Anticarcinogenic and Antiplatelet Effects of Carvacrol S

Experimental Oncology �� ne �� Exp Oncol �� ANTICARCINOGENIC AND ANTIPLATELET EFFECTS OF CARVACROL S. Karkabounas1, *, O. K Kostoula1, 5, T. Daskalou1, P. Veltsistas2, M. Karamouzis4, I. Zelovitis1, A. Metsios1, P. Lekkas1, A. M. Evangelou1, N. Kotsis1, I. Skoufos3 1Laboratory of Physiology, Faculty of Medicine, University of Ioannina, Ioannina, Greece 2Laboratory of Analytical Chemistry, Department of Chemistry, University of Ioannina, Ioannina, Greece 3Laboratory of Infectious Diseases and Hygiene of Animals, Department of Animal Production, Technological Education Institute of Epirus, Arta, Greece 4Laboratory of Biological Chemistry, Faculty of Medicine, University of Thessalonica, Greece 5Department of Biological Applications and Technology, University of Ioannina, Ioannina, Greece Aim: To investigate the effect of carvacrol on chemical carcinogenesis, cancer cell proliferation and platelet aggregation, and to find possible correlation between all these processes and the antioxidant properties of carvacrol. Materials and Methods: 3,4-benzopyrene-induced carcinogenesis model using Wistar rats was used. Leiomyosarcoma cells from Wistar rats were used to study carvacrol antiproliferative activity in vitro. The carvacrol antiplatelet properties were investigated with platelet aggregation assay and flow cytometry technique. The production of thromboxane B2, final metabolite of platelet aggregation, was evaluated by radioimmunoassay. Results: Our study revealed significant anticarcinogenic properties of carvacrol. We observed 30% decrease of 3,4 benzopyrene carcinogenic activity in vivo. Antiproliferative activity of carvacrol (IC50) was 90 μM and 67 μΜ for 24 h and 48 h of incubation of cells, respectively. Carvacrol possessed also mild antiplatelet effect, inducing the decrease of thromboxane A2 production in platelets and as a result — restrictive expression of the GPIIb/IIIa platelet receptor. Conclusion: Our data demon- strated that carvacrol possesses anticarcinogenic, antiproliferative and antiplatelet properties. Key Words: carvacrol, Origanum vulgaris, experimental carcinogenesis, leiomyosarcoma cells, platelet aggregation, GPIIb/IIIa platelet receptors. Carvacrol [isopropyl-�-cresol CH3�OH)(C3H7] is MATERIALS AND METHODS one of main s�bstances of essential oil from the herb Reagents. 34 benzop�rene �B[a]P was p�rchased Origanum vulgaris subs Hirtum possessing antiseptic from Fl�ca �Germany. Adenosine diphosphate �ADP), antibacterial antiviral and antif�ngal properties [1–3]. platelet activating factor �PAF), creatine phoshate �CP It was reported that carvacrol in low concentrations creatin phoshokinase �CPK), arachidonic acid �ARA), (0.15 mg/ml), inhibits growth of microbes h�man acetylsalicylic acid carvacrol trypan bl�e gingkolide animal or plant pathogens s�ch as bacteria Camby- A gingolide B and tricapryline were p�rchased from lobacter jejuni, Escherichia coli, Salmonella. Enterica Sigma �Germany. Thromboxane A� �TXA� RIA kit was methicillin-resistant staphylococci and several f�ngi. Its provided by Isotop Instit�te of Isotopes Co Ltd Platelet antimicrobial effect is more potent than other Origanum GpIIb/IIIa Occ�pancy kit was provided by American compo�nds s�ch as sineol and camphora [4�7]. Diagnostics Inc �USA. D�lbecco’s Modified Eagle It is also well known that essential oils which are Medi�m �DMEM for the cell c�lt�res was p�rchased rich in carvacrol possess strong antioxidant properties from Fl�ka and Sigma �Germany. eq�ivalent to those of ascorbic acid b�tyl hydroxyl tol�ene Chemically induced carcinogenesis. 4� male �BHT and vitamin E [8, 9]. Since many antioxidants exert Wistar rats � months old �mean weight of �3� ± 15 g antiplatelet [10–12] and anticarcinogenic effects [�3–16] were divided into two gro�ps �� animals per gro�p: it is possible that carvacrol f�nctions in a similar way. Bas- control gro�p and experimental. Animals were kept in ing on this hypothesis and reported data in this st�dy we plastic cages in room with constant temperat�re (21 ± examined carvacrol anticarcinogenic effect in vivo and its � °C), �sing alternate 12 h period of light and dark antiproliferative property in vitro. Also we investigated of and were fed with standard rat chow. Animals were platelet aggregation ind�ced by carvacrol and tried to weighted once a week till the end of experiments. find correlation between mentioned processes. �� ml of carvacrol �ε = ��97 mg/ml ��were mixed with Received: March 28, 2006. �� mg of B[a]P in glass t�be and inc�bated at room *Correspondence: Fax: +30 651097850 temperat�re d�ring �4 h �nder contin�o�s steering. E-mail: [email protected] 200 mg of B[a]P were dissolved in 20 ml of tricapryline Abbreviation used: ADP — adenosine diphosphate; ARA – arachido- in other glass t�be and inc�bated at room temperat�re nate; B[a]P — 3,4-benzopyrene; BHT — butyl hydroxyl toluene;� COX��1 – �nder contin�o�s