Jia-Jia Wang

Stanniocalcin1 (STC1) Inhibits Cell Proliferation and Invasion of Cervical Cancer Cells

STC1 is a glycoprotein hormone involved in calcium/phosphate (Pi) homeostasis. There is mounting evidence that STC1 is tightly associated with the development of cancer. But the function of STC1 in cancer is not fully understood. Here, we found that STC1 is down-regulated in Clinical tissues of cervical cancer compared to the adjacent normal cervical tissues (15 cases). Subsequently, the expression of STC1 was knocked down by RNA interference in cervical cancer CaSki cells and the low expression promoted cell growth, migration and invasion. We also found that STC1 overexpression inhibited cell proliferation and invasion of cervical cancer cells. Moreover, STC1 overexpression sensitized CaSki cells to drugs. Further, we showed that NF-κB p65 protein directly bound to STC1 promoter and activated the expression of STC1 in cervical cancer cells. Thus, these results provided evidence that STC1 inhibited cell proliferation and invasion through NF-κB p65 activation in cervical cancer.

Identification of genes associated with tumor development in CaSki cells in the cosmic space

It is important to understand the mechanisms of tumor development for curing cervical cancer. However, the molecular basis determining the different characteristics of tumor remains unclear. Space environment as a special study model can expand the study field of tumor development. To approach this, after human cervical carcinoma CaSki cells were flown on “Shen Zhou IV” space shuttle mission, the cell morphology and proliferation was investigated after flying to ground. We found that the growth of 48A9 CaSki cell (flight group) became slow compared with ground groups. Observation of cells by light microscopy revealed differences in cell morphology between ground controls and flight groups, and the flight group exhibited morphologic differences, characterized by rounder, smoother, decreased, smaller and low-adhension cells. Transmission electron microscope images showed the structure of the ultrastructural characteristics of 48A9 CaSki cells were clearly distinct from those of the ground CaSki cells in aspects of mitochondrion, cytoplasm, nucleus and ribosomes. MTT and soft agar assay showed that 48A9 CaSki cells grew slowly compared to ground control. Furthermore, suppression subtractive hybridization combining with reverse Northern blot was used to identify differently expression genes between flight and ground groups. These differentially expressed genes included cytoskeleton, cell differentiation, cell apoptosis, signal transduction, DNA repair, protein synthesis, substance metabolism, and antigen presentation. The identification of differently expressed genes which is likely to increase our understanding of the molecular processes underlying tumor development will provide new insight into tumor development mechanisms, and may facilitate the development of new anticancer strategies.

Spaceflight alters the gene expression profile of cervical cancer cells.

Our previous study revealed that spaceflight induced biological changes in human cervical carcinoma Caski cells. Here, we report that 48A9 cells, which were subcloned from Caski cells, experienced significant growth suppression and exhibited low tumorigenic ability after spaceflight. To further understand the potential mechanism at the transcriptional level, we compared gene expression between 48A9 cells and ground control Caski cells with suppression subtractive hybridization (SSH) and reverse Northern blotting methods, and analyzed the relative gene network and molecular functions with the Ingenuity Pathways Analysis (IPA) program. We found 5 genes, SUB1, SGEF, MALAT-1, MYL6, and MT-CO2, to be up-regulated and identified 3 new cDNAs, termed B4, B5, and C4, in 48A9 cells. In addition, we also identified the two most significant gene networks to indicate the function of these genes using the IPA program. To our knowledge, our results show for the first time that spaceflight can reduce the growth of tumor cells, and we also provide a new model for oncogenesis study.

