Anticancer Activity of Camel Milk via Induction of Autophagic Death in Human Colorectal and Breast Cancer Cells

Abstract

Background/Objective
Camel milk is traditionally known for its human health benefits and believed to be a remedy for various human ailments including cancer. The study was aimed to evaluate the inhibitory effects of commercially available camel milk on cancer cells and its underlying mechanism(s).

Materials and Methods
Two cell lines: colorectal cancer HCT 116 and breast cancer MCF-7 were cultured with different doses of camel milk. The effects of camel milk on cell death were determined by MTT assay, viability by trypan blue exclusion assay and migration by in vitro scratch assay. The mechanism was elucidated by western blotting and confocal microscopy was used to confirm autophagy.

Results
Camel milk significantly reduced proliferation, viability as well as migration of both the cells. The accumulation of LC3-II protein along with reduction in expression of p62 and Atg 5-12, the autophagy proteins implied induction of autophagy. The (GFP)-LC3 puncta detected by confocal microscopy confirmed the autophagosome formation in response to camel milk treatment.

Conclusion
Camel milk exerted antiproliferative effects on human colorectal HCT 116 and breast MCF-7 cancer cells by inducing autophagy.

Full text

Asian Pacic Journal of Cancer Prevention, Vol 19 3501
DOI:10.31557/APJCP.2018.19.12.3501
Camel Milk Induces Autophagy in Cancer Cells
Asian Pac J Cancer Prev, 19 (12), 3501-3509
Introduction
Cancer forms one of the leading causes of millions
of deaths worldwide. An increasing incidence in the
prevalence of this fatal disease is being observed in the
Middle East region, with the breast cancer (Al-Kuraya
et al., 2005) and colon cancer (Arafa and Farhat, 2015)
ranking high, especially within the State of Qatar (Rasul et
al., 2001; Bener et al., 2008; Bener et al., 2010; Bener et al.,
2012). Among Arab women, breast cancer is ranked rst
among all cancers and has the highest rate of morbidity and
mortality among women (Bazarbashi et al., 2017). Breast
cancers originating from this ethnic population are found
to have an advanced stage, high grade and tend to affect a
younger population as compared to the west (Al Tamimi et
al., 2010). In the case of colon cancer though the incidence
of occurrence has decreased in the west, it is observed to
be increasing in the gulf region and forms the third most
common cancer in Qatar (Rasul et al., 2001; Arafa and
Farhat, 2015). The standard therapies for managing this
disease includes surgery, chemotherapy, radiotherapy
and immunotherapy (Kittaneh et al., 2013). Although
these treatment strategies had signicantly progressed
Abstract
Background/ Objective: Camel milk is traditionally known for its human health benets and believed to be a remedy
for various human ailments including cancer. The study was aimed to evaluate the inhibitory effects of commercially
available camel milk on cancer cells and its underlying mechanism(s). Materials and Methods: Two cell lines:
colorectal cancer HCT 116 and breast cancer MCF-7 were cultured with different doses of camel milk. The effects of
camel milk on cell death were determined by MTT assay, viability by trypan blue exclusion assay and migration by in
vitro scratch assay. The mechanism was elucidated by western blotting and confocal microscopy was used to conrm
autophagy. Results: Camel milk signicantly reduced proliferation, viability as well as migration of both the cells.
The accumulation of LC3-II protein along with reduction in expression of p62 and Atg 5-12, the autophagy proteins
implied induction of autophagy. The (GFP)-LC3 puncta detected by confocal microscopy conrmed the autophagosome
formation in response to camel milk treatment. Conclusion: Camel milk exerted antiproliferative effects on human
colorectal HCT 116 and breast MCF-7 cancer cells by inducing autophagy.
Keywords: Colorectal cancer- breast cancer- camel milk- autophagy
RESEARCH ARTICLE
Anticancer Activity of Camel Milk via Induction of Autophagic
Death in Human Colorectal and Breast Cancer Cells
Roopesh Krishnankutty1*, Ahmad Iskandarani1, Lubna Therachiyil1, Shahab
Uddin1, Fouad Azizi1, Michael Kulinski1, Ajaz Ahmad Bhat1,2, Ramzi M
Mohammad1,3
through the invention of novel therapeutic targets/drugs,
the management of this disease is still a major challenge,
as there are no medical modalities available until now
that can selectively target and kill cancer cells. For
example, chemotherapy the primary treatment mode for
many cancer types is being characterized by its toxicity
on normal cells resulting in potential lethal side effects
sometimes leading to life-long morbidities (Rossi et al.,
2008). These drawbacks intensify the imperative need
for devising alternative treatment/ management strategies
with minimal side effects.
Naturally occurring bioactive compounds have
contributed effectively into cancer therapeutics paving
way for better disease management, with their source of
origin being either plants or animals (Kommanee et al.,
2012; Mothana et al., 2012). Dietary constituents in the
form of functional food has attracted much attention due
to their ability in providing multitude of human health
benets with less or no toxicity and emerged as a novel
approach to control cancer (Kontou et al., 2011). Camel
milk is such a dietary food with profound nutraceutical
value (Alebie et al., 2017). It forms a high nutritional
source with low cholesterol, low sugar, high minerals
Editorial Process: Submission:04/20/2018 Acceptance:11/27/2018
1Translational Research Institute, Academic Health System, Hamad Medical Corporation, 2Division of Translational Medicine,
Research Branch, Sidra Medicine, Doha, State of Qatar, 3Department of Oncology, Barbara Ann Karmanos Cancer Institute,
Wayne State University School of Medicine, Detroit, MI, USA. *For Correspondence: rkrishnankutty@hamad.qa

