JP4556511B2 - 15d-PGJ2 and method using 15d-PGJ2 - Google Patents

Description

  The present invention relates to 15d-PGJ2 (15-deoxy-delta 12,14-prostaglandin J2) and a method using 15d-PGJ2. More specifically, 15d-PGJ2 that promotes the expression of the per2 gene, a circadian rhythm regulator containing 15d-PGJ2, an anticancer agent containing 15d-PGJ2, and a per2 gene using 15d-PGJ2 Expression promotion method, circadian rhythm adjustment method using 15d-PGJ2, circadian rhythm modulation detection marker and circadian rhythm modulation detection method, cancer onset risk detection marker, cancer onset risk detection method, stress detection marker and The present invention relates to a stress detection method.

  The circadian rhythm (circadian rhythm) is a biological rhythm having a cycle of about 24 hours, and is an in vivo phenomenon that is universally seen in many organisms ranging from single-celled organisms to humans.

  In mammals, including humans, it is thought that there is an “internal clock” in the suprachiasmatic nucleus of the hypothalamus, and the circadian rhythm is adjusted at the individual level in the suprachiasmatic nucleus of the hypothalamus. It is estimated that

  In many organisms, including mammals, circadian rhythm is regulated by “clock genes”. The clock gene is a gene involved in the formation of circadian rhythm. Known clock genes include, for example, a per (period) gene, a clock gene, and a Bmal1 gene. As for the per gene, genes of per1, per2, and per3 have been discovered.

  In mammals, clock gene expression is particularly high in the suprachiasmatic nucleus of the hypothalamus. That is, it is considered that the clock gene is strongly involved in not only the circadian rhythm at the cell level but also the circadian rhythm at the individual level of mammals (refer to Non-Patent Document 1 for the above matters).

  In addition, it has already been clarified that circadian rhythm and cancer are related. For example, Non-Patent Document 2 publishes an experimental report showing that the clock gene (per2) may function as a tumor suppressor gene and that cancer cells grow if the function is hindered. is doing.

Here, “15d-PGJ2” which is highly relevant to the present invention will be described. “15d-PGJ2 (15-deoxy-delta 12,14-prostaglandin J2)” is a metabolite of PGD ₂ (prostaglandin D ₂ ) and is known to exist in vivo. 15d-PGJ2 is known as a ligand of PPARγ (Peroxisome Proliferator Activated Receptor γ), a nuclear receptor that is involved in the regulation of fat metabolism expression. In addition, use of 15d-PGJ2 for anti-inflammatory agents and the like is being studied.

Patent Documents 1 to 3 disclose circadian rhythm adjusting agents. Patent Documents 4 to 6 disclose 15d-PGJ2. Non-Patent Document 1 describes circadian rhythms and clock genes, and Non-Patent Document 2 describes the relationship between clock genes and cancer.
JP 2001-316281 A JP 2003-81829 A JP 2003-335669 A JP-A-11-189537 JP-A-11-199448 JP 2002-27998 A "Molecular biology of clock genes", Springer Fairlark Tokyo, edited by Hitoshi Okamura and Yoshitaka Fukada, published April 5, 2004 Loning Fu, et al “The Circadian Gene Period2 Plays an Important Role in Tumor Suppression and DNA Damage Response In Vivo”; Cell, Vol. 111, 41-50, October 4 (2002)

  The present invention provides a circadian rhythm mechanism, provides a circadian rhythm adjusting agent or a circadian rhythm adjusting method, provides an anticancer agent, and circadian rhythm abnormality The main purpose is to provide a means for detecting stress.

  The present invention first provides 15d-PGJ2 or a salt thereof that promotes the expression of the per2 gene, and a method for promoting the expression of the per2 gene using 15d-PGJ2 or a salt thereof.

  Here, 15d-PGJ2 is an abbreviation for “15-deoxy-delta 12,14-prostaglandin J2” and has the following chemical structure (the same applies hereinafter).

  In the course of reaching the present invention, the present inventors constructed a screening system for identifying a factor that promotes the expression of the clock gene per2 in order to elucidate the circadian rhythm mechanism. Using the screening system, a factor that promotes the expression of the clock gene per2 was searched.

  As a result, 15d-PGJ2 was identified as a factor that promotes the expression of the clock gene per2. That is, it was found that 15d-PGJ2 has an action of promoting the expression of the per2 gene. Therefore, expression of per2 gene can be promoted by using 15d-PGJ2 (including its salt).

