WO2011052749A1 - 抗がん剤の感受性の判定方法 - Google Patents
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- WO2011052749A1 WO2011052749A1 PCT/JP2010/069363 JP2010069363W WO2011052749A1 WO 2011052749 A1 WO2011052749 A1 WO 2011052749A1 JP 2010069363 W JP2010069363 W JP 2010069363W WO 2011052749 A1 WO2011052749 A1 WO 2011052749A1
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- 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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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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
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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
- G01N33/57419—Specifically defined cancers of colon
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4727—Calcium binding proteins, e.g. calmodulin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
Definitions
- the present invention relates to an anticancer drug sensitivity determination marker used for determining whether or not a cancer of a target patient has therapeutic reactivity to an anticancer drug to be used, and its application.
- anticancer agents such as alkylating agents, platinum preparations, antimetabolites, anticancer antibiotics, and anticancer plant alkaloids. These anticancer agents may or may not be effective depending on the type of cancer. However, it is known that some types of cancer that are recognized as effective may or may not be effective depending on the individual patient. Whether or not an anticancer drug shows an effect on such individual patient's cancer is called anticancer drug sensitivity.
- Oxaliplatin (SP-4-2)-[(1R, 2R) -cyclohexane-1,2-diamine- ⁇ N, ⁇ N ′] [ethanedioato (2-)- ⁇ O 1 , ⁇ O 2 ] platinum (IUPAC) is the third It is a next generation platinum complex antineoplastic agent.
- the mechanism of action is thought to be DNA synthesis inhibition and protein synthesis inhibition by cross-linking with DNA bases, as with the preceding drugs cisplatin (CDDP) and carboplatin (CBDCA).
- CDDP cisplatin
- CBDCA carboplatin
- oxaliplatin (L-OHP) exhibits an antitumor effect and exhibits an antitumor spectrum different from that of conventional platinum complex antineoplastic agents.
- ERCC1 Exclusion repair cross-complementing group 1
- NER nucleotide excision repair
- SNPs single nucleotide polymorphisms
- Non-patent Documents 2 and 3 In base excision repair (BER), an X-ray repair cross that encodes a protein that is thought to be involved in efficient repair of DNA single-strand breaks formed by exposure to an alkylating agent or the like. -The relationship between the genetic polymorphism of the complementing group 1 (XRCC1) with the Arg399Gln amino acid mutation and the tumor shrinking effect was reported (Non-patent Document 4). It has been reported that there is no effect (Non-Patent Document 2).
- Non-Patent Document 5 DNA mismatch repair
- Glutathione-S-transferase is one of the enzymes responsible for the phase II reaction of detoxification and metabolism, and catalyzes the formation of a conjugate between a DNA platinum adduct and glutathione. Activate.
- GSTP1 has a high expression level in colorectal cancer and is associated with a genetic polymorphism accompanied by amino acid mutation of Ile105Val and survival time (median survival time: Ile / Ile 7.9 months, Ile / Val 13.3 months and Val / Val 24.9 months) (non-patent document 6).
- Non-patent Document 7 organic cation transporters (OCTs) are involved in the transport and sensitivity of oxaliplatin into cells
- OCTs organic cation transporters
- ATP7A A relationship between transporters involved in transport of copper and heavy metals such as ATP7B and sensitivity has also been reported (Non-Patent Documents 8 and 9).
- clinical studies have not been conducted on the relationship between these expressions and oxaliplatin treatment response.
- Non-Patent Documents 11 and 12 Non-Patent Documents 11 and 12
- oxaliplatin has a different therapeutic response depending on the type of cancer than cisplatin.
- the cellular response of cancer cells to platinum DNA adducts responsible for the cytotoxic activity of oxaliplatin has not been elucidated.
- a clear biomarker that can predict treatment responsiveness to chemotherapy with platin has not yet been established.
- Fluorouracil is a fluorinated pyrimidine anticancer drug developed in 1957 and is still a basic drug for digestive cancer chemotherapy. Fluorouracil taken up by cancer cells is an active metabolite in addition to the main mechanism of action of inhibition of thymidylate synthase (TS) caused by inhibition of thymidylate synthase (TS) by the active metabolite fluorodeoxyuridine-5'-monophosphate (FdUMP). It exerts a cell killing effect due to RNA dysfunction caused by a certain 5-fluoridine triphosphate (FUTP).
