KR101195164B1 - The cell lines for measuring the activity of NF-kappa B and a screening method for the substance to induce or suppress the activity of NF-kappa B using them - Google Patents

Abstract

The present invention relates to a cell line for measuring the activity of NF-κB and a method for selecting a substance that induces or inhibits NF-κB activity using the same, and more specifically, the cell line for measuring the activity of NF-κB, NF-κB binding site, A cell line transduced with an expression vector containing constructs fused in bla gene sequence and its NF-κB bla When NF-κB activated by an external stimulus is translocated into the nucleus, it binds to the NF-κB binding site, and the bla gene is expressed, and the green cells are formed by cleaving CCF4-AM, a substrate of the expressed β-lactamase. Since it becomes blue, it can be used to select substances that induce or inhibit NF-κB activity. In addition, NF-κB activity measurement cell line can be used to measure the concentration and time of NF-κB activity induction or inhibition of a specific drug in living cells. It can be usefully used for screening compounds and dietary supplements.

NF-κB, human Jurkat cells, indole-3-carbitol, fluorescence resonance energy transfer

Description

The cell lines for measuring the activity of NF-kappa B and a screening method for the substance to induce or suppress the NF-κＢ activity cell line and a method for screening a substance that induces or inhibits NF-κＢ activity using the same activity of NF-kappa B using them}

The present invention relates to a cell line for measuring activity of Nuclear Factor kappa B (NF-κB) and a method for selecting a substance that induces or inhibits NF-κB activity using the same.

Nuclear factor kappa B family includes p50, RelA or p65, RelA, RelB and RelC. Of these proteins, p50 / p65 (RelA) heterodimers and p50 / p50 homodimers are currently best identified. NF-κB is activated by various stimuli such as proinflammatory cytokines, oxidant free radicals, inhaled particles, ultraviolet light, microorganisms or viruses. When stimulated by these factors, IKK (IkB kinase) activity, IκBα (inhibitor of kappa Bα) degradation and transcriptional activity is increased (see Figure 1).

For example, binding of tumor necrosis factor α to TNF receptor 1 (TNFR1) rapidly activates IKK and completely eliminates IκBα within 10 minutes. Subsequently, translocation of NF-κB occurs to the nucleus, which binds to a specific DNA motif to which NF-κB binds to induce transcription of a specific gene. Transcriptional activity and specificity of NF-κB are regulated differently depending on the cell type.

NF-κB activity is cell survival (cIAP, XIAP, cFLIP and Bcl-XL), proliferation (TNF, IL-1, IL-6, cyclin-D1, c-myc), angiogenesis (VEGF, TNF, IL-1) , IL-8), inflammation (TNF, IL-1, chemokine), invasion (cox-2, MMP9, uPA) and metastasis (ICAM-1, VCAM-1, ELAM-1) Regulate expression. Recently, the continued activity of NF-κB in cancer cells has been reported, but the cause is not yet fully understood.

Most biological or biochemical NF-κB inhibitors currently in use block the signaling pathways leading to activation of NF-κB or reduce the activity of NF-κB binding to target DNA. Some inhibitors act on specific targets, such as TNF / IL-1 receptors, receptor-related proteins or IKK complexes, while others are less specific, such as ubiquitin-conjugating enzymes and proteasome inhibition. Many antioxidants inhibit NF-κB by interfering with upstream signaling leading to phosphorylation of IκB.

NF-κB is constitutively active in most cancer cell lines, proliferating T cells, B cells, thymic cells, monocytes and astrocytic cells. Our main concern is to inhibit this sustained activity. Usually NF-κB plays the most important role in the immune system. Regulation of the transcriptional response to various other stimuli is important for the proper functioning of the mammalian immune system. NF-κB also regulates gene expression outside the immune system, affecting normal or disease physiology, including embryonic development, and many aspects of tissue physiology, including the mammary gland, bone, skin, and central nervous system. However, as mentioned above, the diverse biological roles of NF-κB are such that one common mechanism in all systems regulates signaling to NF-κB, or that different inputs cause a variety of transcriptional responses that are more specific to specific tissues and organs. Focus on Understanding how NF-κB correlates various stimuli in a complex system to produce unique results for a particular situation is a challenge in the art.

