bix-01294 and Glioma

The present study aimed to investigate the differential expression and clinical significance of histone methyltransferase G9a, histone H3K9me2 and histone H3K9me1 in human brain glioma and adjacent tissue samples. It also aimed to observe the effect and mechanism of BIX‑01294, as an inhibitor of methyltransferase G9a, on the proliferation, apoptosis, methylation of H3K9 and H3K27, and the acetylation in U251 glioma cells in vitro. The differential expression of methyltransferase G9a, histone H3K9me2 and histone H3K9me1 in in human brain glioma and adjacent tissues were analyzed by immunohistochemistry, a growth curve of U251 cells following treatment with BIX‑01294 was determined using the MTT assay. In addition, the apoptosis percentage of U251 cells was analyzed by TUNEL assay and the expression levels of apoptosis‑associated proteins, including B‑cell lymphoma 2 (Bcl‑2), Bcl‑2‑associated X protein (Bax), caspase‑9 and caspase‑3, and the acetylation of histones, including H3K27me1, H3K27me2 and H3 in U251 were analyzed by western blot following BIX‑01294 treatment. The positive rate of G9a in glioma tissues was 86% (43/50), which was significantly different from 42% (21/50) in adjacent tissues (P<0.01). The positive rate of H3K9me2 in glioma tissues was 82% (41/50), which was significantly different from 38% (19/50) in adjacent tissues (χ²=18.38; P<0.01). The expression of G9a and H3K9me2 were associated with the World Health Organization (WHO) glioma grade. The positive rate of H3K9me1 in glioma tissues was 54% (27/50) and 44% (22/50) in adjacent tissues, though this result was not significantly different (χ²=1.21, P>0.05). BIX‑01294 inhibited the proliferation of U251, downregulated expression of Bcl‑2, and upregulated expression of Bax, caspase‑3 and caspase‑9, and induced apoptosis of U251. BIX‑01294 downregulated H3K9me1, H3K9me2, H3K27me1 and H3K27me2, however, it did not affect the acetylation of H3K9me3 and H3. High expression of G9a and H3K9me2 in glioma tissue samples was associated with the WHO grade, which indicated that G9a and H3K9me2 may promote generation and development of glioma. BIX‑01294 inhibited proliferation and induced apoptosis of glioma cells, changes in methylation of H3K9 and H3K27 resulting in conformational changes of chromosome may be an underlying mechanism. BIX‑01294 may be a potential novel therapeutic agent in the treatment of glioma.

Topics: Adult; Apoptosis; Azepines; Cell Line, Tumor; Cell Proliferation; DNA Methylation; Female; Gene Expression Regulation, Neoplastic; Glioma; Histocompatibility Antigens; Histone Methyltransferases; Histone-Lysine N-Methyltransferase; Humans; Male; Middle Aged; Neoplasm Proteins; Quinazolines

Epigenetic modification is crucial to keep the self-renewal and the "stemness" states of stem cells, not letting them to differentiate. The actual roles of Histone 3 Lysine 9 dimethylation (H3K9me2) and its methyltransferase G9a in this process are still unclear, especially in cancer stem cells. In our study, we found an interesting observation that most CD133-positive cells were H3K9me2 negative, both in glioma tissues and in cultured cells, although most cancer cells were detected to be H3K9me2 immunopositive. This implied that the G9a-dependent H3K9me2 was one of the crucial barriers of cancer stem cell self-renewal. To test the hypothesis, we examined the loss-of-function and gain-of-function of G9a. We found that bix01294, the selective inhibitor of G9a, can stimulate the sphere formation rate of glioma cancer stem cells, together with increasing Sox2 and CD133 expressions. The increase of CD133-active stem cells was confirmed by flow cytometry. On the other aspect, overexpression of G9a increased the H3K9me2 and decreased the sphere formation rate as well as the CD133 and Sox2 expressions. Since H3K9me2 modification is the major repressive switch, we predict that the repressive H3K9me2 modification may happen at the CD133 promoter regions. By chromatin precipitation assay, we confirmed that the CD133 and Sox2 promoter regions were modified by the H3K9me2. Therefore, we concluded that the G9a-dependent H3K9me2 repression on CD133 and Sox2 was one of the main switches of the self-renewal in glioma cancer stem cells.

Topics: AC133 Antigen; Antigens, CD; Azepines; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Glioma; Glycoproteins; Histocompatibility Antigens; Histone-Lysine N-Methyltransferase; Histones; Humans; Lysine; Methylation; Neoplastic Stem Cells; Peptides; Promoter Regions, Genetic; Quinazolines; Signal Transduction; SOXB1 Transcription Factors; Transfection

Aberrant epigenetic histone modifications are implicated in cancer pathobiology, therefore histone modifying enzymes are emerging targets for anti-cancer therapy. There is a few evidence for deregulation of the histone modifying enzymes in glioblastomas. Glioma treatment is a clinical challenge due to its resistance to current therapies.. The effect of selected inhibitors on epigenetic modifications and viability of glioma C6 cells were studied using immunofluorescence and MTT metabolism test.. We found that VPA and TSA increase histone H4 acetylation in glioma cells, while chaetocin and BIX01294 at low concentrations reduce H3K9me3, and 3DZNep decreases H3K27me3. Long-term treatment with some epigenetic inhibitors affects viability of glioma cells.. We established the concentrations of selected inhibitors which in C6 glioma cells inhibit the enzyme activity, but do not decrease cell viability, hence allow to study the role of histone modifications in C6 glioma biology.

Topics: Acetylation; Adenosine; Animals; Azepines; Brain Neoplasms; Cell Line, Tumor; Cell Survival; Enzyme Inhibitors; Epigenesis, Genetic; Glioma; Histones; Hydroxamic Acids; Piperazines; Quinazolines; Rats; Valproic Acid