Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Research Paper
In vitro cytotoxic and pro-apoptotic effects of water extracts of Tulbaghia violacea leaves and bulbs Saibu G.Ma, Katerere D.Rb, Rees D.J.Gc, Meyer Ma,n a
Department of Biotechnology, University of the Western Cape, Private Bag X17, Bellville 7535, Cape Town, South Africa Tshwane University of Technology, Faculty of Science, Department of Pharmaceutical Science, Building 4, Arcadia Campus, Pretoria 001, South Africa c Agricultural Research Council, Private Bag X05, Onderstepoort, Pretoria 0110, South Africa b
art ic l e i nf o
a b s t r a c t
Article history: Received 18 September 2014 Received in revised form 25 January 2015 Accepted 28 January 2015
Ethnopharmacological relevance: infusions of Tulbaghia violacea (wild garlic) in water are used in traditional medicine in Southern Africa to treat numerous diseases, including cancer. Several studies have previously demonstrated the cytotoxic activities of extracts of T. violacea in cultured cancer cells. Their findings support the potential anti-cancer properties of this plant. However, these studies made use of organic solvent extraction methods, while the traditional use of the plant involves the preparation of infusions in water. Materials and methods: In the current study, we investigated the potential anti-cancer properties of infusions of T. violacea. We also performed a comparative study investigating the cytotoxic activities of T. violacea bulbs and leaves. A panel of four cancer cell lines (HepG2, MCF7, H157, and HT29) and one noncancerous cell line (KMST6) was treated with the two extracts and the effects of the extracts on the growth of the cells were evaluated. We also investigated whether the growth inhibitory effects were associated with the induction of apoptosis and whether the mechanism of cell death is the result of oxidative stress and the activation of caspase-3. Result: We found that extracts of the leaves and not the bulbs have growth inhibitory effects and that this is the result of the induction of apoptosis, which is associated with the production of Reactive Oxygen Species (ROS) and the activation of caspase-3. The leaf extract demonstrated variable selective toxicity towards the cancer lines. Although the extract also induced cell death in the non-cancerous cell line (KMST6), we found that the levels of toxicity were lower in this cell line. Conclusion: this study confirms that infusions of T. violacea have potential anti-cancer activity and that this bioactivity is contained in the leaf extract. This study lends support to claims that this plant can be used to treat cancer. & 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Tulbaghia violacea Infusions Apoptosis Cancer Caspase-3 Reactive Oxygen Species
1. Introduction Humans have been using plants for millennia as a source of food and fuel, in the fabrication of textiles and cosmetics and as medicine. It is estimated that up to 90% of the population in Africa depend on traditional medicine for their health care needs (Wachtel-Galor and Benzie, 2011). A large number of medicinal plants that are indigenous to Southern Africa and their applications in complementary and alternative medicine have been described (Van Wyk et al., 2004). Traditional medicine is commonly used as an alternative treatment for cancer. Medicinal plants such as Sutherlandia frutescens (Tai et al.,
n
Corresponding author. Tel.: þ 27 21 959 2032; fax: þ27 21 959 3050. E-mail address: [email protected] (M. Meyer).
2004; Van Wyk and Albrecht, 2008), Camptotheca acuminate (Efferth et al., 2007), Taxus baccata (Cragg and Newman, 2005) and Tulbaghia violacea (Bungu et al., 2006; Koduru et al., 2007) have a long history of use in the treatment of cancer. Sixty five per cent of the widely used anti-cancer agents in conventional medicine are derived from natural sources such as plants and marine organisms (Balunas and Kinghorn, 2005). The best known examples are vinca alkaloids derived from Catharanthus roseus, paclitaxel from T. baccata, camptothecin from C. acuminate, monocrotaline from Crotolaria sessiliflora L., and colchicine derived from Colchicum autumnale L. (Cragg and Newman, 2005; da Rocha et al., 2001). T. violacea (Alliceae) commonly known as wild garlic, itswele iomlambo (Xhosa), wilde knoffel (Afrikaans) and isihaga (Zulu) belongs to the garlic family and is indigenous to the Eastern Cape and KwaZulu Natal in South Africa. It is used in traditional medicine as a remedy for the treatment of fever, asthma,
http://dx.doi.org/10.1016/j.jep.2015.01.040 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
Please cite this article as: Saibu, G.M, et al., In vitro cytotoxic and pro-apoptotic effects of water extracts of Tulbaghia violacea leaves and bulbs. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.01.040i
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hypertension and oesophageal cancer (Hutchings et al., 1996; Van Wyk and Wink, 2004). The traditional use of T. violacea involves the preparation of infusions by soaking the plant material (leaves and bulbs) in boiled water. The infusions are taken orally to treat various ailments including cancer. The anti-helmintic, antibacterial (McGaw et al., 2000), anti-fungal activity (Motsei et al., 2003) as well as growth inhibitory and potential anti-cancer activities (Bungu et al., 2006; Lyantagaye, 2013) of T. violacea were previously demonstrated. T. violaea was shown to contain allylsulphur compounds which are typical of the onion/garlic family of which marasmicin has been shown to be the most abundant (Ncube et al., 2011; Aremu and Van Staden, 2013). Several free sugars (e.g. arabinose, rhamnose, xylose) and glycosides (methylα-D-glucopyranoside, in particular) have been isolated from T. violaceae (Burton, 1990; Lyantagaye, 2013). Ncube et al. (2011) have also reported the presence of phenolic compounds such as flavonoids and both gallotannins and condensed tannin, as well as saponins. In the study by Bungu et al. (2006), methanol extracts of the T. violacea leaves and bulbs were investigated for growth inhibitory effects in MCF7, WHCO3, HT29 and HeLa cancer cells. This study found that the anti-proliferative effects of the bulb methanolic-extracts were significantly higher compared to the leaf methanolic-extract. The study also showed that the growth inhibitory effects are associated with the induction of apoptosis as demonstrated by the annexin-V assay and the cleavage of Poly (ADP-ribose) polymerases (PARP). Lyantagaye (2013) also confirmed the pro-apoptotic activities of T. violacea extracts. In the Lyantagaye study, a 50% aqueous methanol extract of the whole plant was effective in inducing apoptosis in CHO, MCF7, and HeLa cells, but not in H157 and MG63 cells (Lyantagaye, 2013). The study also showed that the apoptotic activity was associated with caspase-3 cleavage and the activation of DNA fragmentation. Further fractionation of that extract with various organic solvents (i.e. n-hexane, ethyl acetate, chloroform, dichloromethane and n-butanol) eventually led to the isolation of methyl-α-Dglucopyranoside which was postulated to be responsible for the pro-apoptotic activity though this is still subject to independent confirmation. In the current study, we revisited aspects of the previous studies but focused on water extracts of the plant since this is how the plant is used in traditional medicine. We also performed a comparative study between water extracts of the leaves and bulbs.
