Journal of Functional Foods 33 (2017) 112–121

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Journal of Functional Foods

journal homepage: www.elsevier.com/locate/jff

Carob leaf polyphenols trigger intrinsic apoptotic pathway and induce

cell cycle arrest in colon cancer cells
Fatima Zahra Ghanemi a,b, Meriem Belarbi b, Aurélie Fluckiger a, Abdelhafid Nani a,b,c,⇑, Adélie Dumont a,
Charlotte De Rosny a, Ikram Aboura a,b, Amira Sayed Khan a, Babar Murtaza a, Chahid Benammar b,
Boucif Farid Lahfa d, Danish Patoli a, Dominique Delmas a, Cédric Rébé a,e, Lionel Apétoh a,
Naim Akhtar Khan a, François Ghringhelli a,e, Mickael Rialland a, Aziz Hichami a,⇑
a
INSERM U1231, Université de Bourgogne Franche-Comté, 21000 Dijon, France
b
Laboratory of Natural Products, University of Abou-Bekr Belkaid, Tlemcen 13000, Algeria
c
Department of Natural and Life Sciences, African University Ahmed Draia, Adrar, Algeria
d
Laboratory of Antibiotics Anti-fungals: Physical-Chemistry, Synthesis and Biological Activity, University of Abou-Bekr Belkaid, Tlemcen 13000, Algeria
e
Centre Georges François Leclerc, Dijon 21000, France

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

Article history: Chemoprevention of Colorectal cancer (CRC) is the major concern for improving public health. We inves-
Received 17 October 2016 tigated the protective effects of carob, Ceratonia siliqua L., leaf polyphenols (CLP) against CRC. Phenolic
Received in revised form 17 February 2017 content analysis showed that CLP is enriched with gallic acid and m-coumaric acid. We observed that
Accepted 14 March 2017
CLP exerted a dose dependent cytotoxic effect through the induction of apoptosis on CRC cell lines, with
an IC50 around 20 lg/mL. CLP induced intrinsic apoptotic pathway through the caspase-9 activation and
PARP cleavage in HCT-116 and CT-26 cells. Moreover, CLP induced cell cycle arrest in the G1 phase
Chemical compounds studies in this article:
through p53 activation. Gallic acid and m-coumaric acid exerted similar effect of CLP but at higher con-
Chlorogenic acid (PubChem CID:1794427)
Gallic acid (PubChem CID: 370)
centration to that expected to be present in CLP, suggesting the synergistic effect between polyphenols in
m-Coumaric acid (PubChem CID: 637541) CLP activity. Interestingly, the carob leaf infusion reduced CT-26 tumour growth in BALB/c mice. This
p-Coumaric acid (PubChem CID: 637542) study suggests that CLP can be used for the prevention of CRC.
Quercetin (PubChem CID: 5280343) Ó 2017 Elsevier Ltd. All rights reserved.
Syringic acid (PubChem CID: 10742)

Keywords:
Carob (Ceratonia siliqua L.)
Polyphenols
Colorectal cancer
Caspases
Apoptosis
Cell cycle arrest

1. Introduction about 85% of all cases of CRC (Vasen, Tomlinson, & Castells,
2015). Currently, in addition to antitumor drugs used in
Colorectal cancer (CRC) is the third most common form of can- chemotherapy regimens, there is an increasing interest in plant-
cer which causes one million of worldwide deaths every year derived bioactive compounds that might have a role in the man-
(Siegel, Ma, Zou, & Jemal, 2014). Epidemiological studies have agement or the prevention of cancer or enhancement of sensibility
demonstrated that the sporadic development of colon cancer is to chemotherapy (Stoner & Mukhtar, 1995).
tightly linked to dietary habits and lifestyle changes representing The crucial role of selectivity in cancer chemotherapy may be
found in pharmacological targeting of natural molecules avoiding
cytotoxicity against normal cells (Martin, Goya, & Ramos, 2013).
⇑ Corresponding authors at: INSERM U1231, Université de Bourgogne Franche- Polyphenols were described to be beneficial against human dis-
Comté, 21000 Dijon, France (A. Hichami) and Laboratory of Natural Products, eases such as cancer and/or metastasis (Stoner & Mukhtar, 1995).
University of Abou-Bekr Belkaid, Tlemcen 13000, Algeria (A. Nani).
Indeed, evidences from in vitro and in vivo studies have shown that
E-mail addresses: nani.abdelhafid@univ-adrar.dz (A. Nani), aziz.hichami@
u-bourgogne.fr (A. Hichami).
polyphenol-rich food consumption is associated with a less risk for

http://dx.doi.org/10.1016/j.jff.2017.03.032
1756-4646/Ó 2017 Elsevier Ltd. All rights reserved.
 F.Z. Ghanemi et al. / Journal of Functional Foods 33 (2017) 112–121 113

