Antineoplastic cyclic astin analogues kill tumour cells via caspase-mediated induction of apoptosis
Rosanna Cozzolino1,
Pasquale Palladino1,2,
Filomena Rossi1,2,
Gaetano Calì3,
Ettore Benedetti1,2 and
Paolo Laccetti1,*
1 Department of Biological Chemistry and 2 CIRPEB and IBB-CNR, University of Naples Federico II, Via Mezzocannone 16, 80134 Naples, Italy and 3 CNR-IEOS c/o Department of Cellular and Molecular Biology and Pathology L. Califano, University of Naples Federico II, Via Pansini 5, 80131 Naples, Italy
* To whom correspondence should be addressed at: Department of Biological Chemistry, University of Naples Federico II, Via Mezzocannone 16, 80134 Naples, Italy. Tel: +39 081 253 4587; Fax: +39 081 552 1217; Email: paolo.laccetti{at}unina.it
 |
Abstract
|
---|
Astins, a family of cyclopentapeptides, isolated from the roots of a medicinal plant Aster tataricus (Compositae), show antitumour activity. Their chemical structures consist of a 16-membered ring system containing a unique ß,
-dichlorinated proline [Pro(Cl2)], other non-coded amino acid residues, and a cis conformation in one of the peptide bonds. The ß,
-dichlorinated proline residue is considered to play an important role in their antineoplastic activities in vitro on nasopharynx carcinoma (KB) cells and in vivo on sarcoma 180 ascites and P388 lymphocytic leukaemia in mice. The acyclic astins without Pro(Cl2) do not show antitumour activity against S-180 ascites in vivo, suggesting that the cyclic nature of astins plays an important role in their antitumour activities. We synthesized new astin-related cyclopeptides differing from the natural product for the presence of some non-proteinogenic amino acid residues: Aib, Abu, -S(ß3)-hPhe and a peptide bond surrogate (SO2NH) and we tested for their antitumour effect. We observed cytotoxic effects of the newly synthesized cyclic astins, but not with the acyclic analogue astins. We also observed that the cyclic astin induced apoptosis in a human papillary thyroid carcinoma cell line (NPA cell line) and that apoptotis was associated with activation of caspases. The caspase family inhibitor, ZValAsp(OMe)FMK, protected NPA cells from cyclic analogue astin-induced apoptosis. To determine which caspase was specifically activated, we assayed caspase activity in astin-treated cells in the presence of specific caspase and 8, 9 or 3 inhibitors, i.e. ZIETDFMK, ZLEHDFMK ZDEVDFMK, which inhibit caspases 8, 9 and 3, respectively. The data presented here show selective antineoplastic properties of the newly synthesized cyclic astins, and suggest, for the first time, a mechanism for their antineoplastic action through the activation of apoptotic pathway.
Abbreviations: AFC, 7-amino-4-trifluoromethyl-coumarin; DMSO, dimethyl sulfoxide; FITC, fluorescein isothiocyanate; FMK, fluoromethylketone; Hoechst 33258, 2'(4Hydroxyphenyl) 5-(4-methyl-1-piperazynil) 2,5'-bi (1H-benzimidazol)-6-(1-methyl-4piperazyl)benzimidazole trihydrochloride; MTT, 3-(4,5-dimethyltiazol-z-yl) 2,5-diphenyltetrazolium bromide (thiazolyl blue); PI, propidium iodide; Z-DEVD-FMK, Z-Asp-(OMe)-Glu-(OMe)-Val-Asp(OMe)-fluoromethylketone; Z-IETD-FMK, Z-Ile-Glu-Thr-Asp-(OMe)-fluoromethylketone; Z-LEHD-FMK, Z-Leu-Glu-(OMe)-Asp-(OMe)-fluoromethylketone; Z-VAD-FMK, Z-Val-Asp-(OMe)-fluoromethylketone; Pro(Cl2), ß,
-dichlorinated proline
 |
Introduction
|
---|
The possibility to develop new drugs effective on cancer is one of the main problems connected with cancer therapy. The development of new tumour specific drugs is therefore greatly needed.
