zenaida s revista de oncologie comparata

8/8/2019 Zenaida S Revista de Oncologie Comparata

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SOCIETATEA ROMANA DE ONCOLOGIE COMPARATA1 ROMANIAN SOCIETY OF COMPARATIVE ONCOLOGY


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SOCIETATEA ROMANA DE ONCOLOGIE COMPARATAROMANIAN SOCIETY OF COMPARATIVE ONCOLOGY

REVISTA ROMAN^DE ONCOLOG I E COMPARATAROMANIAN JOURNAL

OF COMPARATIVE ONCOLOGY

EDITURA CERES ICERES PUBLISHING HOUSE


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TESTAREA EFICAGIT~TII UNOR EXTRACTE DIN PLANTEASUPRA EVOLUnEl HEPATOMULUI RS1, INERSPECTIVA

THE TEST OF EFFICACY OF SOME PLANT EXTRACTSIN THE EVOLUTION OF THE RS1 HEPATOMA,IN VIEW TO USE-mS ADJUVANTS IN CHEMOTHERAPYEUGENIATIRIFON',ENAIDA STOENESCU2, M. VASILESCU~, IULIANA GRUIA',MONICA NISTOROIU', MARIETA PANAIT', MADALINA CHE$NOIU', ANA-MARIA BURDU$EL',IOLANDA DUMITRESCU', ANTONELA BU$CX1, DANIELA F M T I ~ ' ,

CRlNA TIRIFON2, N. MANOLESCU', S. CINCX'lInstitutu1 Oncotogic ,,Pro$ DI:Al. 7kestioreanu" B ucur e~t i; 2Universitatea de M ed ic id ~iFamacie,,Carol Davila" B u c u ~ t i ; Ministerul Agriculturii ji Dezvoltirii Rurale, Bu cur e~ ti / 'Institute of OncologyPro$ DI: Alex. Trestioreanu Bucharest, Roma nia; 2University of Medicine a nd Pharmucy ,,Carol Davila"Bucharest, Romania; 3Ministry of Agriculrure and Rural Development, Bucharest, Rom aniaREZUMATfn acest experiment au ost testate efectele unor extractedin rridricini de plante medicinale administrate la ~ o b o -h iRtar cu hepatom RSI , in varianfele-preventiv, era-peutic gi tardiv terapeutic , tn scopul indenf@cdrii norposibile ejecte imunostimulutoare pilsau antitumorale.LuGnd fn considerare u d to a r e l e rezultate: nivel cres-

cut de supraviepire, scriderea sintezei de AND pard lalimite nonnale, diminuarea markerilor de stres oxidativ,valorile concentrafiilorde citocromi P450 ,vi a activitdfiiGST, sa poate stabili ejicacitatea produselor testate.Nivelul scdzut a1 toxicitafii ~i a1 ejectelor secundare su-gereazd cd extractele testate ar putea fi utilizale ca posi-bili adjuvanti in chimioterapia antitumoral6 la anima-lele de laborator.Cuvinte cheie: citostatice,metastaze, chimioterapie, siner-gism, imunostimulutor

- Complica@e chimioterapiei limiteazti utilizareacitostaticelor, deoareceorganismul animalelor devine sen-sibil la infectii, iar sdderea imunitg$i favorized evo-lutia cancem1ui $i apariga metastazelor. Restabilirea ca-pacitgfii do a p h organismului este o problemti ma-jori cu care se confruntiispecYialigtiiin timpul fi dupg te-rapia citostaticg. (4)- fn scopul prevenirii complica@lor chimiokrapiei,s-a testat actiunea imunostimulatoare gilsau antiturnoraltiprin administrarea preventivg $i terapeutid a unei aso-cieri cu efect sinergic de extracte din rgdicini de plank(Acanthopanax senticosus / Eleutherococcus senticosus,

ABSTRACTTo identifi possible new immunostimulating a d o r anti-tumor egects, some extracts from medicinal plant rootswere tested in preventive, therapeutic and tardive thera-peuric mode on 140 Wistar rats with RS1 Hepatoma.T&g into acount the following results: a higher sur-vival rate, decreases the DNA synthesis lo the normallimit, diminution the oxidative stress markers, the valuesof cytocromes P45O concs and GST activities, revealedthe eflciency of studied extracts. The lack of toxicityaiulof secondary eaects, suggerates that the investigatedextracts could represent some possible adjuvants to anti-tumoral chemotherapy in laboratory animals.

