Food Research International 96 (2017) 1218

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Food Research International

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Antiproliferative activity of Ontario grown onions against colorectal

adenocarcinoma cells
Abdulmonem I. Murayyan a, Cynthya M. Manohar a, Gordon Hayward b, Suresh Neethirajan a,
a
BioNano Laboratory, School of Engineering, University of Guelph, Guelph, Ontario N1G 2W1, Canada
b
School of Engineering, University of Guelph, Guelph, Ontario N1G 2W1, Canada

a r t i c l e i n f o a b s t r a c t

Article history: Cancer is the leading cause of mortality in Canada and other industrialized nations; the development of new/im-
Received 9 January 2017 proved cancer therapies is desperately needed and continues to be a major focus of cancer research. Flavonoids,
Received in revised form 6 March 2017 which are found in high levels in onions, have been shown to exert antiproliferative and potentially anti-cancer
Accepted 10 March 2017
activities. To test their therapeutic potential, we assessed the antiproliferative, cytotoxic, apoptosis-inducing, and
Available online 11 March 2017
anti-migratory activities of ve onion varieties grown in Ontario against human adenocarcinoma (Caco-2) cells.
Keywords:
The properties of onion extracts were compared to pure extracts of avonoids known to exhibit antiproliferative
Caco-2 cells effects (quercetin, myricetin, and kaempferol). We compared more than one variety of onion, as agronomic and
Onion extracts genetic factors inuence the composition, as well as the quality of phytochemicals (e.g. avonoids) in plant cul-
Pressurized low polarity water tivars. We found that all onion varieties exhibited antiproliferative activity similar to puried avonoids. The cy-
Apoptosis totoxic effects of the Stanley and Fortress onion varieties were strongest among the selected cultivars, as
Anti-migratory determined via lactate dehydrogenase (LDH) assays, while Safrane extracts showed the weakest activity. The
Stanley and Lasalle cultivar extracts also had strong anti-migratory effects. Altogether these onion extracts
may contain one or more compounds that may be effective anti-cancer therapies, while the Stanley extract
showed the most comprehensive biological activities against Caco-2 cells.
2017 Elsevier Ltd. All rights reserved.

1. Introduction commonly found in plant-based foods include carotenoids, phenolic

Increasing evidence from clinical and epidemiological studies sup-
ports an association between a diet rich in fruits and vegetables and a
reduced incidence of chronic diseases, such as cancer (Hui et al., 2013;
He, Jin, Gong, Zhang, & Zhou, 2014; Liu et al., 2014; Guercio, Turati, La
Vecchia, Galeone, & Tavani, 2016). In 2012, there were 14.1 million
newly reported cases of cancer reported globally (Ferlay et al., 2015),
of which malignant neoplasms were the mainly leading cause of mortal-
ity in Canada (Statistics Canada, 2012). After resection, mainstay cancer
therapeutics remain radiation and chemotherapy, despite their often
severe side effects and mixed successes. Therefore, researchers continue
to search for alternative therapeutics, which ideally may include life-
style changes or other non-invasive mitigation strategies for ghting
this prevalent and chronic disease. One reason a diet rich in fruits and
vegetables may be benecial in preventing cancer is that these foods
are an excellent source of phytochemicals. Phytochemicals are bioactive
compounds, classied by their chemical structures and divided into sev-
eral classes, some of which are more prevalent in a wider range of fruits
and vegetables (Schreiner & Huyskens-Keil, 2006). Phytochemicals
Corresponding author.
E-mail address: sneethir@uoguelph.ca (S. Neethirajan).
acids, organosuldes, polyphenols and avonoids.
Flavonoids represent the most prevalent phytochemical in plants.
