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WO2006129717A1 - Marqueur tumoral pour la detection de cancer pancreatique et kit de detection de cancer pancreatique l’employant - Google Patents

Marqueur tumoral pour la detection de cancer pancreatique et kit de detection de cancer pancreatique l’employant Download PDF

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Publication number
WO2006129717A1
WO2006129717A1 PCT/JP2006/310886 JP2006310886W WO2006129717A1 WO 2006129717 A1 WO2006129717 A1 WO 2006129717A1 JP 2006310886 W JP2006310886 W JP 2006310886W WO 2006129717 A1 WO2006129717 A1 WO 2006129717A1
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WO
WIPO (PCT)
Prior art keywords
cancer
protein
apoc
pancreatic cancer
spleen
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2006/310886
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English (en)
Japanese (ja)
Inventor
Akira Togawa
Shigetsugu Takano
Masaru Miyazaki
Fumio Nomura
Takeshi Tomonaga
Kazuyuki Sogawa
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Chiba University NUC
Original Assignee
Chiba University NUC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Chiba University NUC filed Critical Chiba University NUC
Priority to JP2007519041A priority Critical patent/JPWO2006129717A1/ja
Publication of WO2006129717A1 publication Critical patent/WO2006129717A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/775—Apolipopeptides

Definitions

  • the present invention relates to a marker for detecting knee cancer and a kit for detecting knee cancer using the marker.
  • Spleen cancer is known as a cancer with poor prognosis among cancers of extinct organs. The reasons are as follows: (1) Early detection is difficult, advanced cancer is already diagnosed at the time of diagnosis, and resection rate is low. (2) Peripheral plexus, peritoneal dissemination and liver metastasis have occurred at an early stage when the biological malignancy is high. (3) An effective adjunct therapy is established and it is a cunning thing.
  • splenic cancer is very malignant, and metastasis (distant metastasis) has occurred at a site distant from the spleen. Because of this distant metastasis (especially the liver), many patients are subject to surgical resection. Do not become. Patients with inoperables have a worse prognosis. The 5-year survival rate is almost 0%. For this reason, diagnosis and resection as early as possible is the most effective treatment. Therefore, it is most important to develop a method for detecting splenic cancer patients at an early stage.
  • tumor markers such as CA19-9, DUPAN-2, SPAN-1, and SL X are used for detection of tumors of spleen cancer, which contributes to the diagnosis of spleen cancer (for example, the following) (See Non-Patent Documents 1 to 4).
  • Non-Patent Literature 1 Masakazu Zhang et al., “Clinical Significance of New Tumor Marker CA19-9”, “Knee Cancer Case Medium”, Monthly and Spleen, 1985, 6 ⁇ , 1129–1135
  • Non-Patent Document 2 TAKASAKI et al. “Correlative Study on Expression of CA19 —9 and DU—PAN— 2 in Tumor Tissue and in Serum of Pancreatic Cancer Patients” Cancer Research, 1988, 48, 1435—1438 3: YONG S. CHUNG et al. "The Detection of Human Pancreatic Cancer— Associated Antigen in the Serum of Cancer Patients” Cancer, 1987, 60 ⁇ , 1636-1643
  • Non-Patent Document 4 Hiroyasu Kawakami et al. “Clinical usefulness of serum SLX (sialyl SSEA—1) measurement in various cancers of cancer”, Journal of Japan Society of Surgery for Diseases, 1989, 86 ⁇ , 1141— 1148 Page Disclosure of Invention
  • CA19-9 which is the most specific and highly sensitive among the above tumor markers, and is widely used, the positive rate of early splenic cancer is low. Search for cancer is not possible with this test. Furthermore, in Lewis antigen-negative patients, CA19-9 has the problem that it may become negative even in advanced splenic cancer (Hirano et al., “Loss of Lewis antigen expression on erythrocytes in some cancer patients with high serum). CA19—9 levels ”, J Natl Cancer Inst, 1987, 79 ⁇ , 1261-1268). Furthermore, chronic knee inflammation shows high values even in patients with cholangitis and cholelithiasis V, and there remains a problem as a tumor marker for reliably detecting splenic cancer.
  • the present invention is more reliable for splenic cancer with a higher positive rate of early splenic cancer. It is an object of the present invention to provide a tumor marker that is actually detected and a knee cancer detection kit using the same.