Roopesh Krishnankutty et al
Asian Pacic Journal of Cancer Prevention, Vol 19
3502
(sodium, potassium, iron, copper, zinc and magnesium),
high vitamins (vitamin C, B2, A and E) and high
concentrations of insulin (Haddadin et al., 2008; Alhaider
et al., 2014) compared to the ruminant milk.
Camel milk and its products have been reported to
possess various human health benets and used as a
medicine to treat human diseases such as hepatitis, spleen
problems (Korish and Arafah, 2013), diarrhea (Yagil,
2013), psoriasis (Wernery, 2006) etc. Camel milk has been
reported to be very effective against bacteria: Escherichia
coli, Staphylococcus aureus, Salmonella typhimurium
and also rotavirus (Harrison et al., 2003). Camel milk is
known to exhibit signicant antioxidant effects (Korish
and Arafah, 2013) as well as possess protective proteins
which includes lysozyme, lactoperoxidase and lactoferrin
(Agrawal et al., 2009; Shamsia, 2009).
It is being traditionally believed that drinking camel
milk would help to ght against various human ailments
including cancers, however, this proclaimed health
benets of the camel milk is not well recognized due
to limited scientic study reports. A few of the in vitro
studies have reported the inhibitory effects of camel
milk on cancer cells exerted through the mechanism of
apoptosis (Korashy et al., 2012b; Hasson et al., 2015) and
antioxidant activities (Habib et al., 2013). Some of the in
vivo studies have also shown the potent inhibitory effects
of camel milk on pro-inammatory, pro-angiogenic and
pro-brogenic cytokines (Alhaider et al., 2014) as well
as the ability of camel milk to modulate the expression
of known cancer-activating gene and cancer-protective
genes at the transcriptional and posttranscriptional levels
(Korashy et al., 2012a). However, no studies exist so far,
testifying the effect of a commercially available camel
milk on cancer cells.
The objective of the current study was to evaluate
the inhibitory effects of camel milk on proliferation,
viability and migration of human colorectal HCT 116
and breast MCF-7 cancer cells as well as explore the
underlying molecular mechanism(s). The assays such as
trypan blue exclusion and MTT were used to determine
the effect of camel milk on cancer cells viability as well as
proliferation. An in vitro scratch assay was used to check
the effect of camel milk on migration of cancer cells. We
have also attempted to elucidate the mechanism behind
the inhibitory effects of camel milk on cancer cells by
identifying the various protein markers involved in the
cell death pathway.
Materials and Methods
Reagents
Dulbecco’s Modified Eagle’s Medium (DMEM),
Radioimmunoprecipitation assay (RIPA) buffer, protease
inhibitor cocktail, 3-(4,5-dimethylthia- zol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT), fetal bovine serum
(FBS), trypsin, phosphate buffered saline (PBS) were
purchased from Sigma-Aldrich Chemical Co. (St. Louis,
MO, USA). All the antibodies, primary and secondary
were purchased from Cell Signaling Technology (Danvers,
MA, USA). Lipofectamine 2000 transfection reagent
was purchased from Invitrogen (Carlsbad, USA). Camel
milk and bovine milk were purchased locally from the
supermarket in the State of Qatar.
Cell culture and treatment
Colorectal cancer HCT 116 and breast cancer MCF-7
cells were obtained from the American Type Culture
Collection (ATCC, Manassas, VA, USA). Cells were
maintained in DMEM (Gibco), supplemented with 10%
fetal bovine serum (FBS), 1% penicillin– streptomycin
(100 U/ml) and 2 mmol/L glutamine (Hyclone Logan) at
37°C in a humidied atmosphere of 5% CO2. Cells were
cultured in serum-free medium before treatment with
different doses of camel milk.
Cell viability and proliferation assay
Cells (5.0 x 105 cells/well) were cultured in 6-well
plates in the absence and presence of camel milk at
different doses (25, 50, 100, 150 and 250μL/mL) for
24, 48 and 72h while, with bovine milk at doses: 100
and 250μL/mL for 48h. Cells were trypsinized, washed
with phosphate buffered saline (PBS) , mixed with
trypan blue and counted on TC® automated cell counter
(Bio-Rad) for viability. Cell proliferation was determined
by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
(MTT) assay. (Mosmann, 1983). The cells were seeded
into 96 well plates and maintained at 37°C in a humidied
atmosphere of 5% CO2 for 24 h. The cells were then
treated with varying concentrations (25 to 250μL/mL)
of camel milk and incubated for 24, 48 and 72 h. The
media were then removed and 100µL of serum-free
media with MTT (1.2 mM) was added. The plates were
further incubated at 37°C, 5% CO2 for 4h. The reaction
was stopped by decanting the media by inverting the
plates and the formazan crystals were dissolved in 200μL
of 10% sodium dodecyl sulphate (SDS) prepared in
deionized water by incubating at 37°C for overnight. The
color intensity was measured at 630 nm using microplate
reader (Tecan, BioTek Instruments Inc. Winooski, VT,
USA). Six replicate measurements with three independent
experiments were carried out. The cell proliferation
was determined as percentage in relative to the control
(untreated) wells designated as having 100% proliferating
cells.
In vitro wound healing assay
The effect of camel milk on cell migration was
determined by in vitro scratch assay for wound healing
(Liang et al., 2007). Cells were cultured in 6-well plates
(5.0 x 104 cells/well) yielding confluent monolayer.
Wounds were created by scratching monolayer using a
sterile 200µL pipet tip. After incubation in the absence and
presence of camel milk, plates were photographed under
a Nikon DMCI microscope (Nikon, Tokyo, Japan). The
images were further analyzed quantitatively using Image
J (http://rsb.info.nih.gov/ij/) software.
Western blot analysis
Cells were treated with different doses of camel milk
and were lysed in cold RIPA buffer [1% (w/v) NP40, 1%
(w/v) sodium deoxycholate, 0.1% (w/v) SDS, 0z.15 M
NaCl, 0.01 M sodium phosphate buffer, pH 7.2, 2 mM

