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BACKGROUND: Tumor microenvironment (TME) plays a vital role in determining the outcomes of radiotherapy. As an important component of TME, vascular endothelial cells are involved in the perivascular resistance niche (PVRN), which is formed by inflammation or cytokine production induced by ionizing radiation (IR). Protein kinase CK2 is a constitutively active serine/threonine kinase which plays a vital role in cell proliferation and inflammation. In this study, we investigated the potential role of CK2 in PVRN after IR exposure. RESULT: Specific CK2 inhibitors, Quinalizarin and CX-4945, were employed to effectively suppressed the kinase activity of CK2 in human umbilical vein endothelial cells (HUVECs) without affecting their viability. Results showing that conditioned medium from IR-exposed HUVECs increased cell viability of A549 and H460 cells, and the pretreatment of CK2 inhibitors slowed down such increment. The secretion of IL-8 and IL-6 in HUVECs was induced after exposure with IR, but significantly inhibited by the addition of CK2 inhibitors. Furthermore, IR exposure elevated the nuclear phosphorylated factor-κB (NF-κB) p65 expression in HUVECs, which was a master factor regulating cytokine production. But when pretreated with CK2 inhibitors, such elevation was significantly suppressed. CONCLUSION: This study indicated that protein kinase CK2 is involved in the key process of the IR induced perivascular resistant niche, namely cytokine production, by endothelial cells, which finally led to radioresistance of non-small cell lung cancer cells. Thus, the inhibition of CK2 may be a promising way to improve the outcomes of radiation in nonsmall cell lung cancer cells.
Subject(s)
Humans , Carcinoma, Non-Small-Cell Lung/radiotherapy , Endothelial Cells/radiation effects , Casein Kinase II/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Lung Neoplasms/drug therapy , Endothelium, Vascular/cytology , Blotting, Western , Cytokines/biosynthesis , Anthraquinones/pharmacology , Naphthyridines/pharmacologyABSTRACT
Objective To evaluate the effect of a protein kinase CK2 inhibitor on intracellular levels of reactive oxygen species and DNA double?stand break in human non?small cell lung cancer H460 cells. Methods H460 cells were exposed to 0, 12?5, 25.0, and 50.0μmol/L quinalizarin, a specific inhibitor of protein kinase CK2, for 24 hours. The changes in protein and mRNA levels of CK2 subunits were measured. Flow cytometry was used to measure changes in the intracellular levels of reactive oxygen species in H460 cells after 4 or 24 hours of quinalizarin treatment. Immunofluorescence assays were performed to determine the effect of the CK2 inhibitor onγ?H2 AX expression and the average fluorescent number ofγ?H2 AX foci in H460 cells. Comparison was made by analysis of variance and t test. Results There were no significant differences in protein or mRNA levels of CK2 subunits in H460 cells after quinalizarin treatment ( CK2α,0μmol vs. 12?5 μmol/L, P=0?966;0 μmol/L vs. 25 μmol/L, P=0?355;0 μmol/L vs. 50 μmol/L, P=0?864, CK2α’ , 0 μmol/L vs. 12?5μmol/L,P=0?409;0μmol/L vs. 25μmol/L,P=0?833;0μmol/L vs. 50 μmol/L, P=0?0. 746, CK2β, 0 μmol/L vs. 12?5 μmol/L, P=0?532;0 μmol/L vs. 25 μmol/L, P=0?830;0 μmol/L vs. 50 μmol/L, P= 0?061 ) . The intracellular levels of reactive oxygen species were substantially elevated in H460 cells with the increase in quinalizarin concentration and treatment time. Different concentrations of quinalizarin resulted in dose?and time?dependent increases in the numbers of γ?H2 AX foci after 4 and 24 hours of treatment ( treated by Quianlizarin for 4 or 24 h, 0 μmol/L vs. 12?5μmol/L,12?5 μmol/L vs. 25 μmol/L, 25 μmol/L vs. 50 μmol/L, all P=0?000, concentration is 12?5μmol/L,25 μmol/L or 50 μmol/L, 4 h vs. 24 h, all all P=0?000 ) . Conclusions Quinalizarin can increase the intracellular levels of reactive oxygen species and DNA double?stand break in H460 cells by inhibition of protein kinase CK2 activity. This study provides a theoretical basis for using quinalizarin as a potential radiosensitizer for lung cancer.
