Anti-Tumor Effect of Azadirachta indica (Neem) on Murine Solid Ehrlich Carcinoma

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1 Acdemic Journl of Cncer Reserch 7 (1): 38-45, 2014 ISSN IDOSI Pulictions, 2014 DOI: /idosi.jcr Anti-Tumor Effect of Azdircht indic (Neem) on Murine Solid Ehrlich Crcinom 1 2 Fthey M. Metwlly, Htem A. El-Mezyen, 3 1 Ahmed E. Adel Moneim nd Nevin E. Shrf 1 Environmentl nd Occuptionl Medicine Deprtment, Ntionl Reserch Centre, Dokki, Giz, Egypt 2 Chemistry Deprtment, Fculty of Science, Helwn University, Ciro, Egypt 3 Zoology nd Entomology Deprtment, Fculty of Science, Helwn University, Ciro, Egypt Astrct: Tumor growth cn cuse ntioxidnt disturnces in certin tissues of the tumor host. So, we exmined the ntioxidnt system s possile mechnism through which Neem leves preprtion (NLP) exerts its oncosttic potentil. Femle Swiss Alino mice were inoculted intrmusculrly in the right thigh with Ehrlich scites crcinom (EAC) cells. NLP (500 mg/kg ody weight) ws injected for 20 dys intrperitonelly into mice eginning on dy 5 of post-eac cell inocultion. Tumor growth, lipid peroxidtion (LPx), glutthione (GSH) contents nd the ctivity of the ntioxidnt scvenger enzymes were exmined. Results indicted tht NLP efficiently suppressed the growth of tumors which ws ssocited with normliztion of the LPx levels nd ugmenttion of GSH contents. NLP enhnced the ctivity of the endogenous ntioxidnt scvenging enzymes, superoxide dismutse (SOD), glutthione peroxidse (GPx), ctlse (CAT) nd glutthione-s-trnsferse (GST) in liver nd tumor tissue. The effect of NLP ws more pronounced when treted s erly s dy 5 of post-tumor cell inocultion. In conclusion, NLP induced oncosttic ctivity y modulting lipid peroxidtion, ugmenting the ntioxidnt defense system nd protecting ginst oxidtive stress. Key words: Antioxidnt enzymes Ehrlich Crcinom GSH Neem lef preprtion INTRODUCTION popultion studies, correltion ws found etween high intke/high lood levels of ntioxidnts nd low incidence Tumor growth cn cuse ntioxidnt disturnces of different types of cncer [3]. Antioxidnts lock in certin tissues of the tumor host [1]. One of the crcinogenesis y multiple mechnisms tht include chrcteristics of tumor growth nd invsion is the prevention of procrcinogen ctivtion, inhiition of cell incresed flux of oxy-rdicls nd loss of cellulr redox prolifertion, invsion nd ngiogenesis nd stimultion homeostsis. Cncer cells cn generte lrge mounts of of poptosis [4]. Of lte, medicinl plnts rich in hydrogen peroxide, which my contriute to their ility ntioxidnt phytochemicls hve received growing to mutte, dmge norml tissues nd invde other ttention s potentil chemo-preventive gents. tissues. This suggests tht there is direct correltion Severl modern nticncer drugs hve een developed etween chnges in the rte of cncer cell prolifertion from trditionl medicinl plnts [5]. The Neem nd chnges in the ntioxidnt mchinery. Furthermore, (Azdircht indic) tree contins different ioctive some nticncer gents cn ct s ntioxidnt [2]. compounds, which re of interest for their eneficil Severl iologicl response modifiers (BRMs) hve een helth effects nd considered s n nti-genotoxic nd exmined for nticncer ctivity with limited success due chemo-preventive potentil of ethnol extrct of Neem to toxicity. The need for new cncer therpy with leves ginst orl nd stomch tumors [6-8]. miniml or no side effects is gretly wrrnted. In most Extrcts of Neem leves hve een found to possess Corresponding Author: Fthey M. Metwlly, Environmentl nd Occuptionl Medicine Deprtment, Ntionl Reserch Centre, Dokki, Giz, Egypt. 38