2. Materials and methods 2.1. Collection and authentication of plant material T. violacea plants were kindly donated by Ms. Toos van den Berg (Van den Berg Garden Village, Stellenbosch, South Africa). Plant collections were done in 2009, during the month of May. Mr. Franz Weitz (Department of Biodiversity and Conservation Biology, University of the Western Cape) authenticated the plant material and a voucher specimen (number 6975) was deposited at the herbarium of the University of the Western Cape. 2.2. Preparation and extraction of the plant material The leaves and the bulbs were separated, rinsed with distilled water and dried at room temperature for 7 days. The plant material was cut into small pieces and pulverised in a domestic blender. Two and half (2.5) litres of boiled water were added to the leaf (100 g) and the bulb (100 g) material was soaked overnight, while stirring. The extraction was repeated 3 times. The water extract was then filtered through a Whatmans filter paper (0.45 pm pore size) and
frozen in liquid nitrogen before freeze-drying the samples on a Virtis Sentry freeze dryer (Virtis company, Garner, New York). The dried extract was kept in a desiccator until further use. For applications in cell culture, the dried plant extract was weighed and dissolved in cell culture medium. 2.3. Cell culture All the cell lines used in this study i.e. HepG2 (liver carcinoma), MCF7 (breast carcinoma), H157 (lung carcinoma), HT29 (colon carcinoma), and KMST6 (non-cancerous fibroblast) were kindly provided by Prof Denver Hendricks (Department of Clinical and Laboratory Medicine, University of Cape Town, South Africa). The cells were maintained in Dulbecco's modified Eagle's medium containing 10% foetal bovine serum, and 1% penicillin–streptomycin in a 37 1C humidified incubator with 5% CO2 saturation. All cell culture reagents were supplied by Invitrogen Ltd., Grand Island, New York. Cells were either plated in 6-well cell culture plates at a cell density of 2.5 105 cells per well or in 24-well cell culture plates at a cell density of 1 105 cells per well or in a 96-well cell culture plates at a cell density of 2 104 cells per well. 2.4. Morphological studies The cells were plated in 6-well cell culture plates at a cell density of 2.5 105 cells per well. After 24 h the spent medium was replaced with fresh medium containing 2.5 mg/ml of the plant extracts. As a negative control, cells were left untreated. Images of the cells were taken after 24 h at a 20 magnification using a Nikon microscope fitted with a Leica digital camera. 2.5. MTT cell proliferation assay Cell proliferation was determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay following the methods described by Freimoser et al., 1999. The cells were plated in 96-well cell culture plates at a cell density of 2 104 cells per well. After 24 h the spent medium was replaced with fresh medium containing increasing concentrations (0.06–5 mg/ml) of the plant extracts. A negative control of untreated cells was included. All treatments, including the negative control were done in triplicate. The cells were incubated for a further 24 h, after which a 0.5 mg/ml MTT solution (10 μl per well) was added and the cells incubated for an additional 4 h. The media was removed and dimethyl sulfoxide (DMSO) (100 μl per well) was added to the cells. The cell culture plates were placed on a shaker for 10 min and the absorbance was read at 560 nm using a BMG Labtech Omegas POLARStar multimodal plate reader. The percentage cell viability was calculated using the formulae below: cell viability (%)¼OD560 (Treated sample)/OD560 (Untreated control) 100. A survival curve was constructed and the IC50 values were determined using GraphPad Prism software (GraphPad software, San Diego, CA, USA). 2.6. Evaluation of apoptosis using the APOPercentage™ assay The induction of apoptosis was assessed using the APOPercentage™ assay (Biocolor Ltd., UK). The cells were plated in 24-well cell culture plates at a density of 1 105 cells per well. After 24 h the spent medium was replaced with fresh medium containing increasing concentrations (0.06–2.5 mg/ml) of the plant extracts. A negative control of untreated cells was included while cells treated with 10 mM doxorubicin, a known inducer of apoptosis (Tsang et al., 2003), served as a positive control. All treatments, including the negative control were done in triplicate. The cells were incubated for a further 24 h, harvested by trypsinization then stained with the APOPercentage™ dye and analysed by flow
Please cite this article as: Saibu, G.M, et al., In vitro cytotoxic and pro-apoptotic effects of water extracts of Tulbaghia violacea leaves and bulbs. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.01.040i
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cytometry on a Becton Dickinson FACScan instrument (BD Pharmingen™, USA) as described by Meyer et al. (2008). 2.7. Assessment of oxidative stress The generation of intracellular Reactive Oxygen Species (ROS) in response to treatment with the leaf extract (which had been found to be more active in the foregoing assays) was assessed using the 5-(and-6)-chloromethyl-20 ,70 -dichlorodihydrofluorescein diacetate (CM-H2DCFDA) probe (Life Technologies™). The cells were plated in 6-well cell culture plates. After 24 h the spent medium was replaced with fresh medium containing 2 mg/ml of the leaf extract. A negative control of untreated cells was included while cells treated with 1% hydrogen peroxide (H2O2) served as a positive control. Hydrogen peroxide is known to induce oxidative stress damage in cell cultures (Apel and Hirt, 2004). All treatments including the negative control were done in triplicate. Following the treatments, the cells were recovered by trypsinization and stained for 30 min in the dark at 37 1C with 7.5 μM of the CM-H2DCFDA probe. After the incubation, the cells were analysed at 530 nm on Becton Dickinson FACScan instrument (BD Pharmingen™, USA). A minimum of 10,000 cells per sample was acquired and analysed using CELLQuest PRO software (BD Pharmingen™, USA).
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antibody that is specific for active caspase-3. Cell staining was measured by flow cytometry at 670 nm on a Becton Dickinson FACScan instrument (BD Pharmingen™, USA). A minimum of 10,000 cells per sample was acquired and analysed using CELLQuest PRO software (BD Pharmingen™, USA). 2.9. Selective Index (SI) The degree of selective toxicity of the extracts towards cancer cell lines relative to the non-cancerous cell line was expressed as Selective Index (SI) (Badisa et al., 2009). The SI was calculated as: SI¼IC50 of the extract in a non-cancerous cell line (KMST6)/IC50 of the extract in a cancer cell line. The IC50 values were determined using the MTT assay. 2.9.1. Statistical analysis All the data was analysed using one-way ANOVA using GraphPad Prism software (GraphPad software, San Diego, CA, USA). Differences between the mean7SEM (standard error of the mean) of samples were considered significant at Po0.05. The IC50 values were generated from the MTT results using GraphPad Prism software. The Selective Index was calculated against non-cancerous cell line (KMST6) using the IC50 values obtained from the MTT results.
2.8. Caspase-3 activation assay
3. Results
The activation of caspase-3 was detected using the Caspase3mAb Apoptosis Kit (BD Pharmingen™, USA). The cells (HepG2, HT29 and H157 and KMST6) were plated in 6-well cell culture plates. It has previously been shown that MCF7 cells do not express caspase-3 (Liang et al., 2001) consequently, we did not investigate caspase-3 activation in that cell line. After 24 h the spent medium was replaced with fresh medium containing 2 mg/ml of the leave extract. A negative control of untreated cells was included while cells treated with 10 mM doxorubicin served as a positive control. All treatments, including the controls were done in triplicate. The cells were removed by trypsinization, washed twice with PBS and resuspended in Cytofix/Cytoperm buffer (BD Pharmingen™, USA). Following 20 min incubation on ice, the cells were washed twice with Perm/Wash buffer (BD Pharmingen™, USA) and incubated for 30 min at room temperature with a PE-conjugated monoclonal
The results for the MTT assay demonstrated that the water extract of T. violacea leaves had a greater inhibitory effect on the growth of the cells compared to the water extract of T. violacea bulbs, which showed no activity (Fig. 2). This was supported by an investigation into the effects of extracts prepared from T. violacea leaves and bulbs on the morphology of cells in culture. Fig. 1 shows the effects on the morphology of MCF7 cells treated for 24 h with 2.5 mg/ml of T. violacea leaf and bulb extracts. While cells treated with the leaf extract showed signs of growth inhibition, shrinkage, vacuolisation and detachment from the culture flask, cells treated with the bulb extract were not affected by the treatment and resembled the untreated control cells. The results for HepG2, H157, HT29 and KMST6 cells were similar (data not shown). The growth inhibitory effects of the leaf extract were highest in MCF7 with an IC50 value of 1.270.5 mg/ml, while the effects on
Fig. 1. Morphological changes induced in MCF7 cells by water extracts of T. violacea leaves and bulbs. MCF7 cells were either left untreated (A) or treated for 24 h with 2.5 mg/ml of a water extract of T. violacea bulbs (B) or leaves (C). As a positive control, the cells were treated for 24 h with 10 mM doxorubicin (D). The cells were studied by light microscopy using a Nikon microscope. Images were taken at 20 magnification using a Leica digital camera. The experiments were done in triplicate.
