Targeting NF-κB signaling cascades of glioblastoma by a natural benzophenone, garcinol, via in vitro and molecular docking approaches
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ABSTRACT:
SUBMITTER: Rizvi S
PROVIDER: S-EPMC10904546 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
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Project description:BackgroundGlioblastoma multiforme (GBM) is one of the most refractory and palindromic central nervous system (CNS) neoplasms, and current treatments have poor effects in GBM patients. Hence, the identification of novel therapeutic targets and the development of effective treatment strategies are essential. Alantolactone (ATL) has a wide range of pharmacological activities, and its anti-tumor effect is receiving increasing attention. However, the molecular mechanism underlying the anti-GBM activity of ATL remains poorly understood.MethodsThe biological functions of ATL in GBM cells were investigated using migration/invasion, colony formation and cell cycle/apoptosis assays. The localization of nuclear factor kappa B (NF-κB) p50/p65 and its binding to the cyclooxygenase 2 (COX-2) promoter were determined using confocal immunofluorescence, a streptavidin-agarose pulldown assay and a chromatin immunoprecipitation (ChIP) assay. IKKβ kinase activity was determined using a cell IKKβ kinase activity spectrophotometry quantitative detection kit and a molecular docking study. LC-MS/MS analysis was performed to determine the ability of ATL to traverse the blood-brain barrier (BBB). The in vivo anti-tumor efficacy of ATL was also analyzed in xenografted nude mice. Western blot analysis was performed to detect the protein expression levels.ResultsATL significantly suppressed the growth of GBM in vivo and in vitro. ATL significantly reduced the expression of COX-2 by inhibiting the kinase activity of IKKβ by targeting the ATP-binding site and then attenuating the binding of NF-κB to the COX-2 promoter region. Furthermore, ATL induced apoptosis by activating the cytochrome c (cyt c)/caspase cascade signaling pathway. Moreover, ATL could penetrate the BBB.ConclusionsATL exerts its anti-tumor effects in human GBM cells at least in part via NF-κB/COX-2-mediated signaling cascades by inhibiting IKKβ kinase activity. ATL, which is a natural small molecule inhibitor, is a promising candidate for clinical applications in the treatment of CNS tumors.
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Project description:Garcinia is a significant medicinal plant with many beneficial phytoconstituents, including garcinol. This study investigated the anti-inflammatory effect of garcinol isolated from Garcinia dulcis fruit in LPS-activated THP-1 and Raw 264.7 macrophages. The results demonstrated that the low concentration of garcinol did not alter cell viability. Furthermore, co-incubation of garcinol with LPS inhibited the production of pro-inflammatory cytokines, including TNF-α, IL-8, IL-6, IL-1β, and pro-inflammatory mediators, including iNOS and COX-2 at the mRNA and protein expression levels. Garcinol also decreased the secretion of TNF-α, IL-6, IL-1β, PGE2, and NO. Moreover, the anti-inflammatory effects involved an alteration in the NF-κB signaling pathway. Downregulation of pIKKα/β, pIκBα, and pNF-κB was observed, hence reducing the translocation of pNF-κB from the cytosol into the nucleus, which subsequently decreased the production of pro-inflammatory molecules. Therefore, garcinol isolated from Garcinia dulcis is a potential candidate as an anti-inflammatory agent for inflammation-related disease treatment.
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Project description:Dysregulation of the NF-κB transcription factor occurs in many cancer types. Krüppel-like family of transcription factors (KLFs) regulate the expression of genes involved in cell proliferation, differentiation and survival. Here, we report a new mechanism of NF-κB activation in glioblastoma through depletion of the KLF6 tumor suppressor. We show that KLF6 transactivates multiple genes negatively controlling the NF-κB pathway and consequently reduces NF-κB nuclear localization and downregulates NF-κB targets. Reconstitution of KLF6 attenuates their malignant phenotype and induces neural-like differentiation and senescence, consistent with NF-κB pathway inhibition. KLF6 is heterozygously deleted in 74.5% of the analyzed glioblastomas and predicts unfavorable patient prognosis suggesting that haploinsufficiency is a clinically relevant means of evading KLF6-dependent regulation of NF-κB. Together, our study identifies a new mechanism by which KLF6 regulates NF-κB signaling, and how this mechanism is circumvented in glioblastoma through KLF6 loss.
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Project description:Glioblastoma multiforme (GBM) is the most common and lethal form of intracranial tumor. We have established a lentivirus-induced mouse model of malignant gliomas, which faithfully captures the pathophysiology and molecular signature of mesenchymal human GBM. RNA-Seq analysis of these tumors revealed high nuclear factor κB (NF-κB) activation showing enrichment of known NF-κB target genes. Inhibition of NF-κB by either depletion of IκB kinase 2 (IKK2), expression of a IκBαM super repressor, or using a NEMO (NF-κB essential modifier)-binding domain (NBD) peptide in tumor-derived cell lines attenuated tumor proliferation and prolonged mouse survival. Timp1, one of the NF-κB target genes significantly up-regulated in GBM, was identified to play a role in tumor proliferation and growth. Inhibition of NF-κB activity or silencing of Timp1 resulted in slower tumor growth in both mouse and human GBM models. Our results suggest that inhibition of NF-κB activity or targeting of inducible NF-κB genes is an attractive therapeutic approach for GBM.
