JP7044320B2 - Utilization of the sugar chain structure of disease-specific tissue factor pathway inhibitor 2 - Google Patents

Description

The present invention relates to the utilization of the sugar chain structure of a disease-specific tissue factor pathway inhibitor 2, and more specifically, the tissue factor pathway inhibitor 2 having a clear cell cancer-specific sugar chain structure (Tissue Factor Pathway Inhibitor 2, TFPI2). ) As a diagnostic marker and drug discovery target.

There are various histological types of ovarian cancer, and among them, clear cell cancer has a poor prognosis because anticancer drugs are less effective than other histological types. The incidence of clear cell carcinoma in Japan is higher than in Europe and the United States (Europe and the United States: 5%, Japan: 25%), and in Japan, improvements in early diagnosis and treatment of clear cell carcinoma are required.

The current ovarian cancer marker CA125 is excellent for detecting ovarian cancer in general, but (1) cannot distinguish histological type, (2) has a low positive rate for clear cell cancer, and (3) has a high value even for benign endometriosis. There is a problem such as. In particular, since clear cell carcinoma often develops from endometriosis, it is clinically important to distinguish between these two diseases. However, there is no marker that can distinguish these, and delays in response due to misdiagnosis and burden on patients due to false positive judgment have become problems in clinical practice.

The present inventors have previously found that the placenta-specific serine protease inhibitor TFPI2 is upregulated in a clear cell cancer-specific manner, and evaluated its clinical usefulness as a serum diagnostic marker. I have filed an application (Patent Documents 1 and 2).

TFPI2 has two N-type sugar chain binding sites, but there is no report on the structure of the bound sugar chain, and whether the sugar chain structure of placenta-derived TFPI2 and clear cell cancer-derived TFPI2 is the same. Was of course unknown.

Patent No. 5224309 JP 2013-061321

Since TFPI2, also known as placental protein 5, is also increased in pregnant women's blood, there is a problem that the TFPI2 measurement system cannot be used to differentiate the histological type of ovarian cancer in pregnant women. Therefore, an object of the present invention is to provide a method capable of detecting clear cell cancer type TFPI2 in distinction from placental type TFPI2.

The present inventors purified TFPI2 derived from placental villous trophoblast cells (placental type TFPI2) and TFPI2 derived from ovarian clear cell adenocarcinoma cell line (clear cell cancer type TFPI2) from each cell culture supernatant by immunoprecipitation. After digestion with trypsin, the cells were subjected to a mass analyzer. The sugar chain structure was analyzed from the precise mass and mass spacing of glycopeptide ions corresponding to the two N-type sugar chain binding sites (Asn116 and Asn170) of TFPI2, and the product ion spectrum. As a result, the placenta-type TFPI2 has a bi-antenna-type or tri-antenna-type complex sugar chain having LacNAc (galactose-N-acetylglucosamine, Galβ1-4GlcNAc) as a skeleton, whereas the clear cell cancer-type TFPI2 has LacdiNAc. It was found that the bi-antenna type sugar chain having a structure (N-acetylgalactosamine-N-acetylglucosamine, GalNAcβ1-4GlcNAc) is the main bound sugar chain, and that the placenta type and the clear cell cancer type have different sugar chains. ..

The present invention has been completed based on these findings.

The gist of the present invention is as follows.
(1) A method for measuring the expression of tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc (N-acetylgalactosamine-N-acetylglucosamine) in a biological sample for evaluating and / or differentiating clear cell cancer.
(2) The method according to (1), wherein the biological sample is a cell, tissue or body fluid obtained from a subject.
(3) The method according to (2), wherein the body fluid is blood, ascitic fluid or ascitic fluid.
(4) The method according to (3), wherein the blood is whole blood, serum, plasma or plasmapheresis external fluid.
(5) When the presence of tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc is confirmed in a biological sample, the patient is suffering from clear cell carcinoma, or the histological type of the cancer is clear cell carcinoma. The method according to any one of (1) to (4).
(6) The method according to any one of (1) to (5), wherein the presence of tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc is confirmed by mass spectrometry.
(7) In addition to 366.14 Da, signals of 407.17 Da, 553.22 Da and / or 698.27 Da are included in the glycopeptide ions observed when MS / MS measurement of tissue factor pathway inhibitor 2 or its peptide fragment is performed. If so, the method according to (6), wherein the patient is suffering from clear cell cancer, or the histological type of the cancer is evaluated as clear cell cancer.
(8) The expression of tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc (N-acetylgalactosamine-N-acetylglucosamine) in a biological sample is measured for the therapeutic effect or recurrence determination test of clear cell cancer. Method.
(9) A method for identifying a substance effective in treating and / or preventing clear cell carcinoma, wherein the following steps:
(a) Step of contacting the test substance with clear cell cancer cells,
(b) The step of culturing the clear cell cancer cells contacted with the test substance in step (a) for a predetermined time,
(c) A step of measuring the expression of tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc in v cells cultured in step (b), and
(d) Tissue having a sugar chain structure having LacdiNAc in clear cell cancer cells measured in step (c) Tissue having a sugar chain structure having LacdiNAc in control cells in which the expression of tissue factor pathway inhibitor 2 having a sugar chain structure was not contacted with the test substance. The method comprising the step of evaluating the effect of the test substance on the expression of tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc in clear cell cancer cells by comparing with the expression of factor pathway inhibitor 2.
(10) The method according to (9), further comprising a step of examining the effect of the test substance on the proliferation of clear cell cancer cells.
(11) A method for identifying a substance that reduces the resistance of a clear cell cancer to an anticancer drug, and the following steps:
(a) Step of contacting the test substance with clear cell cancer cells,
(b) The step of culturing the clear cell cancer cells contacted with the test substance in step (a) for a predetermined time,
(c) A step of measuring the expression of tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc in the clear cell cancer cells cultured in step (b), and
(d) Tissue having a sugar chain structure having LacdiNAc in clear cell cancer cells measured in step (c) Tissue having a sugar chain structure having LacdiNAc in control cells in which the expression of tissue factor pathway inhibitor 2 having a sugar chain structure was not contacted with the test substance. The method comprising the step of evaluating the effect of the test substance on the expression of tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc in clear cell cancer cells by comparing with the expression of factor pathway inhibitor 2.
(12) The method according to (11), further comprising a step of examining the effect of the test substance on the anticancer drug resistance of clear cell cancer cells.

