Anti-tumor effects of a recombinant anti-prostate specific membrane antigen immunotoxin against prostate cancer cells.

BACKGROUND: To evaluate anti-prostate cancer effects of a chimeric tumor-targeted killer protein. METHODS: We established a novel fusion gene, immunocasp-3, composed of NH2-terminal leader sequence fused with an anti-prostate-specific membrane antigen (PSMA) antibody (J591), the furin cleavage sequences of diphtheria toxin (Fdt), and the reverse coding sequences of the large and small subunits of caspase-3 (revcaspase-3). The expressing level of the immunocasp-3 gene was evaluated by using the reverse transcription-PCR (RT-PCR) and western blot analysis. Cell viability assay and cytotoxicity assay were used to evaluate its anti-tumor effects in vitro. Apoptosis was confirmed by electron microscopy and Annexin V-FITC staining. The antitumor effects of immunocasp-3 were assessed in nude mice xenograft models containing PSMA-overexpressing LNCaP cells. RESULTS: This study shows that the immunocasp-3 proteins selectively recognized and induced apoptotic death in PSMA-overexpressing LNCaP cells in vitro, where apoptotic cells were present in 15.3% of the cells transfected with the immunocasp-3 expression vector at 48 h after the transfection, in contrast to 5.5% in the control cells. Moreover, LNCaP cells were significantly killed under the condition of the co-culture of the immunocasp-3-secreting Jurkat cells and more than 50% of the LNCaP cells died when the two cell lines were co-cultured within 5 days. In addition, The expression of immunocasp-3 also significantly suppressed tumor growth and greatly prolonged the animal survival rate in vivo. CONCLUSION: A novel fusion gene, immunocasp-3, may represent a viable approach to treating PSMA-positive prostate cancer.

[Construction of a recombinant lentiviral vector of p38 MAPK and establishment of a human prostatic carcinoma cell line stably expressing p38 MAPK].

OBJECTIVE: To construct a recombinant lentiviral vector for p38 MAPK and establish a human prostatic carcinoma cell line that stably expresses p38 MAPK. METHODS: EGFP/p38 fusion gene was subcloned into the lentiviral vector pTYF- EF1α-IRES-EGFP. The recombinant lentiviral vector pTYF-EF1α-EGFP/p38 was indentified by restriction enzyme digestion, and packaged in HEK 293T cells using lipofectamintm2000 with the packaging plasmid psPAX2 and envelope plasmid pMD2.G. The viral titer was tested according to the expression level of GFP. The resulting recombinant lentiviral vector was transduced into human prostatic carcinoma DU145 cells, and stably transduced cells were selected by limiting dilution analysis. The intracellular expression level of total p38 was detected by Western blotting and the cell growth curve was drawn. RESULTS: DNA restriction enzyme digestion demonstrated that the recombinant lentiviral vector of the fusion gene EGFP/p38 (pTYF-EF1α-EGFP/p38) was constructed successfully. The recombinant lentiviral vector was packaged in 293T with a viral titer of 4.7×10(6) TU/ml. A stable cell line, EGFP/p38-DU145, was established, which stably expressed exogenous EGFP/p38 MAPK fusion protein as detected by Western blotting and showed a lowered growth rate compared to the control cells. CONCLUSION: We have successfully constructed a recombinant lentiviral vector of the fusion gene EGFP/p38 and established a stable cell line EGFP/p38-DU145. Overexpression of p38 has a significant inhibitory effect on the proliferation of DU145 cells in vitro.

Milk and dairy consumption and risk of bladder cancer: a meta-analysis.

OBJECTIVE: To explore potential relations between the intake of milk or dairy products and the risk of bladder cancer. METHODS: Eligible studies published up to May 2011 were retrieved via both computer searches and manual review of references. Random-effects models were used to calculate summary relative risk estimates (SRRE) based on high-contrast to low-intake values. Sensitivity and influence analyses were conducted, and heterogeneity among study results was explored through stratified analyses by study design, gender, geographic region, year of publication, or whether or not adjustment for several confounders (ie, age, gender, body mass index, smoking, and total energy intake). RESULTS: We extracted data from 14 studies on milk (involving 4879 cases) and 6 studies on dairy products (3087 cases). The total study population was up to 324,241 individuals. Overall, there was no significant association between milk intake and bladder cancer (SRRE 0.89, 95% CI 0.77-1.02). However, an inverse association was found in the United States (SRRE 0.88, 95% CI .79-.99). In addition, no significant association was observed between consumption of dairy products and risk of bladder cancer (SRRE 0.95, 95% CI .71-1.27), though an inverse association was detected in the Japanese population (SRRE 0.56, 95% CI .40-.80). CONCLUSION: There appears to be enough evidence to support the null hypothesis. The overall result was not statistically significant. The findings of this meta-analysis are not supportive of an independent relationship between the intake of milk or dairy products and the risk of bladder cancer. However, these findings are based on limited research. Further efforts should be made to confirm these findings.

[A comparative analysis of the methods for titering adenoviruses].

OBJECTIVE: To compare different methods commonly used for titering adenovirus and analyze the advantages and limitations of each method. METHODS: Four recombined adenoviruses (Ad-G-AT2R-EGFP, Ad-CMV-EGFP, Ad-mif-shRNA-EGFP and Ad-CBA-GFP) were amplified and purified, and each was titered by optical absorbance, real-time PCR, green fluorescent protein (GFP)-labeled method, immunoassay, and cytopathic effect (CPE). The results were then comparatively analyzed. RESULTS: No significant difference was found in the titer amounts derived from GFP-labeled method, immunoassay, and cytopathic effect method (P>0.1). A positive correlation was noted in the titer amounts determined by real-time PCR and immunoassay (r=0.965), even though the value (vg/ml) obtained by real-time PCR was 10 times higher than that by immunoassay (ifu/ml). CONCLUSION: GFP-labeled method and immunoassay allow rapid determination of the adenoviral titer. Real-time PCR can not directly determine the real infectious titer of the adenovirus, but the result is well correlated to that of immunoassay and reflects, though indirectly, the actual infectious titer of adenovirus. Considering the procedural convenience and shorter time consumption, real-time PCR is still a practical method for adenoviral titration.