ABSTRACT
ObjectiveTo study the effect of Qizhu Kang'ai prescription （QZAP） on the gluconeogenesis enzyme phosphoenolpyruvate carboxykinase 1 （PCK1） in the liver of mouse model of liver cancer induced by diethylnitrosamine （DEN） combined with carbon tetrachloride （CCl4） and Huh7 cells of human liver cancer， so as to explore the mechanism on regulating metabolic reprogramming and inhibiting cell proliferation of liver cancer cells. MethodDEN combined with CCl4 was used to construct a mouse model of liver cancer via intraperitoneal injection. A normal group， a model group， and a QZAP group were set up， in which QZAP （3.51 g·kg-1） or an equal volume of normal saline was administered daily by gavage， respectively. Serum and liver samples were collected after eight weeks of intervention. Serum alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， γ-glutamyltransferase （γ-GT）， and alpha-fetoprotein （AFP） in mice were detected to evaluate liver function changes of mice in each group. Hematoxylin-eosin （HE） staining and Sirius red staining were used to observe pathological changes in liver tissue. In the cell experiment， Huh7 cells were divided into blank group， QZAP low， medium， and high dose groups and/or PCK1 inhibitor （SKF-34288 hydrochloride） group， and Sorafenib group. The corresponding drug-containing serum and drug treatment were given， respectively. Cell counting kit-8 （CCK-8） method， colony formation experiment， Edu fluorescent labeling detection， intracellular adenosine triphosphate （ATP） content detection， and cell cycle flow cytometry detection were used to evaluate the proliferation ability， energy metabolism changes， and change in the cell cycle of Huh7 cells in each group. Western blot was used to detect the protein expression levels of PCK1， serine/threonine kinase （Akt）， phosphorylated Akt （p-Akt）， and cell cycle-dependent protein kinase inhibitor 1A （p21）. ResultCompared with the model group， the pathological changes such as cell atypia， necrosis， and collagen fiber deposition in liver cancer tissue of mice in the QZAP group were alleviated， and the number of liver tumors was reduced （P<0.01）. The serum ALT， AST， γ-GT， and AFP levels were reduced （P<0.01）. At the cell level， compared with the blank group， low， medium， and high-dose groups of QZAP-containing serum and the Sorafenib group could significantly reduce the survival rate of Huh7 cells （P<0.01） and the number of positive cells with Edu labeling （P<0.01） and inhibit clonal proliferation ability （P<0.01）. The QZAP groups could also reduce the intracellular ATP content （P<0.05） and increase the distribution ratio of the G0/G1 phase of the cell cycle （P<0.05） in a dose-dependent manner. Compared with the model group and blank group， PCK1 and p21 protein levels of mouse liver cancer tissue and Huh7 cells in the QZAP groups were significantly reduced （P<0.05，P<0.01）， and the p-Akt protein level was significantly increased （P<0.01）. Compared with the blank group， the ATP content and cell survival rate of Huh7 cells in the SKF-34288 hydrochloride group were significantly increased （P<0.05）， but there was no statistical difference in the ratio of Edu-positive cells and the proportion of G0/G1 phase distribution. Compared with the SKF-34288 hydrochloride group， the QZAP combined with the SKF-34288 hydrochloride group significantly reduced the ATP content， cell survival rate， and Edu-positive cell ratio of Huh7 cells （P<0.05） and significantly increased the G0/G1 phase distribution proportion （P<0.05）. ConclusionQZAP may induce the metabolic reprogramming of liver cancer cells by activating PCK1 to promote Akt/p21-mediated tumor suppression， thereby exerting an anti-hepatocellular carcinoma proliferation mechanism.
ABSTRACT
Malaria is one of the most devastating infectious diseases that caused millions of clinical cases annually despite decades of prevention efforts. Recent cases of Plasmodium falciparum resistance against the only remaining class of effective antimalarial (artemisinin) in South East Asia may soon pose a significant threat. Hence, the identification of new antimalarial compounds with a novel mode of action is necessary to curb this problem. Protein kinase has been implicated as a valid target for drug development in diseases such as cancer and diabetes in humans. A similar approach is now recognized for the treatment of protozoan-related disease including malaria. Few Plasmodium protein kinases that are not only crucial for their survival but also have unique structural features have been identified as a potential target for drug development. In this review, studies on antimalarial drug development exploiting the size of Plasmodium protein kinase ATP gatekeeper over the past 15 years are mainly discussed. The ATP-binding site of Plasmodium protein kinases such as Pf CDPK1, Pf CDPK4, Pf PKG, Pf PK7, and Pf PI4K showed great potential for selective and multi-target inhibitions owing to their smaller or unique ATP-gatekeeper amino acid subunits compared to that of human protein kinase. Hence it is a feasible solution to identify a new class of active antimalarial agents with a novel mode of action and longer clinical life-span.
