KR20090095304A - Use of Peroxiredoxin 6 as target for cancer invasion or metastasis - Google Patents

Abstract

A cancer invasion marker having peroxiedoxin 6 activity is provided to develop an inhibitor of metastasis which suppresses the expression and activation of peroxiedoxin 6. A method for determining invasive cancer cell comprises: a step of measuring the expression of nucleic acid which encodes Prx6 in cancer cell sample from a patient; and a step of determining the invasive cancer cell if the expression level is higher than the expression level of control sample. A kit for determining the invasive cancer cell comprises primer set which is specific to a nucleic acid coding the Prx6, specific antibody which is specific to Prx6 and a substrate to the activation of lipase of Prx6.

Description

Use of Peroxiredoxin 6 as target for cancer invasion or metastasis}

The present invention is a cancer invasive marker having the activity of Peroxiredoxin 6, a method and kit for determining invasive cancer cells using the same, a composition for inhibiting cancer cell invasiveness, a method for inhibiting cancer cell invasiveness, It relates to compositions and kits.

Peroxiredoxin (Prx) is an antioxidant enzyme with peroxidase activity that reduces oxides such as hydrogen peroxide. Six types of mammalian cells, Prx1-6, are known. To date, these studies have focused on the enzymatic and biochemical mechanisms and properties of Prx peroxidase activity, and as a result, their understanding is clearly established (Rhee SG et al. Free) . Radic Biol Med 38: 1543-1552, 2005). On the contrary, research on the possibility that Prx modulates the function and activity of cells, that is, the role of cell biology has only recently been reported.

First, it was speculated that Prx would protect cells from oxidative stress from the point of view of antioxidant enzymes. In fact, these cytoprotective functions have been confirmed using Prx1, Prx2 and Prx6 (Immenschuh S and Baumgart-Vogt E). Antioxid Redox Signal 7: 768-777, 2005).

Further, the substrate is hydrogen peroxide Prx is a view that also can serve as a long term wateuna is considered simply toxic substances come in recent years is a delivery signal that can control the various functions of the dominant cell factor (Rhee SG. Exp Mol Med 31: 53-59, 1999). For example, cell growth factors such as EGF and PDGF induce a small amount of hydrogen peroxide production in the cell, and the hydrogen peroxide thus produced promotes cell growth. Therefore, it was predicted that Prx, a hydrogen peroxide degrading enzyme, might interfere with the action of cell growth factors, and it was proved that Prx2 could actually do this (Choi MH et al. Nature 435: 347-353, 2005).

Recent studies suggest that some Prx may have cytoprotective and cell growth regulation functions, but whether this is a characteristic that is common to all Prx and whether there are other unknown features of Prx. It has not been identified yet. In particular, the question of the role of lipase activity specific to Prx6 has not been solved yet.

In addition, Prx is thought to be involved in pathological conditions such as cancer as well as normal conditions. This is because Prx expression has been reported to be increased in many types of cancer cells. For example, when cancer tissues collected from lung cancer patients were analyzed by immunostaining or RT-PCR, all six Prxs were reported to be expressed in lung cancer cells (Lehtonen ST et al. Int J Cancer 111: 514-). 521, 2004). Therefore, although Prx may be important for the development or progression of cancer, there is no direct research on this.

Prx6 has unique characteristics different from Prx1 to Prx5. Prx6 has peroxidase activity like Prx1 to Prx5, but further has lipase activity that degrades phospholipids. In this regard, Prx6 has been called a bifunctional enzyme (Chen JW et al., J. Biol . Chem . 275: 28421-28427, 2000), but studies on the role of lipase activity of Prx6 Is very incomplete.

Accordingly, the present inventors have studied the expression and function of Prx in cancer cells, inducing the expression of uPA (urokinase-type plasminogen activator), an extracellular hepatic degrading enzyme, through lipase activity in lung cancer cells. Results Confirming that the invasiveness of cancer cells is promoted, the present invention has been completed regarding the use of Prx6 as a target for invasion and metastasis in various carcinomas.

It is an object of the present invention to provide a cancer invasive marker having the activity of peroxyredoxin 6 (Prx6) and a method for determining the invasiveness of cancer cells using the same.

Still another object of the present invention is to provide an invasive composition of cancer cells comprising an inhibitor of peroxyredoxin 6.

Still another object of the present invention is to provide a composition for searching for a substance for inhibiting invasiveness of cancer cells including peroxyredoxin 6 and a method for searching for a substance for inhibiting invasiveness of cancer cells using the same.

In order to achieve the above object, the present invention provides a cancer invasive marker having the activity of peroxyredoxin 6 (Prx6).

The cancer invasive marker of the present invention has the activity of peroxyredoxin 6 having the amino acid sequence of SEQ ID NO: 1 (NCBI Accession No: NP004896), wherein the amino acid sequence of SEQ ID NO: 1 is encoded by the nucleotide sequence of SEQ ID NO: 2 .

According to the present invention, peroxyredoxin 6 promotes invasiveness of cancer cells and has lipase activity unlike other peroxyredoxins. Promoting invasiveness of cancer cells by peroxyredoxin 6 was found to be mediated by lipase activity. Thus, peroxyredoxin 6 may act as a target of cancer cell infiltration or metastasis, and the diagnosis of metastasis following cancer cell infiltration may be performed by quantitative analysis of the expression level of peroxyredoxin 6 in cancer cells or tissues. Can be.

