The effect of PI3K/AKT and MAPK pathways on inhibitory effects of PD in human breast cancer cells.

<p>Exponentially growing cells in 96-well plates were pre-treated with with 10μM Specific PI3K inhibitor wortmannin, ERK1/2 inhibitor PD98059, P38 inhibitor SB203580, and JNK inhibitor SP600125 for 1h. Then MDA-MB-231 and MCF-7 cells were treated by PD (2.5, μM) for 24h (A) (C) and 48h(B) (D),. And then cells were subjected to MTT viability assay. Data are presented as the percentage of DMSO-treated controls (mean ± SD) from three independent experiments. PD, polydatin; MTT, thiazolyl blue tetrazolium bromide.</p

Inhibitory effects of PD on the growth of human breast cancer cells.

<p>Exponentially growing cells in 96-well plates were continuously treated with the indicated concentrations of PD for 24 and 48 h and then subjected to MTT viability assay. Dose-response curves of PD in (A) MDA-MB-231 and (B) MCF-7 cells 24 or 48 h following treatment. Data are presented as the percentage of DMSO-treated controls (mean ± SD) from three independent experiments. PD, polydatin; MTT, thiazolyl blue tetrazolium bromide; DMSO, dimethysulfoxide.</p

Inhibition effect of PD on the phosphorylaion levels of Creb.

<p>(A) Human breast cancer cells were treated with PD for different times. Expression levels of phospho-Creb and Creb were detected by western blot analysis and β-actin was used as a control. (B) phospho-Creb protein bands were quantitied by densitometry and the data are presented as the mean ± SD from three experiments.(C) Creb protein bands were quantitied by densitometry and the data are presented as the mean ± SD from three experiments. *P<0.05, compared with the respective controls. PD, polydatin.</p

Human phosphor-kinase array analysis in response to PD treatment.

<p>Whole cell lysates were prepared from MDA-MB-231 and MCF-7 cell lines, either left untreated or exposed to PD and hybridized with a human Phosphor-Kinase array kit.(A)MDA-MB-231 cell lines were treated with PD for 4h, (B) MCF-7 cell lineswere treated with PD for 4h. (C)MDA-MB-231 cell line were treated with PD for 48h Each kinase is spotted in duplicate. The pairs of dots in each corner are positive controls. Each pair of the most positive kinase dots is denoted by a numeral, with the identity of the corresponding kinases listed as follows: Upon exposure to PD, a decrease in phosphorylated Creb was confirmed by Western blot analysis. As shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176501#pone.0176501.g006" target="_blank">Fig 6</a>, the decrease in the phosphorylation levels of Creb occurred when MDA-MB-231 and MCF-7 cells were treated with PD for 2h. In MCF-7 cells, phosphorylated Crebwas still in a lower level after exposure to PD for 40h. In MDA-MB-231 cells, the phosphorylation levels of Creb began to increase after cells were treated with PD for 16h; however, the rate of increase was still lower than untreated control cells in 40h. At the same time, no obvious change was observed in the level of Creb protein.</p

Cell cycle arrest induced by PD in human breast cancer cells.

<p>Cells were treated with increasing concentrations of PD. Following 24h and 48 h of treatment, cells were labeled with Muse Cell Cycle Reagent and then analyzed by Muse Cell Analyzer. Results indicate the percentage of cells in each phase of the cell cycle. All experiments were performed in duplicate and yielded similar results. (A) Original images of cell cycle distribution in MDA-MB-231 cells are presented. (B) The percentage of cells in S phase is presented as the mean ± SD from three various experiments in MDA-MB-231.*P<0.05, compared with the respective controls. (C) Original images of cell cycle distribution in MCF-7 cells. (D)The percentage of cells in S phase is presented as the mean ± SD from three various experiments in MCF-7 cells. *P<0.05, compared with the respective controls. PD, polydatin.</p

Apoptotic effects of PD on MDA-MB-231 and MCF-7 human breast cancer cells.

<p>Cells were treated with the indicated concentrations of PD for 48 h, stained with Muse Annexin V & Dead Cell reagent and then analyzed for apoptosis by Muse Cell Analyzer. The results indicate the percentage of Annexin V-positive cells (apoptosis). All experiments were performed in duplicate and yielded similar results. (A) Original images of apoptosis in MDA-MB-231 cells. (B) The percentage of cells undergoing apoptotic cell death is presented as the mean ± SD from three separate experiments in MDA-MB-231 cells. *P<0.05, vs. the respective controls. (C) Original images of apoptosis in MCF-7 cells. (D) The percentage of cells undergoing apoptotic cell death is presented as the mean ± SD from three separate experiments in MCF-7 cells. *P<0.05, compared with the respective controls. PD, polydatin.</p