cardiovascular disease and cancers (Blumberg et al., 2013; Pappas this purpose, the extract was dried under nitrogen and re-
& Schaich, 2009). Apoptosis, or programmed cell death, is an suspended in culture medium at desired concentration.
important physiologic process in the normal development, and The infusion was prepared using 1 g of carob leaf powder with
induction of apoptosis is a highly desirable mode as a therapeutic 100 mL of boiled water for 15 min, the solution was then filtered
strategy for cancer control (Hu & Kavanagh, 2003). (Corsi et al., 2002).
Ceratonia siliqua L., commonly known as carob, belongs to the Total phenolic contents in the organic extract and infusion of
family of Leguminosae. The nutritional quality of carob flour carob leaves were determined by Folin-Ciocalteu method (Folin &
encouraged its use as food additive material (Biner, Gubbuk, Ciocalteu, 1927). The phenolic contents were calculated with refer-
Karhan, Aksu, & Pekmezci, 2007). Carob pods have traditionally ence to the standard curve and the results were expressed as mg of
been used for animal and human food purposes, and the seeds gallic acid equivalents (GAE)/g of dry matter (mg GAE/g).
are mainly used for gum extraction. The leaves and fruits of this
plant are frequently used to cure various diseases. Several studies 2.3. HPLC analysis
have been conducted on different parts of carob tree for their con-
tent of bioactive compounds, including polyphenols, which exhibit Phenolic extract and infusion were analyzed by HPLC (Model
antioxidant (Hsouna et al., 2011), hypo-glycemic (Mokhtari, Agilent Technologies 1260, Germany) with reverse phase Zorbax
Sharifi, & Shahamir, 2011), anxiolytic-sedative (Avallone, Eclipse XDB-C18 column (4.6  100 mm) and a diode array UV-
Cosenza, Farina, Baraldi, & Baraldi, 2002), antimicrobial (Aissani, detector (operating at 280 nm). The mobile phase used was 1%
Coroneo, Fattouch, & Caboni, 2012), and nephroprotective proper- acetic acid in (A) water vs (B) methanol. Phenolic acid separation
ties (Ahmed, 2010). was achieved using a 35 min linear solvent gradient at a flow rate
Antitumor and anti-proliferative activities of carob pod extract of 0.5 mL/min, as follows: 0 min 90% A, 5 min 80% A, 10 min 70% A,
has been widely studied; Klenow, Glei, Haber, Owen, and Pool- 15 min 50% A, 20 min 30% A, 25 min 10% A, 30 min 50% A, 35 min
Zobel (2008) and Klenow and Glei (2009) reported that aqueous 90% A. Phenolic compounds from carob were identified with refer-
carob fiber extract inhibited human colorectal cell line prolifera- ence to retention time of authentic standards (i.e. gallic, m-
tion (HT29 and LT97). Custódio et al. (2011) demonstrated that coumaric, p-coumaric, chlorogenic, and syringic acid, quercetin
methanol extract of carob fruit pulps reduced viability of breast and quercetin 3-O- rutinoside) and quantified on the basis of their
cancer (MDA-MB-231) and cervical cancer (HeLa) cell lines. How- peak area.
ever, anticancer properties of carob leaf extract are less docu-
mented. Indeed Custódio et al. (2011) observed that methanol 2.4. Cell lines and cell culture
extract of carob leaf exerted cytotoxic, anti-proliferative and anti-
cancer activities on HeLa cervical cancer cells. Also, Corsi et al. HCT-116, SW-480, HT-29 human cancer cell lines and CT-26
(2002) reported that lyophilised carob leaf infusion induced apop- mouse cancer cell line were purchased from ATCC (USA). Cells
tosis in mouse hepatocellular carcinoma cell line (T1). Since a few were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM)
studies have focused on the inhibitory effect of carob leaf polyphe- high glucose (Dutscher, France) supplemented with 10% (v/v) of
nols (CLP) on colorectal cancer, the present study was designed to heat-inactivated fetal bovine serum, 1% (v/v) mixture of strepto-
investigate the anti-tumour activity of CLP and to characterize the mycin, amphotericin and penicillin (PAN-Biotech, Germany). Cells
molecular mechanisms by which CLP exerted its apoptotic effect were incubated at 37 °C in a 5% CO2 humidified atmosphere.
and cell cycle arrest in colorectal cell lines.
2.5. Cell viability assay

2. Materials and methods The cell viability was investigated by the trypan blue exclusion
analysis. Briefly, 5  104 cells were seeded into 24-well plate. Cells
2.1. Plant material were incubated for 24 h with increasing concentrations of CLP
(from 5 to 100 lg/mL). After the indicated time in normal culture
Carob leaves were collected from the region of Nedroma (56 km conditions, cells were trypsinized and diluted in 0.5% trypan blue.
in the North-West of Tlemcen city, Algeria) in Mars 2014. The plant Cells were then counted with a Malassez hemocytometer under
was recognized by Pr Benabadji Nouri, a botanist in Tlemcen the light microscope ‘‘Nikon”.
University (Algeria). A voucher specimen (CS 1724) was deposited
at Herbarium Center of the Faculty of Pharmacy (Tlemcen). 2.6. Apoptosis assay

