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The p21 activated kinase 4 (PAK4) is serine/threonine protein kinase that is critical for cancer progression. Guided by X-ray crystallography and structure-based optimization, we report a novel subseries of C-3-substituted 6-ethynyl-1H-indole derivatives that display high potential and specificity towards group II PAKs. Among these inhibitors, compound 55 exhibited excellent inhibitory activity and kinase selectivity, displayed superior anti-migratory and anti-invasive properties against the lung cancer cell line A549 and the melanoma cell line B16. Compound 55 exhibited potent in vivo antitumor metastatic efficacy, with over 80% and 90% inhibition of lung metastasis in A549 or B16-BL6 lung metastasis models, respectively. Further mechanistic studies demonstrated that compound 55 mitigated TGF-β1-induced epithelial-mesenchymal transition (EMT).
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Objective To explore the regulatory mechanism of microRNA-224 (miR-224) in the apoptosis and proliferation of human cholangiocarcinoma (CC) cells and examine whether the pathway of miR-224-HOXD10-PAK4 exist in cc cells or not.Methods The expression of miR-224 in CC and adjacent normal tissue were measured by RT-PCR.QBC939 cells were chosen and divided into two groups:cell groups transducted with negative control viruses (NC group) and cell groups transducted with target gene miRNA-down virus (DOMN group).The rate of apoptosis and the number of colonies formed in QBC939 cell lines of DOWN group and NC group were counted.The expression of miR-224,API5,PAK4 and HOXD10 in QBC939 cell lines of DOWN group and NC group were measured by RT-PCR and western blotting.Results (1) MiR-224 expression was higher in CC tissues than that in normal bile duct tissues (P ＜ 0.05).(2) In QBC939 cells lines,miR-224 expression was lower in DOMN group than that in NC group.The clone formation of QBC939 cells in DOWN group was significantly lower than that of NC group.The apoptosis rate of QBC939 cells of DOWN group was significantly higher than that of NC group.(3) The expression of API5 and HOXD10 in QBC939 cells of DOWN group was significantly higher than that of NC group in the RT-PCR and western blotting experiments.(4) The expression of PAK4 in QBC939 cells of DOWN group was significantly lower than that of NC group in the RT-PCR and western blotting.Conclusions MiR-224 has anti-apoptotic effects and can promote the proliferation of QBC939 cells and may serve as an important regulator of QBC939 cell proliferation in vitro.There are miR-224-HOXD10-PAK4 signaling pathways in cholangiocarcinoma.
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PURPOSE: p21-activated kinases (PAKs) are involved in cytoskeletal reorganization, gene transcription, cell proliferation and survival, and oncogenic transformation. Therefore, we hypothesized that PAK expression levels could predict the sensitivity of pancreatic cancer cells to gemcitabine treatment, and PAKs could be therapeutic targets. MATERIALS AND METHODS: Cell viability inhibition by gemcitabine was evaluated in human pancreatic cancer cell lines (Capan-1, Capan-2, MIA PaCa-2, PANC-1, Aspc-1, SNU-213, and SNU-410). Protein expression and mRNA of molecules was detected by immunoblot analysis and reverse transcription polymerase chain reaction. To define the function of PAK4, PAK4 was controlled using PAK4 siRNA. RESULTS: Capan-2, PANC-1, and SNU-410 cells were resistant to gemcitabine treatment. Immunoblot analysis of signaling molecules reported to indicate gemcitabine sensitivity showed higher expression of PAK4 and lower expression of human equilibrative nucleoside transporter 1 (hENT1), a well-known predictive marker for gemcitabine activity, in the resistant cell lines. Knockdown of PAK4 using siRNA induced the upregulation of hENT1. In resistant cell lines (Capan-2, PANC-1, and SNU-410), knockdown of PAK4 by siRNA resulted in restoration of sensitivity to gemcitabine. CONCLUSION: PAK4 could be a predictive marker of gemcitabine sensitivity and a potential therapeutic target to increase gemcitabine sensitivity in pancreatic cancer.
Subject(s)
Humans , Cell Line , Cell Proliferation , Cell Survival , Equilibrative Nucleoside Transporter 1 , p21-Activated Kinases , Pancreatic Neoplasms , Phosphotransferases , Polymerase Chain Reaction , Reverse Transcription , RNA, Messenger , RNA, Small Interfering , Up-RegulationABSTRACT
Objective:To analyze the inhibiting mechanism of microRNA-199a/b-3p ( miR-199a/b-3p) on cell motility of breast cancer cells.Methods:The expression of PAK4 in MDA-MB-231 cells transfected with miR-199a/b-3p was detected by Western blot.The biology behaviors of MDA-MB-231 cells transfected with miR-199a/b-3p or PAK4 SiRNA were analysed by cell migration assay,invasion assay and protrusion dynamics.Results: MiR-199a/b-3p could suppress the expression of PAK 4 in MDA-MB-231 cells.Comparing with normal control ,miR-199a/b-3p or PAK4 SiRNA could suppress the migration ,invasion and membrane protrusion of MDA-MB-231 cells.Conclusion:miR-199a/b-3p could suppress the expression of PAK4,which are considered key breast cancer suppressors and inhibit the cell motility of breast cancer cells.
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Objective:To analyze the effect of PAK4 interruption by microRNA-199a/b-3p (miR-199a/b-3p) on migration and invasion of hepatocellular carcinoma (HCC).Methods: To test targeting of PAK4 by miR-199a/b-3p,we used luciferase assay in HEK293T cells cotransfected miR-199a/b-3p mimcs and pmirGLO-PAK4 3′UTR.The expression of PAK4 in SMMC-7721 transfected with miR-199a/b-3p was detected by Western blot.The biology behaviors of SMMC-7721 cells transfected with miR-199a/b-3p or PAK4 Si were analysed by cell migration assay and invasion assay.Results:MiR-199a/b-3p could suppress the mRNA and protein ex-pression of PAK4 by targeting PAK4 3′UTR,and the downregulating PAK 4 expression suppress the migration and invasion of SMMC-7721 cells.Conclusion: MiR-199a/b-3p could suppress the expression of PAK 4, which are considered key HCC suppressors and inhibit the migration and invasion of HCC cells.
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Objective To construct the expression plasmid of human PAK4 gene and identify its recombinant protein expression.Methods Total RISA was extracted from human breast cancer MCF-7 cells.The hPAKA coding sequence was amplified by polymerase chain reaction (PCR) method and subcloned into pEBG vector.After the target region was sequenced.the plasmid was transfected into HEK293 cell line.The expression of the recombinant plasmid in HEK293 cells was proved by Western blot.Results hPAKA had been constructed into the expressing vector pEBG successfully.The length of the fragment was 1 800 bp,identified by restriction enzymes digestion.The expression of pEBG-hPAK4 fusion protein was detected by Western blot,with a molecular weight 94 KDa and was pulled down by Qutathione Sepharose 4B.Conclusion The recombinant plasmid was successfully cloned into eukaryotic expressing vector,and the expression of pEBG-hPAK4 fusion protein was identified and pulled down by Glutathione Sepharose 4B.