Abstract: Solanum aculeastrum is a medicinal plant used by the traditional healers of the Eastern Cape of South Africa, for the treatment of cancers. The antiproliferative activities of this plant were studied in vitro using three human tumour cell lines (HeLa, MCF7 and HT29). Methanolic extracts of the fruits had the highest antiproliferative activity with IC50 between 17.1 and 41.9 μg mL-1 while the activities of their aqueous extracts ranged between 27.9 and 48.5 μg mL-1. The leaf extracts had no anticancer activity under the experimental conditions tested. Overall, the HeLa and MCF7 cell lines were much more sensitive to both extracts than HT29 cells.
Introduction
Over one million people are diagnosed annually with breast cancer which is one of the primary causes of deaths among women globally (Ferlay et al., 2001). The rate of increase of cancer incidence and lack of anticancer drugs has forced scientists to pharmacological and chemical investigations of medicinal plants in search for anticancer agents. The results of the screening of plant extracts for anti proliferative activity have shown that higher plants are a potential source of antioncogenic agents which can compete favourably with chemotherapy and hormonal treatments (Pezzuto, 1997; Wu et al., 2002).
Solanum aculeastrum Dunal (Solanaceae) is widely used in traditional medicine for the treatment of human and livestock diseases (Hutchings et al., 1996). Both fresh and boiled berries of the plant are used as a cure for jigger wounds and gonorrhea (Agnew and Agnew, 1994). Antimicrobial and antioxidant activity of Solanum aculeastrum using crude extracts has been previously reported (Koduru et al., 2006a, b). Also, ethnomedical information from the indigenous people of the Eastern Cape Province of South Africa revealed that this plant is used for the treatment of breast cancer (Koduru et al., 2006c, d). There is, however, no report on the anticancer property of S. aculeastrum in the literature. Yet, species in the genus Solanum are known to be rich in steroidal alkaloids and flavonoids which are known to induce apoptosis in tumor cell lines (Papamichael, 2000; Esteves-Souza et al., 2002). Indeed, phytochemical investigations of S. aculeastrum have revealed the presence of steroidal alkaloids such as solaculine A, solamargine, β-solamarine, solasonine and solasodine (Drewes and Van Staden, 1995; Wanyonyi et al., 2002).
In vitro studies have provided evidence that chemotherapeutic agents such as plant extracts may induce apoptotic tumour cell deaths in vivo. In this investigation, the in vitro cytotoxic properties of the crude extracts of leaves and berries of S. aculeastrum was tested against three cancerous cell lines viz. HeLa, HT29 and MCF7 using standard procedures.
Materials and Methods
Plant Material
The berries and leaves of S. aculeastrum were collected from trees
naturally occurring in the wild at Kayalethu village in the Eastern Cape Province
of South Africa (latitudes 30°00'- 34°15'S and longitudes 22°45'-30°15'E).
The plant was identified at the Department of Botany, University of Fort Hare
and a voucher specimen (Vedic Med 2005/16) was prepared and deposited in the
Griffen Herbarium.
Preparation of Extracts
S. aculeastrum fruits were oven dried at 60°C
while the leaves were air dried at room temperature. Three equal portions (200
g) of each dried plant material were shaken separately in acetone, methanol
and water for 48 h on an orbital shaker. Extracts were filtered using a Buchner
funnel and Whatman No. 1 filter paper and each filtrate was concentrated to
dryness under reduced pressure at 40°C
using a rotary evaporator. The water extracts were freeze dried.
Human Carcinoma Cell Lines and Culture Medium
HT-29 (colonic adenocarcinoma), HeLa (cervical carcinoma) and MCF7 (breast
adenocarcinoma) cells were cultured in 10 cm culture dishes in growth medium
[antibiotic-free RPMI 1640 medium (Sigma, Germany) containing 10% heat-inactivated
fetal bovine serum (Highveld Biological, South Africa), 25 mM HEPES and 2 mM
glutamine] in a humidified 5% CO2 incubator at 37°C. The cancer
cell lines were obtained from the American Type Culture Collection (ATCC), USA.
In vitro Cytotoxic Assays
For the determination of cell viability, cells were seeded into 96-well
culture plates (Nunclon) at a density of 6000 cells/well in 200 μL aliquots.
Cells were allowed to attach for 24 h in a humidified 5% CO2 incubator
at 37°C. Dried fruit and leaf extracts were solubilized in DMSO before further
dilution with growth medium. The final concentration of DMSO in the wells never
exceeded 0.25%. Cisplatin was used as positive control at concentrations of
10 and 100 μM. Cells were exposed to the extracts or cisplatin for 48 h.
