Proapoptotic activity of Ukrain is based on Chelidonium majusL. alkaloids and mediated via a mitochondrial death pathway
- 17k Downloads
The anticancer drug Ukrain (NSC-631570) which has been specified by the manufacturer as semisynthetic derivative of the Chelidonium majus L. alkaloid chelidonine and the alkylans thiotepa was reported to exert selective cytotoxic effects on human tumour cell lines in vitro. Few clinical trials suggest beneficial effects in the treatment of human cancer. Aim of the present study was to elucidate the importance of apoptosis induction for the antineoplastic activity of Ukrain, to define the molecular mechanism of its cytotoxic effects and to identify its active constituents by mass spectrometry.
Apoptosis induction was analysed in a Jurkat T-lymphoma cell model by fluorescence microscopy (chromatin condensation and nuclear fragmentation), flow cytometry (cellular shrinkage, depolarisation of the mitochondrial membrane potential, caspase-activation) and Western blot analysis (caspase-activation). Composition of Ukrain was analysed by mass spectrometry and LC-MS coupling.
Ukrain turned out to be a potent inducer of apoptosis. Mechanistic analyses revealed that Ukrain induced depolarisation of the mitochondrial membrane potential and activation of caspases. Lack of caspase-8, expression of cFLIP-L and resistance to death receptor ligand-induced apoptosis failed to inhibit Ukrain-induced apoptosis while lack of FADD caused a delay but not abrogation of Ukrain-induced apoptosis pointing to a death receptor independent signalling pathway. In contrast, the broad spectrum caspase-inhibitor zVAD-fmk blocked Ukrain-induced cell death. Moreover, over-expression of Bcl-2 or Bcl-xL and expression of dominant negative caspase-9 partially reduced Ukrain-induced apoptosis pointing to Bcl-2 controlled mitochondrial signalling events.
However, mass spectrometric analysis of Ukrain failed to detect the suggested trimeric chelidonine thiophosphortriamide or putative dimeric or monomeric chelidonine thiophosphortriamide intermediates from chemical synthesis. Instead, the Chelidonium majus L. alkaloids chelidonine, sanguinarine, chelerythrine, protopine and allocryptopine were identified as major components of Ukrain.
Apart from sanguinarine and chelerythrine, chelidonine turned out to be a potent inducer of apoptosis triggering cell death at concentrations of 0.001 mM, while protopine and allocryptopine were less effective. Similar to Ukrain, apoptosis signalling of chelidonine involved Bcl-2 controlled mitochondrial alterations and caspase-activation.
The potent proapoptotic effects of Ukrain are not due to the suggested "Ukrain-molecule" but to the cytotoxic efficacy of Chelidonium majus L. alkaloids including chelidonine.
KeywordsAlkaloid Jurkat Cell Pharmaceutical Preparation Sanguinarine Chelerythrine
List of abbreviations
cellular FADD-like interleukin-1 converting enzyme (FLICE) inhibitory protein
Fas-associated death domain
During the last decades pre-clinical investigations pointed to promising antineoplastic activity of Ukrain. In these studies, Ukrain was suggested to exert selective cytotoxic effects on tumour cells without adverse side effects on normal cells and tissues . Recently, Ukrain was also shown to inhibit tumour growth and metastasis of Lewis carcinoma in C57Bl6 mice . Moreover, a recent report revealed radiosensitizing effects of Ukrain on human colorectal and glioblastoma cell lines in vitro, while normal human fibroblasts were protected against the cytotoxic effects of ionizing radiation . However, the observations on selective cytotoxicity of Ukrain are still subject to controversial discussion . In addition to the above mentioned promising pre-clinical data, some clinical investigations predominantly from Eastern Europe suggested beneficial effects of Ukrain in the treatment of patients suffering e.g. from bladder, breast, pancreatic, rectal or colorectal cancer when given as single drug or in combination with standard chemotherapeutic drugs or ionizing radiation (recently reviewed by ). However, the molecular mechanisms of Ukrain-induced antineoplastic effects are not yet completely understood. Apart from suggested immunomodulatory effects, induction of a growth arrest in the G2/M phase of the cell cycle and/or induction of apoptosis may be involved [7, 9, 10, 11, 12, 13].
