Yun Qian

B7-H4 enhances oncogenicity and inhibits apoptosis in pancreatic cancer cells

B7-H4 is expressed in a variety of tumor cells and functions as a negative regulator of T cells. However, clarification is needed as to whether B7-H4 mediates tumorigenesis through mechanisms, such as apoptosis, in addition to mediating tumor immune escape. We investigate the mechanisms involved in enhanced oncogenicity and the inhibition of apoptosis by B7-H4 in pancreatic cancer cells. Short interfering RNAs (siRNAs) specific for B7-H4 were evaluated for their ability to knockdown B7-H4 mRNA and protein expression in pancreatic cancer cells and the most effective siRNA was selected for investigating the effect of B7-H4 gene silencing in a number of functional assays. The inhibition of B7-H4 increased cell-cell adhesion and decreased the formation of pseudopodia. It also increased the expression of E-cadherin and decreased the expression of vimentin and CD44. B7-H4 siRNA inhibited cell proliferation, colony formation and migration of pancreatic cancer cells. Moreover, increased apoptosis in pancreatic cancer cells following B7-H4 silencing was demonstrated in vitro by using flow cytometry and in a xenograft tumor model and was associated with increased caspase activity and decreased Erk1/2 phosphorylation both in vitro and in vivo. Loss of B7-H4 function thus prevents tumor growth through many processes, including the induction of apoptosis and inhibition of the Erk1/2 signaling pathway indicating that B7-H4 is a cancer promoter and a potentially important therapeutic target. B7-H4 inhibition might offer an exciting opportunity to inhibit the progression of human pancreatic cancers.

Dihydrotestosterone protects human vascular endothelial cells from H2O2-induced apoptosis through inhibition of caspase-3, caspase-9 and p38 MAPK

Oxidative stress is proved to be harmful to the vascular endothelial cells which are important in preventing the formation and progression of atheromatous plaque. This study was designed to investigate the protective effect and potential mechanisms of dihydrotestosterone (DHT) against H(2)O(2)-induced apoptosis of human umbilical vein endothelial cells (ECV-304). ECV-304 cells were pretreated with different concentrations of DHT (0.01, 0.1 and 1 microM) for 2h, followed by exposure to 100 microM H(2)O(2) for 18h. 3-(4,5-dimethylthiazol-2yl-)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to evaluate cell viability. To detect apoptosis, the cells were assessed by morphologic examination and Annexin V-propidium iodide double staining with flow cytometry. Finally, the expression of caspase-3, caspase-9 and phospho p38 MAPK was assayed by Western blot to investigate the possible molecular mechanisms. We found that H(2)O(2) treatment for 18h significantly decrease the viability of ECV-304 cells characterized by a high percentage of apoptotic cells. DHT could antagonize the apoptosis inducing effect of H(2)O(2) in a dose-dependent manner. Consistently, DHT also significantly inhibit the expression of caspase-3, caspase-9 and phospho p38 MAPK induced by H(2)O(2). In summary, pretreatment with DHT can inhibit apoptosis of ECV-304 cells induced by H(2)O(2). The protective effect of DHT was associated with the inhibition of caspase-3, caspase-9 and phospho p38 MAPK expression.

B7-H4 expression in various tumors determined using a novel developed monoclonal antibody

B7-H4, a new member of the B7 family, may participate in the negative regulation of cell-mediated immunity, while aberrant B7-H4 expression is detected in some tumors and it participates in the occurrence and development of the tumors. In this study, we developed one monoclonal antibody (mAb) whose clone No. was 4H8 against the extracellular domains of B7-H4 through immunization of Balb/c mice with the 3T3-mB7-H4 cells that expressed extrinsic B7-H4 stably. And we detected the expression characteristics of B7-H4 in various tumors using 4H8 mAb by enzyme-linked immunosorbent assay (ELISA), immunoprecipitation and immunohistochemistry (IHC) analysis. B7-H4 expression was significantly higher in the tumor tissues derived from uterus, breast, and colon than in their corresponding normal tissues. Further, the B7-H4 expression was related to the stage of the tumors. In contrast, B7-H4 expression did not differ significantly between the tumor tissues derived from the stomach and liver and the normal tissues. Different expression levels of B7-H4 in the tumors indicated that B7-H4 may be involved in tumor formation and development. Specific mAbs against B7-H4 will be useful in studying the role of B7-H4 in tumor pathogenesis and pathological process.