Please cite this article as: Saibu, G.M, et al., In vitro cytotoxic and pro-apoptotic effects of water extracts of Tulbaghia violacea leaves and bulbs. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.01.040i
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the non-cancerous cell line, KMST6 were the lowest (IC50 ¼3.0 7 0.8 mg/ml) (Table 1). The Selective Index for the MCF7 was 2.5 and also confirmed that this cell line was more susceptible to the effects of the T. violacea leaf extract compared to the other cancer cell lines. The SI for H157, HT29 and HepG2 cell lines were 2.0, 1.2 and 1.0, respectively. HepG2 was the most resistant cancer cell line (IC50 ¼ 2.8 70.5 mg/ml). There was also a low margin of selectivity between HepG2 and KMST6. The results from the APOPercentage™ assay demonstrated that the growth inhibitory effects of the T. violacea leaf extract are associated with dose dependent induction of apoptosis (Fig. 2). Generally, it was only concentrations higher than 1 mg/ml that were able to induce significant levels of apoptosis in the cells. However, concentrations as low as 0.06 mg/ml induced significant levels of apoptosis H157 cells. A significant variation was observed in the responses of the different cell lines to treatment with the leaf extract. This is more evident at the highest dose of 2.5 mg/ml, where the percentage of apoptotic cells for HepG2 (2A), HT29 (2B), MCF7 (2C), H157 (2D) and KMST6 (2E) were 60%, 80%, 40%, 60% and 40%, respectively. The T. violacea bulb extract failed to induce any significant levels of apoptosis in the cell lines tested in this study. The caspase activation assay showed that the T. violacea leaf extract induced the cleavage of caspase-3 in H157, HepG2 and HT29 but not in KMST6 cells (Fig. 3). However, the extract induced caspase-3 activation more efficiently in HepG2 cells with 70% of the cells staining positive for the presence of cleaved caspase-3,
Cells positive for cleaved caspase-3 (%)
while approximately 20% of H157 and HT29 cells were positive for cleaved caspase-3. However, compared to the noncancerous cell line (KMST6), no statistically significant difference in caspase-3 activity could be observed in H157 and HT29 cells. The measurement of ROS production in cells treated with the T. violacea leaf extract demonstrated that the induction of apoptosis was associated with oxidative stress. The intracellular levels of ROS were measured using the CM-H2DCFDA probe (Fig. 4). These results also showed a selective response in different cell lines. The increased production of ROS was detected in all the cancer cell lines (H157, HepG2, MCF7 and HT29), but not in noncancerous cell line (KMST6). ROS production was the highest in H157 and HepG2 cells, with 80% and 50% of the cells, respectively
Table 1 IC50 values and Selective Index for leaf extracts as determined by the MTT assay.
*
100
Leaf Extract (mg/ml)
Selective Index
MCF7 H157 HT29 HepG2 KMST6
1.2 7 0.50 1.5 7 0.76 2.6 7 1.50 2.8 7 0.45 37 0.80
2.50 2.00 1.15 1.07 1.00
Apoptosis (%)
80 60
*
*
40 20 0
40
0
*
80 60 40 20
10 µM Doxorubicin
0
*
*
*
*
*
*
Apoptosis (%)
Apoptosis (%)
80
20
H157
2mg/ml Leaf extract
HepG2
HT29
*
100 80 60 *
40
*
20 0
100
40
*
* 20
100
*
a
Fig. 3. Evaluating the activation of caspase-3 in response to treatment with the leaf extract. A panel of four human cancer cell lines (KMST6, H157, HepG2, and HT29) was treated for 24 h with 2 mg/ml of the leaf extract. As a positive control, the cells were also treated with 10 μM doxorubicin. Cell fluorescence was measured by flow cytometry. The bar graph indicates the percentage of cells staining positive for active caspase-3. The experiments were done in triplicate. A significance difference (P o0.05) between untreated and extract treated is represented by (n); (a) represents significant difference between the non-cancerous cell line (KMST6) and the cancer cell line.
0
60
*
60
Untreated
The Selective Index (SI) was determined using the following formulae:SI ¼IC50 of KMST6 cell line/IC50 of cancer cell line.
100
* *
Apoptosis (%)
Cell lines
*
80
KMST6
Apoptosis (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
100
Leaf Extract
80 Bulb Extract
60
* *
40 20
*
0
Fig. 2. Quantification of apoptosis induced by bulb and leaf extracts. Four human cancer cell lines (HepG2, HT29, MCF7 and H157) and one non-cancerous human cell line (KMST6) were treated with increasing concentrations (0.06–2.5 mg/ml) of the leaf and bulb extracts. As a positive control, the cells were also treated 10 mM doxorubicin. Apoptosis was quantified by flow cytometry using the APOPercentage assay. Fig. 2A–D and Fig. 1E show the results for HepG2, HT29, MCF7, H157 and KMST6 cells, respectively. All treatments were performed in triplicate. A significant difference (P o 0.05) between treatments with the leaf extract and tuber extract is indicated by (n).
Please cite this article as: Saibu, G.M, et al., In vitro cytotoxic and pro-apoptotic effects of water extracts of Tulbaghia violacea leaves and bulbs. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.01.040i
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
Cells positive for presence of ROS (%)
G.M Saibu et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
*
100
* 80
*
*
*
a
* 60
*
a
* a
40
* a
20
0 Untreated KMST6
Hydrogen Peroxide MCF7
HepG2
2mg/ml Leaf extract
HT29
H157
Fig. 4. Assessing oxidative stress in response to treatment with the leaf extract. A panel of five human cancer cell lines (HepG2, HT29, MCF7, H157 and KMST6) was treated for 24 h with 2 mg/ml of the leaf extract. As a positive control, the cells were also treated with 1% hydrogen peroxide. Cell fluorescence was measured by flow cytometry. The experiments were done in triplicate. A significance difference (Po 0.05) between untreated and extract treated is represented by (n); (a) represents significant difference between the non-cancerous cell line (KMST6) and the cancer cell line.
being positive for oxidative stress. Less than 20% of HT29 cells were positive for increased levels of ROS.
