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Cancer Investigation
ISSN: 0735-7907 (Print) 1532-4192 (Online) Journal homepage: http://www.tandfonline.com/loi/icnv20
Divergent Anticancer Activity of Free and
Formulated Camel Milk α-Lactalbumin
Vladimir N. Uversky, Esmail M. El-Fakharany, Marwa M. Abu-Serie, Hussein
A. Almehdar & Elrashdy M. Redwan
To cite this article: Vladimir N. Uversky, Esmail M. El-Fakharany, Marwa M. Abu-Serie, Hussein
A. Almehdar & Elrashdy M. Redwan (2017): Divergent Anticancer Activity of Free and Formulated
Camel Milk α-Lactalbumin, Cancer Investigation, DOI: 10.1080/07357907.2017.1373783
To link to this article: http://dx.doi.org/10.1080/07357907.2017.1373783
Published online: 26 Sep 2017.
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CANCER INVESTIGATION
, VOL. , NO. , –
https://doi.org/./..
Divergent Anticancer Activity of Free and Formulated Camel Milk α-Lactalbumin
Vladimir N. Uversky a,b,c, Esmail M. El-Fakharany d, Marwa M. Abu-Seriee, Hussein A. Almehdara,
and Elrashdy M. Redwana,d
aDepartment of Biological Sciences, Faculty of Sciences, King Abdulaziz University, Jeddah, Saudi Arabia; bInstitute for Biological
Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow region, Russia; cDepartment of Molecular Medicine and USF Health
Byrd Alzheimer’s Research Institute, Morsani College of Medicine, University of South Florida, Tampa, FL, USA; dTherapeutic and Protective
Proteins Laboratory, Protein Research Department, Medical Biotechnology Department, Genetic Engineering and Biotechnology Research
Institute, City for Scientific Research and Technology Applications (SRTA-City), Alexandria, Egypt; eMedical Biotechnology Department, Genetic
Engineering and Biotechnology Research Institute, City for Scientific Research and Technology Applications (SRTA-City), Alexandria, Egypt
ARTICLE HISTORY
Received January
Revised August
Accepted August
KEYWORDS
Camel milk; Milk protein;
α-Lactalbumin; Anticancer
activity
ABSTRACT
Alpha-lactalbumin (α-LA), a small milk calcium-binding globular protein, is known to possess notice-
able anticancer activity, which is determined by the ability of this protein to form complexes with
oleic acid (OA). To date, in addition to human and bovine α-LA, the ability to form such anti-tumor
complexes with OA was described for goat and camel α-LA. Although the mechanisms of the anti-
cancer activity of human and bovine α-LA are already well-studied, little is currently known about the
anticancer action of this camel protein. The goal of this study was to ll this gap and to analyze the
anticancer and pro-apoptotic activities of camel α-LA in its free form (α-cLA) and as an OA-containing
complex (OA-α-cLA) using four human cancer cell lines, including Caco-2 colon cancer cells, PC-3
prostate cancer cells, HepG-2 hepatoma cells, and MCF-7 breast cancer cells as targets. The anti-tumor
activities of OA-α-cLA and α-cLA were analyzed using MTT test, annexin/PI staining, cell cycle anal-
ysis, nuclear staining, and tyrosine kinase (TK) inhibition methods. We show here that the OA-α-cLA
complex does not aect normal cells but has noticeable anti-cancer activity, especially against MCF-7
cells, thus boosting the anticancer activity of α-cLA and improving the selectivity of OA. The OA-α-cLA
complex mediated cancer cell death via selective induction of apoptosis and cell-cycle arrest at lower
IC50 than that of free α-cLA by more than two folds. However, OA induced apoptosis at higher extent
than OA-α-cLA and α-cLA. OA also caused unselective apoptosis-dependent cell death in both nor-
mal and cancer cells to a similar degree. The apoptosis and cell-cycle arresting eect of OA-α-cLA may
be attributed to the TK inhibition activity of OA. Therefore, OA-α-cLA serves as ecient anticancer
complex with two functional components, α-cLA and OA, possessing dierent activities. This study
declared the eectiveness of OA-α-cLA complex as a promising entity with anticancer activity, and
these formulated OA-camel protein complexes constitute an auspicious approach for cancer remedy,
particularly for breast cancer.
Introduction
Recent research revealed that, in addition to their tra-
ditional biological roles (e.g., catalysis of various reac-
tions), various milk proteins and peptides can possess
multiple unrelated functions, exerting various moon-
lighting activities, such as anti-microbial, antiviral, and
antifungal activities, immuno-modulatory functions,
as well as playing roles in cancer cell apoptosis, anti-
metastatic activities, and antioxidant eects (1).Sev-
eral milk-derived biologics, such as HAMLET (human
CONTACT Elrashdy M. Redwan redwan@yahoo.com Therapeutic and Protective Proteins Laboratory, Protein Research Department, Medical Biotech-
nology Department, Genetic Engineering and Biotechnology Research Institute, City for Scientific Research and Technology Applications (SRTA-City), New Borg
EL-Arab , Alexandria, Egypt; Vladimir N. Uversky vuversky@health.usf.edu Department of Biological Sciences, Faculty of Sciences, King Abdulaziz
University, P.O. Box , Jeddah, Saudi Arabia.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/icnv.