Apoptosis constitutes a highly regulated, physiological form of cell death involving complex intracellular proteolysis. In this scenario, specialized intracellular cysteine proteases known as caspases constitute central executioners of apoptosis that cleave a multitude of cellular substrates triggering morphological alterations and finally cell death . There is accumulated evidence that caspases can either be activated by the extrinsic, death receptor dependent or the intrinsic, death receptor-independent mitochondrial pathway. Death receptor ligands, such as CD95 or TRAIL, trigger clustering of their respective receptors in the cytoplasmic membrane with recruitment of the adapter molecule FADD (Fas associated Death Domain) and pro-caspase-8 to form a multimeric death receptor-inducing complex (DISC). Proximity of several pro-caspase-8 molecules in the DISC allows autoproteolytic cleavage and thus activation of this initiator caspase with subsequent cleavage of downstream effector caspases such as caspase-3, -6 and -7 . In contrast, application of cellular stress mostly triggers the so-called mitochondrial death pathway that critically involves disruption of the mitochondrial membrane potential with release of proapoptotic proteins including cytochrome c from the mitochondrial intermembrane space into the cytosol . Release of cytochrome c culminates in the activation of pro-caspase-9 within a multimeric cytosolic death inducing complex, the apoptosome. Similar to caspase-8, the initiator caspase-9 triggers activation of the effector caspase-cascade that finally executes cell death [17, 18, 19]. A mitochondrial amplification loop mediated by caspase-8 triggered cleavage of the proapoptotic Bcl-2 protein Bid yielding truncated Bid (tBid) and tBid mediated release of cytochrome c from the mitochondrial intermembrane space constitutes a molecular link between the death receptor and the mitochondrial pathway that assures execution of apoptosis in cells with low initial caspase-8 cleavage upon death receptor stimulation . Inversely, dependent on the cell line, complete activation of mitochondria and subsequent execution of drug-induced apoptosis may rely on a mitochondrial amplification loop mediated by caspase-3 and caspase-8 .
Induction of apoptosis is a common mechanism of the cytotoxic action of most DNA-damaging drugs and irradiation. However, tumour cells are often characterized by alterations in genes coding for proteins involved in apoptosis and survival signalling [22, 23]. In this regard, increased levels of anti-apoptotic or decreased levels of pro-apoptotic proteins can cause acquired or intrinsic resistance against apoptosis induction by DNA-damaging antineoplastic drugs and ionizing radiation thereby contributing to treatment failure and poor clinical prognosis . Thus, novel agents targeting aberrant apoptosis pathways or inducing alternative death pathways may be suited to overcome treatment resistance [16, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34].
The putative proapoptotic and radiosensitizing effects together with its reported selective action on tumour cells make Ukrain an interesting novel agent for cancer treatment as single drug and in combination with radiation therapy. However, data regarding the cytotoxic activity of the drug are still conflicting and the molecular mechanisms of Ukrain-triggered cell death are not yet understood. Moreover, concerns about the chemical purity of this semisynthetic drug emerged .
Therefore, the aim of the present study was to characterize the apoptosis inducing capacity of Ukrain, to elucidate molecular mechanisms of Ukrain-induced apoptosis and to analyse the chemical identity of the drug preparation.
Chemicals and drugs
The pharmaceutical preparation "Ukrain" (NSC-631570) with an indicated concentration of 1 mg/ml "Ukrain-molecule") was obtained from Nowicky Pharmaceuticals (Vienna, Austria). Chelidonine, protopine, allocryptopine, chelerythrine, sanguinarine and thiotepa were purchased from Sigma-Aldrich (Munich, Germany). For in vitro investigations the alkaloids were dissolved in water (allocryptopine, chelerythrine and protopine), DMSO (chelidonine) or methanol (sanguinarine) as 1 mM, 10 mM or 100 mM stock solutions.
A native extract of greater celandine (Chelidonium majus L.) was obtained from Caelo & Loretz GmbH (Hilden, Germany). As indicated by the manufacturer the extract solution contained 0.35% (w/w) alkaloids as calculated from the content of chelidonine as major alkaloid component. For experimental use, the stock solution was adjusted to 1 mM chelidonine.
Etoposide and the pan-caspase-inhibitor zVAD-fmk were purchased from Alexis Biochemicals (San Diego, USA) and dissolved in DMSO. TMRE was obtained from Molecular Probes (Mobitec, Goettingen, Germany) and dissolved as a 25 μM stock solution in water. Hoechst 33342 and propidium iodide were purchased from Calbiochem (Bad Soden, Germany) and from Sigma (Munich, Germany), respectively, and dissolved in distilled water. Trypan Blue was from Sigma (Munich, Germany) and used as 0.04% solution for cytotoxicity assays.