The health-promoting effects of dietary avonoids continue to be an ac-
tive area of research (Caridi et al., 2007). Flavonoids can exert antioxi-
dant radical scavenging activities, limiting the lipid peroxidation of
cell membranes (Peng & Kuo, 2003). Additionally, avonoids have
been demonstrated to have anti-cancer properties against several can-
cer types, including colorectal (He et al., 2014; Linsalata, Orlando,
Messa, Refolo, & Russo, 2010), upper digestive tract (Guercio et al.,
2016), breast (Yamazaki, Miyoshi, Kawabata, Yasuda, & Shimoi, 2014),
and liver cancers (Yang, Meyers, Heide, & Liu, 2004). Dietary sources
of avonoids include berries, onions, garlic, citrus fruits, apples, and
leeks (Manach, Scalbert, Morand, Rmsy, & Jime, 2004). Onions are
one of the most widely produced and consumed vegetables
(Sellappan & Akoh, 2002; Slimestad, Fossen, & Vgen, 2007),
representing one of the most common sources of avonoids in the
human diet (Suleria, Butt, Anjum, Saeed, & Khalid, 2015). Three well
studied avonoids, kaempferol, myricetin, and quercetin, are found in
onions, while quercetin is the most abundant among these in onions
(Sellappan & Akoh, 2002).
Agronomic and genetic factors can inuence the quality of phyto-
chemicals among cultivars (Tomas-Bareberan & Espin, 2001),

http://dx.doi.org/10.1016/j.foodres.2017.03.017
0963-9969/ 2017 Elsevier Ltd. All rights reserved.
 A.I. Murayyan et al. / Food Research International 96 (2017) 1218
negatively impacting the health-promoting benets that these nutri-
ents provide. Therefore, it is possible that the benets reported in on-
ions grown outside of Canada may not necessarily apply to the locally
grown varieties, as the cultivars and growing conditions are different
between locals. Accordingly, an investigation on the health promoting
properties of Ontario grown onions, specically in regards to their cyto-
toxic and cytostatic bioactivity, is merited. Caco-2 as our cell line of in-
terest because it is extensively used as a model for human intestinal
epithelium and is the work horse of the pharmaceutical industry for
in-vitro toxicology studies (Sambuy et al., 2005; Yamazaki et al.,
2014). Therefore, we investigated the bioactive properties of ve Ontar-
io grown varieties of onion and assessed their capacity to exert cytostat-
ic/cytotoxic effects on a relevant human colorectal cancer cell line.
2. Materials and methods
2.1. Materials
Quercetin (purity: pharmaceutical secondary standard), myricetin
(purity 96%), kaempferol (purity N 98%), MEM (minimum essential
medium), FBS (fetal bovine serum), L-glutamine, and D-glucose were
purchased from Sigma Aldrich (St Louis, MO). Phenol free DMEM
(Dulbeccos' modied eagle's medium) and 100 penicillin-streptomy-
cin solution were obtained from GE Healthcare Life Sciences (Logan,
UT). Lactate dehydrogenase (LDH) cytotoxicity and MTS cell prolifera-
tion assay kits were purchased from BioVision Inc. (Milipitas, CA). A
TrypLE express and Image-iT lipid peroxidation kit was purchased
from Invitrogen (Carlsbad, CA). The ORIS cell migration assay kit
was acquired from Platypus Technologies (Madison, WI). Five Ontario
grown onion varieties were graciously donated by the Holland Marsh
Growers' Association (Bradford, Ontario).
2.2. Phytochemical extraction using pressurized low polarity water
Low polarity water extractions of ve Ontario grown onion varieties
(Lasalle, Fortress, Safrane, Stanely, and Ruby Ring) were performed.
Chopped onions were lyophilized, and 5 g of freeze-dried onion powder
was mixed with 80 mL of 0.1% formic acid in milliQ water (v/v). An au-
tomated Speed SFE NP model 7100 instrument (Applied Separation Inc.,
Allentown, PA) equipped with a module 7100 pump and a 10 mL thick-
walled stainless cylindrical extractor vessel was used for the extractions.
Phytochemical extraction of the onions using pressurized low polarity
water extraction was performed under the following conditions: 60 C
at 150 bar for 60 min.
