Polydatin down-regulates the phosphorylation level of Creb and induces apoptosis in human breast cancer cell

Polydatin (PD), a component isolated from Polygonum cuspidatum, has a number of biological functions. However, the antitumor activity of PD has been poorly investigated. In this study, the effect of PD on cell proliferation was evaluated by thiazolyl blue tetrazolium bromide assay. Cell cycle distribution and apoptosis were investigated by flow cytometry. The phosphorylation levels of panel of phosphor-kinases were detected by human phospho-kinase arrays. The expression of several proteins associated with cell cycle and apoptosis were analyzed by Western blot analysis. Results showed that PD effectively inhibited the growth of MDA-MB-231 and MCF-7 breast cancer cell lines. Cell cycle analysis demonstrated that PD induced S-phase cell cycle arrest. Human phosphor-kinase arrays showed that the phosphorylation level of cAMP response element-bingding proteins(Creb) was down-regulated, and the results were further confirmed by Western blot analysis. Western blot analysis showed that the expression of protein of cyclin D1 decreased in a time- and dose- dependent manner. Results suggest that PD is a potential therapeutic natural compound.


Introduction
Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among females worldwide, with an estimated 1.7 million cases and 521,900 deaths in 2012. Breast cancer alone accounts for 25% of all cancer cases and 15% of all cancer death among females [1]. Advances in surgery, radiotherapy, hormonal therapy, and chemotherapy have improved the treatment outcome of breast cancer. However, more than 410,000 women still die from this disease every year [2]. To date, chemotherapy has become the most frequently used therapeutic strategy for breast cancer. Furthermore, the outcome of chemotherapy in a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 patients with advanced breast cancer is poor, thus highlighting the need for novel chemotherapy agent.
As described previously [3], polydatin(PD) is a glycoside of resveratrolin which the glycoside group is bonded in the C-3 position and substitutes a hydroxyl group. (the chemical structure of PD is shown in Fig 1). This substitution leads to conformational changes in the molecule, thus resulting in changes in its biological properties. PD is more efficiently absorbed and more resistant to enzymatic oxidation than resveratrol and is soluble in hot water. In contrast to resveratrol, which penetrates cells passively, PD enters cells via an active mechanism using glucose carriers. These properties provide PD with greater bioavailability than resveratrol. Previous studies have demonstrated the chemo-preventive and anticancer activities of resveratrol [4][5][6][7][8][9][10][11][12][13]. However, few previous studies have analyzed the effects of PD on human cancer cells. In the currentstudy, the effects of PD on the proliferation, cell cycle phase distribution, and apoptosis of human breast cancer cell lines as well as potential underlying mechanisms, were investigated. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Cell lines and cell culture
Cancer cell lines were purchased from American Type Culture Collection (Manassas, VA, USA). The cells were maintained as a monolayer in DMEM or RPMI-1640 medium supplemented with 10% fetal calf serum, 2mM glutamine, 100μg/ml streptomycin, and 100 U/ml penicillin in a humidified atmosphere containing 5% CO 2 . Cells in the logarithmic phase were used in the experiments.

MTT viability assay
The determination of cell viability was performed using MTT assay as described previously [14]. Cells were incubated in flat-bottom 96-well plates (6 × 10 3 cells/well) overnight. Thereafter, cells were treated with DMSO (0.1%) or an increasing dosage of PD. Following 20, and 44 h of treatment, 20 μl MTT (5 mg/ml) was added to each well and further incubated for 4 h. Cells were then solubilized in 150 μl DMSO. The absorbance reading was obtained using a Dynatech 96-well spectrophotometer (Dynatech Laboratories, Chantilly, VA, USA). The amount of MTT dye reduction was calculated on the basis of the difference between the absorbances at 570 and 630 nm. The cell viability in treated cells was expressed as the amount of dye reduction relative to that in untreated control cells.

