ANTICANCER RESEARCH 30: 55-64 (2010)
Anticancer Activity of Novel Plant Extracts from
Trailliaedoxa gracilis (W. W. Smith & Forrest)
in Human Carcinoid KRJ-I Cells
BERNHARD SVEJDA1, VICTOR AGUIRIANO-MOSER1, SONJA STURM2,
HARALD HÖGER3, ELISABETH INGOLIC4, VERONIKA SIEGL1,
HERMANN STUPPNER2 and ROSWITHA PFRAGNER1
1Department
of Pathophysiology and Immunology, Center of Molecular Medicine, Medical University of Graz, Graz, Austria;
of Pharmacy, Center of Molecular Biosciences, Leopold Franzens University of Innsbruck, Innsbruck, Austria;
3Core Unit of Biomedical Research, Division of Laboratory
Animal Science and Genetics, Medical University of Vienna, Himberg, Austria;
4Research Institute for Electron Microscopy and Fine Structure Research, University of Technology Graz, Graz, Austria
2Institute
Abstract. Background: Small intestinal (SI) neuroendocrine
tumors (NETs) are rare neoplasms derived from
neuroendocrine cells presenting distinct clinical symptoms
according to the ability to secrete neuroamines. Nevertheless,
many are asymptomatic and misdiagnosed. As response rates
to chemotherapy are low, surgery remains the only effective
treatment. Because many tumors have metastasized at the
time of diagnosis, curative surgery is rarely achieved.
Consequently, a substantial need for new therapeutic options
has emerged. Materials and Methods: The effects of novel
plant extracts from Trailliaedoxa gracilis (W.W. Smith &
Forrest) were investigated in the SI-NET cell line KRJ-I and
in KRJ-I transplanted mice. Proliferation and viability were
analyzed using cell counting and WST-1 cell proliferation
assay. Apoptosis was determined by DAPI staining and
electron microscopy, and quantified by luminescence assays
for caspases 3/7, 6, 8, 9 and 2. Results: Extracts of
Trailliaedoxa gracilis showed a dose-dependent reduction of
proliferation and induction of apoptosis in the KRJ-I cells.
Normal fibroblasts were not impaired. Tumor growth
inhibition was also observed in heterotransplanted SCID
(severe combined immunodeficiency) mice. Conclusion: The
in vitro and in vivo outcomes suggest a potential clinical
effect of Trailliaedoxa gracilis in SI-NETs.
Correspondence to: Professor Roswitha Pfragner, Department of
Pathophysiology and Immunology, Medical University of Graz,
Heinrichstrasse 31, A-8010 Graz, Austria. Tel: +43 3163804297,
Fax: +43 3163809640, e-mail: roswitha.pfragner@meduni-graz.at
Key Words: Neuroendocrine tumor, carcinoid, cell lines, plant
extracts, bioactive agents, chemoresistance, apoptosis.
0250-7005/2010 $2.00+.40
Small intestinal (SI) neuroendocrine tumors (NETs) are rare
neoplasms originating from neuroendocrine cells that are part
of the diffuse neuroendocrine system (1). These tumors are
characterized by malignant potential and slow growth with an
annual incidence of 4-6 per 100,000 (2, 3). Due to increasing
clinical awareness and more wide-spread use of diagnostics,
e.g. endoscopy, ultrasonography, CT, MRI and the
measurement of biochemical markers, the incidence of SINETs has increased annually by 3 to 10% over the last three
decades (2, 4); neuroendocrine tumors of the gut therefore are
not as rare as once considered. Diagnosis is currently based
on the typical clinical symptoms and imaging methods such
as somatostatin receptor scintigraphy and blood levels of
biochemical markers (5). Specific for the ileal carcinoid
tumors are increased serum levels of serotonin, neuropeptide
K and substance P (3, 4). These bioactive substances are
responsible for the characteristic clinical symptoms of the
carcinoid syndrome characterized by ‘flushing’, diarrhea and
abdominal pain. However, many tumors are so-called nonfunctioning causing no symptoms by the secreted peptides,
while functioning tumors presenting with distinct clinical
symptoms are often misdiagnosed or overlooked. Therefore a
delay of diagnosis (5-7 years) is typical, increasing the
probability of liver metastasis (3) and consequently, more
than 85% of patients show liver metastasis at the time of
diagnosis. Surgery remains the only curative therapeutic
option, but due to high rates of metastasis less than 15% of
patients have the likelihood of a curative intervention (5).
Therefore, clinical management focuses mainly on the
treatment of clinical symptoms using somatostatin analogues
and interferon-α. While biotherapy with somatostatin and
interferon-α appears to have unquestioned tumoristatic
properties, these agents do not yield significant objective
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ANTICANCER RESEARCH 30: 55-64 (2010)
tumor responses (5). Recently, single-agent and multi-agent
chemotherapy regimens have been evaluated with response
rates from 0-20% for single agent and 20-30% for multi-agent
treatment (5). Due to the unchanged prognosis for SI-NETs
over the past 30 years, there is a substantial need for finding
new therapeutic strategies.