Next, in the present invention, a circadian rhythm adjusting agent containing 15d-PGJ2 or a salt thereof, or a 15d-PGJ2 derivative or a salt thereof, and a circadian rhythm adjusting method using 15d-PGJ2 or a salt thereof, I will provide a. Here, the 15d-PGJ2 derivative refers to a compound generated by a chemical change of a small part in the 15d-PGJ2 molecule. For example, arachidonic acid, PGH2 (prostaglandin H), which is a precursor of 15d-PGJ2 in vivo, is used. _2), PGD2 (prostaglandin _D 2), PGJ2 (prostaglandin _J 2), etc. delta 12-PGJ2 (delta 12-prostaglandin _{J 2),} included in the 15d-PGJ2 derivatives (hereinafter the same).

  When the circadian rhythm is modulated, the circadian rhythm can be adjusted by promoting the expression of the per2 gene using 15d-PGJ2. That is, for example, the circadian rhythm of the whole living body can be adjusted by adjusting the circadian rhythm of per2 gene expression using 15d-PGJ2 according to the present invention. Therefore, 15d-PGJ2 (including salts thereof and derivatives and salts of derivatives) according to the present invention can be applied to the adjustment of circadian rhythm.

  When circadian rhythm is modulated, for example, sleep disorder, arousal disorder, jet lag, insomnia, autonomic dysfunction, depression, senile dementia, night shift / shift work, etc. Deterioration of physical condition due to aging, exhaustion due to irregular rhythm of life in autism, etc. In addition, as a disease that is triggered by the modulation of circadian rhythm, for example, there is an increase in the incidence of breast cancer in nurses and the like by night work. The circadian rhythm adjusting agent and the circadian rhythm adjusting method according to the present invention can be applied to these pathological conditions.

  Moreover, in this invention, the anticancer agent containing 15d-PGJ2 or its salt, or 15d-PGJ2 derivative | guide_body or its salt is provided.

  As described above, since the per2 gene also has a function as a tumor suppressor gene, by promoting the expression of the per2 gene using 15d-PGJ2 (including salts thereof and derivatives and derivatives thereof), Can suppress the growth of cancer cells.

  Next, the present inventor made 15d-PGJ2 or a salt thereof as a marker for detecting circadian rhythm modulation, and a method for detecting circadian rhythm modulation using 15d-PGJ2 or a salt thereof as a marker. provide.

  15d-PGJ2 is located upstream of the circadian rhythm mechanism in the living body. That is, 15d-PGJ2 regulates the expression of the per2 gene, and the expression of the per2 gene regulates the circadian rhythm. Therefore, 15d-PGJ2 (including its salt) can be used as a marker for detecting circadian rhythm modulation.

  Moreover, in this invention, the cancer onset risk detection method using 15d-PGJ2 or its salt as a cancer onset risk marker and 15d-PGJ2 or its salt as a marker is provided.

  In vivo, the decrease of 15d-PGJ2 suppresses the expression of per2 gene, and the suppression of per2 gene expression promotes the growth of cancer cells. Therefore, 15d-PGJ2 (or a salt thereof) is used as a marker of cancer development risk. Useful.

  Furthermore, the present invention provides 15d-PGJ2 or a salt thereof as a stress detection marker, and a stress detection method using 15d-PGJ2 or a salt thereof as a marker.

  The present inventors have newly found that there is a correlation between the expression level of the per2 gene and stress. That is, it was newly found that the expression of the per2 gene is promoted when stress is applied. This suggests that there is a mechanism of action that increases 15d-PGJ2 and promotes the expression of the per2 gene in vivo due to stress loading. Therefore, 15d-PGJ2 (or a salt thereof) is useful as a stress detection marker.

  As described above, when 15d-PGJ2 or a salt thereof is used as a marker, for example, there is a method of detecting 15d-PGJ2 in blood, urine, or the like. By detecting 15d-PGJ2 such as blood or urine as a marker, modulation of circadian rhythm, risk of developing cancer, stress, etc. can be easily detected. Further, the present invention has an advantage that the circadian rhythm modulation, cancer onset risk, stress, and the like can be quantified by measuring the amount of 15d-PGJ2 in blood or urine.

  15d-PGJ2 according to the present invention has an effect of promoting the expression of the per2 gene. Moreover, when 15d-PGJ2 is used as a marker, circadian rhythm modulation, cancer risk, stress, etc. can be detected.

