Dichloromethane-methanol Extract from Borassus aethiopum Mart. (Arecaceae) Induces Apoptosis of Human Colon Cancer Ht-29 Cells
Borassus aethiopum MART (Arecaceae) is a plant used in traditional herbal medicine for the treatment of various diseases (bronchitis, laryngitis, antiseptic). In particular, their male inflorescences were reported to exhibit cicatrizing, antiseptic and fungicidal properties. In the present study, the biological activity of E2F2, an apolar extract from Borassus aethiopum male inflorescence was investigated on colon cancer HT29 cells. Phytochemical screening was carried according to methodology for chemical analysis for vegetable drugs. Cells proliferation was determined by the MTT assay and cells cycle distribution was analysed by using laser flow cytometer (Beckman coulter). The cytoskeleton organisation was examined under a laser scanning confocal microscope (Zess). Preliminary phytochemical analysis of E2F2 extract revealed the presence of sterols, triterpenes and saponosids. E2F2 extract (1 μg and 100 μg mL-1) significantly inhibited cell proliferation by blocking cell population in G0/G1 phase. Flow Cytometric analysis of E2F2-treated HT29 cells showed that hypoploïd cell population (sub G1 phase) increased with processing time exposures. Immunofluorescence confocal analysis revealed a disrupt actin microfilaments network in E2F2 treated-cells with a significant reduction in actin stress fibres and appearance of a random, non-oriented distribution of focal adhesion sites. These data indicate that E2F2 extract has anti-proliferative and pro-apoptotic activities. Further studies are required to unravel the mechanisms of action of E2F2 extract.
to cite this article:
J. Sakande, P. Rouet-benzineb, H. Devaud, J.B. Nikiema, M. Lompo, O.G. Nacoulma, I.P. Guissou and A. Bado, 2011. Dichloromethane-methanol Extract from Borassus aethiopum Mart. (Arecaceae) Induces Apoptosis of Human Colon Cancer Ht-29 Cells. Pakistan Journal of Biological Sciences, 14: 578-583.
May 07, 2011; Accepted: October 01, 2011;
Published: October 13, 2011
Borassus aethiopum Mart (Arecaceae), also called Ronier palm, is a tall
dioecious palm widely spread in Tropical and southern African savannah and open
forest. It is a plant at once used in alimentation, in technology and in traditional
medicine. In the field of alimentation, a study showed that the young shoots
of Borassius aethiopum contains key nutriments which can help in nutritional
supplementation in western Africa (Glew et al., 2005).
In technology, the activated charcoal stemming from inflorescences of Borassus
aethiopum is used as adsorbent (Nethaji et al.,
2010). In Burkina faso, almost all parts of the palm are used medicinally
for the treatment of abdominal pain, respiratory and inflammatory diseases.
Combination of powdered Borassus aethiopum male inflorescences with shea
butter is well known as cutaneous lesion remedy (Cassou et
In previous studies, using a bioassay-guided fractionation procedure, five
fractions (E1F1, E2F2, E3F3, E4F4 and E5F5) from powdered Borassus aethiopum
male inflorescences were extracted. Among these, the dichlomethane methanol
extract E2F2 was found to exert significant antibacterial, antifungal, anti-inflammatory
and antipyreti activities (Sakande et al., 2003,
2004a, b). Other studies on
Borassus flabellifer flowers a plant belonging to the same family reported
the isolation of an immunosuppressive 17 alpha substituted dammarane (Revesz
et al., 1999) and a spirostane-type steroid saponins with antimetabolic
activity (Yoshikawa et al., 2007). According
to such important results on the flowers of Borassus, there is growing
interest in the use of phytoextracts of this drug for the treatment of human
diseases including cancer. Indeed, despite modern advancement in diagnosis,
prevention and therapy, cancer is still the single largest cause of death for
human worldwide even poor countries as Burkina Faso. Therefore, it is necessary
for pharmaceutical and alternative medicinal industries to study and develop
new and safe drugs (Yi et al., 2003; Kraft,
2009; Kim et al., 2010).