By examining the sugar chain structure of TFPI2, placental type TFPI2 and clear cell cancer type TFPI2 can be distinguished, and by measuring the clear cell cancer type LacdiNAc-TFPI2, it is possible to diagnose clear cell cancer in pregnant women. Become.

Sugar chain structure of placental type TFPI2 and clear cell cancer type TFPI2. TFPI2 sugar chain modifier detected in each cell. (A) TFPI2 released by heparin treatment was detected by Western blotting after immunoprecipitation. (B) Difference in sugar chain modification state of each band. Each band was gel-extracted, and after trypsin digestion, the presence or absence of sugar chain modification revealed by a mass spectrometer was indicated by a diamond-shaped symbol. (C) Detection results of unmodified peptides and glycopeptides in various bands. Typical product ion spectrum of TFPI2 glycopeptide. A representative example of the product ion spectrum of the sugar chain modified product of KD3-pep detected from the TFPI2α band derived from HVT (A), OVISE (B), and OVMANA (C) is shown. It was confirmed that it was a KD3-pep glycopeptide from KD3-pep + GlcNAc ion and sugar chain diagnostic ion, and further, it was assigned to the illustrated structure from the mass of precursor ion and the detection status of characteristic fragment ion. MS spectrum comparison of KD3-pep sugar chain modified analogs detected from each cell. For the measurement results of the TFPI2α band of each cell, MS chromatograms were extracted in the range of m / z 1040-m / z 1240. "C" is a cell line derived from clear cell cancer of the ovary, and "P" is an HVT cell showing a characteristic signal. Classification of sugar chain structures detected in HVT and ovarian clear cell cancer cell lines. From information such as m / z 407.17, [1] structure with Hex5 HexNAc4, bi-antenna type complex sugar chain (Bi-LN) with LN on both antennas [2] structure with Hex6 HexNAc5, all antennas Tri-antenna type complex sugar chain with LN (Tri-LN) [3] Structure with Hex3 HexNAc6, bi-antenna type complex sugar chain with LDN on both antennas (LN / LDN mixed) [4] It was classified into a bi-antenna type complex sugar chain (Bi-LDN) having a structure having Hex4 HexNAc5, LDN on one side of the antenna, and LN on the other side. KD3 bound sugar chain profile. Among the various sugar chain modifiers of KD3-pep detected from the α and β bands of TFPI2 digested in the gel, the type and signal intensity of the bound sugar chain (peak area of monoisotope ion) for the sugar chain whose structure could be estimated. Value) profile is shown. KD2 bound sugar chain profile. Among the various sugar chain modifiers of KD2-pep detected from the α and β bands of TFPI2 digested in the gel, the type and signal intensity of the bound sugar chain (peak area of monoisotope ion) for the sugar chain whose structure could be estimated. Value) profile is shown. Binding sugar chain profile of TFPI2 in cell culture supernatant and pregnant woman serum. The bound sugar chain profile of TFPI2 in KD2 and KD3 in cultured cells and pregnant woman serum is shown. Expression profile of glycosyltransferase genes in placental and ovarian tissues. The vertical axis shows the relative expression level. Expression profiles of glycosyltransferase genes in various cultured cell lines. The vertical axis shows the relative expression level.

Hereinafter, embodiments of the present invention will be described in more detail.

The present invention is a method for measuring the expression of tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc (N-acetylgalactosamine-N-acetylglucosamine) in a biological sample in order to evaluate and / or differentiate clear cell cancer. I will provide a.

Clear cell carcinoma is a tumor with a peculiar histological type, and it is known that tissue carcinoma mainly composed of clear cells occurs not only in the ovary but also in the kidney, but also in the uterus and lungs.

Tissue factor pathway inhibitor 2 has a molecular weight of 26,934 and exhibits inhibitory activity against proteases such as trypsin, plasmin, and VIIa / tissue factor, and is considered to be involved in matrix remodeling. ing. Highly expressed in the placenta and secreted extracellularly. UniProt KB / Swiss-Prot registration number P48307 (TFPI2_HUMAN).

In the present invention, the biological sample may be a sample derived from the subject, and specifically, cells, tissues, body fluids, etc. obtained from the subject, specifically, tissues collected or excised from the ovary of the subject, blood of the subject (for example, blood of the subject). Whole blood, serum, plasma, plasma exchange external fluid, etc.), ascitic fluid, ascitic fluid, etc. can be exemplified. Whole blood, serum or plasma obtained by a normal blood test (clinical test) may be used as a blood sample.

When the presence of tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc is confirmed in a biological sample, it is evaluated that the patient is suffering from clear cell carcinoma or the histological type of the cancer is clear cell carcinoma. be able to.