  • the tumor marker for detecting knee cancer is characterized by having ApoC-1 protein ability. This makes it possible to more reliably detect splenic cancer, which has a higher positive rate of early splenic cancer than conventional tumor markers. This can be detected using, for example, a protein chip system, ELISA method or the like. Examples of ELISA methods include (Micheal D. Curry, Walter J McConathy, Jim D. Fesmire, and Peter
  • a spleen cancer detection kit is used in a protein chip system V, and can contribute to more reliable detection of spleen cancer.
  • the protein chip system is a system developed for the purpose of efficiently performing functional analysis such as protein expression, interaction, post-translational modification, and purification and identification of the target protein. It consists of a protein chip with various chemical properties suitable for protein analysis on the surface, a protein chip reader used for measurement, and a computer installed with software used for measurement and analysis.
  • the protein chip system can capture the protein of interest from a crude sample such as serum, urine, culture broth and cell lysate using the affinity for the protein chip, and measure its mass number. There is an advantage that protein can be easily analyzed on a protein chip without using labels or tags, and results can be obtained in a short time even with a small amount of sample force.
  • a time-of-flight mass spectrometer (SELDI—TOF—MS) is suitable.
  • the time delinquent mass spectrometer irradiates the substance trapped on the surface of the protein chip with a laser to cause ionization.
  • the ionized material is accelerated in the electric field and flies through the flight tube toward the detector.
  • the flying speed is proportional to the molecular weight (more precisely, the molecular weight divided by the number of ionized charges). Therefore, by measuring the time from when the laser is irradiated until the force reaches the detector, the molecular weight of the substance existing on the surface of the protein chip can be known, and the target protein can be identified.
  • the present invention it is possible to provide a tumor marker for more reliably detecting splenic cancer having a higher positive rate of early splenic cancer and a knee cancer detection kit using the same.
  • a time-flight mass spectrometer (SELDI-TOF-MS) was used as the protein chip reader in the IVa Vb protein chip system.
  • WCX2 cation exchange chip
  • pH 6.5 50 mM sodium phosphate
  • the sample composition is urea; patient serum 20 ⁇ 1 + denatured buffer 20 ⁇ 1 + pH buffer 160 l (lZlO diluted), and 100 1 is administered to each spot on the protein chip, and two spots of the same sample are performed. It was.
  • each Sinapic acid was dissolved in EAM solution (50% acetonotryl ZO. 5% TFA) and applied to the pot.
  • SELDI-TOF—MS a protein chip detector, is used to detect proteins in the serum and perform pre- and post-operative protein comparisons (Serum protein profiling).
  • serum protein profiling we found a peak (protein) that decreased after surgery, and detected a peak of about 6630 Da in mass as a candidate protein.
  • a typical peak example is shown in Fig. 1, and the peak intensity of this protein before and after surgery is shown in Fig. 2.
  • CA19-9 was negative in case 3, which was positive for this protein.
  • this protein can be detected more frequently in early-stage knee cancer, is more sensitive than conventional CA19-9, and has the same strength as the Lewis antigen (a— , b +) It can be used as a tumor marker for detecting knee cancer that can be detected in patients.
  • a specific protein was purified from serum to identify a protein that is expected to be a marker for detecting knee cancer.
  • This specific protein was purified through examination of optimum pH (pi) and optimum salt (NaCl) concentration.
  • FPLC FPLC was performed, and a high peak FPLC fraction was applied to HPLC.
  • the conditions of FPLC are to make a fraction of NaCl separated by 15 mM, 165mM ⁇ : SELDI-TOF-MS analysis was performed with NP 20 chip using fraction every 30mM to LOOOmM.
  • the HPLC conditions were linear-gradient with Buffer; 0.1% (A) to 80% (B) acetonitrile, flow rate: 200 1 / min, and C-18 was used as the separation column.
  • the purified target protein was analyzed by analyzing the amino acid residues up to the 15th amino acid residue using the N-terminal amino acid sequence.
  • the 6proteins of 6630 Da each were mature Apolipoprotein C-1 (57 amino acids) (mature ApoC-1 protein), 6
