JP5455505B2 - junB gene expression promoter - Google Patents

Description

  The present invention relates to an expression promoter for junB gene, which is a factor that suppresses cancer.

Currently, some of the cancers that are the top cause of death among Japanese people, accounting for more than 30%, have established therapeutic drugs and treatment methods, and some cancers have a high cure rate. There are many cancers for which there is no fundamental treatment or treatment. This is because cancer development mechanisms are diverse, but therapeutic agents and treatment methods established so far are often effective only for specific mechanisms.
As a mechanism for developing cancer, a mechanism involving the c-jun gene is known. The JUN protein, the product of the c-jun gene, is known to form a transcriptional regulator AP1 complex with proteins such as FOS and RAF, promote cell differentiation and proliferation, and promote cell carcinogenesis. (Non-Patent Document 1).
The junB gene belongs to the jun gene family like the c-jun gene, and the JUNB protein, which is a product of the junB gene, has a structure similar to that of the JUN protein and also has some common functions (Non-patent Document 2). ). However, the JUNB protein does not have a JNK (c-jun n-terminal kinase) binding site and is therefore not activated by JNK. Therefore, it is known that if the expression level of JUNB protein increases, AP1 complex formation by JUN protein is inhibited and canceration of cells is suppressed (Non-patent Document 3). That is, it has been clarified that the junB gene functions as a tumor suppressor gene (Non-patent Documents 3 and 4). Moreover, in psoriasis which is one of skin diseases, it is also known that the expression of junB gene is reduced in the affected area (Non-patent Document 5). Moreover, in the pathological condition of systemic inflammatory response syndrome (SIRS), it is known that the transcriptional regulatory factor AP1 transcription region is activated by JUN protein or FOS protein (Non-patent Document 6). .

  Glycyrrhizin (glycyrrhizic acid) is a compound composed of glycyrrhetinic acid and two molecules of glucuronic acid, and is known to have anti-inflammatory action (antiallergic action, inhibitory action of arachidonsan metabolic enzyme). The compounds are also known to have gastrointestinal ulcer healing action, immunoregulatory action, hepatocyte damage inhibitory action, detoxification action, virus growth inhibition / inactivation action, and the like. The compound is widely used in the clinical field, particularly as an agent for liver disease (Non-patent Document 7) and an allergy drug. Moreover, this compound is known to suppress liver cancer onset.

Eferl R. et al., Liver tumor development, c-jun antagonizes the proapoptotic activity of p53, Cell, 2003, 112, 181-92 Passegue E. et al., JunB can substitute for Jun in mouse development and cell proliferation, Nat. Genet., 2002, 30, 158-66 Shaulian E. et al., AP-1in cell proliferation and survival, Oncogene, 2001, 20, 2390-400 Zenz R. et al., Jun signaling in the epidermis: From developmental defects to psoriasis and skin tumors, Int, J. Biochem.Cell Biol., 2006, 38, 1043-9 Zenz R. et al., Psoriasis-like skin disease and arthritis caused by inducible epidermal deletion of Jun proteins, NATURE, 2005, 437, 15 Naoyuki Matsuda, Perioperative Inflammation Management Strategy-Gene Therapy for Inflammation, Abstracts of Forum Toyama "Drug Discovery" 22nd Meeting, 2007 (http://www.sugitani.u-toyama.ac.jp/ sangaku / forum / souyaku22 / 2.matsuda.pdf) Suzuki H. et al., Effects of glycyrrhizin on biochemical tests in patients with chronic hepatitis, Double blind trial, ASIAN MED. J., 1983, 26, 423-438

  An object of the present invention is to provide a novel junB gene expression promoter.

In order to solve the above-mentioned problems, the present inventors have studied the influence on the expression level of the junB gene for various compounds. As a result, glycyrrhizin monoammonium (MAG) promotes the expression of the junB gene, and the JUNB protein The present invention was completed by finding that it has an action of promoting the production of.
That is, the present invention is as follows.

(1) A junB gene expression promoter comprising a compound selected from glycyrrhizin and pharmaceutically acceptable salts thereof.
(2) The junB gene expression promoter according to (1), wherein the pharmaceutically acceptable salt is glycyrrhizin monoammonium.

  The junB gene expression promoter of the present invention significantly promotes the expression of the junB gene and increases the expression level of the JUNB protein. On the other hand, the junB gene expression promoter of the present invention does not affect the expression level of the JUN protein that is a product of the c-jun gene. Therefore, the junB gene expression promoter of the present invention can inhibit the formation of the AP1 complex by the JUN protein, and is effective in suppressing cell canceration, treating SIRS, and the like.

