ORIGINAL ARTICLE

Sarcostemma viminale : a potential anticancer therapy

Brestovac Brian & Snook Jessica & Ellison Gaywin &

Phillips Alexander & Townsend David

Received: 23 May 2013 /Accepted: 31 October 2013# Springer-Verlag London 2013

Abstract There is a need for cancer treatments to be selec-tively cytotoxic to cancer cells so as to reduce adverse sideeffects. In this study, a cancer cell line (HeLa cells) and a non-cancer cell line (HF-32) were exposed to an extract from theplant Sarcostemma viminale . Cytopathic effects and apoptosiswere measured by morphological changes, annexin V/propidium iodide (PI) and 4′,6-diamidino-2-phenylindole(DAPI) staining assays. Also, a novel mixed culture flowcytometry assay was performed exploiting the overexpressionof p16INK4a in HeLa cells to demonstrate the change in num-bers of HeLa and HF-32 cells post-exposure to the extract. At1 % (v /v ) after 48 h of exposure, HeLa cells showed >75 %cytopathic effect, 77 % were in apoptosis or dead by theannexinV/PI assay, and 100 % had nuclear changes by DAPIstaining; there was a reduction of 76 % in the number of cellsby mixed culture assay. In contrast, for the HF-32 cells, only5 % showed any cytopathic effect, there were no more cells inapoptosis or dead (34 %) than in the control by the annexinV/PI assay, <1 % of cells had nuclear changes by DAPI staining,and there was a slight increase in cell numbers by the mixedculture assay. Results from these assays clearly demonstratethat the extract from S. viminale destroyed the cancer HeLacells quickly and at a low concentration, whilst the non-cancerHF-32 cells survive. This study indicates that extracts from S.viminale may be a specific anticancer agent.

Keywords Anticancer . Sarcostemma viminale . Cytopathiceffect . Apoptosis . Mixed culture

Introduction

Cancer is a global health and economic burden accounting forover 7 million deaths annually (Jemal et al. 2011). Currentcancer treatments have several limitations including lack ofspecificity leading to numerous side effects; hence, the searchfor more specific therapies, including those derived fromplants, continues. Currently, some 60 % of anticancer thera-peutics have been developed from natural products includingplants (Newman et al. 2003).

The National Cancer Institute of America (NCI) imple-mented a large-scale screening program designed for high-throughput testing of plant compounds. The efforts of the NCIhave been focused on screening plant extracts since 1961(Leiter et al. 1961). In 1990, the program moved fromin vivo testing to in vitro methods to incorporate a panel of60 human tumour cell lines. This change reflected the shift offocus from leukaemia to a broader range of cancers (Corbettet al. 1987). However, only a small percentage of plants testedin this program have shown significant activity (Suffness andDouros 1982). The testing methods varied considerably overtime and used predefined concentrations of the plant com-pounds (Monks et al. 1991). Yet, remarks from Paull et al.(1989) of the National Cancer Institute conceded that the useof different assay methods could result in different results andinterpretations. The clear limitation of the NCI method is theinvariable approach to an array of different compounds andthe single assay by which the plants are being screened.

Despite the small percentage of plants discovered withsignificant anticancer activity in the NCI program, a numberof commonly used anticancer therapeutics were derived fromplants. These include the vinca alkaloids obtained from aMadagascan periwinkle plant (Johnson et al. 1963; Nobleet al. 1958); the epipodophyllotoxin lignans VM-26 and VP-16, derived from the Podophyllum plant species traditionally

B. Brian (*) : S. Jessica : E. Gaywin : P. Alexander : T. DavidSchool of Biomedical Sciences, CHIRI Biosciences ResearchPrecinct, Faculty of Health Sciences, Curtin University,GPO Box U1987, Perth 6845, Western Australia, Australiae-mail: B.Brestovac@curtin.edu.au

Comp Clin PatholDOI 10.1007/s00580-013-1843-0

used in the treatment of cancers (Cragg and Newman 2005;Stahelin 1973); taxanes obtained from plants of the Taxusspecies(Cragg and Newman 2005; Fauzee 2011); and thecamptothecin derivatives obtained from the plantCamptotheca acuminate (Cragg and Newman 2005; Wall1998). There are numerous other plant derivatives and semi-synthetic analogues presently in development, demonstratingthe structural diversity of novel compounds still being discov-ered among plants (Pan et al. 2010).

