Identification of potent anticancer activity in Ximenia americana aqueous extracts used by African...

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Toxicology and Applied Pharma

Identification of potent anticancer activity in Ximenia americana aqueous

extracts used by African traditional medicine

Cristina Voss, Ergul Eyol, Martin R. Berger*

Unit of Toxicology and Chemotherapy, Deutsches Krebsforschungszentrum Heidelberg, E100, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany

Received 17 March 2005; revised 13 May 2005; accepted 31 May 2005

Available online 11 July 2005

Abstract

The antineoplastic activity of a plant powder used in African traditional medicine for treating cancer was investigated by analyzing the

activity of various extracts in vitro. The most active, aqueous extract was subsequently subjected to a detailed investigation in a panel of 17

tumor cell lines, showing an average IC50 of 49 mg raw powder/ml medium. The sensitivity of the cell lines varied by two orders of

magnitude, from 1.7 mg/ml in MCF7 breast cancer cells to 170 mg/ml in AR230 chronic-myeloid leukemia cells. Immortalized, non-

tumorigenic cell lines showed a marginal sensitivity. In addition, kinetic and recovery experiments performed in MCF7 and U87-MG cells

and a comparison with the antineoplastic activity of miltefosine, gemcitabine, and cisplatinum in MCF7, U87-MG, HEp2, and SAOS2 cells

revealed no obvious similarity between the sensitivity profiles of the extract and the three standard agents, suggesting a different mechanism

of cytotoxicity.

The in vivo antitumor activity was determined in the CC531 colorectal cancer rat model. Significant anticancer activity was found

following administration of equitoxic doses of 100 (perorally) and 5 (intraperitoneally) mg raw powder/kg, indicating a 95% reduced activity

following intestinal absorption.

By sequencing the mitochondrial gene for the large subunit of the ribulose bis-phosphate carboxylase (rbcL) in DNA from the plant

material, the source plant was identified as Ximenia americana.

A physicochemical characterization showed that the active antineoplastic component(s) of the plant material are proteins with galactose

affinity. Moreover, by mass spectrometry, one of these proteins was shown to contain a stretch of 11 amino acids identical to a tryptic peptide

from the ribosome-inactivating protein ricin.

D 2005 Elsevier Inc. All rights reserved.

Keywords: New antineoplastic agent; Plant origin; Selective cytotoxicity; CC531 isogenic rat model

Introduction

For many centuries, plants have been a main source for

drug development. Early examples of anticancer agents

developed from higher plants are the antileukemic alkaloids

0041-008X/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.taap.2005.05.016

Abbreviations: NCI, National Cancer Institute (USA); MTT, (3-(4,5-

dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium bromide); IC10, inhibitory

concentration 10; IC50, inhibitory concentration 50; IC90, inhibitory

concentration 90; U, unit for growth-inhibiting activity; T/C%, treated

over control ratio (in percent); TA, total growth-inhibiting activity; rbcL,

ribulose bis-phosphate carboxylase, large subunit; MW, molecular weight;

RIP, ribosome inactivating protein.

* Corresponding author. Fax: +49 6221 423313.

E-mail address: [email protected] (M.R. Berger).

vinblastine and vincristine, which were both obtained from

the Madagascar periwinkle (Vinca rosea). Since the early

1950s, the identification and development of new lead

compounds for anticancer chemotherapy has been partially

driven by broad plant screening programs. This way, new

antineoplastic agents like the taxane derivative paclitaxel,

later known as taxol (from Taxus brevifolia) and the alkaloid

camptothecin (from the Chinese tree Camptotheca acumi-

nata) were identified (De Smet, 1997). Interestingly, an

analysis of plant materials that had been studied at the US

NCI for discovering new anticancer drugs showed that if

ethnopharmacological information had been used, the yield

of plants harboring antineoplastic activity would have been

significantly increased (Spjut and Perdue, 1976). The drug

cology 211 (2006) 177 – 187

mailto:[email protected]

http://dx.doi.org/10.1016/j.taap.2005.05.016

C. Voss et al. / Toxicology and Applied Pharmacology 211 (2006) 177–187178

potential of Chinese plants, where a written tradition of

using medical herbs has existed for thousands of years, is

currently being exploited. Several natural compounds with

antineoplastic activity have been identified. Some of them

have already been tested in clinical trials, like the alkaloids

homoharringtonine from Cephalotaxus species (Kantarjian

et al., 2001) and indirubin from a mixture from 11 Chinese

plants traditionally used to treat chronic myelocytic leuke-

mia (Xiao et al., 2002). Many other natural compounds of

Chinese origin, belonging to substance classes as diverse as

alkaloids, diterpenes, flavones, polysaccharides, and pro-

teins, were reported to show significant antineoplastic

activity in various in vitro or in vivo model systems (Tang

et al., 2003a, 2003b).

The same concept of exploiting the knowledge of

traditional medicine was followed in characterizing the

biological activity of a plant material with an African

ethnopharmacological background: case reports from a

cancer clinic in Tanzania described an unexpected improve-

ment of patients who had been terminally ill from advanced

prostate cancer. On the clinician’s inquiry, these patients

reported that they had been treated with a powder used in

African traditional medicine. This plant material was offered

by the clinician to our group for an initial analysis for

anticancer activity. The surprisingly high antiproliferative

efficacy found in two cell lines prompted a broad

experimental investigation, including assessment of the

antineoplastic activity in a cell line screen, comparison with

standard chemotherapeutic drugs and investigation of the

anticancer activity in vivo. In addition, a detailed phys-

icochemical analysis of the nature of the active compound(s)

was started. Moreover, the source of the material was

identified.