α-lactalbumin [α-LA] made lethal to tumor cells),
demonstrated some promising results in clinical trials
(2). The attractiveness of using milk-derived biologics
as potential drugs relies on the important fact that pro-
teins and peptides are well-tolerated by the organism
and exhibit oral bioavailability. This also suggests that
milk proteins and peptides may complement standard
therapies to boost overall success in cancer treatments
(1). Historically, the journey toward the understanding
of the molecular basis of the anticancer activity of milk
© Taylor & FrancisGroup, LLC
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2V. N. UVERSKY ET AL.
components has started from the identication of a
complex of α-LAandoleicacid(OA)inhumanmilk
(2–5). HAMLET was characterized by a highly selec-
tive apoptotic activity against tumor cells both in vivo
(6–10) and in clinical trials (11–13).Sinceα-LA is
a highly conserved milk protein, it was interesting
to investigate anticancer activities that were ascribed
to the oleic acid-bound forms of α-LAs from other
species,suchasbovine,camel,andgoat,givingraiseto
BAMLET, CAMLET, and GAMLET, respectively (14–
18).Fragmentsofbovineα-LAobtainedbylimited
proteolysis can also bind OA and exhibit anti-tumor
activity (19).
For human and bovine α-LAs as well as for
some other proteins forming HAMLET-like com-
plexes with OA, the molecular mechanisms of HAM-
LET/BAMLET formation are well-understood (18).In
fact, the formation of the molten globule-like inter-
mediate (which can be achieved by the removal of
Ca2+from calcium-binding proteins, moderate heat-
ing, acidic or alkaline pH, etc.) seems to be an impor-
tant prerequisite for the eective interaction of a target
protein with OA, and for the formation of HAMLET-
like complexes (18). Therefore, the formation of the
HAMLET-like complexes by milk and some other pro-
teins involves the OA-induced formation of alterna-
tively folded conformations with unexpected benecial
functions (20).
In a recent study, we conducted a systematic com-
parative analysis of structural properties and con-
formational stabilities of bovine and camel α-LAs
usingasetofspectroscopicandcomputationaltech-
niques (21). These analyses revealed the existence
of several noticeable dierences between these two
proteins, with camel α-LA being more stable toward
thermal and pH-mediated denaturation but less stable
toward guanidine hydrochloride-mediated unfolding
(21). Furthermore, the camel α-LAwasshowntobe
more disordered and possessed stronger aggregation
propensities (21). Although noticeable anticancer and
pro-apoptotic activity was reported for some camel
milk proteins such as α-LA (15) and casein (22),the
mechanisms of the anti-cancer potential of camel
α-LA are poorly understood. The goals of the current
study were to analyze the anticancer activity of camel
α-LA and to validate our hypotheses that, similar to
other milk proteins from dierent species, camel α-LA
is able to form complexes with OA with cytotoxic
potential, and that the anticancer and pro-apoptotic
activities of camel milk α-LA are OA-dependent.
We also conducted a control study assaying the anti-
cancer and pro-apoptotic activities of camel α-LA
or OA alone. The anticancer activities of puried
protein and prepared OA-containing complexes were
assayed using dierent human cancer cell lines as
targets.
Material and methods
Purication of camel α-lactalbumin
Camel skimmed milk was prepared according to the
procedure described in Almahdy et al. (22).Camel
α-lactalbumin (α-cLA) was puried from skimmed
milk according to method of Pettersson et al. (14)
with some modications. In brief, the camel skimmed
milk was exposed to decreased pH to reach the
isoelectric point (pI) of casein to cause precipita-
tion of this protein by adding 1 M HCl to milk
until the pH became 4.6. The casein-free prepara-
tion was treated with ammonium sulfate at concen-
tration of 45%, and after incubation for 24 h at 4°C,
the mixture was centrifuged at 6000 ×gfor 20 min.
The supernatant was collected and dialysed against
20 mM phosphate buer and 35 mM EDTA, pH
7.6. Proteins precipitated from skimmed milk in 45%
ammonium sulfate showed 7 bands on 12% SDS-
PAG E ( Figure 1), and this proteins mixture contained
α-cLA.
Thesamplecontainingα-cLA was applied to the
DEAE–cellulose column equilibrated with 50 mM
phosphate buer, pH 7.6. Both camel albumin (a
protein with a molecular weight of ∼67.0 kDa) and
α-cLA (a protein with a molecular weight of 14.4 kDa)
were eluted by a DEAE-cellulose column using gra-
dient 0.0–0.5 M NaCl. After dialysis of the eluted
samples, the fractions containing α-cLA were applied
into Sephadex-G50 gel ltration column equilibrated
with the same buer to separate α-cLA from albumin.
Homogeneity and purity of α-cLA was estimated by
SDS-PAGE. On 12% SDS-PAGE, puried camel α-LA
wascharacterizedbyasinglebandwithanexpected
molecular weight of 14.4 kDa (as shown in Figure 1).
The fractions contained puried α-cLA were pooled,
lyophilized, and kept at −80°C until use. The protein
concentration of α-cLA was estimated spectrophoto-
metrically at 280 nm using an extension coecient of
ε280 nm =32,470 M−1cm−1(23).Camelα-cLA was
standardized spectroscopicaly as described previously
by Redington et al. (21).
Downloaded by [Professor Vladimir N. Uversky] at 16:39 26 September 2017
CANCER INVESTIGATION 3
Figure . % SDS-PAGE of purified camel α-LA during purifica-
tion steps. Lane , molecular mass marker; lane , skimmed camel
milk after precipitation by % ammonium sulfate; lane , eluted
fractions from DEAE–cellulose column; lane , purified α-LA from
Sephadex G column.