Sarcostemma viminale (Brown 1810) is a semi-succulentplant of the Asclepiadaceae family found in various locationsaround the world. Stems and sap of the plant S. viminale havebeen used as a traditional remedy for various illnesses includ-ing cancer by indigenous people in India, South Africa, theMiddle East and Australia. S. viminale sp. australe (Forster,1992), commonly known as caustic bush or milk bush, islocated in Australia and has been used by Australian Aborig-inal people in traditional medicines. Recent anecdotal evi-dence has emerged that the plant was used successfully forskin lesions in the northwest of Western Australia (D.Townsend, personal communication), and evidence suggestedthat it may be useful in the treatment of skin cancers andblemishes (Semple et al. 1998); however, anticancer testingof the plant that was undertaken by the NCI had no significantfindings (Abbott et al. 1967).

Recent pharmacological screening studies involving S.viminale discovered the presence of two of phytochemicals,alkaloids and saponins, and suggested that further pharmaco-logical studies should be carried out (Evans et al. 2010).Testing for these and other phytochemicals has been used asa screening method for compounds that may possess antican-cer activity (Fabricant and Farnsworth 2001). Alkaloids havebeen shown to exert a number of biological effects, includinganticancer properties such as those found in the group of vincaalkaloids. Saponins have been found to cause autophagy, typeII programmed cell death, in cancerous cells, specifically HeLaand colon cancer cells (Ellington et al. 2005; Sy et al. 2008).

In this study, an ethanol extract from S. viminale was testedagainst the cancerous HeLa cell line and the non-canceroushuman fibroblasts (HF-32) cells. Morphological changes (cy-topathic effect, CPE), a mixed culture assay, annexinV/propidium iodide (PI) assay and 4′,6-diamidino-2-phenylindole (DAPI) staining were used to determine whetherthere is selective cytotoxicity between cancerous and non-cancerous cells.

Methods and materials

Plant collection and extract preparation

Plant samples were collected south of Exmouth in WesternAustralia. Permission was granted from the Department of

Environment and Conservation and the Shire of Exmouth.Equal weights of fresh plant clippings and absolute alcoholwere blended, strained and centrifuged to remove solid matter;the alcohol was evaporated at 37 °C. Residual material wasredissolved in phosphate-buffered saline (pH 7.4) equal involume to one tenth of the original amount of alcohol usedto a concentration of 10 g/mL and stored at −20 °C.

Cell culture

Two cell lines were used in this study. The non-cancerous cellline used were the human embryonic lung diploid fibroblasts(HF-32) and the cancerous cell line used were the HeLa cellsderived from a cervical carcinoma (cells lines supplied by theVirology Department, QEII Medical Centre, Perth, Australia).Cells were cultured in minimal essential media (MEM;Sigma-Aldrich) with 10 % heat-inactivated foetal bovine se-rum (FBS; Sigma-Aldrich) and the antimicrobial agents pen-icillin (0.1 mg/mL), streptomycin (0.1 mg/mL) andamphotericin B (2.5 μg/mL). Cells were incubated at 35 °Cin 5 % CO2. Once cells reached confluence, they were pas-saged or maintained in MEM containing 2 % heat-inactivatedFBS with antimicrobial agents.

Observing morphological changes for CPE

Visible changes in cell morphology such as cell size, shape, adecrease in cell number and a loss of adherence are indicativeof cytopathic effect. Cells were seeded in 12-well trays at aconcentration of 1×106 cells/well and incubated for 24 h at35 °C in 5 % CO2. The medium in individual wells wasreplaced with 1 mL of maintenance media containing 0 %(negative control) and 0.25, 0.50, 1 and 5 % (v /v ) of theplant extract. All tests were performed in duplicate at leasttwice and the changes observed and recorded at 24, 48 and72 h. A scoring system was devised whereby no CPE scored0, <5 % CPE scored 1, 5–25 % CPE scored 2, 25–75 % CPEscored 3, >75 % CPE scored 4 and complete destruction ofthe cell culture scored 5.