For centuries, plants have been used in traditional medicines
for the treatment of different diseases. In recent years, oriental
medicinal herbs have aroused scientific interest as
complementary or alternative medicines (6). Several
chemotherapeutic drugs derived from plants, such as
Vinblastine, Taxol, Camptothecin and Podophyllotoxin are
used in medical tumor management (7). Using modern
analytical and chemical techniques, novel natural compounds
from herbs can be isolated by fractionation and isolation. It
has been estimated that only 5-15% of 250,000 species of
higher plants have been screened systematically for natural
bioactive compounds (8, 9). To study new therapeutic
approaches, cell lines are used to investigate novel compounds
and their effects on the tumor cells. However, few cell lines
from human SI-NETs have been maintained so far, namely
GOT1 (10) and CNDT2 (11), and therefore, we established
four continuous tumor cell lines (KRJ-I, P-STS, L-STS, HSTS) (12, 13) from human malignant carcinoid tumors of the
small intestine, KRJ-I and P-STS being derived from primary
ileal carcinoids. All established cell lines were investigated
morphologically and immunocytochemically and established
to derive from an SI-NET (12-14). After cell lines were used
as cancer models to investigate antiproliferative activities of
novel compounds, findings were confirmed by in vivo testings
in xenograft rodents (15). In recent studies, we have
demonstrated a dose-dependent antitumor effect of bioactive
agents derived from Cautleya gracilis (Smith) Dandy and
extracts of Stemona tuberosa Lour with induction of apoptosis
on medullary thyroid carcinoma cell lines established in our
labaratory (16-19).
Belonging to the family of Rubiaceae, like other Chinese
herbs, Trailliaedoxa gracilis (W.W. Smith & Forrest) was
found and described by the Austrian botanist HandelMazzetti (20). In a previous screening, an antiproliferative
effect of T. gracilis (W.W. Smith & Forrest) was noted in the
medullary thyroid carcinoma cell lines MTC-SK (21) and
SINJ (22). However, the bioactivity of T. gracilis is largely
unclear. Thus, the aim of the present study was to investigate
for the first time antiproliferative activity in vitro using the
SI-NET cell line KRJ-I and in vivo in xenotransplanted mice.
Materials and Methods
Plant extracts and chemicals. The plant material (whole plant) was
provided by E. Stöger (Oberndorf, Austria). A voucher specimen was
deposited at the Institute of Pharmacy/Pharmacognosy at the
University of Innsbruck. The dried material was ground and
macerated with dichloromethane (DCM), filtered and the solvent
56
evaporated under reduced pressure (TG-5). The extract was then
re-dissolved in water/methanol and a liquid-liquid partition with
n-hexane (TG-1), DCM (TG-2) and ethylacetate (EtOAc) (TG-3) was
performed. After evaporation of the solvents under reduced pressure,
all samples were re-dissolved in dimethyl sulphoxide (DMSO, SigmaAldrich, Vienna, Austria) at a concentration of 10 mg/ml and stored at
–20˚C. The fractionations of TG-5 and the subsequent subfractions
were obtained by gel chromatography using a Sephadex LH-20
column and DCM:acetone (85:15) or methanol as the mobile phase.
Ursolic acid was crystallized from TG-F28 and the structure
elucidation was carried out by 1D and 2D NMR and MS. The
concentration of ursolic acid in TG-F28 was evaluated by 1H- NMR
in DMSO-d6. The fifteen obtained subfractions of T. gracilis were
tested at different concentrations on KRJ-I cells and on fibroblasts.
The concentrations of each fraction and subfraction ranged from 2
μg/ml to 50 μg/ml in order to determine the IC50 values.
Camptothecin (CPT) was purchased from Sigma-Aldrich (SigmaAldrich, Vienna, Austria), dissolved in DMSO and stored at a
concentration of 1 mM at 4˚C.
Cell lines and cell culture. A continuous cell line from a human
malignant carcinoid of the small intestine, KRJ-I (12) and the
normal human skin fibroblast cell line HF-SAR (23) were
established in our laboratory. The KRJ-I cells had been
characterized histologically and biochemically to be derived from
the original tumor (12, 14). All the cell lines were Mycoplasma-free.
The KRJ-I cells were cultured in Quantum 263 medium optimized
for tumor cell growth (PAA Laboratories, Pasching, Austria) at
37˚C, 5% CO2 and the HF-SAR were maintained in Eagle’s MEM
supplemented with 10% FBS at 37˚C, 5% CO2. For the in vitro
experiments, the KRJ-I and HF-SAR cells were plated at an initial
density of 2×105 cells/ ml or 1×105 cells/ ml, respectively.
Cell counting. KRJ-I cells were cultured in 24-well plates (Sarstedt,
Wr. Neudorf, Austria) for 24, 48 or 72 h with extracts of T. gracilis
dissolved in DMSO. DMSO alone was used as negative control. The
samples were suspended to disperse cell clusters into single cells
and then counted automatically with a CASY®-1 Cell Counter &
Analyzer TTC (Schärfe System, Reutlingen, Germany). Each
sample was quantified four times and the mean of the sample and
standard deviations (S.D.) were calculated by the cell counter.
WST-1 cell proliferation assay. Cell proliferation and viability were
analyzed using the WST-1 Cell Proliferation Reagent (4-[3-(4iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene
disulfonate) (Roche Diagnostics, Vienna, Austria) according to the
manufacturer’s instructions. The assay principle is based on cleavage
of a tetrazolium salt to a formazan by cellular enzymes, especially
mitochondrial dehydrogenases (24). The number of metabolically
active cells correlates directly to the amount of formazan.