- TS thymidylate synthase
- FdUMP active metabolite fluorodeoxyuridine-5'-monophosphate
- DPD dihydropyrimidine dehydrogenase
- TS thymidylate synthase
- TS expression it has been reported that the amount of TS expression can be a therapeutic effect defining factor of a fluorinated pyrimidine anticancer agent regardless of measurement methods such as enzyme activity, protein and RNA level (Non-patent Documents 14 and 15).
- Non-patent Documents 14 and 15 the results are not necessarily consistent in all reports, and no clear biomarker that predicts treatment responsiveness to fluorouracil at an early stage of treatment is known.
- Non-patent Document 16 It has also been reported that in patients with fluorouracil-resistant metastatic colorectal cancer, the mRNA expression levels of intratumoral ERCC1 and TS are predictors of therapeutic response to 5-FU / L-OHP combination therapy (Non-patent Document 16). ). However, in a recent report from a large prospective clinical trial (FOCUS Trial) regarding treatment-responsive biomarkers for chemotherapy in patients with advanced colorectal cancer, ERCC1 is a significant effect of 5-FU / L-OHP combination therapy It was not a predictor, and only Topoisomerase-1 (Topo1) showed only a weak association (Non-patent Document 17).
- An object of the present invention is to provide an anticancer drug sensitivity determination marker capable of discriminating treatment responsiveness of individual patients and a new cancer treatment means using the same.
- the present inventors cultured human cancer cell lines and measured changes in intracellular protein over time after drug exposure using a surface enhanced laser desorption / ionization time-of-flight mass spectrometer (SELDI-TOF MS).
- SELDI-TOF MS surface enhanced laser desorption / ionization time-of-flight mass spectrometer
- the cancer of the cancer patient can be identified as 5-FU, L-OHP, or 5-FU / It is possible to determine whether or not there is sensitivity to a combination therapy of L-OHP, and if the expression variation of these substances is used as an index, screening of an anticancer drug sensitivity-enhancing agent becomes possible.
- the present inventors have found that the therapeutic effect of the anticancer agent can be drastically improved if the cancer agent sensitivity enhancing agent and the anticancer agent to be enhanced are used in combination, thereby completing the present invention.
- the present invention relates to a protein in which a main body or a fragment thereof is detected as a peak at m / z 5,300-5,400 (hereinafter referred to as protein A), m / z 6,130-6, Protein detected as a peak at 230 (hereinafter referred to as protein B), protein detected as a peak at m / z 7,000-7,080 (hereinafter referred to as protein C), m / z 7,840-7, Protein detected as a peak at 920 (hereinafter referred to as protein D), protein detected as a peak at m / z 8,920-9,000 (hereinafter referred to as protein E), m / z 12,440-12, A protein detected as a peak at 560 (hereinafter referred to as protein G), a peak at m / z 17,100 to 17,270 Protein detected as a peak (hereinafter referred to as protein H), protein detected as a peak at m / z 18,290-18,470 (hereinafter referred
- the present invention also provides a sensitivity determination marker for an anticancer agent comprising fluorouracil or a salt thereof, or a combination of fluorouracil or a salt thereof and oxaliplatin or a salt thereof, comprising calcium-binding protein S100A10 (hereinafter referred to as protein F). It is to provide.
- the present invention also provides a method for determining sensitivity to an anticancer agent, characterized by measuring the above-mentioned proteins A to N in a specimen.
- the present invention also provides a kit for carrying out a method for determining sensitivity to an anticancer agent, comprising a protocol for measuring the above-mentioned proteins A to N in a specimen.
- the present invention provides a method for screening an anticancer agent sensitivity-enhancing agent using the expression variation of the above proteins A to N as an index. Furthermore, this invention provides the anticancer agent sensitivity enhancer obtained by said screening method. Furthermore, this invention provides the composition for cancer treatment which combines said anticancer agent sensitivity enhancer and the anticancer agent used as the object of sensitivity enhancement. Furthermore, the present invention provides the above proteins A to N for determining sensitivity to anticancer agents.