Methods

Materials and extracts

Plant material of African origin was obtained from a

local traditional healer in Tanzania. The powdered material

was supplied in three batches (designated A, B, and C)

within a period of 3 years. Each batch was tested for

antineoplastic activity in vitro in two cell lines (MCF7 and

HT29). For the main cell line screen and the in vivo

experiments, batch B was used.

Extracts were prepared by suspending 50 mg dry powder

per ml solvent and shaking for 10 min. The undissolved

material was separated by centrifugation (2500 � g, 5 min)

and the supernatant stored at �20 -C. All extract concen-trations are given in Ag/ml, with ‘‘Ag’’ representing the

amount of dry raw material which had been used for

preparing the extract.

Primary extracts were prepared from the raw material in

water and water-based buffers (PBS, Tris–HCl) as well as in

organic solvents (ethanol, methanol, and acetone) and

admixtures of water and organic solvents. Secondary

extracts were prepared from pre-extracted dried pellets.

To compare the amount of biological activity which

could be extracted by a specific solvent, a biological activity

unit (U) was defined as the amount of extract (in Al) able toinhibit the growth of MCF7 cells by 50%. The specific

activity of an extract was described by the value U/ml. The

total activity (TA, U/g dry raw material) of an extract was

subsequently calculated by multiplying the specific activity

(U/ml) with the total volume of the respective extract (ml)

and dividing by the amount of raw substance (g) which had

been used for extraction.

Other chemicals were obtained as follows: media for cell

culture from Sigma-Aldrich (Munchen, Germany), MTT

from Serva (Heidelberg, Germany), miltefosine from H.J.

Eibl, Max Plank Institute for Biophysical Chemistry

(Gottingen, Germany), gemcitabine from Eli-Lilly (Bad

Homburg, Germany), cisplatin from Bristol-Meyers (Ber-

gisch-Gladbach, Germany).

In vitro experiments

The cell line panel included 17 tumor cell lines originat-

ing from human (n = 16) and rat (n = 1) neoplasias as well as

four non-tumorigenic, immortalized cell lines. The tumor

cell lines were the human chronic-myeloid leukemia cell

lines AR230 (Wada et al., 1995), BV173 (DSMZ), CML-T1

(DSMZ), K562 (DSMZ), LAMA84 (DSMZ), the human

acute-myeloid leukemia cell line HL60 (DSMZ), the human

acute-lymphoblastic leukemia cell line SKW-3 (DSMZ), the

human breast cancer cell lines MCF7 (estrogen-receptor

positive, DSMZ) and MDA-MB-231 (estrogen-receptor

negative, ATCC), the human glioma cell line U333 (kindly

provided by Dr. Langbein, Dept. of Cell Biology, DKFZ,

Germany), the human glioblastoma/astrocytoma cell line

U87-MG (ATCC), the human epidermoid larynx carcinoma

cell line HEp2 (ATCC), the human large cell lung carcinoma

cell line NCI-H460 (ATCC), the human osteosarcoma cell

line SAOS2 (DSMZ), the human prostate carcinoma (meta-

stasis to bone) cell line PC3 (ATCC), the human colon

adenocarcinoma cell line HT29, and the rat colon carcinoma

cell line CC531 (Saenger et al., 2004; Wittmer et al., 1999).

The non-tumor cell lines included the human breast

epithelial cell line MCF10 (ATCC), the canine kidney

epithelial cell line MDCK (ATCC), the mouse fibroblast

cell line NIH/3T3 (ATCC), and the human prostate cell line

PNT-2 (ECACC). Cells were grown as recommended by the

suppliers in an incubator under standard culture conditions

(humidified atmosphere, 37 -C and 5% CO2 in air). For

experiments, the cells were distributed into 96-well (adher-

ently growing cells) or 24-well (cells growing in suspension)

flat bottom microtiter plates. The initial cell density was

selected for each cell line in a way that logarithmic growth

was maintained throughout the experiment.

The viable cell count was measured by the MTT [3-(4,5-

dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium bromide]

Table 1

Design of in vitro experiments

Type of assay Agent Concentration range Duration of exposure (days) Post-exposure period (days)

MTT assay for

antiproliferative activity

Primary/secondary extract in water

or organic solvents

0.1–1000 Ag/ml 3 –

Organic solvent 0.02–2%

Standard MTT assay

for cell line screen

Primary extract in water 0.1–1000 Ag/ml 3 �

Variation in exposure duration Primary extract in water 1–100 Ag/ml 2, 3, 4 �Recovery after

standard exposure

Primary extract in water 1–100 Ag/ml 3 1, 2, 3, 4, 5

Positive controls Miltefosinea 0.32–160 AM 3 �Gemcitabine 0.001–160 AMCisplatinum 0.32–80 Ag/ml

a Hexadecylphosphocholine (HPC).

C. Voss et al. / Toxicology and Applied Pharmacology 211 (2006) 177–187 179

assay as described by Konstantinov et al. (1998) with some

modifications. In short, cells growing in microtiter plates

were exposed to the treatment as described in Table 1. For

all extracts prepared in organic solvents, a vehicle control

was run in parallel covering the respective range of

concentrations, not exceeding 2% (ethanol and methanol)

or 0.2% (acetone).

For adherent cells, 0.1 � volume MTT solution (10 mg/

ml in PBS) was added to each well at the end of the

incubation period except for the blank. After 3 h incubation,

the culture medium containing the excess MTT was

removed. The formazan crystals were dissolved in 200 Alisopropanol containing 0.4 M HCl. Cells growing in

suspension were pelleted, washed in PBS, re-suspended in

1 ml fresh medium, and distributed into 96-well flat bottom

microtiter plates (100 Al/well). After adding 10 Al MTT

solution and incubating 3 h, the formazan crystals were

dissolved by 100 Al isopropanol containing 0.4 M HCl.