Identification of HTA as a novel-specific marker for human hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is the most common malignancy in the world, especially in China. Early diagnosis of new and recurrent hepatocellular carcinoma, followed by timely treatment, will help decrease mortality. Currently biomarkers are not satisfactory. Better diagnostic methods are highly demanded.MethodsIn this study, we have used in silico identification and RT-PCR test and discovered a hepatoma associated gene (HTA). Knockdown of endogenous HTA expression was performed by small interfering RNA in malignant hepatocyte HepG2. Then we tested the cell proliferative ability of these cells in vitro and in vivo.ResultsHTA was expressed specifically in some kinds of tumors, but not detected in any normal tissues. It was expressed especially high in hepatocellular carcinoma. Knockdown of endogenous HTA expression in HepG2 by small interfering RNA attenuated HCC cell growth.ConclusionsHCA is a very good marker for tumors, especially for HCC. It could play important roles in HCC development and progression and can be a promising molecular target for the development of new diagnostic and therapeutic strategies for HCC.

Immunogenicity and efficacy of a DNA vaccine encoding a human anti-idiotype single chain antibody against nasopharyngeal carcinoma.

G22, an anti-idiotype single chain antibody screened from human nasopharyngeal carcinoma phage anti-idiotype antibody library, has been already identified by He et al. G22 DNA vaccine was produced by cloning G22 gene and inserting the cloned gene into pcDNA3.1. To investigate the immunogenicity of pcDNA3.1-G22, C57BL/6 mice were immunized with the vaccine, pcDNA3.1 and PBS individually and the antibody response, T cell phenocyte at the 15th, 22th, 29th, 36th day after the last immunity were detected. In the tumor protection experiment, the immunized mice were then challenged with CMT-93-G22 cells or CMT-93-mock cells. The tumor size and the survival time of the animals were compared between these groups. The results showed that DNA vaccine pcDNA3.1-G22 could raise G22-specific humoral and cellular immune responses. Furthermore, pcDNA3.1-G22 could prolong the survival time and lessen the tumor size of the CMT-93-G22-bearing mice but had no protection effect on the mice attacked by CMT-93-mock cells. These results were expected to lay foundation for further studies on the clinical application of pcDNA3.1-G22 DNA vaccine.

Identification of Rho GTPase Activating Protein 6 Isoform 1 Variant as a New Molecular Marker in Human Colorectal Tumors

The early diagnosis of colorectal cancer (CRC) is important because it is one of the most readily curable of all cancers, if detected early. However, the sensitivity of current markers is low. Immunostaining intensity for the monoclonal antibody Hb3 in CRC cell lines and tissues was stronger than in controls. Interestingly, this was associated with a low level of tumor differentiation. We used Hb3-coupled affinity chromatography to search for a corresponding Hb3 antigen as a candidate biomarker for early detection, and identified a Rho GTPase activating protein 6 (RhoGAP6) isoform 1 variant as an Hb3 antigen by mass spectrometry. Using reverse transcription polymerase chain reaction and western blot analysis, we confirmed that the expression levels of this variant were elevated in aberrant cells and tissues. Thus, the RhoGAP6 isoform 1 variant might serve as a biomarker for the development and progression of CRC.

The ability of human bispecific anti-idiotype antibody to elicit humoral and cellular immune responses in mice

Our goal is to compare the immunogenicity and the extent of immunologic reactivity between bispecific and mono anti-idiotype vaccines. We previously obtained two human anti-Id antibody fragments fuse5-G22, fuse5-I50 by phage display technology which were mimics of the antigens from nasopharyngeal carcinoma cell line (HNE2). In this study, we developed and characterized a bispecific anti-Id antibody vaccine G22-I50 and its parent monovalent antibody vaccines G22 and I50. The efficacy of G22-I50, G22, and I50 as tumor vaccines was evaluated in Balb/c mice with three injections of these vaccines adjuvanted with Freunds adjuvant. Mice immunized with G22-I50 exhibited comparable levels of antibody titers and stronger binding inhibition capabilities. Spleen cells from G22-I50-immunized mice gave a significant proliferative response and higher expression level of IFN-gamma and IL-2.These results suggested that bispecific anti-Id antibody vaccine was able to induce more powerful humoral and cell-mediated immune responses, which might make it to be a potential vaccine candidate for the therapy of nasopharyngeal carcinoma.