4. Discussion and conclusion This study found that the growth inhibitory effects of water extracts of T. violacea leaves in cell cultures are much higher compared to water extracts of T. violacea bulbs. In fact the bulb extract demonstrated negligible activities (less than 20% cell death) in all the cell lines tested in this study (Fig. 2). This appears to be contrary to the findings of Bungu et al. (2006) which demonstrated that the growth inhibitory effects of extracts of the bulbs were higher compared to the leaves (Bungu et al., 2006). However, it should be pointed out that the study by Bungu et al. (2006) used methanol for extraction while the current study used water only, which is in line with the traditional method of extraction. Water extraction is more suitable for this study given that the objective here was to investigate enthnopharmacological claims of this plant in the treatment of cancer. It is well known that the method of extraction, time of extraction, temperature, solvent type, polarity and concentration of the solvent, can affect the extraction of phytochemical compounds (Prashant et al., 2011). Based on the MTT assay, the growth inhibitory effects of water extracts of T. violacea leaves was higher in the MCF7 and H157 cells compared to the other cell lines (Fig. 2). Interestingly, Bungu et al. (2006) also showed that MCF7 were more susceptible to inhibition by methanolic extracts of T. violacea bulbs compared to WHOC3, HT29, and HeLa cells. In the current study, the non-cancerous cell line (KMST6) was shown to be most resistant which may imply that extracts of T. violacea leaves would be selectively toxic to neoplastic cells (Table 1). Cells treated with water extracts of T. violacea leaves displayed signs of growth inhibition, cell shrinkage, vacuolisation and cell detachment (Fig. 1). These are morphological changes often observed in apoptotic cells (Darzynkiewicz et al., 1992; Desagher and Martinou, 2000). Although the MTT assay can be used to assess cytotoxicity, it does not differentiate between the different modes of cell death. We therefore also investigated the mode of the cytotoxicity observed. Bungu et al. (2006) and Lyantagaye (2013) previously demonstrated
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that T. violacea extracts induced apoptosis (Bungu et al., 2006; Lyantagaye, 2013). Apoptosis is a form of programmed cell death and one of the roles of apoptosis is to prevent the development of cancer (Elmore, 2007; Hengartner, 2000). Deregulation in the control of apoptosis is often associated with the development of cancer (Lowe and Lin, 2000; Vogelstein and Kinzler, 2004). The activation of apoptosis in cancer cells is therefore a corrective strategy and many anti-cancer drugs may exert apoptotic effects in cancer cells (Aggarwal and Shishodia, 2006; Evan and Vousden, 2001). Compounds or extracts with pro-apoptotic activities in cancer cells are therefore potentially useful in anti-cancer drug research. Our study shows that water extracts of T. violacea leaves induce apoptosis in cultured cells thus corroborating previous studies (Bungu et al. 2006; Lyantagaye 2013). However, the effective concentrations of the extracts used in the current study are significantly higher compared to the extracts used in the previous studies. Bungu et al. (2006) showed pro-apoptotic activity at concentrations as low as 250 μg/ml within 24 h while Lyantagaye (2013) showed the induction of apoptosis at 500 μg/ml within 1 h with an aqueous methanol extract of the total plant. We on the other hand, generally only observed significant levels of apoptosis after 24 h of treatment with 1 mg/ml of the water extract. This may be as a result of different extraction methods used in these studies. It appears that the methanolic extracts are more potent than the water extracts. Our study found that the non-cancerous cell line (KMST6) was more resistant to the effects of the extract since the levels of apoptosis were significantly lower for this cell line. This selective toxicity towards cancer cells is a characteristic that is highly sought after in the development of new anti-cancer treatments because much of the adverse effects observed with anti-cancer drugs results from toxicity to normal cells (Sa and Das, 2008). We also showed that the T. violacea leaf extract induced higher levels of apoptosis (80% at a concentration of 2.5 μg/ml) in HT29 cancer cells compared to the other cell lines (H157, HepG2, MCF7 and KMST6) used in this study (Fig. 2). This was a surprising finding since the MTT assay showed that the leaf extract was more toxic to MCF7 cells. The MTT assay measures cell viability as a function of the cell's metabolic activity, while the APOPercentage™ assay detects changes in cell membranes specifically associated with the activation of apoptosis. Cell death is classified into three categories; type I (apoptosis), type II (autophagy) and type III (necrosis) (Clarke, 1990). Type III cell death is a non-apoptotic form of cell death and is associated with organelle dysfunction (e.g. mitochondrial disruption). Loss of cell viability is therefore not always due to apoptosis. It is possible that the plant extract induces multiple forms of cell death, which may explain the apparent disparity in the results obtained for MCF7 cells using the MTT and APOPercentage™ assays. We also investigated the activation of caspase-3 in HepG2, H157, HT29 and KMST6 cells. Although caspase-3 is central to the apoptosis pathways (Porter and Jänicke, 1999), caspase-3 independent apoptosis pathways have also been identified (Liang et al., 2001). MCF7 cells for example do not express caspase-3 but are able to activate caspase-3 independent apoptotic pathways (Liang et al., 2001). It is for this reason that we did not investigate the activation of caspase-3 in MCF7 cells. We found the highest levels of caspase-3 activation in HepG2 cells, with 70% of these cells staining positive for cleaved caspase-3, while less than 20% of HT29 and H157 cells stained positive for cleaved caspase-3 (Fig. 3). Consequently, even though higher levels of apoptosis were observed in H157 cells, these cells demonstrated lower caspase-3 activity compared to HepG2 cells. It should be noted that apoptosis can be activated by multiple pathways and that HepG2, H157, HT29 and MCF7 are cancer cell lines with multiple genetic mutations, which could affect how these
Please cite this article as: Saibu, G.M, et al., In vitro cytotoxic and pro-apoptotic effects of water extracts of Tulbaghia violacea leaves and bulbs. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.01.040i
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cells respond to stimuli that activate the same biochemical pathways (Okamoto et al., 1994; Yoshikawa et al., 1999) and this could possibly explain the diverse responses we observed in the panel of cell lines we used in this study. No significant increase in caspase-3 activity was detected in KMST6 cells, confirming that these cells are resistant to the effects of the T. violacea leaf extract. The fact that the APOPercentage™ assay showed that KMST6 cells treated with this extract die via apoptosis might suggest that apoptosis induced in KMST6 cells is independent of caspase-3 activity. Several anti-cancer drugs, including doxorubicin are known to induce oxidative stress in cancer cells, which in turn can cause damage to DNA, proteins and lipids, resulting in the activation of apoptosis (Klaunig et al., 2010; Orrenius et al., 2007). In this study, we found that the T. violacea leaf extract selectively induced oxidative stress in MCF7, HepG2, HT29 and H157 cells but not KMST6 cells (Fig. 4). A significant increase in ROS was detected in H157 (80%) and HepG2 (50%), while lower levels of ROS were detected in HT29 and MCF7 cells. Interestingly, even though high levels of ROS were detected in HepG2, this did not result in high levels of cell death in this cell line (as shown by the MTT and APOPercentage™ assays). Cancer cells have elevated levels of ROS and therefore also have increased antioxidant capacity to detoxify the cells from ROS (Liou and Storz, 2010). Increased levels of ROS play a key role in tumour development and progression, but can also be toxic to cancer cells (Glasauer and Chandel, 2014). It is possible that HepG2 cells are able to neutralise the damaging effects of oxidative stress more efficiently than some of the other cell lines. In conclusion this study supports the enthobotanical use of T. violacea for possible anti-cancer activity. We demonstrated that this activity is present in water extracts of the leaves and not the bulbs. This suggests that for applications in the treatment of cancer, only the leaves are required. This finding can be beneficial for the sustainable use of this plant in traditional medicine, since this implies that there is no need to harvest the bulbs of the plant as well (Katerere and Eloff, 2008). We also demonstrated the selective toxicity of the plant extract towards cancer cells and that the toxicity is in some cases associated with increased oxidative stress. It is not clear which chemical constituents were responsible for the putative biological activity. However from reports on the phytochemistry of T. violacea, it can be speculated that the allylsulphur, phenolic glycosides and free glycosides could possibly be responsible for the bioactivity. Further studies to isolate and characterise the individual biologically active constituents should be conducted.
Conflict of interest We declare that there is no conflict of interests.
Uncited references (Jänicke et al., (1998); Susan, (2007)).
Acknowledgements We thank the National Research Foundation (South Africa) and the South African Medical Research Council for funding this project. We also thank the PROMEC Unit and Indigenous Knowledge Systems Unit of the Medical Research Council for access to their laboratory facilities.