The present invention also provides a method for diagnosing cancer metastasis, comprising obtaining a protein sample expressed from a tissue or cell sample of an individual, and identifying and quantifying the expression of peroxyredoxin 6 in the sample obtained above. .

According to one embodiment of the present invention, whether or not the expression of peroxyredoxin 6 in a protein sample expressed from a tissue or cell sample of an individual includes activity including luminescence or fluorescence according to reaction with the substrate of peroxyredoxin 6 Can be identified and quantified through the detection of.

The invention also includes measuring the expression level of a nucleic acid encoding Prx6 in a cancer cell sample isolated from a patient; And comparing the expression level with the expression level of the Prx6-encoding nucleic acid in a control sample consisting of normal cells or cells identified as not invasive, wherein if the expression level is higher than the expression level in the control sample, the cancer cells are referred to as invasive cancer cells. It provides a method for determining invasive cancer cells, comprising the step of determining.

The method for determining invasive cancer cells of the present invention comprises measuring the expression level of a nucleic acid encoding Prx6 in a sample. The expression level of the nucleic acid encoding Prx6 can be measured at the nucleic acid or protein level. In addition, the expression level of the nucleic acid encoding Prx6 can be measured at the mRNA level.

In the method for determining invasive cancer cells of the present invention, the expression level of nucleic acid encoding Prx6 can be measured using a substrate of peroxyredoxin 6 at the protein level. The substrate of peroxyredoxin 6 may be a phospholipid such as hydrogen peroxide for peroxidase activity, 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC) for lipase activity (Chen JW). et al., J. Biol . Chem . 275: 28421-28427, 2000).

In the method for determining the invasiveness of cancer cells of the present invention, the cancer cell sample may be any kind of cancer cells, and preferably may be selected from the group consisting of lung cancer, cervical cancer and gastric cancer.

In the method for determining the invasiveness of cancer cells of the present invention, the expression level of Prx6 encoding nucleic acid in cancer cell sample is normal cell or the expression level of Prx6 encoding nucleic acid in control sample composed of cancer cells of the same type as confirmed to be invasive. When increased compared to, the cancer cells may be determined to be invasive cancer cells.

The present invention also provides a kit for determining invasive cancer cells, comprising a substance for measuring the expression level of a Prx6 encoding nucleic acid in a cancer cell sample isolated from a patient.

In the kit for determining invasive cancer cells of the present invention, the substance for measuring the expression level of the nucleic acid encoding Prx6 is a primer set specific for the nucleic acid encoding Prx6, a probe, an antibody specific for Prx6, and lipase activity of Prx6. It can be selected from a substrate for.

According to one embodiment of the present invention, the antibody of Prx6 included in the kit for determining invasive cancer cells of the present invention is reacted with a sample obtained from a subject, for example, a protein extract or plasma of cancer cells or tissues, and an ELISA ( Enzyme Linked Immunosorbent Assay method or peroxyredoxin 6 specific histoimmunostaining method can be used to quantitatively determine the invasiveness of cancer cells in the sample. Invasiveness of cancer cells can also be determined by comparing the expression levels of peroxyredoxin 6 using Western blotting in samples from subjects and samples from normal subjects.

Antibodies to peroxyredoxin 6 having the amino acid sequence of SEQ ID NO: 1 included in the kit of the present invention may be produced through commercial monoclonal or polyclonal antibody production methods well known in the art to which the present invention pertains or are commercially available. You can purchase and use what is available.

According to one embodiment of the present invention, the antibody of peroxyredoxin 6 having the amino acid sequence of SEQ ID NO: 1 is conjugated with an enzyme such as alkaline phosphatase or horseradish peroxidase. It can be quantitated through color reaction using primary antibodies and their substrates. Alternatively, to confirm the expression level of peroxyredoxin 6, an enzyme such as alkaline phosphatase or horseradish peroxidase for detecting a color reaction can be directly conjugated to the antibody of peroxyredoxin 6.

Kits for determining invasive cancer cells of the present invention may also comprise PCR primers or probes for nucleic acids encoding peroxyredoxin 6 having the nucleotide sequence of SEQ ID NO: 2.

According to one embodiment of the present invention, the expression of peroxyredoxin 6 is carried out by performing RT-PCR or quantitative RT-PCR using primers for nucleic acids encoding peroxyredoxin 6 having the nucleotide sequence of SEQ ID NO: 2. Invasiveness of cancer cells can be determined by comparing the amount with the normal group. In addition, Northern blot analysis may be performed using a probe for the nucleic acid encoding peroxyredoxin 6, and the invasiveness of cancer cells may be determined by comparing the expression level of peroxyredoxin 6 with the normal group. .

The present invention also provides a composition for inhibiting invasiveness of cancer cells, comprising a substance that inhibits the expression of Prx6 encoding a nucleic acid in a cell or inhibits the activity of Prx6. Inhibition of invasiveness of cancer cells in the present invention includes the reduction and prevention of cancer cell infiltration and metastasis.

Since peroxyredoxin 6 is expressed at a high rate in cancer invasive or metastatic cancer cells, cancer metastasis can be suppressed by reducing or inhibiting its expression or activity using an inhibitor of peroxyredoxin 6.