Induction of protective and therapeutic anti-cancer immunity by using bispecific anti-idiotype antibody G22-I50 for nasopharyngeal carcinoma.

Increasing evidence has suggested that bispecific and multivalent antibodies which have more antigen binding sites will improve their immunogenicity. The bispecific anti-idiotype antibody vaccine G22-I50 was obtained through genetic engineering to enhance the immunogenicity of anti-idiotype antibody vaccines G22 and I50. G22-I50 vaccination could induce anti-tumor immunity in the Balb/c mouse model. The protective and therapeutic efficacy of G22-I50 was also evaluated using the hu-PBL-SCID mouse model injected three times with G22-I50, G22, or I50 mixed with Freunds adjuvant. Results demonstrated that the protective anti-tumor effect of G22-I50 could be relevant with the production of Ab3 antibody and activation of CD8(+) cytotoxic T-lymphocytes. In preventive and therapeutic experiments, G22-I50 could reduce tumor size and prolong the survival time of HNE2-bearing mice (p<0.05). Human CD8(+) T lymphocytes infiltrated the tumor sites, and high levels of human IFN-γ, TNF-α, and caspase-3 were also detected in the tumors from G22-I50-vaccinated and -treated mice. Therefore, the bispecific anti-idiotype antibody vaccine G22-I50 can induce strong humoral and cell-mediated immune responses. This vaccine can be potentially applied to prevent and treat nasopharyngeal carcinoma.

Preparation of human scFv antibody against nasopharyngeal carcinoma and identification of its specificity.

Abstract Conclusion: The selected scFv antibody could specifically recognize and target nasopharyngeal carcinoma (NPC), and could be applied to clinical diagnosis and therapy. Objective: The aim was to construct and screen fully human anti-NPC single chain Fv fusion phage libraries, and to identify the specificity of the scFv antibody. Methods: Peripheral blood mononuclear cells of patients with NPC were immunized in vitro by NPC cells and transformed by Epstein-Barr virus. The total RNAwas used to construct the scFv libraries. By means of ELISA and immunochemistry, the positively bound scFv was selected and identified. The positive scFv was fused to EGFP, and was then expressed in E. coli strain BL21 (DE3) and purified. Furthermore, we observed the binding bioactivity. Results: The fusion protein has the biological activity of binding the NPC cells and emitting green fluorescence. In targeting experiments in vivo, the results showed that the fusion protein can successfully target the NPC.

Modulatory effects of tumor-derived heat shock protein in DNA vaccination against nasopharyngeal carcinoma

Use of anti-idiotype antibody vaccines is a promising strategy against tumor, however, their immunogenicity still need to be improved. Heat shock proteins (HSPs) have been shown to act as adjuvants when coadministered with peptides or given as fusion proteins and enhance the vaccination efficiency. To evaluate the enhancement of the potency of anti-idiotype antibody immunogenicity by heat shock protein gp96, C57BL/6 mice were immunized with three intramuscular inoculations of the G22 DNA and/or gp96 DNA vaccine. Control was inoculated with empty plasmid pcDNA3.1. The levels of G22-specific antibody and lymphocyte phenotype were measured by ELISA, fluorescence activated cell sorter (FACS) analysis, respectively. In the tumor protection experiment, the immunized mice were then challenged with CMT-93-G22 cells. The tumor size and the survival time of the animals were compared among these groups. The results showed that the efficacy of G22 DNA vaccine could be enhanced by coadministrating with gp96 DNA which might be relevant with activating CD8(+)T cells. Furthermore, co-injection of G22 DNA with gp96 DNA could prolong the survival time and lessen the tumor size of the CMT-93-G22-bearing mice. Our study demonstrates for the first time that G22+gp96 DNA vaccine can induce comparable G22-specific CD8(+)T cell response and is a promising candidate DNA vaccine for nasopharyngeal carcinoma.