Objective: To investigate the relationship of loss of P16 expression with biologlical behaviors and prognosis of gastrointestinal stromal tumors(GISTs). Methods: The expression of P16 protein and mRNA was detected in GISTs tissues by immunohistochemistry,Western blot and RT-PCR.The prognosis was evaluated through follow up. Results: The expression rates of P16 protein and mRNA in GISTs were 53.8%(21/39，Frozen tissue)，51.3%(20/39，Frozen tissues)and 51.4%(37/72，Paraffin-embedded tissues)，respectively.The expression of P16 was significantly different among GISTs of different aggressive risk(P<0.05).With the incease of aggressive risk，the expression of P16 was deceased.The expression of P16 was negtively correlated with Ki-67 and patient survival(P<0.05). Conclusion: The loss of P16 expression has a positive correlation with the infiltration and progression of GIST.Detection of P16 protein and mRNA is helpful for the evalutaion of biological behaviors and prognosis of gastrointestinal stromal tumors.

Objective: To study the expressions of mitochondrial protein (MAB1273), Bcl-2, and Bax in renal cell carcinoma and to investigate their role in the tumorigenesis. Methods: The expressions of mitochondrial protein, Bcl-2 and Bax were detected by immunohistochemistry SP method in 9 tissue samples of renal on-cocytoma, 6 samples of chromophobe carcinoma, 23 samples of clear cell carcinoma and 12 samples of normal renal tissue. Results: The expression of MAB1273 in renal oncocytoma, chromophobe carcinoma and clear cell carcinoma was much higher than that in normal renal tissue (P=0.006). No significant difference was found in MAB1273 expression among the three types of renal carcinoma. The expression of Bcl-2 was higher in the renal carcinoma groups than in the normal renal tissue group, with a significant difference (P=0.008). The expression of Bax was not different among all of the groups (P=0.057). Rank correlation analysis showed a positive correlation between MAB1273 and Bcl-2 expression (r=0.341, P=0.015). The expression of Bcl-2 was negatively correlated with Bax expression (r= -0.287, P=0.043). Conclusion: Overexpression of mitochondrial protein and Bcl-2 and underexpression of Bax may play a part in the genesis of renal oncocytoma, chromophobe carcinoma and the clear cell carcinoma, indicating that overexpression of MAB1273 may be correlated with apoptosis of renal cell carcinoma cells.

Objective To investigate the effects of interferon-gamma (IFN-γ) on migration,invasion and vasculogenic mimicry (VM) formation of human melanoma cell line MUM-2B. Methods MUM-2B cells were divided into three groups, control group (10%FBS in DMEM), treatment group1 (10μg/L IFN-γ) and treatment group2 (100μg/L IFN-γ). Different concentrations of IFN-γ were added in the culture medium of MUM-2B cells. Wound-healing assay and matrigel invasion assay were performed to examine the migration and invasion ability of MUM-2B cells. Three-D culture was used to observe the VM formation. The expression of vascular endothelial growth factor (VEGF) of MUM-2B cells was detected by Western blot assay. Results The result of wound-healing assay showed that the migration distance of cells was decreased in treatment groups compared with that of control group. The migration distance of cells was decreased in treatment group 2 compared with that of treatment group 1(P＜0.05). The result of matrigel invasion assay showed that the number of invaded cells was decreased in treatment groups compared with that of control group, and which was significantly decreased in treatment group2 than that of treatment group1 (P＜0.05). The result of 3-D culture showed that cells in control group can form typical VM tube-like structures, whereas cells in treatment groups cannot. Western blot assay showed that the expression of VEGF protein was significantly decreased in treatment groups compared with that of control group, and the expression of VEGF protein was significantly decreased in treatment group2 than that of treatment group 1(P＜0.05). Conclusion These data suggest that IFN-γinhibits migration and invasion of MUM-2B cells, and inhibits VM formation by down regulating VEGF expression in vitro.

