Li-Xiang Wu

Background: Glioblastoma is one of the most common fatal intracranial malignancies. Lysine-specific demethylase 1 (LSD1) reportedly has therapeutic effects on a variety of tumors. This study explored the therapeutic effect of LSD1 inhibition on glioblastoma cell lines and the possible underlying mechanisms. Methods: The MTT assay was utilized to screen for the sensitivity of U87, U251 and T98G cells to 4, 5-dimethoxycarrageenin-6-one. qRT-PCR and western blot were used to measure the proliferation, apoptosis, and pyroptosis signaling pathway expression to observe the effect of LSD1 inhibition on U251 and T98G cells. Flow cytometry, immunofluorescence, immunohistochemistry, wound scratch, clone formation, and TUNEL assay were used to analyze the effects of 4, 5-dimethoxycanthin-6-one on glioblastoma cells. The effect of 4, 5-dimethoxycanthin-6-one was examined in vivo in BALB/c nude mice injected with U251 cells. HE staining was used to detect the histopathology of the tumor. Results: LSD1 specifically catalyzes the demethylation of monomethylated and demethylated histone H3 lysine at position 4 (h3k4me1, h3k4me2, h3k4me3) and lysine at position 9 (h3k9me1). This regulated the transcriptional activity of proliferation, apoptosis, and pyroptosis signaling pathway genes. In vitro, the proliferation of glioblastoma cells was decreased in the 4, 5-dimethoxycanthin-6-one group. The expression of Caspase1 in glioblastoma cells treated with 4, 5-dimethoxycanthin-6-one increased, and the number of apoptotic cells increased. The tumor volume of mice injected with 4, 5-dimethoxycanthin-6-one decreased significantly. Conclusion: 4, 5-Dimethoxycanthin-6-one could act as a novel inhibitor of LSD1 to regulate glioblastoma, which could inhibit the proliferation of U251 and T98G cells and induce their apoptosis and pyroptosis. It is a potential drug for the treatment of glioblastoma.

Glioblastoma is the most common primary central nervous system malignancy and its unique invasiveness hinders effective treatment. Its high invasiveness may be controlled partly by microRNAs (miRNAs, miRs) and their target genes. In the present study, we found that increased miR-223 expression and reduced PAX6 expression coexisted in glioblastoma as detected by quantitative PCR or tissue microarrays. We confirmed that miR-223 directly targets PAX6 through binding to its 3'-UTR using dual luciferase reporter assay. In U251 and U373 glioblastoma cells, overexpression of miR-223 decreased PAX6 mRNA and protein expression; however, inhibition of miR-223 increased PAX6 mRNA and protein expression. Moreover, overexpression of miR-223 led to effects similar to those of PAX6 knockdown: increased cell viability, increased percentage of cells in the G1 phase and increased cell invasiveness parallel with increased MMP2, MMP9 and VEGFA expression. In addition, inhibition of miR-223 resulted in effects similar to those of PAX6 overexpression: decreased cell viability, decreased percentage of cells in the G1 phase and decreased cell invasiveness parallel with reduced MMP2, MMP9 and VEGFA expression. The data presented here suggest that miR-223 promotes the growth and invasion of U251 and U373 glioblastoma cells by targeting PAX6, which serves as a tumor suppressor in glioblastoma exerting the functions of inhibition of cell cycle transition, and the expression of MMP2, MMP9 and VEGFA. In conclusion, the present study supports miR-223 and PAX6 as novel therapeutic targets for glioblastoma.

Subfragments of amyloid-beta (Aβ) appear to protect neurons from Alzheimer’s disease (AD). The permeability of the blood–brain barrier (BBB) has limited in vivo research. The aim of this study is to explore permeation of the BBB by chitosan nanoparticles loaded with Aβ and to evaluate immunogenicity of these particles. Chitosan microspheres were prepared by mechanical stirring emulsification methods combined with chemical crosslinking. Morphological characteristics of the nanoparticles were examined using high-resolution transmission electron microscopy. The peptide association efficiency was determined by high-performance liquid chromatography. Fluorescently labeled chitosan nanoparticle-intramembranous fragments of Aβ (NP-IF-A) were administered systemically to mice in order to evaluate brain translocation by fluorescence microscopy. The immunogenicity of the nano-vaccine was determined by enzyme-linked immunosorbent assay (ELISA). All nanoparticles analyzed were well-separated, roughly spherical structures with uniform particle size distribution in the range of 15.23 ± 10.97 nm. The peptide association efficiency was 78.4%. The brain uptake efficiency of nano-antigen was 80.6%; uptake efficiency of antigen alone was only 20.6%. ELISA showed that the nano-vaccine had favorable immunogenicity. A chitosan nano-carrier for Aβ allowed permeation of the BBB and was non-immunogenic. These findings indicate that this novel targeted nano-vaccine delivery system can be used as a carrier for Aβ. This system will further research of peptide vaccines for AD.

Immunotherapy holds great promise for Alzheimer’s disease (AD), but meningoencephalitis observed in the first AD vaccination trial, which accompanied T-lymphocytic infiltration, needs to be overcome. This study was aimed to investigate alternative approaches for a safer vaccine to treat AD. We used intramembranous fragment of amyloid-β (IF-Aβ) to immunize Kunming mice for up to 2.5 months and then evaluated the immunization efficacy and potential adverse effects. Immunization of mice with IF-Aβ plus Freund’s adjuvant resulted in moderate levels of Aβ antibodies (IgG), and the anti-sera were able to neutralize Aβ1-42-neurotoxicity in cultured primary cortical neurons. IF-Aβ itself did not show neurotoxicity, and immunization with IF-Aβ did not cause behavioral deficits in Morris water maze or any abnormalities by histological examinations of major organs including the brain. We conclude that vaccination with IF-Aβ may be a potentially safe and effective treatment for AD.