Immediately following the 48 h incubation period, cell numbers were determined
using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)
assay as previously described (Alley et al., 1988; Brauns et al.,
2004). Briefly, cells were incubated with 200 μL MTT (Sigma) (0.5 mg mL
in growth medium) for 4 h at 37°C. The formazan product was then dissolved
in DMSO and plates were agitated on a shaker for 5 mins, before the absorbance
was read at 540 nm on multiwell scanning spectrophotometer (Multiskan MS, Labsystems).
The values obtained were used to determine the percentage inhibition of cell
growth caused by the extracts (Hagopian et al., 1999; Huq et al.,
2004). Cisplatin was used as a standard control.
Calculations and Statistics
Initial screening for cytotoxicity and log dose-dependent responses were
performed in triplicate and quadruplicate, respectively. Results were expressed
as percentage growth inhibition of control and treatment values were compared
to control values using the Two-sample Students t-test. IC50 values
for growth inhibition was derived from a nonlinear regression model (curve fit)
based on sigmoidal dose response curve (variable) and computed using GraphPadPrism
4 (Graphpad).
Results
Antiproliferative activities of the different extracts of leaves and berries from the S. aculeastrum on the growth of three human cancerous cell lines were carried out in vitro using tetrazolium assay. Cell proliferation was analyzed at 48 h after cell lines had been cultured with an extracts of 0, 125 and 250 μg mL-1 in media while cisplatin was used as positive control. Results of the initial screening showed that two of the six crude extracts [Fruit Methanol (FM) and Fruit Water (FW)] inhibited the growth of all three tumour cell lines by more than 80% after 48 h exposure (p<0.001 for both concentrations tested (Fig. 1). Inhibition by the other four extracts (three extracts of leaves and acetone extract of berries) were less than 50%, therefore, they were not considered for further testing.
| Fig. 1: | Initial screening results for six extracts at 125 μg mL-1 (clear bars) and 250 μg mL-1 (hashed bars) against HeLa, MCF7 and HT29 cells. Cisplatin at 10 μM (clear bars) and 100 μM (hashed bars) was used as a positive control. Error bars represent the standard deviation of triplicate determinations. # p<0.05; * p<0.001 compared to control (FM = Fruit Methanol; FA = Fruit Acetone; FW = Fruit Water; LM = Leaf Methanol; LA = leaf Acetone; LW = Leaf Water) |
| Fig. 2: | Log dose-response curves for Fruit Methanol (FM) and Fruit Water (FW) extracts against HeLa, MCF7 and HT29 cells. Error bars represent the standard deviation of quadruplicate determinations |
| Table 1: | IC50 values for fruit methanol and fruit water extract for growth inhibition against HeLa, MCF7 and HT29 carcinoma cell lines |
The methanol and water extracts of the berries which were further tested against three tumour cell lines at lower concentrations, indicated that IC50 values of methanol extract ranged between 17.1 and 41.9 μg mL-1 while those of the aqueous extract ranged between 27.9 and 48.5 μg mL-1 under experimental conditions (Fig. 2 and Table 1). The methanol extract had a slightly lower IC50 than water extract in all the three tumour cell lines tested (Fig. 2 and Table 1). The highest activity was shown on cervical carcinoma (HeLa) and breast adenocarcinoma (MCF7) cell lines and their IC50 values for both cell lines were less than 18 μg mL-1 (Table 1). This indicates that the extracts were anti cancerous based on the criterion set by the National Cancer Institute (Geran et al., 1972). The leaf extracts had no anticancer activity under the experimental conditions tested.
Discussion
The methanol and water extracts of S. aculeastrum was shown to have inhibitory effect on cancer cell lines in the in vitro studies. It was effective in the suppression of proliferation of the three cancerous cell lines, HeLa, MCF7 and HT29 in a dose-dependent pattern. A vast variety of naturally occurring substances have been shown to protect against experimental carcinogenesis (Ju et al., 2004). Thus, it is becoming increasingly evident that certain phytochemicals, particularly those included in our daily diet, many have important cancer chemopreventive properties (Sanaha et al., 1997). In this study, there may be inhibitors and active ingredients in the S. aculeastrum extracts, which can induce cytotoxic action against cancer cells and initiate anti proliferative pathway leading to cancer cell death. Traditionally, S. aculeastrum is used for the treatment of breast cancer in South Africa. The berries of this plant are boiled in water until they burst into pieces. After filtration, the decoction is administered once a day until signs of relief is observed. This study has confirmed the ethnomedical application of S. aculeastrum in the treatment of cancer (Koduru et al., 2006c, d). The active compounds in the plant extracts are not yet known. However, plants belonging to the Solanaceae family have been reported to contain complex glycosides and saponins (Tan et al., 2005), which may be responsible for the observed activity.
Acknowledgment
This research was supported by the National Research Foundation of South Africa. The authors would like to thank Ms Debbie du Plessis for technical assistance with the anticancer screening and JGH du Preez from the Metal Ion Separations Unit, Nelson Mandela Metropolitan University for providing cisplatin.