  In Example 1, a screening system for identifying factors involved in the expression of the clock gene per2 was constructed, and candidate substances were screened. The specific procedure is as follows.

  First, a promoter vector of clock gene per2 was incorporated into a luciferase luminescence vector pGL3 basic (manufactured by Promega) to prepare a recombinant vector. Next, this recombinant vector was transfected into cultured cells (RAT1 cells), and the recombinant cells were isolated and passaged.

  The recombinant vector has a promoter region of per2 and a region encoding luciferase (luminescent enzyme). Therefore, when a transcription factor of the per2 gene binds to the promoter region of per2, luciferase is expressed and the cultured cells emit light.

  Next, a candidate substance was searched (screened) using the recombinant cultured cells. Candidate substances were added to the recombinant cultured cells (1 μM or 10 μM), and the amount of luciferase luminescence was measured every 15 minutes for 3 days. At the time of screening, a candidate substance was added to a serum-free medium and cultured for 1 hour, and then cultured in a 1% serum-added medium. A weak luminescence measuring device (manufactured by Hamamatsu Photonics Co., Ltd.) was used for measuring luciferase luminescence. The candidate substances subjected to the screening are 203 kinds of lipids of Bioactive Lipid library (manufactured by BIOMOL).

  As a negative control, the amount of luciferase luminescence was similarly measured every 15 minutes for 3 days for those cultured only under normal culture conditions. (FIG. 1). As a positive control, serum was added to the recombinant cultured cells so that the final concentration was 50% of the medium, and the amount of luciferase luminescence was similarly measured every 15 minutes for 3 days (FIG. 2). Serum is known to contain a factor that promotes the expression of a clock gene, and is suitable as a positive control.

  1 and 2, the horizontal axis indicates time (minutes), and the vertical axis indicates the light emission amount (number of light emission). In FIG. 2, the amount of light emission increases in about 1440 minutes (about 1 day has passed) compared to FIG. 1. This indicates that addition of serum to a high concentration promoted the expression of clock gene per2 and increased the amount of luciferase luminescence.

  As a result of the screening, 15d-PGJ2 was identified as a factor that promotes the expression of the clock gene per2. That is, in FIG. 3, by adding 15d-PGJ2, the amount of luminescence increases around 1440 minutes as in the positive control of FIG. Therefore, it was found that 15d-PGJ2 promotes the expression of clock gene per2. In FIG. 3, the horizontal axis indicates time (minutes), and the vertical axis indicates the light emission amount (number of light emission).

  Conventionally, 15d-PGJ2 identified in Example 1 is known as a ligand for the nuclear receptor PPARγ. Therefore, in Example 2, as an aid for elucidating the mechanism of action, it was examined whether or not the nuclear receptor PPARγ is involved in promoting expression of the clock gene per2 by 15d-PGJ2.

  The experiment was performed in substantially the same procedure as in Example 1. 1 μL of an agonist of the nuclear receptor PPARγ was added to the recombinant cultured cells, and the amount of luciferase luminescence was measured every 15 minutes for 3 days. Similarly, in the case where 1 μL of the nuclear receptor PPARγ agonist was added after 5 μM of the antagonist of the nuclear receptor PPARγ was added for 1 hour pretreatment, the amount of luciferase luminescence was similarly changed every 15 minutes for 3 days. Was measured.

  In addition to 15d-PGJ2, Az-PAF (manufactured by Cayman chemical) was used as an agonist of the nuclear receptor PPARγ, and GW9662 (manufactured by SIGMA) was used as an antagonist.

  The results are shown in FIG. In FIG. 4, when comparing the upper four graphs (with no antagonist added), when “15d-PGJ2” was added, the amount of luminescence increased before and after 1440 minutes (one day). When “Az-PAF” was added, only the same change as in the case of the negative control (the rightmost graph in the upper part of FIG. 4) was shown.

  Further, in FIG. 4, when the antagonist was not added (upper graph) and the pretreatment with the antagonist (lower graph) were compared, 15d-PGJ2, Az-PAF, positive control (Serum), negative control In both cases, both graphs showed almost the same results.

  The above results indicate that the expression of clock gene per2 by 15d-PGJ2 is promoted by another signal pathway not via the nuclear receptor PPARγ. That is, it is suggested that the action of promoting the expression of the clock gene per2 by 15d-PGJ2 newly found this time is based on a mechanism completely different from the action of the conventionally known 15d-PGJ2.

