Abstract Objective To investigate the spleen-derived tyrosine kinase (spleen tyrosine kinase, Syk) in vitro and in vivo anti-tumor effect. Liposome-mediated full-length Syk cDNA into Syk-negative expression in highly invasive human breast cancer cell line MDA  MB  231, MTT assay, Transwell chambers were used to detect Syk cDNA in vitro anti-tumor cell proliferation and invasion the migration role; BABL / c (nu / nu) nude mice xenograft model, detection transfected and non-transfected tumor cells into a tumor and lung metastasis. Results transfected with full-length Syk cDNA MDA-MB  231 cells transfected with empty vector and untransfected MDA  MB  231 cells compared in vitro proliferation of no significant change, while the transmembrane cell invasion and migration experiments Similarly, inoculation of tumor cells in nude mice subcutaneous tumor xenografts showed no significant difference (P <0.05);, but vaccination transfected tumor cells but significantly reduced lung metastasis rate (total number of migrating cells / 5HPF) but significantly lower than untransfected and empty vector transfected group (P <0.05). Conclusion Syk by changing breast tumor cell invasion and migration ability to participate in its anti-tumor effect.

Key words spleen-derived tyrosine kinase; breast tumors; invasion and metastasis; MDA  MB  231

 Anti  Tumor Effect of Spleen Tyrosine Kinase

    QI Yi  xin1, HU Jie2, SONG Xiao  tian3, SHAN Bao  en3

    1. Department of Surgery, The Fourth Hospital of Hebei Medical University, Shijiazhuang 050011, China; 2. Department of Immunology, Hebei Medical University; 3. Scientific Research Center, The Fourth Affiliated Hospital of Hebei Medical University

    Corresponding Author: SHAN Bao  en, E  mail: baoenshan@yahoo.com.cnAbstract: Objective To investigate the anti  tumor effect of spleen tyrosine kinase (Syk) in vitro and in vivo. Methods Human breast cancer cell line MDA  MB  231 which expressing Syk negatively was cultured in optimum medium and transfected with Syk cDNA by liposome, and cells transfected with blank vector were used as control. The inhibited effect of Syk cDNA on the cell proliferation and the ability of assorting in vitro was detected by MTT and Transwell  ECM methode; the pulmonary metastasis and tumor growth of transformed and untransformed human breast cancer cells were investigated by xenografts in BABL / c (nu / nu) athymic mice. Results There is no obviously change in proliferation in vitro, between the MDA  MB  231 cells transformed Syk cDNA and transformed idling or not transformed, but number of the migrating cells (migrating cells/5HPF) reduces obviously in invasion and immigration experiment (P <0.05); in the same, there is no difference in tumor growth of athymic mice inoculated different cancer cells, nevertheless, there is lower incidence of pulmonary metastasis in the group of inoculated cancer cells transforming Syk cDNA (P <0.05). Conclusion Syk can restrain in tumor by reducing the abilities of invasion and migration of breast cancer cells.

    Key words: Spleen tyrosine kinase; Breast cancer; Invasion and metastasis; MDA  MB  231

Important molecules in the spleen-derived non-receptor protein tyrosine kinase Syk (spleen tyrosine kinase, Syk), as a B cell activation signal transduction process has been widely studied. Recently, more and more evidence shows that, Syk may inhibitory factor is a potential candidate of the breast cancer cell [1]. We have reported [2], Syk reduced expression or absence of breast cancer development and poor prognosis, but its exact mechanism of action is not yet clear. This study by Syk cDNA transfection in vitro expression of Syk-negative invasive breast cancer cell line MDA-MB  231 [3], anti-tumor effect of detection of Syk cDNA for further study of the clinical value of the theoretical and experimental evidence.

    1 Materials and methods

    1.1 Materials

    1.1.1 cell lines and experimental animals in human breast cancer cell line MDA  MB  231 saved by the Scientific Research Center of the Fourth Hospital of Hebei Medical University, DMEM medium containing 10% fetal calf serum (FCS), at 37 ° C, 5% CO2 saturated humidity conditions cultured to logarithmic phase cells for experimental specimens. BALB / c (nu / nu) nude mice 15, female, 4 weeks old, weight 10 ~ 16g, were purchased from the Experimental Animal Center of the Chinese Academy of Medical Sciences.

