Genetic Variants in Double-Strand Break Repair Pathway Genes to Predict Platinum-Based Chemotherapy Prognosis in Patients With Lung Cancer.

Objective: The purpose of this study was to investigate the associations of genetic variants in double-strand break (DSB) repair pathway genes with prognosis in patients with lung cancer treated with platinum-based chemotherapy. Methods: Three hundred ninety-nine patients with lung cancer who received platinum-based chemotherapy for at least two cycles were included in this study. A total of 35 single nucleotide polymorphisms (SNPs) in DSB repair, base excision repair (BER), and nucleotide excision repair (NER) repair pathway genes were genotyped, and were used to evaluate the overall survival (OS) and the progression-free survival (PFS) of patients who received platinum-based chemotherapy using Cox proportional hazard models. Results: The PFS of patients who carried the MAD2L2 rs746218 GG genotype was shorter than that in patients with the AG or AA genotypes (recessive model: p = 0.039, OR = 5.31, 95% CI = 1.09-25.93). Patients with the TT or GT genotypes of TNFRSF1A rs4149570 had shorter OS times than those with the GG genotype (dominant model: p = 0.030, OR = 0.57, 95% CI = 0.34-0.95). We also investigated the influence of age, gender, histology, smoking, stage, and metastasis in association between SNPs and OS or PFS in patients with lung cancer. DNA repair gene SNPs were significantly associated with PFS and OS in the subgroup analyses. Conclusion: Our study showed that variants in MAD2L2 rs746218 and TNFRSF1A rs4149570 were associated with shorter PFS or OS in patients with lung cancer who received platinum-based chemotherapy. These variants may be novel biomarkers for the prediction of prognosis of patients with lung cancer who receive platinum-based chemotherapy.

A preliminary study on the activation and antigen presentation of hepatitis B virus core protein virus-like particle-pulsed bone marrow-derived dendritic cells.

Hepatitis B virus core protein virus-like particles (HBc-VLPs) act as a strong immunogen and are suitable for uptake by dendritic cells (DCs), in which they directly promote DC maturation and migration. To illustrate the utility of global proteomic analysis techniques in elucidating the molecular events that are altered in HBc-VLP-pulsed bone marrow-derived DCs (BMDCs) and to gain a better understanding of the molecular mechanisms of capture and processing of HBc-VLP-pulsed BMDCs, an antigen (Ag) delivery system based on HBc-VLP-pulsed BMDCs was developed. Two-dimensional electrophoresis (2-DE) and tandem mass spectrometry (MS/MS) analyses were utilized to analyze the differential protein expression patterns between HBc-VLP-pulsed and untreated BMDCs. Protein spots with significantly altered expression levels were detected, identified and validated. The results showed that exogenous HBc-VLPs were phagocytosed efficiently by BMDCs and enhanced the efficacy of BMDC maturation and Ag presentation, VLPs also induced high levels of Ag-specific CD8(+) T cells that displayed high cytotoxic T lymphocyte (CTL) activity in vivo. Several differentially expressed proteins, including growth factor receptor bound protein 2 (Grb2) and annexin A2 (AnxA2), were detected by proteomic analysis, identified by mass spectrometry and validated by western blot.

Repression of the miR-17-92 cluster by p53 has an important function in hypoxia-induced apoptosis.

We here report that miR-17-92 cluster is a novel target for p53-mediated transcriptional repression under hypoxia. We found the expression levels of miR-17-92 cluster were reduced in hypoxia-treated cells containing wild-type p53, but were unchanged in hypoxia-treated p53-deficient cells. The repression of miR-17-92 cluster under hypoxia is independent of c-Myc. Luciferase reporter assays mapped the region responding to p53-mediated repression to a p53-binding site in the proximal region of the miR-17-92 promoter. Chromatin immunoprecipitation (ChIP), Re-ChIP and gel retardation assays revealed that the binding sites for p53- and the TATA-binding protein (TBP) overlap within the miR-17-92 promoter; these proteins were found to compete for binding. Finally, we show that pri-miR-17-92 expression correlated well with p53 status in colorectal carcinomas. Over-express miR-17-92 cluster markedly inhibits hypoxia-induced apoptosis, whereas blocked miR-17-5p and miR-20a sensitize the cells to hypoxia-induced apoptosis. These data indicated that p53-mediated repression of miR-17-92 expression likely has an important function in hypoxia-induced apoptosis, and thus further our understanding of the tumour suppressive function of p53.