Astins, A-I, are a family of natural antitumour cyclopentapeptides isolated from the roots of Aster tataricus (Compositae) and characterized by a 16-membered ring system containing a unique ß,
-dichlorinated proline [Pro(Cl2)] and the presence of several uncoded amino acid residues. The antitumour activity of astins was first observed, several years ago, by Morita and coworkers (1,2). The astin backbone conformation together with a cis 3,4-dichlorinated proline residue has been considered to play an important role in the antineoplastic activity in vitro, on nasopharynx carcinoma (KB) cells, and in vivo on sarcoma 180A and P388 lymphocytic leukaemia (3,4). The acyclic astins without Pro(Cl2) did not show antitumour activity against S-180 ascites cells in vivo (2,4), suggesting that the cyclic nature of astins plays an important role in the antitumour activities (5). Although the antineoplastic activity of the natural astins has been screened both in vitro and in vivo, their mechanism of action has never been investigated. In order to improve our knowledge of the structural and conformational properties influencing the bioactivity in this class of compounds, we synthesized new astin-related cyclopeptides differing from the natural product in the presence of non-proteinogenic amino acid residues and a peptide bond surrogate (SO2NH), and analysed for their antineoplastic activity. We have shown earlier that the antitumour activity of one of them, astin 3, is comparable in vitro with that of natural astins A and B used by Morita and coworkers (1,2) when tested on a papillary human thyroid tumour cell line (NPA) (6).
In the present study we have extended these observations to several human tumour cell lines of different origin and degree of malignancy, and also investigated the mechanism used by the astins to kill the cells.
It is well documented that most anticancer agents induce apoptosis, and it has been observed that several peptides (7), in their antineoplastic activity, promote apoptosis. A series of enzymes known as caspases are involved in apoptotic cell death (811). Caspases are cysteine proteases that are expressed as inactive pro-enzymes and are normally classified as signalling or effector caspases (12,13). Caspases are synthesized as relatively inactive zymogens that become activated by scaffold-mediated transactivation or by cleavage upstream proteases in an intracellular cascade (11).
The data presented show, for the first time, that the antitumour effect of the cyclic astins depends on the activation of the apoptotic machinery by the subsequent activation of signalling caspases 8 and 9 and consequently activation of effector caspase 3. We determined the specificity of caspase induction by measuring their activity in the presence of selective inhibitors and showing that the caspase family inhibitor protected cells from apoptotic death.
 |
Materials and methods
|
---|
Astins
We synthesized cyclic analogue astins and linear analogue astin as previously described (6). Natural astins were kindly provided by Dr Morita.
Reagents
Culture media and supplements were purchased from Life Technologies (Ponsley, UK), foetal calf serum from Hyclone Lab (Logan, UT, USA), Trypan blue from Flow Laboratories (Irvine, Scotland), MTT, Hoechst 33342, and Etoposide from Sigma Chemical Company (St Louis, MO, USA). Caspase 3 and caspase 8 fluorometric assay kits were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), caspase 9 fluorometric assay kit, AFC, FMK negative control, and inhibitors: Z-VAD-FMK, Z-IETD-FMK, Z-LEHD-FMK and Z-DEVD-FMK from Alexis Biochemicals (San Diego, CA, USA) and Death Detection Elisa Kit Plus from Roche Diagnostics (Mannheim, Germany), respectively.
Cell lines
NPA and ARO cell lines, a gift of Dr A.Fusco, derived respectively from human papillary and anaplastic thyroid carcinomas are tumorigenic when injected into nude mice (14,15). SK-OV-3 cell line, a gift of Dr C.De Lorenzo, was derived from human ovary adenocarcinoma (16). All these cell lines were grown in the Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% foetal calf serum containing glutamine (2 mM), streptomycin (50 U/ml) and penicillin (50 U/ml). The SK-BR-3, human breast carcinoma cell line (16) and A-431, human epidermoid carcinoma cell line (16), kindly provided by Dr C.De Lorenzo, were cultured in RPMI 1640, supplemented with 10% foetal calf serum containing glutamine (2 mM), streptomycin (50 U/ml) and penicillin (50 U/ml).
Cell viability assays
For cell viability analysis, tumour cell lines (NPA, ARO, SK-BR-3, SK-OV-3, A-431) were seeded in 96-well plates (5 x 103/well in 150 µl of medium) and incubated for different times in the presence or absence of astin 3. Astin was added (to a final concentration of 106 nM) to the culture medium 24 h after plating, and the cells were incubated for an additional 24, 48 and 72 h period. At the end of the treatments, a colorimetric assay based on the ability of live, but not dead, tumour cells to reduce a tetrazolium base compound (MTT) to a blue formazan product was used (17,18). MTT was added and the mixture was allowed to incubate for 4 h. During this period, precipitation of tetrazolium salts was observed under the microscope. Finally, to solubilize the formazan crystals, DMSO (100 µl for each well) was added and allowed to incubate for 5 min. All experiments were performed in triplicate. The values, measured in microplate reader (Biorad, CA, USA), are the mean of three different experiments. The percentage of survival was defined as the reduction in optical density in each test. Fractional absorbance was calculated using the formula [(mean absorbance in three test wells absorbance in background well)/(mean absorbance in three test control wells absorbance in background well)] (19).