Formation of α-LA-oleic acid complex
Lyophilized α-cLA was used for the preparation of the
OA-α-cLA complex by gentle heating. Here, native
α-cLA was dissolved in PBS at a concentration of
120 μM,andoleicacidwasaddeddirectlytothepro-
tein solution at the 50 molar equivalents (OA:α-LA=
50:1). The reaction mixture was incubated for 20 min
at 45°C after vortexing for 30 s. To remove unbound
OA, the mixture was centrifuged at 10,000 rpm fol-
lowed by ultraltration using the Centricone (3000 Da
cut-o membrane) (24).TheamountofOAboundto
α-cLA was estimated to be 318 μM.
Oleic acid determination
ConcentrationofadsorbedOAinproteinsampleswas
determined according to the colorimetric method of
Duncombe (25). In brief, the protein samples were
shaken with chloroform and copper nitrate solution,
OAwasremovedfromitscomplexwithα-LA and
formed copper-fatty acid soap which is soluble in chlo-
roform. The amount of copper in chloroform is equiv-
alent to the amount of oleic acid in the sample that is
assayed by addition of sodium diethylthiocarbamate as
a color developer. Here, 2.5 ml of the copper reagent
(nine volumes of 1 M triethanolamine; one volume of
1 N acetic acid and ten volumes of 10 g% copper sulfate)
were added to 500 μl samples and/or 500 μlstandard
oleic acid. All tubes were then shaken vigorously with
a vortex mixer, after which 5 ml of chloroform were
addedtothesolutionandshakenvigorouslyfor1min.
Then, 3 ml of the lower layer were carefully transferred
to another test tube containing 500 μlof0.1%sodium
diethylthiocarbamate in butanol and absorbencies of a
samples (Asam) and the standard (Ast) were read at
440 nm.
Endotoxin determination
The endotoxin levels in the camel milk, puried α-
cLA, and prepared compounds were tested to avoid its
cytotoxic eects on the cell lines. To this end, protein
samples and solutions of other compounds were mixed
with an equal volume of sample buer containing
20% glycerol or 20% sucrose, β-mercatoethanol, 1.5%
sodium deoxycholate (DOC), and 4 M urea. The
samples were boiled for 5 min at 100°C, then loaded
into the gel and separated at 20 mA/gel. The gels were
then transferred to the clean polyethylene containers
and stained according to the established protocol (26).
First, to remove the ions of the running buer, the gels
were washed with water for 5 min. Then, the gels were
xed in 10 volumes of 45% ethanol or methanol (ana-
lytical grade, Sigma-Aldrich, St. Louis, MO, USA) and
in 0.5% periodic acid for 15 min. Second, the gels were
additionally washed ve times over 5 min with 200 mL
water/wash, since washing less than three times gave a
yellow-brown color after the silver solution addition.
Then, aqueous silver nitrate (0.1%) was added, and gels
were for incubated for 15 min. Next, the stained gels
were rinsed in deionized water for 1 min, and then in
1 volume of developer (100 mL of 37% formaldehyde,
3% sodium carbonate) for just 30–40 s to remove
the silver nitrate ions from the gels surfaces. Then,
thegelsweresoakedin5volumesofdeveloperuntil
the desired color of bands was obtained (∼5 min).
Finally, the developed gels were soaked in water for
5 min and then overnight in 40% ethanol. Application
of this method, which can accurately detect less than
1 ng of bacterial lipopolysaccharide (26),revealedthat
all used proteins and prepared compounds were found
to be free from endotoxin.
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4V. N. UVERSKY ET AL.
Cytotoxicity assay against normal mammalian
epithelial cell line
The hydrogen acceptor 3-(4,5-dimethylthiazol-2-yl)-
2,5 diphenyltetrazolium bromide (MTT) assay is a
rapid and highly accurate colorimetric approach that
is widely used to determine cell metabolic activity in
order to evaluate cytotoxicity of various compounds
(including new drugs). Here, mitochondrial dehydro-
genases of viable cells cleave the tetrazolium ring and
then yield purple MTT formazan crystals. Therefore,
the optical density values of dissolved crystals after
treatment are proportional to the number of viable
cells (27, 28). The eect of samples containing query
compounds on the capability of cells to replicate is
used as an index of compound toxicity. In this study,
the concentration of the compound (e.g., the OA-α-
LA complex) at which 50 percent of the cells do not
multiply is called IC50 (the median inhibitory dose).
Vero (African green monkey kidney normal) cell line
(Sigma-Aldrich, USA) was cultured and seeded in a
DMEM medium (Lonza, USA) supplemented with
10% fetal bovine serum (FBS) as 5 ×103cells per
well (96-well cell culture plate). After 24 h incuba-
tion for cell attachment in 5% CO2incubator, dierent
concentrations of α-LA and OA-α-LA ranging from
25 μM to 100 μM were added. In addition, OA and
5-uorouracil (5-FU, which is a medication used in
the treatment of cancer) were added at dierent con-
centrations as positive control (for investigation the
cytotoxic eect of the fatty acid alone, OA was dis-
solved in ethanol and diluted in PBS, pH7.2). After 72 h
incubation in 5% CO2incubator, 200 μl of 0.5 mg/ml
MTT (Sigma-Aldrich) was dissolved in PBS, added to
each well, and the plate was incubated at 37°Cfor3h.
Then MTT solution was removed, 200 μlDMSOwas
added, and the absorbance of each well was measured
with a microplate reader (BMG LabTech, Germany)
at 570 nm. The half maximal inhibitory concentration
(IC50)andsafedose(EC
100)valuesofOA-α-LA were
analyzed by the Graphpad Instat software. Untreated
cellswereincludedasnegativecontrolandeachtested
sample was performed in triplicate.