Asian Pacic Journal of Cancer Prevention, Vol 19 3503
DOI:10.31557/APJCP.2018.19.12.3501
Camel Milk Induces Autophagy in Cancer Cells
mL for HCT 116 and MCF-7 cells respectively.
Camel milk reduces migration of cancer cells
Cell migration is a property of cancer cells that
contributes to its potential to invade into other tissues
or organs that can result in a condition of metastasis. A
dose-dependent reduction in wound healing was observed
in both the cell types treated with camel milk compared
to their respective (untreated) controls (Figure 3a, c).
A signicant reduction in wound healing was achieved
with 5% of wound closure in case of HCT 116 cells and
4% in the case of MCF-7 cells treated at the highest dose
(Figure 3b, d).
Camel milk did not trigger apoptosis in cancer cells
To assess the mechanism behind the cytotoxicity
effects exerted by the camel milk; the HCT 116 and
MCF-7 cells were cultured in the absence or presence
of camel milk. The protein lysates were immuno-blotted
against the apoptotic protein marker: poly (ADP-ribose)
polymerase (PARP). No PARP cleavage was detected in
both the cell lines treated with camel milk (Figure 4a, e)
indicating that the treatment did not trigger apoptosis.
During apoptosis, the full length PARP protein (116 kD) is
cleaved by caspases into 89 kD fragment which inactivates
the enzyme thereby preventing its catalytic action
against DNA damage repair (D’Amours et al., 2001). To
further corroborate this nding, the protein extracts were
tested for Bcl-2 protein expression. Bcl-2 is a known
anti-apoptotic protein, implicating that Bcl-2 protein will
not favor apoptotic pathway mediated cell death (Brunelle
and Letai, 2009). Bcl-2 family members play a signicant
and pivotal role in regulating apoptosis by maintaining a
balance between anti-apoptotic molecules such as Bcl-2
and pro-apoptotic molecule Bax. Slight imbalance or
disturbance in their levels leads to induction or inhibition
of cell death (Martinou and Youle, 2011). Western blot
analysis detected Bcl-2 protein with no altered expression
in control vs. treated (Figure 4a, e). The cell lysates were
also immuno-blotted against caspase-3 antibody and
no cleaved caspase-3 were detected (data not shown).
Caspases are hallmark of apoptosis that propagates the
death signal by activation of caspase-3 that leads the
activation and cleavage of PARP. Activation and cleavage
of PARP in turn causes DNA fragmentation and cell death
(Hussain et al., 2011). Taken together, these data indicated
that growth inhibitory effects exerted by camel milk on
these cells were not via apoptosis.
Camel milk treatment induced autophagy in cancer cells
The HCT 116 and MCF-7 cells cultured in the presence
of camel milk were observed to exhibit significant
morphological changes characterized mainly by lose of
cell membrane integrity along with extensive vacuolization
(Figure 5a, b). The LC3 (microtubule-associated protein1
light chain 3) protein is a known marker of autophagy.
Studies have indicated that the lipidated form of LC3
transforming from LC3-I to LC3-II isomer can be
correlated with the formation of autophagosomes and
hence, LC3-II/LC3-I ratio forms a readout for the degree
of autophagy (Tanida et al., 2004). A dose-dependent
EDTA, and 50 mM phosphatase inhibitor cocktail]. The
cell lysates were centrifuged at 14,000 x g for 10 min at
4oC and the supernatant (total protein) obtained were
quantied by bicinchoninic acid (BCA) protein assay
(Thermo Scientic Pierce kit) using bovine serum albumin
as standard. Equal amounts of protein were separated
on a 12% SDS gel by electrophoresis (100 V, 2h) and
were transferred to polyvinylidene diuoride (PVDF)
membrane. Membranes were probed with primary rabbit
antibodies against poly (ADP-ribose) polymerase (PARP),
B-cell lymphoma 2 (Bcl-2), microtubule associated protein
1 light chain 3 (LC3), sequestosome 1 (SQSTM1 or p62),
Autophagy (Atg5-12) and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) and the bound antibodies
detected with horseradish peroxidase-conjugated
anti-rabbit Ig antibody using enhanced chemiluminescence
system (BioRad Laboratories, Hercules, CA, USA). The
band intensities were quantied using the software Image
J and normalized against GAPDH.
Green fluorescent protein (GFP)-LC3 detection of
autophagosomes
The HCT 116 and MCF-7 cells were transfected
with pEGFP-LC3 plasmids using lipofectamine 2000
(Invitrogen) according to the manufacture’s protocol. At
24 h of transfection, the cells were treated with or without
camel milk (100µL/mL) and incubated further for 24 h
(37°C, 5% CO2). The uorescence of GFP-LC3-labeled
vacuoles (autophagosomes) visualized as GFP-LC3
puncta was detected using confocal microscopy (A1Rsi
Nikon eclipse Ti confocal microscope, 60x magnication).
Statistical analysis
All the experiments were performed in triplicates
unless otherwise mentioned and independently three times
to ensure reproducibility. The data are represented as mean
± standard error of mean (SEM). The differences between
the groups were analyzed using paired Student’s t-test
and difference with p < 0.05 was considered statistically
signicant.
Results
Camel milk exerts cytotoxicity to cancer cells
Camel milk was found to exhibit a time
and dose-dependent cytotoxicity effects on both the cell
lines tested (Figure 1a, b). Camel milk exerted signicant
cytotoxicity at higher concentrations (100 and 250 µL/
mL) after 48 h in both the cell types as evident from the
decrease in total percent cell viability compared to the
control (untreated). In contrast, treatment of cells with
bovine milk at higher concentrations (250 µL/mL) for 48
h did not show any toxicity on both the cell lines (Figure
1c, d). Similarly, camel milk treatment showed a time
and dose-dependent decrease in cell proliferation (Figure
2a, b). In both the cell types tested, reduction in cell
proliferation was observed at higher doses (100 and 250
µL/mL) after 48 h. In fact, the proliferation of HCT 116
and MCF-7 cells decreased by 55 and 45% respectively, at
72 h after treatment. The half maximal inhibitory constant
(IC50) of camel milk was determined to be 31 and 51 µL/