2.3. Total avonoid content
The total avonoid content assay was performed by aluminum chlo-
ride colorimetric assay. 0.5 mL of the total extracted onion test samples
were mixed with 0.1 mL of 10% aluminum chloride and 0.1 mL of 1 M
potassium acetate. Followed by the addition of 2.8 mL of deionized
water. The test samples were then kept for incubation at room temper-
ature for 30 min. The optical density (OD) was then measured using 96
well plate by a plate reader (ELISA plate reader (Amersham Biosciences
Corp., USA) at 415 nm. The total avonoid content was expressed as mg
quercetin equivalent (QE)/g dry plant sample.
2.4. Cell culture
The Caco-2 strain, a human colorectal adenocarcinoma cell line, was
purchased from the American Type Culture Collection (Rockville, MD).
Cells were maintained in MEM media supplemented with 4 mM L-glu-
tamine, 1% non-essential amino acids, 10% heat inactivated fetal bovine
serum, and 1% 100 penstrep solution (nal concentration of
100 IU/mL penicillin and 100 g/mL streptomycin). D-glucose was sup-
plemented at a nal concentration of 4 g/L. Caco-2 cells were
13
propagated in a 75-cm2 culture ask at 37 C and 5% CO2 in a humidied
atmosphere. Media was refreshed three times per week, and cells were
passaged at approximately 80% conuency. Cells were harvested using
Tryple Express and then collected using centrifugation at 200 g for
5 min. Cells at passage numbers 1820 were used for the experiments.
2.5. Colorimetric quantication of Caco-2 cell proliferation
The cytostatic capacities of the onion extracts were determined by
measuring Caco-2 proliferation using the MTS colorimetric assay. Brief-
ly, Caco-2 cells were seeded in a tissue culture-treated 96-well plate at a
density of 1.5 104 cells per well. Cells were left undisturbed for 24 h to
allow for attachment. After 24 h, a total of 100 L of the sample extracts
containing the growth media were subsequently added. Flavonoids
quercetin, myricetin, and kaempferol were used as positive controls to
assess the cytostatic properties of the onion extracts. The concentration
of pure avonoids used for the present study was 100 M. The avo-
noids were dissolved in DMSO and further diluted in cell culture
media. The nal concentration of DMSO was 0.5% (v/v). The 96-well
plate was incubated for 72 h and was gently washed with PBS prior to
the addition of the MTS dye. Absorbance was measured at 490 nm
using an xMark spectrophotometer (Bio-rad; Hercules, CA) after a 3 h
incubation period with MTS dye.
2.6. Determination of cytotoxicity and cytolysis
Lactate dehydrogenase (LDH) supernatant levels were assessed as
an indicator of Caco-2 cell death and were used as a measure of cytotox-
icity activity. Caco-2 cells were seeded into a tissue culture-treated, 96-
well plate at a density of 1.5 104 cells per well. The plate was undis-
turbed for 24 h to allow for cell attachment. The media was then aspirat-
ed and replaced with media containing onion extracts at different
concentrations. LDH activity was determined 72 h post-treatment. The
plate was centrifuged at 600g for 10 min to precipitate any cells and
cellular debris. Ten L of the debris free medium was transferred to a
96-well plate. To assess LDH levels, 100 L of a reaction mixture solution
containing a water soluble tetrazolium salt dye was added to the super-
natant samples. Absorbance was read using an xMark spectrophotome-
ter (Bio-rad; Hercules, CA) after a 30 min incubation period at 450 nm.
2.7. Induction of apoptosis in Caco-2 cells
Expression of the executioner caspases, caspases 3 and 7, was used
to determine the presence of bioactive properties capable of inducing
apoptosis. Briey, cells were seeded in a black, clear bottom 96 well
plate at 1.5 104 cells per well. The plate was left undisturbed for
48 h, after which the media was aspirated. Media containing the various
extract treatments was added as previously described. After 24 h treat-
ment incubation, the treatment medium was replaced with 100 L of
phenol free RPMI:F12 (1:1 ratio) medium supplemented with 5% FBS,
2 mM L-glutamine, and the CellEvent Caspase-3/7 Green Detection
Agent at a nal concentration of 7.5 M. Cells were incubated for
30 min. High content imaging was performed using a Cytation 5 multi-
mode plate reader (Biotek, Winooski, VT).
