This study is to investigate whether naked plasmid DNA can effectively transfect lung cancer related cells and express human kallistatin, an endogenous protein that inhibits angiogenesis and tumor growth, and to explore the biological activity of the low-level expressed kallistatin to lung cancer in vitro and in vivo. The plasmids were delivered with Lipofectamine 2000 to transfect various lung cancer related cells. Kal expression was determined by ELISA. The biological effects of Kal expression on proliferation, migration and apoptosis rate of the cells were examined. In subcutaneous NCI-H446 xenograft model, pKal was injected directly into tumors, the changes of CD34, Ki-67 and E-cadherin expression were detected with immunohistochemical assay, the tumor apoptosis was analyzed with TUNEL assay. Both the endothelial cell and lung cancer cells could express kallistatin after plasmid transfection. The proliferation and migration of human umbilical vein endothelial cells were inhibited, but the apoptosis rate was not affected. The proliferation rates of all the three tested lung cancer cells, such as NCI-H446, NCI-H460 and A549, were inhibited, and their apoptosis rates were enhanced, but different cells behaved differently. In subcutaneous NCI-H446 xenograft model, intratumor injection of pKal inhibited the growth of lung cancer by reducing angiogenesis and proliferation of tumor cells. In conclusion, this study demonstrated the efficacy of plasmid-mediated expression of kallistatin to lung cancer related cells, thus providing a basis for their clinical application in the treatment of lung cancer.
Animals ; Antigens, CD34 ; metabolism ; Apoptosis ; Cadherins ; metabolism ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Human Umbilical Vein Endothelial Cells ; cytology ; metabolism ; Humans ; Ki-67 Antigen ; metabolism ; Lung Neoplasms ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Neovascularization, Pathologic ; Plasmids ; Serpins ; genetics ; metabolism ; physiology ; Transfection ; Tumor Burden

Kallistatin (Kal) is a negative acute phase endogenous protein which can inhibit tumor angiogenesis, growth and metastasis effectively. To express and purify recombinant human kallistatin (rHKal), and characterize its biological activity, P. pastoris was transformed with pPIC9-Kal/GS115 (His4) to express rHKal. The fermentation was carried out in a 7.5 L bioreactor with high density cell culture. 1%-2% methanol was added to the medium to induce the expression of rHKal. The secretion was purified with phenyl sepharose, G-25 sepharose, heparin sepharose and Sephacryl S-100 chromatography. The biological activity of purified bulk rHKal on HUVEC was evaluated with MTT and tube formation assays. The final expression of rHKal in the supernatant reached 50 mg x L(-1), the purity of bulk rHKal after purification was above 98%. A dose-dependent inhibition of rHKal on HUVEC proliferation was observed, however, a U-shaped dose-response curve of rHKal on capillary formation of HUVEC was revealed. The described protocol provides an effective means for preparing rHKal that could be used for anti-angiogenesis therapy in the future.
Bioreactors ; Capillaries ; drug effects ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Electrophoresis, Polyacrylamide Gel ; Fermentation ; Human Umbilical Vein Endothelial Cells ; Humans ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Serpins ; biosynthesis ; genetics ; pharmacology

OBJECTIVETo explore how NF-κB family members regulate maspin expression in prostate cancer cells.

METHODSThe expression of NF-κB subunits and maspin was detected by Western blot analysis in prostate cancer DU145, PC-3, and LNCaP cell lines. RNA interference was performed to analyze whether RelB- or RelA-deletion affectes cell death as well as the expression of NF-κB subunits and maspin. The impact of RelB-silencing in DU145 cells was investigated by flow cytometry. The regulation of RelB on maspin expression in the prostate cancer PC-3 cells was also examined via stable transfection of RelB expression plasmid.

RESULTSRelA, p50, RelB, and p52 were constitutively expressed in androgen-independent prostate cancer DU145 and PC-3 cells, while RelB had the highest expression in DU145 cells. Low expression of maspin was detected in LNCaP and DU145 cells, but elevated expression in PC-3 cells. RelB-silencing in DU145 cells by siRNA interference upregulated the endogenous expression of maspin and induced cell apoptosis (13.3±4.2)%. Overexpression of RelB in PC-3 cells inhibited the endogenous expression of maspin. RelA-silecing had no significant influence on the endogenous expression of maspin.

CONCLUSIONSThe classical and alternative NF-κB activitions are sustained in androgen-independent prostate cancer cell lines. The expressions of RelB and maspin are inversely correlated in these cancer cells. The expression of RelB negatively regulates the endogenous expression of maspin, then interferes the cell survival. RelA is not involved in the regulation of maspin expression.