Objective: To investigate the influence of TGF-β and IFN-γ on the proliferation, migration and invasion of melanoma cells. Methods: Melanoma cells were cultured in vitro. When tumor cells were confluent about 80% degree, cytokines were added into cell culture media. The concentration of TGF-β and IFN-β was 5ng/mL and 10ng/mL, respectively. Melanoma cells were divided into free-cytokine group, TGF-β group,IFN-γ group, TGF-β and IFN-γ group. Tumor cells in each group were then incubated for 8h, 16h, 24h, 32h,40h and 48h, respectively. After incubation, fixing and staining with SRB, the optical densities and percentage viability were then determined by absorption at 540 nm (A 540). The scarification of tumor cells in each group on the surface was created by a 2001μL pipette tube. The motility of tumor cells in each group was assessed by measuring the distance between scarifications. The speed of the scuffing closure was monitored after 12h.The invasive ability of melanoma cells was observed by transwell cultivation. The tumor cells that invaded through the Matrigel and adhered to the bottom of the outside membrane were determined by absorption at 595 nm (A595). Gelatin zymography assay was used to examine the levels of matrix metalloproteinases-2 (MMP-2) and matrix metalloproteinases-9 (MMP-9) activity when the tumor cells were treated with cytokines after 24h. MMP-2 and MMP-9 activity was demonstrated by gradation in the sodium dodecyl sulfate polyacryl-amide gel electrophoresis (SDS-PAGE) gelatin. MMP-2 and MMP-9 activity was determined by Image analy-sis Software. Results: TGF-β promoted the proliferation, migration and invasion of melanoma cells (P<0.05).However, IFN-γ inhibited the proliferation, migration and invasion of melanoma cells (P<0.05). The effect weakened or disappeared when both of them were used (P>0.05). Conclusion: In vitro, TGF-β may affect the inhibitory effect of IFN-γ on the proliferation, migration and invasion of melanoma cells. This study provided a better understanding of the relationship between tumor and inflammatory factors and established a good ba-sis for future research.

Objective:This study aims to investigate the potential of colon cancer cells to differentiate into vascular endothelial cells in endothelial-induced specific environment. Methods:Three colon cancer cells with different differentiated level HCT116 (poor-ly differentiated), SW480 (moderately differentiated), HT29 (well differentiated) were cultured in the conditioned medium containing the endothelial-inducing factors for 15 days respectively. The expression of vascular endothelial indicators Platelet endothelial cell adhe-sion molecule-1、Endothelial cell adhesion molecule CD34 was detected via western blot. Immunofluorescence staining was performed to examine CD31 and CD34 expression level in HCT116 after cultured in endothelial-inducing medium and ordinary medium for 15 days respectively, and the three-dimensional (3D) culture was used to detect the abililty of in vitro tube-like structure formation. Re-sults:Western blot showed that CD31 and CD34 expression level were negatively correlated with degree of differentiation in colon can-cer cells. CD31 and CD34 expression in endothelial-inducing medium HCT116 cells (poorly differentiated) were higher then in the nor-mal medium, while the CD31 and CD34 expression in SW480 cells (moderately differentiated) and HT29 cells (well differentiated) in the two cultural mediums were not notably changed. Immunofluorescence staining illustrated that CD31 and CD34 expression in HCT116 cells cultured in endothelial-inducing medium increased compared with those cultured in ordinary medium. In vitro three-di-mensional culture demonstrated that ability of tube-like structure formation was notably enhanced after endothelial-inducing cultured. Conclusion:Endothelial-inducing medium could promote colon cancer cells with strong stemness differentiate toward vascular endo-thelial cells.

Objective:To determine the expression of BMP4 in hepatocellular carcinoma (HCC) and to study the role of BMP4 in inducing epithelial-mesenchymal transition (EMT) to analyze the effect of BMP4 on the migration and invasion of HCC cells. Methods: The expression of BMP4 in HCC specimens was examined by immunohistochemistry staining, and the correlations were analyzed between the expression of BMP4 and clinicopathological data. The BMP4 expression plasmid was transfected into HepG2 cells to induce exogenous overexpression of BMP4 protein. The changes of HepG2 cell morphology were detected after BMP4 transfection by using a microscope; the changes of the expression of BMP4, EMT-related protein (E-cadherin, Vimentin) in HepG2 cells were detected by Western blot after transfection of BMP4;the wound healing assay in vitro was used to detect the effects of BMP4 gene transfection on the ability of migration of HepG2 cells;the invasion assay was used to determine the role of transfection of BMP4 on the invasive potential of HepG2 cells. Results: Immunohistochemistry staining method displayed that BMP4 expression was positively associated with age, histological differentiation, stage, and poor prognosis. After BMP4 overexpression, the morphology of HepG2 cells showed significant changes from a paving stone structure with cell-cell adhesion to a fibroblastic shape, which showed typical EMT change; Western blot exhibited that the expression of E-cadherin was downregulated and the Vimentin expression was upregulated in HepG2 cells;the wound healing and invasion assay showed that the migration and invasion potentials of HepG2 cells were significantly enhanced. Conclusion: BMP4, which displayed a high expression in HCC specimens, was closely associated with clinicopathologic data, and BMP4 may promote migration and invasion of HCC cells by inducing epithelial-mesenchymal transition.

