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    Reprint | @ Hippokrates Verlag, Stuttgart 1996 edition forschung Karl und Veronica Carstens-Stiftung Herausgegeben von Henning Albrecht und Ingrid Gerhard Grundlagen der Misteltherapie Aktueller Stand der Forschung und Klinische Anwendung Rainer Scheer, Hans Becker und Peter A. Berg. Comparison of the antitumorous effects of Viscum album lectins and the plain or fractional fresh plant preparation Isorel® NN. Zarkovic, M. Jurin, W. Dittrich, M. Hartleb, S. Sladoljev and D. Kissel 8 Hippokrates Verlag Stuttgart 325 Comparison of the antitumorous effects of Viscum album lectins and the plain or fractional fresh plant preparation Isorel® N. Zarkovié, M. Jurin, W. Dittrich, M, Hartleb, S. Sladoljev and D. Kissel Introduction ‘The use of plants for the preparation of agents used in therapy of different pathological disorders has a long history. There are numerous plant prepara- tions used in combined therapy of cancer, for which the active principle has not been identified, Among these preparations of particular interest are mistle- toe (Viscum album L.) preparations (J7). Namely, several data point to a ceytotoxic action of mistletoc extracts against different tumor cell lines (5, 10, 14, 19), Moteover, application of the mistletoe preparations in vivo might have an immunomodulating effect, mainly stimulating the cytotoxic activity of the lymphocytes (1, 2, 7, 8, 9). The dual activity of the mistletoe preparations (cytotoxicity for the tumor cells and the enhancement of the immune host defense against malignant cells) gives a support forthe practical use of these preparations in human medicine as was also shown for the mistleote prepara- tion Isorel which was used in this study (13, 15, 16, 25). Different procedures used for isolation ofthe active components from the mistletoe extracts, resulted in purification of numerous different substances, among which mistletoe lectins are considered as the most important since they often show similar biological activity as the plain plant extracts (8, 10, 12, 14, 19), Thus, iis surprising that the research on the active components of mistletoe did not lead to the determination of some new phytotherapeutic agents. The major reason why no particular and defined phytotherapeutic ‘agent was isolated from mistletoe so far seems to be the common opinion that the mixture of different active components in the plain mistletoe extract gives better efficiency than any of its active components alone.The aim of this study ‘was to test credibility of such hypothesis. 326 Materi and Methods Viscum album extract ‘The original preparation of the apple tree fresh mistletoe extract Isorel M (Novipharm GmbH, Pértschach, Austria) was used. The preparation is pro duced by the cold water extraction ofthe fresh entire plant, without homog- enisation ofthe plant or the extract fermentation, and was for the purpose of, the research given by the company Novipharm. Separation of the Viscum album extract components by ‘chromatography Mistletoe extract Isorel M was used for purification by anion exchange fast protein liquid chromatography (FPLC) with 280 nm detector wavelength. Anion-FPLC was performed on FPLC, Mod. LC 500 (Pharmacia, Sweden) using a 1 ml Resource-Q-column (Pharmacia) with 10 mM TRIS-buffer of iT 8.0 (solvent A). ‘The plain Viscum album preparation was applied to the column and cluted with 3 ml of TRIS-buffer followed by a continuous salt gradient of 0.5 ml NaCl in the same buffer (solvent B) within 15 min and finally to 1M ‘NaCl in 2 min, The fractions eluted were collected at the volume of 1 ml ‘every minute and were kept on +4°C after being filtered through 0.22 p filter. ‘Membrane ultrafiltration Viscum album preparation was filtered through the 30 kDa cut-off membrane (YM 30 Diaflo, Amicon Co., Danvers, USA) using the Amicon Standard UF- Cell 8050 with the nitrogen gassing. Concentration ofthe proteins in original Visewm album preparation and its modifications containing the higher mo- lecular weight (MW) components (MW>30 kDa, the retentant - F1) and the cluant containing the lower MW componenis (MW<30 kDa - F2) was de- termined using the method of Bradford (3) with the bovine serum albumin as standard, No protein loss was determined. Membrane ultrafiltration retentant and the filtrate (FI and F2 respectively) were also mixed together afterwards upon the vA’ ratio as obtained during ultrafiltration and were added to the cultures as well as the other mistletoe samples. The concentrations required for the addition to the cell culture medium were obtained by diluting the 207 samples with medium and the samples were sterilised by filtering through the 0.22 p filter. Viscum album lectin “The preparation of mistletoe lectin (ML) I of Sigma Chemical Co., St. Luis, USA was used, Before being