Histone deacetylase inhibitors exert anti-tumor effects on human adherent and stem-like glioma cells

Abstract

Background: The diagnosis of glioblastoma (GBM), a most aggressive primary brain tumor with a median survival of 14.6 months, carries a dismal prognosis. GBMs are characterized by numerous genetic and epigenetic alterations, affecting patient survival and treatment response. Epigenetic mechanisms are deregulated in GBM as a result of aberrant expression/activity of epigenetic enzymes, including histone deacetylases (HDAC) which remove acetyl groups from histones regulating chromatin accessibility. Nevertheless, the impact of class/isoform-selective HDAC inhibitors (HDACi) on glioma cells, including glioma stem cells, had not been systematically determined.

Results: Comprehensive analysis of the public TCGA dataset revealed the increased expression of HDAC 1, 2, 3, and 7 in malignant gliomas. Knockdown of HDAC 1 and 2 in human GBM cells significantly decreased cell proliferation. We tested the activity of 2 new and 3 previously described HDACi with different class/isoform selectivity on human GBM cells. All tested compounds exerted antiproliferative properties on glioma cells. However, the HDACi 1 and 4 blocked proliferation of glioblastoma cells leading to G2/M growth arrest without affecting astrocyte survival. Moreover, 1 and 4 at low micromolar concentrations displayed cytotoxic and antiproliferative effects on sphere cultures enriched in glioma stem cells.

Conclusions: We identified two selective HDAC inhibitors that blocked proliferation of glioblastoma cells, but did not affect astrocyte survival. These new and highly effective inhibitors should be considered as promising candidates for further investigation in preclinical GBM models.

Keywords: Cell proliferation; Epigenetic drugs; Glioblastoma; Glioma stem cells; HDAC inhibitors; Histone deacetylase.

Fig. 1

Structures of the HDACi 1–5 tested in the study: 1 mocetinostat, 2 compound 106, 3 MC1746, 4 MC2129, and 5 SAHA

Fig. 2

Expression of HDAC 1–8 and 11 in gliomas. Data were acquired from the TCGA repository as data level 3, FPKM values and include 5 normal brain samples (normal), 248 grade II (G2), 261 grade III (G3), and 160 grade IV (G4) tumor samples. Data were quantile normalized and log₂ transformed. ANOVA test with no assumption of equal variance (Welch one-way test) was performed on all G2, G3, and G4 samples. Significant differences of means between G2, G3, and G4 were observed and denoted as significant after FDR corrections for multiple testing: *p value < 0.05, **p value < 0.01, ***p value < 0.001

Fig. 3

Knockdown of HDAC 1 and HDAC 2 results in reduced cell proliferation. a HDAC 1 and HDAC 2 expression was estimated by qRT-PCR in U-87 MG and LN18 cells after gene silencing using specific siRNAs. b Western blot analysis shows efficacy of HDAC 1 and HDAC 2 knockdown at protein level. c Western blot for acetylated histones H3 and H4 (H3Ac, H4Ac) in HDAC 1 and HDAC 2 depleted U-87MG and LN18 cells 48 h after siRNA transfection. d MTT metabolism test for cell viability 24, 48, and 72 h after transfection with HDAC 1 or/and HDAC 2 siRNAs or a control siRNA. e BrdU incorporation test for cell proliferation 48 h after knockdown of HDAC 1 or/and HDAC 2 in U-87MG and LN18 cells. The respective p values were calculated using type 2 two-tailed t test, and p < 0.05 was considered statistically significant. *p value < 0.05, n = 3

Fig. 4

Effects of 1–5 on glioma cell viability and proliferation. a MTT metabolism test for cell viability of U-87 MG and LN18 cells after exposure to 1–5 for 24 h at indicated concentrations. Values for untreated cells were taken as 100%. b BrdU incorporation test for cell proliferation of U-87 MG and LN18 cells after exposure to 1–5 for 24 h at indicated concentrations. The respective p values were calculated using type 2 two-tailed t test followed by FDR corrections for multiple hypothesis testing and p < 0.05 was considered statistically significant (n = 3)

Fig. 5

Antiproliferative effects of 1–5 on glioma cells. a The effects of compounds 1–5 on cell cycle in glioma cells were determined by propidium iodide (PI) staining and flow cytometry. Quantification of three experiments is presented. The respective p values were calculated using type 2 two-tailed t test, and p < 0.05 was considered statistically significant: *p value < 0.05, **p value < 0.01, ***p value < 0.001. b Total protein extracts were collected from U-87 MG and LN18 cells exposed to HDACi. Representative immunoblot shows results of western blot analysis of PARP-1 cleavage, γ-H2AX, p21, and H4Ac levels in U-87 MG and LN18 cells after exposure to 5 μM 1–3, 5, and 1 μM 4. c Densitometry analysis of western blot of PARP-1 cleavage, γ-H2AX, p21, and H4Ac levels in U-87 MG and LN18 cells after exposure to 5 μM 1–3, 5, and 1 μM 4 (n = 2)

Fig. 6

Long-term effects of 1–5 on glioma cell viability and proliferation. a Scheme of the experiment—the cells were exposed to 1–5 for 24 h, then HDACi were washed out and cells were cultured for another 72 h. b MTT metabolism test for cell viability of U-87 MG and LN18 cells after exposure to 1–5 at indicated concentrations. c BrdU incorporation test for cell proliferation of glioma cell after exposure to 1–5 at indicated concentrations. Values for untreated cells were taken as 100%. The respective p values were calculated using type 2 two-tailed t test, and p < 0.05 was considered statistically significant. n = 3, *p value < 0.05, **p value < 0.01, ***p value < 0.001

Fig. 7

Effects of 1–5 on human glioma CSCs. LN18 cells were grown under sphere forming conditions for 6 days and then inhibitors were added for 24 h. a Representative pictures of LN18-derived spheres show sphere disintegration after exposure to 1–5. b At the end of the experiments, spheres were collected by centrifugation and total protein extracts were prepared. Representative immunoblots show western blot analysis of PARP-1 cleavage, γ-H2AX, p21, and AcH4 levels in glioma spheres after 24 h exposure to 5 μM 1–3, 5, and 1 μM 4. c Densitometry analysis of western blot of PARP-1 cleavage, γ-H2AX, p21, and AcH4 levels in glioma spheres after 24 h exposure to 5 μM 1–3, 5, and 1 μM 4 (n = 2)