To measure the expression of tissue factor pathway inhibitor 2 with a sugar chain structure having LacdiNAc (N-acetylgalactosamine-N-acetylglucosamine) in a biological sample, any known method capable of detecting glycoproteins may be used. For example, mass spectrometry can be used. Signals of 407.17 Da, 553.22 Da and / or 698.27 Da in addition to 366.14 Da in the glycopeptide ions observed when tissue factor pathway inhibitor 2 or peptide fragments thereof, or sugar chains thereof are measured by MS / MS. If is included, it can be evaluated that the patient is suffering from clear cell cancer or the histological type of the cancer is clear cell cancer. The peptide fragment of tissue factor pathway inhibitor 2 is preferably a partial peptide containing one or both of two N-type sugar chain binding sites (Asn116 and Asn170) possessed by tissue factor pathway inhibitor 2, and amino acids of the partial peptide. The number may be 4 to 15, and the sugar chain itself released by the desugar chain reaction may be used. This sugar chain is characterized in that the composition of hexose (Hex) and N-acetylhexosamine (HexNAc) has a biantenna type complex sugar chain composed of Hex4 HexNAc5 or Hex3 HexNAc6. Such a partial peptide can be obtained by purifying tissue factor pathway inhibitor 2 from a biological sample by an immunoprecipitation method or the like and then digesting it with trypsin. As an example of the partial peptide containing Asn116, a peptide consisting of the 113-124 sites of tissue factor pathway inhibitor 2 (113 YFFNLSSMTCEK 124) (SEQ ID NO: 1) can be mentioned, and as an example of the partial peptide containing Asn170, there is an example. Examples include, but are not limited to, a peptide consisting of the site 163-173 of tissue factor pathway inhibitor 2 (163 DEGLCSANVTR 173) (SEQ ID NO: 2). Among the glycopeptide ions observed when MS / MS measurement of tissue factor pathway inhibitor 2 or its peptide fragment, the signal of 407.17Da is a signal derived from LacdiNAc, and 553.22Da and 698.27Da are atypical fucosyl of LacdiNAc. It is a signal derived from LacdiNAc and sialyl LacdiNAc and is characteristically detected by tissue factor pathway inhibitor 2 of clear cell cancer. The signal of 366.14 Da is an oxonium ion common to all structures of N-type sugar chains. In addition, information on a signal characteristically detected in placental TFPI2 (for example, a signal of 657.23 Da derived from sialyl LacNAc) may be used as an auxiliary.

In addition to mass spectrometry, a lectin that recognizes the sugar structure of a glycoprotein and an antibody that recognizes the protein structure may be combined to measure the expression of tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc. In addition, the expression of tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc may be measured by a sandwich assay measurement method using two antibodies. At this time, an antibody that recognizes a protein containing glycoprotein sugar or the like and an antibody that recognizes a glycoprotein protein can be used in combination.

The subject is a patient suspected of having clear cell carcinoma (particularly clear cell carcinoma of the ovary), but may be targeted at all humans at risk of developing the disease.

Treatment should be started for subjects who are suffering from clear cell carcinoma or whose histological type of cancer is evaluated to be clear cell carcinoma by the above measurement. The main treatments for clear cell cancer are surgery and chemotherapy, and chemotherapy includes irinotecan, platinum preparations (cisplatin, carboplatin, nedaplatin, etc.), taxane preparations (pacritaxel, docetaxel, etc.), and combinations thereof (pakuritaxel, docetaxel, etc.). Combination therapy) etc. are used. However, clear cell carcinoma is highly resistant to anticancer drugs, and there is currently no optimal chemotherapy, so it is important to perform a complete resection including lymph node dissection.

The expression of tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc (N-acetylgalactosamine-N-acetylglucosamine) in a biological sample may be measured for the therapeutic effect or recurrence determination test of clear cell cancer. ..

When the presence of tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc is confirmed in a biological sample, it can be determined that there is a therapeutic effect on clear cell cancer or that clear cell cancer has recurred. ..

The present invention also provides a method for identifying substances that are effective in treating and / or preventing clear cell carcinoma. This method has the following steps:
(a) Step of contacting the test substance with clear cell cancer cells,
(b) The step of culturing the clear cell cancer cells contacted with the test substance in step (a) for a predetermined time,
(c) A step of measuring the expression of tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc in the clear cell cancer cells cultured in step (b), and
(d) Tissue having a sugar chain structure having LacdiNAc in clear cell cancer cells measured in step (c) Tissue having a sugar chain structure having LacdiNAc in control cells in which the expression of tissue factor pathway inhibitor 2 having a sugar chain structure was not contacted with the test substance. It comprises the step of evaluating the effect of the test substance on the expression of tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc in clear cell cancer cells by comparing with the expression of factor pathway inhibitor 2.

The test substance may be any substance, including proteins (including antibodies), peptides, vitamins, hormones, polysaccharides, oligosaccharides, monosaccharides, small molecule compounds, nucleic acids (DNA, RNA, oligonucleotides, mononucleotides, etc.). , Lipids, natural compounds other than the above, synthetic compounds, plant extracts, fractions of plant extracts, mixtures thereof and the like.

The clear cell cancer cell used in the identification method of the present invention may be derived from any organism as long as the expression of tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc is observed. Examples thereof include those derived from mammals such as humans, pigs, monkeys, chimpanzees, dogs, cows, rabbits, rats, mice, etc., and human-derived clear cell cancer cells (particularly cells that have been established). Specifically, it is preferable to use OVTOKO, OVISE, OVMANA, OVSAYO, RMG-I, RMG-II (available from JCRB).