Rabbit anti-cleaved caspase-3, rabbit anti-Bad, rabbit anti-PARP- and rabbit anti-cleaved PARP antibodies were obtained from Cell Signaling (Frankfurt, Germany). Anti-caspase-9-antibody and polyclonal rabbit anti-Bak-NT were purchased from Upstate (Biomol, Hamburg, Germany). Monoclonal mouse anti-caspase-8-mouse-antibody directed against the p18 subunit was obtained from BioCheck (Münster, Germany). Monoclonal mouse anti-Bcl-2 mouse-antibody was purchased from Santa Cruz Biotech (Heidelberg, Germany). Polyclonal rabbit anti-Bim was purchased from Pharmingen (Becton Dickinson, Heidelberg, Germany) and rabbit anti-Bid from R & D Systems (Wiesbaden, Germany). Horse-radish peroxidase-conjugated secondary antibodies were purchased from Santa-Cruz-Biotech (Heidelberg, Germany; anti-rabbit and anti-mouse) and Amersham-Pharmacia Biotech (Freiburg, Germany; anti-sheep), respectively. All other chemicals were obtained from Sigma (Munich, Germany) if not otherwise specified.
Cell culture and cellular treatment
Jurkat A3 T-lymphoma cells were from American Type Culture Collection (Manassas, VA, USA). Caspase-9 DN expressing Jurkat cells, caspase-8 and FADD-negative Jurkat cells, CD95/TRAIL-resistant Jurkat A3 as well as Bcl-2 overexpressing Jurkat cells and the respective mock transfected control cells (Jurkat Vector) were used as already described [19, 36, 37]. Jurkat J16 control cells as well as cFLIP-L expressing Jurkat cells were kindly provided by J. Tschopp, Epalinges, Switzerland . Jurkat cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum (Invitrogen-Gibco, Karlsruhe, Germany) and 100 U/ml penicilline and 100 μg/ml streptomycine (Invitrogen-Gibco, Karlsruhe, Germany) and maintained in a humidified incubator at 37°C and 5% CO2.
Irradiation was performed with 6 MV photons from a Siemens Mevatron linear accelerator with a dose rate of 4 Gy per min at room temperature.
Cytotoxicity of Ukrain was measured by standard live-dead staining. To this end, cells were incubated for 10 min with Trypan Blue staining solution (0.04%). The number of viable (transparent) and dead (blue) cells was determined by light microscopic analysis.
Determination of apoptosis
Cell death was analysed by fluorescence microscopy upon combined staining of the cells with Hoechst 33342 and propidium iodide. In brief, cells were incubated for 15 min with Hoechst 33342 (1.5 μM) or with Hoechst 33342 (1.5 μM) and propidium iodide (2.5 μg/ml) where indicated. Cell morphology was then determined by fluorescence microscopy (Zeiss Axiovert 200, Carl Zeiss, Jena, Germany) using an excitation wavelength filter of 380 nm. Cells were analysed with 40fold magnification and documented using a CCD camera device (Zeiss Axiocam MRm). At least 250 cells were counted for each value.
Moreover, cell death was quantified by flow cytometry using light scatter characteristics employing a FACS Calibur flow cytometer (Becton Dickinson, Heidelberg, Germany).
Determination of mitochondrial transmembrane potential
The mitochondrial transmembrane potential (Δψm) was analysed by flow cytometry using the Δψm-specific stain TMRE (tetramethylrhodamine ethylester perchlorate) (Molecular Probes, Mobitech, Göttingen Germany). To this end, cells were loaded for 30 min at 37°C with 25 nM TMRE and subsequently analysed by flow cytometry. Preincubation with 1 μM of the proton ionophore CCCP (carbonylcyanide-m-chlorophenylhydrazone) was used as a positive control for complete depolarisation.
Determination of caspase-activation
Caspase activation was determined by Western blot analysis of cytosolic extracts upon treatment with Ukrain or defined alkaloid preparations. To this end, cells were resuspended in 3fold concentrated lysis buffer (final concentration: 62.5 mM Tris-HCl (pH = 6.8), 2% (w/v) SDS, 10% (v/v) Glycerol, 50 mM DDT, 0.01% (w/v) bromphenol-blue in PBS) and subsequently boiled for 10 min at 100°C.
Ten μl lysate (corresponding to 105 cells) were separated by SDS-PAGE and subsequently blotted onto PVDF-membranes (Pharmacia, Freiburg, Germany). Equal protein loading was confirmed by Coomassie Blue staining.
Blots were blocked for 1 h in TBS buffer containing 0.05% Tween 20 and 5% non fat dried milk (3% non fat dried milk in case of caspase-9 antibody). The membrane was then incubated over night at 4°C with the respective primary antibodies. After repeated washings with PBS/Tween-20 (0.05%) the membrane was incubated with the respective secondary antibody (1:10 000) in TBS/Tween for 1 h at room temperature and again washed several times with TBS/Tween. The detection of antibody binding was performed by enhanced chemoluminescence staining (ECL Western blotting analysis system, Amersham-Pharmacia Biotech, Freiburg, Germany).
