PERIODICUM BIOLOGORUM Vol. 97, No. 1, 61-87, 1995 25 UDC 57:61 a 5 CODEN PDBIAD Fy ISSN 0031-5362 JAS Original scientific paper Comparison of the effects of high and low concentrations of the separated Viscum album L. lectins and of the plain mistletoe plant preparation (Isorel) on the growth of normal and tumor cells in vitro NEVEN ZARKOVIC", MILICA TRBOJEVIC-CEPE®, ZORAN ILIC!"*, MAJA HRZENJAK "5, SHERIFE GRAINCA! ‘and MISLAV JURIN: INTRODUCTION Mistletoe (Viscum album) has a long history of plant source for the preparalion of different extracts used in the therapy of numerous diseases, such as connective tissue disorders, hypertension, diabetes mellitus, epilepsy, and even infertility (1, 3, 21, 29 39). The major break-through in the philosophy and practical use of the mistletoe preparations in the hu- man medicine wes done about 70 years ago by Ru- dolf Stoiner (33). Thus, during the last half of the cen- tury there is an increasing interest to use mistletoe extracts in the therapy of cancer (8, 16, 21, 26). Name. ly, several data point to a cytotoxic action of these ex: tracts against the different tumor cell lines that is based on the induction of the programmed cell death ‘apoptosis" and the inhibition of the cellular protein synthesis (10, 12, 14,17, 19, 28). On the other hand, the application of the mistletoe preparations in vivo might have an immunomodulating effect, mainly stimulating the cytotoxic activity of the lymphocytes and the release of cytokines (6, 8, 9, 1, 19). The dual activity of the mistletoe preparations, ve. cylotoxicity for the tumor cells and the enhancement of the im- mune host defence against the malignant cells, gives support for the practical use of the preparations in the human medicine (1). In spite of that, the nature of the active components of the mistletoe preparations i stil not sufficiently defined, but there are data incl coating that the toxic mistletoe lectins might be the active prinoiple of the mistletoe biological actiaty (5) Mistletoe lectins are complexed proteins of the high molecular weight (30-120 kDa), which achieve a prominent cytotoxic effect if used at microgram con: centrations in vitro (4, 26). Yei, i used at ng/kg doses in vio, mistletoe lectins could stimulate the function Of the immune system (6-8). On the other hand, the recent findings show that the mistletoe lectins could even induce production of the specific antibodies in cancer patients as a consequence of applied treat Period biol, Vo! 97, No 1, 1995 ment with the plain mistletoe preparations (34). How: ever, itis still doubtful, if the mistletoe lectins might be the active principle of mistletoe preparations, since mistletoe preparations contain numerous dif ferent compounds which might show additional bio- logical or even therapeutic elfects (13, 24, 27, 32) Hence, the aim ofthis study was to use a simple ultra: filvaton procedure for separation of the low and high ‘molecular weight (MW) components of the mistletoe preparation Isorel and compare the effects of the Separated components with the effects of the entire, original preparation and the punified mistletoe lectins on the growth of the normal and malignant cells in vitro. MATERIALS AND METHODS Viscum album extract ‘The original preparation of the apple tree mistle toe extract Isorel M (Novipharm GmbH, Portschach, Austria) was used. The preparation is produced by the cold water extraction of the entire plant, without the homogenisation of the plant or the extract fermen: tation, and was for the purpose of the research kindly given by the company Novipharm, Ultrafiltration procedure Viscum album preparation was filtered through the 80 kDa cut-off membrane (YM 30 Diaflo, Amicon Co,, Danvers, USA) using the Amicon Standard UF- Cell 8050 (Amicon Grace Div. Danvers, USA) with the nitrogen gassing . The retentant obtained from 10 ml solution of the original preparation was washed by il tering with saline for three times. Concentration of the proteins in oniginal Viscum alaum preparation and its modifications containing the higher molecular ‘weight (MMW) components (MW>30 kDa, the rete: tant Fl) and the eluant containing the lower MV 6N. Parkovié at al components (MW<80 kDa - F2) was determined us ing the method of Bradford (2) with the bovine serum albumin as standard. Membrane ulttalltation reten- tate and the filtrate (FI and F2 respectively) were also mixed togethor afterwards upon the viv ratio as obtained during ultrafitzation 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 sam- ples with medium and the samples were sterlised by filtering through