Survivin selective inhibitor YM155 induce apoptosis in SK-NEP-1 Wilms tumor cells

  • Yan-Fang Tao1Email author,

    Affiliated with

    • Jun Lu1Email author,

      Affiliated with

      • Xiao-Juan Du2,

        Affiliated with

        • Li-Chao Sun3,

          Affiliated with

          • Xuan Zhao4,

            Affiliated with

            • Liang Peng5,

              Affiliated with

              • Lan Cao1,

                Affiliated with

                • Pei-Fang Xiao1,

                  Affiliated with

                  • Li Pang1,

                    Affiliated with

                    • Dong Wu1,

                      Affiliated with

                      • Na Wang1,

                        Affiliated with

                        • Xing Feng1,

                          Affiliated with

                          • Yan-Hong Li1,

                            Affiliated with

                            • Jian Ni6,

                              Affiliated with

                              • Jian Wang1Email author and

                                Affiliated with

                                • Jian Pan1Email author

                                  Affiliated with

                                  BMC Cancer201212:619

                                  DOI: 10.1186/1471-2407-12-619

                                  Received: 29 August 2012

                                  Accepted: 21 December 2012

                                  Published: 26 December 2012

                                  Abstract

                                  Background

                                  Survivin, a member of the family of inhibitor of apoptosis proteins, functions as a key regulator of mitosis and programmed cell death. YM155, a novel molecular targeted agent, suppresses survivin, which is overexpressed in many tumor types. The aim of this study was to determine the antitumor activity of YM155 in SK-NEP-1 cells.

                                  Methods

                                  SK-NEP-1 cell growth in vitro and in vivo was assessed by MTT and nude mice experiments. Annexin V/propidium iodide staining followed by flow cytometric analysis was used to detect apoptosis in cell culture. Then gene expression profile of tumor cells treated with YM155 was analyzed with real-time PCR arrays. We then analyzed the expression data with MEV (Multi Experiment View) cluster software. Datasets representing genes with altered expression profile derived from cluster analyses were imported into the Ingenuity Pathway Analysis tool.

                                  Results

                                  YM155 treatment resulted in inhibition of cell proliferation of SK-NEP-1cells in a dose-dependent manner. Annexin V assay, cell cycle, and activation of caspase-3 demonstrates that YM155 induced apoptosis in SK-NEP-1 cells. YM155 significantly inhibited growth of SK-NEP-1 xenografts (YM155 5 mg/kg: 1.45 ± 0.77 cm3; YM155 10 mg/kg: 0.95 ± 0.55 cm3) compared to DMSO group (DMSO: 3.70 ± 2.4 cm3) or PBS group cells (PBS: 3.78 ± 2.20 cm3, ANOVA P < 0.01). YM155 treatment decreased weight of tumors (YM155 5 mg/kg: 1.05 ± 0.24 g; YM155 10 mg/kg: 0.72 ± 0.17 g) compared to DMSO group (DMSO: 2.06 ± 0.38 g) or PBS group cells (PBS: 2.36 ± 0.43 g, ANOVA P < 0.01). Real-time PCR array analysis showed between Test group and control group there are 32 genes significantly up-regulated and 54 genes were significantly down-regulated after YM155 treatment. Ingenuity pathway analysis (IPA) showed cell death was the highest rated network with 65 focus molecules and the significance score of 44. The IPA analysis also groups the differentially expressed genes into biological mechanisms that are related to cell death, cellular function maintenance, cell morphology, carbohydrate metabolism and cellular growth and proliferation. Death receptor signaling (3.87E-19), TNFR1 signaling, induction of apoptosis by HIV1, apoptosis signaling and molecular mechanisms of cancer came out to be the top four most significant pathways. IPA analysis also showed top molecules up-regulated were BBC3, BIRC3, BIRC8, BNIP1, CASP7, CASP9, CD5, CDKN1A, CEBPG and COL4A3, top molecules down-regulated were ZNF443, UTP11L, TP73, TNFSF10, TNFRSF1B, TNFRSF25, TIAF1, STK17A, SST and SPP1, upstream regulator were NR3C1, TP53, dexamethasone , TNF and Akt.

                                  Conclusions

                                  The present study demonstrates that YM155 treatment resulted in apoptosis and inhibition of cell proliferation of SK-NEP-1cells. YM155 had significant role and little side effect in the treatment of SK-NEP-1 xenograft tumors. Real-time PCR array analysis firstly showed expression profile of genes dyes-regulated after YM155 treatment. IPA analysis also represents new molecule mechanism of YM155 treatment, such as NR3C1 and dexamethasone may be new target of YM155. And our results may provide new clues of molecular mechanism of apoptosis induced by YM155.

                                  Keywords

                                  YM155 SK-NEP-1 Survivin Apoptosis Real-time PCR array

                                  Background

                                  Wilms tumor (WT) is the most common malignant neoplasm of the urinary tract in children [1]. Although it is curable with long-term survival, the combination of surgery, chemotherapy and often radiotherapy in some cases results in severe complications in adulthood [2]. Therefore, novel therapeutic strategies that would decrease treatment burden and improve outcome for high risk patients are required. We evaluated the efficacy of YM155, an inhibitor of survivin, to inhibit Wilms tumor development in xenografts models.

                                  Overexpressed survivin can be detected in virtually every human tumor, but undetectable or present at very low levels in most normal adult tissues [35]. A ‘tumor-specific’ expression of survivin is predominantly dictated at the level of transcription, and that survivin gene expression may be globally ‘deregulated’ in tumors, in vivo[4, 6, 7]. Accordingly, survivin promoter activity is basically silent in normal cells, but strongly activated in tumor cells, and this occurs independently of cellular heterogeneity, mitotic status, or genetic makeup. The differential expression of the survivin gene in normal versus tumor cells is so dramatic that therapeutic strategies to drive tumor-specific expression of suicidal genes under the control of the survivin promoter have now advanced to preclinical stages in a number of settings [3, 59].