    1.1.2 Main reagents DMEM medium and trypsin from Gibco, USA; the TRIzol and transfected kit PlusTM Reagent Invitrogen Corporation, USA; mouse anti-human Syk antibody United States LAB VISION products; Matrigel U.S. BD products; The Transwell plate is the U.S. the Costar company products; recombinant plasmid pcDNA3.1D/V5  His  TOPO / Syk build the Fourth Hospital of Hebei Medical University research center.

    1.2 Methods

    1.2.1 Syk cDNA transfected MDA  MB  231 the cell preparation restructuring “pcDNA3.1D/V5  His  TOPO / Syk" eukaryotic expression plasmids using recombinant DNA technology of Syk (+) human breast cancer cell line MCF-7 to obtain the desired target gene itself the built (article to be published), and positive clones were screened by liposome-mediated transfection of MDA  MB  231 cells, G418 (800 mg / L) limited cell dilution method.

    1.2.2 Western blotting of Syk protein expression in transfected cells, total protein was extracted line SDS  PAGE electrophoresis (5% stacking gel, 80V, 10% separating gel, 120V, 3h), re-the PVDF mark electrophoresis (90V , 3.5h) transferred to a membrane, Ponceau dye Prestained washing the closure of Syk of mAb, plus mouse anti-human, 4 ° C overnight after washing, plus horseradish peroxidase-conjugated secondary antibody (goat anti-mouse IgG), DAB significantly The color kit color, the camera records the experimental results.

    1.2.3 MTT assay the Syk cDNA transfection of MDA-MB  231 cell proliferation ability of MDA  MB  231 cells collected in logarithmic growth phase, to a concentration of 1 × 104 / well were seeded in 96-well plates, each cells (MDA  MB  231/Syk, MDA  MB  231/pcDNA3.1 and MDA  MB  231 cells) do 6 wells at 37 ° C under 5% CO2 in cultured for 7 consecutive days, every 24h random 6 holes MTT (5mg/ml) was added to 20μl, 37 ° C incubation was continued for 4h discard supernatant; Add 150μl of dimethyl sulfoxide (DMSO), oscillation 10min, fully dissolved crystallization; ELISA measured 492nm wavelengths absorption values ​​(A492nm), and the culture time as the abscissa and the absorbance value on the vertical axis, and the cell growth curve.

    1.2.4 Cell invasion assay to artificial basement membrane Matrigel (50mg / L) plastic coated Transwell chamber (equipped with a polycarbonate membrane, pore size 8μm) the bottom of the upper chamber is added to a final concentration of 5 × 105/ml cell suspension 200μl corresponding to the lower chamber to join in DMEM containing 50ml / L FCS, each repeated three samples routinely cultured 24h, formalin-fixed, HE staining, cotton swab wipe the upper microporous membrane cells, inverted under a microscope to count the number of penetrating cells determines its across the ability to invasion artificial basement membrane. “The total number of migrating cells / 5HPF" to record the results.

    1.2.5 cell migration the experiment without Martrigel package to be remaining operating invasion assay.

    1.2.6 Syk cDNA tumor growth and lung metastasis in nude collection logarithmic phase cell suspension, adjust the concentration of the cell suspension to 5 × 106/ml take 0.5ml inoculated in nude mice subcutaneous tissue within the observation nude mice growth conditions and (or) the tumor time. When MDA-MB  231 tumor grew to about 500mm3, neck breaking, all the animals were sacrificed, tumor tissue, measuring the maximum diameter of the tumor and the corresponding transverse diameter. According to the formula V = π / 6 × tumor diameter (mm) × tumor transverse diameter (mm), the calculation of tumor size and weighed; lung tissue cells was observed in 10% formalin-fixed, paraffin section, HE staining, microscopy.

    1.2.7 statistical methods of data SPSS13.0 software univariate analysis of variance results to ± s pairwise comparison q test.

    2 Results

    2.1 Syk protein expression in MDA  MB  231/Syk cells

    Western blot showed that the MDA  MB  231/Syk cells and expression of Syk protein, MDA  MB  231/pcDNA3.1 and MDA-MB  231 cells did not express Syk protein, as shown in Figure 1, suggesting successful transfection of Syk .