Roopesh Krishnankutty et al
Asian Pacic Journal of Cancer Prevention, Vol 19
3504
increase in the conversion of LC3-I to LC3-II was
observed in both the cell lines cultured in the presence
of camel milk (Figure 4b, f) with approximately 5-fold
increase in the LC3-II/LC-I ratio as signied from the
quantied values (histograms).
To further confirm the induction of autophagy
by camel milk, the protein expression profile of
autophagy markers such as p62 and Atg5-12 were tested.
A signicant (~3-fold) decrease (dose-dependent) of
p62 protein expression was observed in both the cell
lines treated with camel milk (Figure 4c, g) which
implied the stimulation of autophagy. The protein Atg5
(autophagy protein 5) complexes with Atg12 (autophagy
protein 12) and forms an essential component of
autophagy which catalyzes the elongation of vesicular
membrane leading to autophagosome formation. A
concomitant dose-dependent decrease in the expression of
Atg5-12 protein was observed in both the cell lines treated
with camel milk (Figure 4d, h) which further conrms the
elongation process of double vesicle membrane forming
nascent autophagosomes.
Figure 1. Effect of Camel Milk and Bovine Milk on Cell Viability. (a, b) HCT 116 and MCF-7 cells were seeded and
after 2h incubated with varying concentration of camel milk and the number of viable cells were counted after 24, 48
and 72h using trypan blue exclusion assay. Values represent percentage of the control (0 µL/mL) and are shown as
mean ± SEM (n=3), *p <0.03, **p <0.003. (c, d) HCT 116 and MCF-7 cells were seeded and after 24h were incubated
with 100 and 250 µL/mL of bovine milk and the viable cell count was made after 48h using trypan blue.
Figure 2. Effect of camel milk on cell proliferation. (a, b) HCT 116 and MCF-7 cells were seeded and incubated with
various concentration of camel milk for 24, 48 and 72h, thereafter cell proliferation was assessed using MTT assay.
Values are presented as percentage of the control (0 µL/mL) and are shown as mean ± SEM (n=3), *p <0.03, **p
<0.01, ***p <0.001.