2 Acdemic J. Cncer Res., 7 (1): 38-45, 2014 immunomodultory nd nti-inflmmtory properties Experimentl Design: After 2 weeks of dpttion, [8-10]. Therefore, it ws in our interests to exmine mice were rndomly ssigned into four groups whether NLP might suppress solid Ehrlich crcinom (8 mice/group) s follows: (SEC) growth through the ntioxidnt system in vivo. Group (1): mice were fed on stndrd control diet nd MATERIALS AND METHODS injected intr peritonel with phosphte uffer sline (PBS) dily for 20 dys. This group set s helthy control Animls: The current study ws conducted on 32, group. 8 weeks old femle Swiss Alino mice (24 ± 2g). The nimls were otined from the Animl House Colony Group (2): fed stndrd control diet nd injected with 500 of the Ntionl Reserch Centre, Dokki, Giz, Egypt nd mg/kg ody weight NLP dily for 20 dys, ccording to mintined in stndrd lortory conditions. The nimls Blsenthil et l. [13]. were housed in plstic cges t room temperture (25±2 C) nd humidity (55%). Mice were controlled Groups (3-4): were intrmusculrly injected with 0.2ml of constntly with 12h light drk cycle t Ntionl 6 EAC, which contined cell in the right thigh of Reserch Centre, Animl Fcility Breeding Colony. the lower lim for production of solid tumors. After 5 dys They were provided with tp wter nd stndrd of EAC cells injection. lortory diet d liitum. The stndrd lortory diet consists of csein 10%, slts mixture 4%, Group (4): (SEC+NLP) ws treted with 500 mg/kg ody vitmins mixture 1%, corn oil 10% nd cellulose 5% weight NLP dily for 20 dys, while group 3 (SEC) served completed to 100 g with corn strch [11]. Animls were s positive control. llowed to cclimte for two weeks to the housing conditions nd received humn cre in complince with the guidelines of the Ethicl Committee of Medicl Smple Collection: At the end of the experiment (dy 25), Reserch of the Ntionl Reserch Centre, Dokki, Giz, nimls were fsted for 16 h then were nesthetized. Liver Egypt. nd tumor tissues were excised nd wshed in ice-cold norml sline, lotted dry nd weighed. Ech tumor ws Tumor Cell Line: The murine Ehrlich scites crcinom weighted individully (TW/g). A 10% w/v homogente (EAC) cells used in this study were originlly otined ws prepred in ice-cold phosphte uffer (0.1M, ph 7.4) from the Ntionl Cncer Institute, Ciro University, Ciro, using homogenizer for oth liver nd tumor tissues. Egypt nd mintined in vivo y weekly intr-peritonel 6 (i.p.) pssge of cells in femle Swiss lino mice. Body Weight Chnges: Body weight (BW/g) nd ws Viility, ssessed y the trypn lue dye exclusion monitored throughout the experimentl time course. method, ws found to e 95% or more. BW ws exmined for initil, finl nd net BWs t dy 25. The net finl BW = finl BW-tumor weight. BW gin ws Neem Lef Preprtion (NLP): Fresh mtured leves determined s the difference etween initil nd net finl of Neem tree were collected from the grden of BW. Al-Oour City, Ciro, Egypt. The smples were identified in the Botny Deprtment, Fculty of Science, Evlution of the Antioxidnt Activity Helwn University, Ciro, Egypt. The leves were clened, Determintion of Glutthione (GSH): dried nd powdered; the powder ws used for the Reduced glutthione (GSH) ws determined using method preprtion of crude methnolic extrct ccording to the of Ellmn et l. [14]. The method ws sed on the procedure descried y Mnikndn et l. [12] with some reduction of 5, 5` dithiois 2-nitroenzoic cid (DTNB) modifiction. Air-dried powder (100g) of Neem leves with GSH producing yellow compound which cn e were mixed with 100 ml methnol (70%) nd kept in mesured t 405 nm. refrigertor for 24 hours. The extrct of Neem leves ws concentrted nd dried under vcuum evportor. Determintion of Thiorituric Acid Rective The residue ws dissolved in distilled wter, filtered nd Sustnces: Thiorituric cid rective sustnces used in the experiment. (TBARS) were ssyed clorimetriclly in liver nd tumor 39