the 0.22 y filter. Polyacrylamide gel electrophoresis ‘The samples of the Viscum album preparation ‘were heated at 100°C for § min in presence of ‘sod! lum dodeey! sulphate (5%) and applied on continous polyacrylamide gradient gel (6-204). Electrophore 215 vas run for 90 min at constant 25 W power supply using horizontal electrophoresis chamber LKB Mul tiphor 2117 (LKB Pharmacia, Uppsala, Sweden). Al the end, the gel was fixed in solution of 50% methanol and 12% acetic acid and the protein bands were Stained by silver staining, Molecular weight of t Standard used (as indicated by the arrows, Figure | lanes 2 and §) was 94, 69, 43, 30 , 20 and 14 kDa re- spectively, Viscum album lectins ‘The preparation of the Viscum album agglutinin (AA) (Sigma Chemical Co., Si. Luis, USA) was used. Before added to the cell culture medium the lectins were dissolved in saline and filtered through 022 y filter ‘Melanoma B16 Cell Cultures Melanoma BIG cells were harvested from the tu ‘merous tissue obtained from the CSTBLIGo2gr male ‘mouse bearing the tumor in the hind limb, The animal vas sacrificed on the LS" day after sc. injection of the 10° live tumor cells. Grossly intact pieces of the tw mor, removed from the animal under the sterile con ditions, were minced in a beaker containing the cold sterile Hanks’ solution and mixed with a 10 ml sy: tinge. The beaker was placed on ice as toallow large particles to settle down. Five minutes later the super natant was removed using a syringe with 22-cauge needle and passed through a stele gauze into test tubes. The specimen was spun down on 160g during IQ min, the supernatant vias discarded and the pellet resuspended. All visible clumps were removed using a Pasteur pipette so that cells were dispersed as & single cell suspension, After washing the cells with RMPI 1640 medium for three times, at 190 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 wore seeded in vito. he seeding density cf the cells cul- tured in 96-well microcytoplates (Greiner, Fricken: ausen, Germany) was 2 x 10* calls per culture. The cells were incubated in RPMI 1640 medium supple: mented with 89 of the fetal calf serum (Serva, 62 ‘Tho affects of Viscum album extracts on the cell growth tou us es vw sranoaaD = 69 koa = 43 x02 = 30 koa = 14 koe FIGURE 1. Polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulphate of the plain mistletoe preparation Isorel (lane 1) and its membrane (30 kDa cut-of) ultrafiltration retentate FI (MW>30kDa, lane 3) and eluant F2 (MW-<30kDa, lana 9, The samples of the Viscum album prepara: tion were heated at 100°C for § min in presence of sodium dodecy! sulphate (5%) and applied on con: tinuos polyacrylamide gradient gel (5-20%). Electro phoresis was run for 90 min at constant 25 W power Supply using horizontal electrophoresis chamber ‘LK3 Multiphor 2117 (LKB Pharmacia, Uppsala, Swe den). At the end, the gel was fixed in solution of $0% methanol and 12% acetic acid and the protein bands were stained by silver staining. Molecular weight of the standard used (as indicated by the arrows, lanes 2and 5) was 94, 69 43, 30, 20 and 14 kDa, Heildelberg, Germany), and were cultured during the 48 hours at 37°C in a humidified air atmosphere wrth 5% CO,, At the time of cell seeding the onginal Viscum albiim preparation, its preparations contain ing different MW substances and the mistletoe lec- tins were added separately at 2 ug or 0.2 ng total pro. tein/ml medium concentrations to the quadruplicates of cultures. The intensity of *H-thymidine (sp act 30, Cymmol, Amersham International Ble, Buckingham shire, UX) incorporation was determined for the last 24 hours of cell growth in vitro, after the cells were harvested with the flow harvester (PHD cell harvest: er, Cambridge Technology , Inc., Watertown, USA) and the intensity of the cell labelling was measured in B counter (Wallac 1209 Rackbela liquid scintillation ‘counter, Pharmacia, Sweden), Period biol, Vol 97, No 1, 1995N. Zackovié at al ‘Murine lymphocyte cultures Lymphocytes were obtsined from the axillary and inguinal lymph nodes of the sacrificed C3H male mice. The tissue of three mice was minced together and the crude cell suspension was prepared as in case of melanoma B16, by mechanical dispersion of the tissue. After the viability of the cells was checked by the trypan blue exclusion, a number of the live cells per ml of the RPMI 1640 medium was adjusted to 8.x 108, and the cells were seeded in vitro. The seeding density of the lymphocytes cultured in 96- well micracytoplates was 2.5 x 10° cells per culture. ‘The lymphocytes were incubated in