Cells (2  105) were seeded in a 6-well plate. After 24 h, the

2.2. Determination of total phenol content medium was changed and HCT-116 and CT-26 cells were pre-
incubated either with vehicle or with 50 lM of N-benzyloxycarbo
Carob leaf polyphenols (CLP) were obtained using organic sol- nyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) for 1 h, and
vent extraction according to the method described by Liyana- then treated for 24 h with different treatments combined or not
Pathirana and Shahidi (2006) slightly modified by Nani et al. with 5-fluorouracil (5-FU, 10 lM): CLP (10 lg/mL and 20 lg/mL),
(2015). Briefly, 2 g of carob leaf powder were extracted two times (gallic acid 460 lM), m-CA (10 mM). Similarly, HT-29 and SW-
(2 h for each extraction) with 40 mL of methanol–acetone–water 480 were also treated for 24 h with different treatments combined
(7:7:6, v/v/v) at room temperature (25 ± 2 °C) with constant stir- or not with 5-fluorouracil (5-FU, 10 lM): CLP (10 lg/mL and 20 lg/
ring. The mixtures were then centrifuged for 20 min at 4000g mL), (gallic acid 460 lM). Floating and adherent cells were
and supernatants were collected and combined, then extracted detached and harvested into 15 mL Falcon tubes and washed twice
with the same volume of hexane to remove low or non-polar with cold PBS by centrifugation for 5 min at 1500  g. Subse-
hydrophobic compounds including fatty acids and neutral lipids. quently, the supernatant was removed and cell death was deter-
After lipid removal, the mixture was evaporated under reduced mined by APC-Annexin V and 7-amino-actinomycin D (7-AAD)
pressure until dryness, and the dry extract was recovered in abso- staining with BioLegend’s APC-Annexin V Apoptosis Detection Kit
lute methanol, to prevent polyphenol hydrolysis. This organic according to the manufacture’s recommendations. Cells were incu-
extract was stored at 20 °C for further in vitro experiments. For bated in the labelling solution for 20 min at room temperature in
114 F.Z. Ghanemi et al. / Journal of Functional Foods 33 (2017) 112–121