added to the cell culture medium the lectin (MW=115 kDa) was dissolved in saline and filtered through 0.22 u filter. Fibrosarcoma CMC-2 cell cultures ‘Murine methilcholantrene - induced fibrosarcoma CMC-2 cells were har- vested from the tumorous tissue obtained from the CBA/HZgr female mouse bearing the tumor in the hind limb. Grossly intact pieces of the tumor, re- ‘moved from the animal under the sterile conditions, were minced in a beaker containing the cold sterile Hanks’ solution, mixed with a 10 ml syringe and passed through a sterile gauze into est tubes, so that cells were dispersed as a single cell suspension. After washing the cells with RPMI 1640 medium for three times, at 150 g 10 min, each time, viability of the cells was tested by trypan blue exclusion, a number of live tumor cells per ml of medium was adjusted to 4 x 10°, and the cells were seeded in vitro. The seeding density of the cells cultured in 96-well microcytoplates (Greiner, Frickenhausen, Ger- many) was 2 x 104 cells per culture. ‘The cells were incubated in RPMI 1640 medium supplemented with 5 percent of the fetal calf serum (Serva, Heidelberg, Germany), and were cul- ‘tured during the 48 hours at 37°C in a humidified air atmosphere with 5 per- ‘cent CO;. At the time of cell seeding the original Viscum album preparation, its preparations containing different substances (prepared by the anion- exchange FPLC or by membrane ultrafiltration) and the mistletoe lectins VAA were added separately at 2g, 20 ng of 0.2 ng total protein/ml medium, concentrations to the quadruplicates of cultures. The intensity of °H-thymi- dine (sp act 30 Ci/mmol, Amersham Intemational Blc, Buckinghamshire, UK) incorporation was determined for the last 24 hours of cell growth in vitro, after the cells were harvested with the flow harvester (PHD cell har- vester, Cambridge Technology, Ine., Watertown, USA) and the intensity of the cell labelling was measured in 8 counter (Wallac 1209 Rackbeta liguid scintillation counter, Pharmacia, Sweden). 328 Induction of the artificial lung metastasis of fibrosarcoma CMC-2 Fibrosarcoma cells were cultured for 24 hours in tissue culture flasks with 2000 ng/ml of the plain Isorel M preparation, its ultrafiltration prepared ‘modifications or with ML-1. Afterwards, the cells were collected by 0.1 per- cent trypsin solution and washed by centrifuge, No particular difference of the incidence of the live cells (according to the trypan blue exclusion) was ob- served, except the reduction for about 30 percent (data not presented) after the treatment with ML-I and the plain Isorel M preparation. Afterwards, incidence of viable cells for each cell suspension was adjusted to 4x10°#ml and CBA/HZer female mice four months old, weighing 23-25 g were injected into the tail vein with 10° viable tumor cells. The animals were kept in plastic cages without additional treatment, with water and food given ad libitum, until being sacrificed by ether on the 18th day after tumor transplantation, ‘when the number of the tumor nodules in the hungs was counted. The only tumor metastases observed were in the lungs. Statisties ‘The results obtained were analysed using the Mann-Whitney U-test, Results Analysis of the protein - peptide composition obtained by the preparative anion - exchange FPLC of the Viscum album L extract Isorel M is presented in Figure 1. About 35 percent of the total proteins eluted as a sharp peak during the first two minutes after injection of the sample, even before the salt gradient as neutral or cationic compounds. Further 30 percent of the proteins eluted with Tow salt concentration of 10-25 percent solvent B (interrupted-dotted line) and were presented as broad peaks (fractions collected as 6-8). The rest of protein - peptide content of Isorel M was presented with numerous small peaks (1-3 percent of the total protein - peptide amount) eluted at 35-65 per- cent of the solvent B. 328 190 90 60 50 40 Percent of the solvent & 20 10 42.345 67 8 9 10111213141516171819202122 ution volume (m) Fig.1 Chromatogram of the preparative anion - exchange FPLC ofthe fresh plant apple tree Viscum album preparation lsorel M. All the fractions from 1 until 14, except 3 and 4 which did not reveal any protein - peptide content, were afterwards used in vitro to evaluate theit bio- logical efficiency in the treatment of fibrosarcoma CMC-2 cells. The results ‘blaine are presented in Figure 2. While plain preparation showed strong antitumorous activity both if used at 2000 and 20 ngial, some of its FPLC-obtained fractions were equally effective only if used at 2000 ng/ml concentration. However, fractions 7 and 8 even if used at 2000 ng/ml concentration were not as efficient asthe plain preparation (P<0.05), although they inhibited the tumor growth (P<0.02). On the other hand, fractions 2-6 did not affect the tumor growth at all (P>0.1), irrespective of the concentration used. + Slant tte con <0 08) ‘cultures are presented (SD<20%% of the respective mean). 