Roles And Mechanisms Of BRD4 Inhibitor JQ1 In Inducing Cellular Senescence And Apoptosis In Small Cell Lung Cancer Cells

Background: In China,small cell lung cancer(SCLC)affects more than 40,000 patients each year.The five-year survival rate of SCLC is only 6%,and it seriously threatens the patients’ lives.The standard treatment for SCLC patients is radiotherapy combined with chemotherapy,but due to its high malignance,short doubling time and drug resistance,the prognosis of SCLC is relatively poor.Therefore,it is urgent to find effective therapies with fewer side effects.BRD4 is a transcriptional and epigenetic regulator which plays an important role in mediating oncogene expression.Currently,the well-known effective BRD4 inhibitor,JQ1,has shown promising anti-tumor effects in NUT midline cancer,acute leukemia,and some other cancers.But the studies of JQ1 in SCLC are still rare.This thesis aims to find new therapeutic targets for SCLC.Thus,it may provide novel insights for alternative treatments and drug development.Methods: Firstly,MTT assays were used to detect the inhibitory effects of JQ1 on SCLC cell lines H446 and H69.Flow cytometry experiments were used to study the effects of JQ1 on cell cycle and apoptosis of these two cell lines.Then SA-β-Gal staining tests were carried out to detect β-galactosidase activity.EdU incorporation assays were applied to evaluate DNA synthesis changes.To investigate whether DNA damage response is activated or not,immunofluorescence experiments were used.Western blot assays were also carried out to detect expression levels of essential proteins in cellular senescence and apoptosis.The mRNA expression of inflammatory factors were detected using qRT-PCR.Finally,the activity of JQ1 on the growth of subcutaneous xenografts in nude mice,as well as the expression of biomarkers in senescence and apoptosis,were evaluated.Results: The results of in vitro experiments showed that JQ1 can significantly inhibit proliferation of H446 and H69 cells.Further studies demostrated that in both cell lines,JQ1 could induce cell cycle arrest at G0/G1 phase and cell apoptosis.It was also found that JQ1 can induce cellular senescence in H446 cells at 10 and 35 μM.These senescent cells showed increased galactosidase activity.The expression levels of CDK4 were significantly reduced.And DNA synthesis was significantly reduced and DNA damage response was activated in cells.53BP1 and γ-H2AX were recruited to chromatin forming DNA damage foci.It was shown that the protein expression levels of p53,p21 and p16 were increased through Western blot.Treatment with the concentrations at both 35 and 60 μM can activate apoptotic signaling pathway.The expression levels of the apoptosis proteins cleaved caspase 9 and cleaved PARP significantly increased.Subsequent subcutaneous tumor transplantation results in mice indicated that JQ1 can significantly inhibit the growth of tumors,and has no significant toxic effects on mice.According to SA-β-Gal staining,TUNEL experiments,and IHC results of tumor tissues,it was demonstrated that JQ1 has relatively high anti-tumor activity in vivo.Conclusion: The results of in vitro and in vivo experiments have shown that JQ1 can effectively inhibit proliferation of SCLC and induce cellular senescence and apoptosis.In this study,we explored the effects of BRD4 inhibition on SCLC cells and the related mechanisms,and provided new insights for the treatment of SCLC targeting BRD4.