Extinctions were measured by a microplate reader at 540 nm

(reference 690 nm). Before treating a specific cell line, the

range of linearity for the MTT assay (extinction vs. cell

number) was defined. In a single experiment, 8 wells were

used for each concentration. Each experiment was repeated

two to three times.

In vivo experiments

For determining the effect of the aqueous extract in a rat

liver metastasis model, the rat colon cancer cell line CC531-

lac-Z was used, as described before (Saenger et al., 2004;

Table 2

Antineoplastic effect of the aqueous extract of X. americana in the CC531-lac-Z

Group no. No. of animals Treatment

Dosage (mg/kg) Route Total dosea

1 11 Control – –

2 6 100 p.o. 1000

3 9 Control – –

4 6 5 i.p. 50

a Administration started on day 1 and was continued every second day until dab P < 0.05 (Wilcoxon rank sum test).

Wittmer et al., 1999). In short, 4 � 106 CC531-lac-Z cells

were implanted intraportally into male Wag Rij rats (day 0).

Tumor-bearing rats were treated perorally or intraperito-

neally with the aqueous extract starting on day 1, as shown

in Table 2. Three weeks later (day 21), the experiment was

terminated, the liver of the animals was removed, weighed,

and kept at �80 -C until analysis. The number of tumor

cells per liver was determined by the h-galactosidase assay

(Applied Biosystems, Weiterstadt, Germany), as compared

to a standard curve established with a mixture of healthy

liver tissue and rising numbers of tumor cells.

Identification of the source plant

DNA was extracted from the plant material by the

cetyltrimethylammonium bromide (CTAB) method and the

chloroplastic gene for the large subunit of ribulose bis-

phosphate carbixilase (rbcL) was amplified and sequenced

as described by Treutlein and Wink (2002). For the resulting

DNA sequence, a phylogenetic analysis was carried out

with the program ‘‘Path’’ of the program package HUSAR

(DKFZ Heidelberg). The identity of the plant was confirmed

by isolating DNA from fresh plant material and sequencing

of the rbcL gene.

Identification of the active compound

pH-dependency. Aqueous extracts were prepared in differ-

ent buffers (100 mM Na acetate, pH = 4.5; 100 mM Tris–

HCl, pH = 7.0; NaCO3, pH = 9.5) as well as in 100 mM HCl

rat liver metastasis model

Liver weight Tumor cell no./liver

(mg/kg) Mean T SD (g) T/C% Mean T SD T/C%

44.5 T 7.2 – 4.8 T 2.0 � 109 –

15.1 T 9.1 34.0b 1.5 T 1.1 � 109 31.2b

37.6 T 11.0 – 10.8 T 2.5 � 109 –

23.4 T 12.5 62.2 3.7 T 2.5 � 109 34.3b

y 21.


and 100 mM NaCl. After a short (15 min) or long (2 h)

incubation at room temperature, the growth-inhibiting

activity of the extracts was determined in MCF7 cells.

Sensitivity to increased temperature. The aqueous extract

was incubated for 10 or 30 min at 50 -C and for 10 min

at 90 -C and its antineoplastic activity was subsequently

analyzed in MCF7 cells.

Precipitation. Precipitation experiments were performed

by adding 1–4 volumes of ethanol, isopropanol, or

acetone to 1 volume of aqueous extract and subsequent

separating the pellet by centrifugation. The pellet was re-

dissolved in the original volume of water and both pellet

and supernatant were analyzed for antineoplastic activity

in MCF7 cells.

Ultrafiltration. Ultrafiltration of the aqueous extract was

performed on Microcon YM10 (MW cutoff 10 kDa), YM50

(MW cutoff 50 kDa), and YM100 (MW cutoff 100 kDa)

membranes (Millipore, Schwalbach, Germany). Filtrates as

well as supernatants were analyzed for antineoplastic

activity in MCF7 cells.

HPLC assay for tannins. Tannins were identified in

various extract samples with or without preceding alka-

line/acid hydrolysis, by liquid-chromatography electro-

spray–ionization mass spectrometry (Owen et al., 2003).

SDS-PAGE analysis. SDS-PAGE of various extracts was

performed with the NuPAGE SDS-polyacrylamide gel-

electrophoresis system from Invitrogen (Karlsruhe, Ger-

many), under reducing or non-reducing conditions. For

protein detection, gels were stained with silver (Silver

staining kit SDS PAGE, Serva, Heidelberg, Germany) or

Coomassie blue (SimplyBlue, Invitrogen, Karlsruhe, Ger-

many). To detect glycosylated compounds, a periodic acid-

Schiff’s reagent-staining (PAS) was performed (Zacharius et

al., 1969).

Galactose affinity. A matrix containing free terminal

galactose was prepared by partial hydrolysis of Sepharose

4B, as described in Adam and Becker (2000). The

secondary aqueous extract was adjusted to 20 mM

Tris–HCl, 500 mM NaCl, pH = 7.0 and incubated with

hydrolyzed Sepharose for 15 min at room temperature

under gentle shaking. The hydrolyzed Sepharose gel was

subsequently separated by centrifugation and the super-

natant was analyzed for antineoplastic activity in MCF7

cells, as well as by SDS-PAGE. The separated gel was

washed three times with the binding buffer (20 mM Tris–

HCl, 500 mM NaCl, pH = 7.0) and incubated at room

temperature for 15 min with elution buffer (20 mM Tris–

HCl, 500 mM NaCl, 100 mM galactose, pH = 7.0). After

a brief centrifugation, the gel was discarded and the

supernatant containing proteins with galactose affinity was

collected and analyzed for antineoplastic activity in MCF7

cells and by SDS-PAGE.

