High dose concentration administration of ascorbic acid inhibits tumor growth in BALB/C mice implanted with sarcoma 180 cancer cells via the restriction of angiogenesis

To test the carcinostatic effects of ascorbic acid, we challenged the mice of seven experimental groups with 1.7 × 10-4 mol high dose concentration ascorbic acid after intraperitoneal administrating them with sarcoma S-180 cells. The survival rate was increased by 20% in the group that received high dose concentration ascorbic acid, compared to the control. The highest survival rate was observed in the group in which 1.7 × 10-4 mol ascorbic acid had been continuously injected before and after the induction of cancer cells, rather than just after the induction of cancer cells. The expression of three angiogenesis-related genes was inhibited by 0.3 times in bFGF, 7 times in VEGF and 4 times in MMP2 of the groups with higher survival rates. Biopsy Results, gene expression studies, and wound healing analysis in vivo and in vitro suggested that the carcinostatic effect induced by high dose concentration ascorbic acid occurred through inhibition of angiogenesis

A Nucleic-Acid Hydrolyzing Single Chain Antibody Confers Resistance to DNA Virus Infection in HeLa Cells and C57BL/6 Mice

<div><p>Viral protein neutralizing antibodies have been developed but they are limited only to the targeted virus and are often susceptible to antigenic drift. Here, we present an alternative strategy for creating virus-resistant cells and animals by ectopic expression of a nucleic acid hydrolyzing catalytic 3D8 single chain variable fragment (scFv), which has both DNase and RNase activities. HeLa cells (SCH7072) expressing 3D8 scFv acquired significant resistance to DNA viruses. Virus challenging with Herpes simplex virus (HSV) in 3D8 scFv transgenic cells and fluorescence resonance energy transfer (FRET) assay based on direct DNA cleavage analysis revealed that the induced resistance in HeLa cells was acquired by the nucleic acid hydrolyzing catalytic activity of 3D8 scFv. In addition, pseudorabies virus (PRV) infection in WT C57BL/6 mice was lethal, whereas transgenic mice (STG90) that expressed high levels of 3D8 scFv mRNA in liver, muscle, and brain showed a 56% survival rate 5 days after PRV intramuscular infection. The antiviral effects against DNA viruses conferred by 3D8 scFv expression in HeLa cells as well as an <i>in vivo</i> mouse system can be attributed to the nuclease activity that inhibits viral genome DNA replication in the nucleus and/or viral mRNA translation in the cytoplasm. Our results demonstrate that the nucleic-acid hydrolyzing activity of 3D8 scFv confers viral resistance to DNA viruses <i>in vitro</i> in HeLa cells and in an <i>in vivo</i> mouse system.</p></div

Soluble Cytoplasmic Expression and Purification of Immunotoxin HER2(scFv)-PE24B as a Maltose Binding Protein Fusion

Human epidermal growth factor receptor 2 (HER-2) is overexpressed in many malignant tumors. The anti-HER2 antibody trastuzumab has been approved for treating HER2-positive early and metastatic breast cancers. Pseudomonas exotoxin A (PE), a bacterial toxin of Pseudomonas aeruginosa, consists of an A-domain with enzymatic activity and a B-domain with cell binding activity. Recombinant immunotoxins comprising the HER2(scFv) single-chain Fv from trastuzumab and the PE24B catalytic fragment of PE display promising cytotoxic effects, but immunotoxins are typically insoluble when expressed in the cytoplasm of Escherichia coli, and thus they require solubilization and refolding. Herein, a recombinant immunotoxin gene was fused with maltose binding protein (MBP) and overexpressed in a soluble form in E. coli. Removal of the MBP yielded stable HER2(scFv)-PE24B at 91% purity; 0.25 mg of pure HER2(scFv)-PE24B was obtained from a 500 mL flask culture. Purified HER2(scFv)-PE24B was tested against four breast cancer cell lines differing in their surface HER2 level. The immunotoxin showed stronger cytotoxicity than HER2(scFv) or PE24B alone. The IC50 values for HER2(scFv)-PE24B were 28.1 ± 2.5 pM (n = 9) and 19 ± 1.4 pM (n = 9) for high HER2-positive cell lines SKBR3 and BT-474, respectively, but its cytotoxicity was lower against MDA-MB-231 and MCF7. Thus, fusion with MBP can facilitate the soluble expression and purification of scFv immunotoxins

STG90 exhibits antiviral effects against PRV.

<p><b>A</b>. The expression levels of PRV gpD RNA in the muscle and brain of WT-Mock, WT-PRV, STG69-PRV, STG90-PRV, and STG135-PRV mice were investigated. Only live STG90-PRV mice did not show PRV gpD expression. <b>B</b>. Immunohistochemistry was performed to detect PRV in PRV target organs; brain (upper panel: ×100) and femoral muscle (bottom panel: ×400). The PRV gpD protein was stained with a monoclonal anti-PRV antibody and visualized with DAB. The Purkinje layer cells in the WT-PRV group stained brown for the gpD protein, whereas no staining was observed in the other groups.</p

Blood chemistry analysis (mean ± standard error).

<p>Standard blood biochemistry parameters were analyzed in 7-week-old WT C57BL/6 and STG90 mice (n = 9 mice per genotype and gender). ALB, albumin; ALT, alanine transaminase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; Glu, glucose; TG, triglycerides; CK, creatine phosphokinase; BUN, blood urea nitrogen.</p

3D8 scFv inhibits HSV-1 encoded gene expression by DNase and RNase activity.