RPMI 1640 me- dium supplemented with 5% of the fetal calf serum, and § ug/ml of the plant T-cell mitogenic lectin Con- canavalin & (ConA) (Sigma Chemical Co,, St. Luis, USA) and were cultured during the 72 hours at 37°C ina humidified air atmosphere mth 8% CO, As in case of melanoma cultures , at the time of cell seeding the original Viscum album preparation, its preparations containing different MW substances and the mistletoe lectins Were added separately at 2 ig or 0.2 ng total protein/ml medium concentrations to the quadruplicates of cultures. The intensity o corporation of *H-thymidine was determined for the last 24 hours of cell growth in vitro, as in case of mel- anoma BI6 call cultures. Statistics The results obtained were anslysed using the Mann-Whitney test. Results The analysis of the protein composition of the plain preparation and its different MW fraction re- vesled the presence of one major band in all the sam- ples (Figure 1). Since the MW of that band was 60-70 kDa it could be supposed that this band mainly rep: resents the mistletoe lectin content in the prepara- tion, Plain preparation contained numerous other protein components of different MW within the range of <10 kDa to >100 kDa, but the majority of the com: ponents was represented by the dominating bard and 40-60 kDa renge. Crude fraction of higher MW (retentant of 30 kDa cutoff membrane) contained al- most exclusively protein compan: a 30 kDa. Beside the MW range of 40-70 kDa which was also dominating in the native preparation, this crude fraction contained relatively high amount of compo: nents of MW>100 kDa which probably represent misiletoe lectin conjugates. As was expected higher MIW components were not found at all in the crude fraction of lower MW (filtrate of 30 kDa cut-off mer brane). This fraction contained the components of MW<30 kDa, but as in the other no samples the major band was of 60-70 kDa, The most convenient explanation for that finding could be that this band represents the conjugata of some lower MW compo: nents. The effects of plain Viscum album preparation, its different molecular weight (MW) components and the mistletoe lectins on the growth of cultured mela- Period biol, Vol 97, No 1, 1995 ‘The elfects of Viscum album extracts on the cell grown noma B16 cells are presented by Figure 2, The addi. tion of the original mistletoe preparation or misiletoe lectins at 2 pg/ml concentration completely abol ished the incorporation of the labelled thymidine into melanoma cells in vitro (both, for the Viscum album preperation and for the pure Viscum album lectins if compared with the control , P< 0.08). However, there was no difference in the intensity of the thymidine in: corporation between the cultures treated with mistle toe lectins or with the mistlstoe preparation (P>0.1), 8 FIGURE 2. The effects of the plain Viscum album preparation’, Its different MW components and mis Yetoe lectins on the intensity of °H-thymialine incor ‘poration into the melanoma B16 cells ia vitto. The re sults are given as mean CPM values per well ( SD 6. 18 %6 of the respective mean ) obtained for quad plicates of cultures. - *Isorel ifused at2 g/m! concentration. On the other hand, if used at 0.2 ng/ml concentration the preparation sig: nificantly (P<0.05) inhibited thymidine corporation into the melanoma cells, while the lectins did not show any effects (P>0.1). The higher MW compo nents (Fl) of the mistletoe preparation (MW>30 kDa) added at 2 ug/ml concentration inhibited signif icantly the growth of melanoma B16 cells (P<0.05) bbut were not so efficient as the entire preparation or isletoe lectins. [used at low concentration (02 }) these components of the mistletoe prepare: tion did not show any effect on the thymidine meorpo- tion (P>0.1), just ike purified mistletoe lectins, but not like the original mistletoe preparation. Hence, theN. Zarkowié at al, intensity of the thymidine incorporation in melanoma BIG cells in presence of the entire preparation was lower than in presence of only its higher MW compo: nents (P<0.08). Unusual results were obtained for the cell cultures containing the low MW (<30 kDa) components of the Viscum album preparation (F2). In the presence of 2 ug/ml of low MW components of the preparation only a moderate, but significant (P<0.08) inhibition of the thymidine incorporation was observed. Yet, the same intensity of thymidine incorporation, was also determined if the cells were incubated in the presence of 0.2 ng/ml of the low MW components of the preparation. Thus, the intensities of thymidine incorporation in presence of 2 