darkness before samples’ analysis by flow cytometry (FACSCalibur, 2.11. Statistical analysis
Becton Dickinson) and data interpretation using FlowJo software
version 10 (Tree Star). Results were expressed as mean ± SD (standard error deviation)
for a given number of experiments (n). Data were analyzed using
2.7. Detection of caspase-3 and caspase-7 activities Statistica (4.1 version, Statsoft, Paris, France). The significance of
differences between mean values was determined by one-way
FAM-DEVD-FMK FLICA reagent which labels active caspase-3 ANOVA, followed by Fisher’s least-significant-difference (LSD) test.
and -7 was used according to the manufacturer’s protocol Differences with p < 0.05 were considered to be significant.
(Immunochemistry Technologies, Bloomington, MN, USA). Briefly,
CT-26 and HCT-116 cells were seeded at density of (2  105) into 3. Results
6-well plate and treated for 24 h with CLP (10 lg/mL and 20 lg/
mL). Cells were then harvested and stained with caspase 3/7- 3.1. Polyphenols content
FAM-DEVD-FMK solution. After 1 h of incubation at 37 °C, cells
were washed twice with wash buffer, and then pellets were re- Polyphenolic contents of organic extract and infusion of carob
suspended in 1% 7-AAD and incubated for 10 min. Caspase-3 and leaves were 9.215 and 2.25 mg gallic acid equivalents/g dry
caspase-7 activities were detected by flow cytometry (FACS Cal- weight, respectively. HPLC analysis (Fig. 1A) showed that organic
ibur, Becton Dickinson) and data were analyzed using FlowJo soft- extract contained high contents of m-coumaric acid (2192.38 lg/
ware version 10 (Tree Star). g DW) and gallic acid (1445.38 lg/g DW) (Table 1). Whereas, HPLC
analysis of infusion (Fig. 1B) revealed the presence of all polyphe-
2.8. Cell cycle assay nols observed in organic extract and m-coumaric acid represented
the major polyphenol in the infusion (1423.98 lg/g DW) (Table 1).
HCT-116 and CT-26 cells were seeded into a 6-well plate
(1.2  105 cells/well) and treated similarly as described above in
3.2. Cell viability
apoptosis assay. Briefly, cell pellets were fixed in ethanol 70% at
20 °C overnight. Cells were then washed twice with wash buffer,
Trypan blue exclusion test showed that CLP significantly
stained with propidium iodide (Immunochemistry, USA) and trea-
reduced colon cancer cell lines viability in a dose-dependent man-
ted with RNase A (Euromedex, France) for 1 h in darkness at 37 °C.
ner (Fig. 2). IC50 was estimated to be 5 lg/mL for CT-26, 20 lg/mL
Cells were assessed for their DNA contents by cytometry on
for both of HCT-116 and SW-480; and 40 lg/mL for HT-29. More-
FACSCalibur (Becton Dickinson), and data were analyzed using
over, CLP at 50 lg/mL concentration induced more than 90% of CT-
ModFit LT software version 3.3.11.
26, HCT-116 and SW-480 cell death. However, 100 lg/mL concen-
tration of CLP was required to achieve 90% of HT-29 cell death.
2.9. Western blotting analysis
Hence, CLP concentration at 10 lg/mL and 20 lg/mL was preferen-
tially used for subsequent in vitro experiments.
HCT-116 and CT-26 cells (2  106) were seeded in Petri dishes
and treated as described above for 24 h. Cells were lysed in
200 lL RIPA lysis buffer (Thermo Fisher Scientific Inc., Rockford, 3.3. Apoptosis assay
IL, USA). Protein concentration was determined by Bicinchoninic
acid (BCA) assay kit (Sigma-Aldrich, USA). Subsequently, proteins We used Annexin-V/7-AAD staining to investigate the CLP-
(80 lg) were separated on a 12% polyacrylamide gel and elec- induced cell apoptosis. Interestingly, our findings demonstrated
troblotted on a PVDF membrane. The membrane was saturated that CLP treatment induced, in a dose-dependent manner, cell
during 1 h in 5% bovine serum albumin (w/v) in Tris Buffered Sal- death of all CRC cell line mainly via apoptosis (Anx-V positive) with
ine containing 0.1% Tween-20 (TBS-T) and then incubated with pri- a weak necrotic effect (Anx-V negative and 7-AAD positive). Fur-
mary antibodies: anti-CDK2, anti-caspase-9, anti-p53, anti- thermore, HCT-116 and CT-26 cells were more sensitive to CLP
phospho-p53, anti-cleaved PARP, anti-phospho-p38 MAPK (Cell (20 lg/mL) treatment, as more than 40% and 70% of HCT-116 and
Signalling Technology, Inc), anti-p27, anti-cyclin A, anti-cyclin E CT-26 treated cells were in late apoptosis stage, respectively
(Santa Cruz Biotechnology) and anti-p38 MAPK (BioLegend) at (Fig. 3).
4 °C overnight. b-actin (Santa Cruz Biotechnology, Inc) was used For comparison, we investigated the apoptotic effect of two
as internal standard. Primary antibodies were then detected either major polyphenols of carob leaves, gallic acid (GA) and m-
with anti-rabbit IgG-HRP-linked secondary antibody or anti-mouse coumaric acid (m-CA), and 5-FU commonly used for CRC treat-
IgG2b-HRP secondary antibody. Blots were visualized using an ECL ment. In HCT-116 and HT-29 cells, we observed that GA
Kit (Merck Millipore) on Bio-Rad Chemi-Doc XRS+ system. Densit- (460 lM) induced higher apoptotic level than that induced by
ometric analysis was performed on Bio-Rad Image Lab Software CLP 20 lg/mL, whereas m-CA (10 mM, most commonly used con-
(version 4.1). centration) and 5-FU (10 lM) exerted less apoptotic effect than
CLP 20 lg/mL in HCT-116 and CT-26. The co-treatment of 5-FU
2.10. Effect of carob infusion on transplanted mouse colon cancer cells with CLP induced similar apoptotic effects to those exhibited by
(CT-26) growth CLP (20 lg/mL) alone in HCT-116 cells (Fig. 3B).
These cell deaths seem to be a caspase-dependent apoptosis, as
Mouse were bred and maintained according to the Animal we noticed a significant decrease in cell death when HCT-116 and
Experimental Ethics Committee Guidelines (University of Bur- CT-26 were co-treated with z-VAD-fmk, a pan-caspase inhibitor
gundy, Dijon, France). Mouse colon cancer cells CT-26 (0.3  106 - (Fig. 4C). We further explored the molecular mechanism of apopto-
cells/100 lL PBS) were injected into the left flank of BALB/c mice sis induced by CLP in the two sensitive cell lines, CT-26 and HCT-116.
(n = 12). Mice were randomly assigned the day of injection to a
control group receiving tap water in feeding-bottle, and to a trea- 3.4. Detection of apoptosis-related proteins
ted group receiving leaf infusion. Palpable tumour surface was
measured three times a week using a Vernier calliper. At the end p53 activation in cells leads to apoptosis or cell cycle arrest.
of experiment, mice were killed by cervical dislocation. Moreover, p53 phosphorylation could be induced by p38 MAPK.
 F.Z. Ghanemi et al. / Journal of Functional Foods 33 (2017) 112–121 115

Fig. 1. HPLC chromatogram of phenolic compounds present in methanol–acetone–water extract of carob leaves (A) and infusion of 1% carob leaves (B). Detection at
k = 280 nm.

Table 1 In our study, we observed that CLP induced the phosphorylation of

Phenolic acid composition of carob leaves. both p38 MAPK and p53 proteins in HCT-116 and CT-26 cells
Phenolic CLP extract lg/g sample DW Infusion lg/g sample DW (Fig. 4A). Thus, CLP dose-dependently induced caspase-9 activa-
compounds (dry weight) (dry weight) tion, an essential downstream component of p53, and activation
Gallic acid 1445.377 166.872 in both HCT-116 and CT-26 cells. Indeed, CLP triggered pro-
Chlorogenic acid 834.9131 44.499 caspase-9 cleavage into its two active fragments. Caspase-9 activa-
Syringic acid 1216.493 97.342 tion induced by CLP was associated with an important cleavage of
p-Coumaric acid 1197.841 266.996 its downstream substrate, PARP. GA (460 lM) induced similar
m-Coumaric acid 2192.378 1423.980
Quercetin 3-O- 1159.291 250.309
cleavage of caspase-9 and PARP to those induced by CLP (20 lg/
rutinoside mL) (Fig. 4A).
Quercetin 1168.708 –

