EBAG9 inducing hyporesponsiveness of T cells promotes tumor growth and metastasis in 4T1 murine mammary carcinoma.

The estrogen receptor-binding fragment-associated gene 9 (EBAG9) has been identified as an estrogen-responsive gene and was recently identified as a tumor-promoting and prognostic factor for renal cell carcinoma. We investigated whether EBAG9 expression was correlated with primary tumor growth and distant tumor metastasis in a murine breast carcinoma model. Knockdown expression of EBAG9 by small interfering RNA significantly suppressed tumor growth and metastasis in vivo in a highly malignant, spontaneously metastasizing 4T1 mouse mammary carcinoma model. 4T1 cells stably overexpressing EBAG9 developed larger and faster tumor growth and lung metastasis compared with parental 4T1 or 4T1 expressing vector alone. Strong specific cytotoxic T lymphocyte activity and enhanced gamma-interferon and interleukin-2 productions were induced in mice that received EBAG9 small interfering RNA therapy. Gene silencing of EBAG9 prolonged the survival of tumor-bearing mice and induced more intensive infiltration of CD8+ T cells in tumor mass. EBAG9 induced apoptosis of T cells, enhanced glycogen synthase kinase 3beta phosphorylation and inhibited gamma-interferon production of T cells when T lymphocytes were cocultured with 4T1 cells overexpressing EBAG9. Furthermore, overexpression of EBAG9 in 4T1 cells was accompanied with enhanced expression of chemokine (C-X-C motif) receptor 4, which might be involved in tumor metastasis. Taken together, our results suggested that EBAG9 promoted primary 4T1 mammary carcinoma growth and distant metastasis, and EBAG9 small interfering RNA exerted overt regression of tumor growth and metastasis. These findings might provide insights into the mechanism through which tumors evade immunosurveillance and provide a strategy for therapeutic intervention of cancer metastases.

A novel co-polymer based on hydroxypropyl alpha-cyclodextrin conjugated to low molecular weight polyethylenimine as an in vitro gene delivery vector.

A novel co-polymer based on 2-hydroxypropyl-alpha-cyclodextrin cross-linked by low molecular weight polyethylenimine was synthesized as a gene delivery vector. The copolymer could bind and condense DNA tightly. It showed lower cytotoxicity than PEI 25kDa in SK-BR-3 cells. Transfection efficiency was increased over 5.5-fold higher than PEI 25 kDa in SK-BR-3 cells in complete serum medium. It is a potential candidate vector for gene therapy.

FGF receptor-mediated gene delivery using ligands coupled to polyethylenimine.

To obtain new nonviral vectors with high gene delivery efficiency and special cell targeting ability, an attractive strategy is to link ligands to polyethylenimine (PEI). Fibroblast growth factor receptors (FGFRs) are highly expressed on a variety of human cancer cells and are potential targets for cancer gene therapy. In this study, the peptides NH2-Met-Gln-Leu-Pro-Leu-Ala-ThrGly-Gly-Gly-Cys-COOH (MC11) which have been proved to combine specially with the FGFR on cell membrane are coupled to PEI using N-Succinimidyl-3-(2-pyridyldithio) propionate (SPDP) as a linker with different molar ratios (1 : 0.3, 1 : 0.75, 1 : 1.5, and 1 : 3.0) and the new polymer PEI-MC11 is verified by a series of physicochemical methods including 1H-NMR and FTIR. The agarose gel electrophoresis assay, particle size test, zeta potential test, and electron microscope observation show that PEI-MC11 can efficiently condense plasmid DNA into nanoparticles with about 200 nm in diameter and with positive surface charge at the suitable N/P ratio. The MTT assay suggests the decreased toxicity of the polymers. The results of the gene delivery efficiency in vitro show that PEI-MC11/pDNA polyplexes have significantly greater transgene activity than PEI/pDNA in COS-7 and HepG2 cells which express FGFR positively, while no such effect is observed in PC3 cells which have negative FGFR. The enhanced gene delivery efficiency of PEI-MC11 can be blocked by the co-culture of free peptides MC11 before the gene delivery procedure. The synthesized nonviral vector based on PEI with the targeting peptides MC11 for binding FGFR has improved efficiency of gene delivery and targeting specificity in FGFR positive cells. It may have potential application in cancer gene therapy.

