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Qu L.,Nanjing Medical University | Wang Y.,Nanjing Medical University | Gong L.,Nanjing Medical University | Zhu J.,Huadong Medical Institute of Biotechniques | And 2 more authors.
Oncology Reports | Year: 2013

The aim of this study was to investigate the selective killing effect of the herpes simplex virus-thymidine kinase/ganciclovir (TK/GCV) suicide gene system controlled by the survivin promoter on hepatocellular carcinoma (HCC) cells in vitro. Recombinant plasmid vectors driven by the survivin promoter were constructed. HepG2 HCC and LO2 normal human liver cells were transfected with the recombinant plasmids, green fluorescent protein (GFP)/pSURV, TK/pSURV and TAT-TK/pSURV. GFP expression was detected by fluoroscopy and flow cytometry (FCM). TK gene expression was detected using RT-PCR and western blot analysis. The selective killing effects after GCV application were evaluated by tetrazolium assay, FCM and western blot analysis. Statistical analysis was performed by ANOVA. After transfection with GFP/pSURV, TK/pSURV and TAT-TK/pSURV for 48 h, GFP expression was observed in the HepG2 cells, but not in the L02 cells and TK gene expression was evidently detected by RT-PCR and western blot analysis in the HepG2 cells. Three stably transfected cell lines (HepG2/pSURV, HepG2/TK/pSURV and HepG2/TAT-TK/pSURV) were successfully established. Compared with the HepG2/TK/pSURV group, a significant 'bystander effect' was observed in the HepG2/TAT-TK/pSURV group with the incorporation of unmodifed HepG2 cells at different ratios. Following transfection with TK/pSURV and TAT-TK/pSURV, the growth of HepG2 cells in the presence of GCV was markedly inhibited. This finding was further corroborated by FCM and immunoblot analysis revealed the repressed expression of proliferating cell nuclear antigen (PCNA). Our results showed that the plasmid vectors carrying the TK and TAT-TK fusion protein gene driven by the survivin promoter were successfully constructed and their specific expression in HepG2 cells provided the basis for the targeted gene therapy of HCC. © 2013 Spandidos Publications Ltd. All rights reserved.


Chen R.,Nanjing Medical University | Zhang D.,Nanjing Medical University | Mao Y.,Jiangsu Provincial Official Hospital | Zhu J.,Nanjing Medical University | And 9 more authors.
Molecular Cancer Therapeutics | Year: 2012

Nasopharyngeal carcinoma (NPC) is a major cause of cancer-related death in Southeast Asia and China. Metastasis and relapse are the primary cause of morbidity and mortality in NPC. Recent evidence suggests that the Epstein-Barr virus latent membrane protein 1 (LMP1) is exclusively expressed in most NPC and is a potential target for biotherapy. In this study, we successfully prepared a novel human antibody Fab (HLEAFab) against LMP1 extracellular domain, which was subsequently conjugated with mitomycin C (MMC), thus forming an immunoconjugate (HLEAFab-MMC). The effects of HLEAFab-MMC on proliferation and apoptosis inNPCcell lines HNE2/LMP1 and the inhibition rate of growth ofNPCxenografts in nude mice were examined. The inhibition rate of HNE2/LMP1 cell proliferation was the highest for HLEAFab-MMC (76%) compared with MMC (31%) and HLEAFab (22%) at a concentration of 200 nmol/L and showed dose-dependent fashion. The apoptosis rate of HNE2/LMP1 cell lines was 13.88% in HLEAFab-MMC group, 3.04% in MMC group, 2.78% in HLEAFab group, and 2.10% in negative control group at the same concentration, respectively. In vivo, the inhibition rate of growth of NPC xenografts in nude mice was 55.1% in HLEAFab-MMC group, 26.5% in MMC group, and 5.64% in HLEAFab group. In summary, our findings show that HLEAFab-MMC is a unique immunoconjugate with the potential as a novel therapeutic agent in the treatment of LMP1-expressing NPC. ©2011 AACR.