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Project description:Dysregulated activity of the transcription factors of the nuclear factor κb (NF-κB) family has been implicated in numerous cancer types, inflammatory diseases, autoimmune disease, and other disorders. As such, selective NF-κB pathway inhibition is an attractive target to researchers for preclinical and clinical drug development. A plethora of commercially and clinically available inhibitors claim to be NF-κB specific; however, such claims of specificity are rarely quantitative or benchmarked, making the biomedical literature difficult to contextualize. This imprecision is worsened because some NF-κB reporter systems have low signal-to-noise ratios. Herein, we use a robust, defined, commercially available reporter system to benchmark NF-κB agonists and antagonists for the field. We also functionally characterize a RELA fusion-positive ependymoma cell culture with validated NF-κB inhibitor compounds.
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Project description:The nuclear factor kappa B (NF-κB) family of transcription factors orchestrates signal-induced gene expression in diverse cell types. Cellular responses to NF-κB activation are regulated at the level of cell and signal specificity, as well as differential use of family members (subunit specificity). Here we used time-dependent multi-omics to investigate the selective functions of Rel and RelA, two closely related NF-κB proteins, in primary B lymphocytes activated via the B cell receptor. Despite large numbers of shared binding sites genome wide, Rel and RelA directed kinetically distinct cascades of gene expression in activated B cells. Single-cell RNA sequencing revealed marked heterogeneity of Rel- and RelA-specific responses, and sequential binding of these factors was not a major mechanism of protracted transcription. Moreover, nuclear co-expression of Rel and RelA led to functional antagonism between the factors. By rigorously identifying the target genes of each NF-κB subunit, these studies provide insights into exclusive functions of Rel and RelA in immunity and cancer.
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Project description:Glioblastoma (GBM) is one of the most lethal primary brain tumor with a poor median survival less than 15 months. Despite the development of the clinical strategies over the decades, the outcomes for GBM patients remain dismal due to the strong proliferation and invasion ability and the acquired resistance to radiotherapy and chemotherapy. Therefore, developing new biomarkers and therapeutic strategies targeting GBM is in urgent need. In this study, gene expression datasets and relevant clinical information were extracted from public cancers/glioma datasets, including TCGA, GRAVENDEEL, REMBRANDT, and GILL datasets. Differentially expressed genes were analyzed and NEK2 was picked as a candidate gene for subsequent validation. Human tissue samples and corresponding data were collected from our center and detected by immunohistochemistry analysis. Molecular biological assays and in vivo xenograft transplantation were performed to confirm the bioinformatic findings. High-throughput RNA sequencing, followed by KEGG analysis, GSEA analysis and GO analysis were conducted to identify potential signaling pathways related to NEK2 expression. Subsequent mechanism assays were used to verify the relationship between NEK2 and NF-κB signaling. Overall, we identified that NEK2 is significantly upregulated in GBM and the higher expression of NEK2 exhibited a poorer prognosis. Functionally, NEK2 knockdown attenuated cell proliferation, migration, invasion, and tumorigenesis of GBM while NEK2 overexpression promoted the GBM progression. Furthermore, High-throughput RNA sequencing and bioinformatics analysis indicated that NEK2 was positively related to the NF-κB signaling pathway in GBM. Mechanically, NEK2 activated the noncanonical NF-κB signaling pathway by phosphorylating NIK and increasing the activity and stability of NIK. In conclusion, NEK2 promoted the progression of GBM through activation of noncanonical NF-κB signaling, indicating that NEK2- NF-κB axis could be a potential drug target for GBM.
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Project description:The inhibitor of κB (IκB) kinase (IKK) is a central regulator of NF-κB signaling. All IKK complexes contain hetero- or homodimers of the catalytic IKKβ and/or IKKα subunits. Here, we identify a YDDΦxΦ motif, which is conserved in substrates of canonical (IκBα, IκBβ) and alternative (p100) NF-κB pathways, and which mediates docking to catalytic IKK dimers. We demonstrate a quantitative correlation between docking affinity and IKK activity related to IκBα phosphorylation/degradation. Furthermore, we show that phosphorylation of the motif's conserved tyrosine, an event previously reported to promote IκBα accumulation and inhibition of NF-κB gene expression, suppresses the docking interaction. Results from integrated structural analyzes indicate that the motif binds to a groove at the IKK dimer interface. Consistently, suppression of IKK dimerization also abolishes IκBα substrate binding. Finally, we show that an optimized bivalent motif peptide inhibits NF-κB signaling. This work unveils a function for IKKα/β dimerization in substrate motif recognition.
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Project description:Despite extensive study, few therapeutic targets have been identified for glioblastoma (GBM). Here we show that patient-derived glioma sphere cultures (GSCs) that resemble either the proneural (PN) or mesenchymal (MES) transcriptomal subtypes differ significantly in their biological characteristics. Moreover, we found that a subset of the PN GSCs undergoes differentiation to a MES state in a TNF-α/NF-κB-dependent manner with an associated enrichment of CD44 subpopulations and radioresistant phenotypes. We present data to suggest that the tumor microenvironment cell types such as macrophages/microglia may play an integral role in this process. We further show that the MES signature, CD44 expression, and NF-κB activation correlate with poor radiation response and shorter survival in patients with GBM.
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