Journal of Medicinal Plants Research Vol. 5(13), pp. 2766-2774, 4 July, 2011 Available online at http://www.academicjournals.org/JMPR ISSN 1996-0875 ©2011 Academic Journals
Full Length Research Paper
Cytotoxicity and mechanism(s) of action of Hypoxis spp. (African potato) against HeLa, HT-29 and MCF-7 cancer cell lines Gerhardt J. Boukes and Maryna van de Venter* Department of Biochemistry and Microbiology, P. O. Box 77000, Nelson Mandela Metropolitan University, Port Elizabeth 6031, South Africa. Accepted 25 January, 2011
Hypoxis is one of the medicinal plants most frequently used by the indigenous people of South Africa. Although many species are used, most research has focused on Hypoxis hemerocallidea with very little or no scientific evidence on other species. The main aim of this study was to investigate and compare the cytotoxicity and mechanisms of action of three Hypoxis spp., namely H. hemerocallidea, Hypoxis stellipilis and Hypoxis sobolifera var sobolifera. Cytotoxicity was determined against HeLa, HT-29 and MCF-7 cancer cell lines and peripheral blood mononuclear cells (PBMCs). DNA cell cycle arrest in the G1, S or G2/M phase was determined using propidium iodide staining. H. sobolifera, which showed the best cytotoxicity against all three cancer cell lines, was used to investigate caspase-3 and/or -7 activation and DNA fragmentation using fluorescence labelled primary caspase antibodies and the TUNEL-based assay, respectively. DNA cell cycle arrest occurred in the late G1 and/or early S phase, which was confirmed by increased p21Waf1/Cip1 activation; caspase-7 was mainly activated in the HeLa and HT-29 cancer cell lines and DNA fragmentation occurred in all three cancer cell lines. This study provides the first data to show that the cytotoxic mechanism of Hypoxis is exerted through the induction of cell cycle arrest and apoptosis. Key words: Hypoxis, cytotoxicity, mechanism(s) of action. INTRODUCTION With only approximately 15% of the world’s known plant resources screened for their therapeutic values (Louw et al., 2002) and over 60% of the currently used anticancer agents derived from natural sources – including plants, marine organisms and micro-organisms (Cragg and Newmann, 2005), it is clear that plants have, and will play, a crucial role in the development of new anticancer agents. Due to few medical facilities, low income and cultural and religious beliefs, most (~80%) of the people in developing countries use traditional medicines obtained from plants (Louw et al., 2002; Steenkamp, 2003). Scientific evidence of South African medicinal plants used for the treatment of cancer is limited (Steenkamp and Gouws, 2006).
*Corresponding author. E-mail:
[email protected] Tel: +27 41 504 2813. Fax: +27 41 504 2814.
Hypoxis (family: Hypoxidaceae; syn. African potato) is one of the most popular (Dold and Cocks, 2002) and controversial medicinal plants used in South African traditional medicine with a wide distribution in southern Africa (Van Wyk et al., 1997). It is traditionally used for the treatment of a variety of ailments, which include intestinal parasites, urinary infections, common cold, flu, nausea, vomiting, heart weakness, nervous disorders, infertility, depression, wounds, anxiety, and many more (Van Wyk et al., 1997; Drewes et al., 2008). Research has proven its antioxidant, anti-inflammatory, antinociceptive, anticonvulsant and antidiabetic properties (Drewes et al., 2008). Hypoxoside [(E)-1,5-bis(3’,5’-dimethoxyphenyl)pent-4en-1-yne], a glycoside with a characteristic pentynene backbone, has been identified, isolated and characterized in the corms of several Hypoxis spp. (Figure 1A) (MariniBettolo et al., 1982; Drewes et al., 1984, 1989; Nicoletti et al., 1992). Hypoxis hemerocallidea (formerly known as Hypoxis rooperi) has been the most frequently used
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A
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Figure 1. Chemical structures of hypoxoside (A) and β-sitosterol (B) isolated from Hypoxis corms.