Inhibitors of peroxyredoxin 6 contained in the composition for inhibiting invasiveness of cancer cells of the present invention inhibit the activity of a substance or peroxyredoxin 6 that inhibits the expression of a gene encoding peroxyredoxin 6, in particular, lipase activity It may be a substance. Inhibitors of peroxyredoxin 6 contained in the composition for inhibiting invasiveness of cancer cells of the present invention is a monoclonal or polyclonal antibody of peroxyredoxin 6 having the amino acid sequence of SEQ ID NO: 1, a nucleic acid or a sequence encoding the antibody SiRNA for a nucleic acid encoding peroxyredoxin 6 having a nucleotide sequence of the number 2. The substance that inhibits the activity of Prx6 included in the composition for inhibiting invasiveness of cancer cells of the present invention may be an antibody against Prx6, or an active fragment thereof or a nucleic acid encoding the same. In addition, the substance that inhibits the activity of Prx6 contained in the invasive inhibitory composition of cancer cells of the present invention may be a decoy molecule (decoy) molecule for the substrate binding site of lipase activity of Prx6.

 According to one embodiment of the present invention, when administering a monoclonal antibody or polyclonal antibody against peroxyredoxin 6 having the amino acid sequence of SEQ ID NO: 1 or a nucleic acid encoding the same, the activity of peroxyredoxin 6 is inhibited. Can inhibit the infiltration or metastasis of cancer cells.

According to another embodiment of the present invention, when administering siRNA to a nucleic acid encoding a peroxyredoxin 6 having a nucleotide sequence of SEQ ID NO: 2, the expression of peroxyredoxin 6 is inhibited to infiltrate or metastasize cancer cells Can be suppressed.

The composition for inhibiting invasiveness of cancer cells of the present invention may further comprise one or more other anticancer agents. Inhibitors of peroxyredoxin 6 having the amino acid sequence of SEQ ID NO: 1 according to the present invention can be used with chemotherapeutic agents well known in the art. Chemotherapeutic agents that may be included in the composition for inhibiting cancer metastasis of the present invention include cyclophosphamide, aziridine, alkylalfonsulfonate, nitrosourea, dicarbazine, carboplatin, alkylating agents such as cisplatin, Antibiotics such as mitomycin C, anthracycline, doxorubicin, methotrexate, 5-fluorouracil, antimetabolite agents such as cytarabine, gemcitabine, plant derived drugs and hormones such as vinca alkaloids and the like.

Compositions for inhibiting invasiveness of cancer cells of the present invention, in addition to the inhibitory agent of peroxyredoxin 6, pharmaceutically acceptable carriers and excipients, disintegrants, sweeteners, binders, coatings, swelling agents, lubricants, lubricants, or flavoring agents And pharmaceutically acceptable adjuvants.

For compositions formulated into liquid solutions, acceptable pharmaceutical carriers include saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, dextrin solution, glycerol, ethanol or mixtures thereof, as necessary And may include other conventional additives such as antioxidants, buffers, antimicrobials, and the like. In addition, the compositions of the present invention may be formulated into injectable formulations, pills, capsules, granules or tablets, such as aqueous solutions, suspensions, emulsions, etc., with the addition of diluents, dispersants, surfactants, binders and lubricants additionally.

The present invention also provides a composition for screening a substance that inhibits invasiveness of cancer cells, including Prx6. The composition for the search for a substance for inhibiting the invasiveness of cancer cells of the present invention is a substance for inhibiting the transcription or translation of a nucleic acid encoding peroxyredoxin 6 or a substance for inhibiting the activity of peroxyredoxin 6 to infiltrate cancer cells It is possible to find a substance that suppresses.

The composition for searching for a substance for inhibiting invasiveness of cancer cells of the present invention may include one or more sequences selected from all or part of SEQ ID NO: 2, a sequence including polymorphism of SEQ ID NO: 2, wherein the sequence is a prokaryote Or in a form included in a eukaryotic expression vector system.

The composition for searching for a substance for inhibiting invasiveness of cancer cells of the present invention has a amino acid sequence of SEQ ID NO: 1, a protein expressed from a sequence comprising a nucleotide sequence of SEQ ID NO: 2 or a polymorphism of SEQ ID NO: 2, or peroxyre Polypeptide fragments of peroxyredoxin 6 having physiological activity equivalent to toxin 6 may be included as active ingredients.

The present invention also provides a kit for searching for substances that inhibit invasiveness of cancer cells, including Prx6 and reagents for measuring lipase activity of Prx6.

 The kit for searching for a substance for inhibiting invasiveness of cancer cells of the present invention includes a reagent for measuring the lipase activity of Prx6, and the reagent may include a phospholipid such as DPPC, which is a substrate of the lipase activity of Prx6.

The present invention also comprises the steps of contacting the test substance with Prx6 to measure the lipase activity of Prx6; And comparing the measured lipase activity with the control lipase activity of Prx6 measured in the absence of the test substance, and when the lipase activity is lower than the control lipase activity, the test substance inhibits the invasiveness of cancer cells. It provides a method for searching for a substance that inhibits the invasiveness of cancer cells, comprising the step of determining the substance.

