 3). The cytotoxic
efficacy of these conjugates is expressed in terms of IC50
values, as shown in Table 1. Among various esters syn-
thesized, propofol-OLA conjugates showed greater in-vitro
anticancer activity with IC50 values ranging from 2.3 lM
for SK-MEL-1 cells to 3.9 lM for HepG2 cells. Propofol-
RCA conjugates however, moderately inhibited various
cancer cells and showed negligible inhibition against
MDA-MB-361 and A549 (IC50 values ranging from
3.9 lM for SK-MEL-1 cells to 7.4 lM for HL-60 cells).
The propofol-n-9 conjugates were non-toxic to the normal
HFL1 cells up to the highest concentration screened
(15 lM).
Fig. 5 Effect of propofol-n-9 conjugates on migration of SK-MEL-1
cancer cells. Results are the means SD for three experiments. The
level of significance for various data when compared to control
(P \ 0.05) is indicated with an asterisk
reagent) was a pre-requisite for esterification of novel
conjugates. The use of the DCC/DMAP method was found
to be a more suitable synthetic route under milder reaction
conditions, giving a higher yield and providing convenient
product purification strategies. The chemical nature of the
four novel MUFA-esters reported in this study show
absorption peaks in the ultraviolet range, which were quite
distinct from the reactant parent controls (Fig. 1). The
presence of an ester bond, aromatic C–H bond, and absence
of the free –OH group as visualized by FT-IR, confirmed
the formation of conjugates. Elemental analysis of conju-
gates showed the values for ‘CHN’ within 0.4 % of the
theoretical ones.
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Lipids
Fig. 7 a Immunoblots showing relative distribution of cytochrome c,
caspase-3 in SK-MEL-1 (human skin malignant melanoma) cells after
treatment with (1) control; (2) oleic acid; (3) propofol; (4) 2,4P-OLA;
(5) 2,6P-OLA. b-actin expression was manifested as loading control.
b, c Densitographs showing relative density of cytochrome c and
caspase-3 respectively, in the nitrocellulose blots. Results are mean
values SD in triplicates
Since, the conjugates revealed optimum cytotoxicity
against SK-MEL-1 cells; we used this cell line for further
studies. Cell migration and adhesion are essential pro-
cesses in tumor metastasis [51]. In the present study,
cell migration and cell adhesion assay revealed that the
conjugates were able to inhibit the metastatic potential of
SK-MEL-1 cells (Figs. 5, 6) where parent controls were
not effective. Selective apoptosis of malignant cells is the
most commonly analyzed attribute in chemotherapeutic
studies. Modulation of expression of apoptotic factors
must be tightly regulated for normal cellular functioning.
Therefore, we also analyzed the effect of the conjugates
on the induction of apoptosis by assaying for caspase-3
cell death at doses of 15 lM. Our current findings, thus
demonstrate that propofol-OLA conjugates can induce the
apoptosis by induction of a novel pathway through which
n-9 MUFA putatively modulate the aggressive behavior
of skin cancer.
Conclusion
The present study has shown the synthesis of four novel
propofol-n-9 ester conjugates revealing unique cytotoxic
action against various cancer cell lines. The conjugates are
able to inhibit the growth of cancer cells in a dose-
dependent manner. Positive results in the inhibition of
cancer cell migration and adhesion further established their
anticancer potential. Although, propofol-RCA conjugates
are observed to be weak candidates in initiating apoptosis,
the propofol-OLA conjugates induce a cell signaling
pathway, that eventually leads to apoptotic cell death in
skin cancer cells. Our preliminary data strongly suggest
that the synthesized conjugates possess potent anticancer
activity. However, the exact mechanism by which these
conjugates modulate the apoptotic pathway in cancer cells
activation and cytochrome
c release (Fig. 7). The
2,6P-OLA conjugate showed increased expression of both
cytochrome c (36.6 %) and caspase-3 (34.1 %) in com-
parison with 2,4P-OLA (Fig. 7b, c). On the other hand,
very weak signals for cytochrome c were detected when
SK-MEL-1 cells were treated with propofol-RCA conju-
gates (data not shown). Interestingly, no signal of cas-
pase-3 was recorded upon treatment of SK-MEL-1 cells
with propofol-RCA conjugates. Thus, among the four
conjugates, propofol-OLA conjugates promote apoptotic
123

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propofol inhibits invasion of cancer cells. Cancer Lett
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further investigation.
Acknowledgments We are thankful to Prof. M. Saleemuddin,
Co-ordinator, Interdisciplinary Biotechnology Unit, Aligarh Muslim
University, Aligarh, India for providing the central instrumentation
facility to carry out the experiments. We also thank the Sophisticated
Analytical Instrumentation Facility (SAIF) of the Central Drug
Research Institute, Lucknow, India for providing the NMR and mass
spectra facility. The study was conducted with the financial support
given to Mr. Azmat Ali Khan by the University Grants Commission
(India).
Conflict of interest All authors declare that there is no conflict of
interest.
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