Zhang H.,Nanjing Medical University | Qiu J.,Nanjing Medical University | Ye C.,Nanjing Medical University | Yang D.,Nanjing Medical University | And 10 more authors.
Scientific Reports | Year: 2014

The receptor-tyrosine-kinase-like orphan receptor 1 (ROR1) is a transmembrane protein belongs to receptor tyrosine kinase (RTK) family. This study aimed to examine the expression of ROR1 in human ovarian cancer and investigate the relationship between its expression and the prognosis of ovarian cancer patients. In this present study, one-step quantitative reverse transcription-polymerase chain reaction (15 ovarian cancer samples of high FIGO stage, 15 ovarian cancer samples of low FIGO stage and nine normal ovary tissue samples) and immunohistochemistry by tissue microarrays (100 ovarian cancer samples and 50 normal ovary samples) were performed to characterize expression of the ROR1 gene in ovarian cancer. Kaplan-Meier survival and Cox regression analyses were executed to evaluate the prognosis of ovarian cancer. The results of qPCR and IHC analysis showed that the expression of ROR1 in ovarian cancer was significantly higher than that in normal ovary tissues (all p < 0.05). Survival analysis showed that ROR1 protein expression was one of the independent prognostic factors for disease-free survival and overall survival (both p < 0.05). The data suggest that ROR1 expression is correlated with malignant attributes of ovarian cancer and it may serve as a novel prognostic marker in ovarian cancer.


Gu X.,Jiangsu University | Fu M.,Jiangsu University | Ge Z.,Jiangsu University | Zhan F.,Jiangsu University | And 11 more authors.
Scientific Reports | Year: 2015

Melanoma-associated antigens (MAGE)-A9 has been reported to play important roles in the development of human cancers. However, the association between MAGE-A9 expression and the clinicopathological characteristics of hepatocellular carcinoma (HCC) is not well understood. The study was to detect the expression of MAGE-A9 in human HCC and investigate the association between its expression and the clinicopathological characteristics of HCC. Reverse transcription-polymerase chain reaction (RT-PCR), one-step quantitative -PCR (qPCR) and immunohistochemistry (IHC) analyses were performed to characterize the expression of MAGE-A9 in HCC cell lines and tissues. Kaplan-Meier survival and Cox regression analyses were employed to evaluate the prognosis of 100 HCC patients. The results showed that the expression of MAGE-A9 in HCC was significantly higher than that in non-cancerous cells and tissues. Moreover, the expression level of the MAGE-A9 protein in HCC was related to the pathological grade (p = 0.003), portal vein invasion (p = 0.001), distant metastasis (p = 0.022) and TNM stage (p = 0.005). Cox regression analysis further revealed that MAGE-A9 expression is an independent prognostic factor for disease-free survival (p = 0.006) and overall survival (p = 0.022). These data are the first to indicate that MAGE-A9 expression is a valuable prognostic biomarker for HCC and that high MAGE-A9 expression suggests unfavorable survival outcomes in HCC patients.


Zhang D.,Nanjing Medical University | Zhang D.,Huadong Medical Institute of Biotechniques | Mao Y.,Jiangsu Province Official Hospital | Cao Q.,Nanjing Medical University | And 4 more authors.
Viruses | Year: 2013