Asian Pacic Journal of Cancer Prevention, Vol 19 3505
DOI:10.31557/APJCP.2018.19.12.3501
Camel Milk Induces Autophagy in Cancer Cells
Figure 3. Effect of Camel Milk on Cell Migration, in vitro Scratch Wound Healing Assay. (a, c) HCT 116 and MCF-7
cells were grown in DMEM media to conuence, wounded (t=0h) by using a sterile pipette tip and then treated with
various concentration of camel milk. After 21h, the migration of cells into the wound surface were captured under
the microscope (magnication, 40x). Scale bar: 200 µm. (b, d): Percentage of wound healing relative to the distance
measured in (a) and (c) quantied using Image J. Values are represented as mean ± SEM, **p <0.02, ***p <0.001.
Data are representative of triplicate experiments.
Figure 4. Effect of camel milk on apoptotic and autophagy markers. (a, e): HCT 116 and MCF-7 cells were treated
with 100 and 250 µL/mL of camel milk for 48h. The expression of PARP and Bcl-2 proteins in the cell lysates were
analysed by western blotting. HCT 116: (b-d) and MCF-7: (f-h); the protein lysates were tested for the expression
of LC3, p62 and Atg5-12 proteins by western blotting analysis. GAPDH served as the loading control. The band
intensities were quantied using Image J and normalised against GAPDH. The data are from triplicate experiments
and presented as mean ± SEM, *p <0.03, **p <0.01.

Roopesh Krishnankutty et al
Asian Pacic Journal of Cancer Prevention, Vol 19
3506
Camel milk treatment stimulated autophagosome
formation in cancer cells
To characterize the autophagic activity in camel milk
treated cells, green fluorescence GFP-LC3 transient
transfection technique was used to detect the formation
of autophagosomes. In control (untreated) HCT 116 and
MCF-7 cells, a diffused uorescence signal of GFP-LC3
was observed (Figure 5c, 5d) while, in both HCT 116 and
MCF-7 cells cultured in the presence of camel milk, the
GFP-LC3 puncta accumulated indicating the formation
of autophagosomes (Figure 5c, 5d).
Figure 5. Camel Milk Induces Autophagy. (a, b): Light microscopy images showing comparative morphological
changes in HCT 116 and MCF-7 cells untreated or treated with camel milk for 24h. The black arrows indicate the
presence of vesicles . Scale bar: 15 µm. (c, d): Confocal microscopy images showing the localization of GFP-LC3
puncta (white arrows) in HCT 116 and MCF-7 cells treated with camel milk in compared to the diffused uorescence
in the untreated cells. Cells were transiently transfected with pEGFP-LC3 plasmid for 24h and then the GFP-LC3
positive cells were treated with or without 100 µL/mL camel milk for additional 24h. Fluorescence images were
captured at 60x magnication, scale bar: 20 µm.
Figure 6. Schematic Representation of Autophagic Flux and Formation of Autophagosomes Emphasising the Role
of Various Proteins Involved. Microtubule-associated protein 1 light chain 3 (LC3) precursors are processed to form
LC3-I is lipidated by phosphatidyl ethanolamine (PE) forms the active LC3-II which is then localized onto the double
membrane vesicles that forms the nascent autophagosomes. The autophagy proteins such as Atg5 and Atg12 forms
a complex (Atg5-12), then gets directed onto to the double membrane vesicles, further mediates elongation process
leading to the autophagosome formation. The protein p62 (sequestosome 1) co-localizes with the ubiquitinated
proteins (fated to be degraded) gets sequestered into the double membrane vesicles, subsequently engulfs into the
autophagosomes destined for degradation.