Hypoxis spp. for hypoxoside isolation and cancer research. In vitro, non-toxic hypoxoside is converted to cytotoxic rooperol in the presence of β-glucosidase (Theron et al., 1994). In vivo studies have shown the absence of hypoxoside and rooperol in the circulation after oral ingestion, but the presence of rooperol phase II metabolites (non-toxic glucuronides and sulphates, which become cytotoxic at tumours with high glucuronidase/ sulphatase activity) had led to phase I trials of hypoxoside as an oral prodrug for cancer therapy (Albrecht et al., 1995a, b; Smit et al., 1995). Plant sterols, also known as phytosterols or 4demethylsterols, are synthesized in plants and must be consumed through the diet by humans and animals. The most commonly found phytosterols are sitosterol (65%), campesterol (30%) and stigmasterol (5%). Phytosterols are structurally and functionally similar to cholesterol (Moghadasian, 2000). All sterols are derived from hydroxylated polycyclic isopentenoids (Abidi, 2001) and have a characteristic fused 1,2-cyclopentanophenanthrine ring structure, which form the steroid nucleus with a 3β-hydroxyl group and 5,6-double bond. Although the nucleus structures of phytosterols resemble those of cholesterol, they differ regarding the side chain at C-17, double bond at C-22 and substituted methyl or ethyl groups at C-24. β-sitosterol and campesterol have ethyl and methyl groups at C-24, respectively. Stigmasterol is characterized by an ethyl group at C-24 and a double bond at C-22. Phytosterols, especially βsitosterol (Figure 1B), are known for their anticancer, cholesterol lowering and immune stimulating properties (De Jong et al., 2003). Studies on chloroform Hypoxis extracts have shown the presence of β-sitosterol as the major phytosterol present in H. hemerocallidea, Hypoxis stellipilis and Hypoxis sobolifera (Boukes et al., 2008). Hypoxis spp. are sold indiscriminately in herbal shops under the common name of African potato. The objective of this study was to compare the cytotoxicity and mechanism of action of chloroform extracts of three Hypoxis spp. – H. hemerocallidea, H. stellipilis and H.
sobolifera – on HeLa, HT-29 and MCF-7 cancer cell lines.
MATERIALS AND METHODS Materials/chemicals/reagents Cervical (HeLa)-, colorectal (HT-29)- and breast (MCF-7) cancer cell lines were purchased from Highveld Biological, South Africa. p21 Waf1/Cip1 (12D1) rabbit mAb, and cleaved caspase-3 (Asp175) and -7 (Asp198) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The Coulter® DNA PrepTM reagents kit, goat anti-rabbit IgG (H+L chain specific) and rabbit IgG isotype, both labelled with fluorescein (FITC) conjugate, and IsoFlowTM EPICSTM sheath fluid were purchased from Beckman Coulter (CA, USA). MEBSTAIN apoptosis kit direct and IntraPrepTM permeabilising reagent were purchased from Immunotech (Marseille, France). 3-(4,5-dimethyl-2-thiazolul)-2.5-diphenyl-2Htetrazolium bromide (MTT) and β-glucosidase (isolated from almonds) were purchased from Sigma (St. Louis, MO, USA). CellTiter-Blue® reagent was purchased from Promega (Madison, WI, USA).
Plant material and preparation of extracts Corms of H. hemerocallidea (voucher number: PEU 14798) and H. stellipilis (PEU14841) were purchased in Port St Johns and Port Elizabeth (Xhosa traditional medicine shop), respectively, in the Eastern Cape, South Africa. Corms of H. sobolifera var sobolifera (PEU 14840) were collected near Plettenberg Bay in the Southern Cape, South Africa. Corms of the three Hypoxis spp. were planted in the same soil type and exposed to equal amounts of sunlight, humidity and water for at least six months before they were harvested and used fresh. Hypoxis spp. were identified by Dr. Y. Singh from the South African National Biodiversity Institute (SANBI) and voucher specimens were deposited in the Nelson Mandela Metropolitan University herbarium. Corms of H. hemerocallidea, H. stellipilis and H. sobolifera were washed, peeled, grated and crushed using a mortar and pestle. Chloroform was added to the plant material in a 1:1 (v:w) ratio, vortexed (5 min), extracted (15 min) and centrifuged (3645 × g for 5 min) at room temperature. The supernatant was removed and the extracting method repeated with the same plant material. The chloroform was evaporated in vacuo and mass of extracts determined. After mass determination the extracts were redissolved in chloroform until further use. Extracts
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Table 1. β-sitosterol and hypoxoside content in 125 µg/ml of Hypoxis extracts.