  • the body of 420Da was found to be Apolipoprotein C-1 (55 amino acids) with two mature N-terminal amino acids deleted. The results are shown in Figs. That is, it was found that the protein that can be used as a tumor marker for detecting splenic cancer is ApoC-1 protein.
  • a tumor marker for detecting splenic cancer with a higher positive rate of early splenic cancer and a knee cancer detection kit using the same can be realized by using the tumor marker for detecting spleen cancer. Furthermore, the value of this tumor marker is related to the patient's survival time, and is closely related to the malignancy of the knee cancer.Therefore, there is a possibility that it can be a prognostic index. Has no features. A small amount of patient serum is sufficient for the practice of the present invention and can be stored at -30 ° C and measured at any time.
  • a preferable condition for detecting ApoC-1 as a tumor marker for detecting spleen cancer is pH 3 or more and 7 or less, more preferably pH 6 or more and 7 or less.
  • Apolipoprotein C-1 is also present in the serum of healthy individuals. It is involved in lipid metabolism and is abundant in chylomicron and VLDL, which are triglyceride transfer lipoproteins. It is synthesized in the rough endoplasmic reticulum of the liver, and the average blood concentration is said to be about 6 mg / dl.
  • ApoC—1 has many lipid-related functions compared to other apolipoproteins. (1) It changes the form of ⁇ protein and binds LDL receptor (LDLR) or LDLR related protein to lipoproteins. It is suggested that it inhibits, and (2) has the function of promoting LCAT activity. ”(The interaction or human apolipoprotein
  • micellar phospholipid with sub— micellar phospholipid. Benjamin W.
  • the length of the ApoC-lPCR product was designed to be 150 bp.
  • the ApoC-1 gene was clearly expressed more strongly in the cancerous part than in the non-cancerous part.
  • Figure 11 shows the result.
  • siRNA-2 Two types of siRNA for specifically suppressing ApoC-1 gene using RNAi were designed, and siRNA capable of suppressing ApoC-1 gene more effectively was selected.
  • the sequence of this Oligonucleotide is shown below, Target sequence, siRNA-2; CTG GAG GAC AAG GCT CGG
  • a lOxlO 4 cells / ml cell suspension is prepared with a culture solution containing 10% FBS. 6-well plate the seeded cells by 2ml (20xl0 4 cells / well) , confirmed by a microscope, to the goal of the next day 50% confluent. Incubate for 24 hours in a 37 ° C, 5% C02 incubator.
  • FIG. 1 is a diagram showing a typical protein peak (mass 6000 to 7000 Da) of serum before and after splenic cancer surgery using a protein chip system. Top shows pre-operative results and bottom shows post-operative results.
  • FIG. 2 is a graph showing the results of examining the expression of a protein having a mass number of 6630 Da before and after surgery.
  • FIG. 3 is a diagram showing the results when the pH is changed in the analysis using the protein chip system.
  • FIG. 4 A diagram showing the results when the NaCl concentration is changed stepwise from 0 mM to 500 mM in the analysis using the protein chip system!
  • FIG. 5 is a diagram showing the peak of HPLC.
  • FIG. 6 Diagram showing the results of analysis using SELDI-TOF-MS for HPLC fraction with high peak.
  • FIG. 7 is a diagram showing the peak of the second HPLC.
  • FIG. 8 A diagram showing the results of analysis using SELDI-TOF-MS for HPLC fraction with high peak.
  • FIG. 9 is a view showing the analysis results of the N-terminal amino acid sequence.
  • Figure 2 Analysis of recurrence-free survival and survival by Kaplan-Meier method between two groups divided by the median peakintensity of ApoC-1 protein in preoperative serum obtained by protein chip system analysis .
  • FIG. 11 RT-PCR study of ApoC-1 gene expression in spleen cancer tissue, normal spleen tissue, and spleen cancer cell lines.
  • FIG. 12 A figure showing the quantification of ApoC-lmRNA by performing Real-time RT-PCR using frozen specimens of 16 cancerous and non-cancerous specimens of splenic cancer.
  • FIG. 14 shows ApoC-1 protein expression confirmed by Western blotting in normal spleen and cancer tissues and four spleen cancer cell lines.
  • FIG. 15 Immunohistochemical staining was performed. Expression of ApoC-1 protein expression in splenic cancer tissues and The figure which confirmed localization.
  • FIG. 16 A diagram showing that the ApoC-1 gene of a ⁇ cancer cell line was specifically suppressed by ApoC-IsiRNA and confirmed using Real-time RT-PCR and Western blotting.
  • FIG. 17 Cell proliferation assay of ApoC-1 involved in cell proliferation in spleen cancer cell lines.

