Dichloromethane-methanol Extract from Borassus aethiopum Mart. (Arecaceae) Induces Apoptosis of Human Colon Cancer Ht-29 Cells

MATERIALS AND METHODS

Materiel
Chemicals drugs and test agents:

Equipment
Material of extraction:

Material of cells study:

METHODS

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, 1982).

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 Dulbecco’s 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 Student’s t-test with p<0.05 as a criterion of significance.

RESULTS

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.

Fig. 1:	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).

Table 1:	Pro-apoptotic effects of E2F2 on colon cancer HT29 cells

Fig. 2(a-c):

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)

Fig. 3:

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.

DISCUSSION

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., 2003).

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).

CONCLUSION

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.