- the anticancer drug sensitivity determination marker of the present invention it is possible to accurately determine the anticancer drug sensitivity of an individual patient before the start of treatment or early after the start of the treatment. Is possible. Furthermore, unnecessary side effects can be avoided because it is possible to avoid the use of anticancer agents that are not effective. In addition, because treatment schedules using anticancer drugs are long-term, the sensitivity of anticancer drugs to cancer over time can be determined by determining anticancer drug sensitivity for each treatment cycle even during treatment. Evaluation is possible and it is possible to determine whether or not to continue treatment.
- the measurement reagent of the anticancer drug sensitivity determination marker of the present invention is useful as an anticancer drug sensitivity determination reagent.
- the anticancer drug sensitivity determination marker in the present invention is protein A to N, and more specifically, using a cation exchange chip in a surface enhanced laser desorption / ionization time-of-flight mass spectrometer (SELDI-TOF MS).
- SELDI-TOF MS surface enhanced laser desorption / ionization time-of-flight mass spectrometer
- the main body or its fragment is detected as a peak at m / z 5,300-5,400 in the mass spectrometer (protein A), and the main body or its fragment is m / z 6 , Protein (protein B) detected as a peak at 130 to 6,230, body or fragment thereof is detected as a peak at m / z 7,000 to 7,080 (protein C), body or fragment thereof Detected as a peak at m / z 7,840-7,920 Protein (protein D), body or fragment thereof is detected as a peak at m / z 8,920-9,000 (protein E), body or fragment thereof is m / z 11,020-11,120 Protein (Protein F) detected as a peak, main body or fragment thereof is detected as a peak at m / z 12,440-12,560, Protein (protein G), main body or fragment thereof is detected as m / z 17,100- Protein (protein H), main body or fragment thereof detected as a peak at 17,270, m
- protein F is calcium-binding protein S100A10.
- S100A10 is known as a member of the S100 protein family with a calcium binding EF-hand motif. Furthermore, S100A10 is composed of its own dimer and annexin A2 (Annexin-2, Annexin II, Lipocortin II, Calpactin I heavy chain, Chromabindin-8, p36, Protein I, Prandial Anti-Pop. Since it is also known to form a tetramer, annexin A2 may be used as a determination marker for anticancer drug sensitivity, similar to S100A10.
- proteins A to C, F, and H to K showed intracellular protein expression in cultured cancer cells using SELDI-TOF MS. As a result, after exposure to 5-FU, Increased intracellular levels were observed with DLD-1, which is less sensitive to FU. On the other hand, no significant difference was observed in HCT116 which is highly sensitive to 5-FU. Therefore, proteins A to C, F, and H to K are useful as anticancer agent sensitivity determination markers, particularly 5-FU anticancer agent sensitivity determination markers.
- Proteins D and M as shown in Examples below, showed a decrease in intracellular levels in HCT116, which is highly sensitive to 5-FU after exposure to 5-FU. On the other hand, no significant difference was observed in DLD-1, which is less sensitive to 5-FU. Therefore, proteins D and M are useful as anticancer drug sensitivity determination markers, particularly as 5-FU anticancer drug sensitivity determination markers.
- proteins A to C and G to K showed an increase in intracellular levels in DLD-1, which is less sensitive to L-OHP after exposure to L-OHP.
- HCT116 which is highly sensitive to L-OHP. Therefore, proteins A to C and G to K are useful as anticancer agent sensitivity determination markers, particularly as L-OHP anticancer agent sensitivity determination markers.
- protein F calcium-binding protein S100A10
- Proteins D and M as shown in Examples below, showed a decrease in intracellular levels in HCT116, which is highly sensitive to L-OHP after exposure to L-OHP. On the other hand, no significant difference was observed in DLD-1, which is less sensitive to L-OHP. Therefore, proteins D and M are useful as anticancer drug sensitivity determination markers, particularly as L-OHP anticancer drug sensitivity determination markers.
