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@#To identify novel inhibitors targeting the polo-box domain of polo-like kinase 1 (Plk1 PBD), a series of new peptidomimetics (7a-7u) without phosphate group were designed and synthesized, where the phosphate group in the structure of the selective Plk1 PBD inhibitor PLHSpT was replaced by the carboxyl group, and the unnatural amino acids were applied for further modification and optimization. The 21 peptidomimetic compounds designed and synthesized had a strong inhibitory effect on Plk1 PBD, of which compound 7l highly selectively inhibited Plk1 PBD with IC50 of 0.285 μmol/L. The growth inhibition effect of HeLa tumor cell lines in vitro was better than that of compounds containing phosphate group. Moreover, the stability of the compound in rat plasma was improved by unnatural amino acids. Thus it is proved that selective Plk1 PBD inhibitor with improved characters can be obtained by replacing the phosphate group with a carboxyl group and restructuring the peptide chain.
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OBJECTIVE@#To explore the role of miR-593 in regulating the proliferation of colon cancer cells and the molecular mechanism.@*METHODS@#Bioinformatics analysis identified PLK1 as the possible target gene of miR-593. Luciferase assay was employed to verify the binding between miR-593 and PLK1, and qRT-PCR and Western blotting were used to verify that PLK1 was the direct target gene of miR-593. CCK-8 assay was performed to test the hypothesis that miR-593 inhibited the proliferation of colon cancer cells by targeting PLK1.@*RESULTS@#Luciferase assay identified the specific site of miR-593 binding with PLK1. Western blotting showed a significantly decreased expression of PLK1 in the colon cancer cells transfected with miR-593 mimics and an increased PLK1 expression in the cells transfected with the miR-593 inhibitor as compared with the control cells ( < 0.05). The results of qRT-PCR showed no significant differences in the expression levels of PLK1 among the cells with different treatments ( > 0.05). The cell proliferation assay showed opposite effects of miR-593 and PLK1 on the proliferation of colon cancer cells, and the effect of co-transfection with miR-593 mimic and a PLK1-overexpressing plasmid on the cell proliferation was between those in PLK1 over-expressing group and miR-593 mimic group.@*CONCLUSIONS@#miR-593 inhibits the proliferation of colon cancer cells by down-regulating PLK1 and plays the role as a tumor suppressor in colon cancer.
Subject(s)
Humans , Binding Sites , Cell Cycle Proteins , Genetics , Metabolism , Cell Line, Tumor , Cell Proliferation , Colonic Neoplasms , Metabolism , Pathology , Down-Regulation , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor , In Vitro Techniques , MicroRNAs , Genetics , Metabolism , Protein Serine-Threonine Kinases , Genetics , Metabolism , Proto-Oncogene Proteins , Genetics , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sincalide , Metabolism , TransfectionABSTRACT
With the method of fluorescence polarization (FP), we screened small molecule inhibitors for PLK1 PBD to identify the lead compounds for antitumor drugs. FP led to the identification of a potent hit, F083-0063, whose inhibition rate was (99.7±0.4)% at 10 μg·mL-1. The IC50 was calculated to be 1.9±0.1 μmol·L-1 using Graphpad Prism 5. The effect of the compound on cells' multiplication was measured by MTT assay which showed that F083-0063 inhibited the proliferation of many tumor cell lines. Flow cytometry analysis indicated that the F083-0063 promoted cell apoptosis and induced cell G2/M arrest. Migration abilities of cells, evaluated using scratch test, increased significantly in the presence of F083-0063 with the mi-gration rate as low as (37.6±0.7)% at 20 μmol·L-1. Molecular linkage technique found F083-0063 had good affinity with PLK1 PBD. The results of Western blotting showed that the expression of cyclin-dependent proteins was increased after treatment with F083-0063. In summary, F083-0063 has an antitumor activity and is expected to be an antitumor lead compound targeting PLK1 PBD.
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Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. Polo-like kinases (Plks) are a family of conserved serine/threonine kinases involved in the regulation of cell cycle progression through G2 and mitosis. One of them being PLK1, it’s over expression leads to a variety of cancers. Plk1 delta, an uncharacterized protein sequence found in UniProt was studied and found to consist of the N-terminal portion of plk1gene. The 3D protein structure of PLK1 delta was modeled and then the predicted structure was validated. Molecular dynamics simulation was performed to find the stability of the protein. The modelled protein was docked to BI 2536, an inhibitor of Plk1 to obtain conformation of least binding energy. The interacting sites between the protein and inhibitor were also analyzed.