The present invention also comprises the steps of contacting a cell comprising a nucleic acid encoding Prx6 with a test substance, and measuring the lipase activity of Prx6; And comparing the measured lipase activity with the control lipase activity of Prx6 measured in the absence of the test substance, and when the lipase activity is lower than the control lipase activity, the test substance inhibits the invasiveness of cancer cells. It provides a method of searching for a substance that inhibits the infiltration of dark cells, comprising the step of determining the substance.

In the method for searching for a substance that inhibits the invasiveness of cancer cells of the present invention, the cell comprising the nucleic acid encoding the Prx6 may be a cell or cancer cell containing the nucleic acid intrinsically or by transduction.

In a method for screening a substance that inhibits the invasiveness of cancer cells of the present invention, the test substance is assumed to have a function as an inhibitor of cancer cell infiltration or metastasis or randomly selected individual nucleic acids, proteins, extracts, natural substances or synthetic substances Can be.

The present invention also provides a composition for searching for substances that inhibit invasiveness of cancer cells, including cells comprising a nucleic acid encoding Prx6.

The present invention also provides a kit for searching for a substance containing a nucleic acid encoding Prx6 and a substance for inhibiting invasiveness of cancer cells, including a reagent for measuring lipase activity of Prx6.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are intended to illustrate the invention and should be construed as limiting the invention.

Peroxyredoxin 6 of the present invention is a target of cancer invasion or metastasis, and the invasiveness of cancer cells increases according to its expression or activity level, so whether the cancer is infiltrated or metastasized using an antibody against peroxyredoxin 6 or its substrate. Can be diagnosed. Moreover, since cancer metastasis can be suppressed by inhibiting the expression or the activity of peroxy redoxin 6, the inhibitor of peroxy redoxin 6 can be developed and used as a cancer metastasis inhibitor.

Culture and transduction of the cells used in the examples of the present invention was performed as follows.

Cell culture and transduction transfection )

A549 lung cancer cells, H460 lung cancer cells, HeLa cervical cancer cells, and SNU-484 gastric cancer cells were used, of which A549, H460, HeLa cells were from the American Type Culture Collection (Rockville, MD, USA), and SNU-484 cells. Was purchased from Korea Cell Line Bank. The cells were all cultured and maintained in 37 ° C., 5% CO ₂ incubator using RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 0.1% gentamycin. The vectors pcDNA3, pCR3.1 and pCMV used for transduction were obtained from Invitrogen (Carlsbad, CA).

For transduction, cells were prepared at 2 × 10 ⁵ per 60 mm dish and incubated overnight. The next day, 2 μg each of the pcDNA3 vector cloned with the wild type gene or the mutant gene of Prx6 and the control pcDNA3 vector without any clones were taken and mixed in 0.15 ml of RPMI 1640 medium. Separately, 4 μl of Lipofectamine ^™ 2000 (Invitrogen; Carlsbad, Calif.) Was added to 0.15 ml of RPMI 1640 medium and mixed. Thereafter, the medium to which the vector was added and the medium to which Lipofectamine ^™ 2000 was added were mixed to make 0.3 ml of the mixed solution and left at room temperature for 30-40 minutes. 1.7 ml RPMI 1640 medium was further added to the mixed solution to prepare 2 ml of the mixed solution, and then, 2 ml of the mixed solution was evenly spread over the cells washed well with PBS, incubated for 5 hours in a 37 ° C. incubator, and induced transfection. It was. The mixture sprayed on the cells was removed after 5 hours of incubation, incubated for 2-3 days with 4 ml of RPMI 1640 medium containing 10% FBS, and then transduced by adding neomycin at a concentration of 1 mg / ml. Screened. Cells for overexpression of Prx-1 (pCR3.1), Prx-2 (pCR3.1), Prx-3 (pCMV), Prx-4 (pcDNA3), and Prx-5 (pCR3.1) It prepared by the method.

Example  One. Peroxyredoxin  Increased invasiveness of cancer cells by 6

(One) Prx  Increased invasiveness of cancer cells by 6

To investigate the effect of peroxyredoxin on cancer cell invasion, peroxyredoxin (Prx) 1 to Prx6 were introduced into the A549 lung cancer cell line (American Type Culture Collection; Rockville, MD) by transduction. The ability of the cells to pass through a carbonate-coated polycarbonate was compared using a transwell chamber (Corning; Acton, Mass.).

Cell invasion quantitative analysis was performed using a reconstituted basement membrance matrix, Matrigel (BD Biosciences, Bedford, Mass.), Derived from the EHS mouse tumor (Englebreth-Holm-Swarm mouse tumor) (Park MJ. Cancer Res . 62: 6318-6322, 2002).

First, methgel was dissolved at 1 mg / ml in RPMI 1640 medium, and then 25 μl of this solution was coated inside the filter of the insert well. The coated insert wells were inverted and dried at room temperature for 1 hour. The cells were suspended in RPMI 1640 containing 0.1% BSA and dispensed 5 × 10 ⁴ per insert well, and 1 ml of RPMI 1640 containing 10% FBS was added to the outer chamber and incubated for 16 hours. After the incubation, the cells that did not pass through the methagel was wiped off with a cotton swab, and the cells passed through were stained with Microscopy Hemacolor (Merck, Germany) and observed under a microscope and photographed with a digital camera to measure the number of cells. As a result, the invasiveness of A549 cells increased by about 4.5 to 5 times when Prx6 was overexpressed (FIG. 1). Thus, Prx6 was identified as a factor that promotes invasiveness of lung cancer cells, unlike Prx1-5.