g/ml and 0.2 ng/ml of low MW components af mistletoe preparation were ‘equal (P>0 1). Furthermore, there was no difference between the effects of the original preparation and its low MW components if used at 0.2 ng/ml concentra: tion (P<0.1), while in presence of 2 g/ml of the en: tire preparation the intenstty of thymidine corpora: tion was significantly lower than in presence of the same concentration of its separated low MW compo: nents (P<0.08). Hence, it is obvious that if used at high concentration in vitro @ pg/ml), the mistletoe preparation showed the inhibitory effects equal to the affect of the pure mistletce lectins. Neither low.nor high MW components of the preparation separately showed the same inhibitory ellact as the entire prep- aration, ifused at high concentration, Moreover, such a strong inhibiting activity of the plain preparation Could not be restored by mixing Fl and F2 modifica: ons of the Viscum album preparation (P>0.08). However, if used at low concentration (0.2 ng/ml), Viscum album preparation could inhibit aymidine in corporation in cultured melanoma 316 cells, like its, low MW components, but unlike the pure mistletoe lectins or hich MW components of the preparation. Mazing of Fl and F2 did not give any particular out ‘come concerning the low concentration activity of the preparation, since the effect of 0.2 ng/ml of such modification added to the tumor cells was equal 10 the elfects of F2 or the plain Viscum album prepara- tion (P>0.05). The results obtained on murine lym- hooyte cultures are presented by Figure 3. An obw ous inhibitory effect on the lymphocyte growth in presence of ConA could be observed if the calls were cultured with high concentauon @ ug/ml) of the mistletoe lectins or mistletoe preparation (for both the lectins and the Viscum album preparation if compared with the contol, P<0.08). The very same effect was also observed for the lymphocytes cul- tured in presence of high MW components of the preparation (if compared with the control, P<0.08). However, there was no difference in the intensity of the thymidine incorporation between the cultures containing the pure lectins or entire mistletoe prep2 ration (P>0.1) just like there was no difference be. tween the effects of the entire mistletoe preparation andits high MV components (P>0.1) or between the mistletoe lectins and the high MW components of the mistletoe preparation (P>0.1). The intensity of the thymidine incorporation wes also decreased if the 4 ‘The effects of Viscum album extracts on the cell growth 8 8 8 cg, Bia Cesdrant FIGURE 3. The effects of the plain Viscum album preparation”, its different MW components and mis jeloe lectins on the intensity of *H-thymidine incor- poration into the murine lymph node lymphocytes cultured in presence of Cond, The results are given a5 mean CPM values per ell (SD 12-25 % of tte re spective mean) obtained for quadruplicates of cul tures. * -Isorel lymphocytes were cultured with 2 ug/ml of the ow MW components of the mistletoe preparation (P.<0.08), However, in presence of 2 ug/ml of the [ow MW components of the mistletoe preparation the in- tensity of the thymidine incorporation was higher than if the cells were cultured with mistletoe lectins, entire mistletoe preparation or with the high MMW ;ponents of the preparation (for all the cases the fence was significant, P<0.08). On the other hand, mixed Fl and F2 modifications of the Vi album preparation showed low inhibiting activity, ‘equal to the activity of the F2 modification alon (50.08) thermore, only a moderate inhibition of the thy: midina imcorporation could be observed if the lym- ‘es were incubated in the presence of low con. jon (0.2 ng/ml) of the mistletoe samples. Yer, rences were significant f compared with the control for all the cases (P<0.08), except if the cells, with mistletoe lectins or with high MW components of the Viscum album preparation (P>0.1). Although lower intensity of the thyrnidine in- corporation could be noticed if the cells were incu: Period biol, Vol 97, No 1, 1995N. Parkovié at a bated with low MW components of the mistletoe preparation than if cultured with the entire prepara: ton, high MW components of the preparation or with, the mistletoe lectins, in none of the cases the differ ence was significant (P>0.1). Thete was no differ ence observed either if 0.2 ng/m! of mixed Fl and F2 modifications was added to lymphocytes (in compar: {son to the other mistletoe samples, P>0.05). Thus, it is obvious that the mistletoe preparation shows the similay, but not the same, pattern of the concentration depencient