Key words: cytostatics, metastares, chemotherapy, syner-gism, immune-stimulating

- The complications of chemotherapy are limitingthe use of cytostatics because the animal body becomessensible to infections and the lower of immunity favorscancer evolution and the metastasis. (4)- For prevention of complications of chemotherapy,the irnmunestimulating andlor antitumoral action by pre-ventive and therapeutic administering of an associationof some plant roots extracts with synergic effect (Acan-t h o p m senticosus / Eleutherococcus senticosus, Ca-lendula oficinalis, Chelidonium rnajus), to Wistar ratswith RS 1 hepatoma.


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REVISTAR O ~ UE ONCOLOGIE COMPARAT~I OMANIAN JOURNAL OF COMPARATIVEONCOLOGY NO. 1512008Calendula oficinalis, Chelidonium mujus), la ~obolaniWistar cu hepatom RS 1.

Experimentul s-a efectuat pe 140 qobolani Wistarinoculati cu hepatom RS1 (indus prin administrarea sub-cutanat2 a 2.500.000 - 4.000. 000 celule tumorale/0,5 mlsolutie 0,9% NaCI).Extractele din plante testate, au fost administrate inciteva variante, astfel:Loturile experimentale:- Lot I (preventiv) - extractele administrate preven-tiv, cu 2 luni inainte de inocularea tumorii;- Lot II (terapeutic) - extractele administrate con-comitent (in acelqi timp) cu inocularea tumorali;- Lot III (terapeutic tardiv) - extractele administratela 25 de zile dupi inocularea tumorali;- Lot IV - martor inoculat cu tumorg $i netratat;- Lot V - martor netratat, neinoculat tumoral;

- Lot VI - martor tratat $i neinoculat cu tumora;- La toate animalele, extractele au fost administrateat& in hrani, c k $i prin aplicafii periturnorale.S-au determinaturmitorii paramehi: spor de crevterein greutate, evaluarea tumorali (volum, greutate .yi aspectmacroscopic), rata de supraviewire$i mortalitate, concen-tratia dialdehidei malonice, activitatea ceruloplasminei,concentrapa de tioli, concentraoa citocromilor P450, ac-tivitatea GST,fracoa de celule aflate?nsintei5, indicele deproliferare $i tndicele ADN, indice apoptotic. (1,2)REZULTATE $I DISCUW

1.Morfometria gi rata de supraviepire. xaminarea macroscopici a evolutiei tumorale re-levi c i in cazurile administrkii preventive a extractelor,se produce reducerea evolufiei tumorale.in cazul adrninistrZrii terapeutice, se produce lizatumorali.

MATERIAL AND METHODThe experiment was made on 140 Wistar rats withRS1 Hepatoma (induced by administration, subcuta-neous of 2,500,000 - 4,000,000 tumor cells 10.5 ml0,9%NaCl) and tested extracts were administrated in severalmodes.Experimental lots:- Lot I (preventive) - extracts administered previ-ously to tumor innoculation (2 months before);- Lot II (therapeutic) - extracts administered con-comitantly (at the same time) with tumour innoculation- Lot ILI (tardy therapeutic) - extracts administeredat 25 days after tumor inoculation;- Lot IV - ,,control" without treatment and inoculat-ed with tumor;- Lot V - ,,control" without treatment and tumor;- Lot VI - ,,controlu reatment and without tumor;- To all animals, extracts were administered both infood and in peritumoral applications.There were surveyed: whole body weight, tumorvolums, survival rates, and after sacrifications: tumorweight, macroscopic aspects, as well as some molecularmarkers: malondialdehyde, thiols, P450 cytochromesconcs, ceruloplasrnin and GST activities, S-phase cellfractions, prolipherative index. (1,2)

$ESULTS AND DISCUSSIONS1.Morphometry and survivalThe Macroscopic Exam of the tumor evolution re-veals, for the cases under previously or therapeutic treat-ment, a reduced tumor evolution.In therapeutic cases appear the tumor lysis.1.a. SurvivalMean TimeThe extracts administration increases survival rates:

Fig. 1 - Lot I (preventiv)/ (preventive)Extractele din plante administrate cu 2 luni inaintede inocularea tumorali; aspect macroscopic la 25 ziledupi inoculare-/Extracts administeredpreviouslyto tumor innoculation (2 months before); macroscopicaspect at 25 days afe r innoculation

