KR20060027135A - A method for the diagnosis of cancers by measuring the changes of glycosylation of transferrin and the diagnosis kit using the same - Google Patents

Abstract

The present invention relates to a method for diagnosing cancer using transferrin, a protein that expresses a sugar chain change during cancer development and metastasis, and a diagnostic kit using the same. Specifically, β1,6-N-, an N-linked sugar chain of transferrin, is used. The present invention relates to a method for diagnosing cancer by measuring the presence or absence of acetylglucosamine and a diagnostic kit using the same. Since the diagnostic method and the diagnostic kit of the present invention are accurate and simple compared to the conventional cancer diagnosis method, it is useful for diagnosing various cancers such as colon cancer, stomach cancer, lung cancer, liver cancer, uterine cancer, breast cancer and pancreatic cancer in Koreans. Can be used.

Description

A method for the diagnosis of cancers by measuring the changes of glycosylation of transferrin and the diagnosis kit using the same}             

1 is a schematic diagram showing six sugar transfer enzymes and N-linked sugar chains obtained by catalyzing them,

2 shows a reaction in which β 1,6 N-acetylglucosamine (GlcNAcβ 1) is added by the sugar transfer enzyme GnT-V.

FIG. 3 is a two-dimensional electrophoresis of only glycoproteins from normal serum (N, normal) and gastric cancer serum (T, tumor) and stained with Coomassie brilliant blue (①, ② The patient from which each serum sample was derived),

Figure 4 is a lectin blotting after the two-dimensional electrophoresis of the glycoproteins collected from normal serum and gastric cancer patient serum,

5 is a comparison of Coomassie Brilliant Blue staining and lectin blotting of each sample for transferrin (1, 2 are patients from which serum samples are derived),

6 is a comparison of Coomassie Brilliant Blue staining and lectin blotting of each sample for haptoglobin (①, ②, ③ are patients from which serum samples are derived),

7 is an analysis of the transferrin amino acid sequence using Electrospray Ionization (ESI) / Quadrupole Time of Flight (Q-TOF),

8 is a schematic diagram showing the principle of a diagnostic kit using a transferrin antibody,

Figure 9 is a test for the detection of cancer in the serum of human or mouse using a diagnostic kit using a transferrin antibody.

1, 2: normal serum

3, 4, 5, 6: cancer patient serum

The present invention relates to a method for diagnosing cancer by using transferrin, a protein that expresses a sugar chain change during cancer development and metastasis, and a diagnostic kit using the same. More specifically, β1,6- which is an N-linked sugar chain of transferrin The present invention relates to a method for diagnosing cancer by measuring the presence of N-acetylglucosamine and a diagnostic kit using the same.

In the past, two-dimensional electrophoresis methods have been used to analyze protein functions, but recently mass spectrometers such as matrix-assisted laser desorption ionization- time of flight (MALDI-TOF) have been developed and N-terminal amino acid sequences have been determined. Easier to do, proteomics, defined as 'proteinology', has emerged as a method of analyzing protein function in the post-genome era. However, since proteomics selects and analyzes only one stationary time point in a dynamically moving living body, there is a limit in studying cancers resulting from complex signal transmission.

Cancer development and metastasis are generally seen as a result of increased signal expression or post-translational modification as a result of signaling rather than the production of new proteins. In particular, the protein appears during the two-dimensional electrophoresis is very small, it is difficult to upgrade the analysis by simple staining alone.

One solution to this problem is the analysis of glycosylation of proteins using lectins. Even when electrophoresis does not reveal a difference in spots compared to the control group, it is significantly different between the two groups. Appears. Analyzing the change in sugar chains in response to the proteomics is called glycomixes, which is considered to be a step-up method that overcomes the difficulties of the ongoing proteomics analysis.

Looking at the cellular biological changes in the development and metastasis of cancer, many kinds of glycoproteins or glycolipids on the surface of the cell membrane are signaled by the oncogenes of ras and raf. "Aberrant glycosylation" and the resulting sugar chain changes alter the adhesion and recognition between cells, leading to cancer development and metastasis (Hakomori and Kannagi, J. Natl. Cancer Inst . 71: 231-251, 1983; Feizi , Nature, 314: 53-57, 1985). Once in the endoplasmic reticulum (ER), the core sugar chain of glycoprotein is made and then moved to Golgi apparatus, an intracellular glycosylation plant, and various sugar chains are added according to various stimuli and signals inside and outside cells. The catalytic sugar transfer enzyme is shown in FIG. 1 . By external stimulation, various sugar transfer enzymes of FIG. 1 are activated through signal transduction of specific oncogenes such as ras and raf and transcription factor etes.