Two novel non-viral gene delivery vectors: low molecular weight polyethylenimine cross-linked by (2-hydroxypropyl)-beta-cyclodextrin or (2-hydroxypropyl)-gamma-cyclodextrin.

Two novel polymers of low molecular weight polyethylenimine cross-linked by (2-hydroxypropyl)-beta-cyclodextrin or (2-hydroxypropyl)-gamma-cyclodextrin showed lower cytotoxicity and higher transfection efficiency for the delivery of plasmid DNA compared with those of polyethylenimine (PEI, 25 kDa).

Receptor-mediated gene delivery using polyethylenimine (PEI) coupled with polypeptides targeting FGF receptors on cells surface.

OBJECTIVE: To construct a novel kind of nonviral gene delivery vector based on polyethylenimine (PEI) conjugated with polypeptides derived from ligand FGF with high transfection efficiency and according to tumor targeting ability. METHODS: The synthetic polypeptides CR16 for binding FGF receptors was conjugated to PEI and the characters of the polypeptides including DNA condensing and particle size were determined. Enhanced efficiency and the targeting specificity of the synthesized vector were investigated in vitro and in vivo. RESULTS: The polypeptides were successfully coupled to PEI. The new vectors PEI-CR16 could efficiently condense pDNA into particles with around 200 nm diameter. The PEI-CR16/pDNA polyplexes showed significantly greater transgene activity than PEI/pDNA in FGF receptors positive tumor cells in vitro and in vivo gene transfer, while no difference was observed in FGF receptors negative tumor cells. The enhanced transfection efficiency of PEI-CR16 could be blocked by excess free polypeptides. CONCLUSION: The synthesized vector could improve the efficiency of gene transfer and targeting specificity in FGF receptors positive cells. The vector had good prospect for use in cancer gene therapy.

[Construction of recombinant GST-RCAS1 fusion gene and its expression in E. Coli].

OBJECTIVE: To construct the recombinant plasmid of RCAS1, to express and purify its fusion protein GST-RCAS1, and to investigate its biological function. METHODS: RCAS1 encoding gene was amplified by RT-PCR from total RNA extract of MCF-7 cells and was ligated with expression plasmid vector pGEX-2T by T4 DNA ligase after digested by the restricted endonucleases BamH I and EcoR I. Then the ligated products were inserted into competence JM109 E. Coli and the positive recombinants were identified by restriction endonuclease digestion assay and DNA sequencing. The GST-RCAS1 fusion protein expression was induced by IPTG in BL21 E. Coli and was purified with GST column and identified by SDS-PAGE and Western blotting with anti-GST monoclonal antibody, anti-RCAS1 (N-18) and anti-RCAS1 (C-20) polyclonal antibody. The apoptosis of activated T cells induced by GST-RCAS1 fusion protein was detected by flow cytometry with Annexin V and propidium iodide (PI) staining. RESULT: A 642 bp product was cloned by RT-PCR and the recombinant plasmid was constructed successfully. The GST-RCAS1 fusion protein was recognized by GST monoclonal antibody and RCAS1(N-18 and C-20) polyclonal antibody. FACS analysis showed that GST-RCAS1 fusion protein induced apoptosis in activated T cells. CONCLUSION: The recombinant plasmid of RCAS1 has been successfully constructed and the GST-RCAS1 fusion protein expressed and purified. The apoptosis inducing effect of GST-RCAS1 fusion protein on activated T cells is demonstrated.

[Study on lysosomes degradation of ricin A chain].

OBJECTIVE: To study lysosomes involvement in the degradation of ricin A chain. METHODS: A lysosome-targeted singal KFERQ was added to the C terminus of rRTA by DNA recombinant technology. A pKK223.3 expression system in E. coli was used to produce recombinant ricine A chain (rRTA) and rRTA-KFERQ. Recombinant proteins were purified by affinity chromatography using Blue-Sepharose 6B. The cytotoxicity of recombinant proteins was measured by the MTT method. RESULTS: Recombinant RTA-KFERQ was 49.87%, 54.18% and 88.68% less cytotoxic than RTA itself on the three cell lines HEPG2, Hela and A549, respectively. CONCLUSION: Lysosomes can degrade, but not completely inactivate RTA in different cells, suggesting cells may have other degradation pathways for RTA.

[Gene transfer by novel non-viral vector polyethylenimine].