Cell counts were determined in triplicate. Cell viability was routinely checked by the trypan blue exclusion test. A 50% cell suspension was mixed with 50% trypan blue isotonic solution. Viable cells cannot absorb the dye that is instead absorbed by the dead cells. All the cells were transferred and counted with a haemocytometer under a microscope. Cell survival was expressed as the percentage of viable cells in the presence of peptide under test with respect to control cultures grown in the absence of the peptide.
Detection of apoptosis
Apoptotic cells are associated with phosphatidylserine exposure (20). Apoptotic and necrotic cells were distinguished using the annexin V-FITC/PI staining assay (21). NPA cells were plated in 6-well plates (106/well in 2 ml of medium) and 24 h later astin 3 or etoposide (VP-16) (50 µM) was added as a positive control (22,23). After an additional 24 h, cells were washed twice with annexin V-binding buffer containing 140 mM NaCl, 5 mM CaCl2, 10 mM HEPES, pH 7.4, resuspended in 1 ml of the same buffer, and incubated in ice for 30 min with 2 µl of 140 nM annexin V-FITC. At the end of the incubation, 5 µl of PI (50 mg/ml) was added to each sample and analysed by flow cytometry (Becton Dickinson, San Jose, CA, USA).
For apoptotic analyses, 104 NPA cells were plated in triplicate on different cover glasses inserted in separate wells of a 24-well plate; 24 h after plating, cyclic astin 3 (106 nM) and linear astin 4 (106 nM) were added. After 24, 48 and 72 h of additional treatment, the medium was removed, the cells fixed for 20 min in 4% formaldehyde, washed in phosphate-buffered saline (PBS) and permeabilized for 5 min in 0.1% Triton X-100. The cells then were washed with PBS and stained for 30 min with Hoecht 33258 immunofluorescent reagent at a concentration of 0.5 µg/ml in PBS. The stained cells were observed under a Zeiss Axiophot microscope and photographed.
To analyse the time dependency of apoptotic DNA fragmentation, NPA cells were plated in 96-well plates (103/well in 100 µl medium) and 24 h later, astin 3 was added to the culture medium (106 nM) and incubated for an additional 24, 48 and 72 h. Etoposide was used as a positive control. Apoptosis was measured in triplicate samples using The Cell Death Detection Elisa Plus Kit. This photometric sandwich enzyme-immunoassay determines cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes) upon induction of cell death. During incubation, the antihistone antibody binds to the histone component of the apoptotic nucleosomes and links the immunocomplex to the steptavidin-coated plates through its biotinylation. The amount of nucleosome material by peroxidase activity was determined spectrophotometrically at 405 nm with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic-acid) (ABTS) as a substrate (Microplate reader, Biorad, Milano, Italy).
Determination of caspase activation
Cells were treated with cyclic and linear analogue astins for various time durations. At the end of each treatment, activities of caspase 3, 8, and 9 were tested with fluorometric protease assay kits according to the manufacturer's instructions. Briefly, after induction of apoptosis, NPA cells were harvested and collected by centrifugation. The pelleted cells were taken up in lysis buffer. Lysates were incubated for 1 h at 37°C with the specific fluorescent substrate. The caspase 3, 8 and 9 kits assay the activity of caspase 3, which recognizes the AspGluValAsp (DEVD) sequence, the activity of caspase 8, which recognizes the IleGluThrAsp (IETD) sequence, and the activity of caspase 9, which recognizes the LeuGluHisAsp (LEHD) sequence, respectively. Fluorescence derived from release of AFC was followed using a spectrofluorometer at 400 nm excitation and 505 nm emission.
Caspase inhibitors
Caspase inhibitors irreversibly bind to the caspase active sites because the peptide recognition sequence is linked to a fluoromethylketone (FMK). A dose of 50 µM ZValAlaAsp(OMe) fluoromethylketone (ZVADFMK, added every 24 h) was used as a general upstream caspase inhibitor for DNA fragmentation assay. A dose level of 10 µM, ZIETDFMK, ZLEHDFMK and ZDEVDFMK, was used as caspase 8, 9 and 3 selective inhibitors, respectively. All samples were analysed with a spectrofluorometer at 400 nm excitation and 505 nm emission.
 |
Results
|
---|
Cytotoxic effect of cyclic astin 3
The antitumour effect of different classes of natural astins (chlorine- and Thr-containing cyclic pentapeptides) has been previously observed (2) but the mechanism of action has never been evaluated.