ABSTRACT
Malaria is one of the most devastating infectious diseases that caused millions of clinical cases annually despite decades of prevention efforts. Recent cases of Plasmodium falciparum resistance against the only remaining class of effective antimalarial (artemisinin) in South East Asia may soon pose a significant threat. Hence, the identification of new antimalarial compounds with a novel mode of action is necessary to curb this problem. Protein kinase has been implicated as a valid target for drug development in diseases such as cancer and diabetes in humans. A similar approach is now recognized for the treatment of protozoan-related disease including malaria. Few Plasmodium protein kinases that are not only crucial for their survival but also have unique structural features have been identified as a potential target for drug development. In this review, studies on antimalarial drug development exploiting the size of Plasmodium protein kinase ATP gatekeeper over the past 15 years are mainly discussed. The ATP-binding site of Plasmodium protein kinases such as Pf CDPK1, Pf CDPK4, Pf PKG, Pf PK7, and Pf PI4K showed great potential for selective and multi-target inhibitions owing to their smaller or unique ATP-gatekeeper amino acid subunits compared to that of human protein kinase. Hence it is a feasible solution to identify a new class of active antimalarial agents with a novel mode of action and longer clinical life-span.
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Objective To investigate the effects of three mitogen-activated protein kinase (MAPK) inhibitors on the expressions of transforming growth factor-β1 (TGF-β1),α-smooth actin (α-SMA) mRNA and protein in human liver stellate cells (LX-2 cells) activated by sodium arsenite.Methods Cultured in vitro LX-2 cells in the logarithmic growth stage were exposed to sodium arsenite at 0.0 (control),2.5,5.0,10.0,20.0,40.0,80.0 μmol/L for 24 h,respectively,and the cell survival rate was determined by CCK-8 assay.According to the results of the study,LX-2 cells were divided into 5 groups:control group,sodium arsenite group,extracellular signal regulation kinase (ERK) inhibition group,c-Jun amino-terminal kinase (JNK) inhibition group,and p38 inhibition group.LX-2 cells were pre-treated with 10.0 μmol/L ERK,JNK,p38 kinase inhibitors (PD98059,SP600125,SB203580) for 30 min in the 3 inhibition groups,and then 20.0 μmol/L sodium arsenite for 24 h.The control group was not treated with sodium arsenite and inhibitors.Sodium arsenite group was not treated with inhibitors.Then mRNA and protein expression levels of TGF-β1 and α-SMA in LX-2 cells were determined by Western blotting and real-time PCR,respectively.Results The survival rates of LX-2 cells in 5.0,10.0,20.0,40.0,80.0 μmol/L sodium arsenite groups were [(92.35 ± 0.92)％,(84.06 ± 0.84)％,(74.27 ± 0.74)％,(59.57 ± 0.60)％,(27.77 ± 0.23)％],which were significantly lower than that of the control group [(100.00 ± 0.00)％,P ＜ 0.05].It was found that the expressions of TGF-β1,o-SMA mRNA and protein of sodium arsenite group were higher than those of the control group (P ＜ 0.01).The expressions of TGF-β1,α-SMA mRNA and protein of the three inhibition groups were lower than those of the sodium arsenite group (P ＜ 0.05).Conclusions Arsenic exposure can cause abnormally high expressions of TGF-β1,α-SMA mRNA and protein in LX-2 cells.Intervention with three MAPK inhibitors can improve the effects of arsenic induced LX-2 cells activation on the expressions of TGF-β1,α-SMA mRNA and protein.
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@#Glaucoma is a leading cause of irreversible blindness, which is characterized by characteristic optic atrophy and visual field defects. Elevated intraocular pressure(IOP)is a primary risk factor of glaucoma, while the main cause of elevated IOP lies in the increased aqueous outflow resistance in pathological trabecular meshwork(TM), which is the conventional outflow pathway of aqueous humor. Rho-associated protein kinase inhibitor(ROCKi)is the IOP-lowering drug that is directly acting on the TM. The TM cell morphology, cell movement, cytokinesis and cell contraction by alteration of cytoskeleton can be changed by ROCKi to increase aqueous humor outflow facility and decrease IOP. ROCKi is now approved for clinical use in the United States and Japan. Meanwhile, it might play a role in optic nerve protection through increasing retinal vascular perfusion and promoting optic nerve regeneration. In addition, it decreases the possibility of filtration bleb scarring. Therefore, ROCKi has become a new pharmacological option to treat glaucoma. This article reviews the Rho-Rho kinase signaling pathway, the mechanism of ROCKi and its clinical application.