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Objective:To investigate the expression of Plk1 ( Polo-like kinase 1 ) and Chk1/2 ( Checkpoint kinase 1/2 ) in primary hepatic carcinoma tissue and HepG2 cell. Methods: Using immunohistochemistry chemical method detected expression of Plk1,Chk1/2 protein in 40 cases of primary hepatic carcinoma tissue and 16 cases of non-tumor tissue of liver. Western blot was applied to detect the expression of Plk1 and Chk1/2 protein in HepG2 cells, and gray value was measured by using the quantitative analysis. Results:The positive rate of Plk1,Chk1/2 protein expression in primary hepatic carcinoma was 57. 5%,75. 0% and 22. 5%respectively,compared with positive rate in the liver of non-tumor tissue were 0%,25. 0% and 56. 3%. The expression of Plk1 and Chk1 protein in primary hepatic carcinoma tissue is higher than that in non-tumor tissue of liver,and the difference was statistically sig-nificant( PChk1>Chk2. Conclusion: Plk1,Chk1 protein in primary hepatic carcinoma was up-regulated,while Chk2 protein was down-regulated in these tissues. The expression degree was Plk1> Chk1>Chk2. There were relatively selective expression in primary hepatic carcinoma tissue of Plk1,Chk1 protein,then Plk1 and Chk1 might be ideal targets for therapy of primary hepatic carcinoma.
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Purpose To investigate the expression of polo-like kinase 1 (Plk1) and Cyclin B1, p21WAF1in cervical carcinoma, and to determine the relationship between the expression of the three proteins and tumor clinicopathological features. Methods The expres-sion of Plk1, Cyclin B1 and p21WAF1 was detected in 102 cases of cervical carcinoma, 20 cases of (cervical intraepithelial neoplasia, CIN) , and 20 cases of nomal cervical tissues by the technique of tissue chip and immunohistochemical staining of EliVision. Statistical analyses of the data were performed with SPSS 19. 0 software. Results The positive rates of Plk1 in cervical carcinoma and CIN were 70. 5%, 55. 0%, respectively, which were significantly higher than normal cervical tissues (10%) (P<0. 01);The positive rates of Cyclin B1 in cervical carcinoma and CIN were 52. 9% and 30. 0%, respectively, which were significantly higher than normal cervical tissues (10%)(P <0.01); The positive rates of p21WAF1 in cervical carcinoma and CIN were 23.5% and 10.0%, respectively, which were significantly higher than normal cervical tissues ( 0 ) ( P<0. 01 ) . There were no significant differences between cervical carcinoma and CIN in the positive rates of Plk1, Cyclin B1 and p21WAF1. The expression of Plk1 was associated with the depth of carci-noma invasion (P<0. 05), that of Cyclin B1 was associated with lymph node metastases and the depth of carcinoma invasion (P<0. 05)and that of p21WAF1 in cervical carcinoma was associated with histological grade (P<0. 05). In cervical carcinoma, the expres-sion of Plk1 was positively correlated with Cyclin B1 (rs =0. 297, P=0. 002) and negatively correlated with p21WAF1(rs = -0. 403, P<0. 001). Conclusion The expression of Plk1, Cyclin B1 and p21WAF1 is involved in the occurrence and development of cervical carcinoma, and the former two are also related with prognosis of cervical carcinoma. The combination of the three would provide a new target for clinical treatment.
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Polo-like kinase 1 (PLK1) is a highly conserved serine/threonine protein kinase that has attracted research attention be-cause it plays a critical role in mitosis regulation. PLK1 is overexpressed in 80%of human tumors, which indicates a poor prognosis in most tumors. PLK1 is one of the most promising targets for antitumor therapy because it is upregulated in castrate-resistant prostate can-cer (CRPC). This review focused on the basic structure and function of PLK1, the relationship between PLK1 and CRPC occurrence and progression, and CRPC treatment by inhibiting PLK1. This study provides a theoretical basis for the targeted molecular therapy of CRPC.