In this regard, the present inventors construct a cell line in which a pLenti- bsd / NFκB bla vector is safely transduced to measure NF-κB activity caused by various stimuli, and erythromycin (Ery) that suppresses continuous activity of NF-κB. By comparing inhibitors such as Bay 11-7082 (Bay11) and indole-3-carbitol (I3C), revealing that indole-3-carbitol (I3C) significantly inhibits sustained NF-κB function in Jurkat cell lines. The present invention has been completed.

An object of the present invention is to provide a cell line for measuring NF-κB activity in order to measure NF-κB activity by a number of factors, and to provide a method for selecting a substance that induces or inhibits NF-κB activity using the same.

In order to achieve the above object, the present invention is for measuring NF-κB activity, characterized in that the animal cells transduced (transfection) with an expression vector comprising a fused construct in the NF-κB binding site, bla gene sequence. Provide a cell line.

The present invention also provides a method of selecting a substance that induces or inhibits the activity of NF-κB using the cell line for measuring the activity of NF-κB.

Using the cell line for measuring the activity of NF-κB of the present invention, the concentration and time of inducing or inhibiting NF-κB activity of a specific drug can be measured in living cells, and the assay is performed using a high throughput search. It can be usefully used for screening new drug candidates and dietary supplements.

The present invention provides a cell line for measuring NF-κB activity, which is an animal cell transduced with an expression vector comprising a construct fused to an NF-κB binding site, bla gene sequence.

The NF-κB bla system binds to the NF-κB binding site when NF-κB activated by an external stimulus is translocated into the nucleus, whereby the bla gene expresses β-lactamase. The expressed β-lactamase cleaves the substrate, CCF4-AM (the acetoxymethyl (AM) ester of coumarin cephalosporin fluorescein 4). CCF4-AM is excited at 408 nm, causing fluorescence resonance energy transfer (FRET), which is emitted at 530 nm, and becomes green.When CCF4-AM is cleaved by β-lactamase, it is excited at 408 nm. It is emitted at 460 nm and becomes blue. Therefore, when NF-κB is activated and translocated into the nucleus by stimulation such as TNFa, β-lactamase is expressed by cleaving CCF4-AM by binding to the NF-κB binding site.

In the same principle as described above, NF-κB activity can be easily measured using a cell line transduced with an expression vector including a construct fused in the order of NF-κB binding site, bla gene.

 The NF-κB binding site preferably has a nucleotide sequence set forth in SEQ ID NO: 1, but is not limited thereto. The gene is preferably selected from the group consisting of a lentiviral vector and an adenovirus vector as an expression vector including a construct, but is not limited thereto. All expression vectors for animal cells known to those skilled in the art are available. The animal cell is preferably an animal cell selected from the group consisting of Jurkat, NIH-3T3, RAW264.7, HELA or cancer cells obtained from each tissue. The present invention is not limited thereto, and those skilled in the art can use any animal cell to be examined for NF-κB activity.

In addition,

1) transducing an animal cell with an expression vector comprising a fused construct in NF-κB binding site, bla gene sequence;

2) selecting the cell line transduced by step 1);

3) treating the test compound to the cell line selected in step 2);

4) measuring NF-κB activity in the cell line treated with the test compound in step 3); And

5) Selecting a substance that induces or inhibits the activity of NF-κB comprising the step of selecting a test compound that significantly changes the NF-κB activity in the cell line treated with the test compound compared to the control group not administered the test compound To provide a way.

Examples of the test compound of step 3) include, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts or plasma, and these compounds may be novel compounds, A well-known compound may be sufficient. Such a test compound may form a salt. Salts of the test compound include salts such as physiologically acceptable acids (eg, inorganic acids) and bases (eg, organic acids, etc.), and of these, physiologically acceptable acid addition salts are preferable. Such salts include, for example, salts of inorganic acids (e.g. hydrochloric acid, phosphoric acid, hydrobromic acid or sulfuric acid) or organic acids (e.g. acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid). , Malic acid, oxalic acid, benzoic acid, methanesulfonic acid or benzenesulfonic acid).