Mass spectroscopy. After separating the proteins from the

fraction with galactose affinity by SDS-PAGE and staining

with Coomassie blue, the desired protein band was cut out

from the gel and an in-gel digestion was performed with

trypsin as described in Kinter and Sherman (2000). The

eluted tryptic peptides were analyzed by electrospray–

ionization mass spectrometry on a hybrid Q-TOF mass

spectrometer type Q-Tof2 (Waters Micromass, Manchester).

For the obtained mass spectra, a database search was

performed using the program Mascot search from Matrix

science (Perkins et al., 1999).

Evaluation and statistics

Means and respective standard deviations were calcu-

lated from the microtiter plate readings. For replicate

experiments, the results were averaged and characterized

by the standard error. Dose–response curves were plotted

for each cell line. Treatment effects were given as percent of

control (T/C%). The sensitivity of each cell line was

characterized by the values IC10 (concentration inhibiting

the growth by 10%), IC50 (concentration inhibiting the

growth by 50%), and IC90 (concentration inhibiting the

growth by 90%). As for all concentrations in this paper,

the IC values are given in Ag/ml, with ‘‘Ag’’ representingthe amount of dry raw material which had been used for

preparing the extract.

A mean IC50 was calculated for the cell panel used. For

comparison of cell lines, a ratio between individual and

mean IC50 values was calculated for each cell line.

Data from the in vivo experiments (liver weight and

tumor cell number/liver) were presented as mean T standard

deviation. A non-parametric rank sum test was used to

compare treated vs. control groups (Wilcoxon rank sum test,

ADAM statistics program, DKFZ, Heidelberg). P values <

0.05 were considered significant.

Results

Antineoplastic activity in various solvents

The material to be investigated appeared as a reddish-

brown powder of plant origin. Primary and secondary

extracts prepared in various solvents or solvent–water

admixtures were tested for antineoplastic activity in MCF7

breast und HT29 colon cancer cell lines (Fig. 1). In both cell

lines, the highest cell growth-inhibition levels were

achieved by extracts prepared in water or water-based

buffers. Primary extracts in organic solvents (methanol,

ethanol and acetone) contained distinctly less activity: in

HT29 cells, the methanol extract showed a higher growth-

inhibitory activity than the corresponding ethanol extract

Fig. 1. Concentration–effect curves of various primary and secondary extracts of the plant powder in HT29 (a) and MCF7 (b–d) cells. The cytotoxic activity

was determined by MTT assay. Each concentration was tested in six replicates. Vertical bars denote standard deviation of the mean.


(Fig. 1a). In MCF7 cells, methanol and ethanol extracts

showed slight growth-stimulatory effects, which were

antagonized by a growth-inhibitory activity at high concen-

trations only (Fig. 1b). The antineoplastic activity of

primary extracts in water and methanol admixtures

decreased with increasing methanol content (Fig. 1c). The

acetone extract showed only stimulatory activity in the

concentration range tested. Interestingly, this effect was

even increased for the primary extract prepared in an

admixture of 70% acetone in water (Fig. 1d). The influence

of the respective solvents was insignificant, as their effects

did not differ from normal experimental variation (T6%).

The total activity of the primary aqueous extract was

0.6 � 106 U/g. Secondary extracts in water or water-

based buffers prepared following extraction with ethanol

and acetone showed an almost identical activity (Fig. 1b

and d, 0.5 � 106 U/g each). The activity of the

secondary extract following methanol extraction was

diminished by 88% (Fig. 1b, 0.07 � 106 U/g) and that

following 70% acetone extraction was increased by 143%

(Fig. 1d, 1.4 � 106 U/g).

Antineoplastic activity in a panel of tumor and non-tumor

cell lines

For the cell panel screen, a primary extract in water was

used throughout. An overview on the cell growth

inhibition is given in Table 3. The cell lines tested showed

different sensitivities, as illustrated in Fig. 2 for both solid

tumor (a) and leukemia-derived cell lines (b). The IC50

values ranged by a factor of 100, from 1.7 Ag/ml (MCF7

cells) to 170 Ag/ml (AR230 cells). The origin of a tumor

cell line did not predict its sensitivity, as evidenced by

BV173 (IC50 = 1.8 Ag/ml) and CML-T1 (IC50 = 160 Ag/ml) chronic myeloid cells. The shape of the respective

concentration–activity curve is reflected by the ratio

between the IC90 and IC10 values (Table 3). A low ratio

represents a dose–response curve with a steep slope, a

high ratio a curve with a lower slope. There was no

obvious relationship between the IC50 value and the slope

of the dose–response curve.

The average IC50 over the whole cell line panel was 49

Ag/ml. A classification of the cell lines as sensitive or less

sensitive was based on the ratio of the individual IC50 to the

average IC50 (Fig. 3). The majority of cell lines (11 out of

17) were classified as sensitive with a degree of sensitivity

that varied over two orders of magnitude. Three of these

sensitive cell lines (MCF7 breast cancer, BV173 CML, and

CC531 rat colon carcinoma) showed a particularly high

sensitivity, with ratios lower than 0.1 of the average IC50.