- Proteins A to C, F, and H to K are DLD-1, which is less sensitive to 5-FU / L-OHP combination after exposure to 5-FU / L-OHP combination as shown in Examples below. Increased intracellular levels were observed. On the other hand, no significant difference was observed in HCT116, which is highly sensitive to the 5-FU / L-OHP combination. Therefore, proteins A to C, F, and H to K are useful as anticancer agent sensitivity determination markers, particularly as anticancer agent sensitivity determination markers when 5-FU / L-OHP is used in combination.
- protein F is useful as an anticancer drug sensitivity determination marker for L-OHP, but the protein F is an anticancer drug sensitivity marker when used in combination with 5-FU / L-OHP. This is the first knowledge obtained this time.
- proteins E and G showed increased intracellular levels in HCT116, which is highly sensitive to 5-FU / L-OHP, after exposure to 5-FU / L-OHP. .
- DLD-1 which is less sensitive to 5-FU / L-OHP. Therefore, proteins E and G are useful as anticancer agent sensitivity determination markers, particularly as anticancer agent sensitivity determination markers when 5-FU / L-OHP is used in combination.
- proteins D and M showed a decrease in intracellular level in HCT116, which is highly sensitive to 5-FU / L-OHP, after exposure to 5-FU / L-OHP. .
- no significant difference was observed in DLD-1, which is less sensitive to 5-FU / L-OHP. Therefore, proteins D and M are useful as anticancer agent sensitivity determination markers, particularly as anticancer agent sensitivity determination markers in combination with 5-FU / L-OHP.
- Proteins B, H, L, and N are pre-drug-exposed to HCT116, which is highly sensitive to any of 5-FU, L-OHP, and 5-FU / L-OHP combination, as shown in Examples below ( Intracellular levels (when not exposed) were significantly higher than DLD-1, which is less sensitive to any of the above drugs. Therefore, proteins B, H, L, and N are useful as anticancer drug sensitivity determination markers, particularly as anticancer drug sensitivity determination markers in combination with 5-FU, L-OHP, and 5-FU / L-OHP. .
- an anticancer agent used as the target of the anticancer agent sensitivity determination marker of this invention for example, oxaliplatin, cyclophosphamide (ifosfamide), thiotepa (thiotepa), melphalan ( melphalan, busulfan, nimustine, ranimustine, dacarbazine (da), procarbazine, templatin, cisplatin, cisplatin, cisplatin, cisplatin, cisplatin, cisplatin methotrexate , Pemetrexed, fluorouracil, tegafur / uracil (tegaful erac), doxyfluridine, tegafur / gimeracil / citabin, tegafur / citabin enocitabine, gemcitabine, 6-mercaptopurine, fludarabine, pentostatin, cladribine, hydroxyurea, hydroxyurea
- fluorinated pyrimidine-based anticancer agents and platinum complex-based anticancer agents are preferable, and fluorouracil, oxaliplatin, or salts thereof are particularly preferable. Furthermore, it can be suitably used in a combination agent of fluorouracil or a salt thereof and oxaliplatin or a salt thereof.
- the proteins A to N in the sample may be measured.
- a biological sample derived from a subject having cancer such as blood, serum, plasma, a cancer tissue biopsy specimen, a cancer excision surgical specimen, stool, urine, ascites, pleural effusion, Although cerebrospinal fluid, sputum, etc. are mentioned, serum is particularly preferable.
- lip, oral and pharyngeal cancer typified by pharyngeal cancer
- digestive organ cancer typified by esophageal cancer
- stomach cancer colon / rectal cancer
- lung cancer Respiratory and intrathoracic organ cancer, bone and joint cartilage cancer, cutaneous malignant melanoma, squamous cell carcinoma and other skin cancers, mesothelioma and mesothelioma Tissue cancer, breast cancer, uterine cancer, female genital cancer represented by ovarian cancer, male genital cancer represented by prostate cancer, urinary tract cancer represented by bladder cancer, brain tumor Representative eye, brain and central nervous system cancer, thyroid and other endocrine adenocarcinoma, non-Hodgkin lymphoma, lymphoid leukemia, lymphoid tissue, hematopoietic tissue and related tissue cancer, and these as the primary focus Cancer of metastatic tissue that does, especially gastric cancer and colorectal cancer
- the measuring means for the proteins A to N in the specimen can be measured by, for example, SELDI-TOF MS, immunological measurement method, or the like.