We synthesized novel analogue astins, similar to the natural products, to better define the structural and conformational properties influencing the bioactivity and analysed them for their antineoplastic activity. The cyclic astin analogues exerted antineoplastic activity whereas the linear astins did not show any activity (5), confirming that the cyclic conformation is crucial for their antineoplastic effect. Among the different novel astins the most effective was astin 3 (or peptide III) in which the carbonyl in the HPhe-Abu peptide bond was replaced by a SO2 group showing an increased backbone flexibility (6) as shown in Figure 1.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 1. Structure of cyclic synthetic astin 3: c[ProAbuSer(S) ß3-hPhe (CH2SO2NH)Abu] (A), structure of natural astin B: c[ProCl2ThrSer(S)ß3PheAbu] (B).
|
|
Figure 2 shows the antiproliferative effect exerted, in vitro, by astin 3 on several human neoplastic cell lines of different origin and different degree of malignancy, when compared with untreated control cells at the same point. As shown, the antitumour effect of astin 3 resulted in 4879% of cell growth inhibition depending on the tumour cell line analysed.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 2. Cytotoxic effect of astin 3 estimated by the MTT assay. NPA (black triangles), ARO (white triangles), SK-BR-3 (black circles), SK-OV-3 (white circles), A-431 (white squares) cell lines were incubated for the indicated time duration with astin 3. The percentage of cell survival was determined by MTT assay as discussed in Materials and methods. Each value represents the mean (±SD) of triplicate determinations in three independent experiments.
|
|
To better define the antitumour effect of the astins, cell viability analysis has been performed on the same cell lines comparing the effect of cyclic versus linear astins. Figure 3 shows that linear astin 4 did not inhibit the survival of NPA cell line. On the other hand, cyclic natural astin B or newly synthesized astin 3 significantly inhibited survival and growth. The cytotoxic effect has been evaluated using the trypan blue exclusion analysis. Basically, similar results were observed using, in parallel, the MTT assay (data not shown).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 3. Effect of cyclic and linear astins estimated by trypan blue analysis. NPA cells were treated for 72 h with cyclic astin 3 (white circles) or linear astin 4 (black circles) at the indicated concentrations, and cell survival was determined by hemocytometric cell count after trypan blue staining. Natural astin B (black triangles) was used as a positive control. The data shown represent the mean (±SD) of triplicate determinations of three different experiments.
|
|
Cyclic astin 3 induces apoptosis and a general caspase inhibitor protects thyroid carcinoma cells from cell death
Since the mechanism of antineoplastic action of astins has never been investigated, and since it is well documented that most anticancer agents induce apoptosis, we investigated the action of our newly synthesized astins on modulating apoptosis. To identify the mode of cell death, we treated NPA cells with cyclic astin 3, linear astin 4 and natural astin B. Etoposide was used as a positive control because it has been reported to facilitate apoptosis (22). Figure 4 shows the effect of annexin V-FITC/PI that discriminates between apoptotic and necrotic cells; annexin V binds only the phosphatidylserine of apoptotic cells. The apoptotic cell count with astin 3 and natural astin B after 24 h treatment is comparable with the count observed after treatment with etoposide, whereas the apoptotic cell count after the ineffective linear astin 4 treatment is similar to that of untreated control cells.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 4. Tumour cells sensitized to apoptosis. NPA cells were incubated for 24 h with cyclic astin 3 (black bar), linear astin 4 (grey bar) and natural astin B (white bar with horizontal stripes), and the percentage of apoptotic cells was determined with Annexin V-FITC and PI as described in Materials and methods, and analysed by flow cytometry. Untreated (white bar) and etoposide (white bar with vertical stripes) treated cells were used as a negative and a positive control, respectively. Each value represents the mean (± SD) of triplicate determinations of three independent experiments.
|
|
To confirm that cell death was attributable to activation of apoptosis pathways, we treated NPA cells with astins 3 and 4, using again etoposide as a positive control. Cells were examined for the presence of apoptotic nuclear bodies by fluorescence microscopy using Hoechst 33258 staining. A clear induction of apoptosis was observed in NPA cells treated with cyclic astin 3, as revealed after 24 and 48 h of treatment in the form of a large number of nuclei with apoptotic bodies (Figure 5B and C). In contrast, no sign of apoptosis was detectable in untreated cells (negative control, Figure 5A) or in NPA cells treated with linear astin 4 (data not shown). A similar degree of apoptotic cells was observed for NPA cells treated with etoposide, which was employed as a positive control (Figure 5D and E).