                                  YM155, a novel small-molecule survivin suppressant, has been shown to suppress survivin with little effect on expression levels of other IAP family or Bcl-2 related proteins [10]. YM155 has been demonstrated antitumor activity, with survivin suppression and tumor cell apoptosis, in various human cancer models [6, 8, 1017]. Survivin is the smallest member of the Inhibitor of Apoptosis (IAP) gene family. Originally described as cell survival factors that target caspase, we now know that IAPs have a much broader portfolio of functions, encompassing signaling pathways, cell division, metabolism and adaptation to unfavorable environments. Survivin embodies this multifunctional diversity, and compelling data accumulated over a decade have elucidated many of its essential roles as a regulator of mitosis [17], a broad cytoprotective factor [18, 19], and an effector of cellular adaptation to stress [20, 21]. These disparate functions rely on hosts of regulated interactions that involve survivin and multiple protein partners, including tubulin [22, 23] and various chromosomal passenger proteins in the control of mitosis, other IAP family members to counteract apoptosis, and Heat Shock Proteins in the modulation of the cellular stress response. These ‘survivin networks’ are dramatically exploited in cancer, and survivin is unanimously viewed as one of the most prominent cancer genes.

                                  In the present study, the antitumor effect of YM155 have been evaluated in human SK-NEP-1 Wilms tumor cells and xenograft models to further characterize its preclinical efficacy, and the molecular mechanism was exploited with real-time PCR arrays.

                                  Methods

                                  Cell and culture conditions

                                  SK-NEP-1 Human kidney (Wilm's Tumor) cell line obtained from the American Type Culture Collection (ATCC) was maintained in the Maccyo’5 (Life Technologies Inc., Gaithersburg, MD, USA) supplemented with 20% heat-inactivated fetal bovine serum (Invitrogen Co., NY, USA) in a humidified incubator with 5% CO2 at 37°C. YM155 (Cat: S1130 Selleck Chemicals, West Paterson, NJ, USA) was dissolved in DMSO (Cat: D4540 Sigma–Aldrich, St. Louis, MO, USA)

                                  Cell proliferation

                                  Sk-NEP-1 cells (2×104) were seeded in 96-well plates overnight and incubated with DMSO, 1 nM YM155, or increasing concentrations of YM155 (0.005, 0.01, 0.02, 0.04, 0.08, 0.16, 0.32, 0.64 or 1.28 μM) for 24 hours. The volume of DMSO added to the vehicle treated wells was the same as that added to the drug treated wells. Each drug concentration was performed in four replicate wells. 20uLMTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide) solution (5 mg/ml) was added to each well and incubated at 37°C for a further 4 hours. Then 200 uL of DMSO was added to each well after the medium was removed. The optical density (OD) values were measured at 490 nm on a scanning multi-well spectrophotometer (BioRad Model 550, USA). Compared with the control group, the relative survival rate of remained cells was calculated from the absorbance values. Cell proliferation was calculated as a percentage of the DMSO- treated control wells with IC50 values derived after plotting proliferation values on a logarithmic curve.

                                  Cell cycle analysis

                                  Cells were collected and washed with PBS for 5 minutes by centrifugation at 125 × g. Cells were fixed with paraformaldehyde and transparented with 0.5% Triton X-100. Then cells were resuspended in a staining solution containing 1.5 μmol/L propidium iodide (P4170, Sigma–Aldrich, St. Louis, MO, USA) and 25 μg/ml RNase A and incubated for 30 minutes in 37°C. The samples (10000 cells) were analyzed by fluorescence-activated cell sorting with a Beckman Gallios™ Flow Cytometer.

                                  Apoptosis assay

                                  Apoptosis assay was according to the manual operation of BD Annexin V Staining Kit (Cat: 556420, BD Biosciences, Franklin Lakes, NJ USA). Briefly, wash cells twice with cold PBS and then resuspend cells in 1×Binding Buffer at a concentration of ~1 ×106 cells/ml. Transfer 100 μl of the solution (~1×105 cells) to a 5 ml culture tube. Add Annexin V and PI 5 μl/test. Gently mix the cells and incubate for 15 min at RT in the dark. Add 400 μl of 1×Binding Buffer to each tube. Analyze by flow cytometry as soon as possible (within 1 hour).

                                  Western blot analysis

                                  For western blot analysis, cellular proteins were extracted in 40 mM Tris–HCl (pH 7.4) containing 150 mM NaCl and 1% (v/v) Triton X-100, supplemented with a cocktail of protease inhibitors. Equal amounts of protein were resolved on 12% SDS-PAGE gels, and then transferred to a PVDF membrane (Millipore, Bedford, MA). Blots were blocked and then probed with antibodies against Caspase 3 (1:1000,

                                  Cell Signaling Technology, Inc. Danvers, MA), GAPDH (1:5000, Sigma, St. Louis, MO). After washing, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies and visualized by enhanced chemiluminescence kit (Pierce, Rockford, IL). Protein bands were visualized after exposure of the membrane to Kodak X-ray film.

                                  Xenograft assays the treatment effect of YM155 in nude mice

                                  This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Soochow university (Permit Number: 2011-10-21). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Female nu/nu mice, aged 4–6 weeks, obtained from the Jackson Laboratory (Vitalriver, China), were kept in Class 10000 Clean Room at the Laboratory Animal Center of Soochow University (http://​dwzx.​suda.​edu.​cn/​pages/​index.​aspx.) SK-NEP-1 cells were subcutaneously injected into five nude mice every group. 10 days after injection, mice were treated with PBS, DMSO, YM155 5 mg/kg and 10 mg/kg dose. During the next six weeks these mice were examined for subcutaneous tumor growth. The tumor volumes were calculated according to the following formula: volume = length × width2/2. After the last treatment, the mice were killed and the tumor weight was measured.