    2.2 Syk cDNA transfected MDA  MB  231 cell proliferative capacity in vitro

    MDA  MB  231/Syk, MDA-MB  231/pcDNA3.1 and MDA-MB-231 cells continuously cultured for 7 days in vitro cell proliferation activity, the difference was not statistically significant (P> 0.05) (data not display), prompted Syk cDNA transfected MDA  MB  231 cell proliferative capacity in vitro growth.

    2.3 Syk cDNA transfection  231 cells in vitro invasion and migration ability of MDA-MB

    MDA  MB  231/Syk MDA-MB  231/pcDNA3.1 and MDA-MB  the transmembrane cell count 231 cells compared to the invasion assay and migration assay (total number of migrating cells / 5HPF) were significantly reduced ( P <0.05), Figure 2,3.

    2.4 Syk cDNA transfection  231 cells were inoculated subcutaneously into nude mice transplanted tumor growth of MDA-MB

    Nude mice inoculated subcutaneously with different of MDA  MB  231 tumor cells after three days of tumor growth, to 30 days, neck breaking, all mice were sacrificed, the tumors, measuring the size and weighed no statistically significant differences among the groups 0.05)(数据未显示), 提示转染Syk cDNA对MDAMB231细胞在裸鼠体内增殖无明显影响。">no effect on proliferation (P> 0.05) (data not shown), suggesting that the Syk cDNA transfected MDA  MB  231 cells in nude mice.

    2.5 Syk cDNA transfected MDA  MB  231 cells transplanted into nude mice lung metastases

    All five mice inoculated subcutaneously with MDA  MB  231 cells occurrence of lung metastases (100%); the cell groups inoculated with MDA  MB  231/pcDNA3.1 5 mice, 4 lung metastasis (80%); MDA  MB  231/Syk group of five mice, only two of lung metastases (40%), the results suggest that Syk cDNA transfected MDA  MB  231 cells in nude mice can significantly reduce pulmonary metastasis incidence (P <0.05), as shown in Figure 4.

    3 Discussion

    2000, Coopman et al [1] for the first time may prove spleen-derived protein tyrosine kinase Syk inhibitor of human breast cancer cell potential growth regulator, which sparked the attention of many scholars. Our previous research results have been of Syk reduced expression or absence of breast cancer development and poor prognosis, but its exact mechanism of action is not yet clear. The occurrence of the full-length Syk cDNA transfection in vitro expression of Syk-negative invasive breast cancer cell line MDA-MB  231, MTT and transplanted into nude mice experimental breast cancer is complex and multifactorial. Recently reported that the number of new molecular therewith related to the development, such as CBFA2T3 15PGDH FBXO31, Wwox, HIN  1, Syk [4-11]. The results of this study show that, the Syk cDNA transfected MDA  MB  231 tumor cells in vivo, growing outside the proliferative capacity had no effect.

    Furthermore, we took advantage of the Transwell chamber and Matrigel glue invasion and migration in vitro model. Matrigel is an extract of rat the ESH sarcoma extracellular matrix composed of collagen type IV, laminin, sulfate heparin proteoglycans and integrins, etc., similar to the composition and organization of the basement membrane. The Transwell chamber is divided into upper and lower levels, the middle of the polycarbonate membrane (pore diameter of 8μm) apart. Microporous filters allows the tumor cells through the migration of cells can be adhered to the film underlayer, counting and comparing the number of penetrating cell, can react to a certain extent for the migration ability of tumor cells. Matrigel as adhesion matrix, laying on the polycarbonate filters, can be analog basement membrane structure, the same can count and compare the number of penetrating cells, the evaluation of tumor cell invasion ability. The results showed that the MDA  MB  231 cells transfected with Syk cDNA penetrating significantly less than the number of cells transfected with empty vector group and untransfected cells, indicating that the the Syk cDNA can be transfected cells significantly inhibited the migration and invasion abilities. Nude pulmonary metastasis model experimental results also proved that Syk cDNA transfection can significantly reduce the incidence of lung metastases in nude mice subcutaneously transplanted tumor cells of MDA  MB  231.

    In summary, this study further confirmed Syk role of anti-tumor cell invasion and metastasis may be important factor associated with poor prognosis of breast cancer.