Objective: To investigate differentially expressed protein profiles in B16-F10 grafted melanoma and its metastasis in the lung in order to identify molecular markers of melanoma metastasis. Methods: Differentially expressed proteins in B16-F10 grafted melanoma and its metastatic lesion in the lung were isolated and identified by fluorescence two-dimensional differential gel electrophoresis(2D-DIGE)coupled with matrix assisted laser desorption ionisation time-of-flight mass spectrometry(MALDI-TOF-MS).Some of identified proteins were further confirmed by Real-time PCR analysis. Results: High resolutional images of differential gel electrophoresis were obtained and 9 of 30 differentially expressed proteins (IRatiol≥2,P<0.01)were identified by MALDI-TOF-MS.The expression of Myoglobin(MB),vimentin(VIM),phosphoglycerate kinase 1(PGK1),Triosephosphate isomerase(TPI or TIM),heavy-chain binding protein(BiP),α-enolase,β-actin,γ-actin,and laminin-binding protein were up-regulated in the experimental group compared with the control group.These proteins were involved in the cytoskeletal formation,glycolysis and so on.Real-time PCR analysis showed up-regulation of mRNA expression of PGK1 and TPI in the experimental group(P=0.001 and 0.003),which was in consistent with the resuits of proteomic analysis. Conclusion: A variety of abnormally expressed proteins contribute to the metastasis of mice melanoma.Glycolytic enzymes PGK1 and TPI may be involved in this process.

Objective: To study the prognostic significance of the subtype of SYT-SSX fusion gene, E-cadherin, β-Catenin and clinicopathologicel parameters for the metastasis of synovial sarcomas. Methods: A total of 98 synovial sar-coma patients with complete clinical and follow-up data were reviewed. RT-PCR was used to detect the subtype of SYT-SSX fusion geneo The expression of E-cadherin and β-catenin was detected by immunohistochemistry. Univariate and multivariate analyses were performed to analyze the influence of the above factors and clinicopathological parameters on the metastasis free survival to explore the factors affecting the metastasis of synovial sarcoma. Results: Of all the pa-tients, 69.4% (68/98) had metastasis during follow-up. The median metastasis free survival was 48 months. The metastasis free 1-, 2-, 3-, 4-, and 5-year survival rate after surgery was 97.5%, 75.5%, 63.5%, 54.0%, and 48.5%, respectively; 31.6% (31/98) patients were found with SYT-SSX1 and 68.4% (67/98) patients with SYI-SSX2. The positive rate of E-cadherin ex-pression was 38.8% (38/98), the positive rate of β-catenin expression was 39.8% (39198) on cellular membrane and 53.1% (52/98) in cellular nucleus/cytoplasm. Univariate analysis showed that age (P=0.003), mitotic figure (P=0.002), histological grade (P=0.001), the subtype fusion gene of SYT-SSX (P=0.014), E-cadherin expression (P=0.015) and β-catenin expres-sion on cellular membrane (P=0.020) were significantly correlated with metastasis free survival of synovial sarcoma pa-tients. Sex (P=0.190), tumor location (P=0.105), tumor size (P=0.180), histological type (P=0.354), necrosis (P=0.451), β-catenin expression in cell nucleus/cytoplasm (P=0.911), radiotherapy (P=0.193), and chemotherapy (P=0.249) had no sig-nificant correlation with metastasis free survival of synovial sarcoma patients. Multivariate analysis revealed that the sub-type of SYT-SSX1 fusion gene (RR=2.505, P=0.003), negative expression of E-cadherin (RR=3.282, P=0.000), patient age (RR=2.157, P=0.004), and grade Ⅲ (RR=1.784, P=0.030) were independent risk factors for metastasis of synovial sarco-ma. Conclusion: The subtype of SYT-SSX, expression of E-cadherin, histological grade and the age of patients are impor-tant factors for evaluating the metastasis and prognosis of synovial sarcoma.

