bix-01294 and Brain-Neoplasms

Glioblastoma (GBM) contains rare glioma stem-like cells (GSCs) with capacities of self-renewal, multi-lineage differentiation, and resistance to conventional therapy. Drug-induced differentiation of GSCs is recognized as a promising approach of anti-glioma therapy. Accumulating evidence suggests that unique properties of stem cells depend on autophagy. Here we demonstrate that BIX01294, an inhibitor of a G9a histone methyltransferase (introducing H3K9me2 and H3K27me3 repressive marks) triggers autophagy in human glioma cells. Pharmacological or genetic inhibition of autophagy decreased LC3-II accumulation and GFP-LC3 punctation in BIX01294-treated cells. GSCs-enriched spheres originating from glioma cells and GBM patient-derived cultures express lower levels of autophagy related (ATG) genes than the parental glioma cell cultures. Typical differentiation inducers that upregulate neuronal and astrocytic markers in sphere cultures, increase the level of ATG mRNAs. G9a binds to the promoters of autophagy (LC3B, WIPI1) and differentiation-related (GFAP, TUBB3) genes in GSCs. Higher H3K4me3 (an activation mark) and lower H3K9me2 (the repressive mark) levels at the promoters of studied genes were detected in serum-differentiated cells than in sphere cultures. BIX01294 treatment upregulates the expression of autophagy and differentiation-related genes in GSCs. Pharmacological inhibition of autophagy decreases GFAP and TUBB3 expression in BIX01294-treated GSCs suggesting that BIX01294-induced differentiation of GSCs is autophagy-dependent.

Topics: Autophagy; Azepines; Brain Neoplasms; Cell Differentiation; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glioblastoma; Histone Methyltransferases; Histone-Lysine N-Methyltransferase; Histones; Humans; Neoplastic Stem Cells; Quinazolines; Tumor Cells, Cultured

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