Apoptosis ; Cell Line, Tumor ; Gene Silencing ; Humans ; Male ; NF-kappa B ; genetics ; metabolism ; NF-kappa B p50 Subunit ; genetics ; metabolism ; NF-kappa B p52 Subunit ; genetics ; metabolism ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; RNA, Small Interfering ; genetics ; Serpins ; metabolism ; Transcription Factor RelA ; genetics ; metabolism ; Transcription Factor RelB ; genetics ; metabolism ; Transfection

Toxoplasma gondii penetrates all kinds of nucleated eukaryotic cells but modulates host cells differently for its intracellular survival. In a previous study, we found out that serine protease inhibitors B3 and B4 (SERPIN B3/B4 because of their very high homology) were significantly induced in THP-1-derived macrophages infected with T. gondii through activation of STAT6. In this study, to evaluate the effects of the induced SERPIN B3/B4 on the apoptosis of T. gondii-infected THP-1 cells, we designed and tested various small interfering (si-) RNAs of SERPIN B3 or B4 in staurosporine-induced apoptosis of THP-1 cells. Anti-apoptotic characteristics of THP-1 cells after infection with T. gondii disappeared when SERPIN B3/B4 were knock-downed with gene specific si-RNAs transfected into THP-1 cells as detected by the cleaved caspase 3, poly-ADP ribose polymerase and DNA fragmentation. This anti-apoptotic effect was confirmed in SERPIN B3/B4 overexpressed HeLa cells. We also investigated whether inhibition of STAT6 affects the function of SERPIN B3/B4, and vice versa. Inhibition of SERPIN B3/B4 did not influence STAT6 expression but SERPIN B3/B4 expression was inhibited by STAT6 si-RNA transfection, which confirmed that SERPIN B3/B4 was induced under the control of STAT6 activation. These results suggest that T. gondii induces SERPIN B3/B4 expression via STAT6 activation to inhibit the apoptosis of infected THP-1 cells for longer survival of the intracellular parasites themselves.
Animals ; Antigens, Neoplasm/genetics/*metabolism ; *Apoptosis ; Cell Line ; DNA Fragmentation ; Humans ; Macrophages/*cytology/metabolism ; Mice ; Mice, Inbred BALB C ; STAT6 Transcription Factor/genetics/*metabolism ; Serpins/genetics/*metabolism ; Toxoplasma/genetics/*physiology ; Toxoplasmosis/genetics/*metabolism/parasitology/*physiopathology

OBJECTIVETo explore the interaction between SerpinB5 and MAFbx in gastric cancer cell and to identify the interaction sites.

METHODSThe interaction between SerpinB5 and MAFbx was screened and validated by yeast two-hybrid screening and co-immunoprecipitation. The expression of MAFbx was analyzed after SerpinB5 expression being modified by RNA interference and pGBKT7-SerpinB5 transfection. The impact of SerpinB5 on the expression of MAFbx was studied in gastric cancer cell line SUN-16. A model of MAFbx was constructed by homology modeling. The related residues for interaction were analyzed by Autodock4.0.

RESULTSThe interaction between SerpinB5 and MAFbx was validated. The expression of MAFbx changed along with SerpinB5 expression. Amino acids including PRO261, ASN361, and LYS362 were key residue in the interaction of SerpinB5 and MAFbx.

CONCLUSIONSerpinB5 interacts with MAFbx in gastric cancer cell. Amino acids including PRO261, ASN361, and LYS362 are potential binding sites.

Cell Line, Tumor ; Humans ; Immunoprecipitation ; Muscle Proteins ; genetics ; metabolism ; RNA Interference ; SKP Cullin F-Box Protein Ligases ; genetics ; metabolism ; Serpins ; genetics ; metabolism ; Stomach Neoplasms ; genetics ; metabolism ; Two-Hybrid System Techniques

OBJECTIVETo study the prevalence of germinal center B-cell-like (GCB) and non-GCB-like types of diffuse large B-cell lymphoma (DLBCL) in Chinese patients.

METHODSImmunohistochemical study for CD10, bcl-6, MUM1, GCET1 and FOXP1 was performed on 124 cases of DLBCL from Shanghai, China. The Hans algorithm and Choi algorithm were applied to classify DLBCL into GCB and non-GCB-like types. Fluorescence in-situ hybridization (FISH) for t (14;18) and bcl-6 gene rearrangement was also carried out on 118 cases.

