Anticancer effect of ethanol Lycium barbarum (Goji berry) extract on human breast cancer T47D cell line

Abstract The anticancer activity of ethanol extract isolated from Goji berry (EEGB) on T47D human breast cancer cell line has been reported. Cell viability and cell proliferation were examined with the use of BrdU, MTT and NR methods. Induction of apoptosis was assessed by propidium iodide and Hoechst 33342 staining. Expression of genes involved in cell proliferation, apoptosis, cell cycle control and regulation of transcription was estimated using Western blotting analysis. EEGB inhibited the proliferation of breast cancer cells in a time-, and dose-dependent manner. The study confirmed the lack of EEGB cytotoxic activity to normal human skin fibroblasts. Western blot analysis demonstrated an increase in pro-apoptotic and a decrease in anti-apoptotic proteins’ expression in cells treated with the extract. Anticancer activity and lack of toxicity against normal cells indicate a chemopreventive potential of Goji berries in breast cancer treatment.


Introduction
Healthy diet has been long proposed as an alternative strategy to reduce the risk of cancer (Peuhu et al. 2010;zhang et al. 2014). Evidence suggests that bioactive phytochemicals derived from fruit play an important role in prevention of this disease (van Duijnhoven et al. 2009). In the recent years, there has been an increasing interest in the anticancer activity of exotic berry-type fruit, including the Goji berry (GB) (Lycium barbarum), and their products (Seeram 2009;li et al. 2013). Therefore, in the present study, we investigated antiproliferative and pro-apoptotic activity of ethanol extract of Goji berries (EEGB) in the T47D human breast carcinoma cell line in order to evaluate its efficacy as a potential anticancer and chemopreventive agent.

Results and discussion
Our experiments confirmed the antiproliferative properties of the EEGB in a dose-and time-dependent manner negatively affecting both cancer cells' DNA synthesis and proliferation pathways. The antiproliferative effect of EEGB was determined in T47D and human normal skin fibroblasts (HSF) cells using a MTT cell viability assay. The EEGB strongly decreased human T47D cell proliferation ( Figure S1A). At the highest concentration (1 mg/ml), the extract reduced cell proliferation to 70, 55.7 and 51.4% of control cells after 24, 48 and 96 h, respectively. IC50 was 0.75 mg/ml (96 h). In contrast to the T47D, HSF cells were unaffected by the EEGB treatment even at a concentration of 2.5 mg/ml ( Figure S1B). T47D cells treated with 0.001-1 mg/ml EEGB proliferated slower than control cells and showed decline of BrdU incorporation during the DNA synthesis ( Figure S1C). The most pronounced effect was observed when T47D cells were treated with 1 mg/ml EEGB, which suppressed proliferation up to 75%. IC50 (0.7 mg/ml) was determined by the original programme of the Medical University of lublin 'ED50' . The cytotoxic effect of EEGB was determined using NR cell viability assay. The extract slightly decreased T47D cell viability in a dose-dependent manner ( Figure S1D). Recent work by Shen and Du (2012) suggests that polysaccharides isolated from Goji fruit can inhibit proliferation of another breast cancer cell line, MCF-7, in a dose-and time-dependent fashion.
The anticancer mechanism of GB action may be attributed mainly to the decreased expression of cell cycle-regulated proteins, including p53, p21 waf1 , cyclins A, B, D and CDK's (Mao et al. 2011). Thereby, using Western blotting technique, we analyzed expression of p21, p27 and p53 proteins, which control cell cycle check points at G1/S and G2/M phases. The treatment of T47D cells with the extract for 48 h resulted in a significant increase in p21 expression even at a concentration of 0.05 mg/ml. There were no significant changes in the expression of p27 protein in cancer cells relative to control treated with extract concentrations of 0.1-2 mg/ml. A slight increase in p53 protein expression was observed at 2 mg/ml EEGB concentration compared to the control ( Figure S3C). Shen and Du (2012) demonstrated similar results -a marked increase in the level of activity of p53 and p21 waf1/cip1 proteins in MCF-7 cancer cell line treated with polysaccharide from Goji berries at the concentration range 10-300 μg/ml. The analyses of other proteins involved in cell cycle progression showed a slight decrease in the expression of the cyclin-dependent kinase 6 (CDK 6) and cyclin D1 at higher concentration of EEGB (1 and 2 mg/ml) ( Figure S3D).
In our study, we have shown the apoptotic and necrotic effects of EEGB on T47D cells using propidium iodide and Hoechst solutions. At a concentration of 0.1 mg/ml, EEGB induced significant apoptotic changes in the cells (bright blue fluorescence) (37% of cancer cells) ( Figure S2A). The intensity of these changes increased with the concentration of the extract used (61.6 and 88% of cancer cells treated 0.5, and 1 mg/ml EEGB, respectively). At a concentration of 0.5 mg/ml EEGB, a statistically significant change in the number of necrotic cells was first observed ( Figure S2B). These findings suggest that EEGB induces cancer cell death mainly through apoptosis. In the present study, we have also shown an EEGB-dependent induction of apoptosis, which could be connected with observed clear increase in the expression of pro-apoptotic Bax protein, and decrease in the expression of anti-apoptotic Bclxl protein, suggesting an EEGB-mediated apoptosis through the mitochondrial pathway. To assess whether EEGB modulates changes in pro-and anti-apoptotic proteins, the expression of Bax and Bclxl was evaluated by Western blotting. The treatment of T47D cells with the extract for 48 h resulted in a dose-dependent significant increase in Bax protein expression, and decrease in Bclxl protein expression in relation to control, observed even at a concentration of 0.05 mg/ml ( Figure S3A). Similar observations were made in polysaccharide-treated PC-3 and DU-145 prostate cancer cells, showing that lBPs isolated from GB regulated the expression of Bax to induce apoptosis (luo et al. 2009). EEBG-mediated changes in pro-and anti-apoptotic proteins expression as well as in the level of p53 and p21 proteins observed in our studies suggest a potential involvement of the NF-kB pathway, as shown in liver cancer cells (zhang et al. 2013). EEBG treatment resulted in a slight decrease in the expression of NF-κB, and an increase in the expression of IκB in T47D cell line. This transcription factor plays an important role in carcinogenesis; however, the conditions that determine whether NF-κB will protect cells or direct them on the path of apoptosis are not yet fully understood (Karin and lin 2002).

Conclusions
The use of natural therapeutic agents has been pursued in the last decade for different indications with emphasis on cancer treatment. In this manuscript, we described a potent in vitro anticancer effect of the Goji berry extract on T47D breast cancer cells and explored the mechanisms underlying this action. Evident antitumourogenic actions of GB together with the lack of toxic effects on normal cells make GB constituents attractive chemopreventive agents.