The sugar transfer enzyme GnT-V (N-acetylglucosaminyltransferase) is an enzyme that catalyzes the reaction of adding N-acetylglucosamine to the β1,6 position in the glycoprotein's basic sugar chain, which is directly involved in cancer attack and metastasis. It is known to be relevant (Dennis et al., Science , 236: 582-585, 1987). In general, glycoproteins are synthesized after protein synthesis, basic sugar chains are made in ER (endoplasmic reticulum), and then go to Golgi body to add sugar by several sugar transfer enzymes. Six types of N-acetylglucosamine are produced primarily (I It is known that GnT-V, which is made by catalyzing the sugar transfer enzyme of -VI) and which makes β1,6-N-acetylglucosamine, is most involved in the development and metastasis of cancer.

A cell line of an embryonic stem cell (ES cell) of a mouse knocked out of the GnT-V gene was made, and the cell line contained a middle T antigen (hereinafter referred to as “PyMT) of polyomavirus. Viral oncogenes were introduced to cause cancer. As a result, it was confirmed that overexpression of PyMT in mice lacking the GnT-V gene significantly inhibited cancer development and metastasis compared to overexpression of PyMT in normal mice (Granovsky et al. Nature Med. , 6: 306-312, 2000).

In a recent study, GnT-V activity was analyzed in 33 types of hepatocellular carcinoma (HCC) tissues. As a result, enzyme activity was 50 times higher than normal tissue and 4 times higher than surrounding tissue. (Yao et al ., J Cancer Res. Clin. Oncol., 124: 27-30, 1998) and it has been reported that branching of β1,6 in animals activates metastasis of rat breast cancer.

As GnT-V is known to be closely related to cancer development and metastasis, studies on GnT-V target proteins, ie, proteins to which β1,6-N-acetylglucosamine sugar chains are added, are also being actively conducted. The cell surface protein, gp130 (glycoprotein 130), is also one of the target proteins of GnT-V. It has already been shown that the addition of β1,6 N-acetylglucosamine sugar chains to the protein activates cancer metastasis. In addition, GnT-V It is believed that a large number of target proteins exist.

Accordingly, the present inventors have found a protein that shows a sugar chain change during cancer development and metastasis by using proteomics technique, confirming that the protein is transferrin, and measuring the sugar chain change of the protein to diagnose cancer occurrence and metastasis. The present invention was completed by confirming.

Disclosure of Invention An object of the present invention is to provide a method for diagnosing cancer using transferrin, a protein that expresses a sugar chain change during cancer development and metastasis, and a diagnostic kit using the same.

In order to achieve the above object, the present invention provides a method for diagnosing cancer by measuring the sugar chain change of transferrin.

The present invention also provides a diagnostic kit for diagnosing cancer by measuring the sugar chain change of transferrin.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for diagnosing cancer by measuring the sugar chain change of transferrin.

The cancer may be selected from the group consisting of colon cancer, stomach cancer, lung cancer, liver cancer, uterine cancer, breast cancer and pancreatic cancer, and may include all kinds of cancers.

Glycoproteins or glycolipids present on the surface of the cell membrane receive signals from cancer genes, resulting in “aberrant glycosylation”, and the resulting sugar chain changes in cell adhesion and recognition. Cancer development and metastasis are indicated. Glycoprotein is the basic sugar chain in ER (endoplasmic reticulum) and then moves to Golgi body to add sugar. N-acetylglucosamine, which is made primarily, is made by six kinds of sugar transfer enzymes (I-VI). Β1,6-N-acetylglucosamine, produced by GnT-V, is known to be most involved in the development and metastasis of cancer.

The present inventors have found a glycoprotein that shows a sugar chain change during cancer development and metastasis. As a result, the protein binds to two molecules of trivalent iron ions (Fe ³⁺ ) absorbed into the serum, thereby receiving iron necessary for cell proliferation or hemoglobin production. It was confirmed that it was transferrin having a molecular weight of about 75,000 supplied intracellularly under each medium.

Specifically, the present inventors obtained serum from the blood of a large number of gastric cancer patients in Korea, removed a large amount of albumin, gamma-globulin and eluted with LCA ( Lens Culinaris Agglutinin) -agarose (agarose) column (SIGMA) Two-dimensional electrophoresis was performed using only glycoproteins. The gel was subjected to Coomassie Brilliant Blue staining (see FIG. 3 ) and lectin blotting (see FIG. 4 ) to identify proteins exhibiting sugar chain changes (see FIGS . 5 and 6 ).