                                  Real-time PCR array analysis

                                  For RNA extraction, cells were immediately submerged in 2 ml Trizol (Invitrogen Co., NY, USA), stored at −80°C until further processed. A volume of 1 ml of each sample was spun at 4°C for 15 min at 12,000 g to remove debris and DNA, 1 ml of supernatant was mixed with 200 ul chloroform, shaken for 15 seconds, incubated at Room Temperature for 2–3 minutes and spun for 10 minutes at 12,000 g at 4°C. RNA was precipitated by adding 500 ul of the aqueous phase to an equal volume of isopropanol and spun at 14,000 g at 4°C for 10 minutes. RNA was washed with 75% ethanol, spun at 14,000 g at 4°C for 10 minutes, dried and resuspended in 40 ul DEPC-treated H2O . The final RNA concentration was determined using a spectrophotometer (Nanodrop 2000) and the purity was assessed by agarose gel electrophoresis. CDNA synthesis was performed on 4 ug of RNA in a 10 ul sample volume using SuperScript II reverse transcriptase (Invitrogen Co., NY, USA) as recommended by the manufacturer. The RNA was incubated with 0.5 ug of oligo(dT)12–18mers primers (Invitrogen Co., NY, USA) for 7 minutes at 70°C and then transferred onto ice. Then, 9 ul of a master mix containing 4 ul of SuperScript II buffer, 2 ul of 0.1 M DTT, and 1 ul each of dNTPs stock (10 mM), Rnasin (40 UI) and SuperScript II were added to the RNA sample, spun and incubated at 42°C for 60 min followed by 5 min at 70°C to inactivate the enzyme. CDNA was stored at −20°C. Real-time PCR array (SABioscience Human Apoptosis PCR Array PAHS-3012) analysis was performed in a total volume of 20 ul including 2ul of cDNA, primers (0.2 mM each) and 10 ul of SYBR Green mix (Roche Co., Basel, Switzerland.). Reactions were run on an Light cycler 480 using the universal thermal cycling parameters (95°C 5 min, 45 cycles of 10 sec at 95°C, 20 sec at 60°C and 15 sec at 72°C; melting curve: 10 sec at 95°C, 60 sec at 60°C and continues melting). Results were obtained using the sequence detection software Light cycler 480 and analyzed using Microsoft Excel. For all samples melting curves were acquired for quality control purposes. For gene expression quantification, we used the comparative Ct method. First, gene expression levels for each sample were normalized to the expression level of the housekeeping gene encoding Glyceraldehydes 3-phosphate dehydrogenase (GAPDH) within a given sample (−ΔCt); the relative expression of each gene was calculated with106 *Log2(−ΔCt ).The difference between the YM155 treatment samples compared to the control samples was used to determine the106 *Log2(−ΔCt ). Statistical significance of the gene expression difference between the YM155 treatment and the control samples was calculated with the T-test using SPSS 11.5 software.

                                  Ingenuity pathway analysis (IPA)

                                  Datasets representing genes with altered expression profile derived from Real-time PCR array analyses were imported into the Ingenuity Pathway Analysis Tool (IPA Tool; Ingenuity H Systems, Redwood City, CA, USA; http://​www.​ingenuity.​com). In IPA, differentially expressed genes are mapped to genetic networks available in the Ingenuity database and then ranked by score. The basis of the IPA program consists of the Ingenuity Pathway Knowledge Base (IPKB) which is derived from known functions and interactions of genes published in the literature. Thus, the IPA Tool allows the identification of biological networks, global functions and functional pathways of a particular dataset. The program also gives the significance value of the genes, the other genes with which it interacts, and how the products of the genes directly or indirectly act on each other, including those not involved in the microarray analysis. The networks created are ranked depending on the number of significantly expressed genes they contain and also list diseases that were most significant. A network is a graphical representation of the molecular relationships between molecules. Molecules are represented as nodes, and the biological relationship between two nodes is represented as an edge (line). All edges are supported by at least 1 reference from the literature, from a textbook, or from canonical information stored in the Ingenuity Pathways Knowledge Base.

                                  Statistical analysis

                                  At least three replicates for each experimental condition were performed, and the presented results were representative of these replicates. All values are presented as means ± SEM. Student’s paired t-test was applied to reveal statistical significances. P values less than 0.05 were considered significant. Statistical analyses were performed using SPSS Software for Windows (version 11.5; SPSS, Inc., Chicago, IL).

                                  Results

                                  Growth inhibitory effect of YM155 on SK-NEP-1 cells

                                  YM155 treatment resulted in inhibition of cell proliferation of SK-NEP-1cells in a dose-dependent manner (Figure 1B). 93%, 85%, 70%, 55% and 30% cells were viable when 10nM, 20nM, 40nM, 80nM and 160nM YM155 treated 24 hours. So the IC50 of YM155 for SK-NEP-1 cells was about 100nM. The adherence of SK-NEP-1 cells was much inhibited by YM155-treatment. YM155 induced detachment of the cells from the dishes (Data not shown).
                                  http://static-content.springer.com/image/art%3A10.1186%2F1471-2407-12-619/MediaObjects/12885_2012_3592_Fig1_HTML.jpg
                                  Figure 1

                                  Growth inhibitory effect of YM155 on SK-NEP-1 cells. (A) Molecular Structure of YM155. (B) Proliferation and IC50 analysis of YM155. Sk-NEP-1 cells (2 × 104) were seeded in 96-well plates overnight and incubated with DMSO, 1 nM YM155, or increasing concentrations of YM155 (0.005, 0.01, 0.02, 0.04, 0.08, 0.16, 0.32, 0.64or 1.28 μM) for 24 hours. Compared with the control group, the relative survival rate of remained cells was calculated from the absorbance values. Cell proliferation was calculated as a percentage of the DMSO treated control wells with IC50 values derived after plotting proliferation values on a logarithmic curve. Experiments were performed in quadruplicate and repeated two times.

                                  YM155 induced apoptosis in SK-NEP-1 cells

                                  To confirm whether YM155 induces apoptosis in SK-NEP-1 cells, we further investigated Annexin V assay, cell cycle, and activation of caspase-3 in SK-NEP-1 cells after YM155 treatment. The result showed that cells treated with YM155 50nM and 100nM for 6 and 12 hours, much more cells showed apoptotic feature compared with control group (Figure 2A). These analyses were repeated three times, and the apoptotic cells in 6 hours was YM155 50nM: 5.9% ±2.3%,YM155 100nM 42.4% ±8.9% compared to DMSO group: 1.5%±0.4%; apoptotic cells in 12 hours was YM155 50nM: 31.5% ±5.7%, YM155 100nM 45.1% ±11.3% compared to DMSO group 6.5% ±2.1%. Cell cycle was confirmed by Cell cycle assay (Figure 2B). As expected, DNA fragmentation was observed after 12 hours treatment and increased in a time dependent manner. These analyses were repeated three times, and the apoptotic cells in YM155 50nM group: 12 hours 8.7% ±1.4%, 24 hours 23.2% ±6.7%, 36 hours 74.0%±9.2%; apoptotic cells in YM155 100nM group: 12 hours 18.5% ±3.7% , 24 hours 25.4% ±9.2% , 36 hours 60.9% ±14.2%. Moreover, to clearly demonstrate that YM155 causes apoptosis in SK-NEP-1 cells, we assessed the molecular aspects of apoptosis, caspase-3, well recognized as a marker of apoptosis by western blot. After 6 and 12 hours treatment with 10nM, 50nM, 100nM and 200nM YM155, cleaved caspase-3 was observed (Figure 2C). This result is consistent with the data of Annexin V assay and cell cycle analysis, demonstrating that YM155 induced apoptosis in SK-NEP-1 cells. The results from both studies suggest that YM155 has promising antitumor activity against SK-NEP-1 cells.
                                  http://static-content.springer.com/image/art%3A10.1186%2F1471-2407-12-619/MediaObjects/12885_2012_3592_Fig2_HTML.jpg
                                  Figure 2

