Therapeutic effects of human monoclonal PSMA antibody-mediated TRIM24 siRNA delivery in PSMA-positive castration-resistant prostate cancer.

Background and Aims: Prostate specific membrane antigen (PSMA) is specifically expressed on prostate epithelial cells and markedly overexpressed in almost all prostate cancers. TRIM24 is also up-regulated from localized prostate cancer to metastatic castration-resistant prostate cancer (CRPC). Because of the high relevance of TRIM24 for cancer development and the universal expression of PSMA in CPRC, we investigated the efficacy of human monoclonal PSMA antibody (PSMAb)-based platform for the targeted TRIM24 siRNA delivery and its therapeutic efficacy in CRPC in vivo and in vitro. Methods: The therapeutic complexes were constructed by conjugating PSMAb and sulfo-SMCC-protamine, and encapsulating TRIM24 siRNA. Flow cytometry, immunofluorescence, and fluorescence imaging were performed to detect the receptor-binding, internalization, and targeted delivery of PSMAb-sulfo-SMCC-protamine (PSP)-FAM-siRNA complex (PSPS) in vitro and in vivo. CCK-8, plate-colony formation, apoptosis, cell cycle, and Transwell assays were performed to evaluate the therapeutic potential of the PSP-TRIM24 siRNA complex in vitro, whereas the in vivo therapeutic efficacy was monitored by small animal imaging, radiography, and micro CT. Results: We confirmed that PSP could efficiently protect siRNA from enzymatic digestion, enable targeted delivery of siRNA, and internalize and release siRNA into PSMA-positive (PSMA+) prostate cancer cells in vitro and in vivo. Silencing TRIM24 expression by the PSP-TRIM24 siRNA complex could dramatically suppress proliferation, colony-formation, and invasion of PSMA+ CRPC cells in vitro, and inhibit tumor growth of PSMA+ CRPC xenografts and bone loss in PSMA+ CRPC bone metastasis model without obvious toxicity at therapeutic doses in vivo. Conclusion: PSMAb mediated TRIM24 siRNA delivery platform could significantly inhibit cell proliferation, colony-formation, and invasion in PSMA+ CRPC in vitro and suppressed tumor growth and bone loss in PSMA+ CRPC xenograft and bone metastasis model.

T-vector and in vivo recombination as tools to construct a large antibody library of breast cancer.

The emergence of phage antibody libraries is an important advance in the field of antibody engineering. It provides a useful methodology to produce human antibodies and has the potential to replace traditional hybridoma technology. In our research, we used T-vector and in vivo recombination to construct a large antibody library from breast cancer patients. The use of T-vector considerably increased the cloning efficiency, and the diversity of the library could be increased easily using in vivo recombination. Taken together, a combination of these two techniques might be valuable in constructing a large antibody library.

[Expression, purification and activity analysis of human single-chain antibody against hepatocellular cancer].

AIM: To express and characterize an active form of a single-chain antibody (scFv) from the gene of human phage antibodies which is specific for hepatocellular cancer. METHODS: The complementary DNAs encoding the variable regions of the light chain (V(L)) and heavy chain (V(H)) were connected by a (Gly(4)Ser)(3) linker using a splicing by overlap extension polymerase chain reaction. The resultant scFv gene was cloned to the pET28a(+) vector and expressed in E.coli as inclusion bodies. Then the inclusion bodies were solubilized, denatured and renatured. Finally, the affinity constant of scFv was determined by noncompetitive enzyme immunoassay. RESULTS: The target protein amounted 26% of the total protein in the condition of A(600) at 0.8 for 6 hours. After renatured, the purity of target protein was 95% and the affinity constant of the scFv was 3.6x10(7) mol/L. CONCLUSION: An active form of scFv which is specific for hepatocellular cancer can bind selectively with hepatocellular cancer cells, which provides a theoretical basis for immunological detection and clinical use of scFv.

[Construction of a large human scFv library against SARS virus].

AIM: To construct a large human scFv library against SARS virus by using in vivo recombination. METHODS: Total RNA was isolated from the lymphocytes of 6 patients recovered from SARS. mRNA was isolated and reverse transcribed into cDNA. The V(H) and V(L) fragments were amplified from the cDNA and then assembled into scFv genes. The scFv genes were amplified and ligated into phagemid pDAN5. The primary library was constructed by transforming the recombinant phagemid into E.coli TG1. The secondary library was generated by in vivo recombination in E.coli BS1365 following the infection of BS1365 by primary library phages. RESULTS: A primary library of 3x10(9) and a second library of 3x10(11) were constructed. CONCLUSION: A large human scFv library against SARS virus with good diversity was constructed, which may be used for screening antibodies to SARS virus antigens.

[Apoptosis of human hepatoma HepG2 cells induced by emodin in vitro].

BACKGROUND & OBJECTIVE: Emodin (3-methyl-1,6,8-trihydro- xyanthrax-quinone) is the main effective composition of some Chinese herbs. Previous studies showed that emodin could inhibit the proliferation of some kind of tumor cells, such as breast cancer and lung cancer, while the mechanism(s) by which emodin suppresses tumor growth remains unknown. The study was designed to investigate the inhibitory effects and mechanisms of emodin-induced cell death in human hepatoma cell HepG2. METHODS: MTT assay was used to evaluate the IC(50) of emodin on HepG2 cells. Through soft agar assay, the ability of cell proliferation when exposed to emodin at various dosages was detected. DNA fragmentation (ladder) and flow cytometry analysis were applied to investigate the effects and mechanisms of emodin on HepG2 cells. RESULTS: Emodin could inhibit the growth of HepG2 cells significantly with IC(50) of 36+/-2.6 microg/ml; and could inhibit the colony formation of the cells in soft agar. After treatment of emodin,extraction of cancer cells exhibited typical DNA fragmentation, and flow cytometry analysis showed apoptosis in a dosage- dependent manner. As the concentration of emodin raised from 10 microg/ml to 20 microg/ml,the ratio of apoptotic cells increased from 27.3% to 59.6%. Under the concentration of 40 microg/ml, there were almost no living cells detected. CONCLUSION: Emodin may inhibit the growth and proliferation of HepG2 cells through the way of apoptosis introduction.

[Construction and significance of directional expression cDNA library from myeloid leukemia cell line U937].

To construct the cDNA expression library from human U937 cell, total RNA and purified mRNA in myeloid leukemia cell line U937 were extracted. The first and second strand of cDNA were synthesized through reverse transcription. After blunting the cDNA termini, the cDNA fragments were connected with EcoR I adapters, and the end of EcoR I adapters was phosphorylated. Then the cDNAs were digested by Xho I, and the fragments smaller than 400 bp were removed by Sephacryl-S400 spin column, the fragments longer than 400 bp were ligated with lambdaZAP vector. The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E coli XL1-Blue-MRF' for titration. The recombinants were examined by color selection. In order to evaluate the size of cDNA inserts and the diversity of library, the pBK-CMV phagemid was excised from the ZAP expression vector by using ExAssist helper phage with XLOLR strain, and then the pBK-CMV phagemid was digested by Xho I and EcoR I. The results showed that the U937 cell line cDNA library consisting of 2.87 x 10(6) recombinant bacteriophages was constructed. The average size of exogenous insert in the recombinants was about 1.7 kb. It is concluded that the constructed cDNA library can be used to screen target clones.