Latent Membrane Protein 1 (LMP1) is a primary target for controlling tumorigenesis in Epstein-Barr virus related malignancies; in this study, we aimed to develop a specific antibody against the TES1 domain of the oncogenic LMP1. We screened a full human naïve Fab phage library against TES1 peptide, which consisted of C terminal-activating regions proximal 44 amino acids. After three rounds of panning, enrichment and testing by phage ELISA and further analyzed by DNA sequencing, we selected a phage clone with the highest affinity to LMP1-TES1 and designated it as htesFab. The positive clone was expressed in Escherichia coli and the purified htesFab was characterized for its binding specificity and affinity to LMP1. ELISA, immunofluorescence and FACS analysis confirmed that htesFab could recognize LMP1 TES1 both in vitro and in LMP1 expressing HNE2-LMP1 cells. Furthermore, MTT assay showed that htesFab inhibited the proliferation of HNE2-LMP1 cells in a dose-dependent manner. In summary, this study reported the isolation and characterization of human Fab, which specifically targets the C terminal region/TES1 of LMP1, and has potential to be developed as novel tool for the diagnosis and therapy of Epstein-Barr virus related carcinoma. © 2013 by the authors; licensee MDPI, Basel, Switzerland.


Han L.,Nantong Tuomor Hospital | Jiang B.,Nantong Tuomor Hospital | Wu H.,Nantong University | Wang X.,Nantong University | And 4 more authors.
Medical Oncology | Year: 2012

The laryngeal squamous cell carcinoma (LSCC) is one of the most common cancers threatening people's life. CXC-chemokine receptor type 2 (CXCR2) was reported to play critical roles in angiogenesis, tumorigenesis, and metastasis of several cancers such as colon cancer, melanoma, lung cancer, and so on. However, the expression of CXCR2 in LSCC and its association with clinical characters of LSCC remain unclear. Quantitative real-time reverse transcription-PCR and immunohistochemistry were used, respectively, to analyze the mRNA level and protein level of CXCR2 in 109 cases of LSCC tissues and 28 cases of tumor-adjacent normal tissues. The expression of CXCR2 in LSCC was significantly higher than that in tumor-adjacent tissues. Moreover, the expression level of CXCR2 protein in LSCC was significantly related to lymph node metastasis (P = .022), histopathological grade (P = .038), and 5 years' survival (P = .007). Cox regression analysis revealed that CXCR2 expression (P = .031), as well as lymphatic metastasis (P = .026) and TNM classification (P = .042), is an independent prognostic marker of LSCC. High expression of CXCR2 is also associated with short survival of LSCC patients. Our data indicate that the expression of CXCR2 is associated with the development and progression of LSCC. CXCR2 expression may serve as an independent prognostic marker for LSCC patients. © 2012 Springer Science+Business Media, LLC.


Wu H.,Nantong University | Xu H.,Nantong University | Zhang S.,Nantong University | Wang X.,Nantong University | And 4 more authors.
Head and Neck | Year: 2013

Background: The human trophoblastic cell surface antigen 2 (TROP2) gene is associated with the development of malignancies, but its expression in laryngeal squamous cell carcinoma (SCC) and its relationship with clinical characteristics of the disease remain undetermined. Methods: Expression of TROP2 protein was detected by immunohistochemistry with a self-made anti-TROP2 antibody in laryngeal SCC tissue microarrays. Results: Elevated expression of TROP2 was detected in laryngeal SCC tissues compared with adjacent noncancerous tissues. TROP2 expression in laryngeal SCC was related to tumor differentiation (p =.0001) and lymph node metastasis (p =.0352). Cox regression analyses confirmed that TROP2 expression (p =.015), lymph node metastasis (p =.001), degree of differentiation (p =.002), tumor site (p =.021), and T classification (p =.003) were independent prognostic factors. Conclusions: TROP2 can be used as an independent prognostic indicator for laryngeal SCC. © 2012 Wiley Periodicals, Inc.