2.8. Cell migration
Measurements of cell motility were used to determine the efcacy of
extract treatments in inhibiting the invasive progression of Caco-2 cells.
The Oris cell migration assay kit consists of a 96-well plate with round
bottom silicone stoppers which create a 2 mm diameter exclusion zone.
Caco-2 cells were seeded at 1 105 cells/well and were allowed to at-
tach for 24 h. After the incubation period, the silicone stoppers were
gently removed, and the plate was imaged to capture the exclusion
zone at time 0. Media containing the extract treatments was then
added to the growth media. The plate was incubated for another 72 h,
14 A.I. Murayyan et al. / Food Research International 96 (2017) 1218
after which high content imaging of the exclusion zones was performed
using the Cytation 5 multimode plate reader.
2.9. Statistical analysis
One-way ANOVA using R Open Source Statistical Programming soft-
ware was performed to assess the difference between the groups. Com-
parisons among treatments were calculated using Tukey's honestly
signicant differences as a post hoc test. P-values b 0.05 were consid-
ered signicant. The data was reported as mean standard deviation.
3. Results and discussion
3.1. Total avonoid content assay
We have recently characterized the avonoid proles as well as an-
tioxidant activities of each of the ve onion varieties (Manohar, Xue,
Murayyan, Neethirajan, & Shi, 2017). We found that the Stanely and
Ruby Red varieties contained the highest avonoid content, while all va-
rieties contained readily detectable amounts of avonoids (Fig. 1). For-
tress, a yellow onion variety, showed the highest total avonoid content
at 0.33 mg of quercetin equivalent per gram of dried onion (P b 0.05). A
2-fold difference was observed in the total avonoid content between
the tested onion varieties Stanley (highest ranked) and Lasalle (lowest
ranked). These results are consistent with prior observations that on-
ions are rich in avonoid compounds.
3.2. MTS cell proliferation assay
To then assess the potential anti-cancer properties of these onion a-
vonoids, we treated a relevant and commonly used immortalized
human cancer cell line (Caco-2) with our prepared onion extracts. Be-
cause unchecked proliferation is a hallmark of cancer and a chief prop-
erty contributing to the spread of and damage caused by human
cancers, we rst assessed the ability of the onion extracts to slow or in-
hibit cellular proliferation by Caco-2 cells. We tested three concentra-
tions of onion extract (a 1:10, 1:50, and 1:100 dilutions of each
extract), because it was unclear what concentration may be necessary
to inhibit proliferation. We found that higher dilutions (1:50 and
1:100) were unsuccessful in inhibiting Caco-2 cell proliferation (data
not shown). However, a 1:10 dilution (10 L of sample extract with
90 L of growth media) of each extract showed a signicant effect of
proliferation as determined by MTS assay (Fig. 2). A 6980% decrease
in absorbance was observed compared to the untreated control wells,
indicating a strong antiproliferative effect of the onion extracts against
Fig. 1. Total avonoid content of the Lasalle (LAS), Fortress (FOR), Ruby Ring (RR), Stanley
(STA), and Safrane (SAF) varieties; (n = 5; P b 0.05).
Fig. 2. Cell proliferation assay for UNTRT: Untreated; LAS: Lasalle; RR: Ruby Ring; FOR:
Fortress; STA: Stanley; SAF: Safrane; QUE: Quercetin (100 M); MYR: Myricetin
(100 M); KAE: Kaempferol (100 M); Data shown as means S.D. (n = 3).
Treatments not sharing a common letter differ signicantly from each other (P b 0.05)
by one-way ANOVA followed by Tukey's post hoc test.