Hypoxis spp. H. hemerocallidea H. stellipilis H. sobolifera
β-sitosterol (µM) 1.77 0.61 4.50
Hypoxoside (µg/ml) 0.31 0.20 None
were again dried in vacuo to remove all traces of chloroform before addition to cells. Chloroform was used as organic solvent to ensure extraction of non-polar compounds, especially phytosterols and sterolins. Previous studies used water, ethanol and methanol (Louw et al., 2002), which are more effective in extracting hypoxoside. Cell culture conditions HeLa, HT-29, MCF-7 cancer cell lines were routinely maintained in 10 cm culture dishes without antibiotics in RPMI 1640 cell culture medium containing 25 mM Hepes, 2 mM glutamine (Lonza, Walkersville, MD, USA) and 10% fetal bovine serum (Gibco, Grand Island, NY, USA) in a humidified 5% CO2 incubator at 37°C. Cytotoxicity of Hypoxis extracts against HeLa, HT-29 and MCF7 cancer cell lines HeLa, HT-29 and MCF-7 cancer cells were seeded at 30 000 cells/ml in 96-well plates and left to attach overnight at 37°C in a humidified incubator and 5% CO2. PBMCs were isolated from venous blood of a healthy donor using heparinised Vacutainer ® CPTTM cell preparation tubes (Beckton Dickinson, Plymouth, UK) within 30 min of collection. PBMCs were seeded at 500 000 cells/ml in round bottomed 96-well plates. Chloroform extracts of Hypoxis spp. were dried in vacuo (no solvent was present in the extracts) on the day of the assay using a SpeedVac SC100 (Savant Instruments, NY, USA), resuspended in 0.25% (v/v) dimethyl sulphoxide (DMSO), sonicated for 15 min and cell culture medium, containing 100 µg/ml β-glucosidase, added to reach concentrations of 125 to 500 µg/ml of Hypoxis extract. DMSO (0.25%; v/v) was used as vehicle control. Cisplatin (10 and 100 µM) was used as positive control. Cells were treated for 48 h and cell viability assays performed. MTT assay was performed (Holst-Hansen and Brünner, 1998) for HeLa, HT-29 and MCF-7 cancer cells, and absorbance read at 540 nm using a BioTek® PowerWave XS spectrophotometer (Winooski, VT, USA). CellTiter-Blue® assay was performed for the PBMCs and fluorescence read at 544Ex/590Em using a Fluoroskan Ascent FL fluorometer (ThermoLabsystems, Finland).