The KRJ-I cells were cultured in 24-well plates for 24, 48 or 72 h
with different concentrations of solvent plant extract or DMSO as
negative control. After incubation, cell clusters were dispersed
carefully into single cells. After 100 μl suspension of KRJ-I cells had
been transferred into 96-well plates, 10 μl WST-1 cell proliferation
reagent was added to each sample well followed by 2 h incubation at
37˚C. Cell proliferation and viability were quantified by measuring
absorbance of the formazan product spectrophotometrically using a
microplate ELISA reader (Molecular Devices Corporation,
Sunnyvale, CA, USA) between 420 nm to 480 nm. The HF-SAR
cells were seeded directly into 96-well plates. After adherence, the
Svejda et al: Anticancer Activity of T. gracilis Extract on KRJ-I Cells
cells were treated with plant extracts for 24, 48, 72 or 96 h. The cell
viability was measured as described above and the mean of six
parallel samples and standard deviation were calculated.
DAPI (4’-6-diamidino-2-phenylindole) staining. This method of
identifying typical apoptotic alterations is based on the ability of 4’6-diamidino-2-phenylindole to form fluorescent complexes with
double-stranded DNA, showing fluorescence specificity for
adenosine-thymidine clusters. KRJ-I cells were cultured in 24-well
plates with different concentrations of plant extract or DMSO alone
as negative control for 24, 48 or 72 h. The cell suspensions were
centrifuged, overlap exhausted and the cell pellet washed with DAPI
methanol twice. The KRJ-I cells were then incubated with 2 ml
DAPI methanol (Sigma-Aldrich) for 15 min at room temperature.
After spinning down, the DAPI methanol was removed and the
remaining pellet was resuspended with PBSA, mounted on a
microscope slide and fixed with glycerin-aldehyde. The cells were
examined using an inverted phase-contrast fluorescence microscope
(Nikon eclipse TE300, Tokyo, Japan) with ultraviolet (UV)
excitation at 300-500 nm. Cells with condensed chromatin or
fragmented nuclei were considered as apoptotic.
Caspase -3/7, -6, -8, -9, and -2 activity assays. The caspase activities
were quantified using Promega luminescent assay kits (Promega,
Mannheim, Germany). KRJ-I cells were treated with different fractions
of plant extracts at a concentration of 10 μg/ml. Camptothecin (5 μM)
(25) was used as positive control. The samples were incubated at 37˚C
and caspase activities were measured every two or three hours for up
to 36 h. Each sample was suspended to disperse cell clusters into
single cells. Afterwards, 50 μl aliquots of cell suspension were
transferred into a 96-well white walled plate (Nunc, Roskilde,
Denmark) and Caspase-Glo Reagent was added at a ratio of 1:1. After
gently mixing on a plate shaker for 30 s, the samples were incubated
at room temperature (22˚C) for 1 h in the dark. Finally, luminescence
was measured with a luminometer (Mediators Phl; Mediators
Diagnostika GmbH, Vienna, Austria) according to the manufacturer’s
instructions. Each sample was quantified twice and the mean value and
standard deviations were calculated.
Since these caspases are characterized by similar substrate
specificities to those of caspase -3 and -7, which causes cross
reactions in the measurement, the specific caspase -3/7 inhibitor AcDEVD-CHO (Promega) (18) was added at a concentration of 10 nM,
5 nM and 60 nM for caspase -9, -6 and -2 measurements,
respectively (26, 27). The effect of the specific inhibitor was tested
on KRJ-I cells by evaluating caspase 3/7 activity with or without the
added inhibitor. For this purpose the KRJ-I cells were seeded into a
24 well plate and treated with 25 μg/ml plant extract. One group was
co-treated with specific caspase -3/7 inhibitor and compared to the
control group without the specific inhibitor. The caspase -3/7 activity
was measured as described above using the Promega luminescent
assay kit according to manufacturer’s instructions.
Transmission electron microscopy. KRJ-I and HF-SAR cells were
cultured in a 6-well plate and treated with TG-F28 10 μg/ml for
48 h at 37˚C, negative controls were treated with PBSA (control 1)
or DMSO (control 2). Additionally, the specific caspase -3/7
inhibitor Ac-DEVD-CHO (60 nM) was added to half of the treated
samples to inhibit apoptotic changes. Harvested cells were fixed in
3% glutaraldehyde in 0.1 M cacodylate buffer, ph 7.2, (Plano,
Wetzlar, Germany) on ice for 24 h. The cells were postfixed in 1%
Figure 1. Antiproliferative effects of T. gracilis extracts on KRJ-I cells.
Cell proliferation was evaluated using the CASY®-1 Cell Counter &
Analyzer. A: Subfractions of T. gracilis, TG-F25 and TG-F28. B:
Different concentrations of T. gracilis subfraction TG-F28; Control:
DMSO alone. All data are expressed as mean±SD.
osmium tetroxide (Sigma-Aldrich) in 0.1 M cacodylate buffer and
processed by modified routine methods. Samples were viewed and
photographed with a FEI Technai12™ (FEI Europe, Eindhoven, the
Netherlands) equipped with a Gatan CCD Camera Bioscan (Gatan,
Munich, Germany).
Treatment of carcinoid tumor xenografts in SCID mice with TG-F28.
In order to create transplantable tumors, 14-week-old female severe
combined immunodeficiency (SCID) mice (n=15) (Division of
Laboratory Animal Science and Genetics, Medical University of
Vienna, Himberg, Austria), were injected with 3×107 KRJ-I cells/
mouse suspended in 0.3 ml PBSA/mouse and transplanted
subcutaneously into the flanks (12). Tumors originated at the site of
injection, but no metastases were found. Fragments of these tumors
(3 mm diameter) were serially transplanted. After the tumor
diameters had reached approximately 10 mm, compounds were
injected intratumorally: a) 9 mice were injected with 100 μg/100 μl
TG28 dissolved in DMSO/PBSA per tumor, b) 5 mice were injected
with DMSO/ PBSA (control). Twice a week, the diameters of the
tumors were measured at three sites of each tumor by using a
sliding caliper and tumor volume was calculated, respectively. The
observation period was 21 days. The mice were killed at this time
for ethical reasons when the tumors grew extremely rapidly and
when necroses of the skin became apparent.