Anti-cancer eect of the α-cLA-OA complexes
Cytotoxicity assay against human cancer cell lines
using MTT assay
The anti-cancer eect of OA-α-cLA was assayed
using four human cancer cell lines. Colon cancer cell
line (Caco-2) and prostate cancer cell line (PC-3) were
maintained as adherent cell cultures in DMEM (Lonza,
USA) containing 10% FBS (Gibco, Life Technologies,
Grand Island, NY, USA) while hepatoma cell line
(HepG-2) and breast cancer cell line (MCF-7) were
cultured in RPMI-1640 (Lonza, USA) supplemented
with 10% FBS. All cancer cell suspensions (2.5 ×103
cells/well) were seeded into sterile 96-well plates and
allowed to attach for 24 h. Then serial concentrations
of α-LA, OA-α-LA, OA, and 5-FU were added to
four cancer cell lines, and the plates were incubated
at 37°Cina5%CO
2incubator. After incubation
for 72 h, the sensitivity of tumor cells to OA-α-LA
was evaluated using MTT assay, as described above.
Moreover the anti-cancer activity of OA-α-LA was
investigated by phase contrast microscope in com-
parison with untreated cells as well as α- LA-, OA-,
and 5-FU-treated cells. The IC50 values was calculated
using Graphpad Instat software and the selectivity
index (SI) that was dened as the ratio of the IC50 on
normal mammalian cells (Vero) versus four cancer cell
lines was also calculated.
Flow cytometry analysis of cell death
Human cancer cell lines (HepG2, Caco-2, MCF-7, and
PC3) and normal mammalian cells (Vero) were treated
with IC50 concentration of α-cLA and OA-α-cLA for
72 h in control with IC50 of OA and 5-FU. The cells were
harvested by trypsinization, washed three time with
cold PBS, and resuspended in 200 μlof1×PBS buer.
Then, the cells were incubated with 5 μlofannexin
V-biotin (Molecular ProbesTM,USA)and5μlofPIfor
15 min in the dark. After staining, the cells were washed
with 1 ×PBS and xed with 4% paraformaldehyde in
PBS for 10 min. After washing twice with cold PBS,
5μg/ml of streptavidine-uorescein (Sigma-Aldrich)
was added to the cells for 15 min; the cells were then
centrifuged and resuspended in PBS. The cell death
ratesweredetectedbyowcytometry(Ex=488 nm;
Em =530 nm) using FITC a signal detector (FL1)
and PI staining by a phycoerythrin emission signal
detector (FL2).
Flow cytometry analysis of the cell cycle
The cell cycle of untreated and treated cancer cells was
analyzed by ow cytometry according to the method
of Jass et al. (29). After treatment of cancer cells with
α-cLA and OA-α-cLA at IC50 concentration as
described above, about 1.0 ×106cells were de-attached
Downloaded by [Professor Vladimir N. Uversky] at 16:39 26 September 2017
CANCER INVESTIGATION 5
and suspended in 300 μlofPBS.Then,thecellswere
centrifuged at 1200 rpm at 4°Cfor5min,followedby
xation using the drop-wise addition of 700 μlofcold
ethanol (70%) to each tube while vortexing gently. The
cells can be left at 4°C (up to a few days) after xation.
The cells were centrifuged and washed three time with
cold PBS, resuspended in 250 μl of PBS containing
5μg/ml RNase A (Sigma-Aldrich), and incubated at
37°Cfor1h.Then10μl of 1 mg/ml PI (Sigma-Aldrich)
in deionized water was added and kept in the dark at
4°C until analysis on FACS (Partec, Germany) by read-
ing at 488 nm using Cell Quist and Mod Fit software.
Nuclear staining analysis of anti-cancer effect
The anti-cancer eect of OA-α-cLA was assayed by
uorescent nuclear dye PI (30, 31).Caco-2cellswere
seeded in 12 well plates and treated with OA-α-cLA
for 1 day under suitable conditions as described above.
Thetreatedcellswerewashedthreetimeswithcold
1.0 ×PBS, and xed with cold 4% paraformalde-
hyde for 10 min. After the cells permeabilization with
3% paraformaldehyde and 0.5% Triton X-100, the
cells were stained with PI dye (10 μg/ml). The cells
were viewed and counted after 15 min with a uo-
rescence inverted microscope with an excitation lter
(480/30 nm) and all tested samples were done in tripli-
cate. Untreated cells were involved as negative control
cells,andthecellshavingcondensedandfragmented
nuclei were considered apoptotic.
Tyrosine kinase (TK)–inhibition assay
The antibody BeaconTM TK assay kit (Molecular
Probes, USA) provides a simple and robust assay for
measuring the activity of TK and their inhibitors
and modulators. According to the instructions from
the manufacturer of the corresponding analytical kit,
12.5 μlofserialdilutionsofα-cLA, OA-α-cLA,OA,
and 5-FU were incubated with 12.5 μlofTK(25U/ml
in reaction buer) for 20 min at 37°C. The Antibody
Beacon TK detection complex (62.5 nM Oregon Green
488 ligand and 125 nM antiphosphotyrosine antibody),
500 μg/ml kinase substrate (poly(Glu:Tyr), 4:1), and
5μlof5mMATPwerethenaddedtoeachwell,
andthereactionswereincubatedat37°C. After 1 h,
the quenched uorescence was measured in a uo-
rescence microplate reader (BMG LabTech, Germany)
using excitation at 485 nm and emission at 535 nm for
estimation of TKi (the concentration required to cause
half maximum inhibition of enzyme activity).