<p><b>A</b>. Schematic diagram for identification of viral gene expression pattern by HSV-1 challenging. <b>B</b>. Wild-type HeLa, SCH07072, and muSCH cells were infected with HSV::GFV at an MOI of 0.5 and then incubated for 2, 6.5, and 25 hr in the presence or absence of PAA 1 hr after virus challenge. ICP4 was used for the immediate early stage, UL9 for the early stage, and UL19 for the late stage of viral infection. Data bars show mean ± standard error. ** and *** indicate significant differences from HSV-1 viral DNA at <i>p<0.01</i> and <i>p<0.001</i>, respectively (one-way analysis of variance and Tukey's post hoc <i>t</i>-test). <b>C</b>. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to measure the expression of immediate early genes (ICP0 and ICP4), early genes (UL9 and UL29), and late genes (UL19 and UL38). The relative concentrations of HSV mRNAs were calculated after normalization to the GAPDH gene using the delta delta C_T method. Data bars represent mean ± standard error. *, **, *** indicate significant differences from HeLa cells at <i>p<0.05, p<0.01</i>, and <i>p<0.001</i>, respectively (one-way analysis of variance and Tukey's post hoc <i>t</i>-test).</p

3D8 scFv cannot hydrolyze methylated DNA or protein-bound DNA.

<p><b>A</b>. HeLa methylated genomic DNA and non-methylated DNA (NEB) were treated with 3D8 scFv and DNase I at four concentrations: 10×10⁻⁴ U/µl, 8.3×10⁻⁴ U/µl, 1.4×10⁻⁴ U/µl, and 0 U/µl. The DNA samples were harvested and analyzed by electrophoresis at 0, 1, 2, and 3 hr after treatment. At a concentration of 8.3×10⁻⁴ U/µl, 3D8 scFv digested more non-methylated DNA than DNase I 1 hr after treatment. <b>B</b>. Chromatin was prepared using the EZ-Zyme Enzymatic Chromatin Prep kit. Prepared chromatin DNA and naked DNA were treated with 3D8 scFv and DNase I (10×10⁻⁴ U, 8.3×10⁻⁴ U, and 0 U/µl). The DNA samples were harvested and analyzed by electrophoresis at 0, 1, 2, and 3 hr after treatment. Neither 3D8 scFv nor DNase I hydrolyzed histone-bound DNA at concentrations of 10×10⁻⁴ U/µl and 8.3×10⁻⁴ U/µl. When naked DNA was treated with 3D8 scFv at concentrations of 8.3×10⁻⁴ U/µl, 3D8 scFv could digest naked DNA. But, DNase I could not digest naked DNA at 8.3×10⁻⁴ U/µl. <b>C</b>. The relative quantitative values of each DNA of methylated, non-methylated, histone-bound, and naked HeLa genomic DNA were measured for 3 hr after each DNA was treated with 3D8 scFv (8.3×10⁻⁴ U/µl). The bar graphs indicate the average values of each sample calculated from three individual experiments.</p

New antiviral mechanism by 3D8 scFv protein.

<p><b>A</b>. Model of the HSV-I replication cycle. Virus infection begins with binding of the virus to the cell surface. The viral envelope fuses with the cell membrane and delivers the viral capsid into the cytoplasm. Viral DNA synthesis begins shortly after the appearance of the beta proteins and the temporal program of viral gene expression ends with the appearance of the gamma or late proteins, which constitute the structural proteins of the virus. Finally, the virus undergoes a lytic cycle. <b>B</b>. Expression model of 3D8 scFv proteins. 3D8 scFv proteins were localized in cytosol using a vector system and targeted in the nucleus by nuclear localization signal. Therefore, 3D8 scFv proteins in SCH07072 were present in both the cytosol and nucleus. <b>C</b>. 3D8 scFv has a unique dual and stereoscopic protection mechanism that includes DNase activity in the nucleus and RNase activity in the cytoplasm. 3D8 scFv acts by inhibiting (1) viral DNA replication and RNA transcription in the nucleus via viral DNA degradation and (2) translation in the cytoplasm via viral RNA degradation. In other words, 3D8 scFv targets the viral DNA genome itself or its RNA transcripts spatially in two different subcellular spaces (nucleus and cytoplasm) and at two different times.</p

Construction of 3D8 scFv-expressing HeLa cells.

<p><b>A</b>. Schematic diagrams of the plasmid constructs. Construction of the (a) pcDNA3.1/V5-HisB-3D8 scFv vector and (b) pcDNA3.1/V5-HisB-mu3D8 scFv vector. Yellow line is the mutation site (His→Ala) <b>B</b>. 3D8 scFv expression levels were analyzed by quantitative real-time polymerase chain reaction (PCR) in transgenic cell lines. The relative concentrations of 3D8 scFv were calculated after normalization to the GAPDH gene using the delta delta C_T method. Data bars represent mean ± standard error. The expression levels of each cell line were compared to SCH07072. <b>C</b>. Identification of three 3D8 scFv cell lines (SCH07041, SCH07071, and SCH07072) by flow cytometry. Transgenic and wild-type HeLa cells were stained with 3D8 scFv Ab and TRITC-anti-rabbit Ig for flow cytometry. <b>D</b>. Localization of 3D8 scFv in transgenic and wild-type HeLa cells by immunocytochemistry. Nuclei were detected by DAPI staining (blue). 3D8 scFv expression was monitored by immunofluorescence using a polyclonal anti-3D8 scFv antibody that was visualized with TRITC (Rhodamine Red). 3D8 scFv proteins were localized in both the cytosol and nucleus of SCH07072 and muSCH cells.</p