View larger version (46K):
[in this window]
[in a new window]
|
Fig. 5. Micrographs representing nuclear change in NPA cells. NPA cells exposed for 24 and 48 h to astin 3 were fixed and stained with Hoechst 33258 immunofluorescent reagent to reveal nuclear signs of apoptosis. Normal morphology of untreated cells (A) was used as a negative control. The data show apoptotic bodies from NPA cells treated with astin 3 for 24 h and 48 h (B and C) and, as a positive control, the effects of induction of apoptosis with etoposide (50 µM) (D and E) were evalutated. Three experiments were performed with similar results. Magnification 1:630.
|
|
To measure apoptosis we also used an ELISA-based assay. As shown in Figure 6, apoptotic nucleosomes were detected after treatment of NPA cells with cyclic astin 3 or with etoposide as a positive control; the maximum effect occurred 48 h after the treatment with etoposide or astin 3. If astin 3 induced a specific apoptosis, then DNA fragmentation should be partially or fully prevented in cells cotreated with astin 3 and caspase family inhibitors. To address this question, we cotreated NPA cells with cyclic astin 3 and ZVADFMK, a general upstream inhibitor of caspases. The inhibitor was replenished daily when the treatment exceeded 24 h. ZVADFMK fully decreased astin 3 or etoposide induced DNA fragmentation showing that the caspase family inhibitor protected NPA treated cells from apoptotic death.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 6. DNA fragmentation of NPA papillary thyroid carcinoma cells. NPA carcinoma cells were untreated or treated with astin 3 for the indicated time duration without or with a general upstream caspase inhibitor ZVADFMK. Etoposide was used as a positive control. At the end of each treatment, DNA fragmentation was measured with a Cell Death detection Elisaplus Kit as reported in Materials and methods. The results are the mean (±SD) of triplicate determinations of three independent experiments.
|
|
Astin 3 triggers caspase cascades in NPA cell line
Since apoptosis is executed by caspases, to assess its role in astin-induced apoptosis, we analysed the kinetics of caspase activation using substrates specific for caspases 8, 9, and 3. In these assays the spectrophotometer detection of AFC fluorophore, cleaved from the specific synthetic substrate, is a measure of caspase activity. Caspase 8 activity increased at 6 h and peaked at 24 h. Its activity declined thereafter and reached the baseline at 48 h, as shown in Figure 7A. Caspase 9 increased slowly at 6 h and peaked at 36 h and this protease decreased progressively until 72 h, as shown in Figure 7B. Caspase 3 activity consistent with its downstream role, increased slowly at 24 h, peaked at 48 h and declined drastically at 72 h, as shown in Figure 7C. No caspase activation was detected with untreated cells (control) or in cells treated with linear astin 4 (data not shown). These results suggest that apoptosis, induced by cyclic astin 3, in the NPA cells, is associated with activation of the caspase pathway.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 7. Caspase activation. (A) Time course (3, 6, 12, 24, 36, 48, 72 h) of caspase 8; (B) of caspase 9 and (C) of caspase 3 substrate hydrolysis in cells exposed to astin 3. Enzymatic activity of caspases was determined by protease assays using the specific chromogenic substrates as reported in Materials and methods. The release of the chromophore AFC was monitored spectrofluorometrically at 505 nm. The data shown represent (±SD) triplicate determinations of three different experiments.
|
|
To further assess the specificity of caspases in cyclic astin-induced apoptosis, selective inhibitors of the three caspases were used as a control of specificity. As shown in Figure 8 the specific caspase inhibitors ZIETDFMK, ZLEHDFMK and ZDEVDFMK stopped the activation of caspases 8, 9, and 3 respectively.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 8. Action of caspase inhibitors. Caspase activity was measured in the presence of caspase 8 inhibitor ZIETDFMK at 24 h (A), caspase 9 inhibitor ZLEHDFMK at 36 h (B), or caspase-3 inhibitor ZDEVDFMK at 48 h (C). Untreated cells were used as a negative control. Each point represents the mean (±SD) of triplicate determinations of three independent experiments.
|
|
To determine whether caspase-8 activated procaspase 9 directly, we blocked the activation of caspase 8 by specific ZIETDFMK inhibitor and evaluated the activation of both caspases 9 and 3. As shown in Figure 9 (A and B), inhibition of caspase 8 prevented activation of caspase 9 or 3, indicating that caspase 8 was upstream of caspases 9 and 3.