RESULTSOf the 124 DLBCL cases studied, 27 cases (22%) showed a GCB-like type and 97 cases (78%) showed a non-GCB-like type, when using Hans algorithm. On the other hand, 34 cases (27%) belonged to GCB-like type and 90 cases (73%) belonged to non-GCB-like type when applying Choi algorithm. The prevalence of GCB-like type was significantly lower than that of non-GCB-like type (P=0.0001). Only four cases (3%) were positive for t (14;18), and three of them were classified as GCB-like type. bcl-6 rearrangement was found in 46 cases (39%) and more frequently encountered in the GCB-like type. There is no relationship between bcl-6 gene rearrangement and bcl-6 protein expression.

CONCLUSIONSThe GCB-like type of DLBCL is significantly less common than non-GCB-like type in Chinese population. This phenomenon is possibly related to the low frequency of t (14;18).

Adult ; Aged ; Aged, 80 and over ; B-Lymphocytes ; pathology ; China ; epidemiology ; Chromosomes, Human, Pair 14 ; Chromosomes, Human, Pair 18 ; DNA-Binding Proteins ; genetics ; metabolism ; Female ; Forkhead Transcription Factors ; metabolism ; Germinal Center ; metabolism ; pathology ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Lymphoma, Large B-Cell, Diffuse ; classification ; epidemiology ; genetics ; pathology ; Male ; Middle Aged ; Neoplasm Proteins ; metabolism ; Neprilysin ; metabolism ; Prevalence ; Proto-Oncogene Proteins c-bcl-6 ; Repressor Proteins ; metabolism ; Serpins ; metabolism ; Translocation, Genetic ; Young Adult

In order to research the bioactivity of kallistatin (Kal), we obtained the recombinant Kal using Pichia pastoris expression system. Kal cDNA was amplified from pAAV-Kal and inserted into pPIC9 vector to generate a recombinant vector of pPIC9-Kal. Then, pPIC9-Kal was linearized and transformed into Pichia pastoris strain GS115 (His4) by electroporation. The positive transformants were selected by MD plate and confirmed by PCR. High level of Kal was obtained in BMMY medium (pH 7.0) after 96 hours induction of 29 degrees C and 2% methanol, with the highest yield of 14 mg/L in shake flask culture. Kal protein was purified from the supernatant with Phenyl Superose and Heparin Sepharose FF chromatograph. The recombinant Kal had a molecular weight of 58 kDa with 98% purity, showing by SDS-PAGE. Moreover, it had a high peroxidase activity (163+/-4) U/(mgmin), which could protect LX-2 cell against oxidation of H2O2. Recombinant Kal also effectively inhibited HUVEC proliferation. In this report, we successfully established the expression system using Pichia pastoris and obtained the bioactive recombinant human Kal. It lays a foundation for its further anti-cancer therapy.
Antioxidants ; pharmacology ; DNA, Complementary ; Electroporation ; Genetic Vectors ; genetics ; Humans ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Serpins ; biosynthesis ; genetics

BACKGROUNDMany studies have suggested that the imbalance of angiogenic factor and anti-angiogenic factor expression contributes significantly to the development of choroidal neovascularization (CNV), and ultrasound microbubble combination system can increase the gene transfection efficiency successfully. This study was designed to investigate whether ultrasound-mediated microbubble destruction could effectively deliver therapeutic plasmid into the retina of rat, and whether gene transfer of pigment epithelium-derived factor (PEDF) could inhibit CNV.

METHODSHuman retinal pigment epithelial cells were isolated and treated either with ultrasound or plasmid alone, or with a combination of plasmid, ultrasound and microbubbles to approach feasibility of microbubble-enhanced ultrasound enhance PEDF gene expression; For in vivo animal studies, CNV was induced by argon lasgon laser in rats. These rats were randomly assigned to five groups and were treated by infusing microbubbles attached with the naked plasmid DNA of PEDF into the vitreous of rats followed by immediate ultrasound exposure (intravitreal injection); infusing liposomes with the naked plasmid DNA of PEDF into the vitreous (lipofectamine + PEDF); infusing microbubbles attached with PEDF into the orbit of rats with ultrasound irradiation immediately (retrobular injection); infusing microbubbles attached with PEDF into the femoral vein of rats with exposed to ultrasound immediately (vein injection). The CNV rats without any treatment served as control. Rats were sacrificed and eyes were enucleated at 7, 14, and 28 days after treatment. Gene and protein expression of PEDF was detected by quantitative real-time RT-PCR, Western blotting and immunofluorescence staining, respectively. The effect of PEDF gene transfer on CNV was examined by fluorescein fundus angiography.