Measurement of NF-κB activity in the cell line treated with the test compound of step 4) was performed using Gene blazer method as a cell analysis method, which is β-lactamase based on fluorescence resonance energy transfer (FRET) ( bla ) Reporter analysis. When β-lactamase substrate CCF2 or CCF4 enters the cell, the cytosolic esterase cleaves the substrate and changes it into a negatively charged substrate. The stimulated or activated cell then produces a blue product. CCF4-AM, which is non-polar, diffuses through the plasma membrane and enters the cytoplasm, and esterase cleaves the ester so that CCF4-AM becomes CCF4. β-lactamase hydrolyzes CCF4 and loses FRET resulting in blue fluorescence. In the absence of β-lactamase (bla ), a green fluorescence signal is emitted at 530 nm, but this enzymatic cleavage separates the dye, stops the FRET phenomenon and is excited at 408 nm to produce a blue fluorescence signal (460 nm). do.

In addition, the present invention is an NF-κB activity inducer to increase or decrease the expression of cell survival, proliferation, angiogenesis, inflammation, invasion and metastasis genes regulated by NF-κB using the cell line for measuring the NF-κB activity Or activity inhibitor screening methods.

In the present invention, two cell lines for measuring NF-κB activity, human T- lymphoma Jurkat cells and mouse fibroblasts NIH-3T3, the effect of the inhibitors Bay11, Ery and I3C of the NF-κB activity pathway to the cell analysis method It was investigated using.

First, NIH-3T3-NF-κB- bla constructed by the method of the present invention Treatment with Bay11 and Ery inhibited TNFa-induced NF-κB activity, but treatment with two inhibitors in Jurkat cell line did not significantly inhibit NF-κB activity.

As a result of treatment with another inhibitor of I3C to Jurkat cell line stimulated by TNFa, it was confirmed that NF-κB activity was significantly inhibited (see FIGS. 2 and 3).

Inhibition of NF-κB activity caused by several factors by I3C correlates with inhibition of IKK, IκBα phosphorylation, ubiquitation and degradation and p65 phosphorylation, nuclear translocation and acetylation. I3C also reduced reporter gene transcription and production of gene products associated with cell proliferation, anti-apoptosis and invasion regulated by NF-κB. Therefore, it can be seen that the cell line for measuring the NF-κB activity of the present invention is very useful for monitoring the change of NF-κB activity by the existing NF-κB activator and NF-κB inhibitor.

As a result, the present invention utilizes cell lines for NF-κB activity that can easily monitor NF-κB activity to increase the expression of cell survival, proliferation, angiogenesis, inflammation, invasion and metastasis genes regulated by NF-kB or Substances that induce or inhibit reducing NF-kB activity can be selected.

In addition, NF-κB inhibitors can be easily selected to treat or prevent cancer, inflammation, rheumatoid arthritis (RA) and osteoarthritis (OA) caused by the continuous activity of NF-κB.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example 1 Culture of Human T-lymphoma Jurkat Cell Line and Mouse Fibroblast NIH-3T3 Cell Line

Human T-lymphoma Jurkat cells (American Type Culture Collection, Manassas, VA) and mouse fibroblast NIH-3T3 (American Type Culture Collection, Manassas, VA) contained 10% FBS, 100 IU / ml penicillin (Gibco Life Technologies, UK) and 10 ug / ml streptomycin (Gibco Life Technologies, UK) were incubated at 37 ℃, 5% carbon dioxide conditions. However, Jurkat cells and NIH-3T3 cells were cultured using RPMI 1640 medium (Gibco Life Technologies, UK) and Dulbeccos modified Eagle medium (DMEM): (Gibco Life Technologies, UK), respectively.