Fig. 2 - Lot 11(terapeutic) (therapeutic)Extractele administrate concomitent cu inoculareatumoral5i; aspect macroscopic la 25 zile dupginocularea tumorali / Extracts administered concomi-tently with tumour innoculation; macroscopicaspect at 25 days after innoculation


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REVISTAROWE ONCOLOGIE COMPARAT~I RONlANlAN JOURNAL OF COMPARATIVE ONCOLOGY No. 1512008

Fig. 3 - Lot I1 (terapeutic) (therapeutic)Aspect macroscopic a1 tumorii izolate la 25 ziledupi inocularea tumorali /Macroscopic aspectof isolated t u m r at 25 days afrer innoculation1.a. Timpul mediu de supraviefuireAdministrarea extractelor din plante favorizeazicre$terea ratei de supravietuire, astfel: 42 zile - variantapreventivi, 38 zile - terapeutic $i 37 zile - terapeutic tar-div, comparativ cu martorul- 31 zile (graficul 1) .1.b. Greutate/volum tumoralValorile greut2~ii/volurnuluiumoral sunt semnifica-tiv reduse, attit fn varianta preventivi - 1,39 cm3 / 1,48 g,c3t $i fn cea terapeutici - 3,83 cm3 / 4,64 g, comparativcu administrarea tardivi - 12,55 cm3 / 14,27 g, sau cucele ale martorului - 23,05 cm3 / 24,31 g (graficul2).2.Analiza fazelor ciclului celular yi a indicilor deproliferare celularii prin metoda citometrieiin fluxTumorile RS1 sunt agresive (S = 19,93%, $i neardi-ploide (indice AND = 1,22).Sensibilitatea celulelor tumorale la extractele dinplante, diferi in func@e e momentul administririi pro-duselor testate.

Fig. 4 - Lot IV - Martor - inoculat cu tumora $inetratat / ,,Control1'(with tumor and without treatment)Aspect macroscopic a1 evolufieitumorale la 25 ziledupi inocularea tumorali / Macroscopic aspect oftumor evolution at 25 days after the tumor innoculation

Fig. 5 - Lot IV - Martor - inoculat cu tumora $inetratat / ,,Controlu (with tumor and without treatment)Aspect macroscopic a1 evolutiei tumorale la 25 ziledupi inocularea tumorali / Macroscopic aspect otumor evolution at 25 days afrer the tumor innoculation42 days fo r preventive, 38 d for therapeutic, and 37 daysfor tardive therapeutic, comparing to 3 1 d fo r control lot.(graphic 1).

LOT 1 -2 months before tumorinnoculation

I LOT I I - conncomitently withtumor innoculationI LOT I l l - 25 days after tumorinnoculationd LOT IV - "control" withouttreatment and inoculated withtumor

Graficul 1 / Graphic 1 - Tmpul mediu de supraviefuire(zile) /Mean time survivaWdays


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REVISTA ROMBN~~E ONCOLOGIE COMPARAT~~ ROMANIAN JOURNAL OF COMPARATIVEONCOLOGY NO. 1512008

hic 2 - Media volumului

Channels (FU-A)Fig. 6 - Lot I - preventiv / Lo t I - preventive

J

I LOT 1 -2months before tumorinnoculation

I LOT I I - conncomitently withtumor innoculationI LOT I l l - 25 days after tumorinnoculation' ' ^T IV - "control" without3tment and inoculated withtumor---- ---

tumoral /lotlcm3/Mean volume

Fig. 7 - Mot IV - r ioculat cu tumori$i f81-5tratament / Lot IV - ,,controla withouttreatnzent and innoculated with tumor1.b. Tumor weight/volumeThe weight/volurne tumor values are significduced in both the preventive - 1,39 cm3 / 1,48 gconcomitant phase - 3,83 cm3/ 4,64 g, if com

the late administration - 12,55 cm3 / 14,27ones of the control lot - 23,05 cm3124,3 1 g2. The analysis of cell cycle phases "*rproliferative indices, determined by the hW@ume-tric methodRS 1 tumors are aggressive (S= 19,93%), and near-diploid (DNA index = 1,22)

o The sensitivity of tumor cells to the plant extractstreatment varies depending on the moment when the testedFig. 8 - Lot I - terapeutic /Lot 1 therapeutic lot product is administrated.At the analyzed tumors, the antitumor effect reachesLa tumorile analizate, efectul antitumoral atinge ni- the maximum level when the plant extracts are previouslyvele maxime, in varianta administriirii preventive a extrac- administrated, with decreasing of DNA synthesis to the1002