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Aim To investigate the effect of PLK1 on epithelial-mesenchymal transition (EMT)of human e-sophageal squamous cell carcinoma (ESCC)cells TE-1 5 and its relevant molecular mechanisms.Methods PLK1 overexpressed ESCC cells and control vector were used as the experimental cells.The expression of EMT-related protein markers E-cadherin and vimentin were measured by Western blot.vimentin mRNA was measured by Real-time PCR.Total cellular protein and nuclear protein were respectively extracted,and then they were used to detect the expression of β-catenin by Western blot.β-catenin siRNA and non-specific siR-NA were transiently transfected into the cell clones overexpressed PLK1 ,and then vimentin was detected by Western blot.β-catenin protein degradation com-plex was detected by immunoprecipitation and Western blot.Results The mesenchymal marker vimentin was distinctively upregulated and the epithelial marker E-cadherin was distinctively downregulated in the cell clones overexpressed PLK1 ,compared with those in the vector clones.This indicated that EMT occurred in ESCC cells.vimentin mRNA was also markedly in-creased.In the cell clones overexpressed PLK1 ,β-catenin were both elevated from the total cells and the nucleus.The expression of vimentin was reduced whenβ-catenin was knocked down.APC and GSK-3βwere both reduced from Axin immunoprecipitate in the cell clones overexpressed PLK1 .Conclusion PLK1 up-regulates vimentin and promotes EMT in ESCC cells probably by inhibiting the formation of protein degrada-tion complex and stabilizing β-catenin.
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Purpose To detect the expression of Nek2, Plk1 and Cdk1 in gastric carcinoma and to explore their correlation with clini-copathological factors. Methods We used immunohistochemistry to detect the protein expression of Nek2, Plk1 and Cdk1 in 80 cases of gastric carcinoma. Results The positive rates of Nek2, Plk1 and Cdk1 expressions in 80 cases of gastric carcinoma were 64%, 79% and 85% respectively. While the positive rates of Nek2, Plk1 and Cdk1 expressions in 20 cases normal gastric tissue were 15%, 10% and 20% respectively,with statistical significance (P0. 05). The expression of Plk1 and Cdk1 had a correlation with tumor size, differentiation of gastric cancer, invasion depth, lymph node metastasis and TNM stage (P0. 05). There were significant associations between Nek2 and Plk1, Plk1 and Cdk1, Nek2 and Cdk1 expression (P <0. 01). Conclusions Abnormal expression of Nek2, Plk1 and Cdk1 might be one of the mechanisms of tumorigenesis, especially of abnormal tumour proliferation. They may represent new potential targets for therapeutic intervention.
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Objective To investigate the effects of down-regulation of polo-like kinase-1 (PLK1) gene by RNA interference (RNAi) on the invasion of a human malignant melanoma cell line A375 and their possible mechanisms.Methods Cultured A375 cells were classified into several groups:blank control group receiving no treatment,liposome group transfected with lipofectamine only,and three siRNA groups transfected with three concentrations of a small interference RNA (siRNA) targeting PLK1 respectively.After additional culture,real time quantitative PCR and Western blot analysis were performed to quantify the expressions of PLK1 mRNA and protein in A375 cells respectively,Transwell invasion assay to evaluate the invasive capacity of A375 cells,agarose gel electrophoresis and terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labelling (TUNEL) to detect anoikis in A375 cells.The colony-forming capacity was also evaluated for A375 cells.Statistical analysis was carried out by one-factor analysis of variance.Results There was a significant decrease in PLK1 mRNA and protein expressions as well as in colony-forming units in the siRNA groups compared with the blank control group (all P ＜ 0.05).The invasive capacity of A375 cells was significantly inhibited in the siRNA groups with the number of migrating cells in Transwell assay being 39 ± 5,19 ± 5 and 9 ± 3 in A375 cells transfected with 3.125,6.250 and 12.500 nmol/L siRNAs respectively,compared to 56 ± 5 in the blank control group (all P ＜ 0.05).A characteristic DNA ladder was observed on agarose gel electrophoresis in the siRNA (6.250 nmol/L) group.Compared with the blank control group and liposome group,the three siRNA groups showed increased apoptotic index (3.86％ ± 0.35％ (3.125 nmol/L siRNA),7.35％ ± 0.36％ (6.250 nmol/L siRNA) and 17.56％ ± 0.38％ (12.500 nmol/L siRNA) vs.1.15％ ± 0.25％ (blank control group) and 1.18％ ± 0.22％ (liposome group),all P ＜ 0.05).Conclusions PLK1 siRNA can inhibit the invasion of malignant melanoma cells,likely by inducing anoikis in these cells.