inhibitory activity on the cell growth in vit. ro as the pure mistletoe lectins. The actiity of the entire preparation could not be obtained by using only its high or low MW components, as well as by using the pure mistletoe lectins alone DISCUSSION Comparison of the effects of the com; toe preparation and its different MW fractions on the growth of melanoma B16 cells in vitro indicate that either low MW components nor high 1 nents of the preparation could inhibi growth as much as the original preparaticn. Howey. x, ifused at high concentration (2 pg/mi) te effect of the original preparation was similar to the eifact of its high MW components alone. Thus obtained inhi tion was also similar to the inhibiting e: pure mistletoe lectins, and could be exp: relatively high MW of the mistlstce lectins (>30 kDa) (@), 80 the lectins should be mainly presented in the high MW fraction of the preparation obteined after ultrafiltration. On the other hand, the effect of the en: tire Viscum album preparation used at low concen: tration (0.2 ng/m)), was similar to the effect of the low ‘MW fraction of the preparation. Thus, it seems that if the preparation was used at such low concentration, more related of the practical dosage in vivo, its act ity might result rom the effect of the low MW compo- nents more likely than from the high MW compo. ents, such as lectins. Moreover if the pure lectins were applied at low concentration, they did not ence the tumor growth at all. [11s known that the mis tletoe preparations contain lower MW components, viscotoxins, which could have the similar cytotoxic activity lie the lectins (31). However, the viscotoxins should be used at much higher concentration in vitro (up to hundred fold) than the lectins to achieve the same biological effects (23). Hen: sahibitory efect on the growth of cultured ma cells caused by the low MW components tion could not be explained as of viscotoxins because the eff increased, but by decreased concentration other hand, it might be possible, that th fraction of the preparation cor: tide complexes or proteins, des It could be possible that the | proteins or glycopeptide coma! ly of physically decompased Plexed mistletoe lectins, a3 wa: Period biol, Vol 97, No 1, 1995 ‘The effects of Viscum album extracts onthe cell growth ly found (23). Yer, since the nature of the viscotoxins is, stil not completely defined, it is an open question if the low MW glycopeptide complexes in ihe mislletoe preparation could be decomposed subunits of the mistletoe lectins. On the other hand, the finding of a broad major 60-70 kDa MW protein band in the plain mistletoe preparation as well as ints Fl and F2 mod iftcations indicates not only the presence of mistletoe Jectns in the preparation but the presence of compo- nents showing high ability of spontaneous or physt- cally induced dis(re)aggregation. This possibility is also indicated by the results which showed that the growth inhibiting components probably exist in some form of conjugate sensitive to membrane filtration, since the activity of the native preparation could net be restored by mixing together low and high MW crude fractions obtained after membrane ultrailtra ton: However this possibilty could be considered only as a speculation as long as the nature of the com- plexed interactions between the different compo. nents of the mistletoe extract will be verified, Inter- estingly, the band of the same MW was also found in the medium of mononuclear cells cultured in pres- ence of Chinese mistletoe extract, and appeared to be the major factor secreted by the mistletoe activat ed mononuclears (3). This factor was found to be dif st from the well known cytokines, but able to in duce differentiation of HL- 60 leukemic cells into ma: ture monocytes (4). The possibility that mistletoe preparation ISOREL itself contains factor similar 10 the one released by mistletoe treated mononuclear cells seems provocative bul requires further studies, ‘The results obtained on the lymphocyte cultures indi- cate again that if used at high concentration in vitro the mistletoe preparation shows equal efficiency as the pure lectins. Itis interesting that the lymphocytes were more sensitive to the high MW components of the preparation than were the melanoma cells, while the original preparation and the pure lectins shawed the same efficiency in the growth inhibition for both types of cells if used at high concentration, Unusual finding was the moderate inhibitory effect of the pure lectins on the lymphocyte growrh if used at low con: centration. This could be the