3 Acdemic J. Cncer Res., 7 (1): 38-45, 2014 homogente ccording to the method of Ohkw et l. dichloro-2-hydroxyenzene sulfonic cid (DHBS) nd [15]. In riefly, 1ml of trichlorocetic cid 10% nd 1ml of 4-minophenzone (AAP) to form chromophore with thiorituric cid 0.67% were heted in oiling wter color intensity inversely proportionl to the mount of th for 30 min. TBARS ws formed nd the sornce CAT in the originl smple. mesured t 535 nm. Assy for Superoxide Dismutse Activity: Assy for Glutthione-S-trnsferse Activity: Superoxide dismutse (SOD) ctivity ws ssyed Glutthione-S-trnsferse (GST) ctivity ws ssyed y the method of Nishikimi et l. [20]. This ssy ccording to method of Hig et l. [16]. Tht method ws relies on the ility of the enzyme to inhiit the phenzine sed on conjugtion of 1-chloro-2, 4-dinitroenzene methosulphte-medited reduction of nitrolue (CDNB) with reduced glutthione. The conjugtion is tetrzolium dye. ccompnied y n increse in sornce t 340 nm. The rte of increse is directly proportionl to the GST Sttisticl Anlysis: Dt were expressed s the ctivity in the smple. men ± stndrd error of the men (SEM). For the comprison of significnce etween groups, Assy for Glutthione Peroxidse Activity: Mnn Whitney U-test ws used. Significnce level ws Glutthione peroxidse (GPx) ctivity ws mesured y recorded t p<0.05. All sttistics were done ccording to the method of Pgli nd Vlentine [17]. The ssy is n the sttisticl progrm softwre "Medicl version 11.0". indirect mesure of the ctivity of GPx. Oxidized glutthione (GSSG), produced upon reduction of orgnic RESULTS peroxide y GPx, is recycled to its reduced stte y the enzyme glutthione reductse. The oxidtion of NADPH Effect of NLP Tretment Tumor Weight (TW): + to NADP is ccompnied y decrese in sornce t Dt in Tle 1 showed tht, on dy 25 of the experiment, 340 nm. the men tumor weights of untreted group (SEC) ws mrkedly increse (4.54 ± 0.24 g) compred to tumor Assy for Glutthione Reductse Activity: ering niml nd treted with NLP (SEC + NLP) which Glutthione reductse (GR) ctivity ws ssyed y the record 3.43 ± 0.43g. Tretment with NLP recorded method of Fctor et l. [18]. GR ctlyses the reduction of 24.44% reduction in TW. glutthione in the presence of NADPH, which ws + oxidized to NADPH. The decrese in sornce ws Effect of NLP Tretment on Body Weight (BW): mesured t 340 nm. The effect of NLP tretment on ody weight (BW) ws investigted weekly, for 25 dys fter tumor chllenge. Assy for Ctlse Activity: Ctlse (CAT) ctivity ws As shown in Tle 1, control mice hd BW chnge of ssyed y the method of Aei [19]. CAT rects with 13.4%. In ddition, mice not inoculted with EAC nd known quntity of H2O 2. The rection ws stopped fter treted with NLP hd BW gin of 5.25%. Menwhile, exctly one minute with ctlse inhiitor. In the presence untreted mice ering SEC nd treted mice ering SEC of peroxidse (HRP), remining H O rects with 3,5 showed BW loss of 13.9% nd 5.27%, respectively. 2 2 Tle 1: Effect of dministrtion of NLP on ody weight (gm) in mice-ering Ehrlich solid crcinom Prmeter Control NLP SEC SEC + NLP Initil ody weight (g) ± ± ± ± 0.85 Lst ody weight (g) ± ± ± ± 0.43 % of chnge % Net finl ody weight (g) ± ± ± ± 0.54 Body weight chnge (g) % of chnge from the initil BW % % % % Ech vlue represents the men ± SE Net finl ody weight = (Lst ody weight-tumor weight) Body weight chnge = (Net finl ody weight-initil ody weight) : p < 0.01 s compred with control group : p < 0.01 s compred with SEC group 40

