RG108 affects the proliferation and apoptosis of NSCLC cells by regulating TFPI-2 methylation and TMPRSS4 expression / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the effect of RG108 on the proliferation and apoptosis of human non-small cell lung cancer (NSCLC) cell lines (A549, H1299) and explore its molecular mechanism. Methods: A549 and H1299 cells were cultured in vitro and treated with different concentrations of RG108. The cell proliferation, cell cycle and apoptosis were detected by MTT assay and Flow cytometry, respectively. qPCR and Western blotting (WB) were used to detect the TFPI-2 mRNA and protein expressions as well as the expression of TMPRSS4 in cells. Meanwhile, the methylation status and degree of TFPI-2 promoter in cells were detected with Methylation-specific PCR (MSP) and colorimetry. Finally, siRNA-TFPI-2 and pcDNA3.0-TMPRSS4 plasmids were used to silence TFPI-2 or overexpress TMPRSS4, and then the changes in cell proliferation and apoptosis were detected. Results: After treatment with RG108, the proliferation rate of A549 and H1299 cells were significantly decreased (all P<0.05), while the apoptosis rate were significantly increased(P<0.05), the cell cycle were arrested in G1/S phase (P<0.05), and the intracellular mRNA and protein expressions of TFPI-2 were significantly increased (P<0.01 or P<0.05). Meanwhile, the methylase degree in TFPI-2 promoter region and the expression of TMPRSS4 in cells were all significanly decreased ( all P<0.05). After TFPI-2 silence, the proliferation levels of A549 and H1299 cells were significantly increased(all P<0.05); however, the apoptosis rate of A549 and H1299 cells were significantly reduced after transfection with pcDNA3.0-TMPRSS4(all P<0.05). Conclusion: RG108 can inhibit proliferation of A549 and H1299 cells and promote apoptosis by inhibiting the methylation of TFPI-2 and negatively regulates the expression of TMPRSS4.

Expressions of TFPI-2 and MMP-9 in esophageal squamous cell carcinoma tissue and their relationship with vasculogenic mimicry / 吉林大学学报(医学版)

Objective To investigate the expressions of tissue factor pathway inhibitor-2 (TFPI-2 ) and matrix metalloproteinase-9 (MMP-9)in esophageal squamous cell carcinoma(ESCC)tissue and their relationship with vasculogenic mimicry (VM).Methods 162 cases of esophageal squamous cell carcinoma tissues were collected. CD34/periodic acid-schiff double staining was performed to observe the distribution of VM in ESCC tissue,and immunohistochemical staining was used to detect the expressions of TFPI-2 and MMP-9 in ESCC tissue. The relationship between VM and the clinicopathologic parameters of ESCC, the expressions of TFPI-2 and MMP-9 were analyzed.Results The positive rate of VM in ESCC tissue was 20.37%.The positive rate of VM in poorly differentiated group(40.38%)was significantly higher than those in moderately differentiated group(11.76%)and well differentiated group (7.14%)(χ2 = 20.915, P < 0.01 ). The positive rate of VM in TNM Ⅰ-Ⅱ(11.59%)group was significantly lower than that in TNM Ⅲ group(26.88%)(χ2=5.707,P=0.017).The positive rate of TFPI-2 in VM(+)group(33.34%)was significantly higher than that in VM(-)group(6.45%) (χ2=4.582,P=0.032)in poorly differentiated ESCC.The positive rate of MMP-9 in VM(+)group(78.79%) was significantly higher than that in VM(-)group(44.96%)(χ2=12.05,P=0.001).The expression level of TFPI-2 in poorly differentiated group was positively correlated with VM (r= 0.166,P= 0.032 ), and the expression level of MMP-9 was positively correlated with the VM(r=0.183,P=0.018).The five-year survival rate in VM (-) group was significant higher than that in VM (+) group (χ2 = 22.84, P < 0.001 ). Conclusion VM exists in ESCC tissue,especially in poorly differentiated and advanced ESCC tissue.VM is related to poor prognostis of ESCC.TFPI-2 and MMP-9 might involve in the formation of VM in ESCC.

Research progress of tissue factor pathway inhibitor-2 gene in non-small-cell lung cancer / 肿瘤研究与临床

Tissue factor pathway inhibitor-2 (TFPI-2) that inhibits plasmin,trypsin and matrix metalloproteinases is a broad-spectrum inhibitor of serine proteinase.TFPI-2 can accommodate the invasion and metastasis of human non-small-cell lung cancer by maintaining the integrity of extracellular matrix and regulating the angiogenesis and apoptosis of tumor cells.It has been a new target for gene therapy of cancers.