Ding G.,Nanjing Medical University | Chen X.,Nanjing Medical University | Zhu J.,Huadong Medical Institute of Biotechniques | Duesbery N.S.,Van Andel Research Institute | And 2 more authors.
Clinical and Developmental Immunology | Year: 2013

Human anthrax infection caused by exposure to Bacillus anthracis cannot always be treated by antibiotics. This is mostly because of the effect of the remaining anthrax toxin in the body. Lethal factor (LF) is a component of lethal toxin (LeTx), which is the major virulence of anthrax toxin. A murine IgG monoclonal antibody (mAb) against LF with blocking activity (coded LF8) was produced in a previous study. In this report, a human/murine chimeric Fab mAb (coded LF8-Fab) was developed from LF8 by inserting murine variable regions into human constant regions using antibody engineering to reduce the incompatibility of the murine antibody for human use. The LF8-Fab expressed in Escherichia coli could specifically identify LF with an affinity of 3.46×107 L/mol and could neutralize LeTx with an ECof 85 g/mL. Even after LeTx challenge at various time points, the LF8-Fab demonstrated protection of J774A.1 cells in vitro. The results suggest that the LF8-Fab might be further characterized and potentially be used for clinical applications against anthrax infection. © 2013 Guipeng Ding et al.


Ding G.,Nanjing Medical University | Chen X.,Nanjing Medical University | Zhu J.,Huadong Medical Institute of Biotechniques | Cao B.,Nanjing Medical University | Cao B.,Van Andel Research Institute
Cellular and Molecular Immunology | Year: 2010

Murine monoclonal antibodies (mAbs) are widely used but have limitations if administered in humans. The use of chimeric or humanized mAbs can reduce immunogenicity. The first step in producing such mAbs is to clone murine variable genes from a hybridoma, but it is possible to amplify both functional and aberrant variable genes, as they coexist in the hybridoma. During the development of a murine-human chimeric antibody, we have cloned from a hybridoma the functional heavy chain variable region (VH) and light chain variable region (VL) genes of a mAb that blocks the binding of anthrax lethal factor to protective antigen. In this study, we report the detection of two aberrant transcripts from a hybridoma produced using myeloma cell line OUR-1, the development of a method to distinguish between the functional and abundant aberrant VL transcripts, and the origins of these aberrant genes. The aberrant VL gene is derived from OUR-1 cells, while the aberrant VH gene might derive from antibody repertoires in B cells or from gene rearrangement in the hybridoma cells. The aberrant VH and VL genes in this study may facilitate discrimination between the functional and aberrant variable genes from hybridoma cells. © 2010 CSI and USTC. All rights reserved.


PubMed | China Pharmaceutical University, Nanjing Medical University, Huadong Medical Institute of Biotechniques, Anhui Medical University and 2 more.
Type: | Journal: Frontiers in immunology | Year: 2017

Sepsis is a major cause of death for hospitalized patients and is characterized by massive overreaction of immune responses to invading pathogens which is mediated by cytokines. For decades, there has been no effective treatment for sepsis. Sialic acid-binding, Ig-like lectin-9 (Siglec-9), is an immunomodulatory receptor expressed primarily on hematopoietic cells which is involved in various aspects of inflammatory responses and is a potential target for treatment of sepsis. The aim of the present study was to develop a human anti-Siglec-9 Fab fragment, which was named hS9-Fab03 and investigate its immune activity in human macrophages. We began by constructing the hS9-Fab03 prokaryotic expression vector from human antibody library and phage display. Then, we utilized a multitude of assays, including SDS-PAGE, Western blotting, ELISA, affinity, and kinetics assay to evaluate the binding affinity and specificity of hS9-Fab03. Results demonstrated that hS9-Fab03 specifically bind to Siglec-9 antigen with high affinity, and pretreatment with hS9-Fab03 could attenuate lipopolysaccharide (LPS)-induced TNF-, IL-6, IL-1, IL-8, and IFN- production in human PBMC-derived macrophages, but slightly increased IL-10 production in an early time point. We also observed similar results in human THP-1-differentiated macrophages. Collectively, we prepared the hS9-Fab03 with efficient activity for blocking LPS-induced pro-inflammatory cytokines production in human macrophages. These results indicated that ligation of Siglec-9 with hS9-Fab03 might be a novel anti-inflammatory therapeutic strategy for sepsis.

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