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Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Research Paper
In vitro cytotoxic and pro-apoptotic effects of water extracts of Tulbaghia violacea leaves and bulbs Saibu G.Ma, Katerere D.Rb, Rees D.J.Gc, Meyer Ma,n a
Department of Biotechnology, University of the Western Cape, Private Bag X17, Bellville 7535, Cape Town, South Africa Tshwane University of Technology, Faculty of Science, Department of Pharmaceutical Science, Building 4, Arcadia Campus, Pretoria 001, South Africa c Agricultural Research Council, Private Bag X05, Onderstepoort, Pretoria 0110, South Africa b
art ic l e i nf o
a b s t r a c t
Article history: Received 18 September 2014 Received in revised form 25 January 2015 Accepted 28 January 2015
Ethnopharmacological relevance: infusions of Tulbaghia violacea (wild garlic) in water are used in traditional medicine in Southern Africa to treat numerous diseases, including cancer. Several studies have previously demonstrated the cytotoxic activities of extracts of T. violacea in cultured cancer cells. Their findings support the potential anti-cancer properties of this plant. However, these studies made use of organic solvent extraction methods, while the traditional use of the plant involves the preparation of infusions in water. Materials and methods: In the current study, we investigated the potential anti-cancer properties of infusions of T. violacea. We also performed a comparative study investigating the cytotoxic activities of T. violacea bulbs and leaves. A panel of four cancer cell lines (HepG2, MCF7, H157, and HT29) and one noncancerous cell line (KMST6) was treated with the two extracts and the effects of the extracts on the growth of the cells were evaluated. We also investigated whether the growth inhibitory effects were associated with the induction of apoptosis and whether the mechanism of cell death is the result of oxidative stress and the activation of caspase-3. Result: We found that extracts of the leaves and not the bulbs have growth inhibitory effects and that this is the result of the induction of apoptosis, which is associated with the production of Reactive Oxygen Species (ROS) and the activation of caspase-3. The leaf extract demonstrated variable selective toxicity towards the cancer lines. Although the extract also induced cell death in the non-cancerous cell line (KMST6), we found that the levels of toxicity were lower in this cell line. Conclusion: this study confirms that infusions of T. violacea have potential anti-cancer activity and that this bioactivity is contained in the leaf extract. This study lends support to claims that this plant can be used to treat cancer. & 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Tulbaghia violacea Infusions Apoptosis Cancer Caspase-3 Reactive Oxygen Species
1. Introduction Humans have been using plants for millennia as a source of food and fuel, in the fabrication of textiles and cosmetics and as medicine. It is estimated that up to 90% of the population in Africa depend on traditional medicine for their health care needs (Wachtel-Galor and Benzie, 2011). A large number of medicinal plants that are indigenous to Southern Africa and their applications in complementary and alternative medicine have been described (Van Wyk et al., 2004). Traditional medicine is commonly used as an alternative treatment for cancer. Medicinal plants such as Sutherlandia frutescens (Tai et al.,
n
Corresponding author. Tel.: þ 27 21 959 2032; fax: þ27 21 959 3050. E-mail address: [email protected] (M. Meyer).
2004; Van Wyk and Albrecht, 2008), Camptotheca acuminate (Efferth et al., 2007), Taxus baccata (Cragg and Newman, 2005) and Tulbaghia violacea (Bungu et al., 2006; Koduru et al., 2007) have a long history of use in the treatment of cancer. Sixty five per cent of the widely used anti-cancer agents in conventional medicine are derived from natural sources such as plants and marine organisms (Balunas and Kinghorn, 2005). The best known examples are vinca alkaloids derived from Catharanthus roseus, paclitaxel from T. baccata, camptothecin from C. acuminate, monocrotaline from Crotolaria sessiliflora L., and colchicine derived from Colchicum autumnale L. (Cragg and Newman, 2005; da Rocha et al., 2001). T. violacea (Alliceae) commonly known as wild garlic, itswele iomlambo (Xhosa), wilde knoffel (Afrikaans) and isihaga (Zulu) belongs to the garlic family and is indigenous to the Eastern Cape and KwaZulu Natal in South Africa. It is used in traditional medicine as a remedy for the treatment of fever, asthma,
http://dx.doi.org/10.1016/j.jep.2015.01.040 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
Please cite this article as: Saibu, G.M, et al., In vitro cytotoxic and pro-apoptotic effects of water extracts of Tulbaghia violacea leaves and bulbs. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.01.040i
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hypertension and oesophageal cancer (Hutchings et al., 1996; Van Wyk and Wink, 2004). The traditional use of T. violacea involves the preparation of infusions by soaking the plant material (leaves and bulbs) in boiled water. The infusions are taken orally to treat various ailments including cancer. The anti-helmintic, antibacterial (McGaw et al., 2000), anti-fungal activity (Motsei et al., 2003) as well as growth inhibitory and potential anti-cancer activities (Bungu et al., 2006; Lyantagaye, 2013) of T. violacea were previously demonstrated. T. violaea was shown to contain allylsulphur compounds which are typical of the onion/garlic family of which marasmicin has been shown to be the most abundant (Ncube et al., 2011; Aremu and Van Staden, 2013). Several free sugars (e.g. arabinose, rhamnose, xylose) and glycosides (methylα-D-glucopyranoside, in particular) have been isolated from T. violaceae (Burton, 1990; Lyantagaye, 2013). Ncube et al. (2011) have also reported the presence of phenolic compounds such as flavonoids and both gallotannins and condensed tannin, as well as saponins. In the study by Bungu et al. (2006), methanol extracts of the T. violacea leaves and bulbs were investigated for growth inhibitory effects in MCF7, WHCO3, HT29 and HeLa cancer cells. This study found that the anti-proliferative effects of the bulb methanolic-extracts were significantly higher compared to the leaf methanolic-extract. The study also showed that the growth inhibitory effects are associated with the induction of apoptosis as demonstrated by the annexin-V assay and the cleavage of Poly (ADP-ribose) polymerases (PARP). Lyantagaye (2013) also confirmed the pro-apoptotic activities of T. violacea extracts. In the Lyantagaye study, a 50% aqueous methanol extract of the whole plant was effective in inducing apoptosis in CHO, MCF7, and HeLa cells, but not in H157 and MG63 cells (Lyantagaye, 2013). The study also showed that the apoptotic activity was associated with caspase-3 cleavage and the activation of DNA fragmentation. Further fractionation of that extract with various organic solvents (i.e. n-hexane, ethyl acetate, chloroform, dichloromethane and n-butanol) eventually led to the isolation of methyl-α-Dglucopyranoside which was postulated to be responsible for the pro-apoptotic activity though this is still subject to independent confirmation. In the current study, we revisited aspects of the previous studies but focused on water extracts of the plant since this is how the plant is used in traditional medicine. We also performed a comparative study between water extracts of the leaves and bulbs.