Apoptosis assays and cell cycle distribution analysis
The percentage of cells that actively underwent apoptosis was analyzed using annexin V-

Human phosphor-kinase arrays
The phosphorylation profiles of kinases after treatment with PD were analyzed using the Human Phosphor-Kinase Array kit according to the manufacturerreatment with PD were analyzed using the Human Phosphor. incubated forusing a Muse Cell Analyzer (Millipore Corporation, Haywarf biotinylated detection antibodies, streptavidin-HRP, and chemiluminescent detection reagents were used to detect the phosphorylated cells. The relative expression of specific protein was determined by following the quantification of scanned images.

Western blotting
Western blot assays were performed as described earlier [15]. Cells were treated with DMSO (0.1%) or PD. After 24-48h of treatment, cell lysates were prepared, and equal aliquots of protein extract were electrophoresed by SDS-PAGE. After transferring the lysates to nitrocellulose membranes, blots were blocked with 5% milk protein and were incubated using these primary antibodies for 2h or overnight: P-Creb(Catalog#: 9198,Cell Signaling 1:1000 dilution); Creb (Catalog#: 9197,Cell Signaling 1:1000 dilution); cyclinD1(Catalog#:SC-8396, Santa Cruz 1:500 dilution); cyclinA (Catalog#:SC-596, Santa Cruz 1:500 dilution); cyclinE (Catalog#:SC-247, Santa Cruz 1:1000 dilution). The blots were then reincubated with the corresponding antibodies for 1h. Blotted proteins were visualized using the enhanced chemiluminescence system, with color markers as molecular size standards. As an internal control for the amount of protein loaded, the same filter was also immunoblotted with a monoclonal β-actin(C-4, Santa Cruz 1:1000 dilution) antibody. The protein assay kit was purchased from Bio-Rad (Hercules, CA) and the enhanced chemiluminescent Western blotting detection reagents were purchased from Pharmacia Biotech (Piscataway, NJ).

Statistical analysis
Data were expressed as mean±SD. Comparisons between the DMSO-treated control cells and PD-treated cells were made using Student's t test. Comparisons were considered statistically significant when P<0.05.

PD inhibits theproliferation ofbreast cancer cells
The cytotoxicity of PD on breast cancer cells MDA-MB-231 and MCF-7 was determined using MTT assay. The decrease in absorbance in this assay can be due to either a consequence of cell death or reduction in cell proliferation. As shown in    for 4h (Fig 5). Western Blot analysis showed that the levels of Phospho-AKT and Phospho-Erk increased first and then decreased. At the same time, no obvious change was observed in the level of the AKT and Erk proteins(datanot shown). To explore the role of phosphatidylinositol 3-kinase (PI3K)/protein kinase B9 (AKT) and mitogen-activated protein kinase (MAPK) pathways in the inhibitory effects PD on cell proliferation, breast cancer cells MDA-MB-231 and MCF-7 were pre-treated with 10μM Specific PI3K inhibitor wortmannin, ERK1/2 inhibitor PD98059, P38 inhibitor SB203580, and JNK inhibitor SP600125 for 1h. The cells were then treated by PD (2.5, μM) for 24 and 48h. As shown in Fig 7, the inhibitory effect of PD on cell proliferation was not affected by the pre-treatment of wortmannin, SB203580, SP600125, orPD98059. The cell cycle distribution and apoptosis induced by PD were not affected by wortmannin, SB203580, SP600125, or PD98059, either (data not shown).

PD down-regulates cyclin D1 expression in breast cancer cell lines
To explore the mechanism underlying the effects of PD on S-phase cell cycle arrest, the expression levels of cell cycle-related protein cyclin D1 were examined. The results showed (Fig 8) that the expression of protein cyclin D1 in MDA-MB-231and MCF-7 cells began to decrease following the treatment with PD for 8h, and continued after 40h of treatment. However, the expression of protein cyclin A didn't change significantly. Furthermore, the results showed that PD induced the decrease incyclin D1 in a dose-dependent manner (Fig 8C).

