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Anti-proliferative effects of carvacrol on a human metastatic breast cancer cell line, MDA-MB 231.


Purpose: Although the anti-tumor effects of carvacrol have been demonstrated earlier, the exact underlying molecular mechanisms involved in its action have not been defined and in the present study an attempt has been made to identify the mechanism of carvacrol induced cell death in human metastatic breast cancer cells, MDA-MB 231.

Methods: Apoptosis induced by carvacrol was determined based on different assays like MTT assay, Annexin V, mitochondrial membrane potential assay, multicaspase activation assay and cell cycle analysis by flow cytometer. Cleavage of PARP, cytochrome c release and modulation of Bax and Bcl2 ratio by Western blot analysis were also studied.

Results: The study clearly showed induction of apoptosis by carvacrol in MDA-MB 231 cells dose dependently at an [IC.sub.50] of 100 [micro]M with a decrease in the mitochondrial membrane potential of the cells resulting in release of cytochrome c from mitochondria, caspase activation and cleavage of PARP.

Conclusion: The data in the present study clearly demonstrated anti-tumor effects of carvacrol on human metastatic breast cancer cells, MDA-MB 231, and that the compound could have a potential therapeutic significance in treating cancer.

Keywords: Carvacrol MDA-MB 231 cells Apoptosis Bax/Bcl2 ratio Cytochrome c PARP


In recent years major research has been focused on the biologically active derivatives of medicinal plants for the development of novel potential drugs for several pathologies with significant social impact (Hedberg, 1993; Heinrich and Gibbons, 2001). The use of natural products from the extracts of medicinal plants in the treatment of skin, respiratory, neuromuscular and mental health disorders and also in obstetrics and gynecology is already known (Abo et al., 2000; Ahmad et al., 1998; Ankli et al., 2002; Dutta et al., 1998; Pinn, 2001). The potential anti-tumor activity of the medicinal plants has been recently described in many studies (Aponte et al., 2008; Manosroi et al., 2006; Yoo et al., 2007).

Extensive research on biologically active compounds from essential oils has proved them to be potent anti-bacterial, anti-fungal and anti-oxidant agents (Albuquerque et al., 2007; Ao et al., 2008; Baik et al., 2008; Bakkali et al., 2008; Lampronti et al., 2006). Carvacrol, (2-methyl-5-(l-methylethyl)-phenol), is a major component of the essential oils of oregano and thyme (Kisko and Roller, 2005; Lampronti et al., 2006). Generally recognized as a safe food additive, carvacrol is used as a flavoring agent in baked foods, sweets, beverages and chewing gum (Fenaroli, 2002). Carvacrol-containing essential oils are biostatic and/or biocidal against many bacteria, yeast and fungi in laboratory media and consequently have attracted considerable research attention as potential food preservatives (Burt, 2004). Studies have shown that carvacrol exhibits biocidal activity against human, animal or plant pathogens by damaging the membrane and thus resulting in an increase in the membrane permeability to protons and potassium ions, depletion of the intracellular ATP pool and disruption of the proton-motive force (Ultee et al., 1999).

Carvacrol possesses strong antioxidant properties equivalent to those of ascorbic acid, butyl hydroxyl toluene (BHT) and vitamin E (Aeschbach et al., 1994; Mastelic et al., 2008). Although the anti-proliferative properties of carvacrol on non-small cell lung cancer cells, A549, chronic myeloid leukemia cells, K562, murine B16 melanoma cells have been shown (He et al., 1997; Horvathova et al., 2007; Karkabounas et al., 2006; Koparal and Zeytinoglu, 2003; Lampronti et al., 2006) the molecular mechanisms involved in its action remains elusive.

Therefore the present study is aimed to evaluate and identify the underlying molecular mechanism involved in the antic-arcinogenic effects of carvacrol using human metastatic breast cancer cell line MDA-MB 231. Curcumin at an [IC.sub.50] value of 30 [micro]M was used as positive control to assess the efficacy of carvacrol. The study demonstrated that carvacrol is a potent anti-cancer compound with an [IC.sub.50] of 100 [micro]M at 48 h inducing apoptosis by depletion in mitochondrial membrane potential, cytochrome c release, decrease in Bcl2/Bax ratio and PARP cleavage.

