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Inhibition of DNA synthesis by Carvacrol in mouse myoblast cells bearing a human N-RAS oncogene.

Summary

Monoterpenes are dietary components found in the essential oils of a wide variety of plants. A number of these monoterpenes have antitumor activity. We have investigated the effects of carvacrol obtained by fractional distillation of Origanum onites L. essential oil, on DNA synthesis of N-ras transformed myoblast cells, C025. Incubation of the cells with different doses of carvacrol prevented DNA synthesis in the growth medium and ras-activating medium, which contains dexamethasone. This result demonstrates that carvacrol inhibits growth of myoblast cells even after activation of mutated N-ras oncogene, suggesting the possibility that carvacrol may find application in cancer therapy.

Key words: Carvacrol, inhibition of DNA synthesis, mouse myoblast cells, human N-RAS oncogene

Introduction

Carvacrol, the predominant monoterpenic phenol which occurs in many essential oils of the family Labiatae including Origanum, Satureja, Thymbra, Thymus and Corydothymus species used through the ages as a source of flavour in food (Krimer et al., 1995). Substantial antibacterial, antifungal and insecticidal effects of carvacrol on different organisms have been described in several studies (Didry et al., 1994; Rice and Coats, 1994; Thomson, 1996; Ultee et al., 1998). Some other activities such as analgesic (Aydin et al., 1996) and antioxidant (Aeschbach et al., 1994) activities also have been reported. In specifically, Ultee et al., (1999) showed the interaction of this compound with the cytoplasmic membrane by changing its permeability for protons and potassium ions and inhibition of diarrheal toxin production from B. cereus by carvacrol has been found (Ultee and Smid, 2001).

Effects of several terpenes have been shown in various types of tumorigenesis. Famesol from this group showed a selective inhibitory effect on the growth of human neoplastic cell lines by preventing of phosphatidyicholine biosynthesis (Melnykovych et al., 1992; Adany et al., 1994). Treatment of Hep-2 cells derived from a human larynx carcinoma induced the apoptotic phenotype (Stammatia et al., 1999). Recently, inhibitory effects of carvacrol on DMBA induced tumorigenesis in rats and on the growth of melanomas in vitro were reported (Zeytinoglu et al., 1998; He et al., 1997). A well-studied monoterpene, limonene, was also shown to have chemopreventive and chemotherapeutic activity against chemically induced mammary, lung and stomach cancer in rodents (Elegbede et al., 1984; Wattenberg and Coccia, 1991; Gould et al., 1994; Gelb et al., 1995). Limonene and its metabolites were shown to inhibit isoprenylations of [p2l.sup.ras], product of ras proto-oncogenes, which are well documented for their activity in carcin ogenesis (Zeytinoglu et al., 1993, 1995; Bos, 1989; Marshall, 1991), by disrupting the biological activity in NIH3T3 cells (Crowell et al., 1991).

In our study, a mouse muscle cell line (CO25) bearing a glucocorticoid inducible mutated human N-ras oncogene was used. The cell line when grown in a weakly mitogenic medium (containing 10% horse serum) proceeds to fuse after 4 days and forms branched myotubes after 7-10 days. On the other hand, the cells fail to differentiate when grown in the presence of dexamethasone (Gossett et al., 1988). Here, the cytotoxicities of carvacrol and range of effective doses on DNA synthesis were determined in C025 cells before or after N-ras activation. Induction of ras protein was analysed by Western blotting. We report that carvacrol has an inhibitory effect on the proliferation of CO25 cells.

* Materials and Methods

Plant Extract

The plant extract examined in this study, carvacrol (2-methyl-5- (1-methyl ethyl) phenol), was isolated from stem distillated essential oil of Origanum onites L. collected from West Anatolia (Fig. 1). A fractional distillation was performed using a lab-size glass fractional distillation unit containing column packed with S/S Knit Mesh packing material (2.8 cm x 1.35 m). Reflux ratio was adjusted at 10/1 to 20/1 and the medium pressure was 8-10 mm Hg. Carvacrol-rich fractions were bulked to obtain carvacrol with 99% purity (GCMS) (Azcan et al., 2000).

Cell Culture

Dr. I. Gibson (University of East Anglia, UK) kindly provided the mouse skeletal muscle cell line (CO25). Cells were maintained in Dulbecco Modified Eagle Medium (DMEM) (Sigma) supplemented with 10% (v/v) of foetal calf serum (FCS) (Gibco), penicillin/streptomycin at 100 units/ml and 1% L-glutamine as adherent monolayers. Cells were incubated at 37 [degrees]C under 5% [CO.sub.2]/95% air in a humidified atmosphere.