Annexin V and propidium iodide assay

The annexin V protein binds to the phospholipidphosphatidylserine that becomes exposed to the external cellsurface during apoptosis, making it a marker for apoptosis,whilst PI selectively stains membrane-damaged cells; in con-junction, the two can be used to differentiate between apopto-sis and necrosis by flow cytometry (King 2000; Özdemir et al.2003). Cells were seeded in 12-well trays at a concentration of1×106 cells/well and incubated for 24 h at 35 °C in 5 % CO2.The media was then replaced with 1 mL of growth mediacontaining dilutions of 0, 1 and 5 % of the plant extract. Afteran incubation time of 24, 48 or 72 h, the adherent and

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suspended cells were harvested. Cells are washed twice incold PBS at 3,000 rpm for 3 min and resuspended in 1×binding buffer (PBS and 3 % FBS) at a concentration of 1×106 cells/mL. Cell suspension (100 μL) was transferred to aFACS tube with 5 μL of annexin V conjugated to FITC(exCitation at 488 nm, emission at 519 nm) and 1 μL of PI(exCitation at 488 nm, emission at 617 nm). Samples weremixed by vortex and incubated for 15 min at room tempera-ture in the dark before the addition of 400 μL of 1× bindingbuffer and analysed on the FACSCantoII flow cytometer(Becton Dickinson).

Mixed culture assay

The internal cell cycle regulator protein p16INK4a isoverexpressed in the cytoplasm of cervical cancer-derivedHeLa cells whilst having a much lower expression in humanfibroblasts (Klaes et al. 2001). This difference was exploitedin a mixed culture assay using an anti-p16INK4a monoclonalantibody labelled with APC-Cy7 (Santa Cruz, CA, USA) withexcitation at 633 nm and emission at 785 nm. HeLa and HF-32 cells were seeded in equal amounts (1×106 cells/well) intothe same wells of a 12-well tray and the plant extract added atconcentrations of 0, 1 and 5 % (v /v ) in maintenance media.After 24 and 48 h, the mixed cell cultures were harvested,centrifuged (5 min at 3,500 rpm), the supernatant removedand the ThinPrep fixative (Hologic Inc.) solution added topermeabilise the cells for a minimum of 15 min. The stainingpreparation involved washing the fixed cells twice in blockingagent (3 % foetal calf serum in PBS) for 3 min with centrifu-gation of 3,000 rpm between. The cells were resuspended inblocking agent at a concentration of 1×106 cells/mL. To thestaining test tube, 100 μL of cell suspension and 5 μL of anti-p16INK4a monoclonal antibody conjugated to APC-Cy7 wereadded, mixed by vortex and incubated at room temperature, inthe dark, for 20 min. Cells were washed twice in PBS andresuspended in 0.5 mL PBS ready for analysis on theFACSCantoII flow cytometer (Becton Dickinson).

DAPI staining

The chemical DAPI is used for staining DNA (Kapuscinski1995).When used to stain cell preparations, apoptotic changessuch as nuclear condensation (pyknosis) and fragmentation(karyorrhexis) can be observed (Zhivotovsky and Kroemer2004). Cells for staining were grown onto coverslips previ-ously sterilised by autoclaving in PBS. Two coverslips wereplaced into 60-mm Petri dishes and seeded with approximate-ly 1×106 cells, followed by the addition of approximately3 mL of growth medium. The cells were incubated at 35 °Cin 5%CO2 until confluent. Cells were then treated with 1% S.viminale extract or left untreated. Staining with DAPI wasdone at 24 and 48 h post-treatment as follows: removal of