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BACKGROUND: Protein kinase CK2 is widely expressed in eukaryotic cells, and plays an important role in cell proliferation, migration, apoptosis, etc. The aim of the current study is to explore how Quinalizarin, a specific CK2 inhibitor, affects the cell proliferation, migration, and apoptosis of different pathological and genetic types of human lung cancer cell lines. MATERIALS AND METHODS: MTT assays were performed to evaluate the cell viability after being treated by Quinalizarin. Transwell migration assays were used to assess whether Quinalizarin could suppress cell migration. Flow cytometry was employed to test the apoptosis rate of different cells. RESULTS: After being treated by Quinalizarin, the viability of different pathological types of lung cancer cells (H446, H460, A549) were significantly suppressed in a time and dose‑dependent manner. More interestingly, in a serial of human lung adenocarcinoma cell lines with different epidermal growth factor receptor (EGFR) mutation status, Quinalizarin was shown to have a much better ability to reduce the viability of cells with EGFR sensitive mutation than those with resistance mutations. Meanwhile, we also found that the cell migration of different pathological types of lung cancer cells (H446, H460, A549) was significantly decreased by Quinalizarin dose‑dependently. In addition, the apoptosis rates in those cells were proved to be increased after exposed to Quinalizarin. CONCLUSIONS: Quinalizarin, the specific CK2 inhibitor, could reduce cell viability with emphasis on adenocarcinoma cells harboring EGFR sensitive mutation, suppresses migration, and accelerates apoptosis in different human lung cancer cell lines.
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Objective To observe the effect of 7 flavonoids on recombinant human protein kinase CK2 holoenzyme activity and investigate their structure-activity relationship. Methods Recombinant hu-man protein kinase CK2 α' and β subunits were mixed at equal molar ratio to reconstitute CK2 holoen-zyme. The CK2 activity was assayed by detecting incorporation of 32p of [γ-32P] ATP into the substrate for the inhibitory effect by flavonoids and calculation of IC50 was performed according to probability unit (PROBIT) method. Results Myricetin, quercetin, morin, luteolin, kaempferol, apigenin, and chrysin were shown to obviously inhibit recombinant CK2 holoenzyme activity in a concentration-dependent man-ner with IC50 values of 1.18, 0.51, 16.16, 0.86, 1.88, 1.72, and 13.63 umol/L, respectively. Myricetin, quercetin, luteolin, kaempferol, and apigenin were more effective than DRB and A3, which were known as CK2 inhibitors in vitro. Whereas morin and chrysin displayed a similar effect to DRB. Structure-activity study indicated that the major structural requirements for the potent inhibition of CK2 by these flavonoids were hydroxyl group at position 6, 3' and 4'. Different from these requirements, absence of a hydroxyl group at position 3 did not modify their inhibitory potency, while addition of hydroxyl groups at positions 2' or 5' was detrimental to the inhibitory effect on CK2. Conclusion The inhibitory effect of flavonoid on protein kinase CK2 in vitro may be determined by the position of their hydroxyl groups.
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AIM To study the expression, purification and activity assay of recombinant mouse protein kinase CK2? subunit from Escherichia coli. METHEDS The recombinant plasmid containing mouse protein kinase CK2? subunit cDNA constructed successfully was transformed into Escherichia coli BL21 (DE3) and specifically induced by IPTG. The recombinant mouse CK2? subunit was sequentially purified by DE 52, P11 phosphocellulose and Heparin Sepharose chromatography. The purified recombinant protein was analysed by SDS PAGE. RESULTS One protein with molecular mass of 42 ku was overexpressed by inducing ITPG. The recombinant protein was composed of approximately 30 6% of the total bacterial proteins. From 278 mg soluble proteins, the yield of the CK2? protein was 4 7 mg. SDS PAGE analysis of the purified recombinant protein showed only one band in agreement with native mouse CK2? subunit. The recombinant mouse CK2? and ? subunits were mixed at the same molar ratio. The produced CK2 holoenzyme displayed full activity. The characteristics and functions of reconstituted CK2 holoenzyme were consistent with those of the given native CK2. CONCLUSION The recombinant protein is mouse protein kinase CK2? subunit.
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Research of cell cycle is the bas is of investigating the organism growth,development,heredity and other medical associated events. The molecular basis of cell cycle control is the regulator control system with CDK-Cyclin-CKI as the core.Protein kinase CK2 is one of the most conservative protein kinase during evolution and more and more researches have proved that protein kinase CK2 plays an important role in cell cycle control.