Fig. 2. Effects of CLP on the viability of HCT-116, CT-26, SW-480, and HT-29 cells. The cells (5  104 cells/well) were incubated with different concentrations of CLP for 24 h as
described in Experimental Section. Cell viability was assessed by trypan blue exclusion test. Cell numbers were determined by a hemocytometer. Data represent the mean ± S.
D. of three independent experiments performed in triplicate (n = 3). ** and *** represent p < 0.01 and p < 0.001 respectively as compared to untreated cells. p values were
obtained by one-way ANOVA, followed by Fisher’s LSD test.
116 F.Z. Ghanemi et al. / Journal of Functional Foods 33 (2017) 112–121

Fig. 3. Effect of CLP on induction of apoptosis in CRC cells. (A) Annexin-V and 7-AAD staining cytograms of CRC cells treated or not with CLP (20 lg/mL). (B) Rate of early or
late apoptosis and necrosis of the same cell lines treated with CLP (10 and 20 lg/mL) and/or 5-FU (10 lM) or GA (460 lM) or m-CA (10 mM) for 24 h then labeled with
Annexin-V and 7-AAD. Cell death was assessed by flow cytometry and data were analyzed using FlowJo software. Data represent the mean ± S.D. (n = 4). *, ** and *** represent
p < 0.05, p < 0.01 and p < 0.001 respectively as compared to untreated cells. $, $$ and $$$ represent p < 0.05, p < 0.01 and p < 0.001 respectively as compared to 5-FU-treated
cells. p values were obtained by one-way ANOVA, followed by Fisher’s LSD test.

3.5. Detection of caspase-3 and caspase-7 activity induced cell cycle arrest in S phase in both cell lines with a disap-
pearance of cells in G2/M phase.
We observed that CLP increased, in a dose-dependent manner, The increase of p27, a cyclin-dependent kinase (CDK) inhibitor,
caspase-3 and caspase-7 activities in colon cancer cells. At 20 lg/ expression is likely responsible in G1 phase arrest. Western blot
mL, HCT-116 (28.4%) and CT-26 (35.4%) cells were positive for analysis showed that CLP dose-dependently increased p27 expres-
CLP-induced active caspase-3/-7 (Fig. 4B). sion when compared to untreated cells. We also observed that CLP
decreased CDK2 expression but increased the expression of cyclin
3.6. Cell cycle assay A in both HCT-116 and CT-26 cells. Nevertheless, CLP increased
cyclin E only in HCT-116 cells (Fig. 5C).
It has been shown that induction of apoptosis was correlated to
the deregulation of cell cycle, thus; we further analyzed the effect
of CLP on cell cycle progression by using propidium iodide staining.
We observed that CLP (20 lg/mL) induced cell cycle arrest of CRC 3.7. Tumour growth
lines in the G1 phase. As shown in Fig. 5A and B, the repartition
of cell populations between different phases of cell cycle in HCT- The anti-proliferative properties of carob leaf infusion were
116 was as follow: 46.96% in G1, 35.58% in S and 17.45% in G2/ evaluated in vivo in mouse cancer model. After subcutaneous
M. CLP (20 lg/mL) treatment increased G1 population to 57.43% transplantation of CT-26 colon cancer cells in BALB/c mice, we
and decreased the cell population in G2/M to 8.93%. In CT-26, the monitored colon tumour progression and observed that carob leaf
repartition of cell population between the phases of cell cycle infusion given in the feeding-bottle reduced the growth of
was as follows: 40.18% in G1, 41.26% in S and 18.54% in G2/M. implanted CT-26 cells compared to control. This reduction in
CLP (20 lg/mL) treatment increased G1 population to 53.56% and tumour growth was significant at 11 days after CT-26 injection;
decreased the cell population in G2/M to 9.33% (Fig. 5A and 5B). this inhibition was maintained until the day 19 where CLP reduced
Also, GA and m-CA induced cell cycle arrest in G1. However 5-FU 42% tumour-size when compared to controls (Fig. 6).
 F.Z. Ghanemi et al. / Journal of Functional Foods 33 (2017) 112–121 117

Fig. 4. Effect of CLP on induction of apoptosis in HCT-116 and CT-26 cells. A (upper panel) western blot analysis of p38 and p53 in HCT-116 and CT-26 cells treated with CLP
10 and 20 lg/mL for 24 h. A (lower panel) Western blot analysis of cleaved-caspase 9 and PARP in cells treated with CLP (10 or 20 lg/mL) or GA (460 lM) for 24 h. (B)
Caspase-3/7 activation expressed in percentage of FLICA caspase-3/7-AAD positive in HCT-116 and CT-26 cells after 24 h of treatment with CLP (10 or 20 lg/mL). Data
represent the mean ± S.D. (n = 4). (C) Cells were incubated (or not) with z-VAD-fmk for 1 h, and then treated with CLP (20 lg/mL). After 24 h, cells were stained with Annexin
V and 7-AAD for 20 min. Cell death was assessed by flow cytometry and data were analyzed using FlowJo software. Data represent means ± SD (n = 4). *, ** and *** represent
p < 0.05, p < 0.01 and p < 0.001 respectively as compared to untreated cells (ctr). $, $$ and $$$ represent p < 0.05, p < 0.01 and p < 0.001 respectively for the comparison
between CLP-treated and z-VAD-fmk co-treated cells. p values were obtained by one-way ANOVA, followed by Fisher’s LSD test.