The contact between the test substance and the clear cell cancer cell may be performed by any method, and examples thereof include a method of adding the test substance to the clear cell cancer cell. Further, the test substance may be administered after transplanting clear cell cancer cells into a living body such as a mammal other than human (for example, mouse, rat, guinea pig, rabbit, pig, etc.).

The culture time of the clear cell cancer cells after contact with the test substance is not particularly limited, and may be any time as long as the presence or absence of the effect of the test substance on the expression of the above protein in the clear cell cancer cells can be confirmed.

The control cells that have not been contacted with the test substance to be compared may be clear cell cancer cells before the contact with the test substance, or the clear cells that have undergone the same treatment except that the test substance is not contacted. It may be a cancer cell.

As one example of the present invention, in clear cell cancer cells contacted with a test substance, the expression level of tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc is decreased as compared with a control cell, and the test substance is used. If it can be evaluated that it has an effect of reducing the expression of the above protein, this test substance can be identified as a substance effective in the treatment and / or prevention of clear cell cancer.

Further, it may include a step of examining the effect of the test substance on the proliferation of clear cell cancer cells. The effect of the test substance on the proliferation of clear cell cancer cells can be examined, for example, by contacting the test substance with the clear cell cancer cells, culturing the test substance for a predetermined time, and then measuring the number of living cells.

As an example of the present invention, in clear cell cancer cells contacted with a test substance, when the growth of the clear cell cancer cells is inhibited as compared with the control cells, this test substance is used for treating clear cell cancer. And / or the certainty that it is effective for prevention is considered to increase.

Since clear cell cancer has high anti-cancer drug resistance, it is highly possible that the substance identified by the above method that is effective in treating and / or preventing clear cell cancer is a substance that reduces anti-cancer drug resistance. Therefore, the present invention also provides a method for identifying a substance that reduces anticancer drug resistance of clear cell cancer. This method has the following steps:
(a) Step of contacting the test substance with clear cell cancer cells,
(b) The step of culturing the clear cell cancer cells contacted with the test substance in step (a) for a predetermined time,
(c) A step of measuring the expression of tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc in the clear cell cancer cells cultured in step (b), and
(d) Tissue having a sugar chain structure having LacdiNAc in clear cell cancer cells measured in step (c) Tissue having a sugar chain structure having LacdiNAc in control cells in which the expression of tissue factor pathway inhibitor 2 having a sugar chain structure was not contacted with the test substance. It comprises the step of evaluating the effect of the test substance on the expression of tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc in clear cell cancer cells by comparing with the expression of factor pathway inhibitor 2.

The test substance may be any substance, including proteins (including antibodies), peptides, vitamins, hormones, polysaccharides, oligosaccharides, monosaccharides, small molecule compounds, nucleic acids (DNA, RNA, oligonucleotides, mononucleotides, etc.). , Lipids, natural compounds other than the above, synthetic compounds, plant extracts, fractions of plant extracts, mixtures thereof and the like.

The clear cell cancer cell used in the identification method of the present invention may be derived from any organism as long as the expression of tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc is observed. Examples thereof include those derived from mammals such as humans, pigs, monkeys, chimpanzees, dogs, cows, rabbits, rats, mice, etc., and human-derived clear cell cancer cells (particularly cells that have been established). Specifically, it is preferable to use OVTOKO, OVISE, OVMANA, OVSAYO, RMG-I, RMG-II (available from JCRB).

The contact between the test substance and the clear cell cancer cell may be performed by any method, and examples thereof include a method of adding the test substance to the clear cell cancer cell. Further, the test substance may be administered after transplanting clear cell cancer cells into a living body such as a mammal other than human (for example, mouse, rat, guinea pig, rabbit, pig, etc.).

The culture time of the clear cell cancer cells after contact with the test substance is not particularly limited, and may be any time as long as the presence or absence of the effect of the test substance on the expression of the above protein in the clear cell cancer cells can be confirmed.

The control cells that have not been contacted with the test substance to be compared may be clear cell cancer cells before the contact with the test substance, or the clear cells that have undergone the same treatment except that the test substance is not contacted. It may be a cancer cell.

As one example of the present invention, in clear cell cancer cells contacted with a test substance, the expression level of tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc is decreased as compared with a control cell, and the test substance is used. If it can be evaluated that there is an effect of reducing the expression of the above protein, this test substance can be identified as a substance that reduces the anticancer drug resistance of clear cell cancer.

Further, it may include a step of examining the effect of the test substance on the anticancer drug resistance of clear cell cancer cells. The effect of the test substance on the resistance of the clear cell cancer cells to the anticancer drug is, for example, to measure the viable cell number before, after, or at the same time when the clear cell cancer cells are brought into contact with the test substance, after adding the anticancer drug and culturing for an appropriate time. Can be investigated by. If the number of viable cells of the clear cell cancer cells contacted with the test substance is smaller than the number of viable cells of the control cells, it can be considered that the anticancer drug resistance of the clear cell cancer cells is reduced.

As an example of the present invention, in the case of clear cell cancer cells contacted with the test substance, when the anticancer drug resistance of the clear cell cancer cells is lower than that of the control cells, the test substance is the clear cell cancer. It is believed that the certainty of reducing anticancer drug resistance will increase.

Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.
[Example 1]
1. 1. Sample preparation To analyze the sugar chain structure of TFPI2 in clear cell cancer and placenta, four ovarian clear cell cancer cell lines (OVISE, OVMANA, OVASAYO, RMG-II) with strong TFPI2 expression, and human placental villous nutrition A membrane (Human Villous Trophoblast, HVT) was used. Culturing of these cells followed the recommended method. To release TFPI2 bound to the extracellular matrix of each cell, each cell reaching 90% density was washed 3 times with PBS, and 150 μg / mL of heparin sodium was added to the medium containing 10% FBS. The treatment was carried out at 37 ° C. for 300 minutes. After the treatment, the culture supernatant was collected.
The obtained culture supernatant was subjected to an immunoprecipitation reaction using antibody beads to which the anti-human TFPI2 mouse monoclonal antibody TS-TF04 and Dynabeads M-280 activated Tosyl beads (Life Science) were bound. The culture supernatant and antibody beads were reacted in a 1.5 mL microtube for 3 hours with stirring with a vortex mixer. The antibody beads were collected, washed 3 times with PBS containing 0.1% Tween, and then 30 μL of 0.1 M HCl was added and allowed to stand for 5 minutes to elute TFPI2. After elution, it was recovered immediately and neutralized by adding 3 μL of 1 M Tris-hydrochloric acid.

2. 2. Western blotting
Eluted samples after IP were separated using a polyacrylamide gel SuperSep ^TM Ace 12.5%, 13 well. After SDS-PAGE was completed, the protein was transferred to the PVDF membrane according to a conventional method. The PVDF membrane after transfer was washed with deionized water and methanol, and then shaken with Bullet Blocking One for Western Blotting (Nacalai Tesque) at 25 ° C for 10 minutes for blocking treatment. Then, the PVDF membrane was reacted with anti-TFPI2 antibody 28Aa diluted 3000 times with Blocking One (Nacalai Tesque) at 25 ° C. for 18 hours. After the primary antibody reaction, the PVDF membrane was washed 5 times with PBS-T [0.05% Tween] and reacted with a peroxidase-labeled anti-mouse IgG antibody diluted 10000 times with Blocking One for 1 hour. After the reaction, the PVDF membrane was washed 5 times with PBS-T [0.05% Tween] and detected using ECL Select reagent (GE Healthcare).
FIG. 2A shows the results of comparative analysis of TFPI2 obtained from HVT cells and four types of clear cell cancer-derived cell lines by IP-Western blotting. In all cells, TFPI2 was detected as triplet bands of α, β, and γ of different sizes.

3. 3. Sugar chain analysis of cultured cell-derived TFPI2 In order to investigate the sugar chain structure of α, β, and γ bands, the gel after SDS-PAGE was washed with deionized water three times for 5 minutes, and the SimplyBlue ^TM Safe Stain staining reagent (Thermo). ) Was used for staining. The detected gel sections (α, β, γ) are extracted, shaken in 25 mM ammonium bicarbonate buffer (ABB) / 50% acetonitrile (ACN) for 15 minutes, then buffered to 50 mM ABB 100 μL. Shake for another 5 minutes. Then, 100% ACN was added, and after shaking for 15 minutes, the solution was discarded and dried by a centrifugal evaporator. After gel drying, the reduction reaction was carried out with 10 mM DTT / 25 mM ABB / 0.2 M guanidine hydrochloric acid at 56 ° C for 45 minutes, replaced with 96 mM monoiodoacetic acid in 25 mM ABB, and allowed to stand in the dark at 25 ° C for 30 minutes. Alkylated by placing. After washing with 200 μL of 25 mM ABB / 50% ACN, it was dehydrated with 100 μL of 100% ACN and dried with a centrifugal evaporator. A trypsin solution was added and the solution was recovered after 18 hours at 37 ° C. The recovered solution was dried on a centrifugal evaporator and then redissolved in Solution A (0.1% (v / v) formic acid / water).

4. LC / MS / MS measurement The tryptic digested peptide was desalted using Pierce® C18 Spin Columns (Thermo), and the Q Exactive ^TM mass spectrometer linked with the EASY-nLC 1000 nano-LC system. Analyzed by (Thermo Scientific). The separation conditions for the EASY-nLC 1000 were set as follows. Solution A (0.1% (v / v) formic acid / water), Solution B (0.1% (v / v) formic acid / ACN), flow rate 300nL / min, Gradient (solution B, 0-40 min: 0-35%, 40-43min: 35-100%), Q Exactive ^TM analysis conditions are Run time: 50min, positive 2 kV, MS1 Resolution: 70,000, scan charge: 350-2000m / z, MS2 Resolution: 17,500, NCE: 27. did.