media from coverslips and fixing cells in 2 % formaldehyde insaline (stored at 4 °C if required); cells were washed twice inwater, rinsed in PBS, and then 300 μL of DAPI diluted to0.3 μM in PBS was added to each coverslip and incubated atroom temperature in the dark for 5 min; cells were rinsed twicein PBS. The coverslips were allowed to drain and weremounted on slides using an anti-fade mounting media (poly-vinyl , Tris–PO4 buffer, phenol red glycerol andchlorobutanol). Slides were viewed using a Zeiss Axiovert200M fluorescent microscope with DAPI filter (exCitation,358 nm; emission, 461 nm) and photographed with anAxiocam MRM camera. Pictures were optimized and record-ed using AxioVision software from Zeiss.

Results

Cytopathic effects of plant extracts on tissue culture cell lines

CPEs seen in the cell lines are demonstrated in Fig. 1. No CPEwas detected at 0.25 and 0.5 % plant extract over 72 h ofincubation for either of the cell lines. Scores for the CPE wereplotted at 1 and 5% concentrations of the plant extract for bothHF-32 and HeLa cells (Fig. 2). The human fibroblast HF-32cells were affected to a lesser extent at 1 % over 72 h thanHeLa (Fig. 2a). The HeLa cells show upwards of 25–75 %CPE after 48 h at 1 % concentration and at 72 h were >75 %CPE. In contrast, HF-32 cells showed only approximately 5 %CPE with 1 % extract over 24 and 48 h, and only after 72 hwas there greater than 25 % CPE (Fig. 2a). At 5 %, the extractcaused >75 % CPE of the HeLa cells after 24 h. However, theextent of CPE seen in HF-32 cells, at 5 % extract exposure,was only 25 % after 24 h and did not reach >75 % even after72 h (Fig. 2b).

Mixed culture assay

In a single cell culture, flow cytometry confirmed that HeLacells have a high expression of p16INK4a (Fig. 3a), whilst HF-32 cells display low-level expression (Fig. 3b). This differencein p16INK4a expression was exploited in a flow cytometry-based assay to test for selective toxicity of the plant extractwith a mixture of HF-32 and HeLa cells. Even numbers ofcells were cultured and then this mixture was tested forp16INK4a expression. After 24 h of incubation, the controlmixed cell culture (with no extract) had approximately evennumbers of both cell lines (47 % HeLa and 51 % HF-32;Fig. 3c), whilst the mixed culture of cells exposed to 1 %extract had a reduced proportion of HeLa cells (41 % HeLaand 57 % HF-32; Fig. 3d). After 48 h, the proportion of thetwo cell populations in the control culture changed. HeLa cellsnow comprised approximately 59 %, whilst HF-32 propor-tionally was reduced to 39 % of the cell numbers (Fig. 3e).

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This probably reflects the more rapid growth rate of the HeLacells as compared to the HF-32 cells and was not unexpected.In contrast to the control, the mixed culture at 48 h with 1 %extract showed a dramatic decrease in the proportion of HeLacells (25 %) and a corresponding increase in the proportion ofHF-32 cells (72 %; Fig. 3e). At the 5 % concentration ofextract, the mixed culture after 24-h exposure contained28 % HeLa cells and 72 % HF-32 cells and after 48 hcontained 23 % HeLa cells and 77 % HF-32 cells.

The percentage change in the cell counts of HeLa and HF-32 cells in mixed culture as compared to the control cellcultures is presented in Table 1. At 24 h, the cell counts ofboth HeLa and HF-32 reduced compared to the control withthe 1 % extract. However, the HeLa cells decrease by 32 %,whilst HF-32 cells decreased by only 12 %. At 48 h ofexposure, the HeLa cell count decreased by 76 % whilst theHF-32 cell count increased by 6 %, suggesting a recovery ofthe HF-32 cells. At 5 % concentration, there was a consider-able decrease in the cell numbers overall at both 24 and 48 hfor both cell types. The HeLa cells were again more affected,with a 98% decrease in cell number after 24 h compared to the49 % decrease seen in HF-32 cells; after 48 h, there was a99 % decrease in HeLa cell number, whilst HF-32 cellsdecrease by 72 % (Table 1).