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REVISTAR O M ~E ONCOLOGIE COMPARAT~I ROM ANI AN JOURNAL OF COMPARATIVE ONCOLOBY NO. 1512008

iI OT 1 -2 months before tumorinnoculation

LOT II - conncomitently withtumor innoculationL O T ll - 25 days after tumorinnoculation

1 w LOTIV - "control. withouttreatment and inoculatedwithtumor. - . . - . . . -Graficul 3 /Graphic 3 - Valorile Indicelui de Stres Oxidativ / I S 0 Value

telor din plante, prin scriderea sintezeiADN pin3 la limitenormale (S = 4%) ~i acumularea celulelor in faza G I.Distribuirea cantitativg a AND-ului in fazele ciclu-lui celular pentru lotul preventiv.Distribuirea cantitativi a AND-ului in fazele ciclu-lui celular pentru lotul martor.Distribuirea cantitativg a AND-ului in fazele ciclu-lui celular pentru lotul I1 (extractele administrate con-comitent cu inocularea turnoralg).

normal limit (S = 4%) and accumulation of cells in G1phase.DNA quantitativedistribution in the cell cycle phas-es for the preventive lotThe DNA quantitative distribution in cell cyclephases for the control lotDNA quantitative distribution in cell cycle phasesfor lot I1 (extracts administered concomitently with tu-mour innoculation)3.Valorile Indidui de Sh.es Oxidativ (ISO)Produsele testate prezinti un efect protector asuprastresului oxidativ, prin scgderea valorilor indicelui (ISO),

in special ?n varianta administr8rii preventive (graficul3).

13.The oxidative stress index ( IS 0 Value)The products have a oxidative stress protectioneffec t, decreasing the oxidative stress index, especially inthe prevention phase (graphic 3).

4. Markerii biologici ai sistemului de detoxifiereP450 prezintil cea mai sernnificativg descrevtere(cca 40%) Fn cazul asocierii sinergice dintre evolutia tu-moral3 ~i administrarea preventivl a extractelor dinplante (lot I), deoarece acestea actioneazi in primele fazeale evolutiei tumorale (graficul4).

4. Bilogical markers for the detoxification systemP450 - the most signifficant degrease appears (cca40%) in the case of the synergic association of the tumorevolution, with the preventive administration of extracts(lot I), because these act in the very early stages of tumorevolution (graphic 4).0.45 / LOT 1 -2 months before tumor0.401 innoculation0,4 II W LOT II - conncomitentlywith0,35 ' I tumor innoculation

W LOT I11 - 25 days after tumor

ILOT IV - "control" without: treatment and inoculatedwith; tumorIW LOT V - "control" withoutI treatment and tumorI LOT Vl - "control" treatment and, ithout tumor

Graficul4 / Graphic 4 - Activitatea citocromilor P450 / The activity of P450 c y t o c hmm~


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REVISTAROWE ONCOLOGIE COMPARAT~ ROMANIAN JOURNAL OF COMPARATIVE ONCOLOGY NO.1512008IA... .OT I - 2 months before tumorinnoculationLOT I I - conncomitently with tumorinnoculationLOT I l l - 25 days after tumorinnoculationLOT IV - "control"withouttreatment and inoculated withtumorLOT V - "control"without treatmentand tumor

I LOTVI - "control"treatment andwithout tumor

Graficul5 /Graphic no. 5 - Activitatea glutationtransferazei (GST)/GST activitiesGS T - activitatea enzimaticri cre$te cu aproximativ GST - the enzyme activity aproximatively grows50% la animalele shitoase qi tratate (Lot IV) datorid efec- with 50% at the healthy animals (VI) due to the inductivetului inductiv al extractelorasupra GST-ului,comparativ cu effect of plants extract on GST, compared with the healthyanimalele shi toase $i netratate (Lot V) (graficul5). Nive- and nontreated ones (lot V) graphic no 5).lul concentratiei P450 scade, in timp ce nivelul activititii P450 concentration degreases, while GST activity isGST crqte, ca rezultat a1 actiunii enzimaticeprotectoare a1 increased as a result of the plants protective enzymesextractelor din plante aspura organismului animal. action.