This study is also based on the fact that apoptosis mechanism is now well established
as a novel strategy in cancer treatment (Ward et al.,
2008; Tan et al., 2009; Yang
et al., 2009).
In The present study, the cytotoxic and apoptogenic effect of dichloromethane-methanol E2F2 extract on human colon cancer HT29 cells lines was investigated.
MATERIALS AND METHODS
Chemicals drugs and test agents:
||Extraction solvents: Dichloromethane and methanol (Prolabo,
||Phytochemical screening reagents from Prolabo, France: Potassium
isobismuthite, KOH 2%, Ferric chloride d = 1,45, Sodium diphenylborate
||Cells and test agents from Laboratories Eurobio and AbCys,
||Human colon cancer cells HT29
||Dulbeccos minimal medium
||Dimethyl Sulfoxyde (DMSO)
||TRITC (tetramethylrhodamine isothiocyanate)-conjugated phalloidin
Material of extraction:
||Column of chromatography (Kieselgel 63-160 μ, Prolabo,
||Lyophilisator Alpha chris 1-2
Material of cells study:
||Hematocytometer (Beckman Coulter, USA)
||Multiscan MCC 340 microplate reader (Titertek, USA)
||Confocal microscope (Zeiss, LSM510)
||Flow cytometer (Beckman Coulter, USA)
Plant material and extraction: Borassus aethiopum male inflorescences
were collected and identified by Professor Sita Guinko, Institute of natural
products research of Ouagadougou (Vegetal Biology and Ecology Laboratory of
UFR SVT). A voucher specimen AA1522 was deposited at the Herbarium.
The inflorescences were air-dried in the shade and powdered. The powder was
exhaustively extracted by percolation with 3 L of dichloromethane-methanol (50:50).
The fraction obtained (E2F2 extract) was evaporated under reduced pressure to
obtain 10 g of residue (scheme 1). Phytochemical screening was carried out according
to the methodology for chemical analysis for vegetable drugs (Ciulei,
Study on human colon cancer cells HT29: The study of E2F2 extract effect
on human colon cancer cells HT29 was carried from February 2008 to may 2009.
To study the ability of chemotherapeutic to induce apoptosis it was essential
to accurately define this mode of cell death (Oh et al.,
2001). Generally, the following traditional criteria are requested as morphological
changes including chromatin condensation, cell nuclear fragmentation and DNA
ladder. For this study following methods were used.
Cell culture: The human colon cancer HT29 cells were seeded at 3.105
cells/25 cm2 flasks and cultured in Dulbeccos minimal
medium supplemented with 20% fetal calf serum under a 5% CO2 humidified
atmosphere at 37°C and left to grow exponentially (Rouet-Benzineb
et al., 2004).
Cell proliferation and viability: The cells were seeded in 96-well microplates and incubated overnight. The cells were treated with 1 and 100 μg mL-1 of the E2F2 extract or its vehicle, methanol (0.1%) for 24 h. The cells were harvested by trypsin digestion and cell viability was determined by trypan blue exclusion with a hematocytometer. The level of cell viability was measured at 0.5, 1, 2, 4 and 24 h.
Cell proliferation was determined by the MTT assay as described previously
by Hu et al. (2002) and Kim
et al. (2009).
Confocal visualization of actin cytoskeleton organization: Cells were
grown on glass coverslips and washed twice with serum-free medium. The cells
were then treated with different concentration of E2F2 for 4 h and 24 h. The
treated cells were fixed with 3.7% paraformaldehyde in Phosphate Buffer Saline
(PBS) at room temperature and permeabilized with 0.1% Triton X-100 as described
by Rao and Li (2004). The cells were stained for actin
with TRITC (tetramethylrhodamine isothiocyanate)-conjugated phalloidin and examined
under a laser scanning confocal microscope (Zeiss, LSM510).
Cell cycle analysis: Cell cycle analysis was performed to determine
the proportion of apoptotic sub G1 hypoploid cells (Nicoletti
et al., 1991).