The IC50 of non-tumor cell lines ranged from 20

(PNT-2) to over 100 (MCF10) Ag/ml (Table 3). Based on

the classification for tumor cell lines, the immortalized

cell lines should be grouped as marginally to less

Table 3

Antiproliferative activity of an aqueous extract from X. americana in 16

human and one rodent tumor cell lines, as well as in four immortalized non-

tumor cell lines

Cell line IC10a

(Ag/ml

medium)

IC50b

(Ag/ml

medium)

IC90c

(Ag/ml

medium)

IC90/IC10d

(ratio)

Tumor cell lines

MCF7 0.6 1.7 10 16.7

BV173 0.4 1.8 7 17.5

CC531 0.8 3.3 12 15.0

U87-MG 1.0 9 100 100

K562 5 11 180 36

SKW-3 3.1 20 700 226

HEp2 5 21 100 20

NCI-H460 4 21 150 38

PC3 3.5 26 >1000 >300

MDA-MB231 5 33 100 20

HT29 8 40 350 44

U333 7 65 300 43

SAOS2 20 66 1000 50

LAMA84 10 90 600 60

HL60 30 90 1000 33

CML-T1 2.5 160 1000 400

AR230 17 170 700 41

Non-tumor cell lines

MCF10 35 >100 >100 >2

MDCK II 12 27 60 5

NIH/3T3 2 33 >100 >50

PNT-2 2 20 >100 >50

a Inhibitory concentration 10 (concentration inhibiting the cell growth by

10%), as assessed by MTT assay.b Inhibitory concentration 50 (concentration inhibiting the cell growth by

50%), as assessed by MTT assay.c Inhibitory concentration 90 (concentration inhibiting the cell growth by

90%), as assessed by MTT assay.d Ratio of IC90 and IC10 values.

Fig. 2. The aqueous extract of the plant powder in adherent (a) and

leukemic (b) tumor cell lines. Four cell lines were selected, respectively,

including those with highest and lowest sensitivity. For comparison,

concentration–effect curves are shown for a non-tumorigenic immortalized

cell line (MDCK) seeded at different cell densities (c). The cytotoxic

activity was determined by MTT assay. Each concentration was tested in six

replicates and each experiment was repeated one or two times. Vertical bars

denote standard error of the mean.


sensitive (Fig. 3). The growth inhibition of these cell lines

strongly depended on the initial cell density, as shown in

Fig. 2c for MDCK II cells, with low cell densities being

distinctly more susceptible to the extract than high cell

densities.

Variation in exposure duration and recovery

When comparing the dose–response curves after 2, 3,

and 4 days of exposure, the ratio of surviving cells (treated/

control) decreased with increasing exposure duration for

MCF7 breast cancer cells over the whole time period

investigated (Fig. 4a). For U87-MG glioblastoma/astrocy-

toma cells, the treated over control ratio decreased for up to

3 days but remained constant thereafter (Fig. 4b).

In the recovery experiments, concentrations effecting

more than >70% growth inhibition in MCF7 (Fig. 4c) or

�80% in U87-MG (Fig. 4d) cells were associated with

permanent growth suppression. Lower concentrations were

inversely related to an increasing degree of cell prolifer-

ation, indicating recovery.

Comparison with positive controls

A comparison of the antineoplastic activity of the extract

with that of three clinically used agents is given in Table 4.

The cytotoxicity profiles of four cell lines are illustrated by

Fig. 3. Sensitivity plot of all cell lines. The ratio of individual and average (based on tumor cell lines only) IC50 was used to rank the panel into more (ratio < 1)

and less (ratio > 1) sensitive cell lines. Cell lines were grouped according to their origin as well as their tumorigenicity. The ratio of the MCF10 cells (*) is an

underestimate, since the IC50 was not reached at 100 Ag/ml.


the respective IC10, IC50, and IC90 values, as well as by the

corresponding IC90 to IC10 ratio, describing the slope of the

concentration–effect curve. Most prominently, the ranking

Fig. 4. Kinetic and recovery experiments based on the aqueous extract of the plan

response to the varied exposure duration in MCF7 (a) and U87-MG (b) cells. The

tested in six replicates and each experiment was repeated one or two times. Vertic

and U87-MG (d) cells following replacement of the extract containing medium af

determined by MTT assay. Each concentration was tested in six replicates. Vertic

in sensitivity differed between the extract and the positive

controls. In variance to the extract, which resulted in the

lowest IC50 and IC90/IC10 ratio in MCF7 cells, miltefosine

t powder in MCF7 and U87-MG cells. (a,b) Concentration–effect curves in

cytotoxic activity was determined by MTT assay. Each concentration was

al bars denote standard error of the mean. (c,d) Growth curves of MCF7 (c)

ter 3 days of exposure with standard growth medium. The absorbance was

al bars denote standard deviation of the mean.

Table 4

Cytotoxicity profiles of the extract and three standard antineoplastic agents

in a subpanel of cell lines

Cell line Treatment IC10 IC50 IC90 IC90/IC10

MCF7 Extract (Ag/ml) 0.6 1.8 10 16.7

Miltefosine (AM) 6.5 40 80 12.3

Cisplatinum (Ag/ml) 0.22 2.2 10 45

Gemcitabine (AM) 0.001 0.012 >100 >105

U87-MG Extract (Ag/ml) 1.0 9 100 100

Miltefosine (AM) 4.7 27 70 14.9


Gemcitabine (AM) 0.002 0.014 >100 >5 � 104

HEp2 Extract (Ag/ml) 5 21 100 20

Miltefosine (AM) 1.2 2.8 8 6.7

Cisplatinum (Ag/ml) 0.09 0.4 1.4 15.6

Gemcitabine (AM) 0.2 0.47 17 85

SAOS2 Extract (Ag/ml) 20 66 1000 50

Miltefosine (AM) 5.0 40 120 24


Gemcitabine (AM) 0.007 0.034 >100 >104


and cisplatinum caused the lowest IC50 and IC90/IC10 ratio

in HEp2 cells. Similar to the extract, the lowest IC50

following gemcitabine exposure was seen in MCF7 cells.