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AIM:To study the effect of Aurora protein kinase inhibitor VX-680 on homogeneous adhesion and migration ability in human hepatocellular carcinoma cell line HepG 2.METHODS:The HepG2 cell were divided into ex-perimental group and control group, respectively.VX-680 was used in experimental groups at 3 concentrations(3.125 μmol/L group,6.25 μmol/L group and 12.5 μmol/L group).DMSO was used in the control group.The effects of VX-680 at different concentrations on the adhesion ability of human hepatocellular carcinoma HepG 2 cells were observed by cell slow aggregation test and separation experiment.The effects of VX-680 at different concentrations on the migration ability of HepG2 cells was detected by wound healing assay.The expression of E-cadherin in HepG2 cells was detected by Western blot.RESULTS:The results of the slow aggregation test showed that compared with the control group,the number of cell clumps formed in experimental groups was significantly decreased(P<0.01).The results of separation experiment showed that the ratio of NTC/NTEgradually decreased with the increased concentration of VX-680.The results of wound healing as-say showed that as the concentration of VX-680 increased, the cell scratch healing ability gradually weakened compared with control group.The results of Western blot showed that the protein expression of E-cadherin in the HepG2 cells in-creased with the increased concentration of VX-680(P<0.05).CONCLUSION:VX-680 increases the homogeneous ad-hesion and inhibits the migration of HepG 2 cells.
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Protein kinases are key components of cell signaling networks and thereby regulate fundamental biological processes such as cellular growth, proliferation, metabolism and survival. Kinome refers to all kinases in cells or tissue and “kinomics”is the global analysis of kinome with respect to abundance, activity, substrate specificity, phosphorylation pattern and mutational status. Human kinome currently contains 568 members, nearly half of which can be mapped to disease loci and deregulation of kinase activity by gene amplifica-tion or mutations has been implicated in diseases such as inflammation, diabetes and cancer. Therefore, human kinome is being recognized as a potentially rich source of drug targets. Kinase inhibitors have been successfully used to treat many kinds of advanced cancers. Chemi-cal proteomics is emerging as a novel comprehensive kinome approach that combines an immobilized inhibitor affinity pull-down approach with mass spectrometry-based proteomics for kinase identification, quantification and phosphorylation analysis under physiological condi-tion. Commonly, one or multiple broad-spectrum kinase inhibitors are covalently immobilized on a biocompatible matrix such as sepharose to enrich all kinases in cells or tissue and then the kinases are identified and quantified by mass spectrometry analysis. It can be used to study the specificity of kinase inhibitor drug, drug candidate or drug resistance mechanism, which can help to understand the mechanism and find combinational drug target. Large-scale unbiased kinome and cancer kinome study will facilitate new drug target discovery and correlate tumor tissue kinome profiles with response to therapy and therefore may be used for future therapy selection in personalized medicine. In this paper, the human kinome, kinase, kinase inhibitor and cancer, chemical proteomics based kinome study progress and its applications in drug discovery are reviewed.
ABSTRACT
Protein kinases are key components of cell signaling networks and thereby regulate fundamental biological processes such as cellular growth, proliferation, metabolism and survival. Kinome refers to all kinases in cells or tissue and "kinomics" is the global analysis of kinome with respect to abundance, activity, substrate specificity, phosphorylation pattern and mutational status. Human kinome currently contains 568 members, nearly half of which can be mapped to disease loci and deregulation of kinase activity by gene amplification or mutations has been implicated in diseases such as inflammation, diabetes and cancer. Therefore, human kinome is being recognized as a potentially rich source of drug targets. Kinase inhibitors have been successfully used to treat many kinds of advanced cancers. Chemical proteomics is emerging as a novel comprehensive kinome approach that combines an immobilized inhibitor affinity pull-down approach with mass spectrometry-based proteomics for kinase identification, quantification and phosphorylation analysis under physiological condition. Commonly, one or multiple broad-spectrum kinase inhibitors are covalently immobilized on a biocompatible matrix such as sepharose to enrich all kinases in cells or tissue and then the kinases are identified and quantified by mass spectrometry analysis. It can be used to study the specificity of kinase inhibitor drug, drug candidate or drug resistance mechanism, which can help to understand the mechanism and find combinational drug target. Large-scale unbiased kinome and cancer kinome study will facilitate new drug target discovery and correlate tumor tissue kinome profiles with response to therapy and therefore may be used for future therapy selection in personalized medicine. In this paper, the human kinome, kinase, kinase inhibitor and cancer, chemical proteomics based kinome study progress and its applications in drug discovery are reviewed.
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OBJECTIVE: To evaluate the effect of protein kinase inhibitor on luteolin glucuronidation. METHODS: LS174T cells were treated with either curcumin or calphostin C of various concentrations. The glucuronidation metabolites of luteolin, catalyzed by the S9 of treated LS174T cells, was measured by HPL C. The expression of UGT1A in treated LS174T cells was determined by realtime PCR and western blot. RESULTS: A rapid down-regulation of luteolin glucuronidation catalyzed by LS174T cells following curcumin and calphostin C treatment was observed. However, there was no effect on the expression of UGT1A in treated LS174T cells. CONCLUSION: The glucuronidation of luteolin can be suppressed by protein kinase inhibitor indirectly. The suppression may be caused by abnormal phosphorylation of UGTs, which are catalyzed by protein kinase.