BIX01294, an inhibitor of histone methyltransferase, induces autophagy-dependent differentiation of glioma stem-like cells

Abstract

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.

Hydrogel Environment Supports Cell Culture Expansion of a Grade IV Astrocytoma

“…The LN-18 cells also formed compact distinctive spheroids in hydrogel. Interestingly, previous studies have demonstrated the formation of neurospheres by LN-18 cells in the presence of growth factors [38]. These combined results are consistent with research that shows that the morphology of cells in culture is responsive to hydrogel environments [8, 9] and warrant future experiments examining the impact of serum-free media on cell growth in monolayer and hydrogel cultures [38, 39].…”

Temozolomide induces autophagy in primary and established glioblastoma cells in an EGFR independent manner

“…A protein complex can be pulled down with one or two antibodies depending on whether only the interaction is a matter of interest, or the expression of the two individual proteins is being examined (18). In this case, Beclin-1 and EGFR are established as being expressed in LN18 cells (19,20). For that reason, co-immunoprecipitation was performed using Beclin-1 alone for pull-down.…”

Mapping chromatin accessibility and active regulatory elements reveals new pathological mechanisms in human gliomas

“…To validate experimentally the discovered regulatory link, we evaluated ANXA2R and FOXM1 expression levels in different glioma cells: LN18, LN229, U87 established glioma cell lines, patient-derived WG4, IPIN glioma cell cultures (Ciechomska et al, 2016) and normal human astrocytes (NHA) using quantitative PCR (qPCR). Relatively to normal human astrocytes, the expression of ANXA2R in those cells was elevated only in patient-derived WG4 glioma cells; while FOXM1 expression was significantly elevated in 4 out of 5 cell lines ( Figure 6H).…”

“…Human malignant U-87 MG, LN18 and LN229 glioblastoma cells were purchased from American Type Culture Collection (ATCC). WG4 and IPIN are primary glioma cell lines developed from GBM patient surgical samples as described before (Ciechomska et al, 2016).…”