Example 2 Transduction and Selection of Cell Lines

Transfection is performed according to the microporation procedure (iNCYTO, KR) using the pLenti- bsd / NFκB- bla vector (Invitrogen, USA). First, the vector was transformed into Stbl2 competent cells (Invitrogen, USA) and mass produced. Microporator MP-100 (Digital Bio Technology co.) Was used to transduce the pLenti- bsd / NF κB- bla vector into the Jurkat cells of Example 1. A 1 × 10 ⁵ cell suspension was collected in 1 × PBS (Phosphate Buffer Saline), centrifuged at 1,000 rpm, 5 minutes, and 4 ° C. to discard the supernatant, and 40 μl of microporator solution (iNCYTO, KR) was added. Then 10 μl of NF-κB plasmid (Invitrogen, USA) was added to 40 μl of suspension and mixed gently. Parameters for microporation of Jurkat cells are plus voltage (v) 1380, plus width (ms) -30, plus number 1 and tip type 10 μl, and NIH-3TS cells The parameters for the microporation of are plus voltage (v) 1740, plus width (ms) -10, plus number 3 and 10 μl tip shape. Both cells were selected by treatment with 20 μg / ml blastine cydine S hydrochloric acid (Invitrogen) because the vector contains blasticidin resistance ( bsdR ) genes.

Example 3 Cell Analysis

Cell based assays were performed using the Gene blazer technique (Invitrogen, USA), which is a β-lactamase ( bla ) reporter assay based on Fluorescence Resonance Energy Transfer (FRET). When β-lactamase substrate CCF2 or CCF4 enters the cell, the cytosolic esterase cleaves the substrate and changes it into a negatively charged substrate. The stimulated or activated cell then produces a blue product. In the absence of β-lactamase ( bla ), a green fluorescence signal is emitted at 530 nm, but substrate cleavage of these enzymes separates the dye, stops FRET, and is excited at 408 nm, resulting in a blue fluorescence signal (460 nm). Will generate

In the present invention, two cell lines constructed in Examples 1 and 2 (NIH-3T3-NF-κB- bla and Jurkat-NF-κB- bla ) were 200-μl, 96 × well at 1 × 10 ⁴ cells / well concentration. TNFα (Sigma-Aldrich, USA) and NF-κB activity inhibitor Erythromycin (Ery) :( Sigma-Aldrich, USA), Bay 11-7082 (Bay11) :( Calbiochem, UK), indole-3-carbitol (I3C): (Sigma-Aldrich, USA) were treated at different times and doses. Then, using a β-lactamase substrate, CCF4-AM loading solution (Invitrogen, USA) was incubated for 90 minutes at 37 ℃, 5% carbon dioxide and fluorescence microscopy. As a result, the effects of the inhibitors Bay11, Ery and I3C on the NF-κB activity pathway were examined using the cell assay as follows.

One. NIH -3 T3 - NF with κB Jurkat - NF Cytokine Treatment on κB Cell Lines

Baud et al. Reported that pre-inflammatory cytokines, TNFα, can activate two transcription factors, NF-κB and AP-1, through the trimerization of TNF receptors. In the present invention, the effect of TNFα in cells was analyzed by Gene blazer technique using a phase contrast microscope.

As a result, NIH-3T3 showed 37% activity after 5 hours of TNFα (10 ng / ml) treatment, but Jurkat cells showed more than 80% activity.

2. In the cell Ery  And Bay11 Effect

To confirm the functional significance of the inhibitors Ery and Bay11, the effect of these inhibitors on translocation of the NF-κB subunit to the nucleus was examined at different concentrations and times. According to Pierce et al., Bay11 irreversibly inhibits TNFα-induced phosphorylation of IκBα, preventing subsequent ubiquitin-mediated IκBα degradation, eventually disrupting NF-κB translocation.

In the present invention, 12.71% of TNFα-activated NIH-3T3-NF-κB treated with TNFα and Bay11 (10 ng / ml and 5 μg / ml, respectively) was treated with TNFα (10 ng / m). One cell showed 18.55% of activated blue cells and 27% and 16.17% of the cells after 5 hours of treatment with TNFα and Ery (10 ng / ml and 5 ng / ml) and Ery (5 ng / ml), respectively. It showed activity and 37% activity when treated only with a positive control TNFα (Fig. 2).

Thus, both inhibitors have the ability to inhibit TNFα-induced NF-κB activity.