Caco-2 cells. All onion extracts (at a 1:10 dilution) exerted a strong
and signicant anti-proliferative effect on Caco-2 cells (N65% reduction
in absorbance). Furthermore, we determined that DMSO alone (the a-
vonoid delivery vehicle) had no impact on proliferation, indicating the
effects of the onion extracts were due to the compounds present in
the onions themselves. Despite the differences in phytochemicals com-
position (e.g. varying avonoid contents), no statistical differences in
proliferation inhibition among the ve onion varieties were observed.
Furthermore, the crude onion extracts were as successful in inhibiting
proliferation as the commercially obtained puried avonoids (e.g.
myricetin). Isolation of pure compounds from food products can be ex-
tremely costly and time consuming, generating large volumes of waste
materials, the end result of which is high production costs and a great
expense to patients (Ovadje et al., 2015). Thus, an ideal natural prod-
uct-derived agent would require minimal processing to exert its impact.
Our results suggest that these onion extracts may be successful in
inhibiting proliferation without further renement, while further explo-
ration into diet modication may reveal other effective strategies for re-
ducing cancer risk.
Our ndings that onion extracts impede cellular proliferation in
Caco-2 cells are consistent with previous reports. It is highly likely
that treatment with onion extracts impacts cell cycle progression, thus
accounting for the cytostatic properties of these extracts. Bioactive a-
vonoid and organosulfur compounds found in onions have been report-
ed to impact signal transduction pathways, leading to cell cycle arrest in
the G1 and G2/M phases when used to treat various cancer cell lines
(Herman-antosiewicz & Singh, 2004; Nicastro, Ross, & Milner, 2015).
Several studies have elucidated the different mechanisms causing cell
cycle arrest for different cancer cell lines, which include the down-reg-
ulation of cyclin (Jeong, An, Kwon, Rhee, & Lee, 2009) and M-phase in-
ducer protein expression (Xiao, Pinto, Gundersen, & Weinstein, 2005),
the disruption of microtubule formation (Xiao et al., 2005), and the de-
creased polyamine synthesis necessary for cellular proliferation
(Linsalata et al., 2010).
3.3. Cytotoxic assay
We went on to explore the potential cytotoxic effects of our onion
extracts using lactate dehydrogenase assays. The detection of lactate de-
hydrogenase (LDH), an enzyme intrinsic to the cytosol, is commonly
used as an indicator of compromised membrane integrity and
 A.I. Murayyan et al. / Food Research International 96 (2017) 1218
ultimately cell viability. In the MTS proliferation assay, MTS is converted
by viable cells into a compound measureable by absorbance reading.
Therefore, a reduction in absorbance in the MTS assay with onion ex-
tract treatment could also denote changes in cell viability. As expected,
we found that LDH levels increased with onion extract treatment (ver-
sus untreated controls), indicating that these extracts also possess cyto-
toxic effects. Similar to results from the proliferation assays, the extracts
were most effective at a 1:10 dilution (Fig. 3), showing no signicant
differences at a 1:50 or 1:100 dilutions (data not shown). A 125228%
increase in LDH release was observed for treatments prepared at a
1:10 dilution. However, unlike the proliferation assays, we found
measureable differences in the cytotoxic effects among the ve onion
varieties, which may reect extract composition. A 228% increase in
the ratio of LDH release was induced by Fortress and Stanley extracts,
which represented the most potent cytotoxic extracts. This ratio was
similar to that exhibited by the control avonoids quercetin and
kaempferol. The remaining three onion variety extracts exhibited a
lower LDH release ratio, which was similar to the myricetin-treated
cells, although all extracts induced a signicant amount of cytotoxicity
compared to untreated controls.
