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 55 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. 57 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 59 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. 61 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