(2) Prx6 Stimulation of cancer cells by lipase activity

All six types of peroxyredoxin (Prx) have a peroxidase activity that decomposes hydrogen peroxide, but Prx6 is known to have a lipase activity that degrades lipids in addition to the peroxidase activity (Manevich Y and Fisher). AB.Free Radic Biol Med 38: 1422-1432., 2005). Unlike Prx1-5 having only peroxidase activity, only Prx6 having lipase activity could promote cancer cell invasiveness. Therefore, it was assumed that the role of lipase activity in cancer cell invasion of Prx6 was important. To confirm this directly, serine 32, which is essential for lipase activity of Prx6, was substituted with alanine to prepare Prx6 mutant (Prx6-M) without lipase activity (Chen JW et al., J Biol Chem 275: 28421-28427). , 2000). As a result of comparing the invasion of A549 cells overexpressed with Prx6-M and A549 cells overexpressed with normal wild-type Prx6 in the same manner as in (1), unlike Prx6, cancer cells were overexpressed with Prx6-M. Did not increase the invasiveness (Fig. 2). Therefore, it was confirmed that Prx6 promotes cancer cell invasion through lipase activity.

(3) Prx6 Invasion of various cancer cells by

We investigated whether Prx6 increases the invasiveness of cancer cells in cancer cells other than A549 cells. To this end, vectors containing Prx6 were cultured and transduced with H460 lung cancer cells, HeLa cervical cancer cells, and SNU-484 gastric cancer cells as described above to overexpress Prx6. 6 shows the invasiveness of the transduced cells. As shown in FIG. 6, Prx6 was found to increase not only lung cancer but also invasiveness of other cancer cells.

Example  2. Peroxyredoxin  UPA by 6 ( Urokinase - type Plasminogen  Increased expression and activity of activators

(One) Prx6 On by uPA Expression and activity

To investigate the mechanism by which Prx6 increases the invasiveness of cancer cells, the expression and activity of extracellular hepatic degrading enzymes necessary for cancer cell invasion were investigated.

Because cells are surrounded by extracellular liver material, cancer cells must break down the extracellular liver material in order to move to other tissues or organs. To this end, cancer cells are known to express and secrete extracellular hepatic degrading enzymes such as matrix metalloproteinase (MMP) and urokinase-type plasminogen activator (uPA). Therefore, the expression and activity of MMP-2, MMP-9 and uPA according to the overexpression of Prx-6 were examined by Western blotting and zymography.

First, Western blotting was performed to measure the expression levels of extracellular hepatic degrading enzymes, MMP-2, MMP-9 and uPA secreted from A549 cells transduced with Prx-6. At this time, HT1080 (American Type Culture Collection), a highly heterologous human fibrosarcoma cell line that is well known to express all MMP-2, MMP-9 and uPA, was used as a positive control.

A549 cells were prepared at 3 × 10 ⁵ per 6-well plate and incubated overnight, followed by two washes with PBS followed by addition of 750 μL of RPMI 1640 medium without serum. After incubation overnight, the culture solution was recovered, and the supernatant obtained by centrifugation at 10,000 rpm for 5 minutes was used for analysis. The supernatant obtained as above was mixed with dye and boiled for 5 minutes, then developed on a 10% SDS-PAGE gel and blotted with nitrocellulose paper (Whatman, Germany). Stir for 1 hour at room temperature using 5% skim milk to prevent non-specific reaction of the antibody, then wash three times for 5 minutes with PBS / T (0.1% Tween-20), MMP-2, MMP-9, and Primary antibody to uPA (Calbiochem, Germany) was diluted 1: 1,000 and reacted at room temperature for 1 hour. After washing 3 times with PBS / T for 5 minutes, the secondary antibody, anti-mouse IgG-HRP (Pierce; Rockford, IL), was diluted 1: 10,000, treated at room temperature for 1 hour, and then again with PBS / T. Wash three times for 10 minutes. Thereafter, in the dark, the reaction was performed with an ECL (Enhanced Chemi Luminenscence) solution (Amersham, UK) and developed on an X-ray film (AGPA, Belgium) to identify the band.

Meanwhile, zymography was performed using the supernatant obtained above. Zymography is a method of measuring the enzymatic activity of gelatin, separating proteins on polyacrylamide gels containing gelatin, and then incubating the band containing gelatinase at 37 ° C. to coarse the degraded gelatin band. This is a method of identifying negative bands through coomassie blue staining.

A549 cells were prepared at 3 × 10 ⁵ cells per 6-well plate and incubated overnight. After washing twice with PBS, 750 μl of RPMI 1640 medium without serum was added. After incubation overnight, the culture solution was recovered, and the supernatant obtained by centrifugation at 10,000 rpm for 5 minutes was used for analysis. Supernatants were quantified and electrophoresed using 10% SDS-PAGE gel containing 0.1% gelatin of the same amount of protein. After electrophoresis, the gel was stirred in a 2.5% Triton X-100 solution for 1 hour to remove SDS, followed by a buffer solution for enzymatic reaction (50 mM Tris-HCl, 20 mM NaCl, 10 mM CaCl ₂ , 1 uM ZnCl ₂ , 0.1 % NaN ₃ , pH 7.5) was washed at room temperature for 30 minutes, and then replaced with the same solution. After the reaction, the gel was stained with 0.25% Coomassie-blue and decolorized (25% ethanol, 8% acetic acid) to confirm the degree of MMP-2 and MMP-9 activity.

