Oridonin Up-regulating PLK1 To Induce G₂/M Phase Arrest In Jurkat Cells

Background and objectiveThe protein level of Polo-like kinase 1（Polo-like kinase 1,PLK1）is strictly cell cycle dependent.Activating PLK1 helps to promote the transition of G₂/M phase,mitosis,and cytokinesis.Because PLK1 participates in various key cell cycle events,it is considered to be one of the targets of effective cancer treatment drug development.Oridonin is a kaurene diterpene and it can inhibit the growth through a variety of mechanisms..We treated Jurkat cells of the acute T lymphocytic leukemia cell line with Oridonin and found that the cells were arrested in the G₂/M phase.Further research found that Oridonin can up-regulat PLK1 protein levels and kinase activity.In this study,we preliminarily explored the effect and mechanism of Oridonin in inducing Jurkat cell cycle arrest through up-regulating PLK1 protein level and kinase activity.We want to provides a theoretical basis for the application of Oridonin in the treatment of targeting acute T lymphocyte leukemia.Methods1.CCK-8 cell proliferation assay was used to detecte the effect of Oridonin on the proliferation of Jurkat cells.Flow cytometry was applied to detect the effect of Oridonin on Jurkat cell cell cycle changes and apoptosis.Swiss Giemsa stain was used to detect the morphological changes of Jurkat cells by Oridonin.Western blot was used to detect the changes of cyclin and mitosis-related protein kinase after oridonin treatment.We constructed PLK1 knockdown and overexpression Jurkat cells,and used flow cytometry to detect cell cycle changes induced by Oridonin.Western blot was used to detect the effect of Oridonin on the phosphorylation level of PLK1 downstream protein.2.Real-time fluorescent quantitative PCR was used to detect the relative expression level of PLK1 in Jurkat cells treated with Oridonin.We used immunoprecipitation and western blot to detect the effect of Oridonin on the level of ubiquitination modification of exogenously expressed PLK1 protein.CETSA assay was used to detect the effect of Oridonin on the stability of PLK1 protein at different temperatures and different concentrations.3.We used AUTODOCK（version 4.2）software to simulate the docking model of Oridonin and PLK1protein and use Py MOL software to analyze their possible binding sites.We constructed two PLK1plasmids with corresponding site mutation based on the prediction results.Immunoprecipitation and western blot were used to detect the effect of Oridonin on the ubiquitination modification level of PLK1 protein after mutation.Then we used CETSA to detect the effect of Oridonin on the stability of PLK1 protein after mutation.Results1.Oridonin can induce G₂/M phase arrest in Jurkat cells and up-regulate PLK1 protein content and kinase activity.2.Oridonin promotes the transcription of PLK1 gene.Oridonin directly binds to PLK1 protein to inhibit its degradation through the ubiquitin proteasome pathway.3.The mutation of Cys67 or Cys133 amino acid residues of PLK1 protein inhibits its binding to oridonin and inhibits the effect of oridonin in stabilizing PLK1.Conclusions1.Oridonin directly binds to PLK1 protein to inhibit its ubiquitination modification and degradation and promote its activity.Oridonin induces G2/M arrest in Jurkat cells2.The Cys67 and Cys133 sites of PLK1 protein are the key sites for PLK1 protein to bind to Oridonin.