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Isoliquiritigenin suppresses human T Lymphocyte activation via covalently binding cysteine 46 of I?B kinase.

ABSTRACT: The efficacious practice of precision personalized medicine requires a more exact understanding of the molecular mechanisms of drug, hence then it is necessary to identify the binding site of the drugs derived from natural sources. In the study, we investigated the suppressive effect and underlying mechanism of isoliquiritigenin (2',4',4-trihydroxychalcone; ILG), a phyto-flavonoid, on human T lymphocyte activation in vitro and in vivo. The results showed that ILG dose-dependently suppressed human T cell activation via suppressing I?B? phosphorylation and degradation, NF-?B nuclear translocation and IKK? activity. Molecular docking results predicted that cysteine 46 (Cys-46) is probably the binding site of ILG on IKK?, and this prediction has been validated by competition assay and kinase assay. To further verify the binding site of this compound in vivo, IKK?C46A transgenic (IKK?C46A) mice were generated. We found that ILG had a less potent immune-suppressive effect in homozygous IKK?C46A mice than IKK? wild type (IKK? wt) littermates with the delay-type hypersensitivity (DTH), suggesting that ILG cannot significantly suppress the inflammation due to the mutation of Cys-46 in the transgenic mice. Collectively, our findings indicate that the ILG inhibited T cell activation in vivo and in vitro via directly binding to IKK? Cys46.

SUBMITTER: Yan F

PROVIDER: S-EPMC5470962 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Isoliquiritigenin suppresses human T Lymphocyte activation via covalently binding cysteine 46 of IκB kinase.

Yan Fenggen F Yang Fen F Wang Rui R Yao Xiao Jun XJ Bai Liping L Zeng Xing X Huang JiaJun J Wong Vincent Kam Wai VKW Lam Christopher Wai Kei CWK Zhou Hua H Su Xiaohui X Liu Juan J Li Ting T Liu Liang L

Oncotarget 20170501 21

The efficacious practice of precision personalized medicine requires a more exact understanding of the molecular mechanisms of drug, hence then it is necessary to identify the binding site of the drugs derived from natural sources. In the study, we investigated the suppressive effect and underlying mechanism of isoliquiritigenin (2',4',4-trihydroxychalcone; ILG), a phyto-flavonoid, on human T lymphocyte activation in vitro and in vivo. The results showed that ILG dose-dependently suppressed huma ...[more]

PMID: 27626700

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Project description:BackgroundIsoliquiritigenin (ISL), a natural flavonoid isolated from the root of licorice (Glycyrrhiza uralensis), has shown various pharmacological properties including anti-oxidant, anti-inflammatory and anti-cancer activities. MicroRNAs (miRNAs), a class of small non-coding RNAs, have been reported as post-transcriptional regulators with altered expression levels in melanoma. This study aims to investigate the anti-melanoma effect of ISL and its potential mechanism.MethodsWe investigated the effect of ISL on the proliferation and apoptosis of melanoma cell lines with functional assays, such as CCK-8 assay, colony formation assay and flow cytometry. The protein level of apoptosis related genes were measured by western blotting. High-throughput genome sequencing was used for screening differentially expressed miRNAs of melanoma cell lines after the treatment of ISL. We performed functional assays to determine the oncogenic role of miR-301b, the most differentially expressed miRNA, and its target gene leucine rich repeats and immunoglobulin like domains 1 (LRIG1), confirmed by bioinformatic analysis, luciferase reporter assay, western blotting and immunohistochemical assay in melanoma. Immunocompromised mouse models were used to determine the role of miR-301b and its target gene in melanoma tumorigenesis in vivo. The relationship between miR-301b and LRIG1 was further verified in GEO data set and tissue specimens.ResultsFunctional assays indicated that ISL exerted significant growth inhibition and apoptosis induction on melanoma cells. MiR-301b is the most differentially expressed miRNA after the treatment of ISL and significantly downregulated. The suppressive effect of ISL on cell growth is reversed by ectopic expression of miR-301b. Intratumorally administration of miR-301b angomir enhances the inhibitory effect of ISL on tumor growth in vivo. Bioinformatic analysis showed that miR-301b may target LRIG1, miR-301b suppresses the luciferase activity of reporter constructs containing 3'UTR of LRIG1 as well as the expression level of LRIG1. And the anti-cancer effect of ISL is mitigated when LRIG1 is silenced in vivo and in vitro. Analysis of the melanoma samples obtained from patients shows that LRIG1 is negatively correlated with miR-301b.ConclusionsISL may inhibit the proliferation of melanoma cells by suppressing miR-301b and inducing its target LRIG1.

Project description:The aim of the present study was to investigate whether an increase in cyclin-dependent kinase 2 (CDK2) activity is involved in apoptosis of human bladder cancer T24 cells induced by isoliquiritigenin (ISL). The viability of T24 cells was estimated using a sulforhodamine B assay. Cell morphological changes were examined using Hoechst 33258 staining. The apoptotic rate was determined by staining cells with Annexin V-fluorescein isothiocyanate and propidium iodide labeling. The mitochondrial membrane potential (ΔΨm) was measured using 5,5,6,6-tetrachloro-1,1, 3,3-tetraethyl benzimidazole carbocyanine iodide. Alterations in the apoptosis-related regulators B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), Bcl-2-interacting mediator of cell death (Bim), apoptotic protease-activating facter-1 (Apaf-1), caspase-9 and caspase-3 were determined using reverse transcription-polymerase chain reaction (PCR) and quantitative PCR methods. Western blot analysis was used to detect the expression of Bcl-2, Bax and caspase-3. CDK2 activity was measured using a spectrometric assay. Following treatment with ISL (between 30 and 70 µg/ml) for 24 h, typical apoptotic morphological changes were observed in T24 cells, exhibiting an edge set of chromosomes, nuclear condensation, nuclear fragmentation and other morphological features. Treatment with ISL increased the apoptotic ratio of T24 cells in a concentration-dependent manner and induced a decrease in the ΔΨm in a time-dependent manner. Treatment with ISL upregulated the expression of Bax, Bim, Apaf-1, caspase-9 and caspase-3, downregulated the expression of Bcl-2, and increased CDK2 activity. MK-8776 (an inhibitor of CDK2) antagonized the apoptosis induced by ISL, and, compared with treatment with ISL alone, pretreatment with MK-8776 inhibited the decrease in ΔΨm, downregulated the mRNA expression of Bax, Bim, Apaf-1, caspase-9 and caspase-3, and upregulated Bcl-2 mRNA expression. Western blot analysis demonstrated that, with increasing ISL concentration, the Bcl-2 expression level was significantly decreased (P<0.05), whereas caspase-3 and Bax expression levels were significantly increased (P<0.01). These results indicated that ISL treatment caused a significant decrease in the proliferation rate and increase in apoptosis of T24 cells. The mechanism by which ISL induces T24 cell apoptosis in vitro may be associated with an increase in CDK2 activity, downregulation of the ΔΨm and activation of caspase-3/caspase-9-mediated mitochondrial apoptotic signaling pathways.

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Isoliquiritigenin suppresses human T Lymphocyte activation via covalently binding cysteine 46 of I?B kinase.

Publications

Isoliquiritigenin suppresses human T Lymphocyte activation via covalently binding cysteine 46 of IκB kinase.

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