The differences in total avonoid content among the tested onion
varieties cannot fully account for why the Fortress variety, with contains
only half as much total avonoid content as the Stanley variety, has a
similar effect on cell viability as assessed by LDH assay. Furthermore,
the Ruby Ring extract performed similarly to varieties with the lowest
total avonoid content, although it contains one of the highest concen-
trations of avonoids. Therefore, it is very likely that other bioactive
compounds, such as organosulfur and/or phenolic compounds contrib-
ute to avonoid effects or may have a more pronounced effect than the
avonoids themselves. Due to the heterogeneous nature of the extracts,
it may also be possible that the presence of a currently unidentied phy-
tochemical may play a role in exerting these effects. Future studies could
be performed to isolate the active components in these extracts, which
may have more potent effects when administered in higher concentra-
tions as pure compounds. However, as far as a therapeutic is concerned,
isolated compounds may be signicantly less cost effective and may not
show greater efcacy; some natural product extracts are more effective
as mixtures, due to the potential synergistic or additive effects of the in-
dividual compounds present within the mixture (Liu et al., 2014).
Fig. 3. Cytotoxic effect for UNTRT: Untreated; LAS: Lasalle; RR: Ruby Ring; FOR: Fortress;
STA: Stanley; SAF: Safrane; QUE: Quercetin (100 M); MYR: Myricetin (100 M); KAE:
Kaempferol (100 M); Data shown as means S.D. (n = 3). Treatments not sharing a
common letter differ signicantly from each other (P b 0.05) by one-way ANOVA
followed by Tukey's post hoc test.
15
3.4. Induction of apoptosis in Caco-2 cells
Although LDH assays are reasonable measures of cell viability, they
do not directly quantify the number of apoptotic cells/events. Apoptosis,
or programmed cell death, is a highly organized and tightly regulated
sequence of biochemical events leading to morphological changes and
eventually cell death. The activation of executioner protease caspases,
namely caspases 3 and 7, is key path shared by both the intrinsic and ex-
trinsic apoptotic pathways (Reed, 2003; Boatright & Salvesen, 2003).
The evasion of programmed cell death, due to oncogenic mutations in
malignant cells, is one of the hallmarks of cancer (Hanahan &
Weinberg, 2011). These mutations result in defects in the signaling
transduction pathway of the apoptosis signaling cascade, giving rise to
the chemotherapeutic resistance of malignant cells (Johnstone, Ruei,
& Lowe, 2002; Lowe & Lin, 2000). Consequently, induction of apoptosis
by a therapeutic agent would likely be instrumental in controlling the
proliferation and metastasis of cancer cells. If a greater number of can-
cerous cells are pushed down the apoptotic pathway, this would help
to stop proliferation and neoplastic growth. Therefore, we also assessed
the ability of our onion extracts to induce the apoptosis of Caco-2 cells.
We found that all extracts were successful in inducing apoptosis at a di-
lution of 1:10 compared to the untreated or DMSO control (Fig. 4). Con-
sistent with previous assays, 1:50 and 1:100 dilutions of our extracts did
not signicantly induce apoptosis (data not shown). Overall, a 3.54-
fold increase in the number of apoptotic cells was observed for nearly
all treatments (versus untreated control). The Stanley extract was
most potent at inducing cell death and was followed by the Lasalle,
Ruby Ring, Fortress, and Safrane extracts. A very promising nding
was that on average, the number of apoptotic events induced by the ex-
tract treatments was higher than the number of events induced by our
positive controls quercetin and myricetin.
Several proposed mechanisms of actions for phytochemicals, which
affect the transduction signaling pathways involved in apoptosis, have
been documented in literature and are dependent not only on the cellu-
lar model, but on the phytochemical involved in the study. Dietary a-
vonoids found in onions have been shown to down-regulate the
elevated levels of fatty acid synthase expression in cancer cells,
compromising their ability to convert acetyl-CoA to fatty acids and neg-
atively impacting cell membrane functions leading to cell death
Fig. 4. The number of apoptotic cells after treatment with onion extracts (1:10 dilution).
Wells were seeded with 1.5 104 cells and left undisturbed for 48 h. Following this
incubation period, the number of apoptotic cells were measured after a 24-h dosing
period. UNTRT: Untreated; LAS: Lasalle; RR: Ruby Ring; FOR: Fortress; STA: Stanley; SAF:
Safrane; QUE: Quercetin (100 M); MYR: Myricetin (100 M); Data shown as means
S.D. (n = 6). Treatments not sharing a common letter differ signicantly from each
other (P b 0.05) by one-way ANOVA followed by Tukey's post hoc test.