Objective: To study the prognostic significance of cell proliferation and apoptosis, MVD and clinicopathologi-cal parameters for the recurrence of synovial sarcoma. Methods: We analyzed the clinical and follow-up data of 56 synovial sarcoma patients without metastasis. RT-PCR was used to detect the subtype of SYT-SSX fusion gene. The expression of Ki67 and MVD was detected by immunohistochemistry. Univariate analysis was employed to analyze the influence of the above factors and clinicopathological parameters on the recurrence free survival and to explore the influencing factors for the recurrence of synovial sarcoma. Results: Of all the patients, 73.2% (41/56) had recurrence during the follow-up. The median recurrence free survival was 19.5 months. The recurrence free 1-, 2-, 3-, 4-, and 5-year survival rates after surgery were 45.0%, 41.0%, 34.0%, 28.0%, and 28.0%, respectively. Ki-67 labeling index (LI) was 19.98%±11.64% and MVD was 51.83±21.92 per ×400. There was no significant difference in apoptotic index (AI) between the two groups (P=0.607). Χ~2 analysis showed that histological type (P=0.000) and MVD (P=0.045) were significantly correlated with the recurrence of sy-novial sarcoma. Univariate analysis showed that Ki67 LI (P=0.009), histological type (P=0.012) and radiotherapy (P= 0.014) were significantly correlated with the recurrence free survival of synovial sarcoma patients. Sex (P=0.015), tumor lo-cation (P=0.411), tumor size (P=0.801), necrosis (P=0.486), MVD (P=0.454), chemotherapy (P=0.272), and apoptotic grade (P=0.899) were not correlated with the recurrence free survival of synovial sarcoma patients. Multivariate analysis re-vealed that higher expression of Ki67 (RR=1.944, P=0.045), radiotherapy (RR=0.482, P=0.04), and histological type (RR= 0.207, P=0.031) were independent risk factors for the recurrence of synovial sarcoma. Conclusion: The expression of Ki67, radiotherapy and histological type are important factors for evaluating the recurrence and prognosis of synovial sarcoma.

Objective: To study the relationship of LPPCN with neovascularization and to analyze its clini-copathologic significance, in an effort to find a new target for anti-vascular therapies. Methods: Sixty-eight ma-lignant melanoma specimens were analyzed to observe the distribution of LPPCN and to examine the expres-sion of CD105 and TGFβ1 using immunohistochemistry. The distribution of vasculogenic mimicry (VM) was observed by immunohistochemical and histochemical double staining of CD31 and PAS. Results: (1) The tu-lines and networks. Of the 68 cases of melanoma, 55.89% (38/68) were recognized as having LPPCN. (2) In malignant melanoma specimens, the rate of vasculogenic mimicry density (VMD) and microvessel density (MVD) labeled by CD105 in LPPCN-positive group were higher than those in LPPCN-negative group, with sig-nificant differences (P<0.05). VMD and MVD were positively-correlated with the density of LPPCN. The posi-tive expression of TGFβ1 in LPPCN-positive group was higher than that in LPPCN-negative group and its ex-pression in the regions of LPPCN was obviously higher than that in circumambient tumor cells, with a signifi-cant difference (P<0.05). The expression of TGFβ1 was positively correlated with MVD labeled by CD105. (3) There was no relationship between LPPCN and gender, age, site, tumor embolus, lymph node metastasis or distant metastasis (P>0.05), but LPPCN was correlated with tumor size, mitosis figure count and Breslow depth (P<0.05). Kaplan-Meier survival analysis showed the survival rate of patients with LPPCN was lower than that of patients without LPPCN, with a statistical significance (P<0.05). The presence of LPPCN indicat-ed poor prognosis. Conclusion: LPPCN exists in malignant melanoma and is associated with VM and angio- genesis. Some tumor cells undergoing LPPCN have a spacial foundation for VM and angiogenesis. LPPCN can be an index for the evaluation of the prognosis of melanoma patients.