                                  YM155 induced apoptosis in SK-NEP-1 cells. YM155 induced apoptosis, DNA fragmentation and activation of caspase 3 in SK-NEP-1 cells. (A) Annexin V analysis showed the cells treated with YM155 50nM and 100nM for 6 and 12 hours, much more cells showed apoptotic feature compared with control group. These analyses were repeated three times, and the apoptotic cells in 6 hours was YM155 50nM: 5.9% ±2.3%,YM155 100nM 42.4% ±8.9% compared to DMSO group: 1.5%±0.4%; apoptotic cells in 12 hours was YM155 50nM: 31.5% ±5.7%, YM155 100nM 45.1% ±11.3% compared to DMSO group 6.5% ±2.1%. (B) Cell cycle analysis the cells treated with YM155 after 12, 24 and 36 hours. As expected, DNA fragmentation was observed after 12 hours treatment and increased in a time dependent manner. These analyses were repeated three times, and the apoptotic cells in YM155 50nM group: 12 hours 8.7% ±1.4%, 24 hours 23.2% ±6.7%, 36 hours 74.0%±9.2%; apoptotic cells in YM155 100nM group: 12 hours 18.5% ±3.7% , 24 hours 25.4% ±9.2% , 36 hours 60.9% ±14.2%. (C) Western-blot analysis the activation of caspase 3 in cells treated with YM155. After 6 and 12 hours treatment with 10nM, 50nM, 100nM and 200nM YM155, cleaved caspase-3 was observed.

                                  YM155 treatment inhibited growth of SK-NEP-1 xenograft tumor in nude mice

                                  We next assessed the impact of YM155 on the cell growth of SK-NEP-1 cells in nude mice. SK-NEP-1 cells were subcutaneously injected into five nude mice every group. 10 days after injection, mice were treated with PBS, DMSO, YM155 5 mg/kg and 10 mg/kg dose. During the next six weeks these mice were examined for subcutaneous tumor growth. After the last treatment, the mice were killed and the tumor weight was measured. YM155 significantly inhibited growth of SK-NEP-1 xenografts (YM155 5 mg/kg: 1.45 ± 0.77 cm3; YM155 10 mg/kg: 0.95 ± 0.55 cm3) compared to DMSO group (DMSO: 3.70 ± 2.4 cm3) or PBS group cells (PBS: 3.78 ± 2.20 cm3, ANOVA P < 0.01 Figure 3A, 3B). YM155 treatment decreased weight of tumors (YM155 5 mg/kg: 1.05 ± 0.24 g; YM155 10 mg/kg: 0.72 ± 0.17 g) compared to DMSO group (DMSO: 2.06 ± 0.38 g) or PBS group cells (PBS: 2.36 ± 0.43 g, ANOVA P < 0.01 Figure 3C). We also observed that the body weight of nude mice treated with YM155 was almost same with control group (YM155 5 mg/kg:20.12 ± 1.66 g; YM155 10 mg/kg: 19.92 ± 1.72 g) compared to DMSO group (DMSO: 20.88 ± 1.83 g) or PBS group cells (PBS: 20.66 ± 1.37 g, ANOVA P >0.05 Figure 3D). These studies support the view that YM155 had significant role and little side effect in the treatment of SK-NEP-1 xenograft tumors.
                                  http://static-content.springer.com/image/art%3A10.1186%2F1471-2407-12-619/MediaObjects/12885_2012_3592_Fig3_HTML.jpg
                                  Figure 3

                                  YM155 treatment inhibited growth of SK-NEP-1 xenograft tumor in nude mice. SK-NEP-1 cells were subcutaneously injected into five nude mice every group. 10 days after injection, mice were treated with PBS, DMSO, YM155 5 mg/kg and 10 mg/kg dose. During the next six weeks these mice were examined for subcutaneous tumor growth. The tumor volumes were calculated according to the following formula: volume = length × width2/2. After the last treatment, the mice were killed and the tumor weight was measured. (A) SK-NEP-1 xenograft tumors from the treatment experiment. (B) Growth curve of SK-NEP-1 cells treated with YM155, DMSO and PBS. YM155 significantly inhibited growth of SK-NEP-1 xenografts (YM155 5 mg/kg: 1.45 ± 0.77 cm3; YM155 10 mg/kg: 0.95 ± 0.55 cm3) compared to DMSO group (DMSO: 3.70 ± 2.4 cm3) or PBS group cells (PBS: 3.78 ± 2.20 cm3, ANOVA P < 0.01). (C) Tumor weight of the treatment experiment. YM155 treatment decreased weight of tumors (YM155 5 mg/kg: 1.05 ± 0.24 g; YM155 10 mg/kg: 0.72 ± 0.17 g) compared to DMSO group (DMSO: 2.06 ± 0.38 g) or PBS group cells (PBS: 2.36 ± 0.43 g, ANOVA P < 0.01). (D) Body weight of nude mice in the treatment experiment. Body weight of nude mice treated with YM155 was almost same with control group (YM155 5 mg/kg:20.12 ± 1.66 g; YM155 10 mg/kg: 19.92 ± 1.72 g) compared to DMSO group (DMSO: 20.88 ± 1.83 g) or PBS group cells (PBS: 20.66 ± 1.37 g, ANOVA P >0.05).