In addition, caspase activation was quantified using the CaspACE® FITC-VAD-fmk in situ marker which consists of a FITC-conjugate of the cell permeable pan-caspase-inhibitor VAD-fmk (Promega, Mannheim, Germany). Within the cell the inhibitor binds to activated caspases and serves as in situ marker for caspase-activation. CaspACE®-staining was performed according to the manufacturer's guidelines.
Chromatographic separation and mass spectrometric investigations of Chelidonium majusextract and the pharmaceutical preparation Ukrain (NSC-631570)
Dissolution of extract and Ukrain prior to analysis
Before analysis both the Chelidonium majus L. extract and Ukrain were diluted with methanol/water. Ten μl of Chelidonium majus extract were diluted with 500 μl methanol/water (90/10, v/v), stirred for 10 min, centrifuged and the brown supernatant was used for analyis. Twenty μl of Ukrain were diluted with 100 μl methanol/water (90/10, v/v), and the light green solution was used for further investigations. In each case 10 μl of the particle free methanolic/water solutions were injected into the HPLC system coupled to ESI-MS interface.
The HPLC system consisted of a Surveyor quaternary pump (Thermo Finnigan; San Jose, California; USA), Surveyor PDA detector (UV-range scanned: 200–800 nm in 5 nm intervals) and Surveyor autosampler. Reversed-phase chromatography was carried out on a Grom Saphir 110 C8, (3 μm, 150 × 2 mm) column (Grom Analytik; Rottenburg, Germany). The mobile phase consisted of water + 0.1% formic acid (eluent A) and acetonitrile + 0.1% formic acid (eluent B); flow rate was 200 μl/min. Formic acid was necessary for improving peak shape. The gradient was applied as follows: 0 min: 100% A – 5 min: 100% A – 1 min: 95% A – 50 min: 50% A – 55 min: 50% A – 56 min: 100% A. The column was washed with pure methanol and pure acetonitrile after each run for 15 min and equilibrated with 100% A for 3 min.
Electrospray and MS settings
All mass spectra were obtained using a Thermo Finnigan (San Jose, California; USA) TSQ Quantum triple quadrupole mass spectrometer equipped with an electrospray interface coupled to the Surveyor HPLC-system and operated in positive ionisation mode. For data processing the Xcalibur 1.3 software was used. The ESI-interface was locked in a 90° position towards the orifice and operated at 3.8 kV. Capillary temperature and sheath gas were set to 320°C and 30 (arbitrary) units, respectively. Auxillary gas was set to 12 (arbitrary) units to enhance signal intensity.
Full scan MS scans
Conventional (non MS/MS) full scans in centroid mode using the third quadrupole as mass selector (Q3 full scan) were recorded in the range of m/z 100–1000 within a dwell time of 0.7 s. Source CID (collision induced fragmentation) voltage was 10 V, which minimizes analyte-solvent adducts.
MS/MS product ion scans
Within one LC-MS run up to four product ion scans for different precursor masses were combined. Source CID voltage was set to constant value of 10 V and collision energy to 35 V (for 354.1 m/z and 370.1 m/z) and to 40 V (for 332.1 m/z and 348.2 m/z), respectively and the argon collision gas was maintained at a pressure of 1.3· 10-6 bar. Product ion spectra were recorded with a scan time of 0.5 s for each mass, a peak width of 0.7 for Q1 and Q3 and within a range of m/z 40 to m/z of the moleclular ion + 10 mass units.
Mass spectrometric fragmentation behaviour of reference compounds
For each reference compound (Fig. 10), a solution with an approximate concentration of 0.1 mg/ml was prepared by dissolving the dry substances in methanol. Ten μl of these standard solutions were injected separately and analysed by LC-MS and LC-MS/MS under the same conditions as the Chelidonium majus extract and Ukrain. The data were then evaluated with respect to ionisation efficiency, compound stability in the electrospray source and adduct formation.
Experiments were at least performed in triplicates. Data were represented as means ± SD. Where appropriate, one-way ANOVA test or paired t-test were performed using GraphPad InStat version 3.00 for Windows 95, GraphPad Software, San Diego California USA, http://www.graphpad.com.
Ukrain induces apoptosis in Jurkat T-lymphoma cells
Signalling pathways of Ukrain-induced apoptosis
Up to now our data indicated that Ukrain triggers activation of caspases and alteration of mitochondrial function. To gain further insight into the molecular requirements for Ukrain-induced apoptosis we subsequently analysed the contribution of death receptor-dependent and mitochondrial apoptosis signalling events.
