Tumor Restrictive Suicide Gene Therapy for Glioma Controlled by the FOS Promoter.

Effective suicide gene delivery and expression are crucial to achieving successful effects in gene therapy. An ideal tumor-specific promoter expresses therapeutic genes in tumor cells with minimal normal tissue expression. We compared the activity of the FOS (FBJ murine osteosarcoma viral oncogene homolog) promoter with five alternative tumor-specific promoters in glioma cells and non-malignant astrocytes. The FOS promoter caused significantly higher transcriptional activity in glioma cell lines than all alternative promoters with the exception of CMV. The FOS promoter showed 13.9%, 32.4%, and 70.8% of the transcriptional activity of CMV in three glioma cell lines (U87, U251, and U373). Importantly, however, the FOS promoter showed only 1.6% of the transcriptional activity of CMV in normal astrocytes. We also tested the biologic activity of recombinant adenovirus containing the suicide gene herpes simplex virus thymidine kinase (HSV-tk) driven by the FOS promoter, including selective killing efficacy in vitro and tumor inhibition rate in vivo. Adenoviral-mediated delivery of the HSV-tk gene controlled by the FOS promoter conferred a cytotoxic effect on human glioma cells in vitro and in vivo. This study suggests that use of the FOS-tk adenovirus system is a promising strategy for glioma-specific gene therapy but still much left for improvement

Integrated multi-omics analyses and functional validation reveal TTK as a novel EMT activator for endometrial cancer

Background: Cancer-testis antigens (CTAs) are often expressed in tumor and testicular tissues but not in other normal tissues. To date, there has been no comprehensive study of the expression and clinical significance of CTA genes associated with endometrial cancer (EC) development. Additionally, the clinical relevance, biological role, and molecular mechanisms of the CTA gene TTK protein kinase (TTK) in EC are yet to be fully understood. Methods: Using bioinformatics methods, we comprehensively investigated the genomic, transcriptomic, and epigenetic changes associated with aberrant TTK overexpression in EC samples from the TCGA database. We further investigated the mechanisms of the lower survival associated with TTK dysregulation using single-cell data of EC samples from the GEO database. Cell functional assays were used to confirm the biological roles of TTK in EC cells. Results: We identified 80 CTA genes that were more abundant in EC than in normal tissues, and high expression of TTK was significantly linked with lower survival in EC patients. Furthermore, ROC analysis revealed that TTK could accurately distinguish stage I EC tissues from benign endometrial samples, suggesting that TTK has the potential to be a biomarker for early EC detection. We found TTK overexpression was more prevalent in EC patients with high-grade, advanced tumors, serous carcinoma, and TP53 alterations. Furthermore, in EC tissue, TTK expression showed a strong positive correlation with EMT-related genes. With single-cell transcriptome data, we identified a proliferative cell subpopulation with high expression of TTK and known epithelial–mesenchymal transition (EMT)-related genes and transcription factors. When proliferative cells were grouped according to TTK expression levels, the overexpressed genes in the TTKhigh group were shown to be functionally involved in the control of chemoresistance. Utilizing shRNA to repress TTK expression in EC cells resulted in substantial decreases in cell proliferation, invasion, EMT, and chemoresistance. Further research identified microRNA-21 (miR-21) as a key downstream regulator of TTK-induced EMT and chemoresistance. Finally, the TTK inhibitor AZ3146 was effective in reducing EC cell growth and invasion and enhancing the apoptosis of EC cells generated by paclitaxel. Conclusion: Our findings establish the clinical significance of TTK as a new biomarker for EC and an as-yet-unknown carcinogenic function. This present study proposes that the therapeutic targeting of TTK might provide a viable approach for the treatment of EC

Adipose-derived mesenchymal stem cell therapy for reverse bleomycin-induced experimental pulmonary fibrosis

