The apoptosis inducing effects of Sutherlandia spp. extracts on an oesophageal cancer cell line
Graphical abstract
Introduction
More than 80% of the world's population makes use of complementary and alternative medicines (Mainardi et al., 2009) which include herbalism and botanical medicines. Herbalism is the “medical use of preparations that contain exclusively plant material” (Ernst, 2003). Botanical medicines contain a number of active ingredients which have the same molecular targets as pharmaceutical drugs (Treasure, 2005). Thus herbal and botanical medicines represent an enormous medicinal resource at a fraction of the cost of conventional medicine. Many drugs used for the treatment of cancer have been discovered from medicinal plants (Balunas and Kinghorn, 2005). Vincristine, irinotecan, etoposide and paclitaxel are classic examples of plant-derived compounds that are used in cancer chemotherapy (Nobili et al., 2009).
Sutherlandia frutescens (L.) R. Br. is a shrub indigenous to southern Africa. It has a long history of medicinal use by a number of cultural groups (including the Zulu, Xhosa, Sotho, Khoi-San and Cape Dutch) as a botanical medicine to treat ailments ranging from fevers, coughs and colds to peptic ulcers, dysentery and diabetes. It is locally referred to as the “cancer bush” because of its reported anti-cancer activity (reviewed by van Wyk and Albrecht, 2008). There are unpublished anecdotes of cancer patients who experienced an improved quality of life and survived for much longer than expected (and some that were apparently cured) after treatment with Sutherlandia (van Wyk and Albrecht, 2008). Furthermore, the use of Sutherlandia tomentosa as a medicinal plant is mentioned (van Wyk, 2008), who states that “both species of Sutherlandia have been used in traditional medicine but there are distinct local preferences for particular forms of S. frutescens”. However, these two plants belong to the same genus and are closely related. Because of this close taxonomical relationship (Moshe et al., 1998), it seemed likely that S. tomentosa (a plant also indigenous to South Africa, yet limited to the Cape coastal region) may also have anti-cancer activity and was included in this study.
The anti-tumour activity of S. frutescens was substantiated when it was found that methanolic extracts of S. frutescens inhibited 12-O-tetradecanoylphorbol-13-acetate-induced COX-2 expression in mouse skin (Kundu et al., 2005). The toxicity of Sutherlandia leaf powder has been studied in both adult vervet monkeys and humans and no toxic or other side effects were observed (Seier et al., 2002, Johnson et al., 2007).
A number of studies have been performed to evaluate the effects of S. frutescens on different cell lines. Studies by Tai et al. (2004) and Stander et al. (2007) showed that ethanolic extracts of S. frutescens had concentration-dependent anti-proliferative effects on a variety of human tumour cell lines. Aqueous extracts of S. frutescens were found to inhibit the growth of MCF-7 cells (Steenkamp and Gouws, 2006) and induce cytotoxicity in cervical carcinoma (Caski) and Chinese hamster ovary (CHO) cells (Chinkwo, 2005).
It has been suggested that the vast catalogue of cancer cell genotypes is a manifestation of six essential alterations in cell physiology that collectively dictate malignant growth. One of these is the ability to evade apoptosis, a form of programmed cell death occurring in metazoans (Hanahan and Weinberg, 2000). This acquired resistance to apoptosis is a hallmark of most and perhaps all types of cancer. Therefore the search for compounds that can precisely activate or inhibit molecules that mediate the diverse forms of cell death, including apoptosis, is ongoing and ultimately aims to develop less toxic and more effective chemotherapeutic regimens. Apoptotic cells are typically characterised by morphological features such as cell shrinkage, chromatin condensation, blebbing of the cell membrane and the formation of apoptotic bodies (Kerr et al., 1972), visible through microscopy. The biochemical features of apoptosis are considered to be the fragmentation of DNA in the cell (Cohen, 1997) and the externalisation of phosphatidylserine to the exterior of the cell membrane (Martin et al., 1995).
A study by Chinkwo (2005) found that 3.5 mg/ml of aqueous S. frutescens extracts were able to induce apoptosis in CHO and Jurkat cells. CHO cells treated with S. frutescens extracts showed morphological features consistent with apoptosis. Phosphatidylserine externalisation and DNA fragmentation confirmed that S. frutescens extracts had induced apoptosis in the CHO cells. Flow cytometric analysis of S. frutescens-treated Jurkat cells revealed that more than 84% were apoptotic (Chinkwo, 2005). Stander et al. (2007) also observed morphological characteristics typical of apoptosis in MCF-7 cells treated with 1.5 mg/ml ethanolic S. frutescens extracts. A gene expression profile revealed that genes thought to be involved in apoptosis, growth inhibition and NF-kB signalling were differentially expressed in S. frutescens-treated and vehicle control-treated cells. Further studies by Stander et al. (2009) showed that aqueous extracts of S. frutescens at 5 and 10 mg/ml were more cytotoxic to cancerous (MCF-7) than non-cancerous (MCF-12A) breast cell lines. These extracts activated both autophagic and apoptotic processes in MCF-7 cells.
