Antitumor Effect Of Brd4 Selective Inhibitor JQ1 On Glioblastoma

Background:A bromodomain is a conserved protein domain that selectively recognizes acetylated lysine residues on the N-terminal tails of histones and this recognition makes the bromodomain-containing proteins binding to specific regions of chromatin so that to regulate a wide variety of biological functions. Members of the Bromodomain and extraterminal domain(BET) family include BRD2, BRD3, Brd4 and BRDT and in which the Brd4 subfamily contains Brd4 A, Brd4 B and Brd4 C and is most wildly well-studied. BET family proteins have been identified to be promising therapeutic targets in inflammation and tumor treatment. The oncogenic role of BET protein was first verified in the NUT midline carcinoma.Recently, it was verified that silence of Brd4 gene resulted in down-regulation of c-Myc, reduction of leukaemia stem cell, and suppression of cancer progression.Small molecular BET inhibitors have been developed rapidly in recent years, including JQ1, I-BET151 and CPI203. JQ1 is one of the Brd4 selective small molecule inhibitor which binds to bromodomain of BET competitively leading to substitution of BET protein from the acetylated lysine residues on the chromatin. JQ1 showed significant anti-tumor effect in diverse tumors, such as hematopoietic malignanciesand NUT midline carcinoma both in vitro and in vivo. However, the sensitivity of JQ1 treatment is not well in lung cancer, breast cancer,cervical carcinoma and other solid tumor cell lines. Several studies indicated that JQ1 can dramatically reduce c-Myc expression and c-Myc has been well demonstrated playing vital role in promoting invasion and maintaining the stem cell activity in glioblastoma highlighting the promising potential of JQ1 in glioblastoma.Objective:The aim of this study is to verify Brd4 as the potential drug target in glioblastoma treatment in vitro and to verify JQ1 could effectively suppress proliferation of glioblastoma cells and perform anti-tumor effect in glioblastoma which is finally expected to provide theoretical and experimental basis for the invention of novel therapeutics.Methods:CCK8 assays, cell growth and neurosphere assays were done to determine the effect of Brd4 selective inhibitor JQ1 on mouse glioma tumor-initiating line CSC1589 proliferation; Hochest staining and Flow Cytometry were applied to detect apoptosis of CSC1589 cells caused by JQ1 treatment and cell cycle alteration was evaluated via Flow Cytometry as well; expression of Brd4, oncogene c-Myc as well as apoptosis and cell cycle related genes and proteins were tested through q RT-PCR and Western blot; Immunofluorescence staining was applied toevaluate the effect on Brd4 and stem cell marker levels after JQ1 treatment.Results:Mouse glioma tumor-initiating cell line CSC1589 was subjected to Brd4 selective inhibitor JQ1 at different concentration and cell proliferation was detected via CCK8 at indicated time point 24 h and 48 h and the result showed that JQ1 treatment inhibited cell growth in a dose-dependent manner rather than a time-dependent manner in comparison to the control and accordingly 125 n M/L and 250 n M/L were selected for the following experiments. Cell growth and neurosphere assays showed that cell proliferation was suppressed by JQ1 treatment especially at 250 n M/L. Immunofluorescence staining results indicated induction of stem cell marker Nestin in JQ1 treated group suggesting reduction of stem cell activity of glioblastoma. And it found that cell apoptosis was significantly induced by JQ1 treatment via flow Cytometry assays and Hochest staining. Cell cycle analysis showed that JQ1 induced G1 phase arrest and cell number reduction in S phase.Moreover, Western blot and q RT-PCR indicated increase in pro-apoptosis m RNA and protein level, while decrease in anti-apoptosis and cell cycle related gene and proteins compared with the control group.Conclusion:Brd4 small molecular inhibitor can effectively inhibit cell proliferation of glioma, promote cell apoptosis and induce cell cycle arrest resulting in anti-tumor effect in glioblastoma indicating that Brd4 is a potential epigenetic target in glioblastoma therapy and Brd4 selective inhibitor JQ1 has a significant anti-tumor effect in glioblastoma treatment.