However, this agent differed from all others by its lack in

effecting 90% growth inhibition in three cell lines, including

the most sensitive MCF7 cells. The only cells, in which

gemcitabine induced 90% growth inhibition, were the HEp2

cells; notably, these cells were most resistant to this agent. In

contrast, SAOS2 cells were found to be most resistant to the

extract as well as to miltefosine and cisplatinum.

In vivo experiments

As shown in Table 2, the aqueous extract was adminis-

tered to male tumor-bearing Wag Rij rats via the peroral and

the intraperitoneal route. Preliminary experiments had

shown that the maximum tolerated single dose was 30

mg/kg following i.p. administration. Higher dosages caused

weight loss, hemorrhage, and acute death within 24 h. In

contrast, no toxicity was observed following dosages of up

to 100 mg/kg given p.o.

Peroral administration of 100 mg/kg every second day

effectively reduced the increase in liver weight seen in

untreated tumor-bearing rats (T/C% = 34.0, P < 0.05, Table

2). Concomitantly, the number of tumor cells was

significantly lower in treated rats as compared to the

respective controls (T/C% = 31.2, P < 0.05, Table 2).

Interestingly, intraperitoneal administration of 5 mg/kg

every second day was found to be less effective regarding

the liver weight (T/C% = 62.0, Table 2), but similarly

effective with regard to tumor cell number (T/C% = 34.0,

P < 0.05, Table 2).

Physicochemical characterization of the biological activity

The variation in antineoplastic activity between three

batches of plant material was determined in MCF7 breast

cancer cells. The most recent batch (C) was characterized by

the lowest IC50 value (0.9 Ag/ml, TA = 1.1 � 106 U/g),

followed by the first batch (A, IC50 = 1.1 Ag/ml, TA = 0.9 �106 U/g) and the second batch (B, IC50 = 1.7 Ag/ml; TA =

0.6 � 106 U/g). Storage of the dry material for up to 2 years

at 4 -C did not change the level of the antineoplastic

activity.

The aqueous extract was stable for up to 2 years at

�20 -C but lost 20% of its antineoplastic activity after 24 h

at 4 -C, as determined in HT29 colon carcinoma and MCF7

breast cancer cells.

Incubation for 10 or 30 min at 50 -C resulted in a marked

loss of activity as shown by IC50 values of 2.5 and 5.0 Ag/ml, respectively, compared to 1.2 for the untreated extract

(as determined in MCF7 breast cancer cells). A complete

loss of the antineoplastic activity was observed after 10 min

incubation at 90 -C.Alterations in pH had also a time-dependent effect on the

biological activity of the extract. Short-term exposure (10

min) to pH values of 1.5–12 had no effect. After 2 h, the

extracts exposed to pH values < 5 lost part of their activity

whereas that of the alkaline extracts remained unchanged.

Exposure for 24 h to both acids and alkalis led to a complete

loss of the antineoplastic activity.

Precipitation caused by addition of organic solvents

(methanol, ethanol, isopropanol) to the aqueous extract was

associated with pelleting of the entire biologic activity.

The filtrate obtained after aqueous extract ultrafiltration

through membranes with 10 kDa cutoff was devoid of any

activity. The antineoplastic activity was completely found in

filtrates obtained by ultrafiltration through a 100-kDa cutoff

membrane and partially in that through a 50-kDa cutoff

membrane.

Confining the active compound(s) to a substance class

SDS-PAGE of the raw aqueous extract with subsequent

staining for protein (Coomassie blue) showed this to contain

proteins as well as a Coomassie-negative smear in the high

molecular weight domain. This smear as well as some of the

protein bands were colored by Schiff’s reagent, demonstrat-

ing the presence of polysaccharides in the smear as well as

of glycosylated proteins. Incubation of the raw aqueous

extract with trypsin or proteinase K, however, did not result

in a diminished biological activity.

Tannins were also identified in the raw aqueous extract

by reverse-phase HPLC and were most efficiently

extracted by 70% acetone. This extract showed no

antineoplastic activity in MCF7 cells (Fig. 1d). The

secondary extract, prepared after pre-extraction with 70%

acetone, was devoid of tannins and showed a >2-fold

increased biological activity as compared to the raw

aqueous extract (TA = 1.4 � 106 and 0.6 � 106 U/g for

the secondary and primary aqueous extracts, respectively).

Incubation of this secondary extract with trypsin or

proteinase K led to a 2-fold (trypsin) to 3-fold (proteinase


K) decrease in antineoplastic activity in MCF7 cells (24

T/C% for trypsin, 38 T/C% for proteinase K vs. 12 T/C%

for the undigested extract, for an extract concentration of

10 Ag/ml medium).

Affinity to galactose

Hydrolysis of the cross-linked galactose polysaccharide

Sepharose 4B was used to prepare an affinity matrix

containing free terminal galactose residues. Incubation of

the aqueous extract with this matrix resulted in an almost

complete depletion of the biological activity from the

supernatant (T/C = 80% after Sepharose incubation vs.

18% before, as determined for an extract concentration of 2

Ag/ml in MCF7 cells). This finding led to the assumption

that the active component(s) had bound to the matrix.

Indeed, after separating the supernatant and washing the

hydrolyzed Sepharose gel, an active fraction could be eluted

by using buffer with 100 mM galactose. A non-reducing

SDS-PAGE analysis of this fraction revealed the presence of

two proteins with a molecular weight (MW) of about 50–60

kDa (Fig. 5a). Under reducing conditions, four bands were

seen within the MW range 25–35 kDa (Fig. 5b), hinting at a

two-chain structure for both proteins. The protein with the

higher mass was subsequently processed for mass-spectro-

metric analysis.