Ukrain induced mitochondrial damage and apoptosis in caspase-8- and FADD-negative cells in a concentration- and time-dependent manner to the same extent as in Jurkat A3 control cells (Fig. 4a right and left panel, respectively). Moreover, activation of caspases was clearly detectable in Ukrain-treated caspase-8- and FADD-deficient cells although a slight delay in the activation of caspase-3 and cleavage of the caspase-3 substrate PARP was observed in the FADD-deficient cells (Fig. 4b). Consistent with these findings, cFLIP-L expressing Jurkat cells as well as CD95/TRAIL resistant Jurkat cells that failed to undergo apoptosis upon treatment with 10 ng/ml TRAIL were sensitive to Ukrain-induced apoptosis to the same extent as the control cells (Fig. 5).
These data reveal that lack of caspase-8 or FADD, two essential components of the death receptor pathway, expression of cFLIP-L or resistance to death receptor ligands do not abrogate apoptosis upon Ukrain-treatment. In addtion, Ukrain-treatment induced mitochondrial alterations. Therefore, we subsequently tested the hypothesis that similar to DNA-damaging anticancer drugs, Ukrain may trigger apoptosis via the mitochondrial apoptosis signalling cascade. Mitochondrial death pathways are characterized by early depolarisation of the mitochondrial membrane potential and activation of caspases downstream of the mitochondria. These events are regulated by pro- and anti-apoptotic Bcl-2-like proteins . In this context, anti-apoptotic Bcl-2 and Bcl-xL function as guardians of mitochondrial integrity preventing mitochondrial damage and caspase-activation triggered by pro-apoptotic Bcl-2 family members e.g. Bax and Bak at the level of the mitochondria .
In line with these findings, overexpression of Bcl-2 reduced Ukrain-induced mitochondrial damage while expression of the dominant negative caspase-9 was less effective, at least at increased drug concentrations (Fig. 6b). The moderate effects on apoptosis induction observed by flow cytometric analysis were reflected by determination of caspase-activation using Western blot analysis that revealed reduction but not inhibition of caspase-activation and PARP-cleavage in Jurkat Bcl-2 cells compared to control cells (Fig. 6d). In this regard, the small amount of PARP-cleavage observed in untreated Jurkat Vector and Jurkat Caspase-9 DN cells was attributed to increased levels of spontaneous apoptosis in untreated cells compared to untreated Bcl-2 control cells.
Since expression of Bcl-2, Bcl-xL and the dominant negative caspase-9 reduced Ukrain-induced apoptosis pointing to the contribution of mitochondrial signalling events we subsequently analysed whether Ukrain-treatment would affect pro-apoptotic members of the Bcl-2 family. However, treatment with Ukrain did not alter expression levels or integrity of the pro-apoptotic Bak, Bid or Bad (data not shown).
Mass spectrometric analysis of Ukrain-components
Extracts of Chelidonium majusextracts have similar proapoptotic potency as Ukrain
Chelidonine is a potent apoptosis inducing component of Ukrain
Up to now our data indicated that the pharmaceutical preparation Ukrain and Chelidonium majus extracts both constitute mixtures of diverse Chelidonium majus L. alkaloids and induce apoptosis of Jurkat T-lymphoma cells with almost similar potency. Consequently, we next aimed to define the apoptosis inducing alkaloids among the components of the Ukrain-preparation identified by mass spectrometry.
The alkaloid protopine (≥ 50 μM) also exerted cytotoxic effects on Jurkat T-lymphoma cells. However, increased drug concentrations compared to chelidonine were required to induce similar levels of cell death (approximately 40% of cell death upon treatment with 50 μM protopine compared to 5 μM chelidonine) (Fig. 14b). Interestingly, fluorescence microscopy of protopine-treated cells upon combined staining with Hoechst 33342 and propidium iodide revealed that a further increase in the alkaloid concentration led to increasing rates of necrotic cell death (data not shown).
In contrast to the above mentioned findings the alkaloid allocryptopine had only minor apoptosis inducing potency. At concentrations between 50 and 150 μM the drug almost completely failed to induce mitochondrial damage, caspase-activation, chromatin condensation or nuclear fragmentation (Fig. 15, left panel). Only at increased drug concentrations (250 μM to 1 mM) allocryptopine induced cell death including apoptosis and necrosis (Fig. 15, right panel).
Influence of Bcl-2 on chelidonine-induced apoptosis
Our data clearly show that the antineoplastic drug preparation Ukrain (NSC-631570) is a potent inducer of apoptosis and cell death in human Jurkat T lymphoma cells involving mitochondrial damage and caspase-activation. However, our data also reveal that the cytotoxic efficacy of Ukrain is not due to the suggested "Ukrain-molecule" but instead to apoptosis inducing alkaloids from Chelidonium majus L..