The CO25 cells were derived by transfection of the parental C2 cells with a plasmid containing a mutational activated human N-ras gene (in codon 61) under transcriptional control of the steroid-sensitive promoter of the mouse mammary tumor virus long terminal repeat (MMTV-LTR). Therefore, to induce transformation, 1 [micro]M dexamethasone (Dex) (Sigma) was added to the cells grown in 10% horse serum (HS). To induce differentiation, the cells at approximately 80% confluence were switched to DMEM containing 10% HS with low mitogenic activity as a fusion-promoting medium (Gosset et al., 1988; Gerhart et al., 2001).

Immunoblotting

Cells were washed twice with ice-cold PBS and lysed in a buffer containing 1% NP-40, 50 mM Tris base, pH 8.0, 50 mM NaCl, 50 [micro]g/ml Leupeptin and 1 mM phenylmethyl sulphonyl fluoride. Amount of protein in each sample was estimated by the method of Bradford (1976). Aliqouts of cell extracts containing 40 [micro]g of protein were mixed with a sample buffer (0.4 M Tris, 0.4 M dithiothreitol, 8% sodium dodecyl sulfate, 40% glycerol and 0.04% bromophenol blue, pH 6.8) and then separated by a 12% SDS-PAGE according to Zeytinoglu et al. (1995). The proteins were transferred onto a nitrocellulose membrane in a buffer containing 0.025 MTris, 0.192 M glycine, 20% methanol, pH 8.5.

The blot was incubated with a mouse polyclonal antibody to [p2l.sup.ras] 142E05 (gift from Dr. P. Hawley, UEA, Norwich, UK) and then with a Rabbit anti-mouse Ig (HRP) (Dako Ltd.). The immunoblot was visualized by enhanced chemiluminescence detection system (Amersham).

MTT Dye Reduction Assay

CO25 cells in exponential growth phase were harvested and the cell number was determined using a haemocytometer. Samples were resuspended in fresh medium to give a density of 2.5 x [10.sup.4]/ml and then various concentrations of carvacrol dissolved in DMSO (1 [micro]g/ml, 5 [micro]g/ml, 10 [micro]g/ml, 50 [micro]g/ml, 100 [micro]g/ml, 150 [micro]g/ml) were added to the cells. As a solvent control, 0.1%, 0.5%, 1%, 5%, 10% and 15% of DMSO were added to the parallel cells. Aliquots (200 [micro]1) of the cell suspensions were placed into each of 88 wells of a 96 well microtitre plate and incubated for periods of time. At the end of the exposure time, 20 [micro]1 of MTT dye solution (5 mg/ml in sterile PBSA) was added to each well and the plates were incubated for a further 2 h. The medium was removed and the dye uptaken by cells was then solubilised by addition of 200 [micro]l of DMSO to each well. Absorbance at 540 nm was determined by use of a Dynatech, MR5000 (Dynatech Lab, USA) plate reader with a reference beam of 690 nm. Each dose of carvacrol was repeated 4 times per experiment. The results of repeat wells within the same experiment were averaged.

Analysis of DNA Synthesis

Cell proliferation assay was performed in 96-well plates (Falcon, Beckton Dickinson) and the BrdU colorimetric kit (Boebringer Mannheim) was used to determine the DNA synthesis by the method as given by the manufacturer. CO25 cells cultured as detailed previously (Zeytinoglu et al., 1993) were detached with 0.25% trypsin/EDTA and 1 x [10.sup.3] cells/ml were transferred into each well containing 10% FCS, 10% HS and 10% HS plus 1 [micro]M Dex.

To investigate the effects of carvacrol on DNA synthesis, the cells were incubated with various concentrations (10 [micro]g/ml, 5 [micro]g/ml and 1 [micro]g/ml) of carvacrol for various periods of time those were chosen according to MTT assay as described above. After each day, the cells were labeled with 10 [micro]1 of BrdU solution at 37 [degrees]C for 2 hours and then fixed with the addition of fixdenat solution for 30 min at room temperature. After removing the fixdenat solution, cells were treated with 100 [micro]l of anti-BrdU working solution for 90 min at room temperature. Then the cells were washed three times with PBSA and incubated with substrate solution until the color is sufficient for photometric detection that was predetermined. The absorbance of the samples was measured in an ELISA reader (Organom, Technica) at 492 nm. DMSO as a solvent control was added to the cells during the time course.