The activity of uPA was determined by dyeing and decolorizing with 0.1 M glycine (pH 8.0) solution using 10% SDS-PAGE gel containing 2.5% casein and 10 μg / ml plasminogen to confirm uPA activity. It was.

As shown in Figure 3, Prx6 increased the expression and activity of uPA, but did not significantly increase the expression and activity of MMP-2 and MMP-9. In addition, according to Figure 4, Prx6-M did not increase the expression and activity of uPA, unlike Prx6.

(2) Prx6 For promoting invasiveness of cancer cells uPA siRNA Effect

In order to investigate whether uPA induced expression of Prx6 is essential for cancer cell invasiveness, uPA siRNA of 50 nM was treated in cancer cells into which the vector containing Prx6 was introduced to inhibit uPA expression.

First, transduction was performed using siRNA (Ambion; Austin, TX) to suppress uPA expression in A549 cells. A549 cells were prepared at 2 × 10 ⁵ per 60 mm dish and incubated overnight. The next day, 50 nM uPA siRNA was added to 0.15 ml of RPMI-1640 medium and mixed. Separately, 4 μl of Lipofectamine ^™ 2000 was added to 0.15 ml of RPMI-1640 medium and mixed. Thereafter, RPMI 1640 medium added with uPA siRNA (or control siRNA) and RPMI 1640 medium added with Lipofectamine ^™ 2000 were combined to make 0.3 ml of a mixed solution, which was allowed to stand at room temperature for 30-40 minutes. 1.7 ml RPMI 1640 culture solution was further added to the mixture to complete 2 ml of the mixed solution. Then, 2 ml of the mixed solution was evenly sprayed on A549 cells well washed with PBS, incubated at 37 ° C. for 5 hours, and induced transduction. After incubation for 5 hours, the culture solution was removed, washed once with PBS, and then fed with 4 ml of RPMI 1640 medium to which 10% FBS was added, followed by incubation.

When uPA expression was inhibited by RNA interference by uPA siRNA, no increased invasiveness by Prx6 was observed in Prx6 overexpressed cells (FIG. 5). This shows that uPA is required to increase the invasiveness of cancer cells by Prx6.

Through the above results, it was confirmed that Prx6 increases the expression and activity of uPA through lipase activity, thereby promoting invasiveness of cancer cells.

Example  3. Peroxyredoxin  Cancer Cell Metastasis Inhibitor Screening Using Substrate of 6

Inhibitors that can inhibit the expression of peroxyredoxin 6 and its activity can be screened through high throughput screening (HTS).

The efficacy of the inhibitor is assessed by measuring the activity of peroxyredoxin 6 using DDPC, the substrate of the lipase activity of peroxyredoxin 6.

Specifically, when reacting peroxyredoxin 6 with its substrate, an inhibitor candidate is added to analyze an inhibitory effect of peroxyredoxin 6, thereby inhibiting the expression or activity of peroxyredoxin 6. Screen it.

Substances found to have the activity of increasing the expression of the peroxyredoxin 6 gene or increasing the function of the peroxyredoxin 6 obtained through the screening method of the present invention may be inhibited by a cancer metastasis inhibitor. Substances that can be used as candidates, on the other hand, that have been identified as having activity to reduce the expression of peroxyredoxin 6 gene or inhibit the function of peroxyredoxin 6 are themselves cancer metastasis inhibitor candidates. It can be used as. Such a cancer metastasis inhibitor candidate acts as a leader in the development of cancer metastasis inhibitors in the future, and the structure modifies the leader to inhibit the expression of the peroxyredoxin 6 gene or to inhibit the function of the peroxyredoxin 6 gene. New cancer metastasis inhibitors can be developed.

1 is a transfection of a vector containing Prx (Peroxiredoxin) 1, Prx2, Prx3, Prx4, Prx5 and Prx6 into A459 lung cancer cell lines by transfection, and then the invasiveness of the cells is measured using a Matrigel filter. The result analyzed by the method used is shown.

Figure 2 is introduced into the A459 lung cancer cell line by transduction of a control vector, a vector containing Prx6, and a vector containing Prx6 (Prx6-M) from which the lipase function of Prx6 is removed, followed by invasion of the cell by metagel ( Matrigel) shows the analysis results by the method using a filter.

FIG. 3 shows Western blotting and zymography of expression and activity of uPA, MMP-2 and MMP-9 in A459 lung cancer cell lines transduced with a control vector and a vector containing Prx6, respectively. The result confirmed by) is shown.

Figure 4 shows the results confirmed by Western blotting and zymography, respectively, the expression and activity of uPA in the cells established in Figure 2.

5 shows the effect of siPA (50 mM) of uPA in A459 lung cancer cell lines into which a vector comprising a control vector and Prx6, respectively, was introduced by transduction.

Figure 6 shows the results of introducing the control vector and Prx6 to the lung cancer cell lines (A549, H460), cervical cancer cell line (HeLa) and gastric cancer cell line (SNU-484) and compare the invasiveness.