Asian Pacic Journal of Cancer Prevention, Vol 19 3507
DOI:10.31557/APJCP.2018.19.12.3501
Camel Milk Induces Autophagy in Cancer Cells
Discussion
Camel milk has been reported to possess anti-cancer
activity as proved by a few of the in vitro and in vivo
studies (Korashy et al., 2012a; Korashy et al., 2012b;
Habib et al., 2013; Alhaider et al., 2014; Hasson et al.,
2015). Lyophilized camel milk has been shown to inhibit
growth and proliferation of many cancer cell types
such as human breast cancer BT-474, laryngeal HE-p2
(Hasson et al., 2015), human hepatoma HepG2 (Korashy
et al., 2012b) and murine hepatoma Hepa 1c1c7 cells
(Korashy et al., 2012a). In addition camel milk protein
‘lactoferrin’ has been shown to inhibit the proliferation
of human colorectal cancer HCT 116 cells by exerting
antioxidant and DNA damage activities (Habib et al.,
2013). The present study, was aimed to evaluate the growth
inhibitory effects of camel milk (commercial) in human
colorectal HCT 116 and breast MCF-7 cells and elucidate
the underlying mechanism.
Apoptosis and autophagy represent two distinct
cellular processes that leads to cell death. Apoptosis
(type I programmed cell death) is a cell suicidal program
that occurs in response to various homeostatic as well
as pathological states by which the cell orchestrates to
its death. This cell death process is characterized by
morphological changes such as membrane blebbing,
formation of apoptotic bodies and chromatin condensation,
DNA fragmentation as well as cleavage of poly
(ADP-ribose) polymerase (PARP) protein (Berger and
Petzold, 1985). PARP cleavage by the proteolytic action
of suicidal proteases resulting in signature fragments (89
kD and 24 kD) forms one of the hallmarks of apoptosis.
In our study, no PARP cleavage was detected (Figure 4a,
e) in both the cell lines treated with camel milk. Though
PARP cleavage forms a prominent marker of apoptosis,
this proteolytic cleavage alone cannot be assessed for the
induction of apoptosis. B-cell lymphoma 2 (Bcl-2) is a
known anti-apoptotic protein that regulates the process
of apoptosis. Expression of Bcl-2 blocks the cell death
process of apoptosis and certain types of cancer cells has
been found to rely on Bcl-2 for their survival (Brunelle
and Letai, 2009). In our study, the protein extracts obtained
from both the cell lines treated with camel milk, no
modulated expression of Bcl-2 protein was observed in
the treated compared to untreated (Figure 4a, e). Taken
together, these data suggest that the cytotoxicity effects
exerted by the commercially available camel milk on HCT
116 and MCF-7 cells were not through the mechanism
of apoptosis as per our experimental conditions. Hence,
we looked for the markers of autophagy, the type II
programmed cell death.
Autophagy also known as self-eating/auto-digestion
is a mechanism of the cells by which it dissipates the
non-functional or long-lived proteins and damaged
organelles within the cells (Thorburn, 2008). Autophagy
is normally induced in cells in response to stimuli such as
metabolic stress, but it has been observed that many of the
anti-cancer agents used for therapeutic intervention also
raise the autophagic ux in cells (Mathew et al., 2007).
Autophagy is a highly regulated cellular process with many
of the evolutionarily conserved proteins involved in the
formation of autophagic vesicles called autophagosomes.
A schematic representation of the induction of autophagy
leading to autophagosome formation emphasizing the
role of various proteins involved is depicted in Figure 6.
The HCT 116 and MCF-7 cells treated with the
commercial camel milk were observed to have signicant
morphological changes characterized mainly by lose of
cell membrane integrity along with extensive vacuolization
(Figure 5a, b); atypical characteristics of autophagy.
Microtubule-associated protein 1 light chain 3 (LC3) is a
known marker of autophagy having isoforms, of which:
LC3-I gets lipidated by phosphatidyl ethanolamine (PE)
forms the active isomer: LC3-II, which gets translocated
onto the double membrane vesicles that forms the
nascent autophagosomes. Thus, the ratio of LC3-II/LC3-I
represents the autophagic ux and the LC3-II protein
forms the autophagosome marker, being localized on the
surface of autophagosomes.
Upon initiation of autophagy, portions of the cytoplasm
are sequestered into double membrane vesicles wherein,
the protein p62 (sequestosome 1) co-localizes with the
ubiquitinated proteins (that are destined to be degraded)
also get sequestered into the double membrane vesicles
and gets engulfed into the matured autophagosomes for
degradation. The autophagy proteins: Atg5 and Atg12
forms a complex (Atg5-12), that gets directed onto to
the double membrane vesicles and mediates elongation
process leading to the autophagosome formation.
Our data demonstrated a dose-dependent increase in
the LC3-I to LC3-II conversion in HCT 116 and MCF-7
cells treated with camel milk (Figure 4b, f). This represents
the autophagic ux and the formation of autophagosomes.
The dose-dependent decrease in the expression of p62
(sequestosome 1) protein in both the cell lines treated
with camel milk, evidenced the sequestration process- an
initial step in the formation of double membrane vesicles.
Similarly, the dose-dependent decrease in expression of
Atg5-12 protein in both the cell lines with camel milk
indicated the formation of autophagosomes, as this protein
is involved only in the elongation process during the
autophagosome formation. In addition, the cells transiently
expressing GFP-LC3 when treated with camel milk,
resulted in the localization of green uorescent GFP-LC3
puncta as observed under the confocal microscope implied
the formation of autophagosomes with LC3 protein, a
hallmark of autophagy. In conclusion, the camel milk used
in the current study exerts cytotoxicity effects on human
colorectal HCT 116 and breast MCF-7 cancer cells by the
induction of autophagy.
Camel milk has already been reported to possess
medicinal as well as therapeutic properties due to its
components such as vitamins E and C (Farah et al., 1992),
lysozymes, lactoferrins, lactoperoxidase (el Agamy et al.,
1992) and immunoglobulins (Konuspayeva et al., 2007).
Among the afore-mentioned components lactoferrin,
an iron-binding glycoprotein has been reported to exert
anti-cancer activity in murine melanoma (B16-F10) cells
through the inhibition of cytochrome P450 1A1 activation
(Roseanu et al., 2010). Camel milk’s lactoferrin has also
been shown to inhibit the growth of colon cancer HCT
116 cells and exert antioxidant as well as DNA damage