A database search based on the mass-spectroscopic

results failed to identify a known protein in the database.

However, one tryptic peptide was identified by its identity to

a peptide contained by the ribosome inactivating protein

ricin with the sequence SNTDANQLWTLK.

Identification of the source plant

In a phylogenetic analysis, the new rbcL sequence was

found to cluster together with the rbcL sequence from

Heisteria parvifolia from the Olacaceae family. A compar-

Fig. 5. SDS-PAGE of the cytotoxic, affinity-purified protein fraction (lanes

G) compared to the raw aqueous extract (lane E) under reducing (a) or non-

reducing (b) conditions. M—molecular weight marker (kDa).

ison with unpublished data (courtesy of Dr. D.L. Nickrent,

Department of Plant Biology, Southern Illinois University,

Carbondale, IL, USA) showed a close homology to the

Ximenia americana rbcL sequence. After obtaining fresh

plant material from X. americana of US origin as well as

DNA from X. americana of African origin and sequencing

the respective rbcL genes, this plant was confirmed to be the

source of the powder used in this study.

Biological activity of X. americana extracts

Aqueous extracts prepared from powdered dry leaves

from X. americana of US origin showed no antineoplastic

effect in MCF7 breast cancer cells. Conversely, a strong

growth inhibition of MCF7 cells was seen (IC50 = 33 ng/ml,

TA = 30.3 � 106 U/g) following treatment with aqueous

extracts prepared from powdered dry nuts from X. ameri-

cana of US origin. As for the African plant material, a

higher biological activity was seen for the secondary

aqueous extract prepared from US X. americana nut

powder, pre-extracted with 70% acetone (IC50 = 23 ng/ml,

TA = 43.5 � 106 U/g).

Discussion

A lot of work is currently being invested into exploiting

the drug potential of Chinese plants, particularly of those

used in Chinese traditional medicine. However, information

about plants used in African traditional medicine is still

scarce. This is related to different ways of propagating

medical experience: whereas the Chinese ethnopharmaco-

logical knowledge has been passed on for thousands of

years based on written information, the traditional African

medicine’s mode of transmission by word of mouth has

hindered systematic scientific investigation (Okpako, 1999).

Case reports on the improvement of cancer patients after

treatment with a plant material used in African traditional

medicine prompted in vitro tests of this powder for antineo-

plastic activity. Since the patients had used the powder by

suspending it in water and ingesting the supernatant, a water

extract was initially used. A surprisingly high growth

inhibition upon extract treatment was found in two tumor-

derived cell lines. This observation gave rise to an extended

study on the aqueous extract’s effect in 17 tumor cell lines

originating from eight different organs/tissues.

Although no typical sensitivity of a certain organ was

discernible, the differences in sensitivity between cell lines

ranked as ‘‘highly sensitive’’ (MCF7 and BV173) and those

classified as ‘‘less sensitive’’ (AR230, CML-T1) point to the

fact that the cytotoxic activity of the aqueous extract is

selective. However, the molecular basis for this selectivity is

so far unknown and is not directly related to the tumor-

igenicity of cell lines, since some of the immortalized, non-

tumorigenic cell lines were ranked as marginally sensitive.

Interestingly, immortalized, non-tumorigenic cells showed a


3- to 4-fold higher sensitivity when seeded as single cells as

compared with higher densities. We speculate that this

phenomenon is related to the proliferation rate, since the

slowly proliferating MCF10 cells were the most resistant of

this subgroup.

The slope of the dose–response curves, as reflected by

the IC90/IC10 ratio, differed also by factor of 25. Moreover,

the shape of these curves showed a shoulder in some cases

(AR230) or a growth stimulation at low concentrations

(SAOS2). Both observations could reflect such diverse

properties as stimulation concomitant with cytocidal effects

both caused by extract components, or subpopulations of

more resistant cells within the same cell line.

The IC50 averaged over all cell lines was surprisingly

low, as compared with other plant extracts tested in our

laboratory (unpublished results). Remarkably, the aqueous

extract prepared from X. americana nuts showed an IC50

almost 100-fold lower than that of the powder extract in

MCF7 cells. This finding leads to the assumption that the

powder obtained from Tanzania was blended with plant

parts containing low cytotoxic activity.

To asses the kinetics of the cytotoxic activity, exposure

duration and recovery experiments were performed in two

cell lines. The decreasing ratio of surviving cells with in-

creasing exposure duration describes an improved effective-

ness of the extract treatment with time. This effect might be

related to various cellular processes triggered by the aqueous

extract, which finally lead to cell death or growth delay. The

highly sensitive MCF7 cells showed this effect for a longer

period than the less sensitive U87-MG cells. In addition, both

MCF7 and U87-MG cells showed no recovery after being

exposed to sufficiently high extract concentrations.

The ‘‘signature’’ of the aqueous extract’s activity in four

cell lines differed from that of three standard chemo-

therapeutic drugs used as positive controls. This implies

that the mechanism of action differs from that of these

agents, including alkylating agents, antimetabolites, and

signal transduction modifiers.

In addition to the antineoplastic activity, a growth

stimulation was seen following exposure to the primary

aqueous extract in three cell lines (MCF7, SAOS2, and

HL60). In MCF7 cells, this stimulating effect was increased

in response to extracts prepared in ethanol, methanol, and

acetone. The highest increase in MCF7 cell proliferation

was observed after treatment with an extract containing a

high tannin concentration (70% acetone primary extract).

Moreover, when separating by size, the stimulating compo-

nents passed through a 10-kDa cutoff membrane, proving

the stimulating components to be physically distinct from

the cytotoxic components. The fact that none of these

extracts had any stimulatory activity in HT29 cells leads us

to speculate that the growth stimulation observed in MCF7

cells was caused by compounds with hormonal activity,

such as phyto-estrogens (De Naeyer et al., 2005).