Development of a novel monoclonal antibody to B7-H4: characterization and biological activity

ObjectiveB7-H4, a member of the B7 family of immunoregulatory receptors, may participate in the negative regulation of cell-mediated immunity. Aberrant B7-H4 expression is detected in some tumors and it plays a role in the occurrence and development of tumors. The aim of this study was to elucidate the functional and structural properties of B7-H4.MethodsWe developed a monoclonal antibody (mAb) against the extracellular domain of B7-H4 through immunization of Balb/c mice with 3T3-mB7-H4 cells which expressed extrinsic B7-H4. A stable hybridoma cell line was established. Then, we analysised the characterization of the mAb through Enzyme linked immunosorbent assay (ELISA), Immunoprecipitation (IP), western blotting, Immunohistochemical (IHC), and tested the biological activity of the mAb.ResultsELISA, IP, and western blotting analyses indicated that the mAb specifically recognized B7-H4. In addition, flow cytometry demonstrated that the mAb exhibits excellent reactivity when applied to leukemic cells. IHC staining revealed that the mAb stained in a predominantly diffuse plasmalemmal or cytoplasmic pattern when applied to certain tumor tissues. The preliminary results of the mAbs biological activity showed that the mAb could effectively inhibit the function of B7-H4 in the inhibition of T cell, while promotingg the growth of T cells and the secretion of Interleukin-2 (lL-2), Interleukin-4 (IL-4), Interleukin10 (IL-10) and Interferon-γ (IFN-γ).ConclusionThis mAb will be a valuable tool for the further investigation of B7-H4 function.

The Protective Effect of Intrasplenic Transplantation of Ad-IL-18BP/IL-4 Gene-Modified Fetal Hepatocytes on ConA-Induced Hepatitis in Mice

Background Concanavalin A (ConA)-induced hepatitis is an experimental murine model mirroring the pathology of human autoimmune hepatitis. Aim To investigate the effects of intrasplenically transplanted fetal hepatocytes (BNL.CL2) transfected with recombinant adenovirus vector expressing the IL-18 binding protein (IL-18BP) and IL-4 fusion protein on ConA-induced hepatitis in mice. Methods Ad-IL-18BP/IL-4 was used to infect BNL.CL2 cells. IL-4 and IL-18BP fusion protein expression were detected by ELISA and Western blotting. BNL.CL2 cells infected with Ad-IL-18BP/IL-4 were intrasplenically transplanted into mice. After 10 days, mice were injected with ConA (15 mg/kg), and sacrificed 18 hours later. Liver injury was assessed by serum transaminase and liver histology. TNF-α, IL-18, IL-4, IL-10, IL-12p70 and monocyte-chemoattracting protein (MCP)-1 levels in serum and liver homogenates were detected by ELISA. Signaling molecules in liver homogenates were analyzed by Western blotting. Results Ad-IL-18BP/IL-4 effectively expressed the IL-18BP/IL-4 fusion protein for more than 14 days in BNL.CL12 cells. Treatment of mice with Ad-IL-18BP/IL-4-BNL.CL2 before ConA injection significantly reduced the elevated plasma levels of transaminases compared with ConA control groups. TNF-α, IL-18, IL-12p70 and MCP-1 levels in serum and liver homogenates from mice transplanted with Ad-IL-18BP/IL-4-BNL.CL2 were lower and IL-4 and IL-10 levels were higher than control groups. Phosphorylation levels of NF-κB p65, AKT, p38 and JNK1/2 in liver homogenates were markedly suppressed by Ad-IL-18BP/IL-4. Conclusions Ad-IL-18BP/IL-4 was effectively transfected into mouse BNL.CL2 cells. Intrasplenic transplantation of Ad-IL-18BP/IL-4-BNL.CL12 cells alleviated the severity of inflammation in ConA-induced experimental hepatitis and provides a useful basis for the targeted gene therapy of liver disease.