                                  Real-time PCR array analysis the dyes-regulated genes implicated into YM155 treatment

                                  In order to identify apoptosis and/or programmed cell death molecules implicated into the treatment with YM155, we used the SABioscience Human Apoptosis PCR Array PAHS-3012.We analyzed and clustered the expression of 370 key genes involved in apoptosis, or programmed cell death with this PCR Array (Additional file 1). This array includes the TNF ligands and their receptors; members of the bcl-2 family, BIRC (baculoviral IAP repeat) domain proteins, CARD domain (caspase recruitment domain) proteins, death domain proteins, TRAF (TNF receptor-associated factor) domain proteins and caspases. We can easily and reliably analyze the expression of a focused panel of genes related to apoptosis with this array. Comparison of PCR results between Test group and control group showed that 32 genes were significantly up-regulated and 54 genes were significantly down-regulated after YM155 treatment (Table 1 and Table 2).
                                  Table 1

                                  Genes up regulated in SK-NEP-1 cells treated with YM155 compared with DMSO control group

                                   

                                  Gene

                                  Symbol

                                  +DMSO

                                  +YM155

                                  Ratio

                                  P value

                                  1

                                  TNF

                                  Tumor necrosis factor

                                  0.052

                                  3.274

                                  62.850

                                  0.0085

                                  2

                                  FOXO1

                                  Forkhead box O1

                                  3.109

                                  59.967

                                  19.287

                                  0.0091

                                  3

                                  IER3

                                  Immediate early response 3

                                  53.221

                                  795.891

                                  14.955

                                  0.0093

                                  4

                                  PEA15

                                  Phosphoprotein enriched in astrocytes 15

                                  85.200

                                  642.789

                                  7.545

                                  0.0105

                                  5

                                  CD5

                                  CD5 molecule

                                  0.079

                                  0.584

                                  7.405

                                  0.0105

                                  6

                                  NDUFS3

                                  NADH dehydrogenase (ubiquinone) Fe-S protein 3,

                                  127.186

                                  919.431

                                  7.229

                                  0.0106

                                  7

                                  TNFAIP3

                                  Tumor necrosis factor, alpha-induced protein 3

                                  69.132

                                  449.671

                                  6.505

                                  0.0109

                                  8

                                  NFKB1

                                  nuclear factor of kappa gene enhancer in B-cells 1 

                                  58.200

                                  377.771

                                  6.491

                                  0.0109

                                  9

                                  CRYAB

                                  Crystallin, alpha B

                                  7.704

                                  49.315

                                  6.401

                                  0.0109

                                  10

                                  DDIT3

                                  DNA-damage-inducible transcript 3

                                  134.710

                                  768.681

                                  5.706

                                  0.0113

                                  11

                                  CRADD

                                  CASP2 domain containing adaptor with death domain

                                  10.127

                                  56.007

                                  5.531

                                  0.0114

                                  12

                                  BNIP1

                                  BCL2/adenovirus E1B 19 kDa interacting protein 1

                                  89.770

                                  475.609

                                  5.298

                                  0.0115

                                  13

                                  BBC3

                                  BCL2 binding component 3

                                  1.388

                                  6.896

                                  4.969

                                  0.0118

                                  14

                                  NOX5

                                  NADPH oxidase, EF-hand calcium binding domain 5 

                                  0.213

                                  1.057

                                  4.953

                                  0.0118

                                  15

                                  CASP7

                                  Caspase 7, apoptosis-related cysteine peptidase

                                  20.274

                                  95.136

                                  4.693

                                  0.0120

                                  16

                                  PIM2

                                  Pim-2 oncogene

                                  29.093

                                  118.888

                                  4.087

                                  0.0127

                                  17

                                  CEBPG

                                  CCAAT/enhancer binding protein (C/EBP), gamma

                                  11.867

                                  47.745

                                  4.023

                                  0.0128

                                  18

                                  EDA

                                  Ectodysplasin A

                                  0.710

                                  2.846

                                  4.009

                                  0.0128

                                  19

                                  SOCS2

                                  Suppressor of cytokine signaling 2

                                  0.258

                                  1.018

                                  3.953

                                  0.0129

                                  20

                                  CDKN1A

                                  Cyclin-dependent kinase inhibitor 1A (p21, Cip1)

                                  124.431

                                  479.383

                                  3.853

                                  0.0131

                                  21

                                  IL1A

                                  Interleukin 1, alpha

                                  146.968

                                  423.629

                                  2.882

                                  0.0155

                                  22

                                  BIRC8

                                  Baculoviral IAP repeat containing 8 

                                  0.248

                                  0.692

                                  2.785

                                  0.0159

                                  23

                                  SERPINB2

                                  Serpin peptidase inhibitor, clade B member 2

                                  0.136

                                  0.362

                                  2.659

                                  0.0165

                                  24

                                  CASP9

                                  Caspase 9, apoptosis-related cysteine peptidase

                                  330.442

                                  853.550

                                  2.583

                                  0.0169

                                  25

                                  LTB

                                  Lymphotoxin beta (TNF superfamily, member 3)

                                  0.052

                                  0.134

                                  2.580

                                  0.0170

                                  26

                                  PMAIP1

                                  Phorbol-12-myristate-13-acetate-induced protein 1 

                                  258.441

                                  642.950

                                  2.488

                                  0.0175

                                  27

                                  NUPR1

                                  Nuclear protein, transcriptional regulator, 1

                                  25.295

                                  61.632

                                  2.437

                                  0.0179

                                  28

                                  BIRC3

                                  Baculoviral IAP repeat containing 3

                                  97.141

                                  232.642

                                  2.395

                                  0.0183

                                  29

                                  DIABLO

                                  IAP-binding mitochondrial protein 

                                  178.419

                                  417.832

                                  2.342

                                  0.0187

                                  30

                                  LTBR

                                  Lymphotoxin beta receptor

                                  120.546

                                  280.744

                                  2.329

                                  0.0188

                                  31

                                  COL4A3

                                  Collagen, type IV, alpha 3

                                  5.993

                                  12.916

                                  2.155

                                  0.0208

                                  32

                                  FOXO3

                                  Forkhead box O3

                                  34.671

                                  71.488

                                  2.062

                                  0.0222

                                  Table 2

                                  Genes down regulated in SK-NEP-1 cells treated with YM155 compared with DMSO control group

                                   

