Targeting NF-κB Signaling by Calebin A, a Compound of Turmeric, in Multicellular Tumor Microenvironment: Potential Role of Apoptosis Induction in CRC Cells

Increasing lines of evidence suggest that chronic inflammation mediates most chronic diseases, including cancer. The transcription factor, NF-κB, has been shown to be a major regulator of inflammation and metastasis in tumor cells. Therefore, compounds or any natural agents that can inhibit NF-κB activation have the potential to prevent and treat cancer. However, the mechanism by which Calebin A, a component of turmeric, regulates inflammation and disrupts the interaction between HCT116 colorectal cancer (CRC) cells and multicellular tumor microenvironment (TME) is still poorly understood. The 3D-alginate HCT116 cell cultures in TME were treated with Calebin A, BMS-345541, and dithiothreitol (DTT) and examined for invasiveness, proliferation, and apoptosis. The mechanism of TME-induced malignancy of cancer cells was confirmed by phase contrast, Western blotting, immunofluorescence, and DNA-binding assay. We found through DNA binding assay, that Calebin A inhibited TME-induced NF-κB activation in a dose-dependent manner. As a result of this inhibition, NF-κB phosphorylation and NF-κB nuclear translocation were down-modulated. Calebin A, or IκB-kinase (IKK) inhibitor (BMS-345541) significantly inhibited the direct interaction of nuclear p65 to DNA, and interestingly this interaction was reversed by DTT. Calebin A also suppressed the expression of NF-κB-promoted anti-apoptotic (Bcl-2, Bcl-xL, survivin), proliferation (Cyclin D1), invasion (MMP-9), metastasis (CXCR4), and down-regulated apoptosis (Caspase-3) gene biomarkers, leading to apoptosis in HCT116 cells. These results suggest that Calebin A can suppress multicellular TME-promoted CRC cell invasion and malignancy by inhibiting the NF-κB-promoting inflammatory pathway associated with carcinogenesis, underlining the potential of Calebin A for CRC treatment.

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Mobility-Shift DNA-Binding Assay Using Gel Electrophoresis

Neurodegeneration Methods and Protocols ◽

10.1385/0-89603-612-x:89 ◽

2003 ◽

pp. 89-98

Author(s):

Yang Xiao ◽

Keith Pennypacker

Keyword(s):

Dna Binding ◽

Gel Electrophoresis ◽

Binding Assay ◽

Mobility Shift ◽

Dna Binding Assay

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Detection of Human Serum Antibodies to the BFRF3 Epstein-Barr Virus Capsid Component by Means of a DNA-Binding Assay

The Journal of Infectious Diseases ◽

10.1093/infdis/172.5.1367 ◽

1995 ◽

Vol 172 (5) ◽

pp. 1367-1370 ◽

Cited By ~ 7

Author(s):

D. Shedd ◽

A. Angeloni ◽

J. Niederman ◽

G. Miller

Keyword(s):

Dna Binding ◽

Human Serum ◽

Epstein Barr Virus ◽

Binding Assay ◽

Serum Antibodies ◽

Virus Capsid ◽

Barr Virus ◽

Epstein Barr ◽

Dna Binding Assay

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GT198 Is a Target of Oncology Drugs and Anticancer Herbs

Frontiers in Oral Health ◽

10.3389/froh.2021.679460 ◽

2021 ◽

Vol 2 ◽

Author(s):

Junfeng Pang ◽

Jie Gao ◽

Liyong Zhang ◽

Nahid F. Mivechi ◽

Lan Ko

Keyword(s):

Dna Binding ◽

Natural Product ◽

Tumor Angiogenesis ◽

Drug Target ◽

Binding Assay ◽

Organic Chemical ◽

High Efficacy ◽

Compound Identification ◽

Dna Binding Assay

Tumor angiogenesis is a hallmark of cancer. Therapeutic drug inhibitors targeting angiogenesis are clinically effective. We have previously identified GT198 (gene symbol PSMC3IP, also known as Hop2) as an oncoprotein that induces tumor angiogenesis in human cancers, including oral cancer. In this study, we show that the GT198 protein is a direct drug target of more than a dozen oncology drugs and several clinically successful anticancer herbs. GT198 is a DNA repair protein that binds to DNA. Using an in vitro DNA-binding assay, we tested the approved oncology drug set VII from the National Cancer Institute containing 129 oncology drugs. Identified GT198 inhibitors include but are not limited to mitoxantrone, doxorubicin, paclitaxel, etoposide, dactinomycin, and imatinib. Paclitaxel and etoposide have higher binding affinities, whereas doxorubicin has higher binding efficacy due to competitive inhibition. GT198 shares protein sequence homology with DNA topoisomerases, which are known drug targets, so that GT198 is likely a new drug target previously unrecognized. To seek more powerful GT198 inhibitors, we further tested several anticancer herbal extracts. The positive anticancer herbs with high affinity and high efficacy are all clinically successful ones, including allspice from Jamaica, Gleditsia sinensis or honey locust from China, and BIRM from Ecuador. Partial purification of allspice using an organic chemical approach demonstrated great feasibility of natural product purification, when the activity is monitored by the in vitro DNA-binding assay using GT198 as a target. Together, our study reveals GT198 as a new targeting mechanism for existing oncology drugs. The study also delivers an excellent drug target suitable for compound identification and natural product purification. In particular, this study opens an opportunity to rapidly identify drugs with high efficacy and low toxicity from nature.