- an immunoassay using anti-protein A to N antibodies is preferable.
- the anti-protein A to N antibody used may be a monoclonal antibody or a polyclonal antibody. More specifically, for example, radioimmunoassay, enzyme immunoassay, fluorescent immunoassay, luminescence immunoassay, immunoprecipitation method, immunoturbidimetric method, Western blot, immunostaining, immunodiffusion method, etc. can be mentioned, preferably Western blot Alternatively, an enzyme immunoassay is particularly preferred, such as Western blotting and enzyme-linked immunosorbent assay (ELISA) (for example, sandwich ELISA).
- ELISA enzyme-linked immunosorbent assay
- the cancer is not anticancer drug sensitive and the If the protein concentration after administration does not change or decreases compared to, the cancer can be determined to be sensitive to anticancer agents. That is, if the concentrations of proteins A to C, F, and H to K have a concentration that is determined to be lower than a predetermined standard concentration at an early stage after administration, the cancer is compared to the anticancer drug of interest.
- the cancer In the early stage after administration, if the concentration of protein A to C, F, or H to K has a concentration that is determined to be higher than a predetermined standard concentration, the cancer is classified as a target anticancer agent. It can be determined that it is not sensitive. If there is no sensitivity to the targeted anticancer drug, its efficacy cannot be expected, and if the administration of an anticancer drug that cannot be expected to have such efficacy is continued, There is concern about progression and increased side effects.
- the anticancer drug sensitivity determination marker in the present invention is not only a determination of anticancer drug treatment responsiveness, but also an increase in cancer progression and side effects associated with continuous administration of an anticancer drug that cannot be expected to have a medicinal effect. It can also be used as a marker for avoiding.
- the target anticancer agent for proteins A to C and G to K is L-OHP
- the target anticancer agent for proteins A to C, F and H to K is 5-FU / L.
- the sensitivity to the anticancer agent can be determined in the same manner as described above.
- the target anticancer agent is 5-FU
- biological samples derived from cancer patients before and after the administration of the anticancer agent
- the cancer is sensitive to the anticancer drug, and the protein concentration after administration is lower than before administration of the anticancer drug. If the concentration does not change or rises, it can be determined that the cancer is not anticancer drug sensitive. That is, if the concentration of protein D or M has a concentration that is determined to be lower than a predetermined standard concentration at an early stage after administration, it is determined that the cancer is sensitive to the target anticancer agent.
- the cancer in the early stage after administration, when the concentration of protein D or M has a concentration that is determined to be higher than a predetermined standard concentration, the cancer is not sensitive to the target anticancer agent. Can be judged. If there is no sensitivity to the targeted anticancer drug, its efficacy cannot be expected, and if the administration of an anticancer drug that cannot be expected to have such efficacy is continued, There is concern about progression and increased side effects.
- the anticancer drug sensitivity determination marker in the present invention is not only a determination of anticancer drug treatment responsiveness, but also an increase in cancer progression and side effects associated with continuous administration of an anticancer drug that cannot be expected to have a medicinal effect. It can also be used as a marker for avoiding.
- the target anticancer agent is L-OHP
- the target anticancer agent is a combination of 5-FU / L-OHP
- Sensitivity to cancer drugs can be determined.
- the target anticancer agent is a combination of 5-FU / L-OHP
- the cancer is sensitive to the anticancer drug and If the protein concentration after administration does not change or decreases compared to, the cancer can be determined not to be sensitive to anticancer agents. That is, if the concentrations of protein E and G have a concentration that is determined to be higher than a predetermined standard concentration at an early stage after administration, it is determined that the cancer is sensitive to the target anticancer drug.
- the cancer in the early stage after administration, if the concentration of protein E or G has a concentration determined to be lower than the predetermined standard concentration, the cancer is not sensitive to the target anticancer agent. Can be judged. If there is no sensitivity to the targeted anticancer drug, its efficacy cannot be expected, and if the administration of an anticancer drug that cannot be expected to have such efficacy is continued, There is concern about progression and increased side effects.
- the anticancer drug sensitivity determination marker in the present invention is not only a determination of anticancer drug treatment responsiveness, but also an increase in cancer progression and side effects associated with continuous administration of an anticancer drug that cannot be expected to have a medicinal effect. It can also be used as a marker for avoiding.