                                  Gene

                                  Symbol

                                  +DMSO

                                  +YM155

                                  Ratio

                                  P value

                                  1

                                  HIPK2

                                  Homeodomain interacting protein kinase 2

                                  31.640

                                  11.619

                                  0.367

                                  0.0353

                                  2

                                  CD27

                                  CD27 molecule

                                  1.462

                                  0.538

                                  0.368

                                  0.0353

                                  3

                                  PCBP4

                                  Poly(rC) binding protein 4

                                  9.419

                                  3.481

                                  0.370

                                  0.0334

                                  4

                                  PAK7

                                  P21 protein (Cdc42/Rac)-activated kinase 7

                                  0.120

                                  0.044

                                  0.370

                                  0.0334

                                  5

                                  PPP1R13B

                                  Protein phosphatase 1, regulatory subunit 13B

                                  75.280

                                  27.926

                                  0.371

                                  0.0322

                                  6

                                  TP73

                                  Tumor protein p73

                                  9.738

                                  3.619

                                  0.372

                                  0.0321

                                  7

                                  RASA1

                                  RAS p21 protein activator 1

                                  138.406

                                  49.465

                                  0.357

                                  0.0315

                                  8

                                  CRYAA

                                  Crystallin, alpha A

                                  0.210

                                  0.079

                                  0.375

                                  0.0272

                                  9

                                  TIAF1

                                  TGFB1-induced anti-apoptotic factor 1

                                  243.057

                                  91.076

                                  0.375

                                  0.0242

                                  10

                                  CARD14

                                  Caspase recruitment domain family, member 14

                                  37.791

                                  13.412

                                  0.355

                                  0.0241

                                  11

                                  NOD1

                                  Nucleotide-binding domain containing 1

                                  163.081

                                  61.858

                                  0.379

                                  0.0214

                                  12

                                  TNFRSF1B

                                  Tumor necrosis factor receptor superfamily 1B

                                  45.110

                                  17.323

                                  0.384

                                  0.0174

                                  13

                                  HIP1

                                  Huntingtin interacting protein 1

                                  14.568

                                  4.941

                                  0.339

                                  0.0174

                                  14

                                  UTP11L

                                  UTP11-like, U3 small nucleolar ribonucleoprotein

                                  267.624

                                  104.245

                                  0.390

                                  0.0118

                                  15

                                  SPP1

                                  Secreted phosphoprotein 1

                                  2.389

                                  0.801

                                  0.335

                                  0.0107

                                  16

                                  NME5

                                  NME/NM23 family member 5

                                  0.217

                                  0.073

                                  0.335

                                  0.0092

                                  17

                                  MYO18A

                                  Myosin XVIIIA

                                  164.894

                                  54.821

                                  0.332

                                  0.0087

                                  18

                                  PPP2R1B

                                  Protein phosphatase 2, regulatory subunit A

                                  9.768

                                  3.887

                                  0.398

                                  0.0033

                                  19

                                  BAG1

                                  BCL2-associated athanogene

                                  70.273

                                  28.001

                                  0.398

                                  0.0033

                                  20

                                  DAPK2

                                  Death-associated protein kinase 2

                                  1.549

                                  0.509

                                  0.329

                                  0.0023

                                  21

                                  TNFSF10

                                  Tumor necrosis factor (ligand) superfamily 10

                                  2.596

                                  0.844

                                  0.325

                                  0.0022

                                  22

                                  TNFRSF25

                                  Tumor necrosis factor receptor superfamily 25

                                  10.685

                                  4.340

                                  0.406

                                  0.0022

                                  23

                                  FOXL2

                                  Forkhead box L2

                                  11.954

                                  4.886

                                  0.409

                                  0.0021

                                  24

                                  CASP10

                                  Caspase 10, apoptosis-related cysteine peptidase

                                  13.465

                                  4.252

                                  0.316

                                  0.0021

                                  25

                                  CARD8

                                  Caspase recruitment domain family, member 8

                                  3.835

                                  1.209

                                  0.315

                                  0.0020

                                  26

                                  ALOX12

                                  Arachidonate 12-lipoxygenase

                                  9.725

                                  3.039

                                  0.312

                                  0.0020

                                  27

                                  OPA1

                                  Optic atrophy 1 (autosomal dominant)