It is a figure which shows the mRNA copy number of junB and c-jun in a glycyrrhizin monoammonium (MAG) addition group. It is a figure (photograph) which shows the expression level of a JUNB protein and a JUN protein in a MAG addition group. It is a figure which shows the junB gene promoter activity (result of a luciferase assay) in a MAG addition group. It is a figure which shows the influence of the addition of the MAPK inhibitor which gives to junB mRNA copy number in a MAG addition group. It is a figure which shows the influence of the addition of a JAK inhibitor on the junB mRNA copy number in a MAG addition group. It is a figure which shows the influence of the addition of a PKC inhibitor on the junB mRNA copy number in a MAG addition group.

  The junB gene expression promoter of the present invention contains a compound selected from glycyrrhizin and pharmaceutically acceptable salts thereof.

In the junB gene expression promoter of the present invention, glycyrrhizin can be extracted from licorice by a conventional method and used. Moreover, you may use a commercial item.
Examples of the pharmaceutically acceptable salt of glycyrrhizin include glycyrrhizin monoammonium and glycyrrhizin diammonium; alkali metal salts such as glycyrrhizin monosodium, glycyrrhizin disodium, glycyrrhizin monopotassium, and glycyrrhizin dipotassium; or glycyrrhizin choline and the like. In addition to these, calcium salts, magnesium salts, aluminum salts, and the like can also be used. Preferably, glycyrrhizin monoammonium is used.
Moreover, the compound chosen from glycyrrhizin and its pharmaceutically acceptable salt can be used individually or in combination of 2 or more types.

  The dosage form of the junB gene expression promoter of the present invention is not particularly limited, and is non-oral such as tablets, powders, granules, capsules, fine granules, liquid medicines, inhalants, suppositories or injections. It can be an oral preparation. These can be produced by conventional methods.

  The junB gene expression promoter according to the present invention is optionally mixed with an excipient, a lubricant, a plasticizer, a surfactant, a binder, a disintegrant, a wetting agent, a stabilizer, a corrigent, an antiseptic, and a coloring agent. , A fragrance | flavor, a buffer, etc. can be mix | blended and it can formulate.

  The content of a compound selected from glycyrrhizin and a pharmaceutically acceptable salt thereof in the junB gene expression promoter of the present invention is appropriately adjusted depending on the administration subject.

  The administration method of the junB gene expression promoter of the present invention may be either oral or parenteral. The dose can be appropriately selected depending on the age, symptoms, etc. of the patient.

  As described above, if the expression level of JUNB protein is increased by promoting the expression of junB gene, the formation of AP1 complex containing JUN protein is inhibited, and thus the canceration of cells can be suppressed. Already known. Therefore, the junB gene expression promoter of the present invention can be used as a carcinogenesis inhibitor. The target cancer is cancer caused by the expression of JUN protein, and includes breast cancer, skin cancer, colon cancer, and leukemia. Of course, the junB gene expression promoter of the present invention is also effective in suppressing the onset of liver cancer. However, the mechanism by which glycyrrhizin suppresses the development of liver cancer is known, although the mechanism has not been elucidated (Non-Patent Document 7), and preferably, a liver cancer inhibitor is excluded from the scope of the present invention. It is burned. In addition, in psoriasis, which is one of the skin diseases, since it is known that the expression of junB gene is reduced in the affected area (Non-patent Document 5), the junB gene expression promoter of the present invention is It can also be used as a preventive or therapeutic agent for psoriasis. In addition, in the pathology of SIRS, it is known that the transcriptional regulatory factor AP1 transcription region is activated by JUN protein and FOS protein (Non-patent Document 6). Therefore, the junB gene expression promoter of the present invention can also be used as a preventive or therapeutic agent for SIRS.

1. The test substance glycyrrhizin monoammonium (hereinafter referred to as MAG) was examined for effects on junB mRNA expression level, c-jun mRNA expression level, JUNB protein expression level, JUN protein expression level, and junB gene promoter activity.
2. Cell The cell line HepG2 derived from human hepatoblastoma was used. Using a Dulbecco's modified Eagle's medium (DMEM: Nissui) containing 10% FBS with an appropriate amount of 10% sodium bicarbonate, a final concentration of 60 μg / ml kanamycin, and a final concentration of 8 mM glutamine, 37 ° C The cells were cultured in a CO ₂ incubator under 5% CO ₂ conditions. A 100 mm cell culture dish (FALCON) was used for the culture.