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 9. Caspase 9 (A) and 3 (B) activity were measured in the presence of caspase 8 inhibitor ZIETDFMK, respectively at 36 and 48 h. Caspase 8 (C) and 3 (D) activity were determined in the presence of caspase 9 inhibitor ZLEHDFMK, at 24 and 48 h respectively. Untreated cells were used as a negative control. The data shown represent (±SD) triplicate determinations of three different experiments.
|
|
Moreover, by blocking caspase 9, by specific ZLEHDFMK inhibitor, no activation of effector caspase 3 was observed (Figure 9C and D). Caspase 8 was activated, under the same conditions showing further that, in our NPA cell system, the main apoptotic pathway activated included sequential activation of caspases 8, 9 and 3.
 |
Discussion
|
---|
Peptides from different natural or newly synthetic sources have been used as therapeutic, diagnostic and vaccines for a long time. Plant extracts or products as the active ingredient are normally used as a source of novel compounds from which drugs active against several different tumours are derived. It is well documented that most anticancer agents induce apoptosis, raising the intriguing possibility that a defect in apoptotic programmes contributes to treatment failure (25). Apoptosis was initially described by its morphological characteristics including cell shrinkage, membrane blebbing, chromatin condensation and nuclear fragmentation (26). Apoptosis is now thought to be a gene-directed programme regulated by factors that influence cell survival as well as those that control proliferation and differentiation. It can be considered an intrinsic suicide programme of the cell, and for this reason it is viewed as a mechanism to limit tissue injury (27).
Cyclic astins, a family of cyclopentapeptides isolated from the roots of a medicinal plant, A.tataricus (Compositae), have been implicated as antitumour substances and this activity depends on their cyclic nature (2). To assess if the structure and conformational properties of astins influence their bioactivity we synthesized new astin related cyclopeptides (6) and analysed them for their antineoplastic activity.
We used a series of malignant cell lines derived from human tumours of different histogenesis to analyse the effect of the newly synthesized cyclic astin 3, and observed that all cell lines analysed were inhibited in their growth by the action of astin 3, whereas no toxic effect was observed with newly synthesized linear astin 4.
To understand the nature of the selective cell death induced specifically by the action of astin 3, the most sensitive NPA cell line was analysed for apoptosis. We observed DNA fragmentation, chromatin condensation and exposure of phosphatidylserine, all typical signs of apoptosis activation. The presence of specific apoptotic nuclear bodies was analysed by fluorescence microscopy after Hoechst 33258 staining (Figure 5B and C). A quantitative estimation of the percentage of cyclic astin induced apoptosis gave values of
40% of apoptotic cells (Figure 4) and these data were confirmed by flow cytometry (data not shown). To confirm that the phenomenon observed was specific, the cells, after treatment with astins, were also analysed for the presence of apoptotic nucleosomes (Figure 6). When the same cells were treated with the acyclic analogue astin, according with the data reported in literature, they did not show any change in the normal morphology and number, suggesting that the cyclic nature of astins plays an important role in their antitumour activities (5).
Since different approaches have shown the activation of the apoptotic programme we tried to understand the mechanism of this activation involved in the analogue astin treated tumour cells. Apoptosis a genetically programmed, morphologically distinct form of cell death can be triggered by a variety of physiological and pathological stimuli. Studies performed over the past 10 years have demonstrated that proteases play critical roles in initiation and execution of this process (1113). The caspases, a family of cysteine-dependent aspartate-directed proteases, are responsible for many of the biochemical and morphological changes associated with apoptosis (28). Initiation of the apoptosis response involves initiation caspases, such as caspase 8 which may induce apoptosis directly activating effector caspases such as caspases 6, 7 and 3 (29,30) or caspase 9 subsequent to the involvement of mitochondria that can then activate the same effector caspases 6, 7 and 3 (3032). Moreover, it has also been reported that chemotherapeutic drugs can activate caspase 8, which produce mitochondrial responses, suggesting that crosstalk between apoptotic signals can occur (3336).
The data presented show that, in our NPA cell system, the activation of cyclic analogue astin follows the mechanism involving the subsequent caspases 8, 9 and 3 activation and the specificity of their activity has been demonstrated using the specific caspases 8, 9 and 3 inhibitors or the caspase family inhibitor, ZVADFMK that blocked caspases activation and protected NPA cells from apoptotic death. In addition on blocking caspase 8 no activation of caspase 9 or 3 has been observed and on blocking activated caspase 9 no activation of caspase 3 occurred while caspase 8 is still active, showing again that in our system the main apoptotic pathway activated by the cyclic analogue astin is the pathway involving the sequential activation of caspases 8, 9 and 3. It is possible that the cascade originated by activation of caspase 8 induced by astins may propagate to mitochondria (caspase 9) and other organelles (caspase 3).