331 Neither plain mistletoe extract nor any of its fractions influenced CMC-2 growth if used at 0.2 ng/ml concentration (P>0.1 for all the samples). Thus, it is obvious that none of the FPLC-obtained components of the Viscum album cextract Isorel could achieve the same concentration dependent antitumorous efficiency as the plain preparation, while there are numerous fractions of the preparation which have similar tumor inhibiting activity. To obtain less fractional” active components we used the membrane ultrafiltration (with, 30 kDa cut-off membrane) and compared antitumorous activity of such ‘modifications (F1>30 kDa and F2 <30 kDa) with the plain Isorel preparation and with the purified Viscum album: lectins (ML-1). The results obtained are presented in Figure 3. 10000. Sample concanntn coo mie! B20 age ezeat Fig.2. Influence of the separated mistletoe lectins (ML-1), of the plain Viscum album extract (\sorel M) and its preparations of different’ molecular Weight (F1>30kDa, F2<30kDa) on the murine fibrosarcoma CMC-2 ‘growth in vitro. Mean values obtained for quacruplicates of cultures are presented (SD<15% of the respeciive mean). ‘All the mistletoe samples used at 2000 ng/ml concentration achieved antitu- ‘morous activity, but only plain Isorel preparation showed the same inhibiting, activity as purified ML-1 if used at this particular concentration (Isorel 10 ML-I, P>0.1, for the other samples comparison to ML-1 - P<005). Interest- ingly, strong antitumorous "lectin-like” activity of Isorel observed at 2000 ng/ml concentration could not be reconstituted by mixing again high and low 332 MW modifications of the preparation since F1+F2 mixture was less efficient in inhibiting tumor growth at this concentration than the original preparation (P<0.05), but still equally efficient as Fl ot F2 preparations alone (for both, P>0.05). On the other hand, if used at concentration of 20 ng/ml, all the mistletoe samples inhibited the tumor growth (P<0.05), but it should be men- tioned that the inhibition observed for FI preparation was weaker than for any other mistletoe preparation (P<0.05 if compared to any other sample). Finally, if used at 0.2 ng/ml concentration only plain Isorel anc its low MW modification F2 inhibited significantly CMC-2 growth in vitro (P<0.05), ‘Thus, tumor growth inhibiting activity appears to be “Iectin-like” ifthe prepa ration was used at the high concentration (2000 ng/ml), but if used at low concentration (0.2 ng/ml) antitumorous effect of Isorel is achieved through the activity of the low MW mistletoe components, not through the activity of the mistletoe lectins. ‘To analyse if observed tumor inhibiting activity could be associated with the ability of malignant cells to form tumor in vivo, CMC-2 cells were treated for 24 hours with 2000 ng/ml of the different mistletoe preparations and the cells were afterwards injected into the tail vein of singeneie recipient mice 10 allow development ofthe artificial ling metastasis (Figure 4), Figsé The effects of in vitro pretroatmant with 2000 ng/ml of the mistletoe lectins (ML-1), of the plain Viscum album extract (Isore! M) and its preparations of different molecular weight (F1>30kDa, F2<30kDa) on the murine fibrosarcoma CMC-2 ability to develop as artical lung metasta- ‘ses in syngeneic CBA/HZgr mice. Mean values obtained for seven mice er group aro presented (SE<14% of the respective mean). 333, Artificial lung metastasis count was significantly lower than for the control mice for all the groups injected with fibrosarcoma cells pretreated in vitro with Viscum album preparations (P<0.05). Evenmore, there was no differ- ence of the antitumorous activity of the different preparations (for all the comparisons, P>0.05). Hence, it could be concluded that in spite of the differ- ences in the activity of purified ML-L, plain fresh mistletoe plant preparation Isorel, and its different MW components on CMC-2 fibrosarcoma growth in vitro, all these Viscum album preparations used were equally able to inhibit ‘fibrosarcoma CMC-2 cells to form tumors in vivo. Discus: Comparison of the effects of the complete mistletoe preparation Isorel and its diferent fractions on the growth of fibrosarcoma cells in vitro indicates that there are no components of the preparation that could inhibit the tumor growth as much as the original preparation, which showed activity equal t0 the pure mistletoe lectins (ML-1). Hence, if used at high concentration (2 tugiml) the effect of the original preparation was almost the same as was the effect of ML-1, although lectin content in Isorel is below 1% of total protei peptide amount (

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