Statistical analysis
Data were expressed as mean ±standard error of
the mean (SEM) for three measurements by the mul-
tiple comparisons Fisher’s least signicant dierence
(LSD)posthoctestoftheonewayanalysisofvari-
ance (ANOVA) using the SPSS16 software program,
with probability (p)-values <0.05 considered statisti-
cally signicant.
Results
In vitro cytotoxicity eects of OA-α-cLA complex on
mammalian Vero cells
We evaluated the viability of Vero cells after treatment
with α-cLA, OA, OA-α-cLA, and uorouracil (5-FU).
The results of these analyses are given in Table 1 ,which
clearly shows that the safe dose (EC100)fortheα-cLA
protein and OA-α-cLA complex were signicantly
(p<0.001) higher than the corresponding values
determined for OA and 5-FU. Furthermore, for OA
and 5-FU, the viability of Vero cells was decreased to
50 percent (IC50)atconcentrationslessthan18μM,
respectively, whereas for α-cLA and OA-α-cLA, the
IC50 values were 288.9 ±1.9 μM and 170.2 ±9.6 μM,
respectively (shown in Tab l e 1). These ndings indi-
cated a highly signicant (p<0.001) increase (by more
than 19 folds) in the safety of using the OA-α-cLA com-
plex instead of OA alone toward normal Vero cells.
In vitro cytotoxicity of OA-α-cLA complex on dierent
cancer cell lines
In vitro cytotoxicity of OA-α-cLA complex was inves-
tigated using dierent human cancer cell lines after
treatment for 72 h and using treatment with α-cLA,
OA, and 5-FU as a control. The cell viability and levels
of the compound cytotoxicity were estimated by the
MTT method. All tested cancer cell lines were found
Tab le . IC (µM) and EC (µM) values for effect of camel α-LA
and OA- α-LA on normal epithelial cell line (Vero) in control with
OA and -FU.
Sample EC IC
α-LA ±.a. ±.a
OA- α-LA . ±.b. ±.b
OA . ±.c. ±. c
-FU . ±.d. ±. d
All values were expressed as mean±SEM. Different letters are significantly dif-
ferent within the same column at p<..
Downloaded by [Professor Vladimir N. Uversky] at 16:39 26 September 2017
6V. N. UVERSKY ET AL.
Tab le . IC (µM) and SI values for cytotoxic effect of camel α-LA and OA-α-LA complex against Caco-, HepG-, PC and MCF- cell lines
in control with OA and -FU.
Caco- HepG- PC- MCF-
IC SI IC SI IC SI IC SI
α-LA . ±.a. ±.a. ±.a. ±.a. ±a. ±. a. ±.a. ±. a
OA- α-LA . ±.b. ±.a. ±.b. ±.b. ±.b. ±. b. ±. b. ±. b
OA . ±.c. ±.b. ±.c. ±.c. ±.c. ±.c. ±. c. ±. c
-FU . ±.d. ±.b. ±.d. ±.c. ±.d. ±. c. ±. d. ±. c
All values were expressed as mean ±SEM. Different letters are significantly different within the same column at p<..
to be sensitive to OA-α-cLA complex at low concen-
trations, whereas free α-cLA showed cytotoxicity at
high concentrations (p<0.01). Tabl e 2 represents
the concentrations of OA-α-cLA complex and α-cLA
alone that were required to kill or reach 50% inhibition
the cells were varied according to the type of the cancer
cells. The MCF-7 cells were the most sensitive to the
treatment with OA-α-cLA, with the IC50 of 45.9 μM
and with substrate inhibition (SI) of 3.7, followed by
the HepG-2 cells, with IC50 of 70.5 μMandSIof
2.41, whereas the PC-3 and Caco-2 cells were the least
sensitive to the OA-α-cLA treatment, showing the IC50
value >80 μM and SI of 2 and 1.45, respectively. Free
α-cLA had a cytotoxic eect on all tested cancer cells
at high concentrations, with the IC50 values ranging
from 118.4 to 254.8 μMandtheSIvaluesrangingfrom
1.18 ±0.22 to 2.5 ±0.1. Our results revealed that the
oleic acid and 5-FU showed the highest cytotoxicity
against all tested cells including normal and cancer
cells, being characterized by the lowest IC50 values
(>66 and 20 μM, respectively) and the lowest SI values
below 0.85.
Figure 2 represents the results of the comparative
morphological analysis of human cancer cells of four
dierent cell lines untreated and treated with the IC50
concentrations of α-cLA, OA-α-cLA complex, OA, and
Figure . Morphological changes of human cancer cell lines (І) Caco- cells, (ІІ) HepG- cells, (ІІІ)PC-and(ІV) MCF- cells. (A) Untreated
cells, (B) cells after treatment with α- LA, (C) OA-α-LA, (D) OA, and (E) -FU (magnification ×).
Downloaded by [Professor Vladimir N. Uversky] at 16:39 26 September 2017
CANCER INVESTIGATION 7
Figure . Annexin/PI staining of cell lines (І) Caco- cells, (ІІ) HepG- cells, (ІІІ) PC-, (ІV) MCF- cells and (V) Vero cells. (A) Untreated cells,
cells after treatment with (B) α-LA, (C) OA-α-LA, (D) OA, and (E) -FU.