Statistical analysis: Data were collected and analyzed by using Expo32 software. The results were expressed as Mean±SD from at least 3 independent experiments in tables and figures. Statistical analysis was performed using Students t-test with p<0.05 as a criterion of significance.
Phytochemical screening: The phytochemical analyses of the dichloromethane methanol E2F2 extract from Borassus aethiopum male inflorescences revealed the presence of sterols, triterpens and saponoids.
E2F2 extract activity on HT29 cell viability and proliferation: Incubation of HT29 cells with E2F2 extract (Fig. 1) showed a significant inhibition of proliferation from the first hour with a 100 μg mL-1 concentration compare to control group (p<0.01). This inhibition was observed from 2 h of incubation with 1 μg mL-1 concentration (p<0.05). This inhibition was dose dependent. The cytotoxic effect on HT29 cells was accompanied with cell detachment.
||Dose-response effect of E2F2 on HT29 proliferation. HT29 cells
(105) were sealed in 12 well placed in triplicate samples. The
cells were serum stored for 24 h and medium were supplemented with E2F2
(100 μ mL-1) for different time period. Values are expressed
as number of viable cells and each column represents Meean±SD of
four experiments *p<0.05, **p<0.01 vs. control
Cell cycle analysis: Cell cycle distribution analyzed by using laser
flow cytometer showed that E2F2 induces hypoploidic cells (Table
1, Fig. 2).
|| Pro-apoptotic effects of E2F2 on colon cancer HT29 cells
||E2F2 extract induces hypoploid peak in HT29 cells. (a) Control
24 h, (b) E2F2 (1 μg mL-1) and (c) E2F2 (100 μg mL-1).
Panels represent cytometric flow analysis. Shown a typical cell cycle profile
of control-or E2F2-treated cells. Cell were seeded at low density, grown
at 70-80% confluence and were incubated with or without 1 μg mL-1
E2F2. Cells were subjected to Propidium Iodide (PI) staining as described
in experimental procedure and 10,000 events were counted (X axis: FL3 log,
Y axis: Cells count)
||E2F2 induces reorganization of actin cytoskeleton. HT29 cells
were grown at 70-80% confluence and were incubate with or without 1 μg
mL-1 E2F2. Cells were fixed and stained with TRITC-conjugated
phalloidin for detection of F-action cyoskeletal organization using immunoflourescence
micros copy. Confocalvies of colon cancer HT29 cells show that stress fibers
are well defined in control cell whereas actin network is precisely disorganized
in E2F2-treated cells
The percentage of hypoploidic cells increased from 1.86% after 1 h exposure
with 1 μg mL-1 concentration to 11, 59% after 24 h. With 100
μg mL-1 of E2F2 extract exposure the percentage of hypoploidic
cells was 25, 86% after 24 h. In HT29 cells treated with E2F2 extract there
was an appearance of hypoploidic peaks (Fig. 2). This apoptotic
effect of E2F2 on human colon cancer HT29 was dose dependent. Moreover, SubG1
cell population appeared and increased as function of time. suggesting a genotoxic
role of E2F2 extract on cancer HT29 cells.
This cytometric analysis revealed that E2F2 fraction induced cell death by producing hypoploidic cells.
Confocal visualization of actin cytoskeleton organization: Exposure of human colon cancer HT29 cells to 100 μg mL-1 of E2F2 extract, led to a reorganization of actin filaments, a characteristic feature of apoptosis (Fig. 3). Confocal analysis of phalloidin-staining of F-actin showed that attached cells become more flattered and elongated in E2F2-treated than control cells.