4 Acdemic J. Cncer Res., 7 (1): 38-45, 2014 Tle 2: Effect of NLP dministrtion on MDA nd GSH content in liver nd tumor tissue of different experimentl groups MDA GSH Animl groups Liver nmol/g tissue Tumor nmol/g tissue Liver µmol/g tissue Tumor µmol/g tissue NLP 49.23± ± % of chnge -19% % -- SEC ± ± ± ±0.01 % of chnge 71.2% % -- SEC+ NLP c 71.01±2.79 c 67.74± ±0.05 c 1.81±0.04 % of chnge % % 13% 81% Ech vlue represents the men ± SE : p < 0.01 s compred with control group : p < 0.05 s compred with control group c: p < 0.01 s compred with SEC group : percentge of chnge from control : percentge of chnge from SEC group Tle 3: Effect of NLP dministrtion on ntioxidnt enzymes, SOD nd ctlse ctivity in liver nd tumor tissue of different experimentl groups. SOD Ctlse Animl groups Liver U/g tissue Tumor U/g tissue Liver U/g tissue Tumor U/g tissue Control 2.17± ± NLP 2.62± ± % of chnge 20.7% -- 25% -- SEC 1.87± ± ± ±0.03 % of chnge -13.8% % -- SEC+ NLP 3.43± ± ± ±0.06 % of chnge 83.4% 34.5% 87.5% 51.5% Ech vlue represents the men ± SE : p < 0.01 s compred with control group : p < 0.01 s compred with SEC group : percentge of chnge from control : percentge of chnge from SEC group Tle 4: Effect of NLP dministrtion on ntioxidnt enzymes, GPx, GST ctivity in liver nd tumor tissue of different experimentl groups Gpx GST Animl groups Liver U/g tissue Tumor U/g tissue Liver µmol/h/g tissue Tumor µmol/h/g tissue Control 0.87± ± NLP 1.17± ± % of chnge 34.5% % -- SEC 0.76± ± ± ±0.01 % of chnge -12.6% % -- SEC+ NLP 1.18± ± ± ±0.06 % of chnge 55.3% 27.8% 23.3% 72.2% Ech vlue represents the men ± SE : p < 0.01 s compred with control group : p < 0.01 s compred with SEC group : percentge of chnge from control : percentge of chnge from SEC group Effect of NLP on Antioxidnt Sttus: Lipid Peroxidtion (Mlondildehyde) Content: The effect of NLP tretment on the level of lipid peroxidtion ws mesured in term of mlondildehyde (MDA) in liver nd tissues of tumor ws depicted in Tle 2. Untreted mice ering solid tumor (SEC group) showed mrked elevtion in MDA level in liver (71.2%, p < 0.01) s compred to the norml nimls. Tretment with NLP on dy 5 post-tumor cell inocultion in SEC-ering mice returned MDA levels to e close to control vlues in liver (-31.79%) nd tumor (-25.54%), s compred with the untreted control SEC-ering mice. 41

5 Acdemic J. Cncer Res., 7 (1): 38-45, 2014 Glutthione (GSH) Level: As summrized in Tle 2, [23, 24] vi scvenging free rdicls [25] nd inhiition of tretment with NLP solely reveled significnt chnge in lipid peroxidtion [21]. Antitumor ctivity of flvonoids, liver GSH level (18.9%, p>0.01) when compred with isolted from severl sources other thn Neem, hs een norml control vlues. A significnt depletion in GSH reported y Horvthov et l. [26]. Tht compounds hve level ws recorded in the liver of niml ering tumor een shown to hve nti-prolifertive effects on humn group (-27.36%, p<0.01) s compred to their squmous cell crcinom [27]. Six phenolic compounds corresponding norml controls. Tretment with NLP including gllic cid, enzoic cid, p-coumric cid, significntly restored GSH content in the liver to the p-hydroxyenzoic cid, vnillic cid nd trns-cinmic norml vlues nd elevted its level in tumor tissue cid were isolted nd identified in oth Neem rk nd ove the vlues of the untreted mice ering SEC leves. Polyphenolics re known for their potent (81%, p<0.01). ntioxidnt nd free rdicl scvenging properties [28]. The present results reveled mrked depletion in GSH The Activities of Antioxidnt Scvenger Enzymes: content s well s the ctivities of the ntioxidnt The effect of NLP on the ctivity of ntioxidnt scvenger enzymes, GPx, GST, SOD nd CAT in the liver scvenger enzymes in norml nd SEC tumor-ering mice nd tumor tissues of tumor-ering mice. The reltionship ws exmined. These enzymes include glutthione mong cncer growth, GSH