It has been shown earlier that Ukrain induces apoptosis in tumour cell lines from human solid tumours [9, 10, 12, 13]. Here, we show for the first time that Ukrain is also effective in a Jurkat T-Lymphoma cell model. Interestingly, the concentrations required for efficient induction of apoptosis in Jurkat cells (1–50 μg/ml) were similar to the concentration required for the reported radiosensitizing effects of Ukrain on human colorectal and glioblastoma cell lines in vitro (0.1–50 μg/ml) .
However, up to now the mechanism of Ukrain-induced apoptosis remained unclear. In our hands, apoptosis induction did not require the presence of caspase-8 and FADD, two integral signalling molecules of the death receptor pathway. In line with these findings, expression of the caspase-8 inhibitor cFLIP-L or resistance to the death receptor ligands CD95 and TRAIL failed to abrogate Ukrain-induced apoptosis. These findings implicate that Ukrain induces apoptosis independently from death receptor signalling. The delayed activation of caspase-3 and cleavage of the caspase-3 substrate PARP in the FADD-deficient cells could be indicative for the contribution of a FADD-dependent signalling event.
In contrast, overexpression of Bcl-2, Bcl-xL and expression of a dominant negative caspase-9 reduced Ukrain-induced apoptosis revealing an involvement of the mitochondrial signalling cascade that is controlled by anti-apoptotic members of the Bcl-2 family. A similar protective effect of Bcl-2 on Ukrain-induced apoptosis has also been reported for human keratinocytes . However, the moderate inhibitory action of Bcl-2, Bcl-xL and caspase-9 DN on Ukrain-induced mitochondrial damage and caspase-activation suggests that some factors different from Bcl-2, Bcl-xL and caspase-9 may be required for regulation of mitochondrial damage and apoptosis execution upon Ukrain-treatment. In this regard, our data reveal that altered expression levels or integrity of the proapoptotic Bcl-2 family members Bak, Bid or Bad were not involved in Ukrain-induced apoptosis.
Our findings on the composition of the pharmaceutical preparation implicate that several distinct alkaloid compounds contribute to the proapoptotic signalling of Ukrain. In this regard, mass spectrometric analyses of the pharmaceutical preparation identified several well-known alkaloids from Chelidonium majus L. including chelidonine, chelerythrine, sanguinarine, protopine and allocryptopine as major constituents of the drug preparation without evidence for the presence of the postulated "Ukrain-molecule" or some dimeric or monomeric condensation products of thiotepa with two or one chelidonine moieties.
Interestingly, the same alkaloids were detected in a commercially available Chelidonium majus L. extract. Of these, chelerythrine, sanguinarine, chelidonine and to a lesser extent protopine turned out to potently induce apoptosis in Jurkat T-lymphoma cells, while allocryptopine was almost ineffective. Together with the observation that extracts from Chelidonium majus L. induced apoptosis of Jurkat T-lymphoma cells in a similar dose range as the reference alkaloid chelidonine and Ukrain these findings implicate that the pharmaceutical preparation Ukrain may mainly constitute purified extracts from Chelidonium majus L. and that the Chelidonium majus L. alkaloids chelidonine, chelerythrine, sanguinarine and protopine are responsible for the reported cytotoxic effects of the drug preparation. Our findings are consistent with a recent publication from Panzer and co-workers that similarly failed to confirm the presence of the specified "Ukrain-molecule" by thin-layer chromatography, high-performance liquid chromatography and liquid chromatography-mass spectrometry and gave raise to the assumption that Ukrain preparations may consist of a mixture of several Chelidonium majus L. alkaloids .
Chelidonium majus L. and its alkaloids are well-known to exert antitumour, antiinflammatory and antimicrobial effects and to affect diverse cellular processes [42, 43, 44, 45, 46, 47]. It has been shown that chelerythrine, sanguinarine and chelidonine exert cytotstatic and cytotoxic effects on human tumour cell lines [48, 49, 50, 51, 52, 53, 54]. In this context, the common antimicrotubule properties of chelidonine, chelerythrine and sanguinarine have been suggested to contribute to their observed antineoplastic activity [49, 55]. Moreover, chelerythrine was found to function as an antagonist of antiapoptotic Bcl-xL and to inhibit, similar to its structural homologue sanguinarine, the anti-apoptotic mitogen-activated protein kinase phosphatase as well as protein kinase C [52, 56, 57, 58, 59].
Up to now only single reports revealed growth inhibitory and pro-apoptotic effects of chelidonine [49, 51]. In the present investigation, we identified chelidonine as a potent proapoptotic component of Ukrain and Chelidonium majus L. extract. Thus, in addition to chelerythrine and sanguinarine chelidonine may contribute to the reported cytotoxic efficacy of both preparations. Interestingly, while protopine, chelerythrine and sanguinarine induced high levels apoptosis and necrosis, the predominant mode of cell death upon chelidonine-treatment was apoptosis. This is consistent with recent data of Kemeny-Beke and co-workers that observed similar effects in a primary human uveal melanoma cell line .