5. Glycopeptide analysis Human TFPI2 has N-type sugar chain binding sequences (NXS / T) in KD2 (Asn116) and KD3 (Asn170), which are commonly conserved in mammals except rodents. Therefore, we focused on tryptic digestive peptides containing the binding site, ¹¹³ YFFNLSSMTCEK ¹²⁴ (KD2-pep) and ¹⁶³ DEGLCSANVTR ¹⁷³ (KD3-pep), and used the values of ions in which GlcNAc was bound to these peptides to use the MS chromatogram. The MS / MS spectrum of the glycopeptide was searched by extracting. By detecting the diagnostic ions and confirming whether the spacing between the major fragment ions matches the mass spacing of the monosaccharide, it was determined whether the spectrum was MS / MS derived from the glycopeptide.
FIG. 3 is a typical product ion spectrum of KD3-pep detected from the α band. Characteristic trivalent glycopeptides with m / z values of 1192.13, 1219.48, and 1122.79 were detected in HVT, OVISE, and OVMANA. In all of these, the signal (m / z 1425.62 ^1- ) added by GlcNAc ion to KD3-pep (m / z = 1221.53) is clearly detected on MS / MS, and the adjacent major fragments. These are because the ions match the mass spacing of the monosaccharide, the signal with m / z value 366.14 ^{1-, which is a sugar chain diagnostic ion, and the signal with m / z value 528.19 1- are} detected. Was determined to be a glycopeptide of KD3-pep. The other bands were analyzed in the same manner, and the sugar chain modification states of KD3-pep and KD2-pep and the detection status of each glycopeptide in each band are summarized in FIGS. 2B and C. In all cells, KD3-pep and KD2-pep sugar chain modified substances were detected in the α band, but unmodified substances were not detected. On the other hand, both the sugar chain modified form and the unmodified form were detected in the β band, but only the unmodified form was detected in the γ band. From the above results, the α band is a sugar chain modified product of both KD3 and KD2, the β band is a mixture of sugar chain modified products of either KD3 or KD2, and the γ band is an unsugar chain modified product. It turned out to be a body.

6. Sugar chain structure analysis of TFPI2 Based on the mass of the detected typical KD3-pep sugar chain modifier, the composition and structure of the sugar chain were first calculated and structurally predicted using the GlycoMod tool and the CPG database. The mass of the bound sugar chain of the glycopeptide (calculated value: 3527.346 Da) typically detected from HVT was calculated to be 2350.82 Da, and the sugar chain was calculated by GlycoMod tool (http://web.expasy.org/glycomod/). As a result of calculating the composition, this was in agreement with the mass of Hex5 HexNAc4 dHex1 NeuAc2 (theoretical value: 2350.83 Da). As a result of collating this composition with the CPG database (http://www.functionalglycomics.org/glycomics/molecule/jsp/carbohydrate/searchByComposition.jsp), the structure is NeuAc-Gal-GlcNAc-Man- (NeuAc-Gal-GlcNAc). It was expected to be a typical bi-antenna type complex sugar chain with a Gal-GlcNAc structure called -Man-)-Man-GlcNAc- (Fuc-) GlcNAc and LacNAc (LN). On the other hand, the bound sugar chains of typical glycopeptides of OVISE (calculated value: 3654.426 Da) and OVMANA (calculated value: 3364.326 Da) were calculated as 2432.90 Da and 2142.80 Da, respectively, and Hex3 HexNAc6 dHex1 NeuAc2 (theory). Values are consistent with the composition of Hex3 HexNAc6 dHex3 (theoretical value: 2142.81 Da), and the expected sugar chain structures are NeuAc-GalNAc-GlcNAc-Man- (NeuAc-GalNAc-GlcNAc-Man-) Man, respectively. -GlcNAc- (Fuc-) GlcNAc, GalNAc- (Fuc-) GlcNAc-Man- (GalNAc- (Fuc-) GlcNAc-Man-) Man-GalNAv- (Fuc-) GalNAc, both with LacdiNAc (LDN) A structure having a so-called GalNAc-GlcNAc sequence in the antenna was speculated. In support of this inferred structure, in the product ion spectrum of FIG. 3, a signal of m / z 407.17 ^1- corresponding to the mass of the LDN group (GalNAc-GlcNAc + H ⁺ ) is commonly seen in OVISE and OVMANA. In contrast, it was not found in HVT cells. Therefore, it was considered that the signal of m / z 407.17 ^1- could be attributed not to the signal derived from the deepest chitobioscore (GlcNAc-GlcNAc) common to N-type sugar chains, but to the signal derived from the LDN group. Furthermore, signals corresponding to the sialylated form, which is a variant of LDN, the sialyl LDN group (LDNS group, NeuAc-Gal-GlcNAc), which is a fucosylated form, and the fucosyl LDN group (LDNF group, GalNAc- (Fuc-) GlcNAc). As a result, signals with m / z value 657.23 ^1- and m / z value 553.22 ^1- were detected characteristically in the corresponding glycopeptides (Fig. 3), and the estimated structure was correctly detected by the MS / MS pattern. Confirmed that it can be attributed.
As a result of a series of analyzes on the α-band and β-band of TFPI2 in all cells, KD3-pep and KD2- are preferentially detected from the α-band and β-band of TFPI2 in ovarian clear cell cancer cell lines. Most of the sugar chain modifiers of pep always detect a signal with an m / z value of 407.17 ^1- , and some have a signal with an m / z value of 657.23 ^1- and a signal with a 553.22 ^1- . It was found that TFPI2 derived from clear cell cancer has a high proportion of LDN structure because it could be assigned to a bi-antenna type sugar chain having an LDN sequence in both or one of the antennas. (table 1).

Table 1. Types of typical TFPI2 bound sugar chains detected in clear ovarian cell cancer cells and HVT cells and characteristic fragment ions

図6および7は、TFPI2のαおよびβバンドから検出されたKD2-、およびKD3-pepの結合糖鎖の種類とシグナル強度（モノアイソトープイオンのピークエリア値）に基づいて作成した糖鎖プロファイルである。KD3-pep（図6）およびKD2-pep（図7）の結合糖鎖プロファイルは、同一の細胞であれば、αバンドとβバンドで、きわめてよく似たパターンを示した。

7. 7. Comparison of types of TFPI2-bound sugar chains and their distribution In order to clarify all TFPI2-bound sugar chains detected from the α-band and β-band of each cell and their distribution, the precursor ion of the glycopeptide whose structure was determined was used. Chromatograms were extracted with m / z values, and glycoforms with the same valence as the precursor ion valence of glycopeptides were searched for from the precise mass and mass interval. FIG. 4 shows a mass chromatogram extracted in the range of KD3-pep sugar chain modified m / z value 1040-1240 whose structure of each cell was determined. Precursor ions of various masses were detected in each cell, and it was inferred from the mutual mass spacing that they were sugar chain modifiers with different KD3-pep. In fact, it was confirmed that it has a sugar chain that can be attributed to the structure inferred from the corresponding product ion spectrum. In this study, all the detected sugar chain modifiers were able to classify the major sugar chains that bind to TFPI2 into the following four types of structures based on the composition of Hex and HexNAc (Fig. 4).