Effect of 5-Aza-dC on the methylation and expression of TFPI-2 gene In pancreatic cancer PANCI cell line / 中华胰腺病杂志

ObjectivesTo investigate the effects of 5-aza-2-deoxycytidine(5-Aza-dC),a methylation inhibitor,on the expression and methylation of tissue factor pathway Inhibitor (TFPI-2) gene in PANC1 cell line of pancreatic cancer.MethodsPANC1 cell lines were treated with different dosages of 5-Aza-dC ( 1× 10-7,5 × 10-7,1× 10 -6 mol/L).The status of TFPI-2 methylafion and expressions of TFPI-2 mRNA and protein were determined by MSP,RT-PCR,and Western blot.Results TFPI-2 gene CpG island was completely methylated,and there was no expression of TFPI-2 mRNA and protein without 5-Aza-dC treatment.After treatment with different dosages of 5-Aza-dC( 1 × 10-7,5 × 10 -7,1 × 10-6 mol/L),TFPI-2 gene CpG hypermethylation was reversed from incomplete methylated to complete non-methylated.The relative expressions of TFPI-2 mRNA were 0.211± 0.087,O.327 ± 0.068,0.609 ± 0.017; and the relative expressions of TFPI-2 protein were O.429 ± O.121,O.675 ± O.044,1.132 ± O.124 in a dose-dependent manner ( P ＜0.05 ).ConclusionsThe hypermethylatien of promoter region may be the primary reason for TFPI-2 gene expression down-rogtdation and inactivation.5-Aza-dC may reverse the hypermethylation of TFPI-2 gene,and induce the m-expression of TFPI-2 mRNA and protein.

Expressions of tissue factor pathway inhibitor-2 and synuclein gamma in esophageal cancer and their correlation with local invasion, lymph node metastasis and apoptosis / 肿瘤

Objective:To investigate the expression of tissue factor pathway inhibitor-2 (TFPI-2) and synuclein gamma (SNCG) in esophageal cancer and their correlation with local invasion, lymph node metastasis and apoptosis. Methods:The expression of TFPI-2, SNCG, and matrix metalloproteinase-9 (MMP-9) was detected by immunohistochemical SP methods in 82 cases of esophageal cancer tissues, 20 cases of atypical hyperplasia tissues, and 54 cases of para-cancerous tissues. The apoptosis of esophageal cancer cells was detected by TUNEL staining and apoptosis index (AI) was calculated. Results:The positive rates were 30.4%, 60.0%, and 87.0% for TFPI-2 protein and 63.4%, 30.0%, and 3.7% for SNCG protein in the tumor tissues, atypical hyperplasia tissues,and tumor-adjacent normal tissues, respectively. There was a significant difference between the three groups(P0.05). The expression of TFPI-2 and MMP-9 was negatively correlated (r=-0.636, P=0.000). The expression of SNCG and MMP-9 was positively correlated(r=0.393,P=0.000). AI was related with TFPI-2 and SNCG expression (P<0.05). Conclusion:TFPI-2 not only inhibited the expression of MMP-9 but also induces apoptosis of esophageal cancer to prevent tumor invasion and metastasis, however, SNCG plays a contradictory role in cancer development. TFPI-2 and SNCG might serve as new tumor markers and the new targets for tumor gene therapy.

Expression and methylation status of tissue factor pathway inhibitor-2 in renal cell carcinoma / 中华泌尿外科杂志

Objective To investigate the effect of tissue factor pathway inhibitor-2(TFPI-2)on invasiveness and apoptosis of renal cell carcinoma,to analysis the relationship between promoter methylation and expression of TFPI-2.Methods Immunohistochemistry,Western blot and real-time RT-PCR were performed to detect the expression of TFPI-2 in 37 renal cell carcinoma tissues and 11 normal kidney tissues.TUNEL was used tO study the apoptosis status of renal cell carcinoma.Real-time methylation specific PCR was performed to analysis the methylation status of TFPI-2 gene promoter.Results The optical density of Western-blot strip in renal cell carcinoma and normal kidnev was 0.92±0.36,1.6l±0.13.The relative expression of TFPI-2 mRNA in renal cell carcinoma and normal kidney was 0.0019±0.0011,0.0065±0.0008.Compared with normal kidney,lower expression of TFPI-2 was detected in renal cell carcinoma.The correlation between expression of TFPI-2 and stage of renal cell carcinoma was negative.Apoptosis index of renal cell carcinoma specimens was 2.41%(TFPI-2 expression:grade 1),3.90%(TFPI-2 expression:grade 2),6.78%(TFPI-2 expression:grade 3),9.57%(TFPI-2 expression:grade 4).The expression of TFPI-2 was positively correlated with the apoptosis index of renal cell carcinoma.The relative expression of TFPI-2 mRNA in methylated and unmethylated tumors was 0.0015±0.0011,0.0024±0.0009.The optical densitv of Western-blot strip in methylated and unmethylated tumors was 0.82±0.35,1.04±0.34.Expressions of TFPI-2 mRNA and protein were significantly lower in methylated tumors than those in unmethylated tumors.Conclusions The expression of TFPI-2 is negatively correlated with the invasiveness of renal cell carcinoma.Overexpression of TFPI-2 may induce tumor cell apoptosis in renal cell carcinoma.Lower expression of TFPI-2 in renal cell carcinoma is partially due to hypermethylation of gene promoter.