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ANTICANCER RESEARCH 30: 55-64 (2010)
Statistical evaluation. All the statistical analyses were performed
using Microsoft Excel and Prism 4 (GraphPad Software, San Diego,
CA, USA). Sigmoidal dose responses were calculated to identify
half-maximal inhibitory (IC50) concentration for the tested
compounds.
Results
Effects of T. gracilis on cell proliferation in KRJ-I cells. In
a preliminary screening, a DCM-extract of T. gracilis (TG5) as well as three fractions obtained by liquid-liquid
partition, TG-1, TG-2 and TG-3 were evaluated for their
antiproliferative potential by cell counting. A strong
antiproliferative effect had been noted from fraction TG-2
at a concentration of 25 μg/ml (data not shown) and 15
subfractions of crude TG-2 were then separated for further
investigation. To determine the most active fraction, each
subfraction was tested for antiproliferative effects at a
concentration of 10 μg/ml and 25 μg/ml (data not shown),
and with TG-F24, TG-F25 and TG-F28 the subfractions
with the most antiproliferative activity were identified and
studied in more detail at different plant extract
concentrations. KRJ-I cells treated with TG-F28 (7.5
μg/ml) and TG-F25 (7.5 μg/ml) reached a cell density of
4.7×105 cells/ml and 6.3×105 cells/ml after 72 h of
incubation, while a cell density of 9.3×105 cells/ml was
noted in the control cells treated with DMSO alone (Figure
1A). A dose-dependent antiproliferative effect was evident
in the KRJ-I cells treated with TG-F28 for 24, 48 and 72 h
(Figure 1B).
To confirm the antiproliferative effects, cell viability was
quantified using the WST-1 cell proliferation reagent. A
dose-dependent reduction in mitochondrial activity was
noted in the KRJ-I cells treated with TG-F24, TG-F25 and
TG-F28 at a concentration of 7.5 μg/ml (Figure 2A, 2B) and
the IC50 of the most active subfractions, TG-F24
(IC50=9.1×10–6) and TG-F28 (IC50=7.12×10–6), was
determined (Figure 2C).
Using light microscopy, alterations in KRJ-I cell
morphology were observed after 48 h of treatment with TGF24 (7.5 μg/ml) and TG-F28 (7.5 μg/ml). Besides a
dissociation of cell aggregation, an increase in both dead
cells and cell debris was noted in the treated KRJ-I cells
compared to the untreated control (Figure 3 A-F).
Induction of apoptosis in KRJ-I cells by T. gracilis TG-2
subfractions. To examine whether the apoptotic pathway was
involved, DAPI staining was performed in KRJ-I cells
treated with all fifteen subfractions. After 48 h treatment with
the plant extracts, chromatin condensation, nuclear pyknosis,
increased number of nuclear body fragments and irregular
edges around the nucleus were observed in treated KRJ-I
cells, while round, clear edged, uniformly stained cell nuclei
were noted in the untreated control (Figure 4 A-D).
58
Figure 2. Effects of T. gracilis extracts on KRJ-I cell viability quantified
using the WST-1 assay. A: T. gracilis subfractions TG-F24, TG-F25 and
TG-F28; control: DMSO alone. B: Dose-dependent effects of TG-F28;
control: DMSO alone. C: IC50 values of subfractions TG-F28
(IC50=7.12×10–6) and TG-F25 (IC50=9.1×10–6) quantified after 72 h
of treatment. All data are expressed as mean±SD.
Induction of caspase activity in KRJ-I cells by T. gracilis TG2 subfractions. To confirm the involvement of the apoptosis
pathway in inhibition of KRJ-I cell proliferation, activated
forms of the effector caspases 3, 6 and 7 and initiator
caspases 8, 9 and 2 were analyzed.
Caspase 3/7: Activated forms of caspase -3/7 were
evaluated in KRJ-I cells treated with the fifteen subfractions.
An increase of caspase -3/7 activity was noted in the KRJ-I
Svejda et al: Anticancer Activity of T. gracilis Extract on KRJ-I Cells
Figure 3. Effects of T. gracilis extracts on KRJ-I cell morphology. A-B: Control (DMSO alone): KRJ-I cells growing in clusters with only few single
cells, no dead cells and little cell debris. C-F: T. gracilis subfractions: KRJ-I cells treated with 7.5 μg/ml TG-F28 (C, D) or 7.5 μg/ml TG-F24 (E,
F) for 48 h (bar=50 μm).
Figure 4. Effects of T. gracilis extract TG-F28 on apoptosis in KRJ-I cells. Nuclear staining was performed after 48 h treatment using DAPI. A, B:
Control (DMSO alone): Round and clear edged cell nuclei. C, D: TG-F28 (10 μg/ml): Increased number of nuclear body fragments and irregular
edges around the nucleus were noted (bar=50 μm).
cells treated with TG-F24, TG-F25 and TG-F28 (10 μg/ml)
for 4, 8, 12 or 24 h compared to the untreated control (Figure
5). With TG-F28, the strongest subfraction was determined
and used for studying the time response of effector as well as
initiator caspases.
