The Antineoplastic Mechanism Of CDK7 Inhibitor THZ1 In Cervical Cancer

Objective:1)To observe the antineoplastic effects of CDK7 inhibitor THZ1 in cervical cancer cell lines.2)To investigate the effects of THZ1 on the cell cycle and transcriptional regulation in cervical cancer cells in vitro.3)To determine the anti-tumor role of THZ1 in cervical cancer mouse xenograft model in vivo.Methods:CCK8 assay was used to assess cell viability and determine the IC50 values of cervical cancer cell lines after THZ1 treatment.Cell apoptosis was measured using flow cytometry analysis after THZ1 treatment at the indicated concentration in cervical cancer cells.Western blot was employed to assess cell apoptosis/necrosis associated protein PARP/Cleaved PARP after THZ1 treatment.CRISPR-Cas9 systems were performed to knockdown CDK7 in cervical cancer cell lines,and CDK7 knockdown was detected by q RT-PCR and Western blot analysis.CCK8 assay revealed the cell proliferation after knockdown of CDK7 in cervical cancer cells.Cell cycle was assessed using flow cytometry analysis of propidium iodide stained cells in THZ1-treated cervical cancer cells.Western blot was subjected to measure the protein level of CDK1/p-CDK1 and cyclin B1 in cervical cancer cells after THZ1 treatment.Western blot was used to assess protein RNA Pol II and its phosphorylation at Ser2,Ser5,Ser7in cervical cancer cells after THZ1 treatment.Gene expression microarray analysis revealed gene expression changes after THZ1 treatment in cervical cancer cells compared with negative control.Gene Ontology enrichment analyzed the gene transcripts inhibited after THZ1 traetment.q RT-PCR revealed the expression of the main oncogenes in the development of tumorigenesis,including c-MYC、h TERT、RAD51、BCL-2 and HPV viral oncogenes E6 and E7 after THZ1 treatment.To further investigate the anti-tumor effect of THZ1 in cervical cancer,we assessed the therapeutic potential of THZ1 in xenotransplantation models in vivo.When tumor volumes reached100-150mm~3,mice were randomly separated into two groups(5 mice each group)and treated with 10 mg/kg THZ1 or vehicle(10%DMSO in 5%glucose)that was administered intraperitoneally twice daily.The volume of the tumors was measured every 4 days for 28 days.At the end of experiments,we calculated the tumor volumes and weights of each group for tumor growth analysis.Immunohistochemistry(IHC)was used to detecte the cell proliferation marker Ki67,and TUNEL was used to detect the cell apoptosis.Results:Cervical cancer cells were highly sensitive to THZ1,with a low concentration of THZ1 could achieve IC50 value,exhibiting time-and dose-dependency on THZ1treatment.Flow cytometry analysis demonstrated that inhibition of CDK7 through THZ1 caused cell apoptosis in a dose-dependent manner.Furthermore,we observed an increase in cell apoptosis/necrosis as assessed by PARP/Cleaved PARP Western blot analysis.The cells that knockdown of CDK7 using CRISPR-Cas9 systems exhibted significantly growth inhibition compared to their respective nontargeting controls in cervical cancer cells.Cell cycle analysis was performed that CDK7 inhibition through THZ1 caused cell cycle arrest at G2/M phase after THZ1 treatment.Western blot revealed that THZ1 led to a striking reduction in phospho-CDK1 and cyclin B1,which are indispensable cell cycle regulators required for the G2/M transition.Western blot revealed that the cells receiving THZ1 treatment showed a striking reduction in the phosphorylation of RNA Pol II at Ser2,Ser5 and Ser7.Microarray analysis showed that massive oncogene transcripts,especially the genes involved in transcriptional regulation,proliferation and DNA repair process,were preferentially suppressed after THZ1 treatment.In addition,THZ1 exhibited a profound reduction in the transcription of c-MYC、h TERT、RAD51、BCL-2 and HPV viral oncogenes E6 and E7 in cervical cancer cell lines.THZ1 exhibited substantial antineoplastic effects against cervical cancer in xenotransplantation models,exhiting tumor growth inhibitory with cell proliferation marker Ki67 decrease and the cell apoptotic marker TUNEL increase.Conclusions:CDK7 inhibitor THZ1 exhibited substantially anti-tumor effect on cervical cancer cell lines,and a low concentration of THZ1 could induce substantial cell apoptosis.THZ1 could inhibit cell cycle,inducing cell cycle arrest at the G2/M phase and inhibiting cell proliferation in cervical cancer cell lines.Moreover,microarray analysis showed that massive oncogene transcripts,especially those associated with tumorigenesis,were preferential suppressed after THZ1 treatment.In addition,THZ1 exhibited substantial antineoplastic effects against cervical cancer in vivo without inducing obvious side effects.These findings indicated that the CDK7inhibitor THZ1 is a potential option for cervical cancer treatment.