16 A.I. Murayyan et al. / Food Research International 96 (2017) 1218
(Brusselmans, Vrolix, Verhoeven, & Swinnen, 2005). In other studies,
avonoids have been shown to promote the expression of pro-apopto-
tic genes (Wenzel, Kuntz, Brendel, & Daniel, 2000) as well as down-reg-
ulate the expression and activation of nuclear transcription factor-,
essential for inhibiting apoptosis (Wenzel et al., 2000; Ban et al.,
2007). A review by Herman-Antosiewicz and Singh (2004) on allium-
derived organosulfur compounds nicely summarizes the possible
modes of apoptosis induction, including those suggested by avonoid
studies. The authors cited the disruption of intracellular calcium homeo-
stasis, the down-regulation of the anti-apoptotic Bcl-2 family of genes,
increased expression of pro-apoptotic genes, and the generation of reac-
tive oxidation species facilitating the release of cytochrome c (and sub-
sequent activation of cytochrome c and caspase 3) as possible modes of
apoptosis induction by organosulfur compounds. Further characteriza-
tion of our extracts would be necessary to determine the precise
modes of actions of their constituent parts. Here, we provide clear evi-
dence that our onion extracts induce apoptosis, but additional study
would be warranted to determine the mechanisms of action. Because
these onion extracts are a heterogeneous mixture of different bioactive
molecules (e.g. avones, organosulfur compounds, and phytosterols), it
is possible that any 1 or a combination of these is responsible for trigger-
ing apoptosis.
Another biological hallmark of cancer cells is their ability to invade
and metastasize from primary neoplasms, which can go on to colonize
other (even, more distant) body sites (Hanahan & Weinberg, 2011).
Ninety percent of all cancer-related mortalities are a consequence of
the metastasis of primary tumors to other parts of the body (Spano,
Heck, De Antonellis, Christofori, & Zollo, 2012; Monteiro & Fodde,
2010). The series of events where primary neoplasms colonize distal
sites in the body is called the metastatic cascade (Nguyen, Bos, &
Massaque, 2009; Mehlen & Puisieux, 2006). While some phytochemi-
cals have been shown to possess cytostatic and cytotoxic properties in
vitro, not all exert a bioactive effect in modulating the mechanisms re-
sponsible for the metastasis of cancer cells (Caltagirone et al., 2000).
Limiting the ability of cancer cells to migrate within their microenviron-
ment, as well as from their primary sites, would greatly reduce the risk
of mortality associated with metastasis. Therefore, we investigated the
abilities of our onion extracts to impair the cellular motility of Caco-2
cells.
3.5. Anti-migratory assay
We found that the onion extracts elicited different anti-migratory ef-
fects against Caco-2 cells. Fig. 5 shows the number of cancer cells detect-
ed in the exclusion zone after a 72 h dosing period. Stanley extracts
performed the best at inhibiting cell migration with a 60% decrease in
the number of cells in the exclusion zone compared to the untreated
control. This was followed by Lasalle (56% decrease), Fortress (49% de-
crease), Ruby Ring (47% decrease), and Safrane (40% decrease).