Materials and methods


Carvacrol (98%) and curcumin ([greater than or equal to] 94%) (curcumin was used as positive control) were purchased from Sigma-Aldrich (Bangalore, India). Phosphate-buffered saline (PBS), RPMI 1640 medium, fetal bovine serum (FBS) were purchased from Gibco BRL (CA, USA). MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide) was from Sigma-Aldrich (Bangalore, India). ECL reagent kit from GE Amersham. Nitrocellulose membrane was from Millipore (Bangalore, India). Mouse monoclonal antibody against cytochrome c was from Chemicon (CA, USA). Monoclonal antibodies of PARP, Bcl2, Bax were from Upstate (Charlottesville, VA, USA). All the other chemicals and reagents were purchased from local companies and are of molecular biology grade.

Cell culture and treatment

MDA-MB 231 cells were grown in RPMI-1640 supplemented with 10% heat inactivated fetal bovine serum (FBS), 100 IU/ml penicillin, 100 mg/ml streptomycin and 2mM L-glutamine. Cultures were maintained in a humidified atmosphere with 5% [CO.sub.2] at 37[degrees]C. The cultured cells were subcultured twice each week, seeding at a density of about 2 x [10.sup.3] cells/ml. Cell viability was determined by the trypan blue dye exclusion method.

Analyses of cell viability and apoptosis

Cell viability was determined by MTT assay. MDA-MB 231 cells (5 x [10.sup.3] cells/well) were seeded to 96-well culture plate and cultured with or without carvacrol (20, 40, 80 & 100 [micro]M) or curcumin (20, 40, 80 & 100 [micro]M) for 24 h or 48 h in a final volume of 200 [micro]l. After treatment, the medium was removed and 20 [micro]l of MTT (5 mg/ml in PBS) was added to the fresh medium. After 2 h incubation at 37[degrees]C, 100 [micro]l of DMSO was added to each well and plates were agitated for 1 min. Absorbance was read at 570 nm on a multi-well plate reader (Victor3, Perkin Emler). Percent inhibition of proliferation was calculated as a fraction of control (without carvacrol).

Annexin V assay, Cell cycle analysis, Mitochondrial membrane potential analysis and multicaspase activity analysis of carvacroltreated cells were performed on Guava Easy Cyte Flow cytometer. Briefly, MDA-MB 231 cells were seeded at a density of 1 x [10.sup.5] cells/ml in 6-well culture plates, cultured in 10% FBS with carvacrol (0, 20, 40, 80 & 100 [micro]M) for 24 h. After treatment, cells were harvested, washed with PBS, stained and analyzed by flow cytometry according to manufacturer's protocol.

DNA fragmentation into 180 bp ladder was detected using the SDS/proteinase K/RNase A extraction method (Herrmann et al., 1994). Briefly, MDA-MB-231 cells (5 x [10.sup.6] cells) were incubated with carvacrol at different concentrations (0, 20, 40, 80 & 100 [micro]M) for 24 h. After treatment, cells were washed in cold PBS and lysed in a buffer containing 50 mM Tris-HCl (pH 8.0). 1 mM EDTA, 0.2% Triton X100 for 20 min at 4 [degrees]C. After centrifugation at 14,000 x g for 15 min, the supernatant was treated with proteinase K (0.5 mg/ml) and 1% SDS for 1 h at 50 [degrees]C. DNA was extracted twice with phenol and precipitated with 140 mM NaCl and 2 vol. of ethanol at - 20 [degrees]C overnight. DNA precipitates were washed twice in 70% ethanol, dissolved in TE buffer, and treated for 1 h at 37 [degrees]C with RNase A. Finally, DNA preparations were electrophoresed in 1% agarose gels, stained with ethidium bromide and visualized under UV light.

Immunoblot analysis

For immunoblot analysis, cells were lysed in a lysis buffer containing 20 mM Tris pH 8.0, 1 mM EDTA, 150mM NaCl, 1% NP- 40, 0.5% sodium deoxycholate, 1 mM [beta]-glycerophosphate, 1 mM sodium orthovanadate. 1 mM PMSF, 10[micro]g/ml leupeptin, 20 [micro]g/ml aprotinin and phosphatase inhibitors with 100-fold dilution. After 30 min of shaking at 4 [degrees]C, the mixtures were centrifuged (10,000 x g) for 10 min, and the supernatants were used as the whole-cell extracts. The protein content was determined according to the Bradford method (Bradford, 1976). Proteins (100 [micro]g) were separated on 8-12% sodium dodecyl sulphate (SDS)-polyacrylamide gels along with protein molecular weight standards and electrotransferred to nitrocellulose membrane. The membranes were blocked with 5% (w/v) nonfat dry milk after checking the transfer using 0.5% Ponceau S in 1% acetic acid and then probed with a relevant antibody (Bax, Bcl2, PARP at 1:1000 dilution) for 8-12 h at 4 [degrees]C followed by detection using peroxidase-conjugated secondary antibodies and chemiluminescence. Equal protein loading was detected by probing the membrane with [beta]-actin antibodies.