                                  172.500

                                  71.720

                                  0.416

                                  0.0020

                                  28

                                  ERN2

                                  Endoplasmic reticulum to nucleus signaling 2

                                  2.851

                                  1.187

                                  0.416

                                  0.0020

                                  29

                                  STK17A

                                  Serine/threonine kinase 17a

                                  111.835

                                  46.583

                                  0.417

                                  0.0020

                                  30

                                  CARD9

                                  Caspase recruitment domain family, member 9

                                  5.195

                                  1.578

                                  0.304

                                  0.0020

                                  31

                                  GRM4

                                  Glutamate receptor, metabotropic 4

                                  0.958

                                  0.408

                                  0.426

                                  0.0020

                                  32

                                  SON

                                  SON DNA binding protein

                                  547.934

                                  234.409

                                  0.428

                                  0.0019

                                  33

                                  PLAGL2

                                  Pleiomorphic adenoma gene-like 2

                                  69.972

                                  30.027

                                  0.429

                                  0.0019

                                  34

                                  BIRC5

                                  Baculoviral IAP repeat containing 5

                                  59.315

                                  25.502

                                  0.430

                                  0.0019

                                  35

                                  FADD

                                  Fas (TNFRSF6)-associated via death domain

                                  323.082

                                  93.703

                                  0.290

                                  0.0019

                                  36

                                  NAIP

                                  NLR family, apoptosis inhibitory protein

                                  44.019

                                  12.731

                                  0.289

                                  0.0008

                                  37

                                  BCL2

                                  B-cell CLL/lymphoma 2

                                  6.950

                                  3.020

                                  0.435

                                  0.0008

                                  38

                                  ZNF443

                                  Zinc finger protein 443

                                  8.149

                                  3.583

                                  0.440

                                  0.0003

                                  39

                                  CASP8AP2

                                  Caspase 8 associated protein 2

                                  43.673

                                  19.445

                                  0.445

                                  0.0003

                                  40

                                  CUL5

                                  Cullin 5

                                  134.969

                                  60.117

                                  0.445

                                  0.0002

                                  41

                                  DAPK1

                                  Death-associated protein kinase 1

                                  15.806

                                  4.237

                                  0.268

                                  <0.0001

                                  42

                                  NKX3-2

                                  NK3 homeobox 2

                                  0.830

                                  0.371

                                  0.447

                                  <0.0001

                                  43

                                  SEMA4D

                                  Itransmembrane domain semaphoring 4D

                                  9.709

                                  2.589

                                  0.267

                                  <0.0001

                                  44

                                  SST

                                  Somatostatin

                                  0.694

                                  0.179

                                  0.258

                                  <0.0001

                                  45

                                  ALOX15B

                                  Arachidonate 15-lipoxygenase, type B

                                  0.263

                                  0.063

                                  0.242

                                  <0.0001

                                  46

                                  NOTCH2

                                  Notch 2

                                  930.886

                                  223.933

                                  0.241

                                  <0.0001

                                  47

                                  NLRC4

                                  NLR family, CARD domain containing 4

                                  20.624

                                  4.653

                                  0.226

                                  <0.0001

                                  48

                                  PRKCA

                                  Protein kinase C, alpha

                                  30.560

                                  6.439

                                  0.211

                                  <0.0001

                                  49

                                  APOE

                                  Apolipoprotein E

                                  1.075

                                  0.210

                                  0.195

                                  <0.0001

                                  50

                                  NTF3

                                  Neurotrophin 3

                                  0.295

                                  0.044

                                  0.150

                                  <0.0001

                                  51

                                  CARD17

                                  Caspase recruitment domain family, member 17

                                  0.363

                                  0.047

                                  0.131

                                  <0.0001

                                  52

                                  MAPK8IP2

                                  Mitogen kinase 8 interacting protein 2

                                  0.432

                                  0.049

                                  0.114

                                  <0.0001

                                  53

                                  F2

                                  coagulation factor II

                                  3.794

                                  0.104

                                  0.027

                                  <0.0001

                                  54

                                  CUL3

                                  cullin 3

                                  1.228

                                  0.059

                                  0.048

                                  <0.0001

                                  Ingenuity pathway analysis the pathway regulated by YM155

                                  To investigate possible biological interactions of differently regulated genes, datasets representing genes with altered expression profile derived from real-time PCR array analyses were imported into the Ingenuity Pathway Analysis Tool. The list of differentially expressed genes analyzed by IPA revealed significant networks. Figure 4A represents the list of top 5 networks identified by IPA. Of these networks, cell death was the highest rated network with 65 focus molecules and the significance score of 44 (Figure 4D). The score is the probability that a collection of genes equal to or greater than the number in a network could be achieved by chance alone. A score of 3 indicates a 1/1000 chance that the focus genes are in a network not due to random chance. The IPA analysis also groups the differentially expressed genes into biological mechanisms that are related to cell death, cellular function maintenance, cell morphology, carbohydrate metabolism and cellular growth and proliferation (Figure 4B). Death receptor signaling (3.87E-19), TNFR1 signaling (2.34E-13), induction of apoptosis by HIV1 (2.67E-12), apoptosis signaling (6.56E-12) and molecular mechanisms of cancer (2.13E-11) came out to be the top four most significant pathways (Figure 4C). IPA analysis also showed top molecules up-regulated were BBC3,BIRC3,BIRC8,BNIP1,CASP7,CASP9,CD5,CDKN1A,CEBPG and COL4A3, top molecules down-regulated were ZNF443, UTP11L, TP73, TNFSF10, TNFRSF1B, TNFRSF25,TIAF1,STK17A,SST and SPP1, upstream regulator were NR3C1, TP53, dexamethasone , TNF and Akt ( Additional file 2). These upstream regulators such as TP53, TNF and Akt have already been reported as important regulators for the surviving network. TP53 and Akt have been widely investigated and there are hundreds of papers about the important roles in surviving pathway. But there is still no report about the relationship between NR3C1, dexamethasone and survivin.
                                  http://static-content.springer.com/image/art%3A10.1186%2F1471-2407-12-619/MediaObjects/12885_2012_3592_Fig4_HTML.jpg
                                  Figure 4

                                  Ingenuity Pathways Analysis (IPA) summary. To investigate possible interactions of differently regulated genes, datasets representing 86 genes with altered expression profile obtained from real-time PCR arrays were imported into the Ingenuity Pathway Analysis Tool and the following data is illustrated: (A) The list of top five networks with their respective scores obtained from IPA . (B) The list of top five molecular and cellular functions with their respective scores obtained from IPA. (C) Toxicology pathway list in IPA analysis. The x-axis represents the top toxicology functions as calculated by IPA based on differentially expressed genes are highlighted and the y-axis represents the ratio of number of genes from the dataset that map to the pathway and the number of all known genes ascribed to the pathway. The yellow line represents the threshold of p value, 0.05 as calculated by Fischer’s test. (D) Most highly rated network in IPA analysis. The network representation of the most highly rated network. The genes that are shaded were determined to be significant from the statistical analysis. A solid line represents a direct interaction between the two gene products and a dotted line means there is an indirect interaction.

                                  This work indicated firstly that NR3C1and dexamethasone may be upstream regulators in the survivin pathway. These results may provide new clues of molecular mechanism of apoptosis induced by YM155.

                                  Discussion

                                  Survivin is highly expressed in a broad range of solid tumors and hematological malignancies. Increased survivin expression in cancer patients is an unfavorable prognostic marker correlating with decreased overall survival in several malignancies, including non-small cell lung [2426], gastric [2731], colorectal [3234], and breast carcinomas [35], neuroblastoma [36], prostate cancer [37], pancreatic cancer [38] , hepatocellular carcinoma [39] and hematologic malignancies [4044]. Increased survivin expression was also associated with increased risk of recurrence, lymph node invasion and metastasis. Finally, survivin overexpression may be a predictive factor to determine response to chemotherapy and radiotherapy in patients with bladder cancer, breast cancer, multiple myeloma and lymphoma. Studies have shown that survivin suppression induces tumor cell apoptosis and enhances sensitivity to apoptosis induced by existing anticancer drugs and other apoptotic stimuli. This work indicated that survivin also be an important target for human Wilms tumor cells.

                                  YM155 is a novel survivin suppressant that is currently in clinical development by Astellas Pharma, Inc. YM155 inhibited the growth of 119 human cancer cell lines, with the greatest activity in lines derived from non-Hodgkin’s lymphoma, hormone-refractory prostate cancer, ovarian cancer, sarcoma, non-small-cell lung cancer, breast cancer, leukemia and melanoma. The mean log growth inhibition of 50% (GI50) value was 15 nM. A preclinical study showed that YM155 suppressed both survivin protein and mRNA expression. In a toxicologic study, short-term exposure at high blood concentrations caused cardiotoxicity in the form of atrioventricular. In this phase I study, YM155 seemed to be safe and well-tolerated, with a maximum tolerated dose of 8.0 mg/m2/d. Stable disease was achieved in nine patients. The data in this study indicate that the adverse reactions observed can be well-controlled by taking due caution and suggest that YM155 has more easily controllable toxicities compared with conventional cytotoxic anticancer drugs. This work also supports the view that YM155 had significant role and little side effect in the treatment of SK-NEP-1 xenograft tumors.