Annexin V/PI assay

The annexin V/PI assay detects apoptosis by annexin V stain-ing and cell death by PI staining; the cells showing thisstaining were viewed with flow cytometry (Gillespie et al.2004; Jimenez-Medina et al. 2006). Scatter plots were

generated for analysis and are divided into four quadrants(Figs. 4 and 5). Quadrant 3-1 (Q3-1) indicates no annexin Vor PI staining and so represents live cells. Q4-1 indicatesannexin V staining which represents cells undergoing apopto-sis. Q1-1 and Q2-1 indicate PI staining and so represent celldeath. Visual interpretation was done by observing the migra-tion of healthy cells in Q3-1 through apoptosis in Q4-1 andthen upwards to cell death in Q1-1 and/or Q2-1. This obser-vation was seen for both cell lines, and so apoptosis wasdetermined as the mechanism of cell destruction by exposureto S. viminale plant extract.

At 24 h, 15 % of the control HF-32 cells were in apoptosis,whilst for the 1 and 5 % extract-exposed cells, 31 and 77 %were in apoptosis, respectively (Fig. 4a). Approximately 2 %of HF-32 cells exposed to 1 % extract after 24 h were dead;however, at 5 % extract, it was 14 % (Fig. 4a). HeLa cellsexposed to 1% extract for 24 h did not show amarked increasein apoptosis compared to the control; however, the cells ex-posed to 5 % extract at 24 h were mostly dead (77 %) or inapoptosis (20 %), with only 3 % cells remaining alive (Fig. 4b).

After 48-h exposure to 1 % of the extract, there wereslightly less HF-32 cells undergoing apoptosis (28 %) com-pared to those exposed for 24 h (31 %). However, there weremore dead cells (8%) compared to 2% at 24 h. At 5% extract,82 % of cells were in apoptosis compared to 77 % at 24-hexposure. There were 13 % of cells dead (Fig. 5a), which wassimilar to the 24-h exposed cells (14 %). HeLa cells, after48 h, showed a marked increase in the number of cells inapoptosis compared to the control and to the 24-h exposedcells. In the 1 % extract exposed cells after 48 h, the majorityof cells were in apoptosis (67 %), with approximately 10 %

Control Visible CPEa

b

Fig. 1 Changes in cellmorphology indicative of CPE.Control cell cultures demonstrateadequate confluency andappropriate cell morphology. CPEcauses a visible decrease in cellconfluency and a change in cellsize, shape and appearance. aHeLa cell line. b HF-32 cell line

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dead, whilst in the 5 % extract, after 48 h, 95 % of HeLa cellswere dead and 4 % were in apoptosis (Fig. 5b), leaving onlyapproximately 1 % of the HeLa cells alive.

DAPI staining

Nuclear morphological changes were viewed with the DAPIfluorescent stain after exposure to S. viminale extract. Vital,healthy cells have ovoid nuclei (elongated for HF-32 cells)with even staining. Cells undergoing apoptosis exhibit chro-matin condensation, crescent-shaped nuclear condensation,fragmented nuclei or tightly condensed nuclei without frag-mentation. For control HeLa cells (no exposure to extract),<1 % showed nuclear apoptotic changes (Fig. 6a), whilst cellsexposed to 1 % extract for 24 h had approximately 33 % ofcells with nuclear apoptotic changes (Fig. 6b). At 48 h, all(100 %) HeLa cells were either dead or had nuclei withapoptotic changes (Fig. 6c).

At 24 and 48 h post-exposure to 1 % extract, HF-32 cellshad no discernible difference from the control, with <1 % of

cells showing apoptotic changes (Fig. 6e, f). Even after 72 hpost-exposure with 1 % extract, there were only approximate-ly 10 % of nuclei showing apoptotic changes (Fig. 6g).

