Cancer Letters

Cancer Letters

Volume 238, Issue 2, 18 July 2006, Pages 304-313
Cancer Letters

Cytotoxic response of breast cancer cell lines, MCF 7 and T 47 D to triphala and its modification by antioxidants

https://doi.org/10.1016/j.canlet.2005.07.013Get rights and content

Abstract

The cytotoxic effects of Triphala (TPL), an Indian Ayurvedic formulation with known anti-cancer properties, has been investigated on two human breast cancer cell lines differing in their p53 status. In vitro studies showed that MCF 7 with wild type p53 was more sensitive to TPL than T 47 D, which is p53 negative. TPL induced loss of cell viability was determined by MTT assay. After 72 h incubation, the IC 50 values for MCF 7 was found to be ∼8 μg/ml and that for T 47 D was ∼26 μg/ml. Moreover, TPL inhibited the clonogenic growth of MCF 7 cells, which was significantly recovered by pifithrin-α, the p53 inhibitor. However, pifithrin-α, did not modify TPL induced cytotoxicity in T 47 D cells. Exogenous addition of antioxidants, glutathione (GSH) and N-Acetyl-Cysteine (NAC) inhibited the anti-proliferative ability of TPL in both MCF 7 and T47 D. Annexin-V and propidium iodide double staining of cells treated with TPL for 2 h revealed that TPL induced significant apoptosis in both the cell lines in a dose dependant manner but magnitude of apoptosis was significantly higher in MCF 7 than in T 47-D cells. TPL was also found to induce dose and time dependent increase in intracellular reactive oxygen species in both the cell lines. Present results have demonstrated that MCF 7 and T 47 D cells exhibited differential sensitivity to TPL, which seems to be dependant on their p53 status. Inhibition of anti-proliferative ability of TPL by antioxidants suggests a role for TPL induced ROS in the induction of apoptosis. It is concluded that p53 status of cancer cells formed an important factor in predicting the response of cancer cells to prooxidant drugs.

Introduction

Breast cancer is the second most common cancer in Indian women. Every year, over 80,000 new cases of breast cancer are diagnosed in India and is one of the leading causes of death in women. This implies that there is a continuing need for development of new anticancer drugs, drug combinations and treatment modalities to effectively treat breast cancer patients. Natural products, especially plant-based products have frequently been examined as anti cancer agents [1]. There exists high hope for effective treatment of different cancers by systematic screening of a variety of natural products. Ayurveda, the ancient Indian science of health, is based on natural products including several phytochemicals for treatment of a variety of diseases. Reports have appeared showing that plants extracts displayed anti-tumor/anti-cancer/anti-proliferative effects on cultured human tumor cell lines [2], [3], [4], [5], [6], [7]. Some of the recent reports, concerned with, purified compounds from herbal extracts with their possible mechanisms of anti-cancer effects [8], [9], [10], [11], [12], [13]. Polyherbal formulations from Chinese and Indian medicinal systems have long been advocated for anticancer properties [14], [15], [16]. One such formulation from Ayurveda, Triphala (TPL), extracted in acetone has been recently reported for its anticancer property [17].

TPL is an herbal drug consisting of the dried and powdered fruits of three plants, Terminalia chebula, Emblica officinalis and Terminalia bellerica in equal proportions. It is an important medicine of the ‘rasayana’ group and is believed to promote health, immunity and longevity [18]. This formulation, rich in antioxidants, is frequently used in Ayurvedic medicine to treat many diseases such as anemia, jaundice, constipation, asthma, fever and chronic ulcers.

We have recently shown that TPL was significantly more toxic to tumor cells in vitro as well as in vivo as compared to normal cells [19]. In addition, it has been shown that TPL induced apoptosis in tumor cells in vitro. This study describes the cytotoxic response of two breast cancer cell lines, MCF 7 (p53+ve) and T 47 D (p53−ve) to TPL as evaluated by MTT and clonogenic assays. It has further been shown that pifithrin, a known inhibitor of p53, markedly removed the TPL induced cytotoxic effect on cells. Antioxidants, such as, GSH and NAC were found to restore the drug induced cellular toxicity. Our results have shown differential sensitivity of MCF 7 and T 47 D cells to TPL in terms of proliferation and clonogenicity. Over all, these results may have implications in developing protocols for effective treatment of cancer in clinic based on the p53 and redox status of tumor cells.

Section snippets

Cell culture and harvesting

Dulbecco's Modified Eagle's Medium (DMEM) was purchased from Gibco Co., USA and fetal bovine serum (FBS) was obtained from Biomedia, France. Pifithrin-α, Glutathione and N-Acetyl-l-Cysteine were purchased from Sigma, USA. MCF 7 and T 47 D cells were purchased from National Centre for Cell Science, Pune, India and they were cultured in DMEM supplemented with FCS (10%) and antibiotics (streptomycin 200 μg/ml and penicillin 100 units /ml) at 37 °C in 5% CO2 environment. Cells were harvested from 80

Effect of TPL on proliferation of MCF 7 and T 47 D

MCF 7 and T 47 D cells were treated with increasing concentration of TPL (0–50 μg/ml) and their loss of viability was assessed by the MTT assay (Fig. 1). It was found that rapid loss of cell proliferation occurred with the increasing concentration of TPL. For each concentration of TPL treatment, cells were incubated for different time periods of 24, 48, 72 and 120 h before evaluation of their proliferation. It can be seen that inhibition of cell proliferation induced by the drug was dependant on

Discussion

Chemotherapeutic drugs are known to induce cytotoxicity in tumor cells through diverse mechanisms, in which signaling events play an important role depending upon the cell type and stimulus. This study was aimed to investigate the effects of TPL on two breast cancer cell lines, MCF 7 and T47 D, which are known to differ in their p53 status. Results have shown that TPL remarkably reduced proliferation of wild-type MCF 7 cells (p53+ve) in a dose and time dependant manner (Fig. 1(A)). The effect

Acknowledgements

Sandhya Thulasidas would like to acknowledge the support of DAE fellowship for conducting her research. The authors would like to acknowledge Ajanta pharmaceuticals and Dr K.I. Priyadarshini for providing the triphala extract. Acknowledgements are also due to Dr B.N. Pandey and Dr K.M. Lathika for critical review of the manuscript.

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      The major active constituents of Triphala are gallic acid, tannin, ellagic acid and ascorbic acid [5,6]. Previous scientific studies of Triphala have reported that it possesses antioxidant [7–10], antihypercholesteremic [11,12], antidiabetic [13,14], anti-obesity [15,16], immunomodulation [2,17,18], antimicrobial [19–27], anti-inflammatory [28–30] and anticancer properties [31–38]. Other studies of the Triphala extract have shown that it did not produce any signs of toxicity at doses of 1750 and 2000 mg/kg during acute toxicity tests [39,40].

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