(Miami, FL, USA). Table 1 summarizes the β-sitosterol and hypoxoside content in 125 µg/ml of Hypoxis extracts as determined from data published by Boukes et al. (2008). p21Waf1/Cip1 expression HeLa, HT-29 and MCF-7 cancer cells were seeded at densities of 40 000 cells/ml in 24-well plates and left to attach overnight at 37°C in a humidified incubator and 5% CO2. Cells were treated with DMSO (0.25%, v/v, vehicle control), H. sobolifera (125 µg/ml, best cytotoxicity against the HeLa, HT-29 and MCF-7 cancer cells) and rooperol [IC50 of 13.01, 29.03 and 18.66 µg/ml for HeLa, HT-29 and MCF-7 cancer cells, respectively (Boukes et al., 2010), positive control] for 15 h. After exposure, cells were washed with PBSA, trypsinized for 10 min at 37°C and resuspended in PBS. Cells were centrifuged at 500 × g for 5 min at room temperature and the supernatant discarded. Cells were washed as described above to remove any traces of trypsin. Cells were fixed and permeabilized using the IntraPrepTM permeabilizing reagent as described in the kit protocol. After permeabilization, cells were washed using cold incubation buffer (0.5% BSA in PBS), supernatant discarded and cells blocked for 10 min in incubation buffer at room temperature. p21 Waf1/Cip1 (12D1) rabbit mAb was added at recommended working dilutions to the cells and incubated for 1 h. Cells were washed twice as described above. After washing, goat anti-rabbit IgG (H+L chain specific) labelled with FITC conjugate was added at recommended working dilutions. Cells were incubated for a further 30 min in the dark and washed as described above. Cells were resuspended in PBS and read on a Beckman Coulter Cytomics FC500. Caspase-3 and/or -7 activation HeLa, HT-29 and MCF-7 cancer cells were seeded at densities of 40 000 cells/ml in 24-well plates and left to attach overnight at 37°C in a humidified incubator and 5% CO2. Cells were treated with DMSO (0.25%, v/v, vehicle control), H. sobolifera (125 µg/ml) and rooperol (IC50 values, positive control) for 48 h. After exposure, cells were washed with PBSA, trypsinized for 10 min at 37°C and resuspended in PBS. Cells were centrifuged at 500 × g for 5 min at room temperature and the supernatant discarded. Cells were washed as described above to remove any traces of trypsin. Cells were fixed and permeabilized using the IntraPrepTM permeabilizing reagent as described in the kit protocol. After permeabilization, cells were washed using cold incubation buffer, supernatant discarded and cells blocked for 10 min in incubation buffer at room temperature. Cleaved caspase-3 (Asp175) or -7 (Asp198) antibodies were added at recommended working dilutions to the cells and incubated for 1 h. Cells were washed twice as described above. After washing, goat anti-rabbit IgG (H+L chain specific) labelled with FITC conjugate was added at recommended working dilutions. Cells were incubated for a further 30 min in the dark and washed as described above. Cells were resuspended in PBS and read on a Beckman Coulter Cytomics FC500.
DNA cell cycle arrest HeLa, HT-29 and MCF-7 cancer cells were seeded at 1.2 × 106 cells/10 ml in 10 cm culture dishes and left to attach overnight at 37°C in a humidified incubator and 5% CO2. Chloroform Hypoxis extracts (125 µg/ml) were added as described above. After 15 and 48 h treatments, cells were trypsinized for 10 min, resuspended in sheath fluid and transferred to polypropylene tubes. The Coulter ® DNA PrepTM reagents kit was used for DNA cell cycle analysis. In brief, lysis reagent (100 µl) was added to each tube, incubated for 5 min at room temperature, 500 µl propidium iodide (50 µg/ml) added and incubated for 15 min at 37°C in the dark. Samples were immediately analyzed using a Beckman Coulter Cytomics FC500
DNA fragmentation HeLa, HT-29 and MCF-7 cancer cells were seeded in 24-well plates at densities of 40 000 cells/ml and left to attach overnight at 37°C in a humidified incubator and 5% CO2. Cells were treated with DMSO (0.25%, v/v, vehicle control), H. sobolifera (125 µg/ml) and rooperol (IC50 values, positive control) for 48 h. Cells were washed with PBSA, trypsinized for 10 min and resuspended in PBS. Cells were washed (centrifuge at 500 × g for 5 min at room temperature) with PBS to remove trypsin, fixed and permeabilized using the IntraPrepTM Permeabilizing kit according to the kit’s protocol. DNA
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fragmentation was investigated using the MEBSTAIN Apoptosis Kit Direct and the protocol followed as described in the product insert. Samples were read on a Beckman Coulter Cytomics FC500. Mean fluorescence intensity (MFI) of treated cells was expressed as a percentage of the MFI of control cells. Statistical analysis Statistical significance was determined using the two-tailed Student’s t-test and p