Additional experiments are necessary to better define the interaction between cyclic astin analogues and tumoural cells and their involvement in the specific activation of the apoptotic pathways. Moreover, these data suggest the possibility of utilizing synthetic analogue astins as a potential new chemotherapeutic drug.
 |
Acknowledgments
|
---|
We gratefully acknowledge the gift of samples of astin B from Dr H.Morita. The authors thank Prof Philip John and Prof Vittorio Enrico Avvedimento for their suggestions and for a critical review of the manuscript. R.C. is recipient of a fellowship in the PhD programme on Biological Chemistry, Cellular and Molecular Biology. This work was supported by grants from AIRC (Associazione Italiana per la Ricerca sul Cancro) and by MIUR (Ministero dell'Università e della Ricerca Scientifica e Tecnologica), Italy.
 |
References
|
---|
- Morita,H., Nagashima,S., Takeya,K., Itokawa,H. and Iitaka,Y. (1995) Structures and conformation of antitumor cyclic pentapeptides, astin A, B and C, from Aster tataricus. Tetrahedron, 51, 11211132.[CrossRef][ISI]
- Morita,H., Nagashima,S., Yuki,U., Takeya,K. and Itokawa,H. (1993) Astin A and B, antitumor cyclic pentapeptides from Aster tataricus. Chem. Pharm. Bull., 41, 992993.[ISI][Medline]
- Morita,H., Nagashima,S., Yuki,U., Takeya,K. and Itokawa,H. (1996) Cyclic peptides from higher plants. XXVIII. Antitumor activity and hepatic microsomal biotransformation of cyclic pentapeptides, astins, from Aster tataricus. Chem. Pharm. Bull., 44, 10261032.[ISI][Medline]
- Itokawa,H., Takeya,K., Hitotsuyanagi,Y. and Morita,H. (2000) Antitumor compounds isolated from higher plants. J. Biochem. Mol. Biol. Biophys., 4, 213222.
- Morita,H., Nagashima,S., Takeya,K. and Itokawa,H. (1995) Structure of a new peptide, astin J, from Aster tataricus. Chem. Pharm. Bull., 43, 271273.[ISI][Medline]
- Rossi,F., Zanotti,G., Saviano,M., Iacovino,R., Palladino,P., Saviano,G., Amodeo,P., Tancredi,T., Laccetti,P., Corbier,C. and Benedetti,E. (2004) New antitumor cyclic astin analogues: synthesis, conformation and bioactivity. J. Pept. Sci., 10, 92102.[CrossRef][ISI][Medline]
- Vesely,B.A., McAfee,Q., Gower,W.R. and Vesely, D.L. (2003). Four peptides decrease the number of human pancreatic adenocarcinoma cells. Eur. J. Clin. Invest., 33, 9981006.[CrossRef][ISI][Medline]
- Johnstone,R.W., Ruefli,A.A. and Lowe,S.W. (2002) Apoptosis: a link between cancer genetics and chemotherapy. Cell, 108, 153164.[ISI][Medline]
- Wolf,B.B. and Green,D.R. (1999) Suicidal tendencies: apoptotic cell death by caspase family proteinases. J. Biol. Chem., 274, 2004920052.[Free Full Text]
- Cryns,V. and Yuan,J. (1998) Proteases to die for. Genes Dev., 12, 15511570.[Free Full Text]
- Budihardjo,I., Oliver, H., Lutter,M., Luo,X. and Wang,X. (1999) Biochemical pathways of caspase activation during apoptosis. Annu. Rev. Cell Dev. Biol., 15, 269290.[CrossRef][ISI][Medline]
- Thornberry,N.A. and Lazebnik,Y. (1998) Caspases: enemies within. Science, 281, 13121316.[Abstract/Free Full Text]
- Earnshaw,W.C., Martins,I.M. and Kaufmann,S.H. (1999) Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu. Rev. Biochem., 68, 383424.[CrossRef][ISI][Medline]
- Fagin,J.A., Matscio,K., Karmakar,A., Chen,D.L., Tang,S.H. and Koeffler,H.P. (1993) High prevalence of mutation of the p53 gene in poorly differentiated human thyroid carcinomas. J. Clin. Invest., 91, 179184.[ISI][Medline]
- Ain,K.B., Tofiq,S. and Taylor,K.D. (1996) Antineoplastic activity of taxol against human anaplastic thyroid carcinoma cell lines in vitro and in vivo. J. Clin. Endocrinol. Metab., 81, 36503653.[Abstract]
- De Lorenzo,C., Palmer,D.B., Piccoli,R., Ritter,M.A. and D'Alessio,G. (2002) A new human antitumor immunoreagent specific for ErbB2. Clin. Cancer Res., 8, 17101719.[Abstract/Free Full Text]
- Carmichael,J., De Graff,W.G., Gazdar,A.F., Minna,J.D. and Mitchell,J.B. (1987) Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res., 47, 936942.[Abstract]