Screen of multifunctional monoclonal antibodies against hepatitis B core virus-like particles.

HBc-VLP can be used in an epitope presentation system to carry foreign epitopes and mimic live virus in order to study viral particle uptake, virion-mediated activation and antigen presentation by dendritic cells. In this study, a multifunctional mAb was produced using a novel research strategy. A truncated HBc-VLP bone vector with a special conformation was used as an immunogen and the target hybridoma cell lines were screened by a series of tests; including ELISA, Western blot, and cellular immunofluorescence based on the epitope presentation system. The screened monoclonal antibody was used to identify the HBc-VLP vector, a fusion HBc-VLP vaccine, and intracellular HBV capsids. The new strategy facilitated acquisition of the desired mAbs and will serve as a reference for other VLP-related research.

Expression of annexin a5 is associated with higher tumor stage and poor prognosis in colorectal adenocarcinomas.

GOALS: To gain an insight into the putative role of annexin A5 (ANXA5) in the tumor stage and its clinical outcome. BACKGROUND: ANXA5 is a calcium-binding protein, which has been implicated in the carcinogenesis of several carcinomas. However, the role of ANXA5 in colorectal cancer (CRC) is unclear. STUDY: We investigated the expression of ANXA5 in colorectal adenocarcinoma. This study included 207 consecutive patients with sporadic CRC. Paired colorectal tissue samples and corresponding nonmalignant tissues were obtained by surgical resection. ANXA5 mRNA and protein expression in each tissue were assessed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemical staining. Data were statistically correlated with pathologic parameters and clinical outcome. RESULTS: Real-time reverse transcriptase polymerase chain reaction showed that there is an up-regulation in the mRNA level of ANXA5 in tumors (P<0.001). Immunohistochemical study revealed that high ANXA5 expression was present in 40.58% (84 of 207) of tumors. Univariate analysis showed increased ANXA5 expression correlated with pT stage (P=0.008), liver metastasis (P=0.024), pathologic tumor-node-metastasis stage (P=0.015), Dukes' stage (P=0.017), recurrence (P=0.024), cancer-related death (P=0.028), recurrence-free probability (P=0.003), and overall survival (P=0.005). Multivariate analysis showed that ANXA5 expression and liver metastasis significantly correlated with recurrence-free probability (P=0.039 and P=0.048, respectively) and overall survival (P=0.012 and P=0.021, respectively) independent of pT stage and pN stage. CONCLUSIONS: From these findings ANXA5 expression seems to be related to the tumor stage and clinical outcome of CRC. Thus ANXA5 could serve as a prognostic marker for tumor progression.

Multiepitope peptide-loaded virus-like particles as a vaccine against hepatitis B virus-related hepatocellular carcinoma.

UNLABELLED: To develop a hepatitis B virus (HBV) therapeutic vaccine that can induce a broad but specific immune response and significant antitumor effects both in vivo and in vitro, we inserted HBV X protein (HBx)-derived epitopes HBx(52-60), HBx(92-100), and HBx(115-123); a novel subdominant cytolytic T lymphocyte (CTL) epitope HBx(140-148); and the universal T helper epitope pan human leukocyte antigen DR-binding epitope into HBV core protein to form multiepitope peptide-loaded virus-like particles (VLPs). CTL responses against epitope-loaded VLPs were elicited by priming with VLP-pulsed dendritic cells in both HLA-A*0201 transgenic (Tg) mice and peripheral blood lymphocytes from HLA-A2(+)/HBx(+) HBV-infected hepatocellular carcinoma (HCC) patients. The multiepitope peptide-loaded VLPs demonstrated significantly higher immunogenicity in Tg mice than any single responsive epitope. Significant antitumor effects were demonstrated both with primary cultured autologous HCC cells in vitro and tumor-bearing Tg mice in vivo in an HLA-A2-restricted and epitope-specific fashion. CONCLUSION: The significant antitumor effects both in vivo and in vitro demonstrate the potential of multiepitope peptide-loaded VLPs as a vaccine against HCC.

Calcium-dependent proapoptotic effect of Taenia solium metacestodes annexin B1 on human eosinophils: a novel strategy to prevent host immune response.