                                  Real-time PCR Array System is the ideal tool for analyzing the expression of a focused panel of genes. The flexibility, simplicity, and convenience of standard SYBR Green PCR detection methodology make the PCR Array System accessible for routine use in any research laboratory [45]. In this study, we analyzed the dyes-regulated genes by YM155 with this powerful platform, Real-time PCR arrays.

                                  Comparison of PCR results between Test group and control group showed that 32 genes were significantly up-regulated and 54 genes were significantly down-regulated after YM155 treatment. Some genes, such as TNF, NFKB1, CDKN1A, CASP9, COL4A3, BIRC5 (survivin), BCL2, and DAPK1 have already been reported with YM155 treatment. There are also some other genes never reported with YM155 treatment and these genes have complicate functions far exceeds the apoptosis. These results consistent with the complicate roles of survivin in cancer biology. Survivin has been implicated in the regulation of the mitotic spindle checkpoint, from kinetic core to spindle assembly; it’s over expression in cancer may allow cells with spindle defects or misaligned kinetic cores to continue through cell division. Recent studies also suggest that survivin plays a role in tumor progression and chemoresistance. Survivin has been shown to inhibit cell death induced by several anticancer agents, including paclitaxel [46], etoposide [47] and Tumor Necrosis Factor-a related apoptosis-inducing ligand [47, 48]. In vitro and in vivo studies showed that inhibiting survivin reduces tumor growth potential and sensitizes tumor cells to chemotherapeutic agents, such paclitaxel, cisplatin [14, 49], etoposide, gamma irradiation and immunotherapy. To explore the molecule mechanism of YM155 treatment, we try to explore new target and “net work” of YM155 with a powerful platform, Ingenuity Pathway Analysis program.

                                  The basis of the IPA program consists of the Ingenuity Pathway Knowledge Base (IPKB) which is derived from known functions and interactions of genes published in the literature. The IPA Tool allows the identification of biological networks, global functions and functional pathways of a particular dataset. The program also gives the significance value of the genes, the other genes with which it interacts, and how the products of the genes directly or indirectly act on each other, including those not involved in the microarray analysis. This work represents cell death was the highest rated network with 65 focus molecules and the significance score of 44. Death receptor signaling, TNFR1 signaling ,induction of apoptosis by HIV1 ,apoptosis signaling and molecular mechanisms of cancer came out to be the top four most significant pathways. IPA analysis also showed top molecules up-regulated was BBC3 (PUMA). PUMA encodes a member of the BCL-2 family of proteins. This family member belongs to the BH3-only pro-apoptotic subclass. The protein cooperates with direct activator proteins to induce mitochondrial outer membrane permeabilization and apoptosis. It can bind to anti-apoptotic Bcl-2 family members to induce mitochondrial dysfunction and caspase activation. Because of its pro-apoptotic role, this gene is a potential drug target for cancer therapy and for tissue injury. IPA analysis also showed upstream regulators were NR3C1, TP53, dexamethasone, TNF and Akt. These upstream regulators such as TP53, TNF and Akt have already been reported as important regulators for the survivin network. TP53 and Akt have been widely investigated and there are hundreds of papers about the important roles in survivin pathway. But there is still no report about the relationship between NR3C1, dexamethasone and survivin. NR3C1 gene encodes glucocorticoid receptor, which can function both as a transcription factor that binds to glucocorticoid response elements in the promoters of glucocorticoid responsive genes to activate their transcription and as a regulator of other transcription factors. This receptor is typically found in the cytoplasm, but upon ligand binding, is transported into the nucleus. It is involved in inflammatory responses, cellular proliferation, and differentiation in target tissues. Dexamethasone gene encodes a member of the Ras superfamily of small GTPases and is induced by dexamethasone. The encoded protein is an activator of G-protein signaling and acts as a direct nucleotide exchange factor for Gi-Go proteins. This protein interacts with the neuronal nitric oxide adaptor protein CAPON, and a nuclear adaptor protein FE65, which interacts with the Alzheimer's disease amyloid precursor protein. This gene may play a role in dexamethasone-induced alterations in cell morphology, growth and cell-extracellular matrix interactions. Epigenetic inactivation of this gene is closely correlated with resistance to dexamethasone in multiple myeloma cells. This work indicated firstly that NR3C1and dexamethasone may be upstream regulators in the survivin pathway. These results may provide new clues of molecular mechanism of apoptosis induced by YM155.

                                  Conclusions

                                  The present study demonstrates that YM155 treatment resulted in apoptosis and inhibition of cell proliferation of SK-NEP-1cells. YM155 had significant role and little side effect in the treatment of SK-NEP-1 xenograft tumors. Real-time PCR array analysis firstly showed expression profile of genes dyes-regulated after YM155 treatment. IPA analysis also represents new molecule mechanism of YM155 treatment, such as NR3C1 and dexamethasone may be new target of YM155. And our results may provide new clues of molecular mechanism of apoptosis induced by YM155.

                                  Declarations

                                  Acknowledgements

                                  This work was supported by grants from the National Key Basic Research Program No. 2010CB933902, National Natural Science Foundation for youth No. 81100371, Natural Science Foundation of Jiangsu Province No. BK2011308, Universities Natural Science Foundation of Jiangsu Province No. 11KJB320014 and Talent’s subsidy project in science and education of department of public health of Suzhou City No. SWKQ1020. Medical innovation team and leading talent of Jiang-su Province No. LJ201126. Major scientific and technological special project for “significant new drugs creation” No.2012ZX09103301-040.

                                  Authors’ Affiliations

                                  (1)
                                  Department of Hematology and Oncology, Children’s Hospital of Soochow University
                                  (2)
                                  Department of Gastroenterology, the 5th Hospital of Chinese PLA
                                  (3)
                                  Department of Cell and Molecular Biology, Cancer Institute (Hospital), Chinese Academy of Medical Sciences, Peking Union Medical College
                                  (4)
                                  Beijing Insititute for Drug Control
                                  (5)
                                  Institute of Clinical Medical Science, China-Japan Friendship Hospital
                                  (6)
                                  Translational Research Center, Second Hospital, The Second Clinical School, Nanjing Medical University

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                                  50. Pre-publication history

                                    1. The pre-publication history for this paper can be accessed here:http://​www.​biomedcentral.​com/​1471-2407/​12/​619/​prepub

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                                  © Tao et al.; licensee BioMed Central Ltd. 2012

                                  This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.