steering. Animals of control gro�p cycloxygenase 1; COX2 – cycloxygenase 2; CP�� — creatine phosphate; were then anesthetized with diethyl ether and � ml CP — carcinogenic potency of B[a]P; CPK — creatin phoshokinase; B[a]P of B[a]P — tricapryline sol�tion containing 10 mg DMEM — Dulbecco’s Modified Eagle Medium; GpIIb/IIIa — glyco- protein IIb/IIIa;�� 5-LOLOX —�� 5-lipoxygenase; MS��T — mean survival time; of B[a]P was injected in dorsal area of each animal Mabs — monoclonal antibodies; NSAD — non steroid anti-inflammatory while each animal of experimental gro�p was injected drug;�� PAF — platelet activating factor; PPP — platelet poor plasma; in dorsal area with � ml of sol�tion B[a]P — carvacrol PRP — platelet rich plasma; TXA2 — thromboxane A2. containing 10 mg B[a]P in 97 mg of carvacrol. 122 Experimental Oncology �� 121–125, �� ne The s�rvived animals of both gro�p were sacrificed possible selective inhibition of carvacrol �or other s�b- on 350th day after B[a]P or B[a]P � carvacrol injection. stances in platelet aggregation pathways [�7 18]. Car- All developed t�mors were caref�lly excised weighted vacrol was added in aggregometer c�vettes with 450 μl of fixed in �% formaldehyde sol�tion and s�bmitted to PRP in increasing doses: �.488 mg �.976 mg �.46 mg histological examination. All animal proced�res were �.952 mg �.44 mg �.928 mg 3.9�4 mg 4.88 mg perforwed �nder strict r�les of recommendations of 9.76 mg �4.4 mg and �9.52 mg. Each dose of carvacrol Ethic committee. was added 2–3 min before the addition of the selected Measurement of B[a]P carcinogenic potency. agonist and then PRP and carvacrol were mixed well by The eval�ation of B[a]P carcinogenic potency �CP B[a]P contin�o�s stirring. ADP arachidonic acid and PAF at was carried o�t on established rat model �sing the concentrations of 12 �M �.7 �M and 15 �M respectively following mathematic form�la [�3]: were �sed as agonists of platelet aggregation. Res�lts CPB[a]P = [Percentage �% of t�mor ind�ction/Mean were expressed as percentage of inhibition of maxim�m s�rvival time of the rats]X100 platelet aggregation by carvacrol via each aggregation The inhibition of chemical carcinogenesis in Wistar pathway. Carvacrol concentration ind�cing 50% inhibition rats by carvacrol we estimated by calc�lating the diffe- of platelet aggregation �IC50), was calc�lated. rence between CPB[a]P of the control gro�p and CPB[a]P Thromboxane A2 production by platelets. Re- of the experimental gro�p: agents were provided by Isotop �Instit�te of Isotopes B[a]P carcinogenesis inhibition = CPB[a]P �cont- Co Ltd. Levels of TXA� were estimated by radioimm�- rol gro�p — CPB[a]P �experimental gro�p X100 noassay as described [�9 20]. Samples were divided Cell cultures. Malignant cells �leiomyosarcoma on three gro�ps: a 450 �l of PRP treated with �.25 mg cells from Wistar rats were �sed for in vitro experi- of indomethacin �resting platelets control; b 450 �l of o ments. Cells were c�lt�red in DMEM at 37 C �% CO� PRP pretreated with agonists and 7 min later treated with and were plated in density of104 cells per plate. Two �.25 mg of indomethacin �agonist control; c) 450 μl of plates were �sed as control ones and to other plates PRP pretreated with different concentrations of carvacrol carvacrol was added at different concentrations that ind�ces complete inhibition of aggregation and then (2 plates per each concentration: �10� ��μΜ �25 � ��μΜ treated with agonists. Seven min�tes after the addition of 50 ��μΜ 7�� μΜ ��100� ��μΜ ��150� ��μΜ 3�00� ��μΜ 600�� μΜ agonist �.25 mg of indomethacin were also added to the 1000 �� 1500� �μΜ�� and 4�� .. Cell growth rate and samples. Indomethacin was added in order to stop the cytotoxicity of carvacrol were estimated after �4 h and prod�ction of TXA� after the end of the platelet aggrega- 4� h of inc�bation �sing Newba�er cytometer and tion experiments by blocking platelet cycloxygenase � staining cells with Trypan Bl�e. �COX1). All samples prepared as described before were Platelet aggregation assay. Veno�s blood from kept for 10 days at –80 oC. ± 10 healthy non smoking male vol�nteers (24 �.5 years For estimation of TXA� concentration in the old was collected in 25 ml t�bes containing citrate as samples each sample after thawing was centrif�ged anticoag�lant (1 ml of citrate per 9 ml of blood. Blood at �� rpm for � min and 4�� l of the s�pernatant samples were then centrif�ged at 900 rpm for 10 min were �sed for lipoids extraction in ethyl acetate on and platelet reach plasma �PRP was isolated as s�- activated minicol�mn [21]. Samples with extracted pernatant. For the calibration of Ca-500 aggregometer lipoids in ethyl acetate were evaporated �nder nitrogen �Chronolog Co USA), the rest of blood samples were stream and were treated with the radiolabeled specific centrif�ged again at 3100 rpm for 15 min and platelet antibody against TXB� final stable metabolite of TXA�.

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