Moreover, we show for the first time that apoptosis induction by chelidonine involves alteration of mitochondrial function and caspase-activation. The observation that overexpression of Bcl-2 interfered with chelidonine-induced apoptosis argues for a role of the intrinsic mitochondrial pathway. These data are reminiscent to recent findings revealing the involvement of the mitochondrial death pathway in sanguinarine-induced apoptosis in immortalised keratinocytes . However, Bcl-2 failed to completely inhibit chelidonine-induced proapoptotic effects pointing to the contribution of Bcl-2-independent signalling events.
Our data implicate that defined proapoptotic alkaloid compounds from Chelidonium majus L. may constitute promising novel anticancer drugs . In contrast, despite numerous reports on beneficial effects of Ukrain in preclinical investigations, case reports and non-randomized as well as randomized clinical trials, up to now there is no solid scientific basis for a rational use of Ukrain in cancer treatment. In this regard, the suggested selective antitumour activity of Ukrain is still controversial . Moreover, a recent systematic review of published clinical trial data revealed that most of the clinical data do not meet stringent criteria for randomized clinical studies and that methodological limitations of the remaining studies (e.g. small sample size, poor or unknown method of randomisation) actually prevent final conclusions on the putative value of Ukrain for the treatment of cancer patients . Our finding that Ukrain represents a mixture of Chelidonium majus L. alkaloids instead of the "Ukrain-molecule" further challenges clinical use of Ukrain and in addition rises concerns on potential adverse side effects of the drug preparation on liver tissue as observed for Chelidonium majus L. extracts [61, 62].
Taken together, our investigation clearly shows that induction of apoptosis contributes to the cytotoxic effects of the pharmaceutical preparation Ukrain. Ukrain induced apoptosis independently from death receptor signalling via a pathway involving mitochondrial damage and caspase-activation that were partially sensitive to overexpression of Bcl-2, Bcl-xL and a dominant negative caspase-9.
Importantly, our mass spectrometric data reveal that instead of the composition indicated by the manufacturer the pharmaceutical preparation Ukrain constitutes a mixture of Chelidonium majus L. alkaloids including chelidonine, protopine, allocryptopine, chelerythrine and sanguinarine. In addition to native Chelidonium majus L. extract the isolated alkaloids sanguinarine, chelerythrine and chelidonine were identified as potent inducers of apoptosis in Jurkat T-Lymphoma cells that induced cell death with similar potency as Ukrain. Consequently, our data strongly suggest that the observed antineoplastic effects of Ukrain are based on proapoptotic Chelidonium majus L. alkaloids instead of the hypothetic "Ukrain-molecule" and implicate that purified Chelidonium majus L. alkaloids should be considered for further preclinical and clinical development as single drugs or in combination with standard therapies including ionizing radiation.
We are thankful to P. Juo and J. Blenis (Boston, MA, USA) for the caspase-8 and FADD negative Jurkat cells, J. Tschopp for the cFLIP-L overexpressing and Jurkat J16 control cells and to E. Alnemri (Philadelphia, USA) for the caspase-9-DN construct. The work was supported by the Doctoral Program of the Deutsche Forschungsgemeinschaft (DFG) "Cellular mechanisms of immune-associated processes", GK 794, a grant of the Federal Ministry of Education and Research (Fö. 01KS9602) and the Interdisciplinary Center of Clinical Research Tuebingen (IZKF) to V.J. and C.B. a grant from the Mildred Scheel Stiftung to (10–1970 Be-III) to C.B. and V. J We are grateful to Nowicky Pharma (Vienna, Austria) for providing us with Ukrain and financial support.