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  • Health & Medical Sciences (AREA)
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Abstract

L’invention renvoie à un marqueur tumoral hautement positif au test pour le cancer pancréatique à un stade précoce et pouvant détecter un cancer du pancréas avec une grande fiabilité. L’invention renvoie également à un kit destiné à la détection d’un cancer du pancréas employant le marqueur tumoral ; à un marqueur tumoral destiné à la détection du cancer pancréatique comprenant une protéine ApoC-1, ainsi qu’à un kit destiné à la détection du cancer pancréatique comprenant une puce à protéines pour détecter la protéine ApoC-1.
PCT/JP2006/310886 2005-05-31 2006-05-31 Marqueur tumoral pour la detection de cancer pancreatique et kit de detection de cancer pancreatique l’employant Ceased WO2006129717A1 (fr)

Priority Applications (1)

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JP2007519041A JPWO2006129717A1 (ja) 2005-05-31 2006-05-31 膵癌検出用腫瘍マーカー及びそれを用いた膵癌検出用キット

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JP2005-160736 2005-05-31

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009075092A (ja) * 2007-08-24 2009-04-09 Univ Kansai Medical 扁平上皮がんに対する放射線治療後における予後の予測方法および予後予測用キット
WO2009088022A1 (fr) * 2008-01-07 2009-07-16 Kagoshima University Nouveau marqueur de cancer et diagnostic l'utilisant

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004531687A (ja) * 2000-09-12 2004-10-14 ユニバーシティ オブ シドニー 診断的検定法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004531687A (ja) * 2000-09-12 2004-10-14 ユニバーシティ オブ シドニー 診断的検定法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KITAMURA A.: "Array Protein System o Mochiita Apolipoprotein Sokutei ni Tsuite", TOKAI YONKEN NOSON IGAKUKAI ZASSHI, October 1989 (1989-10-01), pages 50 - 51, XP003006984 *
RICCI F.: "Stromal Responses to Carcinomas of the Pancreas", CANCER BIOLOGY & THERAPY, vol. 4, no. 3, March 2005 (2005-03-01), pages 302 - 307, XP003006983 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009075092A (ja) * 2007-08-24 2009-04-09 Univ Kansai Medical 扁平上皮がんに対する放射線治療後における予後の予測方法および予後予測用キット
WO2009088022A1 (fr) * 2008-01-07 2009-07-16 Kagoshima University Nouveau marqueur de cancer et diagnostic l'utilisant

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