  In each graph shown in FIG. 4, the horizontal axis indicates time (minutes), and the vertical axis indicates the light emission amount (the number of light emission). In FIG. 4, “Control” is the result of the negative control, “Serum” is the result of the positive control (when the serum is added so that the final concentration is 50% of the medium), “−” (FIG. 4). The middle and lower four graphs) show no added antagonist, and “+” (upper four graphs in FIG. 4) shows the added antagonist.

  Example 3 is an experiment that clarifies the correlation between stress load and per2 gene expression. The procedure is as follows.

  First, the mice were divided into a first group to a sixth group (6 mice in each group). In the experiment, ICR mice (male, 5 weeks old) purchased from SLC were used. The circadian rhythm was then synchronized for all groups of mice. That is, mice were housed in a room under light conditions from 8 o'clock to 20 o'clock and dark conditions from 20 o'clock to 8 o'clock the next two weeks to synchronize the circadian rhythm. Next, all groups of mice were housed in a dark room from 8 o'clock to 36 hours (until 20 o'clock the next day).

  Next, for the first group to the sixth group of mice, a stress load start time (one hour before the blood collection time) and a blood collection time were set, and blood was collected after applying stress for one hour from the stress load start time. By setting the blood collection time (stress load start time) for each group every 4 hours, the circadian change of the stress load can be examined.

  That is, the first group of mice was set to apply stress for 1 hour from 19:00 (1 hour before the end of the dark condition), and the second group was set at 3 o'clock (after the dark condition ended, the same applies hereinafter) Was set to apply stress for 1 hour from 3 o'clock, 4th group from 7 o'clock, 5th group from 11 o'clock, and 6th group from 15:00.

  After 1 hour stress, the first group is 20:00 (at the end of the dark condition), the second group is at 24:00 (after the dark condition is finished), the third group is at 4 o'clock the next time, the fourth group is the next time At 8 o'clock, blood was collected at 12 o'clock in the 5th group and at 6 o'clock in the 6th group. By setting the blood collection time in this way, blood in each time zone of the day can be examined, so that circadian changes in stress load can also be examined.

  The stress load was performed by the following method. Mice were placed in a mouse fixed adjuster (SANPLATEC, product code 13302E) used for tail vein injection for 1 hour. As a result, the mouse was restricted in movement and stressed.

  Next, the collected blood was mixed for each group, and the expression level of the per2 gene in leukocytes in the blood was examined. The specific procedure is as follows.

  First, the collected blood was mixed for each group, and then white blood cells were separated by a density gradient method using NycoPrep 1.077 Animal (NYCOMED PHARMA). Next, total RNA was extracted from the cells using Promega total SV RNA Isolation Kit (Promega). Next, reverse transcription PCR was performed using SuperScript2 (Invitrogen), and the transcription product of per2 was amplified from the extracted total RNA to synthesize cDNA of per2. Then, the expression level of the per2 gene was determined based on the amount of synthesized per2 cDNA.

  In addition, as a control, experiments were also performed on mice not subjected to stress (Group 1 to Group 6) in exactly the same manner as described above. That is, blood was collected without applying stress to the first group to the sixth group of mice, and the expression level of the per2 gene was determined by the above procedure.

  In the above experiment, Prism 7000 (ABI) was used as the PCR device. In the case of PCR, SYBR Green PCR Master Mix (ABI) was used to perform 50 ° C., 2 minutes / 95 ° C., 10 minutes / (95 ° C., 15 seconds / 60 ° C., 1 minute) × 40 cycles. . As the primer for the per2 gene, the sense primer shown in SEQ ID NO: 1 and the antisense primer shown in SEQ ID NO: 2 were used.

  In the above experiment, the expression level of the per2 gene is a relative value calculated based on the expression level of the housekeeping gene. First, during the above experimental procedure, β-actin and G3PDH transcripts were simultaneously amplified from total RNA extracted during reverse transcription PCR to synthesize β-actin and G3PDH cDNA. β-actin and G3PDH are housekeeping genes, and their expression levels (cDNA levels) are almost constant. Therefore, the relative expression level of the per2 gene was calculated by comparing the cDNA amount of the per2 gene with the cDNA amount of β-actin or G3PDH. In calculating the expression level, the expression level of the per2 gene was determined using two types of housekeeping genes (β-actin, G3PDH), and the error when using β-actin and when using G3PDH was corrected. By adjusting, the accuracy of the expression level of the per2 gene was improved.