Annexins are a family of calcium-dependent phospholipid-binding proteins that have been proposed to be involved in a wide range of important biological processes. At present, only a few annexins have been identified in parasites, and the physiological roles of these annexins are obscure. Earlier, we cloned a novel annexin (annexin B1) from Taenia solium metacestodes and found that annexin B1 was detectable in the surrounding host-derived layer with granulomaous infiltration. The objective of this study was to investigate the secretion and physiological function of annexin B1. We expressed a green fluorescent protein-tagged annexin B1 (GFP-anxB1) in living SiHa cells and showed that it was secreted upon stimulation with dexamethasone (Dex). This secretion was not inhibited by brefeldin A but was blocked by pre-treatment with the intracellular calcium-specific chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Furthermore, we describe for the first time that annexin B1 can bind to the extracellular surface of human eosinophils and produce Ca(2+)-influx. The Ca(2+)-influx induced apoptosis in eosinophils, which was inhibited by pre-loading the Ca(2+) channel blocker 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-metho-xyphenethyl]-1H-imidazole, HCl (SKF-96365). In conclusion, these findings represent direct and substantial evidence for the secretion of annexin B1 by living cells; the apoptosis in eosinophil induced by annexin B1 might be a novel strategy for T. solium metacestodes to prevent the host's immune attack.

Translocation of annexin B1 in response to the stimulation of PMA and ionomycin in cervical cancer cells.

Annexin B1 is a novel member of the annexin superfamily which was isolated from a Cysticercus cellulosae cDNA library. To investigate the physiological roles of annexin B1, we firstly performed immunohistochemical analysis on frozen Cysticercus cellulosae sections and found that annexin B1 was present not only in the tegument of the bladder wall, but also in the host-derived inflammatory layer; In addition, ELISA analysis revealed that annexin B1 could be detected in the cystic fluid of Cysticercus cellulosae and the sera of pigs with cysticercosis. These findings indicated that annexin B1 might be a secretary protein. We further constructed a pEGFP-annexin B1 plasmid and transfected it into SiHa cells. We found that GFP-annexin B1 was stimulated to translocate to the plasma membrane by phorbol 12-myristate 13-acetate (PMA). By contrast, it was induced to distribute at the plasma and nuclear membranes by treatment with calcium ionophore ionomycin. PMA increased annexin B1 membrane binding, which might facilitate exocytosis. Moreover, translocation of the protein to the plasma and nuclear membranes after stimulated by ionomycin, was predicted to be related to an additional function.

A novel, cheap and effective fusion expression system for the production of recombinant proteins.

To develop faster, less expensive methods for expression and purification of proteins, the annexin B1-intein fusion expression system was constructed. The interest proteins fused to the annexin B1-intein tag were purified in a single-step method based on the Ca(2+)-binding activity of annexin B1, and the annexin B1-intein fusion tag was removed based on the self-cleaving activity of the intein. Moreover, we found that in some cases, fusion to annexin B1 can promote the solubility of heterologous proteins. The production of soluble and highly active of interleukin-2 and low-molecular single-chain urokinase in our results proved that the system was a novel, cheap and effective fusion expression system for the production of valuable soluble recombinant proteins in Escherichia coli.

Annexin B1 from Taenia solium metacestodes is a newly characterized member of the annexin family.

We previously reported cloning of the Taenia solium annexin B1 gene from a metacestode cDNA expression library and demonstrated that it acts as a protective antigen for effective vaccine development against cysticercosis. In the present study we produced recombinant annexin B1 and antiserum against the protein to investigate its structural and functional properties. Western blotting of metacestode fractions indicated that T. solium annexin B1, similar to vertebrate annexins, associates with acid phospholipids in the presence of Ca(2+). This property was confirmed by the recognition of apoptotic cells by labeled annexin B1. CD spectroscopy results demonstrated that alpha-helices are the main secondary structures of the protein. Ca(2+) binding increases the alpha-helix content and causes significant thermal stabilization with a melting temperature increase of approximately 10 degrees C. Functional Ca(2+)-dependent phospholipid binding sites of annexin B1 were investigated using mutant proteins. By changing a conserved acidic amino acid residue that putatively combines Ca(2+) in each domain of annexin B1 singly or in combination, we found that Ca(2+) binding in the first domain is more important than that at the other Ca(2+) binding sites. Annexin B1 is a metacestode stage-specific antigen, with the protein being mainly localized in the teguments and surrounding cyst wall of T. solium metacestodes, suggesting a role in the parasite-host interaction.