5-FU. These photomicrographs show that the mor-
phologies of the cancer cells were extremely altered as
aresultofthetreatmentwiththeOA-α-cLA complex,
OA-, or 5-FU, in comparison with the untreated cells
or free α-cLA-treated cells. Treatment with the OA-α-
cLA complex caused cells to become smaller in size,
and their cytoplasm to become denser, perhaps due to
cell apoptosis.
Apoptotic eect of OA-α-cLA complex assayed by
ow cytometry
The apoptosis-dependent anticancer activity of OA-
α-cLA complex at IC50 concentration was evaluated
using a double staining annexin-V/PI method using α-
cLA, OA, and 5-FU at IC50 concentrations as controls.
Figure 3 and Table 3 show that the OA-α-cLA complex
has the ability to induce apoptosis in all tested cancer
cell lines. At IC50 concentration, MCF-7 cells were the
most sensitive to OA-α-cLA complex, which induced
early apoptosis in 65.9% and late apoptosis in 8.2%
of the cells, followed by HepG-2 cells, in which early
and late apoptosis was induced in 45.1% and 10.7% of
thecells,respectively.Thepercentageoflateapoptotic
PC-3 cells (28%) was two-fold higher than the percent
of early apoptotic PC-3 cells after treatment with OA-
α-cLA. The colon cancer cells (Caco-2) were the least
sensitive to the treatment with OA-α-cLA complex,
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8V. N. UVERSKY ET AL.
Tab le . Annexin/PI analysis of samples-induced apoptosis in Caco-, HepG-, PC- and MCF- cells as well as normal cell line (Vero).
Caco- HepG- PC- MCF- Vero
Cells
Apoptosis (%) Early Late Early Late Early Late Early Late Early Late
Untreated control . ±.a. ±.a. ±.a. ±.a. ±. a. ±. a. ±. a. ±. a. ±.a. ±.a
α-LA . ±.b,e . ±.b. ±.b. ±. b. ±.b. ±. b. ±.b. ±.b. ±.a. ±.b
OA-α-LA . ±. c. ±.c. ±.b. ±.c. ±.c. ±.c. ±.c. ±.c. ±.a. ±.c
OA . ±.d. ±.d. ±.a. ±.d. ±.d. ±.d. ±.d. ±.d. ±.b. ±.d
-FU . ±.e. ±.e. ±.c. ±.e. ±.e. ±.e. ±.e. ±.e. ±. c. ±.e
All values were expressed as mean ±SEM. Different letters are significantly different within the same column at p<..
since early and late apoptosis was induced in 27.8 and
3% of the cells, respectively. Furthermore, free α-cLA,
being present at IC50 concentrations, was able to induce
apoptosis in all cancer types, although they were less
ecient than the OA-α-cLA complex (see Figure 3 and
Table 3). In fact, at IC50 concentration of α-cLA, the
percentages of early (late) apoptotic cells in MCF-7,
HepG-2, PC-3, and Caco-2 were 57.6% (15.1), 43.9%
(3.7%), 35.9% (7.7%), and 14.4% (10.3%), respectively.
Additionally, OA and 5-FU had the highest ecacy
to induce the early and late stages of apoptosis in all
dierentcancercellsaswellasinnormalVerocells.
Importantly, although OA-α-cLA possessed higher
apoptosis eciency toward all tested cancer cells than
free α-cLA, no signicant dierence was found in the
percentage of early apoptosis (less than 2.5%) between
the OA-α-cLA- and α-cLA-treated Vero cells (Figure 3
and Table 3).
Analysis of the cell-cycle phases by ow cytometry
Analysis of the scanning of cell cycle phases was
conducted to explore the apoptotic and anti-cancer
mechanisms of OA-α-cLA complex, α-cLA, OA,
and 5-FU at their IC50 concentrations. Both G0/G1
andG2/Mphaseshaveasignicantroleandactas
the main check points in cell cycle progression (32).
Figure 4 and Table 4 show the results of the cell cycle
analysis for four human cancer cell lines before and
after their treatment with dierent compounds. The
percentage of G0/G1 and G2/M phases was around
97% in untreated cancer cells, as shown in Figure 4 and
Table 4. The G0/G1 phase was decreased signicantly
after treatment with IC50 doses of OA-α-cLA complex,
α-cLA, OA, and 5-FU. The sub-G1 phase was detected
and became observed (p<0.01), indicating all tested
compounds were able to induce apoptosis. However,
theaccumulationoftheOA-α-cLA complex-treated
cancercellsintheSphaseandtheG2/Mphasewas
suppressed. Our results indicated that the S phase
was suppressed in the Caco-2 and PC-3 cells after
treatment with OA (Tab le 4 ). Figure 4 and Ta b l e 5
show that the MCF-7 cells were the most sensitive to
the apoptosis induction after the treatment with the
OA-α-cLA complex. In fact, for these cells, a signif-
icant increase in the percentage of the sub-G1 phase
(55.8%) was observed in comparison with the other
studied cancer cells. The MCF-7 cells were followed by
the HepG-2 and PC-3 cells, for which 48.4 ±1.5% and
48.2 ±0.3% of the cell population were in the sub-G1
phase, respectively. The least sensitive were Caco-2
cells, with 30.3 ±0.4% of the cell population being in
the sub-G1 phase. Free α-cLA at IC50 dose was also
able to induce apoptosis in all analyzed cells, albeit less
eciently (p<0.001) than the OA-α-cLA complex.
More importantly, this OA-α-cLA complex was able to
reinforce all studied human cancer cell lines to enter
sub-G1 phase by two folds in control with α-cLA.