  The results are shown in FIG. In FIG. 5, “Wild” refers to a group of mice not subjected to stress, and “Stress” refers to a group of mice subjected to stress. In FIG. 5, the horizontal axis indicates the first to sixth groups of mice and each blood collection time. However, in order to show the circadian change, the horizontal axis was rearranged in the order of blood sampling time, and from the left, the fourth group, the fifth group, the sixth group, the first group, the second group, and the third group were arranged in this order.

  In FIG. 5, the bar graph shows the per2 expression level. The vertical axis (left side) in this case represents the per2 expression level (value calculated by the above method). As this bar graph shows, the per2 expression level was significantly higher in the stressed mice group than in the non-stressed mice group in all groups. This indicates that the expression of per2 increases markedly when stress is applied.

  In addition, the correlation between the stress load and per2 gene expression clarified by this experiment indicates that there is a possibility that there is an action mechanism that 15d-PGJ2 increases due to stress load and promotes the expression of per2 gene. Strongly suggest. Therefore, it is suggested that 15d-PGJ2 can be a stress detection marker.

  In addition, in this experiment, as shown in FIG. 5, the difference in the per2 expression level between the group of mice given stress and the group of mice not given stress was particularly remarkable in the fourth group (collected at 8 o'clock). It was. There was also a significant difference in the per2 expression level in the fifth group (collected at 12:00), the sixth group (collected at 16:00), and the seventh group (collected at 20:00). Therefore, for example, when 15d-PGJ2 in blood is detected as a stress detection marker, it is preferable to collect blood from 8 o'clock to 20 o'clock, and it is particularly preferable to collect blood from around 8 o'clock or from 8 o'clock to 12 o'clock. It is done.

  The line graph in FIG. 5 shows the amount of corticosterone in plasma. The vertical axis (right side) in this case represents the plasma corticosterone level (ng / ml). In FIG. 5, “CORT” indicates corticosterone. Corticosterone is a hormone known to increase plasma concentration when stressed.

  As shown in FIG. 5, the amount of corticosterone in plasma was significantly increased in the group of mice given stress compared to the group of mice not given stress. Therefore, the stress loading method used in this experiment is effective as a means for creating a stress loading model.

  The amount of corticosterone in plasma was determined by performing ELISA on the collected blood of each group of mice. For the ELISA method, Enzyme immunoassay kit (manufactured by Assay Designs Inc.) was used. Donkey anti-sheep IgG antibody was used as the primary antibody, and sheep polyclonal antibody was used as the secondary antibody.

  The 15d-PGJ2 (including salts thereof) that promotes the expression of the per2 gene according to the present invention and the method for promoting the expression of the per2 gene using 15d-PGJ2 may be used for, for example, a kit for expressing the per2 gene There is a possibility of industrial use.

  The 15d-PGJ2 (a salt thereof, or a 15d-PGJ2 derivative or a salt thereof) according to the present invention may be applicable to, for example, a prophylactic / therapeutic agent for diseases related to modulation of circadian rhythm, an anticancer agent, etc. In addition, it is applicable to supplements, food additives, laboratory reagents, etc., and is industrially useful.

  The circadian rhythm adjustment method using 15d-PGJ2 or a salt thereof according to the present invention may be applicable to a service industry, health promotion service industry, relaxation service industry, etc. for the purpose of circadian rhythm adjustment. There is a possibility of being available on top.

  15d-PGJ2 (including a salt thereof) as a marker according to the present invention and a method using 15d-PGJ2 or a salt thereof according to the present invention as a marker include, for example, modulation of circadian rhythm, risk of developing cancer, There is a possibility that it can be used for a kit for detecting stress, and there is a possibility that it can be used industrially.

Change in luminescence amount of luciferase in the negative control over time. Change in luminescence amount of luciferase of positive control over time. Change with time of luciferase luminescence amount of 15d-PGJ2. Comparison of changes over time in the amount of luciferase luminescence when an agonist of the nuclear receptor PPARγ is added. The figure which shows the per2 expression level at the time of giving stress.

Claims (1)

A per2 gene expression promoter comprising 15d-PGJ2 or a salt thereof as an active ingredient .

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