content nd the ntioxidnt peroxidse (GPx), glutthione-s-trnsferse (GST), system hs een lso studied. GSH, potent inhiitor of superoxide dismutse (SOD), Glutthione reductse (GR) the neoplstic process, plys n importnt role s n nd ctlse (CAT). Dt in Tle 3 showed tht, endogenous ntioxidnt system tht is found in dministrtion of NLP solely reveled insignificnt chnge prticulrly high concentrtion in the liver nd is known in SOD nd CAT ctivities of liver when compred to the to hve key function in the protective process [29]. norml control group. The ctivities of these enzymes in Reduced ctivities of the GSH nd GSH relted the liver nd tumor tissue of the SEC group ws enzymes such s GPx in cncer ptients were lso significntly lower (p<0.01) thn tht of their reported y Blsurmniyn et l. [30] nd Wong et l. corresponding control. Tretment with NLP to [31]. It ws reported tht during cncer growth, SEC-ering mice elevted SOD nd CAT ctivity in liver glutthione redox (GSH/GSSG) decreses in the lood of to e comprle with the norml levels. In ddition, Ehrlich scites tumor-ering mice, minly due to n tretment with NLP mrkedly ugmented SOD nd CAT increse in lood GSSG levels s result of oxidtive ctivity in the tumor tissue (p<0.01) when compred with stress. This increse my e cused y n increse in the untreted SEC vlues. Tle 4 indicted tht, peroxide production y tumor cells tht cn led to GSH dministrtion of NLP solely reveled significnt increse oxidtion within the red lood cells nd incresed in GPx level in liver (34.5%, p<0.01) when compred with GSSG relese from different tissues into the lood [32]. the norml control. SEC group showed gret depletion GSH-Px plys n importnt role in metolizing lipid in liver ctivities of Gpx nd GST [(-12.6%, p<0.01) nd peroxides in the liver nd this enzyme decrese is (-4.6%, p<0.01), respectively] compred with norml potentilly scrile to inctivtion y the increse in control group. Tretment with NLP mrkedly ugmented ROS or lipid peroxide formtions [33]. In the current study, GPx nd GST ctivity in the tumor tissue (p<0.01) when we lso oserved tht the detoxifying enzyme compred with the untreted SEC vlues [(27.8%, p<0.01) glutthione-s-trnsferse (GST) ctivity ws significntly nd (72.2%, p<0.01), respectively)]. dropped in the liver of the SEC-ering mice. Our results were in ccordnce with others who detected low liver DISCUSSION GST ctivity in SEC-ering mice [34] nd in lung cncer-ering nimls [35]. SOD, CAT nd GPx re Administrtion of NLP t dy 5 post-tumor cell involved in the clernce of superoxide nd hydrogen inocultion to tumor-ering mice showed mrked peroxide. SOD ctlyses the diminution of superoxide into nd progressive suppression of the tumor growth. H2O 2, which hs to e eliminted y GPx nd/or CAT [36]. The ntitumor ctivity is exemplified y significnt The decline in SOD ctivity ws oserved in different reduction in TW. The iologicl ctivity of Neem tissues of SEC-ering mice [2, 37, 38]. It is worth leves ws due to its rich content of flvonoids [21]. mentioning tht SOD ctivity plys n importnt role in Flvnoids hve een reported to possess oth the ntitumor effects of ctive oxygen-forming nticncer ntioxidnt ctivity [22] nd nti-inflmmtory ctivities gents. However, when the oxidtive dmge is extreme s 42