<110> Korea Institute of Radiological & Medical Sciences <120> Use of Peroxiredoxin 6 as target for cancer invasion or          metastasis <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 224 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE (222) (1) .. (224) <223> Peroxiredoxin 6 <400> 1 Met Pro Gly Gly Leu Leu Leu Gly Asp Val Ala Pro Asn Phe Glu Ala   1 5 10 15 Asn Thr Thr Val Gly Arg Ile Arg Phe His Asp Phe Leu Gly Asp Ser              20 25 30 Trp Gly Ile Leu Phe Ser His Pro Arg Asp Phe Thr Pro Val Cys Thr          35 40 45 Thr Glu Leu Gly Arg Ala Ala Lys Leu Ala Pro Glu Phe Ala Lys Arg      50 55 60 Asn Val Lys Leu Ile Ala Leu Ser Ile Asp Ser Val Glu Asp His Leu  65 70 75 80 Ala Trp Ser Lys Asp Ile Asn Ala Tyr Asn Cys Glu Glu Pro Thr Glu                  85 90 95 Lys Leu Pro Phe Pro Ile Ile Asp Asp Arg Asn Arg Glu Leu Ala Ile             100 105 110 Leu Leu Gly Met Leu Asp Pro Ala Glu Lys Asp Glu Lys Gly Met Pro         115 120 125 Val Thr Ala Arg Val Val Phe Val Phe Gly Pro Asp Lys Lys Leu Lys     130 135 140 Leu Ser Ile Leu Tyr Pro Ala Thr Thr Gly Arg Asn Phe Asp Glu Ile 145 150 155 160 Leu Arg Val Val Ile Ser Leu Gln Leu Thr Ala Glu Lys Arg Val Ala                 165 170 175 Thr Pro Val Asp Trp Lys Asp Gly Asp Ser Val Met Val Leu Pro Thr             180 185 190 Ile Pro Glu Glu Glu Ala Lys Lys Leu Phe Pro Lys Gly Val Phe Thr         195 200 205 Lys Glu Leu Pro Ser Gly Lys Lys Tyr Leu Arg Tyr Thr Pro Gln Pro     210 215 220 <210> 2 <211> 675 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (672) <400> 2 atg ccc gga ggt ctg ctt ctc ggg gac gtg gct ccc aac ttt gag gcc 48 Met Pro Gly Gly Leu Leu Leu Gly Asp Val Ala Pro Asn Phe Glu Ala   1 5 10 15 aat acc acc gtc ggc cgc atc cgt ttc cac gac ttt ctg gga gac tca 96 Asn Thr Thr Val Gly Arg Ile Arg Phe His Asp Phe Leu Gly Asp Ser              20 25 30 tgg ggc att ctc ttc tcc cac cct cgg gac ttt acc cca gtg tgc acc 144 Trp Gly Ile Leu Phe Ser His Pro Arg Asp Phe Thr Pro Val Cys Thr          35 40 45 aca gag ctt ggc aga gct gca aag ctg gca cca gaa ttt gcc aag agg 192 Thr Glu Leu Gly Arg Ala Ala Lys Leu Ala Pro Glu Phe Ala Lys Arg      50 55 60 aat gtt aag ttg att gcc ctt tca ata gac agt gtt gag gac cat ctt 240 Asn Val Lys Leu Ile Ala Leu Ser Ile Asp Ser Val Glu Asp His Leu  65 70 75 80 gcc tgg agc aag gat atc aat gct tac aat tgt gaa gag ccc aca gaa 288 Ala Trp Ser Lys Asp Ile Asn Ala Tyr Asn Cys Glu Glu Pro Thr Glu                  85 90 95 aag tta cct ttt ccc atc atc gat gat agg aat cgg gag ctt gcc atc 336 Lys Leu Pro Phe Pro Ile Ile Asp Asp Arg Asn Arg Glu Leu Ala Ile             100 105 110 ctg ttg ggc atg ctg gat cca gca gag aag gat gaa aag ggc atg cct 384 Leu Leu Gly Met Leu Asp Pro Ala Glu Lys Asp Glu Lys Gly Met Pro         115 120 125 gtg aca gct cgt gtg gtg ttt gtt ttt ggt cct gat aag aag ctg aag 432 Val Thr Ala Arg Val Val Phe Val Phe Gly Pro Asp Lys Lys Leu Lys     130 135 140 ctg tct atc ctc tac cca gct acc act ggc agg aac ttt gat gag att 480 Leu Ser Ile Leu Tyr Pro Ala Thr Thr Gly Arg Asn Phe Asp Glu Ile 145 150 155 160 ctc agg gta gtc atc tct ctc cag ctg aca gca gaa aaa agg gtt gcc 528 Leu Arg Val Val Ile Ser Leu Gln Leu Thr Ala Glu Lys Arg Val Ala                 165 170 175 acc cca gtt gat tgg aag gat ggg gat agt gtg atg gtc ctt cca acc 576 Thr Pro Val Asp Trp Lys Asp Gly Asp Ser Val Met Val Leu Pro Thr             180 185 190 atc cct gaa gaa gaa gcc aaa aaa ctt ttc ccg aaa gga gtc ttc acc 624 Ile Pro Glu Glu Glu Ala Lys Lys Leu Phe Pro Lys Gly Val Phe Thr         195 200 205 aaa gag ctc cca tct ggc aag aaa tac ctc cgc tac aca ccc cag cct 672 Lys Glu Leu Pro Ser Gly Lys Lys Tyr Leu Arg Tyr Thr Pro Gln Pro     210 215 220   taa 675 <210> 3 <211> 224 <212> PRT <213> Homo sapiens <400> 3 Met Pro Gly Gly Leu Leu Leu Gly Asp Val Ala Pro Asn Phe Glu Ala   1 5 10 15 Asn Thr Thr Val Gly Arg Ile Arg Phe His Asp Phe Leu Gly Asp Ser              20 25 30 Trp Gly Ile Leu Phe Ser His Pro Arg Asp Phe Thr Pro Val Cys Thr          35 40 45 Thr Glu Leu Gly Arg Ala Ala Lys Leu Ala Pro Glu Phe Ala Lys Arg      50 55 60 Asn Val Lys Leu Ile Ala Leu Ser Ile Asp Ser Val Glu Asp His Leu  65 70 75 80 Ala Trp Ser Lys Asp Ile Asn Ala Tyr Asn Cys Glu Glu Pro Thr Glu                  85 90 95 Lys Leu Pro Phe Pro Ile Ile Asp Asp Arg Asn Arg Glu Leu Ala Ile             100 105 110 Leu Leu Gly Met Leu Asp Pro Ala Glu Lys Asp Glu Lys Gly Met Pro         115 120 125 Val Thr Ala Arg Val Val Phe Val Phe Gly Pro Asp Lys Lys Leu Lys     130 135 140 Leu Ser Ile Leu Tyr Pro Ala Thr Thr Gly Arg Asn Phe Asp Glu Ile 145 150 155 160 Leu Arg Val Val Ile Ser Leu Gln Leu Thr Ala Glu Lys Arg Val Ala                 165 170 175 Thr Pro Val Asp Trp Lys Asp Gly Asp Ser Val Met Val Leu Pro Thr             180 185 190 Ile Pro Glu Glu Glu Ala Lys Lys Leu Phe Pro Lys Gly Val Phe Thr         195 200 205 Lys Glu Leu Pro Ser Gly Lys Lys Tyr Leu Arg Tyr Thr Pro Gln Pro     210 215 220  