- the target anticancer agent is a combination of 5-FU, L-OHP, and 5-FU / L-OHP, determine the sensitivity to the anticancer agent. Measures the concentration of these proteins in a biological sample derived from a cancer patient before administration of an anticancer drug, and if the concentration of these proteins is determined to be lower than a predetermined standard concentration, It can be determined that the cancer is not sensitive to the targeted anticancer drug. When there is no sensitivity to the target anticancer agent, it is not possible to expect its medicinal effect, and it is considered that only side effects due to the anticancer agent are expressed.
- the determination marker can also be used as a marker for avoiding the development of unnecessary side effects or the progression of cancer and the increase in side effects associated with continued invalid treatment.
- concentration of these proteins is determined to be higher than a predetermined standard concentration, it can be determined that the cancer is sensitive to the target anticancer agent, and thus a therapeutic effect is expected. It can also be used as a marker to actively select patients who can do it.
- proteins B and H as a marker during administration of an anticancer agent, when the concentration of the protein after administration does not change or decreases compared to before administration of the anticancer agent, the cancer is anti-cancerous. If it has a concentration that can be determined to be cancer drug sensitivity and is determined to be lower than the prescribed standard concentration as a marker before administration of the anticancer drug, the cancer is not sensitive to the anticancer drug. Can be judged. Since the positioning as a marker is different during the administration of the anticancer agent or before the administration of the anticancer agent, it is preferable to use it while paying attention to that point.
- kits including a protocol for measuring proteins A to N in a specimen.
- the kit includes a measurement reagent for proteins A to N, a method for using the measurement reagent, a standard for determining the presence or absence of anticancer drug sensitivity, and the like.
- the standards include standard concentrations of proteins A to N, concentrations judged to be high, concentrations judged to be low, factors affecting the measurement results, and the degree of their influence, etc. It is possible to set for each anticancer drug. Using the reference, it can be determined as described above.
- the expression fluctuation of these proteins, specifically, suppression of expression can be used as an index.
- Can screen for anti-sensitivity agents That is, a substance that suppresses protein expression in vitro or in vivo enhances anticancer drug sensitivity.
- a substance that lowers the protein concentration in the presence of an anticancer drug in various cancer cell lines is a substance that enhances the sensitivity of the anticancer drug (anticancer drug sensitivity enhancer). is there.
- a substance that enhances the decrease in protein concentration before and after administration of an anticancer drug in a cancer-bearing animal is a substance that enhances the sensitivity of the anticancer drug (anticancer drug sensitivity enhancer).
- anticancer drug sensitivity enhancer When the anticancer agent targeted for the proteins A to D, G to K, and M is L-OHP, the anticancer agent targeted for the proteins A to D, F, HK, and M is 5
- the anticancer agent sensitivity-enhancing agent can be screened in the same manner as described above.
- Sensitizers can be screened. That is, a substance that increases protein expression in vitro or in vivo enhances anticancer drug sensitivity.
- a substance that increases the protein concentration in the presence of an anticancer drug in various cancer cell lines is a substance that enhances the sensitivity of the anticancer drug (anticancer drug sensitivity enhancer).
- anticancer drug sensitivity enhancer a substance that enhances the increase in protein concentration before and after administration of an anticancer agent in a cancer-bearing animal is a substance that enhances the sensitivity of the anticancer agent (anticancer agent sensitivity enhancer).
- anti-cancer drug sensitivity-enhancing agents can be screened using increased expression as an index. That is, a substance that increases protein expression in vitro or in vivo enhances anticancer drug sensitivity.
- a substance that increases the protein concentration in the absence of an anticancer drug in various cancer cell lines is a substance that enhances the sensitivity of the anticancer drug (anticancer drug sensitivity enhancer). It is.
- a substance that enhances the increase in protein concentration before administration of an anticancer drug in a cancer-bearing animal is a substance that enhances the sensitivity of the anticancer drug (anticancer drug sensitivity-enhancing agent). .