2. Materials and methods 2.1. Collection and authentication of plant material T. violacea plants were kindly donated by Ms. Toos van den Berg (Van den Berg Garden Village, Stellenbosch, South Africa). Plant collections were done in 2009, during the month of May. Mr. Franz Weitz (Department of Biodiversity and Conservation Biology, University of the Western Cape) authenticated the plant material and a voucher specimen (number 6975) was deposited at the herbarium of the University of the Western Cape. 2.2. Preparation and extraction of the plant material The leaves and the bulbs were separated, rinsed with distilled water and dried at room temperature for 7 days. The plant material was cut into small pieces and pulverised in a domestic blender. Two and half (2.5) litres of boiled water were added to the leaf (100 g) and the bulb (100 g) material was soaked overnight, while stirring. The extraction was repeated 3 times. The water extract was then filtered through a Whatmans filter paper (0.45 pm pore size) and
frozen in liquid nitrogen before freeze-drying the samples on a Virtis Sentry freeze dryer (Virtis company, Garner, New York). The dried extract was kept in a desiccator until further use. For applications in cell culture, the dried plant extract was weighed and dissolved in cell culture medium. 2.3. Cell culture All the cell lines used in this study i.e. HepG2 (liver carcinoma), MCF7 (breast carcinoma), H157 (lung carcinoma), HT29 (colon carcinoma), and KMST6 (non-cancerous fibroblast) were kindly provided by Prof Denver Hendricks (Department of Clinical and Laboratory Medicine, University of Cape Town, South Africa). The cells were maintained in Dulbecco's modified Eagle's medium containing 10% foetal bovine serum, and 1% penicillin–streptomycin in a 37 1C humidified incubator with 5% CO2 saturation. All cell culture reagents were supplied by Invitrogen Ltd., Grand Island, New York. Cells were either plated in 6-well cell culture plates at a cell density of 2.5 105 cells per well or in 24-well cell culture plates at a cell density of 1 105 cells per well or in a 96-well cell culture plates at a cell density of 2 104 cells per well. 2.4. Morphological studies The cells were plated in 6-well cell culture plates at a cell density of 2.5 105 cells per well. After 24 h the spent medium was replaced with fresh medium containing 2.5 mg/ml of the plant extracts. As a negative control, cells were left untreated. Images of the cells were taken after 24 h at a 20 magnification using a Nikon microscope fitted with a Leica digital camera. 2.5. MTT cell proliferation assay Cell proliferation was determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay following the methods described by Freimoser et al., 1999. The cells were plated in 96-well cell culture plates at a cell density of 2 104 cells per well. After 24 h the spent medium was replaced with fresh medium containing increasing concentrations (0.06–5 mg/ml) of the plant extracts. A negative control of untreated cells was included. All treatments, including the negative control were done in triplicate. The cells were incubated for a further 24 h, after which a 0.5 mg/ml MTT solution (10 μl per well) was added and the cells incubated for an additional 4 h. The media was removed and dimethyl sulfoxide (DMSO) (100 μl per well) was added to the cells. The cell culture plates were placed on a shaker for 10 min and the absorbance was read at 560 nm using a BMG Labtech Omegas POLARStar multimodal plate reader. The percentage cell viability was calculated using the formulae below: cell viability (%)¼OD560 (Treated sample)/OD560 (Untreated control) 100. A survival curve was constructed and the IC50 values were determined using GraphPad Prism software (GraphPad software, San Diego, CA, USA). 2.6. Evaluation of apoptosis using the APOPercentage™ assay The induction of apoptosis was assessed using the APOPercentage™ assay (Biocolor Ltd., UK). The cells were plated in 24-well cell culture plates at a density of 1 105 cells per well. After 24 h the spent medium was replaced with fresh medium containing increasing concentrations (0.06–2.5 mg/ml) of the plant extracts. A negative control of untreated cells was included while cells treated with 10 mM doxorubicin, a known inducer of apoptosis (Tsang et al., 2003), served as a positive control. All treatments, including the negative control were done in triplicate. The cells were incubated for a further 24 h, harvested by trypsinization then stained with the APOPercentage™ dye and analysed by flow
Please cite this article as: Saibu, G.M, et al., In vitro cytotoxic and pro-apoptotic effects of water extracts of Tulbaghia violacea leaves and bulbs. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.01.040i
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cytometry on a Becton Dickinson FACScan instrument (BD Pharmingen™, USA) as described by Meyer et al. (2008). 2.7. Assessment of oxidative stress The generation of intracellular Reactive Oxygen Species (ROS) in response to treatment with the leaf extract (which had been found to be more active in the foregoing assays) was assessed using the 5-(and-6)-chloromethyl-20 ,70 -dichlorodihydrofluorescein diacetate (CM-H2DCFDA) probe (Life Technologies™). The cells were plated in 6-well cell culture plates. After 24 h the spent medium was replaced with fresh medium containing 2 mg/ml of the leaf extract. A negative control of untreated cells was included while cells treated with 1% hydrogen peroxide (H2O2) served as a positive control. Hydrogen peroxide is known to induce oxidative stress damage in cell cultures (Apel and Hirt, 2004). All treatments including the negative control were done in triplicate. Following the treatments, the cells were recovered by trypsinization and stained for 30 min in the dark at 37 1C with 7.5 μM of the CM-H2DCFDA probe. After the incubation, the cells were analysed at 530 nm on Becton Dickinson FACScan instrument (BD Pharmingen™, USA). A minimum of 10,000 cells per sample was acquired and analysed using CELLQuest PRO software (BD Pharmingen™, USA).
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antibody that is specific for active caspase-3. Cell staining was measured by flow cytometry at 670 nm on a Becton Dickinson FACScan instrument (BD Pharmingen™, USA). A minimum of 10,000 cells per sample was acquired and analysed using CELLQuest PRO software (BD Pharmingen™, USA). 2.9. Selective Index (SI) The degree of selective toxicity of the extracts towards cancer cell lines relative to the non-cancerous cell line was expressed as Selective Index (SI) (Badisa et al., 2009). The SI was calculated as: SI¼IC50 of the extract in a non-cancerous cell line (KMST6)/IC50 of the extract in a cancer cell line. The IC50 values were determined using the MTT assay. 2.9.1. Statistical analysis All the data was analysed using one-way ANOVA using GraphPad Prism software (GraphPad software, San Diego, CA, USA). Differences between the mean7SEM (standard error of the mean) of samples were considered significant at Po0.05. The IC50 values were generated from the MTT results using GraphPad Prism software. The Selective Index was calculated against non-cancerous cell line (KMST6) using the IC50 values obtained from the MTT results.
2.8. Caspase-3 activation assay
3. Results
The activation of caspase-3 was detected using the Caspase3mAb Apoptosis Kit (BD Pharmingen™, USA). The cells (HepG2, HT29 and H157 and KMST6) were plated in 6-well cell culture plates. It has previously been shown that MCF7 cells do not express caspase-3 (Liang et al., 2001) consequently, we did not investigate caspase-3 activation in that cell line. After 24 h the spent medium was replaced with fresh medium containing 2 mg/ml of the leave extract. A negative control of untreated cells was included while cells treated with 10 mM doxorubicin served as a positive control. All treatments, including the controls were done in triplicate. The cells were removed by trypsinization, washed twice with PBS and resuspended in Cytofix/Cytoperm buffer (BD Pharmingen™, USA). Following 20 min incubation on ice, the cells were washed twice with Perm/Wash buffer (BD Pharmingen™, USA) and incubated for 30 min at room temperature with a PE-conjugated monoclonal
The results for the MTT assay demonstrated that the water extract of T. violacea leaves had a greater inhibitory effect on the growth of the cells compared to the water extract of T. violacea bulbs, which showed no activity (Fig. 2). This was supported by an investigation into the effects of extracts prepared from T. violacea leaves and bulbs on the morphology of cells in culture. Fig. 1 shows the effects on the morphology of MCF7 cells treated for 24 h with 2.5 mg/ml of T. violacea leaf and bulb extracts. While cells treated with the leaf extract showed signs of growth inhibition, shrinkage, vacuolisation and detachment from the culture flask, cells treated with the bulb extract were not affected by the treatment and resembled the untreated control cells. The results for HepG2, H157, HT29 and KMST6 cells were similar (data not shown). The growth inhibitory effects of the leaf extract were highest in MCF7 with an IC50 value of 1.270.5 mg/ml, while the effects on
Fig. 1. Morphological changes induced in MCF7 cells by water extracts of T. violacea leaves and bulbs. MCF7 cells were either left untreated (A) or treated for 24 h with 2.5 mg/ml of a water extract of T. violacea bulbs (B) or leaves (C). As a positive control, the cells were treated for 24 h with 10 mM doxorubicin (D). The cells were studied by light microscopy using a Nikon microscope. Images were taken at 20 magnification using a Leica digital camera. The experiments were done in triplicate.