4. Discussion Carob leaves are known for their high contents of polyphenols
(Custódio et al., 2011). In our study, we have observed that metha-
The discovery of ideal anti-cancer agents which are non-toxic nol/acetone/water extract contains 4-fold more total phenolic con-
and highly effective, with known mechanisms of action, is a major tent compared to infusion of carob leaves.
challenge in the treatment of cancer (Khan & Mukhtar, 2008). Quick HPLC analysis showed that gallic acid and m-coumaric
Plants contain bioactive constituents and possess antioxidant acid are the major polyphenols present in CLP. The comparison of
properties, which could play an important role in therapeutics. phenolic contents reported in literature showed that the gallic acid
There is an emerging interest in the chemotherapeutic application represents the main and the most detectable component, both in
of natural bioactive plant compounds, especially polyphenols that pods and leaves of carob. Indeed, Aissani et al. (2012) have also
could prevent cancer incidences (Esakkirajan et al., 2014; Vauzour, detected, in addition to GA, the presence of isoquercitin, myricitrin
Rodriguez-mateos, Corona, Oruna-concha, & Spencer, 2010). and epigallocatechin-3-gallate compounds, whereas Custódio et al.
Carob leaf polyphenols have been shown to exert beneficial (2011) identified catechin, gentisic acid and chlorogenic acid as
effects in several diseases including cancer. Custódio et al. (2011) additional compounds in carob leaf methanol extract. Ben
reported that methanol extract of carob leaves strongly decreased Hsouna et al. (2011) reported that acetate ethyl extract of C. siliqua
the viability of human cervical adenocarcinoma cell line (HeLa), leaf contained mainly syringic acid, myricetin glycosides and gallic
but to a less extent of prostate (DU-145), breast (MDA-MB-231) acids derivatives. Also, coumaric acid was identified in aqueous
and colon (HCT-116) cell viability. Corsi et al. (2002) have observed acetone (Fadel et al., 2011; Ortega et al., 2009) and methanol
that aqueous leaf and pod extracts from Ceratonia siliqua L. exhib- (Torun, Ayaz, Colak, Grúz, & Strnad, 2013) extracts of carob-pod
ited antiproliferative effects on mouse hepatocellular carcinoma flour.
cell line. However, to our knowledge, there is no report that deals This discrepancy in phenolic content might be due to different
with the molecular mechanism of CLP in CRC. phytopathogenic factors which are linked directly to different
118 F.Z. Ghanemi et al. / Journal of Functional Foods 33 (2017) 112–121

Fig. 5. Effect of CLP on induction of cell cycle arrest in HCT-116 and CT-26 cells. (A) Cell cycle representative profile of HCT-116, CT-26 in untreated or treated cells with CLP
(20 lg/mL) for 24 h then labelled with IP before analysis by FACS Calibur. (B) Analysis of G1, S and G2/M phases of cell cycle in untreated and treated cells with CLP (10 or
20 lg/mL) or 5-FU (10 lM) or GA (460 lM) or m-CA (10 mM). *, ** and *** represent p < 0.05, p < 0.01 and p < 0.001 respectively as compared untreated cells (Ctr). (C) Western
blot analysis of p27, CDK2, cyclin E and cyclin A expression in HCT-116 and CT-26 treated with CLP (10 or 20 lg/mL) for 24 h by western blot analysis. b-actin was used as the
internal protein control.