Release of cytochrome c from mitochondria to cytosol was measured by Western blot as previously described (Chandra et al., 1998) with some modifications. Briefly, cells were washed once with ice-cold PBS and gently lysed for 30 s in 80 [micro] 1 ice-cold lysis buffer (250 mM sucrose, 1 mM EDTA. 0.05% digitonin, 25 mM Tris, pH 6.8, 1 mM dithiothrietol, 1 mg/ml aprotinin, 1 mg/ml pepstatin, 1 mg/ml leupeptin, 1 mM PMSF and 1 mM benzamidine). Lysates were centrifuged at 12,000 x g at 4 [degrees]C for 5 min to obtain the extracts (cytosolic extracts free of mitochondria). Supernatants were electrophoresed on a 15% SDS-polyacrylamide gel and then analyzed by Western blot using cytochrome c antibody.

Results and Discussion

Carvacrol induced apoptosis in MDA-MB 231 cells

In an effort to gain mechanistic insight into carvacrol-induced apoptosis of metastatic breast cancer cells (MDA-MB 231 cells) first, the anti-proliferative effects of carvacrol on MDA-MB 231 cells were evaluated by MTT assay. As shown in z. 1 a, a dose-dependent decrease in the growth of cells was observed with increasing concentrations of carvacrol. The p[IC.sub.50] of carvacrol for MDA-MB 231 cells was determined to be 100 [micro]M at 48 h. Results from previous studies have shown similar effects of carvacrol on different cancer cells with [IC.sub.50] value ranging between 90-125 [micro]M (He et al., 1997; Karkabounas et al., 2006). As a positive control, curcumin was included in the study and the [IC.sub.50] of curcumin was determined to be 30 [micro]M as reported earlier (Prasad et al., 2009). The assay suggests carvacrol as an apoptotic inducer of MDA-MB 231 cells but ~3 fold less potent compared to curcumin.

In order to evaluate the cause of growth inhibition of MDA-MB cells by carvacrol, characteristic features of apoptosis were studied. Apoptosis or programmed cell death is recognized by characteristic pattern of morphological, biochemical, and molecular changes occurring in a cell (Elmore, 2007). Carvacrol-treated cells showed prominent morphological changes like cell shrinkage with rounding of cells and formation of membrane blebs characteristic of apoptosis as evidenced by microscopic studies (Fig. 1b). This result is similar to that found by Koparal and Zeytinoglu (Koparal and Zeytinoglu, 2003) in carvacrol-treated non small cell lung cancer cells, A549.


One of the biochemical features of apoptotic cells is the expression of cell surface markers achieved by flip-flop movement of the phosphatidylserine from inner membrane to the outer membrane of the plasma membrane (Bratton et al., 1997). Annexin V, a recombinant phosphatidylserine-binding protein, interacts strongly and specifically with phosphatidylserine residues and can be used for the detection of apoptosis (Arur et al., 2003). Results from Annexin V assay using flow cytometer demonstrated a dose-dependent increase in the Annexin V positive cells indicating induction of apoptosis by carvacrol (Fig. 1c).


Quantification of apoptosis induced by carvacrol in MDA-MB 231 cells

Loss of DNA content is a typical characteristic feature of apoptosis and staining of cell with Propidium iodide and analyzing by flow cytometer would help in evaluating the cell viability. Therefore, flow cytometric analysis of carvacrol-treated cells was performed and the results showed the increase of sub G0/G1 phase (apoptotic peak) of cell) cycle and a decrease of cells at S phase in a concentration-dependent manner indicating induction of apoptosis and inhibition of DNA synthesis in S phase (Fig. 2). This result is in accordance with the previous results (Zeytinoglu et al., 2003).