- the combined form of the anticancer agent sensitivity-enhancing agent and the anticancer agent targeted for sensitivity enhancement may be a single composition containing both of these components. There may be. In addition, these components may be different administration routes.
- Anticancer agents to be used here are the same as described above, and are oxaliplatin, cyclophosphamide, ifosfamide, thiotepa, melphalan, busulfan.
- fluorinated pyrimidine-based anticancer agents and platinum complex-based anticancer agents are preferable, and fluorouracil, oxaliplatin, or salts thereof are particularly preferable.
- a combination agent of fluorouracil or a salt thereof and oxaliplatin or a salt thereof can be suitably used.
- Example 1 (1) Method (a) Cells used Two types of human colon cancer cell lines (HCT-116, DLD-1) were obtained from ECACC. The culture was performed under the conditions of ⁇ 100 mm / Tissue Culture Dish (IWAKI), medium (D-MEM, 2 mM Glutamine, 10% Fetal Bovine Serum), 37 ° C., 5% CO 2 .
- IWAKI Tissue Culture Dish
- D-MEM 2 mM Glutamine, 10% Fetal Bovine Serum
- Drug exposure conditions are as follows: Control (0 ⁇ M) as 5-FU single agent, 11 conditions of 0.001 ⁇ M, 0.01 ⁇ M, 0.1 ⁇ M, 1 ⁇ M, 3 ⁇ M, 10 ⁇ M, 30 ⁇ M, 100 ⁇ M, 1000 ⁇ M, and 10,000 ⁇ M, as L-OHP single agent 11 conditions of Control (0 ⁇ M), 0.001 ⁇ M, 0.01 ⁇ M, 0.1 ⁇ M, 0.3 ⁇ M, 1 ⁇ M, 3 ⁇ M, 10 ⁇ M, 30 ⁇ M, 100 ⁇ M, and 1000 ⁇ M were used.
- the survival rate at the time of combination was compared with the survival rate at the time of single agent exposure of each drug at the same concentration as used at the time of combination. If it decreased, it was determined that there was a combined effect.
- (D) Drug exposure experiment Based on the results of (c) above, the drug concentration used in the exposure experiment is the same as that used in the combination exposure of 5-FU + L-OHP: 2 ⁇ M + 1 ⁇ M, 2 ⁇ M + 10 ⁇ M, 100 ⁇ M + 1 ⁇ M, 100 ⁇ M + 10 ⁇ M.
- the exposure experiments were conducted under the exposure of each concentration of each drug alone under the conditions of 5-FU of 2 ⁇ M and 100 ⁇ M, L-OHP of 1 ⁇ M and 10 ⁇ M, and a drug-free control added thereto.
- the drug exposure time was set to 5 types, immediately before exposure (0 hour), 4 hours, 12 hours, 24 hours, and 48 hours, and the number of cells at the end of the exposure was counted to extract intracellular proteins.
- HCT116 For 5-FU / L-OHP combination, HCT116 combined after exposure to 5-FU 2 ⁇ M + L-OHP 1 ⁇ M and after exposure to 5-FU 100 ⁇ M + L-OHP 10 ⁇ M for 48 hours rather than each single agent exposure In some cases, the cell viability was significantly low, and a combined effect was observed, but no significant combined effect was observed in DLD1. Therefore, it was confirmed that HCT116 is a highly sensitive cell for 5-FU / L-OHP combination, and DLD-1 is a low sensitive cell. (FIG. 1).
- HCT116 showed a peak in intracellular level (FIGS. 5 (A) and 14) M / z 7,840-7,920 (protein D) M / z 9,100-9,200 (protein M)
- Example 2 For all peaks found in Example 1, Insulin oxidized B chain (bovine) (m / z 3396.94 + 1H), Insulin (bovine) (m / z 5733.51 + 1H), Cytochrome, as standards with known molecular weights. c (equine) (m / z 123600.96 + 1H), Apomyoglobin (equine) (m / z 16952.27 + 1H), Aldorase (reabbit muscle) (m / z 39212.28 + 1H), and two types sandwiching each target peak The m / z was confirmed by molecular weight correction (internal calibration) using two molecular weights of the standard substance.