Myricetin did not inhibit Caco-2 cell migration into the exclusion
zone. Factors including cell cycle arrest/decreased proliferation and cy-
totoxicity/apoptosis may have contributed to reductions in migration,
as it counts the number of cells migrating to the exclusion zone,
which means non-viable cells will not be counted. Representative im-
ages of Caco-2 cells treated with Lasalle extracts and quercetin are
shown in Fig. 6. This data correlates with our apoptosis data, showing
the same order of efcacy among the different extracts. In support of
our observation, avonoids have an established role in disrupting can-
cer cell migration/metastasis; avonoids, such as quercetin, have been
shown to disrupt microtubule formation (Gupta & Panda, 2002), rear-
range actin microlaments in the cytoskeleton (Psahoulia et al., 2007),
and modulate enzymes and signaling pathways associated with the me-
tastasis cascade (Weng & Yen, 2012; Pan, Lai, Wu, & Ho, 2011; Park et al.,
2005). The complex mixture of compounds presents in the onion ex-
tracts, with varying effects on proliferation and viability, likely contrib-
utes to their observed anti-migratory effects. The anti-migratory effect
Fig. 5. Anti-migratory activity of the onion extracts. UNTRT: Untreated; LAS: Lasalle; RR:
Ruby Ring; FOR: Fortress; STA: Stanley; SAF: Safrane; QUE: Quercetin (100 M); MYR:
Myricetin (100 M); KAE: Kaempferol (100 M); Data shown as means S.D. (n = 4).
Treatments not sharing a common letter differ signicantly from each other (P b 0.05)
by one-way ANOVA followed by Tukey's post hoc test.
of Safrane on Caco-2 cells was similar to the effect of kaempferol, but
was statistically different than the effect of quercetin. However, the
other varieties were more effective in impeding Caco-2 migration
with a 2342% and 1334% reduction in the number of observed cells
compared to quercetin and kaempferol, respectively. Interestingly,
myricetin did not inhibit Caco-2 migration, even though it exerted cyto-
static and cytotoxic effects. Therefore, it is plausible that myricetin can-
not modulate the signaling pathways associated with the metastasis
cascade and/or disrupt the cytoskeletal microstructures responsible
for structural support and cellular motility. It is recognized that the re-
sults of this study is limited to colonic cell line only. Absorption of avo-
noids may be poor in an in-vivo setting and hence the relevance of
transformed cells in peripheral tissues would be unknown. Unabsorbed
compounds may enter large intestine where that may be bio-trans-
formed by microbiota. Such metabolites may be active against trans-
formed cells in colon and absorbed where they may have similar
activity.
4. Conclusions
Here we show that extracts created from ve different Ontario
grown onion varieties are effective in inducing cytotoxic, cytostatic,
and anti-migratory activities in a human colorectal adenocarcinoma
cell line. These extracts signicantly induced apoptosis, reduced the
rate of proliferation, and slowed the migration of cancer cells into an ex-
clusion zone. Importantly, these effects were at least comparable, and
often stronger, than those of the commercially available pure avonoid
compounds. Moving forward, we are continuing to conduct studies to
elucidate the possible mode of action of these extracts against Caco-2
cells and to determine which active agents are most important for
these properties. These data represent an important step in characteriz-
ing the anti-cancer properties of avonoids and provide further evi-
dence for their inclusion in dietary treatments. Although positive
results were observed at the highest tested concentration (1:10 dilution
of onion extracts), the marked success of our crude onion extract sup-
ports the notion that dietary supplementation or even the use of a fairly
inexpensive onion extract could have potent anti-cancer effects. Further
in vivo and clinical trials however, would be required to conrm the ef-
cacy of the onion extracts in preventing tumor development.
 A.I. Murayyan et al. / Food Research International 96 (2017) 1218
Fig. 6. Cell migration assay. Figure shows cells (green) migrating the exclusion zone A) Exclusion zone of untreated cells at 0 h B) Caco2 cells treated with Lasalle at 0 h D) Caco2 cells
treated with Quercetin at 0 h E) untreated cells at 72 h F) Caco2 cells treated with Lasalle at 72 h G) Caco2 cells treated with Quercetin at 72 h. (For interpretation of the references to
colour in this gure legend, the reader is referred to the web version of this article.)
Acknowledgments
The authors sincerely thank the Natural Sciences and Engineering
Research Council of Canada (Grant 400929) and the Ontario Ministry
of Agriculture, Food and Rural Affairs (Grant 300513) for funding this
study. The authors also thank the Holland Marsh Growers Association
for providing Onion samples. The authors appreciate the onion extracts
samples provided by Dr. John Shi of the Agriculture and Agri-Food
Canada.
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