Specifically, the two-dimensional electrophoresis of the metastasized cancer cell culture was attempted, and after obtaining two gels, one was stained with Coomaji Brilliant Blue and the other was subjected to lectin blotting to normal cells. In comparison, proteins causing sugar chain changes in cancer cells and metastasized cells were selected (see FIGS . 5 and 6 ). In the case of lectin blot that recognizes β 1,6 N-acetylglucosamine branch, dark spots appeared in several places in cancer cells compared to the control group, and the protein corresponding to the spots was involved in cancer development and metastasis and thus the sugar chain changes. Is a protein. Corresponding spots were cut on the gel, the protein was digested, and the resulting peptide was confirmed by amino acid sequence using an Electrolysis Ionization (ESI) / Quadrupole Time of Flight (Q-TOF) mass spectrometer (Micromass) ( FIG. 7 ). Comparing the identified amino acid sequence with the existing protein database, it was confirmed that the protein causing the sugar chain change is transferrin.

In the case of cancer patients, there was no change in the expression level of transferrin, whereas in the lectin blot, the pH was shifted toward the basic and the intensity was strong due to the addition of the sugar chain branched β 1,6-N-acetylglucosamine. It was.

Therefore, cancer can be diagnosed by measuring and comparing the sugar chain addition of the protein using transferrin, which shows a difference in sugar chain change between cancer cells and normal cells.

 The method for measuring the sugar chain change of transferrin is characterized in that the immunoassay such as ELISA.

In other words, when the specimen sample is reacted with the transferrin antibody, the biotin-labeled lectin recognizes and binds the sugar chain branch of transferrin included in the sample sample and transfers the transferrin-sugar chain branch by adding a specific enzyme or fluorescent substance and color substrate to the biotin. Lectin-biotin-chromatase or fluorophore will be bound. They interact, resulting in absorbance.

Specific diagnostic method using this principle,

i) adding a sample to the transferrin antibody and adsorbing the sample;

Ii) adding the labeled lectin to step i) to bind it;

Iii) adding chromophores or fluorescent materials to step ii);

Iii) adding a color substrate to color development reaction in step iii); And

V) measuring and comparing the absorbance of the reaction solution of step iii).

The labeled lectin of step ii) is preferably lectin labeled with biotin and more preferably leukoagglutinating phytohemaggl utinin (L ₄ -PHA) labeled with biotin.

Lectin belongs to a group of plant proteins and is combined with glycoproteins or glycolipids of glycolipids to exert effects such as cell aggregation, division, and functional activation. Types of lectins include phytohemagglutinin (PHA), pokeweed mitogen (PWM), and concanavalin A (Con A), depending on the extraction site and the plant from which the lectin is extracted. L ₄ -PHA (leukoagglutinating phytohemagglutinin) is a type of phytohemagglutinin in which four lectins are combined.

Meanwhile, the chromophore of step iii) is selected from the group consisting of peroxidase, alkaline phosphatase, and the fluorescent material is from the group consisting of FITC (fluorescein isothiocyanate) and TRITC (tetramethylrhodamine isothiocyanate). Can be selected.

The present invention also provides a diagnostic kit for diagnosing cancer by measuring the sugar chain change of the transferrin.

Diagnostic kits of the invention include transferrin antibodies, substrates to which the transferrin antibodies are adhered, labeled lectins and chromosomes or fluorescents.

The principle of lectin-detecting sugar chain changes in transferrin in the diagnostic kit is as follows. When a specimen sample is added to a transferrin antibody adhered to a substrate of a diagnostic kit, transferrin binds to the antibody and lectin binds to transferrin having a sugar chain change to which β 1,6-N-acetylglucosamine sugar chain branch is added. The chromophore or fluorescent substance and the chromophore substrate are combined in this order, and the result is shown as absorbance (see FIG. 8 ).

As a substrate of the diagnostic kit of the present invention, a nitrocellulose membrane, a 96 well plate synthesized with a polyvinyl resin, a 96 well plate synthesized with a polystyrene resin, a slide glass made of glass, etc. may be used. In order to adhere the antibody to the substrate, the surface of the substrate must be treated with L-lysine, poly-arylamine or aldehyde. The diagnostic kit of the present invention comprises a labeled lectin, wherein the lectin is a biotin-labeled-lectin labeled by biotin and the lectin is characterized in that L ₄ -PHA (leukoagglutinating phytohemagglutinin).