Roopesh Krishnankutty et al
Asian Pacic Journal of Cancer Prevention, Vol 19
3508
inhibitory activities (Habib et al., 2013). In accordance
with these previous studies, we anticipate that it could be
the component lactoferrin in the camel milk that exerts the
anticancer effects on colon and breast cancer cells tested
in this study. Further research is this direction is highly
under consideration and will be pursued to identify the
active component(s) of the camel milk that can selectively
kill cancer cells. Though the potential component of the
camel milk capable of inducing autophagy in the cancer
cells was not particularly investigated in the current study,
our data clearly has shown the anticancer effect exerted
by the whole camel milk which is of commercial use. The
camel milk used in the current study being a commercial
product, it is safe for public use and in case of cancer
patients, consuming camel milk will certainly not bring
in any side effects but on the other hand could be of some
positive effects. The current study can serve as a base
and does urge for more in vitro as well as in vivo studies
which would help in extending it to some prospective
clinical trials.
Funding Statement
This study was supported by the Medical Research
Center (grant: MRC#16283/16), Hamad Medical
Corporation, Doha, State of Qatar.
Conict of interest
The authors do not have any conict of interest to
declare.
Acknowledgements
The authors are grateful to Prof. Serhiy Souchelnytskyi,
College of Medicine, Qatar University, Dr. Anna Halama,
Weill Cornell Medicine-Qatar and Dr. Jeorge Buddenkotte,
Dept. of Dermatology and Venereology, Translational
Research Institute, Hamad Medical Corporation for their
valuable suggestions and input.
References
Agrawal RP, Dogra R, Mohta N, et al (2009). Benecial effect
of camel milk in diabetic nephropathy. Acta Biomedica,
80, 131-4.
Al-Kuraya K, Schraml P, Sheikh S, et al (2005). Predominance of
high-grade pathway in breast cancer development of Middle
East women. Mod Pathol, 18, 891-7.
Al Tamimi DM, Shawarby MA, Ahmed A, et al (2010). Protein
expression prole and prevalence pattern of the molecular
classes of breast cancer--a Saudi population based study.
BMC Cancer, 10, 223.
Alebie G, Yohannes S, Worku A (2017). Therapeutic applications
of camel’s milk and urine against cancer: current development
efforts and future perspectives. J Cancer Sci Ther, 9, 468-78.
Alhaider AA, Abdel Gader AG, Almeshaal N, et al (2014). Camel
milk inhibits inammatory angiogenesis via downregulation
of proangiogenic and proinammatory cytokines in mice.
Acta Pathologica Microbiologica Et Immunonologica
Scandinavica, 122, 599-607.
Arafa MA, Farhat K (2015). Colorectal cancer in the Arab
world-screening practices and future prospects. Asian Pac
J Cancer Prev, 16, 7425-30.
Bazarbashi S, Al Eid H, Minguet J (2017). Cancer incidence in
Saudi Arabia: 2012 data from the Saudi Cancer Registry.
Asian Pac J Cancer Prev, 18, 2437-44.
Bener A, Ayub H, Kakil R, et al (2008). Patterns of cancer
incidence among the population of Qatar: a worldwide
comparative study. Asian Pac J Cancer Prev, 9, 19-24.
Bener A, Moore MA, Ali R, et al (2010). Impacts of family
history and lifestyle habits on colorectal cancer risk: a
case-control study in Qatar. Asian Pac J Cancer Prev, 11,
963-8.
Bener A, Zirie MA, Kim EJ, et al (2012). Measuring burden of
diseases in a rapidly developing economy: state of Qatar.
Glob J Health Sci, 5, 134-44.
Berger NA, Petzold SJ (1985). Identication of minimal size
requirements of DNA for activation of poly(ADP-ribose)
polymerase. Biochem, 24, 4352-5.
Brunelle JK, Letai A (2009). Control of mitochondrial apoptosis
by the Bcl-2 family. J Cell Sci, 122, 437-41.
D’Amours D, Sallmann FR, Dixit VM, et al (2001).
Gain-of-function of poly(ADP-ribose) polymerase-1 upon
cleavage by apoptotic proteases: implications for apoptosis.
J Cell Sci, 114, 3771-8.
el Agamy EI, Ruppanner R, Ismail A, et al (1992). Antibacterial
and antiviral activity of camel milk protective proteins.
J Dairy Res, 59, 169-75.
Farah Z, Rettenmaier R, Atkins D (1992). Vitamin content of
camel milk. Int J Vitam Nutr Res, 62, 30-3.
Habib HM, Ibrahim WH, Schneider-Stock R, et al (2013). Camel
milk lactoferrin reduces the proliferation of colorectal cancer
cells and exerts antioxidant and DNA damage inhibitory
activities. Food Chem, 141, 148-52.
Haddadin MS, Gammoh SI, Robinson RK (2008). Seasonal
variations in the chemical composition of camel milk in
Jordan. J Dairy Res, 75, 8-12.
Harrison RA, Oliver J, Hasson SS, et al (2003). Novel
sequences encoding venom C-type lectins are conserved in
phylogenetically and geographically distinct Echis and Bitis
viper species. Gene, 315, 95-102.
Hasson SS, Al-Busaidi JZ, Al-Qarni ZA, et al (2015). In vitro
apoptosis triggering in the BT-474 human breast cancer
cell line by lyophilised camel’s milk. Asian Pac J Cancer
Prev, 16, 6651-61.
Hussain AR, Ahmed M, Ahmed S, et al (2011). Thymoquinone
suppresses growth and induces apoptosis via generation of
reactive oxygen species in primary effusion lymphoma. Free
Radic Biol Med, 50, 978-87.
Kittaneh M, Montero AJ, Gluck S (2013). Molecular proling
for breast cancer: a comprehensive review. Biomark Cancer,
5, 61-70.
Kommanee J, Preecharram S, Daduang S, et al (2012).
Antibacterial activity of plasma from crocodile (Crocodylus
siamensis) against pathogenic bacteria. Ann Clin Microbiol
Antimicrob, 11, 22.
Kontou N, Psaltopoulou T, Panagiotakos D, et al (2011). The
mediterranean diet in cancer prevention: a review. J Med
Food, 14, 1065-78.
Konuspayeva G, Faye B, Loiseau G, et al (2007). Lactoferrin
and immunoglobulin contents in camel’s milk (Camelus
bactrianus, Camelus dromedarius, and Hybrids) from
Kazakhstan. J Dairy Sci, 90, 38-46.
Korashy HM, El Gendy MA, Alhaider AA, et al (2012a).
Camel milk modulates the expression of aryl hydrocarbon
receptor-regulated genes, Cyp1a1, Nqo1, and Gsta1, in
murine hepatoma Hepa 1c1c7 cells. J Biomed Biotechnol,
2012, 782642.
Korashy HM, Maayah ZH, Abd-Allah AR, et al (2012b).
Camel milk triggers apoptotic signaling pathways in human
hepatoma HepG2 and breast cancer MCF7 cell lines through