Apoptotic eects of OA-α-cLA complex on colon
cancer cells
Further support to the strong apoptotic and anti-cancer
potential of the OA-α-cLA complex was provided by
the analysis of morphological changes in Caco-2 cells
using PI dye for nuclear staining. PI dye binds to DNA
fragments and is used for identifying the apoptotic
eectofdrugs.AlthoughPIdyebindstoplasmamem-
brane and enters inside the intact cells, the eciency of
its entrance to the nucleus dramatically increases after
the cell integrity becomes disrupted. In the nucleus, PI
forms complexes with DNA which make the nucleus
highly uorescent and easily detectable in uores-
cence microscopy. Figure 5 shows that the Caco-2
cells lose their normal spindle shape and became more
round after treatment with the OA-α-cLA complex
for 24 h, than the cells treated by free α-cLA at the
IC50 concentrations for both compounds. Figure 5 also
shows that the nuclei became more condensed and
the chromatin was fragmented after the cell treatment
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CANCER INVESTIGATION 9
Figure . Cell cycle analysis of cell lines (І) Caco- cells, (ІІ) HepG- cells, (ІІІ)PC-and(ІV) MCF- cells. (A) Untreated cells, (B) cells after
treatment with α-LA, (C) OA-α-LA, (D) OA, and (E) -FU.
Tab le . Cell cycle analysis of samples-treated human cancer cell lines.
Cell lines Cell cycle phase Untreated control α-LA OA-α-LA OA -FU
Caco- Sub G . ±. a. ±. b. ±. c. ±. d. ±. e
G/G . ±. a. ±. b. ±. c. ±. d. ±. e
S.±. a. ±. b. ±. b±c. ±. d
G/M . ±. a. ±. b. ±. b±c. ±. b
HepG- Sub G . ±. a. ±. b. ±. c. ±. d. ±. c
G/G . ±. a. ±. b. ±. c. ±. d. ±. e
S.±. a. ±. b. ±. b. ±. c. ±. d
G/M . ±. a. ±. b. ±. c. ±. d. ±.e
PC- Sub G . ±. a. ±. b. ±. c. ±. d. ±. e
G/G . ±. a. ±. b. ±. c. ±. d. ±. e
S.±. a. ±. b. ±. c±d. ±. e
G/M . ±. a. ±. b. ±. c±d. ±. e
MCF- Sub G . ±. a. ±. b. ±. c. ±. d. ±. e
G/G . ±. a. ±. b. ±. c. ±. d. ±. e
S.±. a. ±. b. ±. c. ±. d. ±. e
G/M . ±. a. ±. b. ±. c. ±. d. ±. e
All values were expressed as mean ±SEM. Different letters are significantly different within the same raw at p<..
Downloaded by [Professor Vladimir N. Uversky] at 16:39 26 September 2017
10 V. N. UVERSKY ET AL.
Figure . Photograph of Caco- cells stained with PI dye under a light microscope (A) control “untreated” cells, cells exposed to IC (μM)
of (B) α-LA, (C) OA- α-LA,(D)OA,and(E)-FU.
with OA-α-cLA complex, whereas the untreated cells
were characterized by a normal cell shape and intact
nuclei, with a very minor number of cells being PI pos-
itive. It was observed that the nuclear condensation,
chromatin fragmentation, and shedding of apoptotic
bodies were the major features of the apoptotic eect in
the case of the treatment with OA-α-cLA complex, and
that these eects more noticeable than those caused by
the treatment with α-cLA or 5-FU.
Inhibition eect of OA-α-cLA complex on tyrosine
kinase
TheeectiveanticanceractivityofOA-α-cLA complex
may be attributed to the inhibition of the TK, as shown
in Table 5. In fact, data presented in Tab l e 5 demon-
strate that all the compounds tested were potent TK
inhibitors, excluding free α-cLA, which had the low-
est in vitro inhibitory activity. According to the Ki val-
ues, OA-α-cLA was found to be nearly 2.5-fold more
potent than α-cLA as a TK inhibitor. In addition, no
Tab le . The estimated Ki (µM) of α-LA and OA- α-LA for tyrosine
kinase inhibition in control with OA and -FU.
Sample Ki
α-LA . ±.a
OA-α-LA . ±. b
OA . ±. b
-FU . ±. c
All values were expressed as mean ±SEM. Different letters are significantly
different within the same column at p<..
signicant dierence between OA-α-cLA and OA was
observed.
Discussion
Although historically best-known for its ability to bind
calcium and regulate the catalysis of the nal step of the
lactose biosynthesis in the lactating mammary gland,
α-LA, which might constitute up to 20% of total milk
protein, is now recognized to have a multitude of bio-
logical activities (33–35),includingfattyacidbinding
(15, 36–38). It was pointed out that complexes of this
protein with some fatty acids (such as oleic or linoleic
acids), which are known now as HAMLET-like anti-
cancer compounds (39) or liprotides; i.e., complexes
consisting of protein and fatty acids (39, 40),possess
selective anticancer activity (6–13, 35, 39).α-LA-based
liprotides are believed to be formed as a result of bind-
ing of fatty acid molecules to the hydrophobic core
of the partially unfolded and aggregated protein (39),
where a micellar OA core is surrounded by a shell com-
prised of the partially unfolded protein molecules (41).
The HAMLET-like anticancer compounds were shown
to possess profound cytotoxicity and anticancer activ-
ity against all of the ∼50 dierent cancer cell types, for
which the representatives of the HAMLET family have
been trialed (39).