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AIM: To study the effects and kinetics of sodium quercetin-7-sulphate (SQMS) on recombinant human protein kinase CK2 holoenzyme. METHODS: The recombinant human CK2 holoenzyme activity was assayed by detecting incorporation of 32p of [γ-32P]ATP into the substrate at various conditions. RESULTS: SQMS was shown to strongly inhibit the holoenzyme activity of recombinant human protein kinase CK2 with an IC50 of 1.37 μmol·L-1, Kinetic studies of SQMS on recombinant human CK2 showed: the inhibition was competitive with ATP and noncompetitive with casein. CONCLUSION: SQMS is an inhibitor of protein kinase CK2, and the inhibitory action of SQMS on CK2 is competitive with ATP and noncompetitive with casein.
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Object To study the direct effect of quercetin on recombinant human protein kinase CK2 holoenzyme and its kinetics. Methods Recombinant human protein kinase CK2? and ? subunits were cloned and expressed by gene engineering, and were purified. The two subunits were mixed at the same molar ratio, thus reconstituting CK2 holoenzyme, which displayed the maximum bioactivity. The CK2 activity was assayed by detecting incorporation of 32 P of [? 32 P] ATP into the substrate in the various conditions. Results The recombinant human protein kinase CK2 was the second messenger (Ca 2+ , cAMP and cGMP) independent protein kinase, the characterization and function of the reconstituted holoenzyme were consistent with those of native CK2. It was found that quercetin strongly inhibited the holoenzyme activity of recombinant human protein kinase CK2 with an IC 50 of 522 nmol/L, which was much more effective than DRB and A3, known as CK2 special inhibitors. Kinetic studies of quercetin on recombinant human protein kinase CK2 showed: the inhibition was competitive with ATP and noncompetitive with casein. Conclusion Quercetin is a potent inhibitor of recombinant human protein kinase CK2. The inhibition may be another molecular mechanism of antitumor effect of quercetin. This study provides a simple and rapid screening method for the development of more effective inhibitors of recombinant human protein kinase CK2.
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AIM To study the effects and kinetics of sodium quercetin 7 sulphate (SQMS) on recombinant human protein kinase CK2 holoenzyme. METHODS The recombinant human CK2 holoenzyme activity was assayed by detecting incorporation of 32 P of [? 32 P]ATP into the substrate at various conditions. RESULTS SQMS was shown to strongly inhibit the holoenzyme activity of recombinant human protein kinase CK2 with an IC 50 of 1 37 ?mol?L -1 , Kinetic studies of SQMS on recombinant human CK2 showed: the inhibition was competitive with ATP and noncompetitive with casein. CONCLUSIONSQMS is an inhibitor of protein kinase CK2, and the inhibitory action of SQMS on CK2 is competitive with ATP and noncompetitive with casein.
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AIM To study the direct effect of amiloride on recombinant human protein kinase CK2 holoenzyme and its kinetics. METHODS Recombinant human protein kinase CK2 ? and ? subunits were cloned,expressed and purified by gene engineering. The two subunits were mixed at the same molar ratio to form CK2 holoenzyme,which displayed the maximum biological activity. CK2 activity was assayed by detecting incorporation of 32 P of [? 32 P]GTP into the substrate in various conditions. RESULTS Amiloride inhibited the holoenzyme activity of recombinant human protein kinase CK2 with an IC 50 of 257 57 ?mol?L -1 . Kinetic studies of amiloride on recombinant human CK2 showed that the inhibition was competitive with GTP with the K i of 236 30 ?mol?L -1 ,and noncompetitive with casein with the K i of 163 63 ?mol?L -1 . CONCLUSION Amiloride inhibits the activity of cAMP or ATP dependent enzymes as well as the kinase with GTP as phosphate donor,and the recombinant human protein kinase CK2 may be used as a molecular target for simple screening and development of effective inhibitors of CK2.
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Protein kinase CK2 is a ubiquitously expressed protein serine/threonine kinase present in all eukaryotes. It can intervene transversally into a multitude of signal pathways.It plays a key role in regulation of cell growth and apoptosis in a hierarchical and vertical manner. It was demonstrated that modest deregulation in the CK2 expression impacts a potent oncogenic potential to the cells.Moreover,inhibition of CK2 with specific chemical inhibitors or CK2-targeting antisense RNA may result in depression of tumor. Thus, studying the regulation mechanism of CK2 in tumorigenesis and developing new anti-cancer drugs by employing specific strategies against CK2 are of significant clinical value.