3. Jurkat - NF in κB cells I3C Effect

Indole-3-carbitol (I3C) was treated due to insufficient inhibitory effects of Ery and Bay11 in Jurkat cells. Takada et al. Reported that I3C is an autolysis product of glucosinolate and glucobrassicin found in Brassica spp. Or cruciferous vegetables (cabbage, broccoli, cauliflower, and brussels spouts). Its effect on expression interferes with the NF-κB active pathway.

In the present invention, it was confirmed that TNFα-induced NF-κB activity was significantly inhibited after I3C treatment.

That is, the results of treatment at different concentrations when I3C alone and TNFα and I3C mixed applications are shown in FIG. 3. In FIG. 3, 5.26% and 8.19% of activated cells were observed after 5 hours of TNFα and I3C mixing (10 ng / ml and 1 mM) and I3C alone (1 mM). Almost 85% of the activated cells were treated with TNFα alone (10 ng / ml) as the positive control, and 82% of the activated cells were shown because the negative control had self-stimulating properties of Jurkat-NF-κB. Inhibition of IF-induced NF-κB activity by various factors, regardless of cell morphology, correlates with inhibition of IKK, IκBα phosphorylation, ubiquitination and degradation, and p65 phosphorylation, nuclear translocation and acetylation. I3C also reduced reporter gene transcription and production of gene products associated with cell proliferation, anti-apoptosis and invasion regulated by NF-κB. By varying the time and dose, 1 mM of I3C (3-4 hours) significantly inhibited the NF-κB activity of Jurkat-NF-κB caused by TNFα (FIG. 4).

We also hypothesized a pretreatment scenario of NF-κB activity caused by TNFα. Nelson et al. Reported that cells treated with 12.5 μM Bay11 showed significant inhibition of p65 translocation 30 minutes prior to stimulation with TNFα, indicating that p65 binds to IκBα in the cytoplasm and is released upon phosphorylation of IκBα. .

In the present invention, 14.33% of the cells were activated by I3C (1 mM) 30 minutes before TNFα treatment and 54.11% of the cells by I3C treatment 30 minutes after TNFα treatment (Table 1).

30 minutes ago I3C  process Cell count Activated Cells (%) I3C + TNFα (1 mM + 10 ng / ml) 132 14.33 I3C + TNFα (100 μM +10 ng / ml) 147 77.55 I3C + TNFα (500 μM +10 ng / ml) 145 85.51 No (I3C + TNFα) 162 82.09 After 30 minutes I3C  process Cell count Activated Cells (%) TNFα + I3C (10 ng / ml + 1 mM) 85 54.11 I3C + TNFα (500 μM + 10 ng / ml) 117 77.78 I3C + TNFα (100 μM + 10 ng / ml) 155 85.80

(Jurkat-NF-κB was treated with different concentrations of I3C before and after 30 minutes of TNFα (5 hours in total). Only 1 mM treatment of I3C showed significant differences.)

This means that I3C (1 mM) treated before TNFα stimulation is involved in the binding of IκBα and NF-κB to slow degradation. However, TNFα treatment stimulated NF-κB translocation and after 30 minutes I3C treatment reduced NF-κB translocation. Thirty minutes prior to stimulation with TNFα, cells treated with 1 mM I3C showed very significant inhibition of NF-κB activity, indicating that p65 binds to IκBα in the cytoplasm and is released upon phosphorylation of IκBα. As a result, it can be seen that I3C is a very effective inhibitor in inhibiting TNFα-induced NF-κB activity.

We also compared I3C with Ery and Bay11. I3C showed significant NF-κB inhibition compared to the other two inhibitors (FIG. 5).

Example 4 Cell Proliferation Assay

PreMix WST-1 (Takara Bio. Inc., JAPAN), a kind of tetrazolium salt, was used for cell proliferation analysis. Tetrazolium salts are cleaved into formazen dyes by succinic acid-tetrazolium reductase present in the mitochondrial circulation chain acting on living cells. Formagen dyes formed by metabolic active cells are quantified by measuring absorbance with an ELISA reader. Cells were cultured by dispensing in 96 well plates at a density of 5 × 10 ³ cells per well, 100 μl. Depending on treatment time and dose, PreMix WST-1 was added and reacted for an additional 4 hours, and then absorbance was measured at 540 ± 20 nm. To analyze cell viability, different concentrations of Bay11 and Ery were applied first.