Although previous studies have investigated the effects of S. frutescens extracts on various cell lines, no studies have involved an oesophageal cancer cell line. In South Africa, oesophageal cancer is the second most common cancer in men and one of the more challenging cancers to treat. The 5-year survival rate of oesophageal cancer sufferers is estimated to be only 10% (Johnstone, 2000). Conventional treatment of oesophageal cancer includes one, or a combination of, surgery, radiation therapy and chemotherapy. These treatments, however, have high rates of morbidity and mortality. In patients with resectable oesophageal cancer, surgery is the treatment of choice (Forshaw et al., 2005, Van Meerten and van der Gaast, 2005). However, patients tend to live only 12–18 months after the surgery and the 5-year survival rate seldom exceeds 25% (Johnstone, 2000). Furthermore it is estimated that, within a year of surgery, the disease will have recurred in almost half of the patients (Forshaw et al., 2005). The 5 year survival rates of patients with oesophageal cancer treated with only radiation therapy has been found to be below 5% and there is a 77% local recurrence rate. A combination of chemotherapy and radiation therapy improves the 5 year survival rate to between 9 and 25% (Neuner et al., 2009). It is therefore important to determine what effects Sutherlandia spp. extracts have on oesophageal cancer cells. This study examined the potential apoptosis-inducing effects of three different Sutherlandia spp. extracts on a South African established oesophageal cancer cell line, the SNO cell line.
Section snippets
Materials
Hank's balanced salt solution (HBSS), Dulbecco's modified Eagle's medium (DMEM), foetal bovine serum (FBS), trypsin/versene, penicillin/streptomycin/fungizone cocktail and gentamycin were supplied by Highveld Biologicals (Lyndhurst, South Africa). Z-VAD-fmk, dimethyl sulfoxide (DMSO), cycloheximide (CHX), hydrogen peroxide (H2O2), Histopaque® and 70% ethanol were supplied by Sigma–Aldrich (Steinheim, Germany).
Cell culture
SNO (ATCC, cat no. CCL-185) oesophageal cancer cells, an adherent cell line, were
Concentration and time trials
In order to determine the optimal treatment conditions, concentration– and time–response studies were initially conducted in a preliminary study. SNO cells were treated with extracts A, B and C at concentrations of between 0.5 and 10 mg/ml for 3, 24 or 48 h. Vehicle, necrotic (10% H2O2) and apoptotic (0.5 mM CHX) controls were included. From these dose– and time–response studies (results not shown), the 24 h treatment with extracts A, B and C at concentrations of 2.5 and 5 mg/ml was elected for
Discussion
S. frutescens has been used as a traditional medicine by a number of different cultural groups to treat a wide variety of diseases. S. frutescens has become known as the “cancer bush” due to its reported use by the Khoi-San and Cape Dutch people for the treatment and prevention of internal cancers (van Wyk and Albrecht, 2008). However, little is known about how the plant exerts its anti-cancer activity. The aim of this study was to investigate the potential apoptosis-inducing effects of three
Conflict of interest
The authors of this study have no conflicts of interest or any financial disclosures to make.
Acknowledgements
The authors would like to acknowledge Professor Ben-Erik van Wyk, for his donation of the Sutherlandia frutescens and Sutherlandia tomentosa materials that were used in this study. Aspects of this project were funded by the National Research Foundation of South Africa, the Faculty of Science Walker Trust Fund, University of Johannesburg, SA and the Cancer Association of SA (CANSA).
References (40)
- et al.
Drug discovery from medicinal plants
Life Sciences
(2005) Sutherlandia frutescens extracts can induce apoptosis in cultured carcinoma cells
Journal of Ethnopharmacology
(2005)The current position of complementary/alternative medicine in cancer
European Journal of Cancer
(2003)- et al.
Neoadjuvant chemotherapy for oesophageal cancer: the need for accurate response prediction and evaluation
Surgeon
(2005) - et al.
The hallmarks of cancer
Cell
(2000) - et al.
Inhibitory effects of the extracts of Sutherlandia frutescens (L.) R. Br. and Harpagophytumprocumbens DC. on phorbol ester-induced COX-2 expression in mouse skin: AP-1 and CREB as potential upstream targets
Cancer Letters
(2005) - et al.
Inhibition of caspase activity induces a switch from apoptosis to necrosis
FEBS Letters
(1998) - et al.
Lack of genetic differentiation between 19 populations from seven taxa of Sutherlandia Tribe: Galegeae, Fabaceae
Biochemical Systematics and Ecology
(1998) - et al.
Natural compounds for cancer treatment and prevention
Pharmacological Research
(2009) - et al.