Discussion
As described previously [3], Polygonum cuspidatum, a traditional Chinese medicinal herb commonly used for its roots and rhizomes, has been officially listed in the Pharmacopoeia for a number of years. PD is one of the main effectivecomponents of P. cuspidatum. Previously, our study [3]demonstrated that PD could inhibit cell proliferation, induce S-phase cell cycle arrest and apoptosis in human lung cancer cells. In the present study, we evaluated the effect of PD on human breast cancer cell lines MDA-MB-231 and MCF-7, and explored the possible mechanisms. The results showed that PD could effectively inhibit the proliferation and induce the S-phase cell cycle arrest apoptosis in human breast cancer cells MDA-MB-231 and MCF-7.
Creb is a well-characterized transcription factor of the basic leucine zipper family [16]. Creb regulates a number of genes with diverse functions, including proliferation, survival, memory, and learning [17]. Recent studies showed that Creb plays an important role in the development and metastasis of different solid tumors. The level of Creb1 in breast cancer patients is elevated and is significantly up-regulated in patients with a poor prognosis, metastatic disease, and nodal involvement [18]. The phosphorylation of Ser133 and/or the nuclear translocation of the transducer of regulated Creb activity co-activators are required for Creb activation [17,19].
In this study, we found that PD significantly decreased the phosphorylation levels of Creb in a dose-dependent manner. However, the levels of Creb protein were not affected. The results indicated that PD inhibited the activation of Creb by decreasing the phosphorylation of Creb,thus inhibiting the proliferation of breast cancer cells.
In recent studies using a genome wide analysis, Creb was found to regulate approximately 4,000 target genes. The list includes genes that are important for cell cycle control such as cyclinD1 and cyclin A [20]. CyclinD1, an important regulator of cell cycle progression, functions as a transcriptional co-regulator [21]. CyclinD1 levels must be high during the G 1 phase to initiate DNA synthesis but must be suppressed to low levels during the S phase for efficient DNA synthesis. To continue cell proliferation, cyclinD1 must be induced once again during the G 2 phase [22]. In the present study, we found that the phosphorylation levels of Creb began to decrease after breast cancer cells were treated with PD for 2h, whereas the decrease in the expression level of cyclinD1 occurred at 8h of treatment. Therefore, we speculate that the mechanism underlying the inhibitory effect of PD might be due to inhibition in Creb phosphorylation which in turn suppresses the transcription of cyclinD1, and causes cell cycle progression arrest at the S phase, eventually leading to apoptotic death of breast cancer cells.
The PI3K/AKT pathway are implicated in the regulating of proliferation, survival, and death of multiple cells. Recent studies showed that the PI3K/AKT pathway plays a key role in promoting cell proliferation and inhibiting cell apoptosis in the genesis and development of tumors, and abnormalities in this pathway were found in a variety of tumors [23][24][25]. In the current study, we found that the levels of Phospho-AKT increased in breast cancer cells after treatment byPD. The PI3K inhibitor wortmannin exhibited an insignificant effect on the cell survival, cell cycle, and apoptosis of PD-treated breast cancer cells. These results indicated that the PI3K pathway was not involved in the effect of PD on cell proliferation.
MAPKs include Erk(extracellular signal-regulated protein kinase), JNK(c-Jun N-terminal kinase),and P38. MAPKs are important signaling pathways in the transduction of extracellular stimuli into intracellular responses, and their functions include regulating cell proliferation, differentiation, and apoptosis [26][27][28]. In this study, Phospho-P38 and Phospho-Erk were increased in breast cancer cells after treatment by PD. However, cell growth inhibition, cell cycle arrest, and apoptosis induced by PD were not affected by Erk inhibitor PD98059 or p38 inhibitor SB203580. These results indicated that the MAPK pathway was not involved in the effect of PD on cell proliferation, either. The increases in levels of Phospho-AKT, Phospho-P38, and Phospho-Erk are likely due to mechanism of cell protection triggered by PD. However, this cell-protecting mechanism is not enough to counter the anti-tumor effect of PD and subsequent cell apoptosis.
In conclusion, we have performed a preliminary investigation into the inhibitory effect of PD on breast cancer cells. The anti-proliferation effect of PD involves the suppression of cell cycle progression and the induction of apoptosis in human breast cancer cells. The phosphorylation of Creb and cyclinD1 might be involved in the antitumor effect of PD. However, the antitumor effect and toxicity of PD in vivo is unknown. Future studies on the in vivo effect of PD are necessary.