Please cite this article as: Saibu, G.M, et al., In vitro cytotoxic and pro-apoptotic effects of water extracts of Tulbaghia violacea leaves and bulbs. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.01.040i
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the non-cancerous cell line, KMST6 were the lowest (IC50 ¼3.0 7 0.8 mg/ml) (Table 1). The Selective Index for the MCF7 was 2.5 and also confirmed that this cell line was more susceptible to the effects of the T. violacea leaf extract compared to the other cancer cell lines. The SI for H157, HT29 and HepG2 cell lines were 2.0, 1.2 and 1.0, respectively. HepG2 was the most resistant cancer cell line (IC50 ¼ 2.8 70.5 mg/ml). There was also a low margin of selectivity between HepG2 and KMST6. The results from the APOPercentage™ assay demonstrated that the growth inhibitory effects of the T. violacea leaf extract are associated with dose dependent induction of apoptosis (Fig. 2). Generally, it was only concentrations higher than 1 mg/ml that were able to induce significant levels of apoptosis in the cells. However, concentrations as low as 0.06 mg/ml induced significant levels of apoptosis H157 cells. A significant variation was observed in the responses of the different cell lines to treatment with the leaf extract. This is more evident at the highest dose of 2.5 mg/ml, where the percentage of apoptotic cells for HepG2 (2A), HT29 (2B), MCF7 (2C), H157 (2D) and KMST6 (2E) were 60%, 80%, 40%, 60% and 40%, respectively. The T. violacea bulb extract failed to induce any significant levels of apoptosis in the cell lines tested in this study. The caspase activation assay showed that the T. violacea leaf extract induced the cleavage of caspase-3 in H157, HepG2 and HT29 but not in KMST6 cells (Fig. 3). However, the extract induced caspase-3 activation more efficiently in HepG2 cells with 70% of the cells staining positive for the presence of cleaved caspase-3,
Cells positive for cleaved caspase-3 (%)
while approximately 20% of H157 and HT29 cells were positive for cleaved caspase-3. However, compared to the noncancerous cell line (KMST6), no statistically significant difference in caspase-3 activity could be observed in H157 and HT29 cells. The measurement of ROS production in cells treated with the T. violacea leaf extract demonstrated that the induction of apoptosis was associated with oxidative stress. The intracellular levels of ROS were measured using the CM-H2DCFDA probe (Fig. 4). These results also showed a selective response in different cell lines. The increased production of ROS was detected in all the cancer cell lines (H157, HepG2, MCF7 and HT29), but not in noncancerous cell line (KMST6). ROS production was the highest in H157 and HepG2 cells, with 80% and 50% of the cells, respectively
Table 1 IC50 values and Selective Index for leaf extracts as determined by the MTT assay.
*
100
Leaf Extract (mg/ml)
Selective Index
MCF7 H157 HT29 HepG2 KMST6
1.2 7 0.50 1.5 7 0.76 2.6 7 1.50 2.8 7 0.45 37 0.80
2.50 2.00 1.15 1.07 1.00
Apoptosis (%)
80 60
*
*
40 20 0
40
0
*
80 60 40 20
10 µM Doxorubicin
0
*
*
*
*
*
*
Apoptosis (%)
Apoptosis (%)
80
20
H157
2mg/ml Leaf extract
HepG2
HT29
*
100 80 60 *
40
*
20 0
100
40
*
* 20
100
*
a
Fig. 3. Evaluating the activation of caspase-3 in response to treatment with the leaf extract. A panel of four human cancer cell lines (KMST6, H157, HepG2, and HT29) was treated for 24 h with 2 mg/ml of the leaf extract. As a positive control, the cells were also treated with 10 μM doxorubicin. Cell fluorescence was measured by flow cytometry. The bar graph indicates the percentage of cells staining positive for active caspase-3. The experiments were done in triplicate. A significance difference (P o0.05) between untreated and extract treated is represented by (n); (a) represents significant difference between the non-cancerous cell line (KMST6) and the cancer cell line.
0
60
*
60
Untreated
The Selective Index (SI) was determined using the following formulae:SI ¼IC50 of KMST6 cell line/IC50 of cancer cell line.
100
* *
Apoptosis (%)
Cell lines
*
80
KMST6
Apoptosis (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
100
Leaf Extract
80 Bulb Extract
60
* *
40 20
*
0
Fig. 2. Quantification of apoptosis induced by bulb and leaf extracts. Four human cancer cell lines (HepG2, HT29, MCF7 and H157) and one non-cancerous human cell line (KMST6) were treated with increasing concentrations (0.06–2.5 mg/ml) of the leaf and bulb extracts. As a positive control, the cells were also treated 10 mM doxorubicin. Apoptosis was quantified by flow cytometry using the APOPercentage assay. Fig. 2A–D and Fig. 1E show the results for HepG2, HT29, MCF7, H157 and KMST6 cells, respectively. All treatments were performed in triplicate. A significant difference (P o 0.05) between treatments with the leaf extract and tuber extract is indicated by (n).
Please cite this article as: Saibu, G.M, et al., In vitro cytotoxic and pro-apoptotic effects of water extracts of Tulbaghia violacea leaves and bulbs. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.01.040i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
Cells positive for presence of ROS (%)
G.M Saibu et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
*
100
* 80
*
*
*
a
* 60
*
a
* a
40
* a
20
0 Untreated KMST6
Hydrogen Peroxide MCF7
HepG2
2mg/ml Leaf extract
HT29
H157
Fig. 4. Assessing oxidative stress in response to treatment with the leaf extract. A panel of five human cancer cell lines (HepG2, HT29, MCF7, H157 and KMST6) was treated for 24 h with 2 mg/ml of the leaf extract. As a positive control, the cells were also treated with 1% hydrogen peroxide. Cell fluorescence was measured by flow cytometry. The experiments were done in triplicate. A significance difference (Po 0.05) between untreated and extract treated is represented by (n); (a) represents significant difference between the non-cancerous cell line (KMST6) and the cancer cell line.
being positive for oxidative stress. Less than 20% of HT29 cells were positive for increased levels of ROS.