Figures 6 and 7 show sugar chain profiles created based on the types and signal intensities (peak area values of monoisotope ions) of KD2- and KD3-pep bound sugar chains detected in the α and β bands of TFPI2. be. The bound sugar chain profiles of KD3-pep (Fig. 6) and KD2-pep (Fig. 7) showed very similar patterns in the α band and β band in the same cell.

8. Verification using human serum samples In order to verify whether the tissue-specific TFPI2 sugar chain modifier detected in cultured cells is also expressed in vivo, we performed a sugar chain structural analysis of TFPI2 in pregnant women's serum. .. For pregnant women's serum, 8 samples purchased from ProMedDx were used. All samples are Western samples that are described as having consented to informed consent. As shown in Table 2, the final TFPI2 concentration is based on the TFPI2 concentration quantified in advance with the TFPI2 measurement reagent (immunassay reagent by sandwich method using two types of anti-TFPI2 monoclonal antibodies labeled with solid phase or enzyme). The reagents were divided into 3 groups so as to have the same level, and 100 μL of each was mixed in a pool and used for the analysis of glycopeptides. For comparison, culture supernatants of HVT, OVISE, and OVMANA cells cultured for 7 days without medium exchange were also used for analysis.

Table 2. Pregnant woman serum sample used for analysis

Since the TFPI2 concentration of the sample prepared for this analysis is lower than that of the sample obtained by heparin treatment, in this analysis, TFPI2 after immunoprecipitation is directly trypsin-digested without gel electrophoresis and acetone. The sugar chain structure of the glycopeptide concentrated by precipitation was analyzed. Reduction alkylation and tryptic digestion were performed on TFPI2 purified by immunoprecipitation using DTT and monoiodoacetic acid. After digestion with trypsin, 5 times the amount of ice-cold acetone as the sample was added, and after stirring, acetone precipitation was carried out at −25 ° C. for 18 hours. After the acetone precipitation, centrifugation was performed at 12,000 × g at 25 ° C. for 10 minutes, the supernatant was discarded, the precipitate was dissolved in Solution A, and the glycopeptide was analyzed.
As shown in FIG. 8, the bound sugar chain profile of TFPI2 in the culture supernatants of HVT, OVISE, and OVMANA is very similar to the results obtained by heparin treatment for both KD2 and KD3, and HVT has an LN structure. Peptides having a biantennary sugar chain with an LDN structure were preferentially detected from biantennary and triantennary complex sugar chains, and OVISE and OVMANA.
In pregnant women's serum, there are two types of structures strongly detected as KD2 bound sugar chains, both of which are biantennary complex sugar chains with fully cialified LN, one of which is the deepest GlcNAc. It was assigned as a sugar chain to which core fucose was bound, and the other was a sugar chain having no core fucose. There are two types of strongly detected structures in KD3, which are also attributed to biantennary with fully cialified LN and triantennary type complex sugar chains, both of which are sugar chains with core fucose. there were. The sugar chain structure distribution pattern of this pregnant woman's serum showed extremely high similarity among the pooled samples of the three groups, but the partially sialylated sugar chain structure detected from the culture supernatant of HVT cells was detected. There wasn't. However, it is consistent with HVT cells in that it is mainly a biantennary or triantennary type sugar chain having LN in the skeleton, and a sugar chain having an LDN structure characteristic of ovarian clear cell cancer cells was detected. There wasn't.

9. Glycosyltransferase gene expression analysis Genes encoding β1,4-N-acetylgalactosyltransferase 3 and 4, which are glycosyltransferases involved in LacdiNAc (LDN) sugar chain synthesis, B4GALNT3 and B4GALNT4, and sugars involved in LacNAc (LN) sugar chain synthesis. The expression levels of the transferases β-galactoside α-2,6-sialyltransferase 1 and 2, ST6GAL1 and ST6GAL2, in tissues and various cancer cell lines were examined by a quantitative RT-PCR method. Extract all RNA and synthesize cDNA from tissues and various cancer cell lines using One Step PrimeScript RT-PCR Kit (Takara Bio Inc.), and compare gene expression levels by quantitative RT-PCR using the PCR primers in Table 3. did.
FIG. 9 shows the results of comparative quantification of gene expression of various glycosyltransferases in human normal placenta and normal ovarian tissue. Among the four glycosyltransferases, B4GALNT3 and B4GALNT4, which are the responsible glycosyltransferase genes for LDN glycosylation, have higher expression levels in the ovary than in the placenta, but there is a significant difference in the expression levels between ST6GAL1 and ST6GAL2. I couldn't see it.
FIG. 10 shows placental villous vegetative membrane cells (HVT), clear-cell ovarian cancer cell lines (OVISE, OVMANA, OVSAYO, RMG-2, ES-2), ovarian serous cancer cell lines (OVSAHO, OVKATE), ovarian mucous. It is the result of comparative quantification of the gene expression of various glycosyltransferases in the cancer cell line (RMUG-S, MCAS) and the prostate cancer cell line (LnCap). Among the four glycosyltransferase genes, B4GALNT3 was found to have high expression specificity in ovarian clear cell cancer cell lines except ES-2. From this result, it was shown that the addition of LDN sugar chain of TFPI2 in clear cell carcinoma of the ovary may be caused by histologically specific expression of B4GALNT.