RESULTSIn vitro cell experiments showed that microbubbles with ultrasound irradiation could significantly enhance PEDF delivery as compared with microbubbles or ultrasound alone. In the rat CNV model, transfection efficiency mediated by ultrasound/microbubbles was significantly higher than that by lipofectamine-mediated gene transfer at 28 days after treatment. The study also showed that with the administration of ultrasound-mediated microbubbles destruction, the CNV of rats was inhibited effectively.

CONCLUSIONSUltrasound-microbubble technique could increase PEDF gene transfer into rats' retina and chorioid, in association with a significant inhibition of the development of CNV, suggesting that this noninvasive gene transfer method may provide a useful tool for clinical gene therapy.

Animals ; Cells, Cultured ; Choroidal Neovascularization ; prevention & control ; Eye Proteins ; genetics ; Female ; Genetic Therapy ; Humans ; Microbubbles ; Nerve Growth Factors ; genetics ; RNA, Messenger ; analysis ; Rats ; Rats, Long-Evans ; Retina ; metabolism ; Serpins ; genetics ; Transfection ; Ultrasonics

OBJECTIVETo evaluate the antiangiogenic property of pigment epithelium-derived factor(PEDF) in heptocarcinoma cell lines and explore its possible application in the gene therapy of human hepatocellular carcinoma (HCC).

METHODSThe gene encoding human PEDF was subcloned into lentiviral vector to generate the recombinant plasmid pLenti-PEDF. The plasmid pLenti-PEDF and two other packaging plasmids were cotransfected to 293T cells by calcium phosphate. Then HepG2 was infected with recombinant lentivirus and the expression efficiency of PEDF was analyzed by western blot. Proliferation and migration assay of human umbilical vein endothelial cells (HUVEC) was used to evaluate the biological activity of PEDF in vitro. Murine subcutaneous tumor model was established to investigate the therapeutic effects of Lenti-PEDF on HCC, and the expression of PEDF mRNA in tumor tissues was analyzed by RT-PCR.

RESULTSRestriction enzyme digestion and DNA sequencing demonstrated that the recombinant plasmid pLenti-PEDF was constructed successfully. HepG2 secreted PEDF in the media effectively after infected with the recombinant lentivirus and this protein exhibited strong inhibitory effects on proliferation and migration of human umbilical vein endothelial cells (P less than 0.01). Intratumoral injection of Lenti-PEDF caused significant inhibition of tumor growth (P less than 0.01), and high level expression of PEDF mRNA was detected in tumor tissues by RT-PCR.

CONCLUSIONSOur data suggest that PEDF may exert an inhibitory effect on tumor angiogenesis and PEDF gene therapy may provide a new approach for the treatment of HCC.

Animals ; Cell Proliferation ; Endothelial Cells ; metabolism ; Eye Proteins ; genetics ; metabolism ; Genetic Therapy ; Genetic Vectors ; Hep G2 Cells ; Humans ; Lentivirus ; genetics ; Liver Neoplasms ; blood supply ; genetics ; therapy ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Neovascularization, Pathologic ; therapy ; Nerve Growth Factors ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Serpins ; genetics ; metabolism ; Transfection ; Umbilical Veins ; cytology

OBJECTIVETo investigate the effect of fetal bovine serum (FBS) on expression of pigment epithelium-derived factor (PEDF) in normal epidermal keratinocytes and dermal fibroblasts.

METHODSKeratinocytes and fibroblasts were incubated with 10% FBS. PEDF protein level in the cells was determined by immunofluorescence and Western blot.

RESULTSPEDF was localized mostly in the cytoplasm,while some in the nuclei. The distribution of PEDF in cytoplasm was in a granular pattern. 10% FBS increased the expression of PEDF both in keratinocytes and fibroblasts,but histamine and Phorbol 12-myristate 13-acetate (PMA) did not interfere the distribution of PEDF in cells.

CONCLUSION10% FBS can upregulate expression of PEDF in epidermal keratinocytes and dermal fibroblasts.

Animals ; Cattle ; Cells, Cultured ; Epidermis ; cytology ; metabolism ; Eye Proteins ; genetics ; metabolism ; Fetus ; Fibroblasts ; cytology ; metabolism ; Keratinocytes ; cytology ; metabolism ; Nerve Growth Factors ; genetics ; metabolism ; Serpins ; genetics ; metabolism ; Serum ; physiology ; Skin ; cytology ; metabolism ; Up-Regulation