3. Influence on expression level of junB gene 3-1: Construction of standard plasmid DNA A primer surrounding the region amplified by junB primer (SEQ ID NO: 3 and 4, TAKARABio) designed for real-time RT-PCR is designed (SEQ ID NO: 1). And 2, Operon), and HotStar HiFidelity Polymerase Kit (QIAGEN). As a template, Human Genomic DNA (ROCHE) was used. After electrophoresis separation on an agarose gel and purification of the desired PCR product using QIAquick Gel Extraction Kit (QIAGEN), the obtained PCR product was subcloned into pGEM-T easy Vector (Promega).
The obtained plasmid was introduced into competent cell DH5α (Promega), cultured in LB Broth (DIFCO), and purified using GenElute Plasmid Miniprep Kit (SIGMA).

3-2: Reverse transcriptase-polymerase chain reaction (RT-PCR)
5 × 10 ⁴ HepG2 cells per well were spread on a 24 well plate (IWAKI) and cultured in a CO ₂ incubator for 18 hours to fix the cells on the bottom of the plate.
The medium in the well is replaced with a medium with MAG added (concentration: 5 mg / ml) and cultured for 24 hours. After aspirating the medium, single-stranded cDNA can be prepared directly from cultured cells without RNA purification. CDNA was prepared from the cells using FastLane Cell cDNA (QIAGEN) and RNeasy Mini Kit (QIAGEN), which removes the genome from the RNA solution. The prepared cDNA was stored at -80 ° C. In addition, as a non-addition group, in the above method, the medium in the well was replaced with a medium not added with MAG and cultured for 24 hours, and cDNA was similarly prepared.

3-3: Real-time PCR
Using Quantitect SYBR Green PCR kit (QIAGEN), the number of copies of junB mRNA in cDNA was measured using junB primers for real-time RT-PCR (SEQ ID NOs: 3 and 4, TAKARABio).
The reaction conditions were 95 ° C / 15 min x 1 cycle, (94 ° C / 15 sec, 60 ° C / 25 sec, 72 ° C / 12 sec) x 30 cycles, followed by melting curve analysis (95 ° C / 0 sec 58 ° C / 15 seconds, 95 ° C / 0 seconds, 0.5 ° C increments).
Similarly, the copy number of c-jun mRNA was also measured using a real-time c-jun primer (SEQ ID NOs: 5 and 6, TAKARABio). The reaction conditions are the same as those for measuring the junB copy number, 95 ° C / 15 minutes × 1 cycle, (94 ° C / 15 seconds, 60 ° C / 25 seconds, 72 ° C / 12 seconds) × 30 cycles, and then melting. Curve analysis (95 ° C / 0 sec, 58 ° C / 15 sec, 95 ° C / 0 sec, 0.5 ° C increments) was performed.
The copy number was corrected using GAPDH gene mRNA as a control measured with LightCycler Faststart DNA Master SYBR Green 1 and LightCycler-Primer Set Human GAPDH (both ROCHE) (reaction conditions conform to protocol).

3-4: Results
FIG. 1 shows the relative values of the junB mRNA and c-jun mRNA copy numbers in the MAG added group to the copy numbers in the non-added group.
The copy number of junB mRNA in the MAG added group was more than 5 times that in the non-added group, and there was a significant difference when t-test was performed. In addition, the copy number of c-jun mRNA in the MAG added group was about twice that of the non-added group.
From the above, it was suggested that MAG has an action of promoting the expression of junB gene. It was also suggested that MAG has an action of promoting the expression of c-jun gene.

4). Influence on the expression level of JUNB protein 4-1: Cell culture and nucleoprotein extraction
Using HepG2, the expression levels of JUNB protein and JUN protein were examined in the MAG addition group.
Since JUNB protein and JUN protein are localized in the nucleus, the nuclear protein was extracted and used in the experiment. The specific method is as follows.
A 60 mm plate (IWAKI) was sprayed with 1 × 10 ⁷ cells per plate and cultured in a CO ₂ incubator for 18 hours to fix the cells on the bottom of the plate. On the next day, the medium in the plate was replaced with 20 ml of 5 mg / ml MAG-added medium, and cultured for 24 hours. Then, the medium was removed and washed twice with ice-cold PBS. After washing, cells were collected in 10 ml PBS using a cell scraper, and nuclear proteins were extracted from the collected cells using Qproteome Nuclear Protein Kit (QIAGEN). In addition, as a non-added group, in the above method, the medium in the well was replaced with a medium not added with MAG and cultured for 24 hours, and nucleoprotein was prepared in the same manner.
The protein concentration in the extracted nucleoprotein was measured using Biorad protein assay (Biorad).