The chromophore included in the diagnostic kit is selected from the group consisting of peroxidase, alkaline phosphatase, and the fluorescent material is selected from the group consisting of fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). It is characterized by.

As a result of confirming the amount of transfer chain-transferrin in normal serum of humans and mice and serum of cancer patients using the diagnostic kit of the present invention, there was a clear difference between normal and cancer patients. Confirmed (see Fig. 9 ).

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

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Experimental Example 1 Glycoprotein Purification and Two-Dimensional Electrophoresis

The present inventors attempted proteomics on the blood of normal patients and the blood of gastric cancer patients to find cancer markers using glycoics directly from the serum of patients. A lectin LCA ( Lens Culinaris Agglutinin) that recognizes the core of sugar chains in N-linked sugar chains is first diluted with normal serum, diluted in 20 mM Tris-HCl (pH7.5) buffer for about 3 ml of serum. The LCA-agarose column (SIGMA Co., Ltd.) was loaded. Glycoprotein was eluted by washing with buffer solution and adding 0.4 M methyl mannoside when the absorbance was less than 0.03 at 280 nm (no more protein flowed). The solution was dialyzed overnight to remove methyl mannoside, and then acetone containing trichloroacetic acid (TCA) was added to a final 10% to precipitate only protein. After washing three times with acetone, residual TCA was removed and dried and dissolved by adding gel loading buffer (8 M Urea, 2% Triton X-100, 20 mM DTT, 0.5% carrier ampholyte, Bromophenol Blue dye) Electrophoresis was performed using a electrophoresis apparatus (Multiphor-II, Pharmarcia) (13 cm drystrip pH 3-10). The developed one-dimensional electrophoretic gel was equilibrated with an equilibration buffer containing SDS and 2-mercaptoethanol and then 12% using a two-dimensional electrophoresis apparatus (Protean II, Bio-Rad). Two-dimensional electrophoresis was performed on polyacrylamide gels. After securing two gels with electrophoresis, one was stained with Coomassie brilliant blue (Biosafe, Bio-Rad) staining solution ( FIG. 3 ). For direct comparison with normal serum, serum of gastric cancer patients was also subjected to one-dimensional (isoelectric point separation) electrophoresis under the same conditions and stained with Coomassie Brilliant Blue ( FIG. 3 ). As a result of three or more two-dimensional electrophoresis attempts and analysis of protein changes using software (PDQUEST, Bio-Rad), no protein showed a significant difference (more than two times) in the expression pattern of the protein. .

Experimental Example 2 Lectin Blotting for Glycoprotein Derived from Serum

Since the staining results of the two-dimensional electrophoresis showed no difference in the expression level of glycoprotein, lectin blotting was attempted to confirm the glycoprotein change of the glycoprotein. Another gel developed under the same conditions was transferred to a polyvinylidene difluoride (PVDF) membrane using a semi-dry transfer (Semi-Dry transfer, Bio-Rad). Biotin labeled L ₄ -PHA (leukoagglutinating phytohemagglutinin) lectin, which recognizes a β 1,6 N-acetylglucosamine sugar chain branch, is added to the glycoprotein having the sugar chain branch and then HRP-labeled. Streptoavidin was attached and photosensitive to the film using ECL fluorescence ( FIG. 4 ). Spot 1 in FIG. 5 was darker and more basic towards the L ₄ -PHA blot for gastric cancer patient serum. This may be due to the formation of poly-lactosamine by the sugar transfer enzyme GnT-V, resulting in a decrease in the total content of sialic acid present at the terminal (Handerson T. and Pawelek JM, cancer research). 63: 5363-5369, 2003). On the other hand the control of Figure 6 Haptoglobin showed no significant difference between cancer patients and normal subjects.

Experimental Example 3 Protein Sequence Analysis Using an ESI / Q-TOF Mass Spectrometer

In order to identify the protein of the spot selected in < Experimental Example 2> , the present inventors stained the PVDF membrane subjected to the lectin blot in <Experimental Example 2> again with Coumaris Brilliant Blue. It was attached to the photosensitive film of <Experimental Example 2> to confirm the exact position of the spot and then cut the corresponding spot on the gel stained with Kumarage brilliant blue from the beginning. After destaining with 30% methanol and 100% acetonitrile, 10 U trypsin (Promega) was added and cleaved overnight at 37 ° C. The cleaved peptide was extracted with acetonitrile and dried with a centrifugal freeze dryer, and the amino acid sequence was determined by ESI / Q-TOF (Electrospray Ionization / Quadrupole Time of Flight) mass spectrometer (Micromass) ( FIG. 7 ).