Carvacrol resulted in cytochrome c release from mitochondria and activation of caspases

A loss of mitochondrial membrane potential ([DELTA][[psi].sub.M]) indicates the loss of cell viability as it reflects the pumping of protons across the inner membrane during processes of electron transport and oxidative phosphorylation that drives the conversion of ADP to ATP (Ly et al., 2003). In the present study, the [DELTA][[psi].sub.M] was measured by flow cytometer and the results demonstrated a dose-dependent decrease in the membrane potential and thus, a dose-dependent increase in the percent apoptotic cells (Fig. 3). A decrease in [DELTA][[psi].sub.M] alters the membrane stability leading to release of mitochondrial apoptosis initiation factors (AIFs), cytochrome c and the apoptosis protease-activating factor (Apaf-1) into the cytosol. In cytoplasm, cytochrome c is known to become associated with caspase-9, Apaf-1 and dATP to form the apoptosome complex (Chinnaiyan, 1999), which in turn activates caspase- 9, -3 and -7. To further explore the apoptotic pathway, the release of cytochrome c from mitochondria into cytosol was analyzed by Western blot analysis in cytosolic fractions of carvacrol-treated cells. There is a dose-dependent increase in the levels of cytochrome c in cytoplasm indicating the execution of apoptosis (Fig. 4a). Further, activation of caspases (multicaspase activation) by cytochrome c was also studied by flow cytometer and the results clearly demonstrated an increased activity with increase in concentration of carvacrol (Fig. 4b).



Oligonucleosomal fragmentation of DNA and PARP cleavage in response to carvacrol Treatment

Another AIF, Caspase Activated DNase (CAD), released from mitochondria, translocates into the nucleus, after getting cleaved by the activated caspase-3, and leads to oligoneucleosomal cleavage of DNA into 180 bp fragments (Enari et al., 1998; Sakahira et al., 1998). In the present study, the fragmentation of DNA into 180 bp ladder was observed in carvacrol-treated MDA-MB 231 cells confirming the apoptosis (Fig. 5a).

PARP (poly(ADP-ribose) polymerase) catalyzes the poly ADP-ribosylation of a variety of nuclear proteins with NAD as substrate. Upon DNA damage, PARP gets activated and depletes NAD and ATP of the cell in an attempt to repair the broken DNA During apoptosis, caspase-3 inactivates PARP by cleaving it into 83 and 24 kDa fragments and thereby preserves ATP resources of the cell for apoptosis (Andrabi et al., 2008). Results from immunoblot analysis using antibody that recognizes uncleaved PARP of 116 kDa and 83 kDa cleaved fragment clearly demonstrated a dose dependent inactivation of PARP in carvacrol-treated MDA-MB 231 cells and curcumin at 30 [micro]M [IC.sub.50] was included as a positive control (Fig. 5b).


Modulation of Bcl2/Bax ratio

The control and regulation of the apoptotic mitochondrial events occurs through members of the Bcl-2 family of proteins which can be pro-apoptotic (Bcl-10, Bax, Bak, Bid, Bad, Bim, Bik, and Blk) or anti-apoptotic (Bcl-2, Bcl-x, Bcl-XL, Bcl-XS, Bcl-w, BAG). Bcl-2 monomers or homodimers favor survival and Bax homodimers favor cell death and their ratio decides the fate of the cell (Thomadaki and Scorilas, 2006; Thomadaki et al., 2007). In the present study, the expression levels of Bax and Bcl2 are studied by Western blot and carvacrol down regulated Bcl2 significantly with a dose dependent increase in Bax levels leading to lowered ratio of Bcl2/Bax followed by induction of apoptosis (Fig. 6).



In summary, the present work demonstrates the antiproliferative effects of carvacrol in metastatic breast cancer, MDA-MB 231, cells with an [IC.sub.50] value of 100 [micro]M. The work also presents the underlying molecular events occurring in presence of carvacrol. Carvacrol induced morphological changes such as cell shrinkage, rounding of cells and membrane blebbing which depict the induction of apoptosis. This induction of apoptosis appears to be mediated by cell cycle arrest at S phase, increase in Annexin V positive cells, decrease in mitochondrial membrane potential and increase in cytochrome c release from mitochondria, decrease in Bcl2/Bax ratio, increase in caspase activity and cleavage of PARP and fragmentation of DNA. Thus, the current work clearly indicates that carvacrol could be a potent anti-tumor molecule against metastatic breast cancer cells.


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K.M. Arunasree *

Institute of Life Sciences, University of Hyderabad Campus, Biology, Hyderabad 500 046, AP, India

* Tel.: +91 40 66571528; fax: +91 40 66571581.

E-mail address:

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doi: 10.1016/j.phymed.2009.12.008
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Author:Arunasree, K.M.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
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Geographic Code:9INDI
Date:Jul 1, 2010
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