Time response of effector caspases: To evaluate the time
response of effector caspases, the activity of caspase -3/7
(10 μg/ml) and -6 (10 μg/ml) was determined every three h.
Activated caspase -3/7 was induced in the treated KRJ-I cells
with a maximum at 19 h; similar levels were noted compared to
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ANTICANCER RESEARCH 30: 55-64 (2010)
Figure 5. Effects of T. gracilis extracts on caspase -3/7 activity in KRJI cells determined by luminescence based assay. Positve control:
Camptothecin (5 μM); negative control: DMSO solvent alone.
CPT (5 μM) (Figure 6A). Additionally, caspase-6 was enhanced,
with a maximum increase at 25 h compared to the untreated
control in the presence of Ac-DEVD-CHO (18) (Figure 6B).
Time response of initiator caspases: An increase of
caspase-8 activity was noted in the KRJ-I cells treated with
TG-F28 (10 μg/ml) for 30 h, with a maximum at 22 h (data
not shown). Using the specific caspase -3/7 inhibitor AcDEVD-CHO, a similar increase in caspase-9 was observed,
with a maximum at 15 h (Figure 7A). TG-F28 induction of
caspases 8 and 9 showed similar levels compared to the
positive control, CPT. Additionally, activated caspase-2 was
evaluated, showing a maximum 1.7-fold increase at 24 h in
the presence of Ac-DEVD-CHO (Figure 7B).
Effects of T. gracilis subfraction TG-F28 on KRJ-I cells
compared to normal human fibroblasts. WST-1 assay and
Caspase -3/7 and -6 activities were quantified in KRJ-I and
HF-SAR cells, both treated with TG-F28 (10 μg/ml) for 24,
48 or 72 h. Treated HF-SAR cells showed no reduction of
cell viability and no induction of caspases 3, 6 and 7
compared to untreated control, whereas a decrease of cell
viability and increase in caspase activities was noted in the
treated KRJ-I cells (Figure 8A-C).
Electron microscopy. KRJ-I cells treated with TG-F28
(10 μg/ml) showed early apoptotic alterations, including
lobulated or deeply cleaved cell nuclei. Additionally, the
cytoplasm contained a large number of vacuoles (Figure 9A).
Treatment with TG-F28 (10 μg/ml) in combination with AcDEVD-CHO (60 nM) showed a lower degree of apoptosis.
Fewer vacuoles were noted and the nuclei were less impaired
compared to the TG-F28 treated KRJ-I cells without the
inhibitor (Figure 9B). The characteristic morphology of
round cells with oval or lobulated nuclei were observed in
PBSA treated KRJ-I cells used as control (Figure 9C).
Identical morphology was noted in KRJ-I cells treated with
60
Figure 6. Time-dependent activation of effector caspases 3/7 (A) and 6
(B) in KRJ-I cells, quantified by luminescence based assay with the
specific caspase -3/7 inhibitor Ac-DEVD-CHO. Positive control:
Camptothecin (5 μM), negative control: DMSO alone.
DMSO (Figure 9D). No modification in ultrastructure was
observed in TG-F28 (10 μg/ml) treated human fibroblast HFSAR cells (data not shown).
Treatment of carcinoid xenografts in SCID mice with TG-F28.
The tumor volumes were evaluated three times (day 0, 9 and
14) in TG-F28 (100 μg)-treated tumor-bearing SCID mice. A
significant inhibition of tumor growth was noted after 9 days
(74±22%) and 14 days (62±18%) of TG-F28 treatment
compared to the untreated control (Figure 10A). Differences
of the measured tumor volumes were calculated and a
significant decrease in tumor progression was noted between
day 0 to 9 (66±21%), day 9 to 14 (43±29%) and day 0 to 14
(55±19%) compared to the untreated control (Figure 10B).
Discussion
The predominant aims of analyzing crude plant extracts are
either to isolate bioactive agents for direct use as drugs or to
identify bioactive compounds that can be used as lead
Svejda et al: Anticancer Activity of T. gracilis Extract on KRJ-I Cells
Figure 7. Time-dependent activation of initiator caspases 9 and 2 in
KRJ-I cells with the specific caspase-3/7 inhibitor Ac-DEVD-CHO.
Positive control: Camptothecin (5 μM), negative control: DMSO alone.
A: Increase of activated caspase 9 with a maximum at 15 h (2.7-fold).
B: Caspase-2 levels in treated KRJ-I cells, with a maximum at 24 h (1.7fold), compared to untreated control.
substance in the preparation of semisynthetic drugs. In this
study, we demonstrate the anticancer potential of T. gracilis
subfractions in a well-characterized SI-NET cell line.
Tumor cell growth was inhibited by all 15 subfractions of
TG-2 in a dose-dependent manner. The fractions with the
strongest tumoristatic activity were the ursolic acid
containing subfraction TG-F24, TG-F25 and TG-F28.
As the cell’s intrinsic cell death program, apoptosis plays a
key role in growth control of cells and tissue homeostasis and
consequently, an imbalance or inactivation of important
pathways can result in tumor formation and progression (28).
Furthermore, cytotoxic therapies in anticancer treatment, e.g.
chemotherapy, γ-irradiation, immunotherapy or suicide gene
therapy, are mainly dependent on the function of cell apoptosis
(29-31) and many tumors develop different escape mechanisms
that subsequently result in drug resistance (32, 33). Therefore,
the induction and recovery of the apoptotic response in tumor
cells are relevant steps in anticancer treatment.
