Elsevier

Phytomedicine

Volume 17, Issues 8–9, July 2010, Pages 581-588
Phytomedicine

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

https://doi.org/10.1016/j.phymed.2009.12.008Get rights and content

Abstract

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 IC50 of 100 μM 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.

Introduction

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-(1-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 anticarcinogenic effects of carvacrol using human metastatic breast cancer cell line MDA-MB 231. Curcumin at an IC50 value of 30 μM was used as positive control to assess the efficacy of carvacrol. The study demonstrated that carvacrol is a potent anti-cancer compound with an IC50 of 100 μM at 48 h inducing apoptosis by depletion in mitochondrial membrane potential, cytochrome c release, decrease in Bcl2/Bax ratio and PARP cleavage.

Section snippets

Chemicals

Carvacrol (98%) and curcumin (≥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

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 Fig. 1a, a dose-dependent decrease in the growth of cells was observed with increasing concentrations of carvacrol. The IC50 of carvacrol for MDA-MB 231 cells was determined to be 100 μM at 48 h. Results from previous studies have shown similar effects of

Conclusion

In summary, the present work demonstrates the antiproliferative effects of carvacrol in metastatic breast cancer, MDA-MB 231, cells with an IC50 value of 100 μM. 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

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