MicroRNA Expression Profiling of Pancreatic Cancer Cell Line L3.6p1 Following B7-H4 Knockdown

Background/Aims: Co-stimulating molecule B7-H4 regulates T cell-mediated immune responses, participates in tumor immune escape, and promotes the proliferation and metastasis of pancreatic cancer cells. However, the specific mechanisms are unclear. MicroRNAs (miRNAs) participated in the pathogenesis and progression of cancer. Methods: In this study, a microarray technique was used to screen B7-H4-related differentially expressed miRNAs in a pancreatic cancer cell line find those associated with pancreatic cancer. Using a miRCURYTM LNA Array approach, we compared the miRNA expression profiles of L3.6p1 pancreatic cancer cells transfected with B7-H4 siRNA for 72 h with those transfected with non-target siRNAs. Results: B7-H4 siRNA significantly up-regulated 57 miRNAs and down-regulated 14 miRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway analysis of predicted miRNA targets showed that these genes were mainly involved in protein binding, pathways in cancer, mitogen-activated protein kinase (MAPK) signaling pathway, and phosphatidylinositol 3-kinase-Akt (PI3K-Akt) signaling pathway. Conclusions: This is the first description of target genes of B7-H4, showing that miRNAs participate in the B7-H4 mediated regulation of oncogenicity and pathogenesis of pancreatic cancer. These results may help us better understand the role of B7-H4 in the progression of pancreatic cancer and its possible mechanisms. We also provide novel biomarkers for potential treatments of pancreatic cancer.

Prognostic significance of B7-H4 expression in matched primary pancreatic cancer and liver metastases

Liver metastasis development in pancreatic cancer patients is common and confers a poor prognosis. Clinical relevance of biomarker analysis in metastatic tissue is necessary. B7-H4 has an inhibitory effect on T cell mediated response and may be involved in tumor development. Although B7-H4 expression has been detected in pancreatic cancer, its expression in liver metastases from pancreatic cancer is still unknown. In this study, overall 43 pancreatic cancer liver metastases (with matched primaries in 15/43 cases) and 57 pancreatic cancer cases without liver metastases or other distant metastases were analyzed for their expression of B7-H4 by immunohistochemistry. Survival curves and log-rank tests were used to test the association of B7-H4 expression with survival. B7-H4 was highly expressed in 28 (65.1%) of the 43 liver metastases and 9 (60.0%) of the 15 matched primary tumors. The expression of B7-H4 in liver metastases was significantly higher than in the matched primary tumors (p < 0.05). Patients with high B7-H4 expression in their primary pancreatic cancer had higher risk of developing liver metastases (p < 0.05). In univariate analysis, B7-H4 expression was significantly associated with the risk of death (p < 0.05). And the multivariate analysis identified that B7-H4 was an independent prognostic indicator (p < 0.05). Our results revealed B7-H4 to be associated with poor prognosis in patients with pancreatic cancer liver metastasis. B7-H4 may promote pancreatic cancer metastasis and was promising to be a potential prognostic indicator of pancreatic cancer.

B7-H4 is a prognostic biomarker for poor survival in patients with pancreatic cancer ☆ ☆☆

B7-H4 belongs to the immune costimulatory B7 family and is thought to negatively regulate T-cell-mediated immunity, and may contribute an important role in tumor immune evasion. Although the expression of B7-H4 has been observed in human pancreatic cancer, the prognostic significance of this expression is poorly understood. This present study explored the prognostic value of B7-H4 in pancreatic cancer. Patients with pancreatic cancer and healthy controls were recruited at the Second Affiliated Hospital to Zhejiang University from January 2011 to December 2014. Expression of B7-H4 was assessed by immunohistochemistry. Immunohistochemical analysis indicated that B7-H4 was expressed in 100% (188/188) of the pancreatic cancer tumor tissue samples, while only in 68% (17/25) of normal pancreatic tissue samples. Furthermore, the expression levels of B7-H4 in pancreatic cancer patients were significantly higher than in controls (P<.01). A significant difference in B7-H4 expression was observed between patients with late tumor-node-metastasis (TNM) stage (III and IV) and early TNM stage (I and II) (P<.01). The expression of B7-H4 was associated with distant metastasis (P<.01) and differentiation (P<.01). In addition, B7-H4 expression (P<.01), distant metastasis (P<.01), TNM stage (P<.01), differentiation (P<.01) and chemotherapy treatment (P<.05) were indicators of poor overall survival time. Multivariate survival analysis indicated that B7-H4 expression, distant metastasis, and chemotherapy treatment (P<.05) were independent prognostic indicators of poor overall survival. In conclusion, B7-H4 is highly expressed in pancreatic cancer, and is an independent predictor of poor prognosis in patients with pancreatic cancer. B7-H4 may represent an immunotherapeutic target in pancreatic cancer.