Discussion

Many cancer therapies lack specificity and so damage non-cancerous tissue, resulting in unwanted and dangerous sideeffects, so the development of specific anticancer therapies isa major priority in cancer research (Guillemard and Saragovi2004). The present study demonstrates the selective antican-cer activity of a plant extract from S. viminale which, byseveral assays, was cytotoxic towards the cancer-derivedHeLa cell line, but was much less cytotoxic to the non-cancer HF-32 cell line.

The first assay used to examine the effects of S. viminaleextract was simply to expose cultures of the cells lines tovarying concentrations of the extract and observe for morpho-logical changes in the cells over time. At the lower concen-trations of 0.25 and 0.5 % (v /v ), there was no effect on eithercell line. However, at 1 and 5% (v /v ), the extract had a greatercytopathic effect on both cell lines (Fig. 2). The non-cancerous HF-32 cell line tolerated the 1 % concentration ofextract with little change even after 48 h of exposure, whilstthe HeLa cells showed much greater morphological changes.At 5 % (v /v ) concentration of extract, there was more cyto-toxic changes to both cell lines, as expected. However, HeLacells had greater morphological changes than HF-32 cells.

To further demonstrate the selective toxicity of the extractagainst the cancerous HeLa cells, a novel assay using a mixedculture of the HeLa and HF-32 cells was set up; this mixedculture was exposed to the extract over time. At a 1 % (v /v )concentration of extract, HF-32 cells survived whilst HeLacells diminished over time (Fig. 3). After 48 h of exposure,HF-32 cells not only survived but increased in number, whilstHeLa cells were all but destroyed (Table 1). The increase inthe HF-32 cell counts greater than the control may also indi-cate a stimulating effect of the plant extract at these lowerconcentrations. Hormesis is a term that describes the biphasicdose–response whereby a toxic or inhibitory substance has abeneficial or stimulatory effect at low doses. It is thought to bedue to an adaptive activation stress response to restore homeo-stasis which overcompensates, resulting in a transient expan-sion of cells (Calabrese 2010; Martins et al. 2011). In thecontrol culture, HeLa cells outgrew HF-32 cells; however,when exposed to 1 % (v /v ) of extract, the culture comprisedonly approximately 25 % HeLa cells and 72 % HF-32 cellsafter 48 h. In this mixed culture assay, it is clear that thecancerous HeLa cells were being destroyed by the plantextract whilst the HF-32 cells survived.

The results of the annexin V/PI assay indicate that celldeath was occurring via apoptosis (Figs. 4 and 5) as cells were

a

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0

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0 24 48 72

Time (hours)

Cyt

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Fig. 2 Cytopathic effect observed in cell cultures with 1 % (a) and 5 %(b) plant extract over 72 h. A scoring system was devised whereby noCPE scored 0, <5 % CPE scored 1, 5–25 % CPE scored 2, 25–75 % CPEscored 3, >75 % CPE scored 4, and complete destruction of the cellculture scored 5. Squares , HeLa; crosses , HF-32

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clearly observed to be stained by annexin V before PI. At 1 %(v /v ) concentration of extract after 24 h of exposure, therewas little difference between HeLa and HF-32 cells. Howev-er, after 48 h of exposure, 77 % of HeLa cells were eitherdead or in apoptosis, whilst only 36 % of HF-32 cells weredead or in apoptosis. At 5% (v /v) concentration, both cell lineswere affected, and after 24-h exposure, 82 % of HF-32 cellsand 97 % of HeLa cells were dead or in apoptosis. It is clearthat the mechanism by which S. viminale extract acts is byinducing apoptosis, and this occurs in both cell lines. It is alsoclear that the HeLa cells were affected to greater extent than theHF-32 cells.