Influence of Sutherlandia frutescens extracts on cell numbers, morphology and gene expression in MCF-7 cells
Journal of Ethnopharmacology
(2007)
In vitro effects of Sutherlandia frutescens water extracts on cell numbers, morphology, cell cycle progression and cell death in a tumorigenic and a non-tumorigenic epithelial breast cell line
Journal of Ethnopharmacology
Cytotoxicity of six South African medicinal plant extracts used in the treatment of cancer
South African Journal of Botany
In vitro culture studies of Sutherlandia frutescens on human tumor cell lines
Journal of Ethnopharmacology
Cycloheximide-induced T-cell death is mediated by a Fas-associated death domain-dependent mechanism
The Journal of Biological Chemistry
Herbal medicine and cancer: an introductory overview
Seminars in Oncology Nursing
A broad review of commercially important southern African medicinal plants
Journal of Ethnopharmacology
A review of the taxonomy, ethnobotany, chemistry and pharmacology of Sutherlandia frutescens (Fabaceae)
Journal of Ethnopharmacology
A novel assay for apoptosis flow cytometric detection of phosphatidylserine early apoptotic cells using fluorescein labelled expression on Annexin V
Journal of Immunological Methods
Cell death independent of caspases: a review
Clinical Cancer Research
Do inducers of apoptosis trigger caspase-independent cell death?
Nature Reviews Molecular Cell Biology
Cited by (18)
Cytotoxicity and <sup>1</sup>H NMR metabolomics analyses of microalgal extracts for synergistic application with Tamoxifen on breast cancer cells with reduced toxicity against Vero cells
2022, HeliyonCitation Excerpt :The 4T1 (Figures 1e and S1) and Vero (Figures 1f and S2) cells treated with TMX and synergistic application also exhibited similar morphologies. These are unique changes in apoptosis where the nucleus becomes anaphase and dense, due to the nuclear chromatin condensation (Skerman et al., 2011). For analyses of apoptosis during the early and late apoptotic stage, Annexin-V attaches to the PS, the apoptotic biomarker.
Geographic-based metabolomic variation and toxicity analysis of Sutherlandia frutescens L. R.Br. – An emerging medicinal crop in South Africa
2019, Industrial Crops and ProductsCitation Excerpt :As an emergent crop, agricultural farming is generally taking place in regions where plants are naturally found and most farmers view this species as a means to generate additional income, even if their main revenue stream is obtained from other agricultural endeavours. An increasing body of scientific research has aimed at testing extracts mainly for: 1) anticancer activity (Tai et al., 2004; Chinkwo (2005); Stander et al., 2007; Skerman et al., 2011; Vorster et al., 2012; Mqoco et al., 2014; Leisching et al., 2015), 2) antidiabetic activity (Chadwick et al., 2007; MacKenzie et al., 2009; MacKenzie et al., 2012; Sia, 2004; Williams et al., 2013); 3) stress modulation (Smith and Myburgh, 2004; Prevoo et al., 2008; Sergeant et al., 2017); and, 4) anti-HIV activity (Harnett et al., 2005). With new information validating its use as a botanical medicine, a rise in commercial interest has seen many different types of extracts being produced from this plant species (Van Wyk, 2011).
Differential cellular interaction of Sutherlandia frutescens extracts on tumorigenic and non-tumorigenic breast cells
2014, South African Journal of BotanyCitation Excerpt :Studies using extracts of this plant have demonstrated that SF possesses pharmacological properties such as antithrombotic (Kee et al., 2008), antibacterial (Katerere and Eloff, 2005), antidiabetic (MacKenzie et al., 2009) and antiproliferative activities (Tai et al., 2004). Recent studies performed in our laboratory (Department of Physiology, University of Pretoria, South Africa) and by other researchers revealed that these extracts inhibit cell growth in cancer cell lines namely the human breast adenocarcinoma cell line (MCF-7, MDA-MB-468), human promyelocyte (HL60), human prostate cancer cell line (DU-145) and esophageal cancer cell line (SNO) (Stander et al., 2009; Tai et al., 2004; Steenkamp and Gouws, 2006; Skerman et al., 2011). Stander et al. (2009) revealed that exposure of MCF-7 cells to an ethanol extract of SF for a period of 48 h yielded a 50% growth reduction.
LC-MS-based metabolomics assists with quality assessment and traceability of wild and cultivated plants of Sutherlandia frutescens (Fabaceae)
2012, South African Journal of BotanyCitation Excerpt :A growing body of evidence confirming its anti-proliferating and apoptotic effects using cancer cell lines and leaf extracts is now available. For examples refer to the work of Tai et al. (2004; human leukemia and breast cancer); Chinkwo (2005; cervical cancer); Stander et al. (2007; 2009); Vorster et al. (2012; breast cancer); and Skerman et al. (2011; oesaphageal cancer). Whereas most of these studies showed some anti-cancer activity at the concentrations tested, it is highly debatable whether these concentrations have physiological efficacy or not.
The Effect of Aqueous Lessertia frutescens Extract on TM3 Leydig Cells Exposed to TNF-α in vitro
2023, Frontiers in Bioscience - LandmarkNatural Products for Esophageal Cancer Therapy: From Traditional Medicine to Modern Drug Discovery
2022, International Journal of Molecular Sciences