Protein Determination

The C025 cells grown in 10% FCS or 10% HS with or without 1 [micro]M Dex were incubated with various concentration of carvacrol or DMSO for periods of time. After each day, the cells were lysed with 0.5 ml of a lysis buffer (50 mM Tris/HCI, pH 7.4, 150 mM NaCI, 1 mM EDTA, 1% Triton X-100, 1 mM PMSF, 1 [micro]g/m1 aprotinin, 1 [micro]g/ml leupeptin). The lysate was scraped off the dish and centrifuged at 13,000 rpm for 10 ml at 4 [degrees]C. Protein determination was performed by Bradford assay (Bradford, 1976).

Statistical analysis

Results of the BrdU incorporation assay were analysed using Student-t test. The level of significance has been given in the text as a p value.

* Results

CO25 mouse myoblast cells were grown in 10% HS with dexamethasone to activate N-ras gene or without dexamethasone to allow differentiation, and then protein extracts prepared at various times were analyzed for p[21.sup.ras] level by Western blotting. The antibody used is a polyclonal anti-ras and recognize all Ras proteins. Incubation of the blot with the antibody showed that a 21 kDa polypeptide was significantly expressed and increased in the presence of Dex over the 1-4 days period compared to samples extracted the cells grown in the absence of Dex (Fig. 2). This result indicates that mainly the level of human p[21.sup.N-ras] was increased, since very weak bands have obtained in the absence of Dex at longer exposure time (data not shown).

Cytotoxicity of the carvacrol used in the cell proliferation assay was determined with a MTT assay as described in Material and Methods. As shown in Fig. 3, 1 [micro]g/ml, 5 [micro]g/ml and 10 [micro]g/ml concentrations of carvacrol used in cell proliferation assay showed between 10-25% toxicity after 3 days. However, 60 [micro]g/ml of carvacrol showed approximately 50% toxicity upon the incubation time. 100 [micro]g/ml and 150 [micro]g/ml doses of carvacrol were found to be significantly toxic and [IC.sub.50] value for C025 myoblast cells was determined as [IC.sub.50] = 60[micro]g.

The effects of carvacrol on DNA synthesis in C025 cells with or without N-ras activation were examined using a specific cell proliferation kit as described in Material and Methods. The cells normally grew in the medium containing 10% FCS until reaching contact inhibition and ceased proliferation, then committed to differentiation in 10% HS after 4 days (Fig. 4), as documented before (Gosset et al., 1988; Zeytinoglu et al., 1993). However cells became transformed and lost contact inhibition after ras activation by the addition of Dex to the differentiation medium.

Basically, the treatment of CO25 cells grown in either 10% FCS (Fig. 5b), 10% HS (Fig. 6b) or 10% HS plus Dex (Fig. 7b) with carvacrol at concentrations ranging from 1-5 [micro]g/ml had no effect on cell proliferation compared to the controls (Fig. 5a, 6a and 7a). In contrast, substantial effects on cell proliferation were obtained when the cells were treated with 10 [micro]g/ml of carvacrol. Thus, as shown in Fig. 5b, there was a significant reduction (p <0.001) in the level of DNA synthesis after 2 days, when compared with the solvent control. This meant that at concentration of 10 [micro]g/ml the DNA synthesis of these cells was reduced about 75% as compared to DMSO treated control cells (Fig. 5a). In the experiment shown there appears to be a similar reduction (by 43%) of DNA synthesis of CO25 grown in differentiation medium (10% HS) (Fig. 6b) as compared to controls (Fig. 6a).

DNA synthesis was also significantly declined (p <0.001) in the ras activated transformed cells in the presence of 10 [micro]g/ml of carvacrol for 3 and 4 days after an elevation on day 2 (Fig. 7b) as compared to the control cells (Fig. 7a). Unlike the other medium conditions, ras-dependent proliferation of the cells appeared to be inhibited by 1 [micro]g/ml and 5 [micro]g/ml of carvacrol treatment as well.

To test these inhibitory effects of 10 [micro]g/ml of carvacrol on DNA synthesis, the total protein levels of cells grown in parallel plates were measured as described in Material and Methods. In contrast to the cells grown in transformation medium, which showed significant increase in the level of total protein (Fig. 7c), Fig. 5c and 6c show that the level of total protein obtained from each culture dish was slightly elevated.

Discussion

CO25 cells provide an excellent model for investigation of the mechanism of differentiation and transformation (Zeytinoglu et aL, 1993, 1995). Here, we have used a mouse myoblast cell line, C025, bearing a mutated human N-ras oncogene, for the first time to test the potency of therapeutical agent. As shown in Fig. 2, ras proteins were detected at high level after addition of Dex, which was used as an inducer of mutated human N-ras oncogene.

