Melatonin Inhibits Glioblastoma Stem-like cells through Suppression of EZH2-NOTCH1 Signaling Axis - PubMed (original) (raw)

. 2017 Feb 6;13(2):245-253.

doi: 10.7150/ijbs.16818. eCollection 2017.

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Melatonin Inhibits Glioblastoma Stem-like cells through Suppression of EZH2-NOTCH1 Signaling Axis

Xiangrong Zheng et al. Int J Biol Sci. 2017.

Abstract

Glioblastoma stem-like cells (GSCs) play essential roles in glioma growth, radio- and chemo-resistance, and recurrence. Elimination of GSCs has therefore become a key strategy and challenge in glioblastoma therapy. Here, we show that melatonin, an indolamine derived from I-tryptophan, significantly inhibited viability and self-renewal ability of GSCs accompanied by a decrease of stem cell markers. We have identified EZH2-NOTCH1 signaling as the key signal pathway that regulated the effects of melatonin in the GSCs. Instead of transcriptionally silencing gene expression by generating a methylated epigenetic mark at histone 3 at lysine 27 (H3K27), EZH2 regulates NOTCH1 expression by directly binding to the NOTCH1 promoter. Moreover, correlation between the expressions of EZH2 and NOTCH intracellular domain 1 (NICD1) was observed in the clinical tumor samples, evidently supporting the existence of EZH2-NOTCH1 interaction in the gliomas and GSCs. Collectively, we demonstrated that melatonin, a potential tumor inhibitor, performs its function partly by suppressing GSC properties through EZH2-NOTCH1 signaling axis.

Keywords: EZH2; Glioblastoma stem-like cells; Melatonin; NOTCH1; self-renewal; viability.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1

Figure 1

Melatonin Inhibited Viability and Self-renewal Ability of GSCs. (A) The representative images of the GSCU251 and GSCT98G (400×). (B) mRNA expression levels of CD133 and SOX2 were determined by qRT-PCR in the GSCU251 and GSCT98G and the original cell lines. Data were presented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01vs. control. (C) The GSCU251 and GSCT98G were treated with 0, 100nM, 100μM, 1mM melatonin for 48 hr. Cell viability was determined by CCK-8 assay. Data were presented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01 vs. results of the control w/o melatonin. (D) Morphological changes were shown in the GSCU251 and GSCT98G treated with 0, 100μM, 1mM melatonin for 10 days (200×). Tumor-sphere numbers were calculated. Data were presented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01 vs. results of the control w/o melatonin.

Figure 2

Figure 2

EZH2 Was Involved in the Inhibitory Role of Melatonin in Regulation of GSC Properties. mRNA (A) and protein (B) levels of CD133, SOX2 and EZH2 were shown in the GSCU251 and GSCT98G treated with 1mM melatonin for 48 hr. Each bar represents mean ± SD from three independent experiments. *P < 0.05, **P < 0.01 vs. control. (C) The GSCU251 transfected with GV230-EZH2 (EZH2) or GV230 alone (Con) were treated with 0, 1mM melatonin for 48 hr. Cell viability was determined by CCK-8 assay. Data were presented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.01. (D) The changes of morphology and tumor-sphere numbers were shown in the indicated cells (200×). Each bar represents mean ± SD from three independent experiments. **P < 0.01. (E) mRNA levels of CD133 were determined by qRT-PCR in the indicated cells. The result represents mean ± SD from three independent experiments. *P < 0.05, **P < 0.01.

Figure 3

Figure 3

NOTCH1 Was an EZH2 Target Gene Involved in the Effects of Melatonin. (A) The GSCU251 and GSCT98G were treated with 1mM melatonin for 48 hr. mRNA expression levels of NOTCH family and NOTCH signaling pathway genes were determined by qRT-PCR. Data were presented as mean ± SD from three independent experiments. *P < 0.05 vs. control. (B) Protein levels of EZH2, NICD1 and HES1 were analyzed by immunoblot in the GSCU251 and GSCT98G treated with 1mM melatonin for 48 hr. β-ACTIN served as a loading control. (C) The GSCU251 were transfected with pGL2-NOTCH1; a vector-encoding EZH2-shRNA (shEZH2) or scrambled-shRNA (shSC), or a vector-encoding wild-type EZH2 (EZH2) or its comparative control (Con); and pSV-Renilla. Values in graphs represent mean of FLuc:RLuc activity ± SD performed in triplicate. *P < 0.05 vs. control. (D) Protein levels of NOTCH intracellular domain 1(NICD1) and HES1 were examined by immunoblot in the EZH2 depleted (shEZH2) or over-expressed (EZH2) GSCs. β-ACTIN was used as a loading control. (E) The GSCU251 were treated with 0, 1mM melatonin for 48 hr. ChIP analysis was performed by using antibodies against EZH2, tri-methylation of histone 3 at lysine 27 (H3K27me3) or suppressor of zeste 12 (SUZ12) with primers targeted to the promoter region of NOTCH1. Isotype matched IgG was used as a negative control. Data are presented as mean ± SD from three independent experiments. *P < 0.05, **P<0.01 vs. control.

Figure 4

Figure 4

Clinical Relevance Existed between EZH2 and NOTCH1 Activation. (A) IHC was performed to detect EZH2 and NOTCH intracellular domain 1(NICD1) levels in the GBM specimens and samples of normal brain specimens (400×). (B) Distribution of EZH2 and NICD1 expression in 67 GBM specimens. (P < 0.001). (C) Correlation between expression of EZH2 and NICD1 in 67 GBM specimens (P < 0.01).

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