6 Acdemic J. Cncer Res., 7 (1): 38-45, 2014 result of tumor growth, ROS scvenging enzymes such 4. Vlko, M., et l., Free rdicls nd ntioxidnts s SOD nd ctlse re degrded [39]. The inhiition of in norml physiologicl functions nd humn CAT ctivity in different tissues of mice ering n disese. Int. J. Biochem. Cell Biol., 39(1): Ehrlich tumor s result of tumor growth ws lso 5. Crgg, G.M. nd D.J. Newmn, Plnts s reported y severl investigtors [2, 40]. source of nti-cncer gents. J. Ethnophrmcol., Results of the present study demonstrted the 100(1-2): ntioxidnt nd free rdicl scvenging property of NLP, 6. Supriy, R. nd S. Ngini, Ethnolic neem lef s exemplified y the ility of NLP to increse ctivity of extrct protects ginst N-methyl-N'-nitro-Nthe ntioxidnt enzymes in the cells of norml nimls nd nitrosogunidine-induced gstric crcinogenesis in in nimls ering tumors. In ddition, NLP normlized the Wistr rts. Asin Pc. J. Cncer Prev., 4(3): level of LPx in liver nd tumor tissue in nimls ering 7. Supriy, R., V. Bhuvneswri nd S. Ngini, tumors. In the present finding, the free rdicl scvenging Ethnolic neem (Azdircht indic) lef extrct GSH content in liver nd tumor tissue of mice treted with induces poptosis in the hmster uccl pouch NLP ws found to e significntly higher thn tht in the crcinogenesis model y modultion of Bcl-2, untreted SEC-ering mice. Elevtion in the ctivity of Bim, cspse 8 nd cspse 3. Asin Pc. J. Cncer GST, help in susequent initition of the poptotic Prev., 6(4): process in tumor cells hve enormous clinicl significnce 8. Supriy, R., et l., Ethnolic lef extrct of for immunotherpy of vrious forms of cncer with neem (Azdircht indic) inhiits uccl pouch completely nontoxic therpy [41, 42]. An ethnolic extrct crcinogenesis in hmsters. Cell Biochem. Funct., of Neem hs een shown to induce cell deth in prostte 23(4): cncer cells (PC-3) y inducing poptosis s evidenced 9. Ry, A., B.D. Bnerjee nd P. Sen, y dose-dependent increse in DNA frgmenttion nd Modultion of humorl nd cell-medited immune decrese in cell viility [43]. Further, their studies responses y Azdircht indic (Neem) in mice. indicted tht tretment with Neem extrct could decrese Indin J. Exp. Biol., 34(7): level of Bcl-2, which is n nti-poptotic protein nd t the 10. Chttopdhyy, R.R., Possile iochemicl sme time, incresed expression of pro-poptotic Bx mode of nti-inflmmtory ction of protein. Moreover NLP ws le to cuse tumor Azdircht indic A. Juss. in rts. Indin J. Exp. regression in SEC-ering mice through the induction of Biol., 36(4): cncer cell poptosis vi its immunomodultory effect 11. Shly, A.B., A.H. Hmz nd H.H. Ahmed, [44]. New insights on the nti-inflmmtory effect of some In conclusion, our results strongly suggest tht, Egyptin plnts ginst renl dysfunction induced NLP represent high potentil for ntitumor ctivity y cyclosporine. Eur. Rev. Med. Phrmcol. Sci. in vivo vi its rdicl scvenging effect y encountering 16(4): free rdicls fter tumor cells inocultion nd in the future 12. Mnikndn, P., et l., Evlution of it my e used s djuvnt chemotherpy. Azdircht indic lef frctions for in vitro ntioxidnt potentil nd in vivo modultion of REFERENCES iomrkers of chemoprevention in the hmster uccl pouch crcinogenesis model. Food Chem. Toxicol., 1. Wenger, F.A., et l., Influence of octreotide on 46(7): liver metstsis nd heptic lipid peroxidtion in 13. Blsenthil, S., et l., Chemopreventive BOP-induced pncretic cncer in Syrin hmsters. potentil of neem (Azdircht indic) on Pncres, 23(3): ,12-dimethylenz[]nthrcene (DMBA) induced 2. Gupt, M., et l., Antitumor ctivity nd hmster uccl pouch crcinogenesis. J. ntioxidnt role of Buhini rcemos ginst Ethnophrmcol., 67(2): Ehrlich scites crcinom in Swiss lino mice 14. Ellmn, G.L., Tissue sulfhydryl groups. [corrected]. Act Phrmcol. Sin., 25(8): Arch. Biochem. Biophys., 82(1): Suzuki, T., et l., Effect of dietry ntioxidnts 15. Ohkw, H., N. Ohishi nd K. Ygi, nd risk of orl, phryngel nd lryngel squmous Assy for lipid peroxides in niml tissues y cell crcinom ccording to smoking nd drinking thiorituric cid rection. Anl Biochem, hits. Cncer Sci., 97(8): (2):

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