Elsevier

Pharmacological Reports

Volume 69, Issue 5, October 2017, Pages 924-928
Pharmacological Reports

Original article
Epigallocatechin-3-gallate promotes apoptosis in human breast cancer T47D cells through down-regulation of PI3K/AKT and Telomerase

https://doi.org/10.1016/j.pharep.2017.04.008Get rights and content

Abstract

Background

Green tea has antioxidant, anti-tumor and anti-bacterial properties. Epigallocatechin-3-gallate (EGCG) in green tea is highly active as a cancer chemopreventive agent. In this study, we designed a series of experiments to examine the effects of EGCG on proliferation and apoptosis of estrogen receptor α-positive breast cancer (T47D) cells.

Methods

Cells were treated with EGCG (0–80 μM) and tamoxifen (0–20 μM), as the positive control, up to 72 h. Cell viability was determined by MTT assay. Apoptosis investigated by real time PCR of apoptosis and survival (Bax, Bcl-2, p21, p53, PTEN, PI3 K, AKT, caspase3 and caspase9 and hTERT) genes and by western blot of Bax/Bcl-2 proteins expressions.

Results

The results showed that EGCG decreased cell viability as concentration- and time-dependently. IC50 values were 14.17 μM for T47D and 193.10 μM for HFF cells, as compared with 3.39 μM and 32.75 μM for tamoxifen after 72 h treatment, respectively. Also, EGCG (80 μM) significantly increased the genes of PTEN, CASP3, CASP9 and decreased AKT approximately equal to tamoxifen. In gene expression, EGCG (80 μM) significantly increased Bax/Bcl-2 ratio to 8-fold vise 15-fold in tamoxifen (20 μM)–treated T47D cells during 72 h. In protein expression of Bax/Bcl-2, EGCG significantly increased 6-fold while this ratio augmented 10-fold in tamoxifen group. EGCG significantly decreased 0.8, 0.4 and 0.3 gene expression of hTERT in 24, 48 and 72 h, respectively.

Conclusions

This study suggests that EGCG may be a useful adjuvant therapeutic agent for the treatment of breast cancer.

Introduction

The most common cancer among women is breast cancer (17.1 per 100,000 person-year), which has mainly affected Iranian women about a decade earlier than western countries [1], [2]. Presently, common treatments for breast cancer are chemotherapy, radiotherapy and surgery. Current systemic therapies for breast cancer are often limited by major organ damage, short-term efficacy due to the emergence of drug resistance and poor prognosis [3]. So, the search for new antitumor agent’s development with improved efficacy and side-effect profile has been continued. Researchers believe that dietary phytochemical agents may influence chemotherapy treatment and help cure patients with cancer. Different natural compounds can improve the efficiency of chemotherapeutic agents, decrease resistance of chemotherapeutic drugs, and lower as well as alleviate adverse side-effects of chemotherapy [4]. As a result, researchers attempt to employ different herbs and their effective agents both in vitro and in vivo for cancer therapy. Most herbs contain antioxidant agents that could be consumed to prevent cancer or potentiate chemotherapy. Experimentally, several medicinal plants and herbal ingredients have been reported to have anticancer effects [5]. Also, a number of phytochemicals isolated from medicinal plants have been shown to decrease cell proliferation, induce apoptosis, retard metastasis and inhibit angiogenesis [6]. Currently, some of these plant-derived compounds are widely used for the chemotherapy of patients with cancer. For example, taxol analogues, vinca alkaloids (vincristine, vinblastine), and podophyllotoxin analogues have played an important role in the treatment of such patients [7]. Green tea is a popular beverage in Asia. Epidemiological studies have suggested that drinking green tea is effective in the treatment of different diseases. Based on many in vivo and in vitro studies, the biological activity of green tea is mediated by its major polyphenolic constituent, epigallocatechin gallate (EGCG), which is a potent antioxidant [8]. The beneficial effects of EGCG are reported in the treatment of cancer, cardiovascular diseases, diabetes, neurodegenerative diseases, and liver diseases. It reduces the risk of cancer developing in the prostate, bladder, stomach, oesophagus and lung [9], [10], [11], [12], [13]. In the pioneer study confirmed that EGCG affected the ERα-positive cells more than ERα-negative breast cancer cells [14]. Therefore, we chose T47D cells (as estrogen receptor α-positive breast cancer cell model) to evaluate the effects of EGCG on proliferation and apoptosis compared with tamoxifen (as positive control). It was also aimed to determine whether antitumor effects of EGCG are associated with altering of PI3K/AKT and telomerase genes expressions.

Section snippets

Cell lines and reagents

The estrogen receptor positive human breast cancer cell line T47D and normal Human Foreskin Fibroblast cell line HFF were obtained from Pasteur Institute of Iran. Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum, trypsin, penicillin and streptomycin were obtained from Gibco BRL Life Technologies (USA). The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium (MTT) 4-, OH-tamoxifen and EGCG (>95%) were purchased from Sigma-Aldrich company (USA). Tripure was purchased from

EGCG decreased cell viability in a concentration-dependent manner

T47D and HFF cell lines were treated with different concentrations of EGCG (10–80 μM) or tamoxifen (2.5–20 μM) for up to 72 h. Then, cell viability was measured by MTT assay. Exposure to EGCG or tamoxifen showed concentration-dependent suppression in cell survival (Fig. 1A). The toxicity of EGCG and tamoxifen was significantly higher in T47D than HFF cells. The results are summarized in Table 2.

The IC50 (concentration of 50% inhibition) values of EGCG in T47D cells were 46.88, 22.31 and 14.17 μM at

Discussion

Breast cancer is the most common cancer and, after lung cancer, is the second cause of cancer death in women. Defects in the apoptotic pathways are responsible for both the disease pathogenesis and its therapy resistance. It is thus a good candidate for treatment by pro-apoptotic agents [1]. Natural products have long been utilized to prevent and treat neoplasms; therefore, searching for natural products directed at the inducing apoptosis of cancer cells may be a great strategy for breast

Funding

This work was supported by a grant from the Vice Chancellor for Research and Technology, Jahrom University of Medical Sciences, Jahrom, Iran.

Authors and contributions

Design study and collection data: M.M.; Analysis and interpretation of data: S.E., H.R.; Writing of manuscript and decision to submit the article for publication: A.H., M.M.

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      Previous literature also explored the anticancer activity of EGCG and quercetin (see Table 4). Besides steroid receptor (Hallman et al., 2017) and PPARγ receptor (Wu et al., 2017) interaction, other assumed mechanisms of action include interaction with the PI3K/Akt/mTOR signaling pathway (Ding et al., 2017), VEGF (Rashidi et al., 2017), the 67-kDa laminin receptor (67-LR) (Li et al., 2017), p53, Bax protein (Moradzadeh et al., 2017) and Bcl-2 (Huang et al., 2017). However, none of the anticancer effects of EGCG have been proven clinically.

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