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Objective To explore the expression and clinical significance of GSK-3β,PTEN and PLK1 in pediatric AML . Methods Experiment group was bone marrows of 33 cases incipient children with AML .Control group was 10 cases normal bone marrows .GSK-3β,PTEN and PLK1 gene expressions in BMMNC of the two groups were tested using semi quantitative reverse transcriptase polymerase chain reaction(RT-PCR)technique .GSK-3βprotein and P-GSK-3βexpressions were tested by ELISA .Re-sults The expression of GSK-3βmRNA ,GSK-3βprotein ,PLK1 mRNA in BMMNC of children with AML was higher than that of control group(P=0 .012 ;P= 0 .014 ;P= 0 .040);The expression of PTEN mRNA ,P-GSK-3β was lower than the control group (P=0 .012 ;P=0 .002);GSK-3βprotein had a negative correlation with PTEN mRNA (r= -0 .415 ,P=0 .016);GSK-3βmRNA , GSK-3βprotein had a positive correlation with PLK 1 mRNA(r=0 .388 ,P=0 .026;r=0 .427 ,P=0 .013) .The expression of GSK-3βprotein was high in which had high peripheral white blood cell counts ;both the expressions of GSK-3βmRNA and GSK-3βpro-tein were high in which had high risk ;but the expression of P-GSK-3βwere low .Conclusion In pediatric AML ,GSK-3βand PLK1 may play a role of oncogene and PTEN may play a role of tumor suppressor gene .
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Polo-like kinase 1(PLK1)is a serine/threonine kinase which is highly conserved, and its activity is elevated in many human tumor cell lines.Lots of reports have shown that PLK1 is critical for all stages of mitosis.The newest report finds that PLK1 is required for DNA synthesis, maintenance of DNA integrity, and prevention of cell death.PLK1 physically binds to the tumor suppressor p53 in mammalian cultured cells, and inhibits its transactivation activity as well as its function in activate checkpoint protein and its pro-apoptotic function.Also PLK1 is involved in the development of tumors, and Polo-like kinase1(PLK1)regulates IFN induction by MAVS, breakdown the innate immunity. Various of inhibitors of PLK1 show the feature of high performance and low toxicity, suggesting that PLK1 may be a feasible target for cancer therapy and immunity therapy.
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Objective To study the significance of polo like kinase1(plk1) mRNA and protein expression in gastric cancer tissues and the relationship between the plk1mRNA and protein expression level in gastric(cancer) tissues and the clinicopathological parameters.Methods Plk1mRNA and protein level were(respectively) measured by real-time PCR and Western blot in fresh tumor tissues and the normal gistric mucosa from 60 patients with gastric cancer.Results plk1mRNA and protein expression level in gastric cancer(tissues) was significantly higher than that in normal gastric tissues(P
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Objective:To investigate the effects of siRNA-mediated Plk1 depletion on COS-7 cells and the therapeutic potential of Plk1 in cancer treatment.Methods:Small interfering RNA(siRNA) were used to specifically deplete Plk1 in COS-7 cells.RT-PCR and immol/Lunoblotting analysis were used to verify the inhibitory effect of siRNA against Plk1.Trypan blue exclusion assay and FACS analysis were conducted to determine the effects of Plk1 depletion on the growth and cell cycle profile of COS-7 cells.Results:Synthetic siRNA duplexes against Plk1 were introduced into COS-7 cells,which subsequently resulted in a significant inhibition in Plk1 expression in the cells.Cell growth assay indicated that siRNA-mediated Plk1 depletion significantly inhibited the proliferation of COS-7 cells.FACS analysis revealed that Plk1 depletion caused cell cycle arrest,resulting into large population of sub-G1 cells.Conclusion:siRNA-mediated Plk1 depletion significantly inhibited the proliferation of COS-7 cells suggesting the therapeutic potential of Plk1 in cancer treatment.