CONCLUZII1. Abinis t rarea extractelor din plante produc creqterearatei de supraviepire: 42 zile pentru varianta preven-tivi, 38 zile pentru cea terapeuticg $i 37 zile pentruvarianta terapeutic tardiv, comparativ cu 3 1 zile pentrulotul martor.2. Indiferent de varianta administririi, la loturile tratate,s-a observat o descrqtere semnificativi a volumuluitumoral (peste 50%), comparativ cu lotul martor.3. Dirninuarea valorilor markerilor oxidativi, indicg efec-tul protector a1produselor asupra stresului oxidativ, in

special in varianta adrninistriirii preventive.4. La tumorile analizate prin citometrie in flux, afectulantitumoral atinge nivele maxime, in varianta admin-istriirii preventive a produselor testate, respectiv, prinsciderea sintezei de ADN, p i h i la limita normali(S=4) $i acurnularea celulelor in faza G I .5. Valorile enzimelor implicate in sistemele de detoxi-fiere, respectiv, concentratia citocromi P450 $i activi-tatea GST, relevti eficienfa produselor testate. S-a ob-servat o creftere semnificativia activitioi GST, at& laanimalele shi toase , c l t $i la cele tratate, in special invarianta preventivri $i terapeutici.

CONCLUSIONS1. The extracts administration increases survival rates: 42days fo r preventive, 38 d for therapeutic, and 37 d fortardive therapeutic, comparing to 3 1 d for control lot.2. A significant tumor volume decrease in extracts cases,(more than 50%) was observed, comparing to tumorcase.3. Diminution of the oxidative markers indicated a pro-tective effect over oxidative stress, mainly in 'preven-tive' administration.4. At the analyzed tumors by flow-citometry, the antitu-mor effect reaches the maximum level when the plantextracts are previously administrated, with decreasingof DNA synthesis to the normal limit (S = 4%) andaccumulation of cells in G 1 phase. *5. Values of cytocromes P4 50 concs and GST activities -enzymes involved in detoxication pathways - revealedthe efficiency of studied extracts. Significant increas-es of GST activities were noticed, both in healthy ani-mals and in tumor cases, especially in preventive andtherapeutic cases.


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REVISTAR O M ~ ~E ONCOLOGIE COMPARAT~I OMANIAN JOURNAL OF COMPARATIVEONCOLOGY No. 15120086. Lulnd in considerare rezultatele mentionate anterior,respectiv: nivel crescut de supraviepire, greutate cor-poralI crescutg, sciderea indicelui de proliferare tu-moralii, efectul protector asupra stresului oxidativ si a1

sistemului de detoxifiere, se poate considera c l produ-sele testate prezintSi efect imunostimulator. Extracteledin plante testate pot fi utilizate ca posibili adjuvanti inchimioterapia la animalele de laborator, datoriti nive-lelor reduse privind toxicitatea qi efectele secundare.

6 . Taking into acount the previous mentioned results: ahigher survival rate, a higher weight, decreases of pro-liferative index, effects on enzymes and systems invol-ved in oxidative stress or detoxication, we assume thatplant extracts exhibit an irnmunostirnulatory effect.We shall mention the lack of toxicity and also of thesecondary effects, suggesting so that the investigatedextracts may represent some possible adjuvants forchemotherapy in laboratory animals.

BTBLIOGRAFIE SELECTIVA 1SELECTIVE REFERENCES

'9 .Habig W.H., Pabst H.J., Jakoby W.B. - GlutathioneS-trunsferuses, thefirstenz>matic step in mercapbric acid formatiopn. J. Biol. Chem. 1974, 249,(22). 7130-9-12. Omllra T, Sa to R - The carbon monoxide - binding pigment of liver micro-somes. Solubilization,purijcation, properties. J.Biol.Chem 1964, 239, (7),2379-85.

13. Voinea-Fanica E ne - Ghid de terapie nauristc, editia a IV-a revizuid, Editura

. ALL, Bucueqti 2004, p a ~ 5 2 1 .P4. Voinea-Fanica E ne - Imunoterapia cancerulzri cu mijloace naturale, extrasdin comunicarea qtii@>ca' la cea de a IX-a Conferinfa' Intema!ionala' deTerapii C?mpfe?q?@re_dee&Arad - Rominia, 2005

6 a@nd*.r7.69. , $11,- . l42, . ,..? . ,-, ,*&,,> $..*, b< .A , ,------

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