Table 1 Change in cell counts in a mixed cell population (HeLa/humanfibroblasts) with the addition of plant extract at 1 and 5 % after 24 and48 h

Plant extract concentration Change in HF-32cell count (%)

Change in HeLacell count (%)

24 h 48 h 24 h 48 h

1 % −12 6 −32 −765 % −49 −72 −98 −99

Cou

nt

p16INK4a

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Fig. 3 Individual cell lines stained with anti-p16INK4a monoclonal anti-body conjugated to APC-Cy7. a HeLa cells stain positive. b HF-32 cellsshow a diffuse weak staining pattern. Effect of S. viminale plant extract ina mixed cell culture after 24 h. c Control cell culture—no extract. d Test

cell culture with 1% extract. Effect of S. viminale plant extract in a mixedcell culture after 48 h. e Control cell culture—no extract. f Test cellculture with 1 % extract

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DAPI staining of both cell lines after exposure to the S.viminale extract at 1 % (v /v ) revealed that HeLa cellsunderwent apoptosis sooner and to a greater extent than theHF-32 cells after 24 and 48 h. At 24 h, approximately 26 % ofHeLa cells showed signs of nuclear condensation or

fragmentation, whereas <1 % of the HF-32 cells were affect-ed. At 48 h, approximately 100 % of HeLa cells were affectedwhilst, again, <1 % of HF-32 cells were. These results wereconsistent with the previous assays, but as these nuclearchanges probably occur earlier than other changes, the

Control 1% 5%a

b

Fig. 4 Annexin Vand propidium iodide staining of cells with 1 and 5 %extract at 24 h. Quadrant (Q) 1-1 represents the PI-positive cells indicat-ing cell death. Q2-1 represents the dual positive population, also

indicating cell death. Q3-1 represents the dual-negative live cells. Q4-1indicates cells are annexin V-positive and in early apoptosis. a HF-32. bHeLa

control 1% 5%a

b

Fig. 5 Annexin Vand propidium iodide staining of cells with 1 and 5 %extract at 48 h. Quadrant (Q) 1-1 represents the propidium iodide-positivecells indicating cell death. Q2-1 represents the dual-positive population,

also indicating cell death. Q3-1 represents the dual-negative live cells.Q4-1 indicates cells are annexin V-positive and in early apoptosis. a HF-32. b HeLa

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difference between the HF-32 and HeLa cells were observedto bemore dramatic. In this assay, HeLa cells were muchmoreaffected by the extract than the HF-32 cells.

Taken together, these assays demonstrated that the extractfrom the plant S. viminale destroyed the cancerous HeLa cellsby apoptosis, whilst the non-cancer HF-32 cells were muchless affected. For example, at 1 % (v /v ) of the extract, HF-32cells showed great resistance to the effects of the extract with

only 5 % CPE, <1 % nuclear changes as shown by DAPIstaining, no change in annexinV/PI staining over the controland an actual increase in number in the mixed culture assayafter 48 h of exposure. In contrast, HeLa cells were greatlyaffected, with >75 % CPE, 77 % apoptosis or dead byannexinV/PI staining, 100% of cells showing nuclear changesby DAPI and a reduction of 76 % in cell numbers by mixedculture assay at 48 h.

Fig. 6 DAPI staining of HeLaand HF-32 cells exposed to 1% S.viminale plant extract. HeLacells: no extract control after 24 h(a); 1 % extract after 24 h (notecells with condensed andfragmented nuclei) (b) and 1 %extract after 48 h (all cells showapoptotic changes) (c). HF-32cells: no extract control after 24 h(d); 1% extract after 24 h (e); 1%extract after 48 h (f) and 1 %extract after 72 h (g)

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Previous large-scale screening tests missed the effects of S.viminale , and this was probably due to the limited range ofdilutions used for all plant extracts. The present findingsdemonstrate a selective cytotoxic action of the plant S.viminale extract towards cancerous cells whilst having lesseffect against non-cancerous cells, by a multiple of assays.This study suggests that the extract could be a potential usefulanticancer therapy, perhaps used in conjunction with othertherapies. Future studies on other cell lines and elucidatingthe active components of the plant are warranted.

Acknowledgments The authors acknowledge the provision of researchfacilities and the scientific and technical assistance of the staff of CHIRIBiosciences Research Precinct core facility, Curtin University.

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