More recently, Crowell (1999) reviewed prevention and therapy of cancer by monoterpenes. The inhibition of cell growth in melanoma by isoprenoids including carvacrol, d-limonene, its metabolites and farnesol has reported by several grubs (Crowell et al., 1991; Adany et al., 1994; He et al., 1997). Dietary limonene also inhibits the development of ras oncogene-induced mammary carcinomas in rats (Gould et al., 1994; Gelb et al., 1995).

In our study, the cytotoxicity of carvacrol and the range of effective doses were determined and its effect on DNA synthesis was assessed using cell proliferation assay. In vitro cytotoxicity of carvacrol reported here is [IC.sub.50] = 60 [micro]g. However, this [IC.sub.50] value is not the same as reported by a previous study (Stammatia et al., 1999), it might be due to cell type and purity of carvacrol. For the inhibition of DNA synthesis, the maximal dose of carvacrol was chosen as 10 [micro]g/ml. Because, the significant effect of carvacrol on the inhibition of DNA synthesis was thought to shown in the least toxic doses, since it is quite important that minimize the cytotoxicity of a predrug for future use.

Result here showed that 10 [micro]g/ml dose of carvacrol is very effective for the inhibition of DNA synthesis in C025 cells. Our findings support the tumor growth-suppressive action of carvacrol as reported by Zeytinoglu et al. (1998) and He et al. (1997). This effect of 10 [micro]g/ml of carvacrol also determined by MTT is not due to cytotoxicity since there was no change in total protein levels of these cells. The inhibition of DNA synthesis during growth phases especially transformation process is very important. However it seems that this inhibitory effect might be specific for only transformation phases or in other words for ras activated transformation since the doses of 1 [micro]g/ml and 5 [micro]g/ml of carvacrol was found to be effective on DNA synthesis.

Here, especially 10 [micro]g/ml of carvacrol was shown to affect the cells in growth or transformation medium. On the other hand, the decrease in DNA synthesis in the absence of ras activation seems to be not as a result of carvacrol treatment, since the cells normally withdraw from the cell cycle and commit to differentiate (Zeytinoglu et al., 1993).

DNA synthesis in the cells grown in the presence of 1 [micro]g/ml and 5 [micro]g/ml of carvacrol seems to be normal for both growth and differentiation medium since it is known that CO25 myoblasts cease proliferation when they commit to differentiate after contact inhibition in the growth medium. In contrast, after activation of N-ras oncogene in the presence of Dex in differentiation medium, the cells enter transformation process; therefore DNA synthesis is elevated (Zeytinoglu et al., 1993). Our results demonstrate for the first time the inhibitory effect of carvacrol on DNA synthesis in N-ras transformed cells. It is well documented that many monoterpenes inhibit isoprenylation of proteins, which occurs at the level of prenyl-protein transferase enzyme (Crowell et al., 1991; Elson, 1995; Geib et al, 1995). Prenylation of Ras enables it to associate with plasma membrane, which is required for its oncogenic activity (Bos, 1989; Marshall, 1991) and impairment of the prenylation of Ras might account for the an titumor activity of monoterpenes. Moreover, like ras proteins, the prenylated proteins TC21, Rho and PRL-1/PTP-CAAX tyrosine phosphatases can be oncogenic (Crowell et al., 1994), thus they may be important cellular targets of protein prenylation inhibitors such as monoterpenes.

Taken together, these data raise the possibility that carvacrol and similar isoprenoids may find application in cancer therapy by preventing prenylation of many proteins including Ras. Therefore these results call for further investigations of the suppression mechanism of the DNA synthesis and ras expression.

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Acknowledgments

We would like to thank Mrs. M. Irmak for technical assistance with ELISA and Department of Pharmacology, Faculty of Medical, University of Osmangazi for providing us their facilities. Anadolu University Research Fund grant 1996-15 and Medicinal and Aromatic Plant and Drug Research Center (TBAM) supported part of the experimental work.

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H. Zeytinoglu (1)

Z. Incesu (2)

K.H.C. Baser (3)

(1.) Department of Biology, Faculty of Sciences, Anadolu University, Eskisehir, Turkey

(2.) Department of Biochemistry, Faculty of Pharmacy, Anadolu University, Eskisehir, Turkey

(3.) Medicinal and Aromatic Plant and Drug Research Center (TBAM), Anadolu University, Eskisehir, Turkey

Address

H. Zeytinoglu, Anadolu University, Faculty of Sciences, Department of Biology, Yunusemre Campus, 26470 Eskisehir, Turkey

Tel.: ++90-222-3350580/ext. 5727; Fax: ++90-222-3353616;

e-mail: hzeytino@anadolu.edu.tr
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Author:Zeytinoglu, H.; Incesu, Z.; Baser, K.H.C.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:7TURK
Date:May 1, 2003
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