geographical, genetic and cultural conditions (Avallone, Plessi, the cell lines differ in their sensitivity to polyphenols. GA is known
Baraldi, & Monzani, 1997). Used techniques and sample prepara- to induce programmed cell death in various tumour cancer cell
tion methods might also be responsible for the observed differ- lines, including leukemia, lung, colon, and stomach cancer (Bin-
ences in phenolic concentrations (Custódio et al., 2011). Chuan et al., 2009; Inoue et al., 1994; Yoshioka et al., 2000). Fur-
We observed that CLP extract induced CRC cell death at low thermore, Giftson, Jayanthi, and Nalini (2010) reported that GA
concentration, with an IC50 around 20 lg/mL. Interestingly, an could exert a significant chemopreventive effect on 1,2-
IC50 of polyphenols lower than 30 lg/mL has been recommended dimethyhydrazine (DMH)-induced colon carcinogenesis. Coumaric
for the search of new anticancer agents (Dos Santos et al., 2010). acid (CA), another major polyphenol in CLP, also exhibited an apop-
CRC cell line sensitivity to CLP treatment, observed in our study, totic affect but to a lesser extent than GA and CLP in both HCT-116
is as follows: CT-26 > HCT-116 > SW-480 > HT-29. This is partially and CT-26 cells. CA has been shown to induce apoptosis in HCT-5
in accordance with Custódio et al. (2011) who observed that colon cancer cells and to exhibit an anti-invasion effect in human
polyphenols in methanol leaf extract reduced HCT-116 cell viabil- lung adenocarcinoma (A549) cells (Jaganathan, Supriyanto, &
ity. However, these authors reported that IC50 value was 20-fold Mandal, 2013; Tsai, Yen, Sun, Yang, & Weng, 2013).
higher (400 lg/mL) than the obtained values in our study. Similarly, Klenow et al. (2008) reported that GA represented the
Induction of apoptosis in cancer cells can be considered as a major constituent of aqueous polyphenol extract of commercially
useful strategy for anticancer drug development (Hu & Kavanagh, available carob fiber (pods). Interestingly, they observed that
2003). We observed that CLP induced cell death mainly via apopto- polyphenols of carob fiber reduced HT29 cell number in a dose
sis as illustrated by a high number of Annexin-V positive cells. dependent manner up to 10 lg/ml, but this effect was not repro-
Indeed, z-VAD-fmk significantly reduced Annexin-V staining duced by equimolar concentrations of GA. Thus, the induction of cell
induced by CLP treatment, confirming the role of caspases in death by CLP can be explained by the synergistic effect between
CLP-induced apoptosis. Necrosis induced by CLP was weak and bioactive compounds, including GA (Bin-Chuan et al., 2009;
represented less than 10% of cell death in HCT-116 and CT-26. Subramanian, Jaganathan, Mandal, Supriyanto, & Muhamad, 2016),
GA, a major polyphenol in CLP, induced more significant apoptotic and CA (Jaganathan et al., 2013; Janicke, Önning, & Oredsson,
effect than CLP in HT-29. Interestingly, CLP induced important 2005), as well as other phenolic compound including chlorogenic
apoptotic effect than GA in CT-26. These observations indicate that acid (Nkondjock, 2009) and quercetin (Gibellini et al., 2011).
 F.Z. Ghanemi et al. / Journal of Functional Foods 33 (2017) 112–121 119

2000; Toshiyuki & Reed, 1995). p53 is a downstream substrate of

p38 MAPK, and polyphenols (such as resveratrol) have been
reported to induce p53 phosphorylation and activation by p38
MAPK (She, Bode, Ma, Chen, & Dong, 2001). Our results also
showed that CLP dose-dependently induced p38 MAPK and p53
phosphorylation. Interestingly, p38 is known to activate p53 which
can induce cell cycle arrest, to allow time for DNA repair (Thornton
& Rincon, 2009).
In some cancers, it has been shown that down-regulation of p53
is associated with down-regulation of p27 which acts as a tumour
suppressor by controlling cell cycle progression. In the current
study, we observed that CLP including GA and modestly CA
induced cell cycle arrest in G1/S phase whereas, as expected, 5-
FU induced cell cycle arrest in the S phase.
The cell cycle is tightly monitored by checkpoints at G1/S and
G2/M (Niculescu et al., 1998) which involve different cyclin-
dependent kinases (CDKs) complexes. The cell cycle progression
is also regulated by the relative balance between the cellular con-
centrations of CDK inhibitors, such as the p21 and p27 proteins of
the Cip/Kip family (Sherr & Roberts, 1999). It has been shown that
Fig. 6. Effect of carob infusion on tumour growth in BALB/c mice bearing CT-26
p27 interacts with cyclin E-CDK2 inducing cell cycle arrest in G1.
carcinoma cells. CT-26 cells (1  106) were injected subcutaneously into the left We have noticed that CLP induced an increase in p27, associated
flank of BALB/c mice (n = 12). Half of these animals received carob leaf infusion (1%) with a decrease in CDK2 in both cell lines. Furthermore, CLP treat-
in the feeders, while tap water was given to the other half (control). Palpable ment increased cyclin A in both cell lines and cyclin E in HCT-116.
tumors were measured using a Vernier calliper for 3 weeks. Results are presented as
CLP induction of G1/S cell cycle arrest could result from the
mean ± SEM. ** and *** represent respectively p < 0.01 and p < 0.001 as compared to
group of mice receiving tap water. p values were obtained by one-way ANOVA, increase in p27 expression. Such correlation between
followed by Fisher’s LSD test. polyphenol-induced p27 accumulation, cyclin A and E dysregula-
tions, and CDK2 down-regulation in G1/S phases arrest has already
been reported (Abdoul-Azize et al., 2013; Pozo-Guisado, Alvarez-
We also observed that 5-FU induced cell death to a lesser extent Barrientos, Mulero-Navarro, Santiago-Josefat, & Fernandez-
than CLP, particularly in HCT-116 and CT-26. We observed that GA Salguero, 2002; Sherr & Roberts, 1999; Subramanian et al., 2016).
potentiated the apoptotic effect of 5-FU in HT-29 and SW-480; Recently, numerous in vitro and in vivo studies have investi-
however, our results did not show an additive effect of CLP and gated the phenolic content of plant infusions for their antiprolifer-
5-FU, suggesting that CLP and 5-FU might share common targets. ative effects against several kinds of cancer including
The mechanisms of apoptosis are complex, involving several hepatocarcinoma and colon carcinogenesis (Moreno-Jimenez
cascades of molecular events. Two main apoptotic pathways are et al., 2015; Yang et al., 2012). To assess, in vivo, the anticancer pro-
usually described: the extrinsic or death receptor pathway and prieties of carob leaves, we maintained mice, injected with CT-26
the intrinsic or mitochondrial pathway (Eum & Lee, 2011). In both cells, on carob leaf infusion. We observed that infusion of carob
cases, when outer mitochondrial membranes become permeable, leaves significantly reduced CT-26 tumour growth. This result sup-
cytochrome c is released into the cytosol (Henry-Mowatt, Dive, ports the potential use of Ceratonia siliqua L. leaves for the chemo-
Martinou, & James, 2004). Cytochrome c binds to Apaf–1 which prevention of CRC.
recruits the initiator caspase, procaspase-9 to apoptosome, where
procaspase-9 is activated and cleaved into its active form. The acti-
vated caspase-9, in turn, cleaves and activates other downstream 5. Conclusions
caspases such as caspase-3 (Green & Reed, 1998) which cleaves
downstream various substrates including PARP (Tewari et al., CLP decreased in dose-dependent manner cell viability and
1995). Indeed, caspase-3 plays a central role in the terminal execu- induced apoptosis in CRC cell lines via the activation of caspase-
tion phase of apoptosis (Hirsch et al., 1998). During the last dec- 9/caspase-3/ PARP pathway. Carob leaf infusion significantly
ades, a growing body of evidence has indicated that polyphenols, reduced CT-26 tumour growth in BALB/c. Hence, both in vitro
like quercetin and luteolin, can directly modulate different path- and in vivo studies demonstrate that CLP exhibit potent anti-
ways of the apoptotic process and/or the expression of regulatory cancer properties and can be used as a functional food with chemo-
proteins, such as the release of cytochrome c with subsequent acti- preventive effect against CRC.
vation of caspase-9 and caspases-3 (Chyesunong, Nguyen, Ong, &
Lee, 2004). Our results revealed that CLP extract, dose- Author contributions
dependently, activated caspase-9, caspase-7 and caspase-3. The
latter cleaved the PARP (116 kDa) protein to generate an 89 kDa Aziz Hichami, Mickael Rialland and François Ghringhelli
fragment. These observations are in agreement with the finding designed the overall research experiments. Meriem Belarbi, Ikram
of Corsi et al. (2002) who reported that polyphenols from aqueous Aboura, Chahid Benammar and Boucif Farid Lahfa prepared plant
extract of carob leaves induced caspase-3 activation in mouse hep- extract and make technical part of polyphenol analysis. Fatima
atocellular carcinoma cell line. Zahra Ghanemi, Aurélie Fluckiger, Adélie Dumont, Charlotte De
There is also an increasing body of evidence which demon- Rosny, Amira Sayed Khan, Danish Patoli, Babar Murtaza and Abdel-
strates that p53 gene inactivation in the majority of human cancers hafid Nani performed the experiments and data analysis. Meriem
results in deregulation of cell birth and death processes (Levine, Belarbi, François Ghringhelli, Dominique Delmas, Cédric Rébé and
1997). Evidences have suggested that p53 death signals lead to Lionel Apétoh analyzed and interpreted the results. Naim Akhtar
pro-apoptotic Bcl-2 family member Bax, caspase-9, and Apaf-1 Khan, Fatima Zahra Ghanemi and Abdelhafid Nani wrote and edi-
activation (Schuler, Bossy-Wetzel, Goldstein, Fitzgerald, & Green, ted the manuscript.
120 F.Z. Ghanemi et al. / Journal of Functional Foods 33 (2017) 112–121