- Denizot,F. and Lang,R. (1986) Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J. Immunol. Methods, 89, 271277.[CrossRef][ISI][Medline]
- Dedoussis,G.V.Z., Mouzaki,A., Theodoropoulou,M., Menounos,P., Kyrtsonis,M.C., Karameris,A. and Maniatis,A. (1999) Endogenous interleukin 6 conveys resistances to cis-diamminedichloroplatinum-mediated apoptosis of the K562 human leukemic cell line. Exp. Cell Res., 249, 269278.[CrossRef][ISI][Medline]
- Vermes,I., Haanen,C., Steffens-Nakken,H. and Retelingsperger,M. (1995) A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on apoptotic cells using fluorescein labelled Annexin V. J. Immunol. Methods., 184, 3951.[CrossRef][ISI][Medline]
- Aubry,J.P., Caron,A.G., Moine,V. and Bonnefoy,J.Y. (1999) Annexin V used for measuring apoptosis in the early events of cellular cytotoxicity. Cytometry, 37, 197204.[CrossRef][ISI][Medline]
- Catchpoole,D.R. and Stewart,B.W. (1993) Etoposide-induced cytotoxicity in two human T-cell leukemic lines: delayed loss of membrane permeability rather than DNA fragmentation as an indicator of programmed cell death. Cancer Res., 53, 42874296.[Abstract]
- Boccellino,M., Cuccovillo,F., Napolitano,M., Sannolo,N., Balestrieri,C., Acampora,A., Giovane, A. and Quagliuolo,L. (2003) Styrene-7,8-oxide activates a complex apoptotic response in neuronal PC12 cell line. Carcinogenesis, 24, 535540.[Abstract/Free Full Text]
- Lowe,S.W. and Lin,A.W. (2000) Apoptosis in cancer. Carcinogenesis, 21, 485495.[Abstract/Free Full Text]
- Kerr,J.F.R. (2002). History of the events leading to the formulation of the apoptosis concept. Toxicology, 181182, 471474.[CrossRef][ISI]
- Kerr,J.F., Winterford,C.M. and Harmon,B.V. (1994) Apoptosis. Its significance in cancer and cancer therapy. Cancer, 73, 20132026.[ISI][Medline]
- Pirnia,F., Scheneider,E., Betticher,D.C. and Borner,M.M. (2002) Mitomycin C induces apoptosis and caspase-8 and -9 processing through a caspase-3 and Fas-independent pathway. Cell Death Differ., 9, 905914.[CrossRef][ISI][Medline]
- Cohen,G.M. (1997) Caspases: the executioners of apoptosis. Biochem. J., 326, 116.[ISI][Medline]
- Srinivasula,S.M., Ahmad,M., Fernandez-Alnemri,T., Litwack,G. and Alnemri,E.S. (1996) Molecular ordering of the Fas-apoptotic pathway: The Fas/APO-1 protease Mch5 is a CrmA-inhibitable protease that activates multiple Ced-3/ICE-like cysteine proteases. Proc. Natl Acad Sci. USA, 93, 1448614491.[Abstract/Free Full Text]
- Felik,S.W. (2000) Insights into programmed cell death through structural biology. Cell, 103, 273282.[ISI][Medline]
- Srinivasula,S.M., Ahmad,M., Fernandez-Alnemri,T. and Alnemri,E.S. (1998) Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Mol. Cell, 1, 949957.[ISI][Medline]
- Pan,J., Xu,G. and Yeung,J.S.C. (2001) Cytocrome c release is upstream to activation of caspase-9, caspase-8 and caspase-3 in the enhanced apoptosis of anaplastic thyroid cancer cells induced by manumycin and paclitaxel. J. Clin. Endocrinol. Metab., 86, 47314740.[Abstract/Free Full Text]
- Wesselborg,S., Engels,H.I., Rossmann,E., Los,M. and Ostholff-Schulze,K. (1999) Anticancer drugs induce caspase-8/FLICE activation and apoptosis in the absence of CD95 receptor/ligand interaction. Blood, 93, 30533063.[Abstract/Free Full Text]
- Schimnich,A.A, Pietras,E.M., Barnhar,B.C., Legembre,P., Vijayan,S., Holbech,S.L. and Peter,M.E. (2003) Two CD95 tumor classes with different sensitivities to antitumor drugs. Proc. Natl Acad. Sci. USA, 100, 1144511450.[Abstract/Free Full Text]
- Barnhart,B.C., Pietras,E.M., Algeciras-Schimnich,A., Salmena,L., Sayama,K., Hakem,R. and Peter,M.E. (2004) CD95 apoptosis resistance in certain cells can be overcome by noncanonical activation of caspase-8. Cell Death Differ., 113.
- Debatin,K.M. and Krammer,P.H. (2004) Death receptors in chemotherapy and cancer. Oncogene, 23, 29502966.[CrossRef][ISI][Medline]
Received July 28, 2004;
revised November 3, 2004;
accepted January 8, 2005.