Table 3. PCR primers used for expression analysis of glycosyltransferase gene

The clear cell cancer evaluation and / or differentiation method of the present invention can be used for diagnosis of clear cell cancer. Further, the substance identification method of the present invention can be used for searching for a therapeutic agent for clear cell cancer.

<SEQ ID NO: 1>
SEQ ID NO: 1 shows the amino acid sequence of the partial peptide KD2-glycopeptide (Asn116) of tissue factor pathway inhibitor 2.
<SEQ ID NO: 2>
SEQ ID NO: 2 shows the amino acid sequence of the partial peptide KD3-glycopeptide (Asn170) of tissue factor pathway inhibitor 2.
<SEQ ID NO: 3-10>
SEQ ID NOs: 3 to 10 are PCR primers (B4GALNT3-F, B4GALNT3-R, B4GALNT4-F, B4GALNT4-R, ST6GAL1-F, ST6GAL1-R, ST6GAL2-F, respectively) used for the expression analysis of the glycosyltransferase gene. The nucleotide sequence of ST6GAL2-R) is shown.

Claims (10)

A method for measuring the expression of a tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc (N-acetylgalactosamine-N-acetylglucosamine) in a biological sample for evaluating and / or differentiating clear cell carcinoma . If the glycopeptide ion observed when measuring tissue factor pathway inhibitor 2 or its peptide fragment for MS / MS contains a signal of 407.17 Da in addition to 366.14 Da, the patient will have clear cell carcinoma. Or said method in which the histological type of cancer is shown to be clear cell cancer . Glycopeptide ions observed during MS / MS measurements of tissue factor pathway inhibitor 2 or peptide fragments thereof further include at least one signal selected from the group consisting of 553.22 Da, 698.27 Da and 495.18 Da. The method according to claim 1, wherein if it is present, it is indicated that the patient is suffering from clear cell cancer or the tissue type of the cancer is clear cell cancer . The method according to claim 1 or 2, wherein the biological sample is a cell, tissue or body fluid obtained from a subject. The method according to claim 3 , wherein the body fluid is blood, ascitic fluid, or ascitic fluid. The method of claim 4 , wherein the blood is whole blood, serum, plasma or plasmapheresis external fluid. It is a method for measuring the expression of tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc (N-acetylgalactosamine-N-acetylglucosamine) in a biological sample for a therapeutic effect or a test for determining recurrence of clear cell cancer. In addition to 366.14 Da, the signal intensity of 407.17 Da is contained in the glycopeptide ions observed when the tissue factor pathway inhibitor 2 or its peptide fragment is measured by MS / MS. The above-mentioned method, which does not change or increases as compared with that before treatment, indicates that there was no therapeutic effect on clear cell cancer or that clear cell cancer recurred . A method for identifying substances that are effective in treating and / or preventing clear cell carcinoma, wherein the following steps:
(a) Step of contacting the test substance with clear cell cancer cells,
(b) The step of culturing the clear cell cancer cells contacted with the test substance in step (a) for a predetermined time,
(c) In the step of measuring the expression of tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc in the clear cell cancer cells cultured in step (b) , tissue factor pathway inhibitor 2 or a peptide fragment thereof is MS / If the glycopeptide ion observed during MS measurement contains a signal of 407.17Da in addition to 366.14Da, it indicates the expression of tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc. The above steps and
(d) Tissue having a sugar chain structure having LacdiNAc in clear cell cancer cells measured in step (c) Tissue having a sugar chain structure having LacdiNAc in control cells in which the expression of tissue factor pathway inhibitor 2 having a sugar chain structure was not contacted with the test substance. The method comprising the step of evaluating the effect of the test substance on the expression of tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc in clear cell cancer cells by comparing with the expression of factor pathway inhibitor 2. The method according to claim 7 , further comprising a step of examining the effect of the test substance on the proliferation of clear cell cancer cells. A method for identifying substances that reduce anticancer drug resistance in clear cell cancer, and the following steps:
(a) Step of contacting the test substance with clear cell cancer cells,
(b) The step of culturing the clear cell cancer cells contacted with the test substance in step (a) for a predetermined time,
(c) In the step of measuring the expression of tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc in the clear cell cancer cells cultured in step (b) , tissue factor pathway inhibitor 2 or a peptide fragment thereof is MS / If the glycopeptide ion observed during MS measurement contains a signal of 407.17Da in addition to 366.14Da, it indicates the expression of tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc. The above steps and
(d) Tissue having a sugar chain structure having LacdiNAc in clear cell cancer cells measured in step (c) Tissue having a sugar chain structure having LacdiNAc in control cells in which the expression of tissue factor pathway inhibitor 2 having a sugar chain structure was not contacted with the test substance. The method comprising the step of evaluating the effect of a test substance on the expression of tissue factor pathway inhibitor 2 having a sugar chain structure having LacdiNAc in clear cell cancer cells by comparing with the expression of factor pathway inhibitor 2. The method according to claim 9 , further comprising a step of examining the effect of the test substance on the anticancer drug resistance of clear cell cancer cells.

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