Correlation of TFPI-2 gene expression with invasion, metastasis, and prognosis of supraglottic carcinoma / 肿瘤

Objective: To investigate the relationship between TFPI-2 gene expression and the invasion, metastasis, and prognosis of supraglottic carcinoma. Methods: Reverse transcriptase polymerase chain reaction and immunohistochemical methods were used to examine the expression of TFPI-2 in supraglottic carcinoma, and the relationship between TFPI-2 expression and the clinicopathological features of supraglottic carcinoma were analyzed. Results: Positive ratio of TFPI-2 expression was significantly increased at protein and mRNA levels in supraglottic carcinoma tissues (P < 0.05). TFPI-2 mRNA and protein expression were decreased with the increase in the invasive depth of supraglottic carcinoma (P < 0.05). TFPI-2 mRNA and protein expression were significantly lower in supraglottic carcinoma tissues with nodal metastasis and at III-IV clinical stage than those without nodal metastasis and at I - II clinical stage (P < 0.05). The staining intensity of TFPI-2 protein was also decreased with increase in invasive depth and nodal metastasis (P < 0.05). The supraglottic carcinoma patients with negative TFPI-2 protein expression had poor prognosis than those with positive TFPI-2 expression. Conclusion: The downregulation of TFPI-2 has a close relationship with invasion and metastasis of supraglottic carcinoma, and it may serve as a helpful marker in evaluating the metastasis and prognosis of supraglottic carcinoma.

Advances in the research on human tissue factor pathway inhibitor / 国际生物医学工程杂志

Human tissue factor pathway inhibitor (TFPI) is a member of the Kunitz-type serine protease inhibitor family, which is divided into TFPI-1 and TFPI-2. The key function of TFPI-1 is anticoagulate, while TFPI-2 is a broad-spectrum serine protease inhibiter. Both of them are composed of three repeated Kunitz-type domains. Even though sharing some structural homology, they are quite different from each other in terms of the coding sequences, tissue origins and distribution, the functions and the mechanisms involved. These differences between TFPI-1 and TFPI-2 lead to the discrepancies in the roles they played in a variety of physiological and pathological processes. The recent progress in the related research is reviewed in this article.

The Influence of TFPI-2 on the Growth and the Invasion of K562 Cells in vitro / 医学研究杂志

Objective To evaluate the influence of tissue factor pathway inhibitor 2(TFPI-2)gene on the proliferation and invasion of K562.Methods The expression vector pcDNA3.0/TFPI-2 was transfected into human leukemia line K562 cells(K562-T)by using liposome,then the mRNA and protein TFPI-2 were detected by real-time RT-PCR and western blot separately.The growth curve and the colony-forming unit assay were used to measure the ability of cells growth and transwell chamber model was employed to test the ability of cell invasion in vitro.Results Expression of mRNA and protein of TFPI-2 was detected in transfected cells.The growth rate and self-replication ability of K562-T cells were lower than those of the two control groups obviously.The number of K562-T cells to traverse a Matrigel-coated membrane was dramatically decreased compared with that of non-expressing cells.Conclusion The gene of TFPI-2 can inhibit the growth,proliferation and invasion of the K562 cells.