- 1.Danilos J, Zbroja-Sontag W, Baran E, Kurylcio L, Kondratowicz L, Jusiak L: Preliminary studies on the effect of Ukrain (Tris(2-([5bS-(5ba,6b,12ba)]- 5b,6,7,12b,13,14-hexahydro-13-methyl[1,3] benzodioxolo[5,6-v]-1-3- dioxolo[4,5-i]phenanthridinium-6-ol]-Ethaneaminyl)Phosphinesulfide.6HCl ) on the immunological response in patients with malignant tumours. Drugs Exp Clin Res. 1992, 18 (Suppl): 55-62.PubMedGoogle Scholar
- 6.Cordes N, Plasswilm L, Bamberg M, Rodemann HP: Ukrain, an alkaloid thiophosphoric acid derivative of Chelidonium majus L. protects human fibroblasts but not human tumour cells in vitro against ionizing radiation. Int J Radiat Biol. 2002, 78 (1): 17-27. 10.1080/09553000110089991.CrossRefPubMedGoogle Scholar
- 19.Belka C, Rudner J, Wesselborg S, Stepczynska A, Marini P, Lepple-Wienhues A, Faltin H, Bamberg M, Budach W, Schulze-Osthoff K: Differential role of caspase-8 and BID activation during radiation- and CD95-induced apoptosis. Oncogene. 2000, 19 (9): 1181-1190. 10.1038/sj.onc.1203401.CrossRefPubMedGoogle Scholar
- 21.von Haefen C, Wieder T, Essmann F, Schulze-Osthoff K, Dorken B, Daniel PT: Paclitaxel-induced apoptosis in BJAB cells proceeds via a death receptor-independent, caspases-3/-8-driven mitochondrial amplification loop. Oncogene. 2003, 22 (15): 2236-2247. 10.1038/sj.onc.1206280.CrossRefPubMedGoogle Scholar
- 24.Mrozek A, Petrowsky H, Sturm I, Kraus J, Hermann S, Hauptmann S, Lorenz M, Dorken B, Daniel PT: Combined p53/Bax mutation results in extremely poor prognosis in gastric carcinoma with low microsatellite instability. Cell Death Differ. 2003, 10 (4): 461-467. 10.1038/sj.cdd.4401193.CrossRefPubMedGoogle Scholar
- 33.Schmelz K, Wieder T, Tamm I, Muller A, Essmann F, Geilen CC, Schulze-Osthoff K, Dorken B, Daniel PT: Tumor necrosis factor alpha sensitizes malignant cells to chemotherapeutic drugs via the mitochondrial apoptosis pathway independently of caspase-8 and NF-kappaB. Oncogene. 2004, 23 (40): 6743-6759. 10.1038/sj.onc.1207848.CrossRefPubMedGoogle Scholar
- 34.Eberle J, Fecker LF, Hossini AM, Wieder T, Daniel PT, Orfanos CE, Geilen CC: CD95/Fas signaling in human melanoma cells: conditional expression of CD95L/FasL overcomes the intrinsic apoptosis resistance of malignant melanoma and inhibits growth and progression of human melanoma xenotransplants. Oncogene. 2003, 22 (57): 9131-9141. 10.1038/sj.onc.1207228.CrossRefPubMedGoogle Scholar
- 39.von Haefen C, Gillissen B, Hemmati PG, Wendt J, Guner D, Mrozek A, Belka C, Dorken B, Daniel PT: Multidomain Bcl-2 homolog Bax but not Bak mediates synergistic induction of apoptosis by TRAIL and 5-FU through the mitochondrial apoptosis pathway. Oncogene. 2004, 23 (50): 8320-8332. 10.1038/sj.onc.1207971.CrossRefPubMedGoogle Scholar
- 42.Chung HS, An HJ, Jeong HJ, Won JH, Hong SH, Kim HM: Water extract isolated from Chelidonium majus enhances nitric oxide and tumour necrosis factor-alpha production via nuclear factor-kappaB activation in mouse peritoneal macrophages. J Pharm Pharmacol. 2004, 56 (1): 129-134. 10.1211/0022357022467.CrossRefPubMedGoogle Scholar
- 51.Kemeny-Beke A, Aradi J, Damjanovich J, Beck Z, Facsko A, Berta A, Bodnar A: Apoptotic response of uveal melanoma cells upon treatment with chelidonine, sanguinarine and chelerythrine.Google Scholar
- 52.Hoffmann TK, Leenen K, Hafner D, Balz V, Gerharz CD, Grund A, Ballo H, Hauser U, Bier H: Antitumor activity of protein kinase C inhibitors and cisplatin in human head and neck squamous cell carcinoma lines. Anticancer Drugs. 2002, 13 (1): 93-100. 10.1097/00001813-200201000-00011.CrossRefPubMedGoogle Scholar
- 57.Vogt A, Tamewitz A, Skoko J, Sikorski RP, Giuliano KA, Lazo JS: The benzo[c]phenanthridine alkaloid, sanguinarine, is a selective, cell-active inhibitor of mitogen-activated protein kinase phosphatase-1. J Biol Chem. 2005, 280 (19): 19078-86 Epub 2005 Mar 7. 10.1074/jbc.M501467200.CrossRefPubMedGoogle Scholar
- 59.Chmura SJ, Mauceri HJ, Advani S, Heimann R, Beckett MA, Nodzenski E, Quintans J, Kufe DW, Weichselbaum RR: Decreasing the apoptotic threshold of tumor cells through protein kinase C inhibition and sphingomyelinase activation increases tumor killing by ionizing radiation. Cancer Res. 1997, 57 (19): 4340-4347.PubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/6/14/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.