As a result, the peptide sequence represented by SEQ ID NO: 1 was determined by ESI / Q-TOF, and it was confirmed that the sequence was transferrin compared with a database of proteins.

<Example 1> Cancer diagnosis using the diagnostic kit and diagnostic method of the present invention

First, a commercial transferrin antibody (Santacruz) was purchased, and then a carbonate buffer solution consisting of 15 mM Na ₂ CO ₃ , 35 mM NaHCO ₃ and 0.2 g / L NaN ₃ (pH9.6) was prepared in an ELISA plate. Attach overnight at 4 ° C, block with bovine serum albumin to a final concentration of 10 mg / ml, and dilute (1/100, 1/400, 100 μl) serum of normal or patient to transferrin antibody at 37 ° C. Attached. Then washed three more times with 10 mM Tris-HCl, pH7.4 (hereinafter TBS-T) containing 0.15 M NaCl and 0.05% Tween-20, and reacted by adding biotin-labeled L ₄ -PHA. . After washing with TBS-T and adding avidin (avidine) is connected to the peroxidase (peroxidase) was added to the substrate OPD (o-phenylenediamine) and hydrogen peroxide. The color reaction was terminated by addition of sulfuric acid solution and the absorbance was measured at 492 nm ( FIG. 8 ). As a result, as in all people, mice appears normal serum and the cancer is the absorbance difference in serum significantly formed (see Fig. 9) confirmed that it is possible to effectively diagnose cancer diagnostics using the kit of the present invention.

As described above, a method for diagnosing cancer by measuring a sugar chain change of transferrin, a protein involved in cancer development and metastasis of the present invention, and a diagnostic kit using the same are more accurate and simple than conventional cancer diagnosis methods. It can be useful for diagnosing various cancers including stomach cancer, colon cancer, stomach cancer, lung cancer, liver cancer, uterine cancer, breast cancer and pancreatic cancer.

Claims (13)

How to diagnose cancer by measuring sugar chain changes in transferrin. The method of claim 1, wherein the sugar chain change of the transferrin is a sugar chain change, characterized in that the transfer of the N-linked β1,6 N-acetylglucosamine (acetylglucosamine) of transferrin. The method of claim 1, wherein the measuring sugar chain change is immunoassay. The method of claim 1, i) adding a sample sample to the transferrin antibody and adsorbing the sample; Ii) adding the labeled lectin to step i) to bind it; Iii) adding chromophores or fluorescent materials to step ii); Iii) adding a color substrate to step iv) to perform color reaction; And Iii) measuring the absorbance of the reaction solution of step iii) and comparing the cancer. 5. The method according to claim 4, wherein the labeled lectin of step ii) is a lectin labeled with biotin. The method of claim 4, wherein the labeled lectin of step ii) is leukoagglutinating phytohemagglutinin (L ₄ -PHA). The method of claim 4, wherein the chromophore of step iii) is selected from the group consisting of peroxidase, alkaline phosphatase, and the fluorescent material is fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC). Diagnostic method, characterized in that selected from the group consisting of. Diagnostic kit for diagnosing cancer by measuring sugar chain changes in transferrin. The diagnostic kit of claim 8 includes an antibody to transferrin, a substrate to which the antibody adheres, a labeled lectin, a chromophore or a fluorescent substance, and a chromophoric substrate. 10. The slide of claim 9, wherein the substrate of the diagnostic kit is a nitrocellulose membrane, a 96 well plate made of polyvinyl resin, a 96 well plate made of polystyrene resin, and a glass slide. Diagnostic kit, characterized in that selected from the group consisting of glass. 10. The diagnostic kit according to claim 9, wherein the labeled lectin is a biotin-labeled-lectin labeled by biotin. 10. The diagnostic kit according to claim 9, wherein the labeled lectin is leukoagglutinating phytohemagglu tinin (L ₄ -PHA). The method of claim 9, wherein the chromophore is selected from the group consisting of peroxidase, alkaline phosphatase, and the fluorescent material is selected from the group consisting of fluorescein isothiocyanate (FITC) and tetramethylrhodamine (TRITC). Diagnostic kit, characterized in that.

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