The effect of TFPI-2 gene on apoptosis of pancreatic cancer cell / 中国癌症杂志

Background and purpose: TFPI-2 is a new serine proteinase inhibitor,it is related to many tumors.In this study,the effect of TFPI-2 gene on cell proliferation and apoptosis of human pancreatic cancer cell line Panc-1 were investigated.Methods:The growth curve was drawn for the 3 groups,Panc-1-TFPI-2、Panc-1-V and Panc-1.DNA strand breaks were used to detect the apoptosis of the 3 groups,and the change of cell cycle and apoptosis index was evaluated by flow cytometry.Results:Compared with nontransfected cells,the growth of Panc-1 cell transfected with TFPI-2 was inhibited significantly.The transfected cells showed a significant increase in G1/G0 phase.The apoptosis of Panc-1-TFPI-2 cell could be identified by DNA strand breaks and flow cytometry in comparison with the control group.The apoptosis index of the Panc-1-TFPI-2 cell(6.9?0.5)% was higher than the group Panc-1-V and group Panc-1[(3.0?0.4)% and(3.5?0.4)%]P

Regulation of TFPI-2 gene expression and its role in diseases / 基础医学与临床

Tissue factor pathway inhibitor-2(TFPI-2) is a matrix-associated Kunitz-type serine proteinase inhibitor,and is considered to play an important role in some pathophysiological processes,including artherosclerosis,tumor metastasis and angiogenesis.Extracellular signals can regulate TFPI-2 gene expression through modulating promoter or signaling pathway or other factors.The mechanism,more over,has become one of the focus in recent years.

Effect of Recombinant Tissue Factor Pathway Inhibitor 2 Gene on The Invasion of Human Pancreatic Cancer in Vitro and in Vivo / 中国普外基础与临床杂志

Objective To investigate the invasion ability of Panc-1 cells in vivo and in vitro after being transfected with tissue factor pathway inhibitor 2 gene(TFPI-2).Methods The expression vector pEGFP-C1-TFPI-2 was transfected into human pancreatic cancer line Panc-1 cells by using liposome.TFPI-2 mRNA and protein of transfected and nontransfected cells were detected by reverse transcription-polymerase chain reaction(RT-PCR)and Western blot respectively.The tumor cells invasive behavior of transfected(Panc-1-TFPI-2)and nontransfected(Panc-1-V and Panc-1-P)cells were assessed in vitro through Boyden Chamber method.The transfected and nontransfected cells were implanted into nude mice to observe its growth and metastasis in vivo.Results Expressions of mRNA and protein of TFPI-2 were confirmed in transfected cells.After TFPI-2 transfection,the number of Panc-1-TFPI-2,Panc-1-V and Panc-1-P cells passing through membrane of Boyden Chamber were 24.4?3.5,61.3?4.1 and 60.2?3.9,respectively.The number of TFPI-2-expressing cells to traverse a Matrigel-coated membrane was obviously decreased compared with that of non-expressing cells,the invasion ability was lower than that before transfection in vitro.The subcutaneous tumor volume of the Panc-1-TFPI-2 group was(438.0?69.8)mm3,the Panc-1-V group was(852.0?102.9)mm3 and the Panc-1-P group was(831.0?78.1)mm3,P

Study on prokaryotic expression and partial biological activities for tissue factor pathway inhibitor-2 / 中华检验医学杂志

Objectives To express human TFPI-2 gene in E.coli and get the recombinant protein;to investigate the recombinant activities of antiplasmin and antitumor.Methods (1) Encoding region of mature protein of human TFPI-2 was amplified by PCR.The 645 bp PCR product was cloned into expression vector pET28a and transformed into BL21 strain to get expression.(2)Identifying the DNA segment of TFPI-2 with enzyme digestion and colony PCR and the TFPI-2 fusion protein with western blot respectively.(3)To establish the kinetics assay for detecting the TFPI-2 and test it′s inhibiting plasmin activities.(4)To assess ovarian tumor cell migratory and invasive behaviors with the Boyden chamber in vitro.Results (1)Expression plasmid of recombinant TFPI-2 was constructed and high-level expression of TFPI-2 was produced as fusion protein. (2) The TFPI-2 fusion protein was identified by western blot. (3) Antiplasmin activity of the TFPI-2 was confirmed in the fluid and on the cell surface. (4) In invasion assay, the number of the A2780 and A2780-TFPI-2 groups transferred the Matriged matrix-coated PVPF membrane was obvious decreased compared with that of A2780 group(P0.05).Conclusion (1) Prokaryotic expression of TFPI-2 gene was got, which can provide the rich experimental material for investigating the role of TFPI-2 in relative fields; (2)Recombinant TFPI-2 has antiplasmin activity, which provides the experiment basis for investigating the role of recombinant TFPI-2 in human ovarian tumor migration and invasion in vitro; (3) The recombinant TFPI-2 inhibits the invasive ability of human ovarian tumor cells in vitro, but has no effect on the migration, which may provide a target basis for treating human ovarian tumor with TFPI-2 protein therapy.