A complex cascade of cell signaling interactions is
involved in the induction of the apoptotic pathway of a cell,
while caspases – the main regulators of apoptotic cell death
(34, 35) – are the most viable approach to determine the
Figure 8. Comparison of T. gracilis effects on normal human fibroblast
HF-SAR and KRJ-I cells. The cells were treated with TG-F28 (10
μg/ml) or DMSO control. Cell viability was analyzed using WST-1
assay, activated caspase-3/7 and -6 were quantified using a
luminescence based assay. A: Decrease of KRJ-I cell viability after
treatment with TG-F28, while HF-SAR were not impaired. B, C:
Caspase-3/7 and caspase 6 activities were induced in treated KRJ-I
cells with a maximum at 24 h (4.1 fold, 2.8 fold), while no increase of
activated caspase-3/7 and -6 was observed in treated HF-SAR cells.
apoptotic changes (36). In the present study, caspase
activation was involved in the antiproliferative effects of the
T. gracilis extracts. The increase of the activated effector
caspases 3, 6 and 7 suggested an efficient induction of the
apoptotic pathway. To ensure that the apoptosis pathway
was functional in the KRJ-I cells, camptothecin, a known
initiator of apoptosis (25), was used as a positive control.
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ANTICANCER RESEARCH 30: 55-64 (2010)
Figure 9. Ultrastructure of T. gracilis-treated KRJ-I cells. A: TG-F-28 (10 μg/ml): Cleavage of the nucleus and the formation of many vacuoles,
indicating an early stage of apoptosis. B: TGF-28 (10 μg/ml) and the specific caspase-3/7 inhibitor Ac-DEVD-CHO (60 nM), showing a lower
number of vacuoles. C: Control treated with PBSA: Intact KRJ-I cell, characteristic oval nucleus with a large single nucleolus. D: Control treated
with DMSO: Morphology unimpaired and identical to the untreated control (C) (bar=2 μm).
Figure 10. Effects of T. gracilis (TG-F28 100 μg/ml) in carcinoid xenograft SCID mice. A: A significant inhibition of tumor growth was noted in tumor
bearing SCID mice after 9 and 14 d treatment with TG-F28. B: Differences of tumor growth within 9-, 5- and 14-day periods compared to untreated
control. Mean±SEM; N=13, *p<0.05.
To further define the mechanism of action of the plant
extracts, levels of the initiator caspases 8, 9 and 2 were
quantified. Initiator caspases are responsible for the initiation of
the caspase cascade and an extrinsic cell receptor mediated
62
pathway can be distinguished from an intrinsic pathway
triggered by the release of apoptogenic factors from intracellular
compartments (37). While ligation of death receptors, e.g.
CD95, TNF-related apoptosis-inducing ligand receptors
Svejda et al: Anticancer Activity of T. gracilis Extract on KRJ-I Cells
(TRAIL) and tumor necrosis factor receptors (TNF-Rs) (38,
39), is followed by activation of initiator caspase 8, the release
of mitochondrial apoptogenic factor (cytochrome c, the bcl-2
family) (40, 41), nuclear damage (42, 43) and endoplasmatic
reticulum stress (44) increase the activity of initiator caspase-9.
Both pathways converge at the proteolytic activation of effector
caspases 3, 6 and 7. The T. gracilis extracts increased activated
caspase-8, -9 and -2 levels in treated KRJ-I cells, suggesting
that the T. gracilis extracts induced the intrinsic pathway via
apoptogenic factors and caspase-9, although activity of
caspase-8 was increased. It is known that caspase-8 amplifies
the apoptotic signal by cleavage of the B-cell lymphoma 2
family member bid (45, 46). As a result, mitochondrial
cytochrome c is released followed by the formation of the
apoptosome, a complex consisting of cytochrome c/ apoptotic
peptidase activating factor 1/ caspase-9. Interestingly, an
increase of caspase-8 was noted in tumor cells after treatment
with camptothecin, which is known to induce the intrinsic
apoptosis pathway via caspase 9 by inhibition of topoisomerase
I (25). Because of a close homology of the specific caspase-8
substrate and the caspase-3/7 inhibitor Ac-DEVD-CHO (26),
active caspase-8 was quantified without the caspase 3/7
inhibitor. These findings suggested an unspecific cross-reaction
of effector caspases 3 and 7 in the measurement of caspase-8.
An ideal anticancer drug should specifically target the
malignant cells while non-malignant cells in the body should
not be impaired. While this is unfortunately rarely achieved,
the present data demonstrated that a pro-apoptotic effect of
the plant subfractions was only noted in the tumor cells
without impairing the normal HF-SAR cells. These findings
suggested a tumor-specific effect.
Ultrastructural observations confirmed the selective induction
of apoptosis in the treated KRJ-I cells, while the two control
groups with PBSA- or DMSO-treatment remained unimpaired.
Especially, the cleavage of the nuclei and the accumulation and
increase of vacuoles indicated the induction of apoptosis in the
TG-F28 treated group. The apoptotic alterations were partially
inhibited by the addition of the specific inhibitor Ac-DEVDCHO. Interestingly, a complete “restitutio ad integrum” was not
noted. This implied that another cellular pathway might be
involved in the antiproliferative effect induced by the
subfractions of T. gracilis.
The TG-F28 treatment of carcinoid xenograft SCID mice
showed a significant reduction of tumor growth compared
to the untreated controls. These in vivo data implicated a
strong antiproliferative effect of T. gracilis subfraction TGF28 and confirmed the in vitro findings in the SI-NET cell
line KRJ-I.