As a result, Bay11 below 10 μg / ml and Ery below 10 ng / ml did not show any negative effects on NIH-3T3-NF-κB and Jurkat-NF-κB.

In addition, the effects of I3C on Jurkat-NF-κB were investigated by mixing TNFα and I3C and I3C alone for 6 hours and premix-Wst-1 for 4 hours.

The result was higher absorbance at higher concentrations of 1 mM compared to the negative control (FIG. 6).

1 is a diagram showing an active pathway of NF-κB.

Figure 2 shows the analysis of NIH3T3-NFκB cell line 5 hours after TNFα and Ery treatment. TNFα (10 ng / ml) stimulation, a positive control, showed 37% activated cells. However, the addition of Ery (5 ng / ml) inhibited the activity (27%) and 16.17% of the cells were activated when only Ery (5 ng / ml) was treated. Each value is represented as mean ± SE in three independent experiments.

3 shows the effect of I3C on Jurkat-NF-κB. After 5 hours of treatment with TNFα + I3C (10 ng / ml + 1 mM) in B and I3C (1 mM) alone in E, 5.26% and 8.19% of activated cells were seen, respectively. Treatment with TNFα (10 ng / ml) alone in A showed nearly 85% of the cells as a positive control and 82% for the negative control (H). TNFα + I 3 C in C (10 ng / ml + 500 μM); D (10 ng / ml + 100 μM); F, I 3 C (500 μM); And G, I 3 C (100 μM) represent 73.36%, 77.24%, 79.04% and 79.09% activated cells.

4 shows the results of I3C and TNFα treatment according to the pattern depending on time and dose. Jurkat-NF-κB activity caused by TNFα (10 ng / ml) was significantly inhibited after 3-4 hours treatment compared to 1 hour treatment (67.32%) of I3C (1 mM), 18.27% and 12.74, respectively. % Cells show activity. The activity was markedly inhibited by treatment with I3C (1 mM) alone, and 6.81% of the cells showed activity after 4 hours of treatment. Each value is represented as mean ± SE in three independent experiments.

5 compares inhibitor treatment results in Jurkat-NF-κB (5 hour treatment). 8.19% of the cells were active upon treatment with I3C (1 mM), which is about 10 times lower than 5 μg / ml Bay11 (78.26%) and 10 ng / ml Ery (87.75%).

6 analyzes cell proliferation. I3C was treated for 6 hours at different concentrations. The absorbance of 1 mM I3C is greater than that of the negative control (0.773) (1.048).

<110> Industry-University Cooperation Foundation Hanyang University          CoreStem          CHUNKBUK TECHONOPARK <120> The cell lines for measuring the activity of NF-kappaB and a          screening method for the substance to induce or suppress the          activity of NF-kappaB using them <130> 7p-09-29 <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> binding sites of NF-kappaB <400> 1 ggggactttc c 11  

Claims (14)

1) An expression vector comprising a fused construct in the order of NF-κB binding site (SEQ ID NO: 1), bla gene (gene encoding β-lactamase) is selected from human T- lymphoma Jurkat cells or mouse fibroblast NIH. Transducing to -3T3; 2) selecting the cell line transduced by step 1); 3) treating the test compound to the cell line selected in step 2); And 4) A method for selecting a substance that induces or inhibits the activity of NF-κB, comprising measuring the activity of β-lactamase in a cell line treated with the test compound in step 3). The method of claim 1, wherein the expression vector is any one selected from the group consisting of lentiviral vectors and adenovirus vectors. delete delete The method of claim 1, wherein the test compound of step 3) is a peptide, protein, non-peptidic compound, synthetic compound, fermentation product, cell extract, plant extract, animal tissue extract or plasma to inhibit the activity of NF-κB Screening for substances that induce or inhibit. The method of claim 1, wherein the activity of β-lactamase is to measure the blue fluorescence signal at 460 nm after treatment with the acetoxymethyl (AM) ester of coumarin cephalosporin fluorescein 4), a substrate of β-lactamase. A method for selecting a substance that induces or inhibits the activity of NF-κB characterized by the above. delete delete delete delete delete delete delete delete

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