Expression of B7-H4 and hepatitis B virus X in hepatitis B virus-related hepatocellular carcinoma

AIM To investigate the expression and clinical significance of B7-H4 and hepatitis B virus X (HBx) protein in hepatitis B virus-related hepatocellular carcinoma (HBV-HCC). METHODS The expression of B7-H4 in the human HCC cell lines HepG2 and HepG2.2.15 were detected by western blot, flow cytometry, and immunofluorescence. The expression of B7-H4 and HBx in 83 HBV-HCC was detected by immunohistochemistry, and the relationship with clinicopathological features was analyzed. Paraffin sections were generated from 83 HBV-HCC patients (22 females and 61 males) enrolled in this study. The age of these patients ranged from 35 to 77 years, with an average of 52.5 ± 11.3 years. All experiments were approved by the Ethics Committees of the Second Affiliated Hospital, Zhejiang University School of Medicine. RESULTS B7-H4 was significantly upregulated in HepG2.2.15 cells compared to HepG2 cells. Specifically, the protein expression of B7-H4 in the lysates of HepG2 cells was more than that in HepG2.2.15 cells. In addition, HBx was expressed only in HepG2.2.15 cells. Similar data were obtained by flow cytometry. The positive rates of B7-H4 and HBx in the tissues of 83 HBV-HCC patients were 68.67% (57/83) and 59.04% (49/83), respectively. The expression of HBx was correlated with tumor node metastases (TNM) stage, and the expression of B7-H4 was positively correlated with HBx (rs = 0.388; P < 0.01). The expression level of B7-H4 in HBx-positive HBV-HCC tissues was substantially higher than that in HBx-negative HBV-HCC tissues. The expression level of B7H4 was negatively related to tumor TNM stage. CONCLUSION Higher expression of HBx and B7-H4 was correlated with tumor progression of HBV-HCC, suggesting that B7-H4 may be involved in facilitating HBV-related hepatocarcinogenesis.

Cloning and Expression of Retinoic Acid-Induced Gene-I and Its Effect on Hepatitis C Virus Replication

OBJECTIVE To explore the influence of the retinoic acid indicible gene-I (RIG-I) on hepatitis C virus (HCV) replication and the molecular mechanism of action of RIG-I. METHODS We constructed an RIG-I expression vector and co-transfected it into Huh-7 cells along with HCV-replicon RNA. We assayed HCV replication and NS5A protein synthesis via real-time polymerase chain reaction (RT-PCR) and western blotting. Also, we performed an enzyme-linked immunosorbent assay (ELISA) to measure the level of interferon (IFN)-alpha/-beta secretion. Additionally, we examined, via western blotting, the phosphorylation state of p38, Erk1/2, and nuclear factor (NF)-kappaB p65. RESULTS Overexpression of RIG-1 in Huh-7 cells co-transfected with an HCV-replicon RNA significantly inhibited HCV replication and NS5A protein synthesis. Co-transfected cells had increased production of IFN-alpha/-beta production and had higher levels of phosphorylated p38, Erk1/2, and NF-kappaB p65. CONCLUSIONS RIG-I significantly inhibits HCV replication and NS5A protein synthesis by inducing type I IFN production. The underlying molecular mechanism for this effect appears to be mediated by increased phosphorylation of NF-kappaB p65, p38-mitogen-activated protein kinases (MAPK), and Erk1/2.