OBJECTIVE: To investigate the influencing factors of polyethylenimine (PEI) in gene transfer in vitro. METHODS: Cytotoxic effects of PEI on in vitro cultured NIH 3T3 cells were quantified by MTT assay. The interaction between PEI and DNA at different charge ratios was analyzed by agarose gel electrophoresis retardation assay. The expression of gene transfer was monitored in Cos-7 cells using pEGFP and pSV beta plasmids as the reporter gene systems. Influences of chloroquine, albumin, serum, salt ion strength, and Mg(2+) ion and other factors on PEI/DNA transfer efficiency were evaluated. RESULT: The survival rate of NIH3T3 cells at 6 mg/L of PEI was 64.2% and at 7 mg/L of PEI was 54.4%. Gel electrophoresis retardation assays showed that PEI completely retarded DNA migration at 3.0 PEI nitrogen per DNA phosphate. Chloroquine enhanced the transfection efficiency of PEI. Albumin and serum in the culture medium decreased the transfection efficiency. HBS(HEPES buffered solution) or 150 mmol/L NaCl as the dilution solution of PEI/DNA was superior over 278 mmol/L glucose solution in the transfection efficiency. Mg(2+) in the dilution solution decreased the transfer efficiency of PEI/DNA. CONCLUSION: PEI is efficient gene transfer agent of eukaryotes in vitro, and can be possibly used in vivo.

[Anticancer therapeutic effect of SEA-linked and membrane-bound HSP70-expressed intestine-carcinoma vaccine].

OBJECTIVE: To develop a novel dual-modified vaccine, the superantigen-linked intestine-carcinoma cells expressing membrane-bound heat shock protein 70 (HSP70), and further examine its anticancer therapeutic effect. METHODS: The pre-established intestine carcinoma CT26 line expressing membrane-bound heat shock protein 70 (HSP70) was amplified and incubated with superantigen fusion protein, staphylococcal enterotoxin A (SEA) fused with transmembrane sequence (SEA-TM), thereby the dual-modified vaccine was prepared after inactivation. The anticancer efficacy of the vaccine was examined. RESULTS: The laser confocal microscopy and flow cytometry showed that there co-existed much HSP70 and SEA on the vaccine membrane surface. Both of the single-modified vaccines, the SEA-linked vaccine and membrane-bound-HSP70-expressing one, displayed marked tumor suppression, a prolonged survival period, augmented lymphocyte proliferation and higher NK and CTL activity in the vaccinated mice when compared with its counterpart. Furthermore, the dually modified vaccine induced lymphocyte proliferation most intensively, generated the highest NK and CTL activity as well as the strongest tumor rejection in the vaccinated mice. The survival period of the mice was further prolonged. CONCLUSION: A new vaccine, SEA-linked and membrane-bound-HSP70-expressing intestine-carcinoma cells can induce more potent anticancer immunity and produce better therapeutic efficacy.

[Antitumor therapeutic effect induced by intestine-carcinoma cells vaccine expressing membrane-bound heat shock protein 70].

OBJECTIVE: To develop a new vaccine expressing membrane-bound heat shock protein 70 (mbHSP70) and further study its antitumor therapeutic effect. METHOD: The pre- established vector expressing mbHSP70 was transfected into CT26 cells of colorectal cancer. After the CT26 cells were incubated with 900 microg/ml G418, the sub-clones resistant to G418 were harvested and the HSP70 positive clones were selected by limiting dilution. The clones were amplified and inactivated, thereby the vaccine expressing mbHSP70 was prepared. Lymphocyte proliferation stimulated by the vaccines, NK and CTL activity was observed. The antitumor efficacy of vaccine was observed in BALB/c mice model with colorectal cancer. RESULTS: The laser confocal microscopy and flow cytometry showed that there existed much HSP70 on the vaccine membrane surface. The HSP70 gene-modified vaccine displayed augmented lymphocyte proliferation and higher NK and CTL activity in vitro,and marked tumor suppression and prolonged survival time of the vaccinated micein vivo, when compared with its counterpart. Furthermore, mbHSP70-expression vaccine elicited lymphocyte proliferation most intensively, generated the highest NK and CTL activity as well as the strongest antitumor effect, and prolonged survival time of the vaccinated mice. CONCLUSION: A new vaccine expressing mbHSP70 has more potent antitumor immunity and better therapeutic efficacy than HSP70 gene-modified vaccine did.