4-2: SDS-PAGE
Add 6 × loading buffer (125 mM Tris-HCl pH6.8 / 4% SDS / 10% DTT / 20% Glycerol / 0.25% BPB) and dH ₂ O to a total volume of 12 μl to 30 μg nucleoprotein extract. The sample was prepared by heating at 100 ° C. for 5 minutes.
Apply the sample to SDS polyacrylamide gel (BIO-RAD) set in the electrophoresis layer and use electrophoresis buffer (25 mM Tris-HCl pH7.5 / 192 mM Glycin / 0.1% SDS) under the condition of constant voltage 150 mV. Electrophoresis was performed.

4-3: Transfer PVDF membrane (Millipore) cut to the size of gel was immersed in methanol. Subsequently, the sample was transferred to a blotting buffer (25 mM Tris-HCl pH 7.5 / 192 mM Glycin / 0.1% SDS / 10% MtOH) and left at room temperature. Moreover, the filter paper cut to the same size as the membrane was previously immersed in the same blotting buffer.
The filter paper (3 sheets), membrane, gel, and filter paper (3 sheets) were superposed in this order on a semi-dry transfer blotting apparatus, set on the blotting apparatus, and transferred at a constant voltage of 100 mA for 80 minutes.

4-4: Blotting In order to prevent non-specific adsorption of antibodies, the protein-transferred membrane is kept at room temperature for 1 hour in skim milk solution (5% skim milk / 1% BSA / 0.5M Tris / 9% NaCl / 0.05% Tween20). Shake and block.
Primary antibodies (anti-JUNB antibody, anti-JUN antibody, anti-SP1 antibody (control) (all santacruz)) were diluted 200, 200, and 100 times with skim milk solution, and reacted with the blocked membrane for 18 hours at 4 ° C. I let you.
After the reaction, the membrane was washed twice with TBST solution (0.5M Tris / 9% NaCl / 0.05% Tween20) for 30 minutes with shaking and diluted to 1000 times with skim milk solution (AP-anti-rabbit antibody (santacruz)). ) At room temperature for 1 hour.
After the reaction, the membrane was similarly washed twice with TBST solution for 30 minutes and then washed with shaking in TBS solution (0.5 M Tris / 9% NaCl) for 5 minutes. After washing, color was developed reaction with BCIP / NBT solution (WAKO), after the band has been detected, to stop the color development reaction with 0.01% acetic acid solution, shaken for 5 minutes in dH ₂ 0, and dried at room temperature.

4-5: Results The results are shown in FIG.
The expression level of JUNB protein in the MAG added group was significantly larger than that in the non-added group. On the other hand, the JUN protein expression level was not significantly different from that of the additive-free group. Further, the expression level of SP1 protein as a control was not different between the NAC added group and the non-added group.

  From this result, it was found that MAG promotes junB mRNA expression, thereby significantly increasing the expression level of JUNB protein. Moreover, although MAG promotes c-jun mRNA expression, it was found that this level was such that it did not affect the expression level of JUN protein. Considering the results obtained together with the conventionally known findings regarding the inhibition of canceration of JUNB protein, it is presumed that MAG is effective in suppressing canceration of cells.

5. Influence on junB gene promoter activity 5-1: Determination of human junB gene promoter region and evaluation of activity To determine the promoter region of human junB gene, Ensemble Genome Browser (http://www.ensembl.org) is a genome database. The 5kb upstream of the mouse junB gene, whose gene sequence is known, is obtained from /index.html) and analyzed using the homology analysis tool VISTA (http://genome.lbl.gov/vista/index.shtml) did.
As a result, it was found that the region up to 3 kbp upstream of the human junB gene has high homology with the mouse. Moreover, it was found that the region of 3 kbp or more upstream of the human junB gene has almost no homology with the mouse.
A luciferase vector pGL4 (luc2CP, Promega) (hereinafter referred to as a 3 kb UP vector) incorporating a region up to 3 kbp upstream of the human junB gene was introduced into HepG2 cells at a concentration of 0.25 to 2 μg / ml, and the activity was measured. As a result, strong luciferase activity was measured, and the activity increased depending on the concentration of the vector.
From the above, it was found that the region up to 3 kbp upstream of the human junB gene has promoter activity.
Based on this finding, in the following experiments, the influence of MAG on junB gene promoter activity was examined using a 3 kb UP vector.