Asian Pacic Journal of Cancer Prevention, Vol 19 3509
DOI:10.31557/APJCP.2018.19.12.3501
Camel Milk Induces Autophagy in Cancer Cells
This work is licensed under a Creative Commons Attribution-
Non Commercial 4.0 International License.
transcriptional mechanism. J Biomed Biotechnol, 2012,
593195.
Korish AA, Arafah MM (2013). Camel milk ameliorates
steatohepatitis, insulin resistance and lipid peroxidation
in experimental non-alcoholic fatty liver disease. BMC
Complement Altern Med, 13, 264.
Liang CC, Park AY, Guan JL (2007). In vitro scratch assay:
a convenient and inexpensive method for analysis of cell
migration in vitro. Nat Protoc, 2, 329-33.
Martinou JC, Youle RJ (2011). Mitochondria in apoptosis:
Bcl-2 family members and mitochondrial dynamics. Dev
Cell, 21, 92-101.
Mathew R, Karantza-Wadsworth V, White E (2007). Role of
autophagy in cancer. Nat Rev Cancer, 7, 961-7.
Mosmann T (1983). Rapid colorimetric assay for cellular growth
and survival: application to proliferation and cytotoxicity
assays. J Immunol Methods, 65, 55-63.
Mothana RA, Al-Musayeib NM, Matheeussen A, et al (2012).
Assessment of the in vitro antiprotozoal and cytotoxic
potential of 20 selected medicinal plants from the island of
Soqotra. Molecules, 17, 14349-60.
Rasul KI, Awidi AS, Mubarak AA, et al (2001). Study of
colorectal cancer in Qatar. Saudi Med J, 22, 705-7.
Roseanu A, Florian PE, Moisei M, et al (2010). Liposomalization
of lactoferrin enhanced its anti-tumoral effects on melanoma
cells. Biometals, 23, 485-92.
Rossi A, Caracciolo V, Russo G, et al (2008). Medulloblastoma:
from molecular pathology to therapy. Clin Cancer Res, 14,
971-6.
Shamsia SM (2009). Nutritional and therapeutic properties of
camel and human milks. Int J Genet Mol Biol, 1, 52-8.
Tanida I, Ueno T, Kominami E (2004). LC3 conjugation system
in mammalian autophagy. Int J Biochem Cell Biol, 36,
2503-18.
Thorburn A (2008). Apoptosis and autophagy: regulatory
connections between two supposedly different processes.
Apoptosis, 13, 1-9.
Wernery U (2006). Camel milk, the white gold of the
desert. J Camel Pract Res, 13, 15-26.
Yagil R (2013). Camel milk and its unique anti-diarrheal
properties. Isr Med Assoc J, 15, 35-6.