Conflicts of interest hydrazine induced rat colon carcinogenesis. Investigational New Drugs, 28(3),
251–259. http://dx.doi.org/10.1007/s10637-009-9241-9.
Green, D. R., & Reed, J. C. (1998). Mitochondria and apoptosis. Science, 281(5381),
The authors have declared no conflicts of interest. 1309.
Henry-Mowatt, J., Dive, C., Martinou, J.-C., & James, D. (2004). Role of mitochondrial
membrane permeabilization in apoptosis and cancer. Oncogene, 23(16),
Acknowledgments 2850–2860. http://dx.doi.org/10.1038/sj.onc.1207534.
Hirsch, T., Xiang, J., Chao, D. T., Korsmeyer, S. J., Scaife, J. F., Colell, A., ... Williamson, J.
R. (1998). Caspases: Enemies within. Science, 281(August), 1312–1316.
The study was supported, in part, by Labex LipSTIC INSERM Ben Hsouna, A., Saoudi, M., Trigui, M., Jamoussi, K., Boudawara, T., Jaoua, S., & El
U1231 University of Burgundy, France and a bilateral Franco- Feki, A. (2011). Characterization of bioactive compounds and ameliorative
Algerian collaborative project ‘‘Tassili” (grant number 30850QG). effects of Ceratonia siliqua leaf extract against CCl 4 induced hepatic oxidative
damage and renal failure in rats. Food and Chemical Toxicology, 49(12),
The funders had no role in study design, data collection and anal- 3183–3191. http://dx.doi.org/10.1016/j.fct.2011.09.034.
ysis, decision to publish, or preparation of the manuscript. Hu, W., & Kavanagh, J. J. (2003). Anticancer therapy targeting the apoptotic
pathway. The Lancet Oncology, 4(12), 721–729. http://dx.doi.org/10.1016/
S1470-2045(03)01277-4.
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