Claims (17)

Cancer invasive marker with the activity of peroxyredoxin 6 (Prx6). Measuring the expression level of nucleic acid encoding Prx6 in a cancer cell sample isolated from the patient; And comparing the expression level with the expression level of the Prx6-encoding nucleic acid in a control sample consisting of normal cells or cells identified as not invasive, wherein if the expression level is higher than the expression level in the control sample, the cancer cells are referred to as invasive cancer cells. And determining the invasive cancer cells. The method of claim 2, wherein the expression level of the nucleic acid encoding Prx6 is measured at the nucleic acid or protein level. The method of claim 3, wherein the expression level of the nucleic acid encoding Prx6 is measured at the mRNA level. The method of claim 2, wherein the cancer cell sample is selected from the group consisting of lung cancer, cervical cancer and gastric cancer. A kit for determining invasive cancer cells comprising a substance for measuring the expression level of a nucleic acid encoding Prx6 in a cancer cell sample isolated from a patient. The kit of claim 6, wherein the agent is selected from a primer set specific for a nucleic acid encoding Prx6, a probe, an antibody specific for Prx6, and a substrate for lipase activity of Prx6. A composition for inhibiting invasiveness of cancer cells, comprising a substance that inhibits the expression of Prx6 encoding a nucleic acid in a cell or inhibits the activity of Prx6. The composition of claim 8, wherein the substance that inhibits the expression of the nucleic acid encoding Prx6 is siRNA for Prx6. The composition of claim 8, wherein the substance which inhibits the activity of Prx6 is a decoy molecule for the substrate binding site of the antibody or active fragment thereof or lipase activity of Prx6. Contacting the test substance with Prx6 to measure lipase activity of Prx6; And comparing the measured lipase activity with the control lipase activity of Prx6 measured in the absence of the test substance, and when the lipase activity is lower than the control lipase activity, the test substance inhibits the invasiveness of cancer cells. A method for searching for a substance that inhibits invasiveness of cancer cells, comprising the step of determining the substance. A composition for searching for a substance that inhibits invasiveness of cancer cells, including Prx6. A kit for screening a substance that inhibits invasiveness of cancer cells, comprising reagents for measuring lipase activity of Prx6 and Prx6. Contacting a cell comprising a nucleic acid encoding Prx6 with a test substance and measuring the lipase activity of Prx6; And comparing the measured lipase activity with the control lipase activity of Prx6 measured in the absence of the test substance, and when the lipase activity is lower than the control lipase activity, the test substance inhibits the invasiveness of cancer cells. A method for searching for a substance for inhibiting invasiveness of cancer cells, comprising the step of determining the substance. The method of claim 14, wherein the cell comprising the nucleic acid encoding Prx6 is a cell or cancer cell comprising the nucleic acid intrinsically or by transduction. A composition for screening a substance that inhibits invasiveness of cancer cells, including a cell comprising a nucleic acid encoding Prx6. A kit for searching for a substance that inhibits invasiveness of cancer cells, comprising a cell comprising a nucleic acid encoding Prx6 and a reagent for measuring lipase activity of Prx6.

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