In conclusion, inducing apoptosis by novel bioactive
compounds of T. gracilis reduces tumor cell proliferation in
a dose-dependent way without impairing normal cells. Thus,
extracts of T. gracilis could potentially be a new therapeutic
option in anticancer treatment for SI-NETs.
Acknowledgements
The investigation was supported by the Austrian Cancer Aid/ Styria
(EF 01/2004) and the Franz Lanyar Foundation (Project # 334). We
thank Maria-Theresia Hammer for carefully reading the manuscript.
References
1 Toni R: The neuroendocrine system: organization and
homeostatic role. J Endocrinol Invest 27: 35-47, 2004.
2 Modlin IM, Lye KD and Kidd M: A 5-decade analysis of 13,715
carcinoid tumors. Cancer 97: 934-959, 2003.
3 Modlin IM, Oberg K, Chung DC, Jensen RT, de Herder WW,
Thakker RV, Caplin M, Delle Fave G, Kaltsas GA, Krenning EP,
Moss SF, Nilsson O, Rindi G, Salazar R, Ruszniewski P and
Sundin A: Gastroenteropancreatic neuroendocrine tumours.
Lancet Oncol 9: 61-72, 2008.
4 Modlin IM, Kidd M, Latich I, Zikusoka MN and Shapiro MD:
Current status of gastrointestinal carcinoids. Gastroenterology
128: 1717-1751, 2005.
5 Modlin IM, Latich I, Kidd M, Zikusoka M and Eick G:
Therapeutic options for gastrointestinal carcinoids. Clin
Gastroenterol Hepatol 4: 526-547, 2006.
6 Lee KH: Research and future trends in the pharmaceutical
development of medicinal herbs from Chinese medicine. Public
Health Nutr 3: 515-522, 2000.
7 Raskin I, Ribnicky DM, Komarnytsky S, Ilic N, Poulev A, Borisjuk
N, Brinker A, Moreno DA, Ripoll C, Yakoby N, O’Neal JM,
Cornwell T, Pastor I and Fridlender B: Plants and human health in
the twenty-first century. Trends Biotechnol 20: 522-531, 2002.
8 Cragg GM and Newman DJ: Discovery and development of
antineoplastic agents from natural sources. Cancer Invest 17:
153-163, 1999.
9 Cragg GM, Newman DJ and Snader KM: Natural products in
drug discovery and development. J Nat Prod 60: 52-60, 1997.
10 Kölby L, Bernhardt P, Ahlman H, Wangberg B, Johanson V,
Wigander A, Forssell-Aronsson E, Karlsson S, Ahren B,
Stenman G and Nilsson O: A transplantable human carcinoid as
model for somatostatin receptor-mediated and amine transportermediated radionuclide uptake. Am J Pathol 158: 745-755, 2001.
11 Van Buren G II, Rashid A, Yang AD, Abdalla EK, Gray MJ, Liu
W, Somcio R, Fan F, Camp ER, Yao JC and Ellis LM: The
development and characterization of a human midgut carcinoid
cell line. Clin Cancer Res 13: 4704-4712, 2007.
12 Pfragner R, Wirnsberger G, Niederle B, Behmel A, Rinner I,
Mandl A, Wawrina F, Luo J, Adamiker D, Höger H, Ingolic E
and Schauenstein K: Establishment of a continuous cell line
from a human carcinoid of the small intestine (KRJ-I):
characterization and effects of 5-azacytidine on proliferation. Int
J Oncol 8: 513-520, 1996.
13 Pfragner R, Behmel A, Höger H, Beham A, Ingolic E, Stelzer I,
Svejda B, Moser VA, Obenauf AC, Siegl V, Haas O and Niederle
B: Establishment and characterization of three novel cell lines P-STS, L-STS, H-STS - derived from a human metastatic midgut
carcinoid. Anticancer Res 29: 1951-1961, 2009.
14 Kidd M, Eick GN, Modlin IM, Pfragner R, Champaneria MC and
Murren J: Further delineation of the continuous human neoplastic
enterochromaffin cell line, KRJ-I, and the inhibitory effects of
lanreotide and rapamycin. J Mol Endocrinol 38: 181-192, 2007.
63
ANTICANCER RESEARCH 30: 55-64 (2010)
15 Itokawa H, Morris-Natschke SL, Akiyama T and Lee KH: Plantderived natural product research aimed at new drug discovery.
Nat Med (Tokyo) 62: 263-280, 2008.
16 Li ZX, Stuppner H, Schraml E, Moser VA, Siegl V and Pfragner
R: The dichloromethane fraction of Stemona tuberosa Lour
inhibits tumor cell growth and induces apoptosis of human
medullary thyroid carcinoma cells. Biologics 1: 455-463, 2007.
17 Rinner B, Siegl V, Purstner P, Efferth T, Brem B, Greger H and
Pfragner R: Activity of novel plant extracts against medullary
thyroid carcinoma cells. Anticancer Res 24: 495-500, 2004.
18 Li ZX, Sturm S, Svejda B, Höger H, Schraml E, Ingolic E, Siegl
V, Stuppner H and Pfragner R: Anticancer activity of novel
extracts from Cautleya gracilis (Smith) Dandy: apoptosis in
human medullary thyroid carcinoma cells. Anticancer Res 28:
2705-2713, 2008.
19 Rinner B, Sturm S, Stuppner H, Siegl V and Pfragner R: Use of
cell lines to define new bioassays for the therapy of medullary
thyroid carcinoma. Ann Endocrinol 76: P29, 2006.
