The pharmacological role of histone demethylase JMJD3 inhibitor GSK-J4 on glioma cells

Abstract

Glioma is regarded as the most prevalent malignant carcinoma of the central nervous system, and lack of effective treatment. Thus, the development of new therapeutic strategies targeting glioma is of significant clinical importance. In the present study, histone H3K27 demethylase jumonji domain-containing protein 3 (JMJD3) was investigated as target for glioma treatment. The mRNA of JMJD3 was overexpressed in glioblastoma tissues compared to normal brain tissues (P<0.05). The content of JMJD3 was also higher in glioma cells than in human brain microvascular endothelial cell (hCMEC), and the corresponding level of H3K27me3 was decreased (P<0.05). The treatment with JMJD3 specific inhibitor GSK-J4 can increase the content of H3K27me3 in glioma cells, which means the activity of JMJD3 was inhibited. GSK-J4 can inhibit glioma cell proliferation in a concentration dependent and time-dependent manner (P<0.05). GSK-J4 also induced glioma cell apoptosis and inhibited cell migration (P<0.05). But there was no obvious effect of GSK-J4 on hCMEC cells. All together, these data suggest that GSK-J4 has important potential in the gliomas treatment.

Keywords: GSK-J4; JMJD3; glioma; histone demethylase; inhibitor.

Figure 1. JMJD3 is overexpressed in brain glioblastoma

Oncomine data mining analysis of JMJD3 mRNA levels between normal brain tissues versus glioblastoma tissues. *, P < 0.05.

Figure 2. JMJD3 is overexpressed in glioma cells

(A) JMJD3 mRNA expression was higher in glioma cells U87 and U251 than endothelial cells hCMEC. (B) The content of JMJD3 protein was higher in U87 and U251 than hCMEC, and the corresponding H3K27me3 level was lower. *, P < 0.05.

Figure 3. GSK-J4 decreases the content of H3K27me3 in glioma cells

U87, U251 and hCMEC cells were treated with 4μM GSK-J4 for 24h, and the H3K27me3 levels were determined with western blotting. Total H3 served as a loading control.

Figure 4. GSK-J4 inhibits the proliferation of glioma cells in a concentration dependent manner

hCMEC (A), U87 (B) and U251 (C) cells were treated with GSK-J4 at indicated concentration for 24h, and the effects on cell proliferation were determined with CCK-8 assay. *, P < 0.05.

Figure 5. The inhibitory effects of GSK-J4 glioma cells in a time dependent manner

Three cells hCMEC (A), U87 (B) and U251 (C) were treated with 4μM GSK-J4 for 24h, 48h and 72h, and the effects on cell proliferation were determined with CCK-8 assay.*, P < 0.05.

Figure 6. GSK-J4 promotes the apoptosis of glioma cells

The three cell lines were treated with 4μM or 8μM GSK-J4 for 24h, and the cell apoptosis changes were determined by flow cytometry analysis. (A) hCMEC, (B) U87, (C) U251, (D) quantitative results of cell apoptosis. **, P < 0.01.

Figure 7. GSK-J4 inhibits cell migration of glioma cells

Both U87 and U251 cells were treated with 8μM GSK-J4 for 24h, and the effects on cell migration were determined with cell scratch test. (A) The cell migration of U87. (B) Quantitative results of U87 cell migration. (C) The cell migration of U251. (D) Quantitative results of U251 cell migration. Values are the mean of triplicate samples from a representative experiment. *, P < 0.05. **, P < 0.01.