Combination of siRNA-directed gene silencing with epigallocatechin-3-gallate (EGCG) reverses drug resistance in human breast cancer cells

Abstract

Elevated expression of NF-E2-related factor 2 (Nrf2), a nuclear transcription factor, is a frequent genetic abnormality seen in this malignancy and is an important contributor to chemoresistance in cancer therapy. In the present study, we investigated if Nrf2 was associated with drug resistance in tamoxifen-resistant MCF-7 (MCF-7/TAM) cells, and whether EGCG, major flavonoid isolated from green tea, could reverse drug resistance in MCF-7/TAM cells. Our results showed that the endogenous expression of Nrf2 as well as its target proteins heme oxygenase-1, NADP (H):quinone oxidoreductase in MCF-7/TAM cells was higher than that in MCF-7 cells. Epicatechin gallate (EGCG) significantly sensitizes MCF-7/TAM cells to tamoxifen and dramatically reduced Nrf2 expression at both the messenger RNA and protein, leading to a reduction of Nrf2-downstream genes. In addition, using siRNA technique, we found that the intracellular Nrf2 protein level was significantly decreased in MCF-7/TAM cells and tamoxifen resistance was partially reversed by Nrf2 siRNA. Combination of siRNA-directed gene silencing with EGCG downregulated the Nrf2-dependent response and partly reversed tamoxifen resistance in MCF-7/TAM cells in a synergic manner. These results suggested that combining the chemotherapeutic effect of EGCG with siRNA-mediated Nrf2 knock-down results in the feasibility of using Nrf2 inhibitors to increase efficacy of chemotherapeutic drugs.

Keywords: Apoptosis; Epigallocatechin-3-gallate; MCF-7 breast cancer; Nrf2 siRNA; Tamoxifen.

Fig. 1

The inhibitory effect of tamoxifen on MCF-7 and MCF-7/TAM cell proliferation. Cells were treated with various concentrations of tamoxifen for 24 h, and then cell viability was determined by the MTT assay. Data were expressed as means ± SEM of three independent experiments

Fig. 2

The expression of total (a) and nuclear (b) protein Nrf2 and its downstream gene HO-1 and NQO1 was assessed by Western blot in MCF-7 and MCF-7/TAM cells. Results represent mean values of three experiments ± SEM

Fig. 3

The effect of EGCG on release of LDH (a), MDA contents (b), SOD (c), and GSH-PX (d) activities in tamoxifen-resistant MCF-7 cells. Results represent mean values of three experiments ± SEM

Fig. 4

Effects of EGCG on Nrf2 expression in MCF-7/TAM cells. MCF-7 and MCF-7/TAM cells were treated with indicated concentrations of EGCG for 24 h, and the change of mRNA (a) and protein expression (b) levels of total Nrf2 and its downstream genes NQO1 and HO-1 in MCF-7 and MCF-7/TAM cells were detected by qRT-PCR and immunoblot analysis, respectively. Results represent mean values of three experiments ± SEM

Fig. 5

Transient knockdown of Nrf2 by siRNA sensitizes MCF-7/TAM cells to tamoxifen. a The Nrf2, NQO1, and HO-1 protein levels as well as the nuclear Nrf2 levels were compared in MCF-7/TAM cells transfected with Nrf2 siRNA or control siRNA. b MCF-7/TAM cells, transfected with Nrf2 siRNA or control siRNA for 48 h, were treated with the indicated doses of tamoxifen for 24 h, and cell viability was determined by the MTT assay. c Apoptosis analysis by flow cytometry of MCF-7/TAM cells, transfected with Nrf2 siRNA and treated with the indicated doses of tamoxifen. The data are presented as means ± SEM

Fig. 6

Knockdown of Nrf2 by RNAi reduces colony formation in soft agar. MCF-7/TAM cells were transfected with Nrf2 siRNA and seeded in 0.3 % agarose containing Dulbecco’s modified Eagle’s medium with 10 % fetal bovine serum. The colony numbers were counted 10 days later. The numbers of colonies of treated cells were standardized against the control cells (set at 100 %). The data shown are means and SD from two independent triplicate experiments. The difference between treatments is statistically significant (p < 0.001)

Fig. 7

The inhibitory effect of EGCG on Nrf2 siRNA transfected MCF-7/TAM cell proliferation. Cells were treated with various concentrations of EGCG 24 h, and cell viability was determined by the MTT assay

Fig. 8

a Apoptosis analysis by flow cytometry of MCF-7/TAM cells, transfected with Nrf2 siRNA and co-treated with the indicated doses of EGCG. b Western blots analysis of apoptotic factors in MCF-7/TAM transfected with Nrf2 siRNA and co-treated with EGCG. The data are presented as means ± SEM