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EVALUATION OF THE NATIVE DNA‐BINDING ASSAY FOR DNA ANTIBODIES IN SYSTEMIC LUPUS ERYTHEMATOSUS AND OTHER CONNECTIVE TISSUE DISEASES

The Medical Journal of Australia ◽

10.5694/j.1326-5377.1974.tb70809.x ◽

1974 ◽

Vol 2 (9) ◽

pp. 324-328 ◽

Cited By ~ 7

Author(s):

John Webb ◽

Keith Whaley

Keyword(s):

Systemic Lupus Erythematosus ◽

Dna Binding ◽

Connective Tissue ◽

Lupus Erythematosus ◽

Binding Assay ◽

Connective Tissue Diseases ◽

Systemic Lupus ◽

Dna Antibodies ◽

Dna Binding Assay

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Short-term modulation of interleukin-1β signaling by hyperoxia: uncoupling of IκB kinase activation and NF-κB-dependent gene expression

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00191.2003 ◽

2004 ◽

Vol 286 (3) ◽

pp. L554-L562 ◽

Cited By ~ 5

Author(s):

Kelli Odoms ◽

Thomas P. Shanley ◽

Hector R. Wong

Keyword(s):

Gene Expression ◽

Signal Transduction ◽

Dna Binding ◽

Nuclear Translocation ◽

A549 Cells ◽

Concomitant Treatment ◽

Simultaneous Treatment ◽

Iκb Kinase ◽

Tnf Α ◽

Respiratory Epithelial

We have been interested in elucidating how simultaneous stimuli modulate inflammation-related signal transduction pathways in lung parenchymal cells. We previously demonstrated that exposing respiratory epithelial cells to 95% oxygen (hyperoxia) synergistically increased tumor necrosis factor-α (TNF-α)-mediated activation of NF-κB and NF-κB-dependent gene expression by a mechanism involving increased activation of IκB kinase (IKK). Because the signal transduction mechanisms induced by IL-1β are distinct to that of TNF-α, herein we sought to determine whether hyperoxia modulates IL-1β-dependent signal transduction. In A549 cells, simultaneous treatment with hyperoxia and IL-1β caused increased activation of IKK, prolonged the degradation of IκBα, and prolonged the nuclear translocation and DNA binding of NF-κB compared with cells treated with IL-1β alone in room air. Hyperoxia did not affect IL-1β-dependent degradation of the interleukin receptor-associated kinase differently from treatment with IL-β alone. In contrast to the effects on the IKK/IκBα/NF-κB pathway, simultaneous treatment with hyperoxia and IL-1β did not augment NF-κB-dependent gene expression compared with treatment with IL-1β alone. Similar observations were made in a different human respiratory epithelial cell line, BEAS-2B cells. In addition, simultaneous treatment with hyperoxia and IL-1β caused hyperphosphorlyation of the NF-κB p65 subunit compared with treatment with IL-1β alone. In summary, concomitant treatment of A549 cells with hyperoxia and IL-1β augments activation of IKK, prolongs degradation of IκBα, and prolongs nuclear translocation and DNA binding of NF-κB. This activation, however, is not coupled to increased expression of NF-κB-dependent genes, and the mechanism of this decoupling is not related to decreased phosphorylation of p65.

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The Development and Applications of Nonradioactive Plate-Formatted DNA-Binding Assay for Ku70/80, a Multifunctional DNA-Binding Protein Complex

Assay and Drug Development Technologies ◽

10.1089/adt.2004.2.483 ◽

2004 ◽

Vol 2 (5) ◽

pp. 483-495

Author(s):

Hongwen Ma ◽

Joseph Thibault ◽

Yali Lu ◽

Carson Whiting ◽

Shinong Long ◽

...

Keyword(s):

Dna Binding ◽

Protein Complex ◽

Binding Assay ◽

Binding Protein ◽

Dna Binding Protein ◽

Dna Binding Assay

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IκB Kinase-Dependent Chronic Activation of NF-κB Is Necessary for p21WAF1/Cip1 Inhibition of Differentiation-Induced Apoptosis of Monocytes

Molecular and Cellular Biology ◽

10.1128/mcb.21.6.1930-1941.2001 ◽

2001 ◽

Vol 21 (6) ◽

pp. 1930-1941 ◽

Cited By ~ 51

Author(s):

Kevin N. Pennington ◽

Julie A. Taylor ◽

Gary D. Bren ◽

Carlos V. Paya

Keyword(s):

Transcription Factor ◽

Cell Differentiation ◽

Molecular Mechanisms ◽

Nuclear Translocation ◽

Dependent Manner ◽

Macrophage Differentiation ◽

Monocyte Differentiation ◽

Cell Functions ◽

Iκb Kinase ◽

Induced Apoptosis

ABSTRACT The molecular mechanisms regulating monocyte differentiation to macrophages remain unknown. Although the transcription factor NF-κB participates in multiple cell functions, its role in cell differentiation is ill defined. Since differentiated macrophages, in contrast to cycling monocytes, contain significant levels of NF-κB in the nuclei, we questioned whether this transcription factor is involved in macrophage differentiation. Phorbol 12-myristate 13-acetate (PMA)-induced differentiation of the promonocytic cell line U937 leads to persistent NF-κB nuclear translocation. We demonstrate here that an increased and persistent IKK activity correlates with monocyte differentiation leading to persistent NF-κB activation secondary to increased IκBα degradation via the IκB signal response domain (SRD). Promonocytic cells stably overexpressing an IκBα transgene containing SRD mutations fail to activate NF-κB and subsequently fail to survive the PMA-induced macrophage differentiation program. The differentiation-induced apoptosis was found to be dependent on tumor necrosis factor alpha. The protective effect of NF-κB is mediated through p21WAF1/Cip1, since this protein was found to be regulated in an NF-κB-dependent manner and to confer survival features during macrophage differentiation. Therefore, NF-κB plays a key role in cell differentiation by conferring cell survival that in the case of macrophages is mediated through p21WAF1/Cip1.

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