result of the caroohy- drate moiety usually present in the puritied lectins, since the biological oligosaccharides are stil not completely dafined (22, 26), Moreover, mist lotds, which might also inhibit the growih of the cul tured cells (16, 18). They appear fo exist as labile glycoconjugates with mistlsiee proteins (including lectins). Yet tis supposed that the loose of such ba: sic nitrogenous compounds from the mistletoe ex. tracts curing the isolation of lectins or seperation of the components according to the MW might be an explanation for the weaker biological activity of :s0- lated components of mistletoe preparation in coz parison with the effects of the entire one (18). Finally, in spite of the lot of uncertainties concerning the na- ture of the active principle of the mistletoe prepara- tions, t could be concluded that the lectins could not be the only or the major active constituents of these 65IN. Zarkowié at al preparations, or at least not of the particular Viscum album preparation used. More likely, the effects of the mistletoe preparation depends on the concentra- tion used, the type of cells treated and the combined effects of he low and the high MW components of the preparation. The mistletoe lectins might be the main high MW active components, but the nature of the low MW active principle of the mistletoe prepara: tions has yet to be explained, Acknowledgements. This study was supported by the research found of Croatian Ministry of Science and Technology. The authors would also like to ex- press their sincere gratitude to Mrs, Nevenka HirS! fo Mrs. Gordana Franz and Mrs. Margareta Cveik ovski for excellent technical assistance’ REFERENCES |. BETUH J, KO HL, TUNGGAL L, BUSS G. 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Kcbagescaanon 8 einer Publishing Co, Landon feels 34, STATIINA SCHULTZE [L, STECHMESSERE, BERG PA. 1000 ‘Anu mistatce leetn anodes ae produced in patons during 36 WAGNER H, JORDAN F FEIL 3 1968 Suds on the suandard tharapy wih an aqueous mistetoe exact desired rom Viscum ‘main ol misletce preparation, Oncabay #9 (supa. 1) ABSTRACT Comparison of the effects of high and low concentrations of the separated Viscum album I. lectins and of the plain mistletoe plant preparation (Isorel) on the growth of normal and tumor cells in vitro Background and purpose: Due to their cytotoxic, but not immunosup: pressive effects various mistletoe (Viscum album L.) preparations are Used for the therapy of human diseases, particularly cancer. However, nature of the active principle of mistletoe is stil uncertain, while itis sup posed that the toxic mistletoe lectins could be the main active component ofthe plant extracts. The aim of this study was to compare the effects of the mistletoe lectins with the mistletoe preparation Isorel and its di MW components (prepared by membrane ultrailtration) on the growth of mu tne melanoma cells and Cond stimulated lymphocytes in vito. Material and methods: The effects were determined for high @ ug. proteins/ml) concentration, which allows the maximal efficiency of the mistletoe lectins in vitro, and for low concentration (02 ng proteins/m)) , which corresponds better to the in vivo applied doses of the mistletoe preparations, The effects of the samples added to the culture medium were determined according to their influence on the intensity of “H-thymi dine incorporation, Results: The results obtained indicate that the effects of used at higher dose could be result of the unspecified toxic activiy of the mistletoe lec. tins, On the contrary, if used at lower concentration, Viscum album prepa. ration showed mainly growth inhibiting effect on tumor ce result of the activity of the low MW components, but ni Conclusions: Hence, itis supposed that the beneficial therapeutic ef fects of the Viscum album preparation might result irom the combined bi- logical activity of the high and the low MW components, since none of them separately was as efficient as the entie preparation. While the high MW cytotoxic components of the preparation might mainly be mistletoe lectins, the nature of the low MW active components is sul ff, Cliical Hospital Centar, Uniecsty of Zagreb, Kapaa O18 12, $1000 Zag re 1, 82010, Graz, £08 * Deparment of Patology and Laborato: xy Medicine, Unversity af Texas Housion, Unrest of Teas, Heat ‘erat San Antone, Depart and Gyneccogy, § Botconé fate at Zag. Key Words: scum album, Missive, Lactns, Mela roma, Limpictes, Coma emisiny, University of Dedicated to the late Mr. Rudolf Weiss, whose ideas inspired us for this research Period biol, Vol 97, No 1, 1995 or