Annexin B1 at the host-parasite interface of the Taenia solium cysticercus: Secreted and associated with inflammatory reaction.

Annexin B1 is a novel member of annexin family firstly cloned by immunological screening a Taenia solium cysticercus library. To investigate the histological distribution and physiological role(s) of this protein, we first prepared a specific monoclonal antibody against annexin B1. Western blot analysis indicated that annexin B1 could be detected in cystic fluid of T. solium cysticercus and sera of pigs/humans with cysticercosis. Thus, annexin B1 might belong to the secreted members of annexins. Immunohistochemical analysis revealed that annexin B1 was mainly present in the tegument of bladders, but not in the scolex and neck; it was also detected in the surrounding host-derived layer with granulomatous infiltration. Together with previous, the presented data suggested that the protein inhibited mammalian PLA2 in vitro, and might down regulate host inflammatory responses.

Design and characterization of a platelet-targeted plasminogen activator with enhanced thrombolytic and antithrombotic potency.

To obtain a thrombus-targeted plasminogen activator with high affinity for activated platelets and enhanced thrombolytic and antithrombotic potency, we engineered a sequence encoding RGDS (Arg-Gly-Asp-Ser) peptide into the loop between domains II and III of the sequence-deleted mutant of annexin B1 and then constructed a chimaeric plasminogen activator gene mAnxB1-RGDS-ScuPA by fusing ScuPA32k [low-molecular-mass single-chain urokinase (32 kDa)] with the N-terminus. The chimaeric protein was expressed in inclusion bodies in Escherichia coli at 25% of the total cellular protein content. Ion-exchange and gel-filtration chromatographies were applied to purify the chimaeric protein, achieving purity greater than 98%. We demonstrated that this chimaera can be expressed and purified in an active form; in vitro testing indicated that the chimaera fully retained the thrombolytic activity, platelet membrane-binding activity and anti-platelet aggregation activity of the parent molecules. The plasma clearance of the chimaera was similar to that of urokinase and ScuPA32k. In vivo experiments in a canine system indicated that animals administered the chimaera presented a decreased time to reperfusion, higher reperfusion ratio and less bleeding effects than treatment with urokinase. These results show that the chimaera is a platelet-targeted plasminogen activator with enhanced thrombolytic and antithrombotic potency that may have advantages over currently available thrombolytic agents.

Single-nucleotide polymorphisms of mismatch repair genes in healthy Chinese individuals and sporadic colorectal cancer patients.

Mismatch repair (MMR) genes are among of the most important genes associated with colorectal cancer (CRC). Single-nucleotide polymorphisms (SNPs) are generally thought to provide important information across a wide spectrum of life sciences; however, no study of association between SNPs of MMR genes and Chinese sporadic colorectal cancer (SCRC) is available. We chose 29 reported single-nucleotide variants that have rarely been verified in a population-based study. We identified SNPs and the genotype-phenotype association in Chinese populations of 150 healthy individuals and 160 SCRC patients. We extracted the genomic DNA from the blood of these individuals and used sequencing to determine these SNPs. Three SNPs (MLH1 394G-->C, 655A-->G, 1151T-->A) occurred with a frequency of 8.8-11.2% in the Chinese population. These SNPs formed a series with combined effects. The haplotype of concurrent MLH1 655 and 1151 SNPs and the haplotype combinations of MLH1 1151, MLH1 394 occurred exclusively in SCRC. None of the other 26 variants were detected in the Chinese population.

Cloning, purification and biochemical characterization of a thrombus-ditargeting thrombolytic agent, comprised of annexin B1, ScuPA-32K and fibrin-adherent peptide.

The development of thrombolytic agent could provide invaluable progress for antithrombotic therapy. In this paper, we reported the cloning, purification and biochemical characterization of AnxB1ScuPAFap, a thrombus-ditargeting chimera composed of annexin B1, low molecular single-chain urokinase (ScuPA-32K) and fibrin-adherent peptide (dodecapeptide, Fap). In vitro test showed that, the chimera was a thrombolytic agent with anticoagulant activity and thrombus-ditargeting with the activated-platelet membrane binding and fibrin clot binding activity. Compared to urokinase, the chimera had less reperfusion time, higher reperfusion ratio, and less bleeding effects on coronary thrombolysis by clot lysis assay in dogs. Thus, the chimera appeared to be suitable for thrombolytic therapy of thrombus diseases.