Concomitantly with the cell line screen, the stability of

the plant material and that of the aqueous extract was

monitored. The biological activity of the powdered plant

material was stable throughout the period of the cell line

screen, as shown by the reproducibility of the results.

Moreover, different batches of plant material effected

comparable degrees of growth inhibition. Aqueous extracts,

however, lost activity in a time-dependent manner when

stored at 4 -C and were therefore always stored at �20 -C.Since the rat colon carcinoma cell line CC531 was ranked

among the three most sensitive cell lines in the panel, it was

tempting to determine whether any antineoplastic activity

would be discernible in a rat model based on this cell line.

The significant antineoplastic activity observed following

peroral administration was surprising since no concomitant

toxicity was detected. This observation is furthermore in line

with the case reports from Tanzania. A 20-fold lower, equi-

toxic dosage administered i.p. was less effective in moderat-

ing the liver weight increase, but equally effective in

controlling the increase in tumor cell number. Several factors

could be responsible for this difference, including degrada-

tion within the gastrointestinal tract, incomplete gastro-

intestinal absorption, differences in tissue distribution,

metabolism, and excretion. Future pharmacokinetic studies

are needed to determine the reason for the observed differ-

ence between peroral and parenteral administration routes.

The promising results of both the in vitro screen and the

in vivo experiments triggered studies aiming at the identi-

fication of the source plant and the active component(s).

The phylogenetic analysis used for plant identification,

allowing the identification of the presumed plant family,

Olacaceae, took advantage of the broad information

available on the rbcL gene sequence in public databases.

The assumed source species, X. americana, was confirmed

by comparison to rbcL sequences from fresh X. americana

plant material, as well as by finding a high cytotoxic activity

in plant parts subsequently obtained from this species.

In order to define the substance class of the active

component(s), experiments were carried out on their

physicochemical properties, including solubility, stability at

varying pH and temperature, size estimation, and precip-

itation. In this process, lipids and lipophilic plant secondary

metabolites could be excluded, since the biological activity

was only extracted by strongly polar solvents.

Large amounts of tannins were identified in the aqueous

extract. However, extracts prepared in methanol or 70%

acetone, both solvents known to efficiently extract tannins

from plant materials, had only a low (methanol) or no (70%

acetone) cytotoxic activity. Remarkably, depletion of tannins

by pre-extraction with 70% acetone led to the release of a

higher total biological activity in the secondary aqueous

extract, when compared to a respective primary extract. This

implies that the biological activity is partially inhibited or

hidden by tannins or other components extracted by 70%

acetone.

Molecules smaller than 10 kDa were excluded by

ultrafiltration experiments, which also allowed estimating

the MW of the active components to be in the range of 50 T


20 kDa. Out of the known classes of plant cell macro-

molecules, DNA and RNA were not found in the aqueous

extract and digestion experiments with DNase or RNase had

no effect on the biological activity. However, proteins and

polysaccharides were shown to be present in the primary

and the secondary aqueous extracts and could not be further

separated by physicochemical methods. Finally, digestion

experiments with trypsin and proteinase K hinted at a

protein being responsible for the cytotoxic activity.

A well-defined family of cytotoxic plant proteins is that

of the type II ribosome-inactivating proteins (RIPs; Battelli,

2004). These proteins with a molecular weight of about 60

kDa consist of two polypeptide chains, termed A- and B-

chain, with an MW of about 30 kDa each, being held

together by a disulphide bridge. The A-RIP chain has the

function of an RNA N-glycosidase and is able to inhibit

protein synthesis in a cell-free lysate. The B-chain has the

function of a lectin with affinity for galactose or other sugar

residues. Binding of the B-chain to oligosaccharides on the

cell membrane surface elicits internalization of the RIP and

results in cytotoxicity (Barbieri et al., 1993).

Based on this background, the biologically active extract

components were tested for their affinity to galactose and

found to bind to Sepharose containing free galactose ends.

When eluted with galactose-containing buffer, the galactose-

binding fraction was associated with high cytotoxic activity.

This fraction consisted of only two proteins (Fig. 5).

The cytotoxic effects, the MW, as well as the two-chain

structure of the proteins in the affinity-purified fraction can

be related to the molecular characteristics of known members

of the type II RIP family. In keeping with this assumption,

one of the mass-spectrometrically sequenced tryptic pep-

tides, originating from one of these proteins, showed identity

with a tryptic peptide in the B-chain of the type II RIP ricin.

However, the lack of further hits when comparing the MS

spectrum with the database implies that the protein is largely

unknown. This is corroborated by the fact that no RIPs are

known to date to exist in the plant X. americana, which has

been identified as the source of the powder used. In

conclusion, this cumulative evidence strongly suggests that

the active components of the plant material are so far

unknown proteins belonging to the type II RIP family.

Acknowledgments

We are indebted to Dr. D.L. Nickrent, Department of

Plant Biology, Southern Illinois University, Carbondale, IL,

USA, for comparing our rbcL sequence with his unpub-

lished data and for providing the rbcL sequence of Ximenia

americana collected in the Bahamas.

We also thank Dr. W. Lehmann, Central Spectroscopy

Unit, DKFZ, Heidelberg, for the mass spectroscopical

analysis of the protein bands and Dr. B. Owen, Department

of Toxicology and Cancer Risk Factors, DKFZ, Heidelberg,

for analyzing our samples by HPLC for tannins.

Finally we are grateful to Captain A. Kurzenhauser

(retired, US Navy), Tampa, FL, USA, for his invaluable help

in tracing and collecting Ximenia americana.

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