4. Discussion and conclusion This study found that the growth inhibitory effects of water extracts of T. violacea leaves in cell cultures are much higher compared to water extracts of T. violacea bulbs. In fact the bulb extract demonstrated negligible activities (less than 20% cell death) in all the cell lines tested in this study (Fig. 2). This appears to be contrary to the findings of Bungu et al. (2006) which demonstrated that the growth inhibitory effects of extracts of the bulbs were higher compared to the leaves (Bungu et al., 2006). However, it should be pointed out that the study by Bungu et al. (2006) used methanol for extraction while the current study used water only, which is in line with the traditional method of extraction. Water extraction is more suitable for this study given that the objective here was to investigate enthnopharmacological claims of this plant in the treatment of cancer. It is well known that the method of extraction, time of extraction, temperature, solvent type, polarity and concentration of the solvent, can affect the extraction of phytochemical compounds (Prashant et al., 2011). Based on the MTT assay, the growth inhibitory effects of water extracts of T. violacea leaves was higher in the MCF7 and H157 cells compared to the other cell lines (Fig. 2). Interestingly, Bungu et al. (2006) also showed that MCF7 were more susceptible to inhibition by methanolic extracts of T. violacea bulbs compared to WHOC3, HT29, and HeLa cells. In the current study, the non-cancerous cell line (KMST6) was shown to be most resistant which may imply that extracts of T. violacea leaves would be selectively toxic to neoplastic cells (Table 1). Cells treated with water extracts of T. violacea leaves displayed signs of growth inhibition, cell shrinkage, vacuolisation and cell detachment (Fig. 1). These are morphological changes often observed in apoptotic cells (Darzynkiewicz et al., 1992; Desagher and Martinou, 2000). Although the MTT assay can be used to assess cytotoxicity, it does not differentiate between the different modes of cell death. We therefore also investigated the mode of the cytotoxicity observed. Bungu et al. (2006) and Lyantagaye (2013) previously demonstrated
5
that T. violacea extracts induced apoptosis (Bungu et al., 2006; Lyantagaye, 2013). Apoptosis is a form of programmed cell death and one of the roles of apoptosis is to prevent the development of cancer (Elmore, 2007; Hengartner, 2000). Deregulation in the control of apoptosis is often associated with the development of cancer (Lowe and Lin, 2000; Vogelstein and Kinzler, 2004). The activation of apoptosis in cancer cells is therefore a corrective strategy and many anti-cancer drugs may exert apoptotic effects in cancer cells (Aggarwal and Shishodia, 2006; Evan and Vousden, 2001). Compounds or extracts with pro-apoptotic activities in cancer cells are therefore potentially useful in anti-cancer drug research. Our study shows that water extracts of T. violacea leaves induce apoptosis in cultured cells thus corroborating previous studies (Bungu et al. 2006; Lyantagaye 2013). However, the effective concentrations of the extracts used in the current study are significantly higher compared to the extracts used in the previous studies. Bungu et al. (2006) showed pro-apoptotic activity at concentrations as low as 250 μg/ml within 24 h while Lyantagaye (2013) showed the induction of apoptosis at 500 μg/ml within 1 h with an aqueous methanol extract of the total plant. We on the other hand, generally only observed significant levels of apoptosis after 24 h of treatment with 1 mg/ml of the water extract. This may be as a result of different extraction methods used in these studies. It appears that the methanolic extracts are more potent than the water extracts. Our study found that the non-cancerous cell line (KMST6) was more resistant to the effects of the extract since the levels of apoptosis were significantly lower for this cell line. This selective toxicity towards cancer cells is a characteristic that is highly sought after in the development of new anti-cancer treatments because much of the adverse effects observed with anti-cancer drugs results from toxicity to normal cells (Sa and Das, 2008). We also showed that the T. violacea leaf extract induced higher levels of apoptosis (80% at a concentration of 2.5 μg/ml) in HT29 cancer cells compared to the other cell lines (H157, HepG2, MCF7 and KMST6) used in this study (Fig. 2). This was a surprising finding since the MTT assay showed that the leaf extract was more toxic to MCF7 cells. The MTT assay measures cell viability as a function of the cell's metabolic activity, while the APOPercentage™ assay detects changes in cell membranes specifically associated with the activation of apoptosis. Cell death is classified into three categories; type I (apoptosis), type II (autophagy) and type III (necrosis) (Clarke, 1990). Type III cell death is a non-apoptotic form of cell death and is associated with organelle dysfunction (e.g. mitochondrial disruption). Loss of cell viability is therefore not always due to apoptosis. It is possible that the plant extract induces multiple forms of cell death, which may explain the apparent disparity in the results obtained for MCF7 cells using the MTT and APOPercentage™ assays. We also investigated the activation of caspase-3 in HepG2, H157, HT29 and KMST6 cells. Although caspase-3 is central to the apoptosis pathways (Porter and Jänicke, 1999), caspase-3 independent apoptosis pathways have also been identified (Liang et al., 2001). MCF7 cells for example do not express caspase-3 but are able to activate caspase-3 independent apoptotic pathways (Liang et al., 2001). It is for this reason that we did not investigate the activation of caspase-3 in MCF7 cells. We found the highest levels of caspase-3 activation in HepG2 cells, with 70% of these cells staining positive for cleaved caspase-3, while less than 20% of HT29 and H157 cells stained positive for cleaved caspase-3 (Fig. 3). Consequently, even though higher levels of apoptosis were observed in H157 cells, these cells demonstrated lower caspase-3 activity compared to HepG2 cells. It should be noted that apoptosis can be activated by multiple pathways and that HepG2, H157, HT29 and MCF7 are cancer cell lines with multiple genetic mutations, which could affect how these
Please cite this article as: Saibu, G.M, et al., In vitro cytotoxic and pro-apoptotic effects of water extracts of Tulbaghia violacea leaves and bulbs. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.01.040i
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cells respond to stimuli that activate the same biochemical pathways (Okamoto et al., 1994; Yoshikawa et al., 1999) and this could possibly explain the diverse responses we observed in the panel of cell lines we used in this study. No significant increase in caspase-3 activity was detected in KMST6 cells, confirming that these cells are resistant to the effects of the T. violacea leaf extract. The fact that the APOPercentage™ assay showed that KMST6 cells treated with this extract die via apoptosis might suggest that apoptosis induced in KMST6 cells is independent of caspase-3 activity. Several anti-cancer drugs, including doxorubicin are known to induce oxidative stress in cancer cells, which in turn can cause damage to DNA, proteins and lipids, resulting in the activation of apoptosis (Klaunig et al., 2010; Orrenius et al., 2007). In this study, we found that the T. violacea leaf extract selectively induced oxidative stress in MCF7, HepG2, HT29 and H157 cells but not KMST6 cells (Fig. 4). A significant increase in ROS was detected in H157 (80%) and HepG2 (50%), while lower levels of ROS were detected in HT29 and MCF7 cells. Interestingly, even though high levels of ROS were detected in HepG2, this did not result in high levels of cell death in this cell line (as shown by the MTT and APOPercentage™ assays). Cancer cells have elevated levels of ROS and therefore also have increased antioxidant capacity to detoxify the cells from ROS (Liou and Storz, 2010). Increased levels of ROS play a key role in tumour development and progression, but can also be toxic to cancer cells (Glasauer and Chandel, 2014). It is possible that HepG2 cells are able to neutralise the damaging effects of oxidative stress more efficiently than some of the other cell lines. In conclusion this study supports the enthobotanical use of T. violacea for possible anti-cancer activity. We demonstrated that this activity is present in water extracts of the leaves and not the bulbs. This suggests that for applications in the treatment of cancer, only the leaves are required. This finding can be beneficial for the sustainable use of this plant in traditional medicine, since this implies that there is no need to harvest the bulbs of the plant as well (Katerere and Eloff, 2008). We also demonstrated the selective toxicity of the plant extract towards cancer cells and that the toxicity is in some cases associated with increased oxidative stress. It is not clear which chemical constituents were responsible for the putative biological activity. However from reports on the phytochemistry of T. violacea, it can be speculated that the allylsulphur, phenolic glycosides and free glycosides could possibly be responsible for the bioactivity. Further studies to isolate and characterise the individual biologically active constituents should be conducted.
Conflict of interest We declare that there is no conflict of interests.
Uncited references (Jänicke et al., (1998); Susan, (2007)).
Acknowledgements We thank the National Research Foundation (South Africa) and the South African Medical Research Council for funding this project. We also thank the PROMEC Unit and Indigenous Knowledge Systems Unit of the Medical Research Council for access to their laboratory facilities.
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