The majority of research on the anticancer activity
of α-LA-basedliprotideswasconductedusinghuman
and, to a much lesser degree, bovine protein, whereas
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CANCER INVESTIGATION 11
anticancer and pro-apoptotic activities of the com-
plexes between OA and camel α-LA are studied to a
muchlesserdegree.Infact,asearchofPubMedforarti-
cles discussing human, bovine, or camel LA and can-
cer generated 248, 55, and 3 hits, respectively. This is
instarkcontrasttothebroaduseofcamelmilkand
related products as an important nutritional source in
several world regions. Previous in vivo and in vitro
studiesreportedthepotencyofmilkproteinsthatwere
obtained from camel in halting breast, liver, and colon
cancer progression (42–44). It is therefore likely that
camel α-LA may have anticancer activity, in contrast to
the anticancer eects of HAMLET and BAMLET that
attributed only to OA (44, 46). Therefore, this study was
conductedtotesttheapplicabilityofcamelα-LA com-
plexed with OA as anticancer compounds with pro-
apoptotic activities against various cancer cells. These
eects were compared with those imposed on nor-
mal Vero and cancer cells by α-LA and OA alone, as
well as by a well-known anti-cancer drug 5-uorouracil
(5-FU).
Our analysis revealed that although OA, 5-FU,
and OA-α-cLA showed noticeable cytotoxic eects
on all studied cancer cells when compared to α-cLA
alone, OA-α-cLA was the only compound that had
the minimal eects on the viability of normal cells
with the highest SI. However, both OA and 5-FU
showed signicant cytotoxicity against Vero cells
with the lowest SI. These results illustrated that this
complex potentiated the cytotoxic activity of protein
portion (α-cLA) against cancer cells and improved
the selectivity of fatty acid portion (OA) to be more
specic to cancer cells rather than normal cells. Beside
amoderateanticancereectofcamelα-LA, previous
studies declared that protein portions of OA-α-LA
complex play an essential role in transporting OA
from membrane to cytoplasm then accumulated in
nucleus, subsequently causing nuclear shrinkage and
damage (47, 48).Aswasshowninthisstudy,PIpen-
etrated and stained the nucleus of treated cancer cells,
particularly those with an OA-α-cLA complex. We also
showed that the OA-α-cLA complex serves as an e-
cientinductorofcelldeathintheinvestigatedcancer
cell lines by causing severe damage in their morphol-
ogy, signicant increase of annexin-stained apoptotic
cell populations, and induction of cell cycle arrest
at lower IC50 than that of α-cLA. Our recent study
proved that OA-α-LA complex mediated cancer cell
death through induction of apoptosis (49).Also,other
previous studies demonstrated the anticancer activi-
tiesofHAMLETandBAMLETmediatedmainlyvia
apoptosis (45, 50).
Here, the observed cell cycle arrest and apoptosis
induction may be related to the capacity of OA-α-
cLAfortheTKinhibitionatlowconcentrationlike
OA. TK activation is required for several fundamen-
tal processes in cancer cells, such as oncogenic activa-
tion, angiogenesis, apoptosis inhibition, uncontrolled
cell cycle, and abnormal proliferation in several tumor
types. Therefore, TK inhibitors are considered as eec-
tive anticancer agents through targeting and disrupting
cancer cell signaling pathways and subsequent apopto-
sis induction (51–53). Khamaisie et al. (54) found that
oleic acid, being one of active component of mushroom
Daedalea gibbosa extract, inhibited TK autophospho-
rylation that mediated its anti-leukemic eect in an ani-
malmodel.OurresultsrevealedthattheOA-α-cLA
complex inhibited the activity of tyrosine kinase as e-
ciently as OA, while free α-cLA showed less inhibition
activity for tyrosine kinase.
Conclusion
We show here that camel α-cLA is able to bind OA
toformacomplex,whichcanbenamedCAMLET
(camel α-lactalbumin made lethal to tumor cells), with
a highly selective anticancer activity against four cancer
cells, associated with tumors in diverse tissues of the
humanbody,andcharacterizedbyavariablesensitivity.
The selectivity and potency of the anticancer activity
of CAMLET are related to α-cLA and OA, respectively.
The OA-α-cLA complex destroys cancerous cells, par-
ticularlybreastcancercells,byenhancingtheselective
apoptosic process and causing arrest of the cell cycle.
Unlike OA, this complex is able to specically induce
apoptosis only in cancer cells. All tested cancer cells in
G0/G1 and G2/M phase were intensely reduced, while
the cells in sub G1 phase were dramatically induced
when treated with IC50 of OA-α-cLA, compared to the
untreated control cells. Furthermore, the OA-α-cLA
complex has the ability to induce apoptosis and cell
cycle arrest through inhibition of tyrosine kinase activ-
ity.Thisstudymayprovidefoundationsforthedevel-
opment of a competent and cost-eective approach
fortheanalysisofproteinsthatformcomplexeswith
small molecules, such as oleic acid, characterized
by new functions and highly promising biological
activities.
Downloaded by [Professor Vladimir N. Uversky] at 16:39 26 September 2017
12 V. N. UVERSKY ET AL.
Declaration of interest
The authors report no declarations of interest.
Acknowledgments
This work was supported in part by a grant from the King Abdu-
laziz University (56-130-35- HiCi). We are thankful to Alexey
Uversky for careful reading and editing this manuscript.
ORCID
Vladimir N. Uversky http://orcid.org/0000-0002-4037-5857
Esmail M. El-Fakharany http://orcid.org/0000-0001-8246-
0075
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