20 Handel-Mazzetti H: Naturbilder aus Südwest-China Erlebnisse
und Eindrücke eines österreichischen Forschers während des
Weltkrieges. Wien - Leipzig Österreichischer Bundesverlag pp.
380, 1927.
21 Pfragner R, Höfler H, Behmel A, Ingolic E and Walser V:
Establishment and characterization of continuous cell line MTCSK derived from a human medullary thyroid carcinoma. Cancer
Res 50: 4160-4166, 1990.
22 Pfragner R WG, Behmel A, Wolf G, Passath GA, Ingolic E
Adamiker D New continuous cell line from human medullary
thyroid carcinoma: SINJ. phenotypic analysis and in vivo
carcinogenesis. Int J Oncol 2: 831-836, 1993.
23 Wolf C, Lederer K, Pfragner R, Schauenstein K, Ingolic E and
Siegl V: Biocompatibility of ultra-high molecular weight
polyethylene (UHMW-PE) stabilized with alpha-tocopherol used
for joint endoprotheses assessed in vitro. J Mater Sci Mater Med
18: 1247-1252, 2007.
24 Mosmann T: Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays.
J Immunol Methods 65: 55-63, 1983.
25 Capranico G, Ferri F, Fogli MV, Russo A, Lotito L and
Baranello L: The effects of camptothecin on RNA polymerase II
transcription: roles of DNA topoisomerase I. Biochimie 89: 482489, 2007.
26 Garcia-Calvo M, Peterson EP, Leiting B, Ruel R, Nicholson DW
and Thornberry NA: Inhibition of human caspases by peptidebased and macromolecular inhibitors. J Biol Chem 273: 3260832613, 1998.
27 O’Brien MA, Daily WJ, Hesselberth PE, Moravec RA, Scurria
MA, Klaubert DH, Bulleit RF and Wood KV: Homogeneous,
bioluminescent protease assays: caspase-3 as a model. J Biomol
Screen 10: 137-148, 2005.
28 Hengartner MO: The biochemistry of apoptosis. Nature 407:
770-776, 2000.
29 Herr I and Debatin KM: Cellular stress response and apoptosis
in cancer therapy. Blood 98: 2603-2614, 2001.
64
30 MacKenzie SH and Clark AC: Targeting cell death in tumors by
activating caspases. Curr Cancer Drug Targets 8: 98-109, 2008.
31 Brown JM and Attardi LD: The role of apoptosis in cancer
development and treatment response. Nat Rev Cancer 5: 231237, 2005.
32 Persidis A: Cancer multidrug resistance. Nat Biotechnol 17: 9495, 1999.
33 Yang XH, Sladek TL, Liu X, Butler BR, Froelich CJ and Thor
AD: Reconstitution of caspase 3 sensitizes MCF-7 breast cancer
cells to doxorubicin- and etoposide-induced apoptosis. Cancer
Res 61: 348-354, 2001.
34 Lawen A: Apoptosis – an introduction. Bioessays 25: 888-896,
2003.
35 Lamkanfi M, Declercq W, Kalai M, Saelens X and Vandenabeele
P: Alice in caspase land. A phylogenetic analysis of caspases
from worm to man. Cell Death Differ 9: 358-361, 2002.
36 Kumar S: Caspase function in programmed cell death. Cell
Death Differ 14: 32-43, 2007.
37 Chen M and Wang J: Initiator caspases in apoptosis signaling
pathways. Apoptosis 7: 313-319, 2002.
38 Ashkenazi A and Dixit VM: Death receptors: signaling and
modulation. Science 281: 1305-1308, 1998.
39 Ashkenazi A, Holland P and Eckhardt SG: Ligand-based
targeting of apoptosis in cancer: the potential of recombinant
human apoptosis ligand 2/tumor necrosis factor-related
apoptosis-inducing ligand (rhApo2L/TRAIL). J Clin Oncol 26:
3621-3630, 2008.
40 Festjens N, van Gurp M, van Loo G, Saelens X and Vandenabeele
P: Bcl-2 family members as sentinels of cellular integrity and role
of mitochondrial intermembrane space proteins in apoptotic cell
death. Acta Haematol 111: 7-27, 2004.
41 Zamzami N and Kroemer G: The mitochondrion in apoptosis:
how Pandora’s box opens. Nat Rev Mol Cell Biol 2: 67-71, 2001.
42 Ewald B, Sampath D and Plunkett W: Nucleoside analogs:
molecular mechanisms signaling cell death. Oncogene 27: 65226537, 2008.
43 Sordet O, Khan QA, Kohn KW and Pommier Y: Apoptosis
induced by topoisomerase inhibitors. Curr Med Chem
Anticancer Agents 3: 271-290, 2003.
44 Rao RV, Ellerby HM and Bredesen DE: Coupling endoplasmic
reticulum stress to the cell death program. Cell Death Differ 11:
372-380, 2004.
45 Li H, Zhu H, Xu CJ and Yuan J: Cleavage of BID by caspase 8
mediates the mitochondrial damage in the Fas pathway of
apoptosis. Cell 94: 491-501, 1998.
46 Luo X, Budihardjo I, Zou H, Slaughter C and Wang X: Bid, a
Bcl2 interacting protein, mediates cytochrome c release from
mitochondria in response to activation of cell surface death
receptors. Cell 94: 481-490, 1998.
Received July 9, 2009
Revised November 13, 2009
Accepted November 23, 2009