5-2: Construction of test vector
The junB gene promoter region was amplified by PCR using primers (SEQ ID NOs: 7 and 8, OPERON) designed so that EcoRV was attached to the 5 'end of the DNA fragment and HindIII site was attached to the 3' end, and agarose gel was used. Electrophoresis and purification using QIAquick Gel Extraction Kit.
The purified DNA fragment and the luciferase vector pGL4.12 (luc2CP, Promega) were treated with restriction enzymes with EcoRV and HindIII, purified using QIAquick PCR Purification Kit (QIAGEN), and subcloned with DNA Ligation Kit (TAKARA).
The obtained plasmid was introduced into competent cells DH5α and TAM1 (PROMEGA), cultured in LB Broth (DIFCO), and purified using plasmid Maxi Kit (QIAGEN) to prepare a test vector.

5-3: Luciferase assay
5 × 10 ⁴ HepG2 per well was spread on a 24 well plate and cultured in a CO ₂ incubator for 18 hours to fix the cells on the bottom of the plate.
The next day, the test vector is diluted with serum-free DMEM to 2.5 μg / ml, and the internal correction vector (a vector that always shows a constant luciferase activity even if the cell condition changes, such as drug exposure) is 50 μg / ml. Then, the lipofection reagent Tfx-20 Reagent (Promega) was added in an amount equivalent to 3 times the vector. The medium in the plate was aspirated, the prepared vector solution was added, cultured in a CO ₂ incubator for 1 hour, serum-added DMEM was added thereto, and cultured in a CO ₂ incubator for 48 hours.
After culturing, add MAG-added medium (concentration is the same as above), further incubate for 6 hours, aspirate the medium, wash cells with PBS, and use luciferase protein lysate using Dual Luciferase Assay System (Promega). Was extracted and the activity was measured (in accordance with the protocol).
Note that the luciferase protein produced using the test vector used in the Dual Luciferase Assay and the luciferase protein produced using the internal correction vector have different luminescent substrates. Therefore, even if both vectors are introduced into cells and the luciferase proteins produced from both vectors are mixed in the cell extract, the activity of the luciferase proteins produced by each vector can be obtained by adding each luminescent substrate solution separately. Can be measured.

5-4: Results
FIG. 3 shows the relative value of the luciferase activity of the MAG added group to the luciferase activity of the non-added group.
MAG decreased the promoter activity of the junB gene. One of the reasons why MAG decreased the promoter activity of junB gene is as follows. Since the DNA incorporated into the luciferase vector does not have a chromatin structure like the endogenous junB gene, it is considered that the expression was different from that of the endogenous junB gene promoter.
It is also possible that MAG does not have an effect of increasing the promoter activity of the junB gene. In other words, MAG does not increase the transcription amount of junB gene, but increases the stability of junB gene mRNA in the cell and makes it difficult to degrade, thereby reducing the amount of mRNA of junB gene and the amount of translation of JUN protein. There is a possibility that it has increased.

6). Search for signal cascade leading to activation of junB gene expression
In order to elucidate the pathway through which MAG promotes junB mRNA expression, we created a test group to which various signal cascade inhibitors were added together with MAG, and determined the junB mRNA copy number by real-time RT-PCR as described above. It was measured. That is, the copy number was internally corrected using the control GAPDH gene mRNA.
Signal cascade inhibitors include UO126 (ERK inhibitor, WAKO), SB203580 (p38 inhibitor, WAKO), JNKI1 (JNK inhibitor, CALBIOCHEM), AG490 (JAK inhibitor, CALBIOCHEM), PKCinhibitorpeptide (PKC inhibitor, CALBIOCHEM) )It was used. The concentration of each inhibitor was determined with reference to past papers and IC50 (50% effective concentration).

The results are shown in FIGS. The copy number of junB mRNA in each graph is represented by a relative value when the copy number in the control is 1.
In all the inhibitor-added groups, there was no tendency for the junB mRNA copy number to decrease as compared to the inhibitor-free group.
From this, it was found that the promotion of junB gene expression by MAG was not inhibited by any of the inhibitors. In other words, it was suggested that the promotion of junB gene expression by MAG occurs via a different route from the previously reported MAP kinase, JAK / STAT pathway and PKC pathway.

  The junB gene expression promoter of the present invention can be used for the purpose of suppressing carcinogenesis. The junB gene expression promoter of the present invention can be used for the purpose of prevention or treatment of SIRS.

Claims (2)

  A junB gene expression promoter comprising a compound selected from glycyrrhizin and pharmaceutically acceptable salts thereof.   The junB gene expression promoter according to claim 1, wherein the pharmaceutically acceptable salt is glycyrrhizin monoammonium.

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