Abstract Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive respiratory disease. Arguably, the complex interplay between immune cell subsets, coupled with an incomplete understanding of disease pathophysiology, has hindered the development of successful therapies. Despite efforts to understand its pathophysiology and develop effective treatments, IPF remains a fatal disease, necessitating the exploration of new treatment options. Mesenchymal stromal/stem cell (MSC) therapy has shown promise in experimental models of IPF, but further investigation is needed to understand its therapeutic effect. This study aimed to assess the therapeutic effect of adipose-derived mesenchymal stem cells in a bleomycin-induced pulmonary fibrosis model. First, MSC cells were obtained from mice and characterized using flow cytometry and cell differentiation culture methods. Then adult C57BL/6 mice were exposed to endotracheal instillation of bleomycin and concurrently treated with MSCs for reversal models on day 14. Experimental groups were evaluated on days 14, 21, or 28. Additionally, lung fibroblasts challenged with TGF-β1 were treated with MSCs supernatant or MSCs to explore the mechanisms underlying of pulmonary fibrosis reversal. Mesenchymal stem cells were successfully isolated from mouse adipose tissue and characterized based on their differentiation ability and cell phenotype. The presence of MSCs or their supernatant stimulated the proliferation and migration of lung fibrotic cells. MSCs supernatant reduced lung collagen deposition, improved the Ashcroft score and reduced the gene and protein expression of lung fibrosis-related substances. Bleomycin-challenged mice exhibited severe septal thickening and prominent fibrosis, which was effectively reversed by MSCs treatment. MSC supernatant could suppress the TGF-β1/Smad signaling pathway and supernatant promotes fibroblast autophagy. In summary, this study demonstrates that MSCs supernatant treatment is as effective as MSCs in revert the core features of bleomycin-induced pulmonary fibrosis. The current study has demonstrated that MSCs supernatant alleviates the BLM-induced pulmonary fibrosis in vivo. In vitro experiments further reveal that MSC supernatant could suppress the TGF-β1/Smad signaling pathway to inhibit the TGF-β1-induced fibroblast activation, and promotes fibroblast autophagy by Regulating p62 expression. These findings contribute to the growing body of evidence supporting the therapeutic application of MSCs in cell therapy medicine for IPF

High Tumor-Specific Transcriptional Activity of the FOS Promoter.

<p>Three glioma cell lines (U87, U251, U373), and normal astrocytes cells were transiently co-transfected with plasmid DNA indicated and pRL-TK, the internal control. At 48 h following transfection, dual luciferase ratio was measured as the ratio of luciferase normalized to the Renilla luciferase internal control. A) Characterization of the plasmids in transiently transfected glioma cells and astrocytes. Relative luciferase activity is plotted on a logarithmic scale on the y-axis. Error bars are SEM. Plasmids used for transfections are noted below the x-axis. B) The ratio of relative luciferase activity is plotted on a percentage scale on the y-axis. Error bars are SEM. The data are means of multiple independent experiments. ** denotes a <i>p</i> value of < 0.01 indicating a significant difference compared with the control cell group.</p

Ad-cfos-HSVtk inhibited tumor growth in a subcutaneous xenograft animal model.

<p>(A)Adenoviral gene therapy was initiated when tumors attained a volume of 500 mm³. Tumor volume was calculated as (length × width²)/2; * and ** denote <i>p</i> values of < 0.05 and < 0.01, respectively, indicating significant differences compared with the control vehicle group. (B) Representative tumor regions in routinely HE stain.</p

Schematic of the promoter-driven luciferase reporter plasmids.

<p>Schematic of the promoter-driven luciferase reporter plasmids.</p

Cell killing activities of Ad-CMV-HSVtk-IRES-GFP and Ad-FOS-HSVtk- IRES-GFP in glioma cell lines and normal cells.

<p>(A) Fluorescence microscopy was used to observe U87, U251, U373 and astrocytes cells 48 h after the cells were infected with Ad-CMV-HSVtk-IRES-GFP or Ad-FOS-HSVtk-IRES-GFP at an MOI of 100 pfu/cell, original magnification 200×. (B) Three glioma cell lines and normal astrocytes were infectedwith Ad-CMV-HSVtk or Ad-FOS-HSVtk plus ganciclovir. After 48 h, the percentage of cell death was evaluated with a Cell Counting Kit-8 (CCK-8) test with the negative control set at 100%. The data shown are the means of three independent experiments.</p

Distribution of luciferase activity in mouse major organs.

<p>Luciferase activity was measured <i>in vivo</i> in five major organs 48 h after intravenous administration of the FOS or CMV promoter-driven luciferase expression viruses. pGL4-CMV, red block; pGL4-CFOS, green triangle; pGL4-BASIC, black dot. The mean value of five samples is shown.</p