The chemical analysis of the extract E2F2 of Borassus aethiopum revealed
the presence of substances such as sterols, triterpens and saponoids whom known
pharmacological activities can justify the use in traditional medicine. The
presence of these same constituents was already reported by Lanza
et al. (1962). Other authors also isolated different steroids saponins
from the flowers of Palmyrah palm (Borassus flabellifer) a plant belonging to
the same family as Borassus aethiopum (Yoshikawa
et al., 2007; Keerthi et al., 2009)
The study of the activity of the extracts of Borassus aethiopum on cancer
cells HT29 showed derangements of cytoskeleton witch may be a cell death signal
as described by Khan (2010). It is well known that disruption
of cytoskeleton actin filaments can affect multiple cell functions including
motility, signal transduction, cell division, with cell death as an ultimate
action (Suria et al., 1999; Yamazaki
et al., 2000; White et al., 2001).
Apoptosis plays an essential role in controlling cell numbers in many developmental
and physiological settings and in chemotherapy-induced tumour cell killing.
The hindrance of this process leads to the development of many severe diseases
and disorders, including tumours. Selective induction of the apoptosis process
may become the fundamental strategy in developing anti-cancer drugs (Kim
et al., 2010). Most available chemotherapy drugs break down tumour
cells by inducing apoptosis as reported by Bremer et
al. (2006), Yang et al. (2009) and Sun
and Peng (2009). But it is important to discriminate between cells that
were experiencing apoptosis and cells undergoing necrosis as a result of the
lethal effects of plant extract. Indeed, the hepatotoxic syndrome of Borassus
flabellifer extract was previously reported due to water soluble saponins
like neurotoxins (Keerthi et al., 2009).
The chemical groups brought to light in the extract E2F2 could explain the
observed apoptosis effect. It is for example the case of phytosterols. Indeed
the β-sitosterol is capable of leading the apoptosis of cancer cells HT116.
The apoptosis activity of the β-sitosterol would be bound to the activation
of the caspase 3 and 9 on one hand and on the other hand it decreases the expression
of anti-apoptosis proteins Bcl-2 and Bax (Choi et al.,
2003). Also glycosides steroids (gitoxigenin) isolated from Digitalis
purpurea are capable of leading the apoptosis of TK-10 which are cancer
cells of renal adenocarcinoma (Lopez-Lazaro et al.,
The extract E2F2 also contains triterpenes which are also quoted among substances
stemming from plants and endowed to be able to apoptosis. It is the case of
colostral that is an isolated triterpen of plants of the family of Celastraceae
endowed with an anticancer and anti-inflammatory activity. The colostral leads
the apoptosis on the cancer cells of leukaemia (HL-60) by fragmentation of the
DNA and the inhibition of the topoisomerase II (Nagase et
al., 2003). Other parts the effect of Spirulina species extracts
containing various terpenes as carotenoid compounds and tocopherols on the viability
of Ehrlich Ascites Carcinoma Cells (EACC) were evaluated. All algae extracts
at different concentration of 200 and 400 ppm significant reduced the cell viability
ranged from 89.11 to 5.25%. These extracts did not induce any significant changes
in DNA fragmentation of treated EACC compared with untreated cells. These finding
suggest that algae extracts may be reduce cell viability by other mechanism
such as membrane lyases instead of apoptosis (Abd El-Baky
et al., 2003; Bin-Meferij, 2009; Sixabela
et al., 2011).
Finally saponins contained in the extract E2F2 could justify also its apoptosis
activity. Indeed the saikosaponines of Bupleurum spp. is endowed with
apoptosis activity on lineages of liver carcinoma cells Hep-2. The apoptosis
mechanism of these saponins is based on the activation of caspases 3 and 7,
the cleavage of the Poly-ADP-Ribose-Polymerase (PARP) and the fragmentation
of the DNA (Chiang et al., 2003).
The powerful apoptosis activity of E2F2 should be bound to the synergy of these various molecules which it contains (phytosterols, triterpens and saponins).
This study clearly demonstrates that E2F2 extract from Borassus aethiopum strongly inhibits cell proliferation and induces apoptosis in HT29 cells. In summary, this study demonstrate for the first time that E2F2 extract from Borassus aethiopum may possess anticancer properties. Further investigations would be necessary to understand this apoptosis mechanism and to evaluate the potential clinical efficacy of E2F2 in apoptosis process for cancer therapy.
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