- Example 1 the peaks found in Example 1 were m / z 5,300-5,400 (protein A), m / z 6,130-6,230 (protein B), m / z 7, respectively. , 000-7,080 (protein C), m / z 7,840-7,920 (protein D), m / z 8,920-9,000 (protein E), m / z 11,020-11, 120 (protein F), m / z 12,440-12,560 (protein G), m / z 17,100-17,270 (protein H), m / z 18,290-18,470 (protein I) , M / z 24,660-24,750 (protein J), m / z 35,980-36,290 (protein K), m / z 8,650-8,750 (protein L), m / z 9 , 100-9, 20 It was confirmed that it was detected between 0 (protein M) and m / z 11,760-11,890 (protein N).
- Example 3 With respect to all the peaks found in Example 1, changes in peak intensity accompanying changes in pH were examined in order to investigate the properties in more detail.
- the pH at which the decrease in peak intensity is recognized is near the isoelectric point (pI) where the ionization of the protein is lost.
- the isoelectric point (pI) of the peak detected in Example 1 is -For m / z 7,000-7,080 (Protein C), pH 3.5-6.5, PH 4.0-7.0 for m / z 8,920-9,000 (protein E) PH 7.0-8.0 for m / z 11,020-11,120 (protein F) -PH 4.5-7.5 for m / z 12,440-12,560 (Protein G) M / z 17,100-17,270 (protein H), pH 5.0-8.0, M / z 18,290-18,470 (Protein I) pH 6.5-9.5, -PH 3.5-6.5 for m / z 24,660-24,750 (protein J), PH 3.0-6.5 for m / z 35, 980-36,
- Example 4 Database search for the peaks found in Example 1 using The ExPASy proteomics server TagIdent tool (http://au.expasy.org/tools/tagident.html) based on the information of Examples 2 and 3. Went. The results were as follows.
- Protein S100-A10 protein F
- protein F calcium binding protein S100A10
- WO2009 / 96196 international publication pamphlet This example can be used not only to predict L-OHP sensitivity before treatment, but also as a marker for determining treatment responsiveness early after the start of treatment.
- 5-FU alone or a combination of 5-FU and L-OHP It was confirmed that the marker can be used sometimes.
- Example 5 As a result of the database search in Example 4, only one kind of protein (Protein S100-A10) was hit at m / z 11,020 to 11,120 (protein F), so expression in HCT116 and DLD-1 was confirmed. did. 1) Method HCT116 and DLD-1 were subjected to SDS-PAGE at a constant voltage of 100 V after extracting intracellular protein by the same method as in Example 1. After electrophoresis, the protein was blotted on a PVDF membrane, blocked, and then anti-S100A10 monoclonal antibody (Purified Mouse Anti-Annexin II light chain monoclonal antibody, BD Transduction Laboratories) was used.
- Anti-S100A10 monoclonal antibody Purified Mouse Anti-Annexin II light chain monoclonal antibody, BD Transduction Laboratories
- CDP-Star TM chemiluminescent substrate was added as a reaction substrate to emit light, and detection was performed with a lumino image analyzer (LAS-4000mini, FUJIFILM).
- Example 2 Results In Example 1, the expression of S100A10 in HCT116 of m / z 11,020-11,120 (protein F) and DLD-1 was confirmed by Western blotting using an anti-S100A10 monoclonal antibody. (FIG. 13).
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Abstract
Description
1)オキサリプラチンによりもたらされる損傷DNAの除去・修復能の亢進
2)細胞内におけるオキサリプラチン(活性化体)の不活化(解毒)
3)オキサリプラチンの細胞内蓄積量の低下
などが関与していると考えられている。大腸癌患者を対象にしたオキサリプラチン+5-FU併用療法における治療反応性や予後予測因子として1)~3)と関連する研究が行われている。
同様に、L-OHP曝露後に、高感受性細胞と低感受性細胞において、細胞内発現レベルの異なる経時的変化が認められるピークを見出し、そのピークが、質量分析計において、本体もしくはそのフラグメントがm/z 5,300~5,400のピークとして検出されるタンパク質、m/z 6,130~6,230のピークとして検出されるタンパク質、m/z 7,000~7,080のピークとして検出されるタンパク質、m/z 7,840~7,920のピークとして検出されるタンパク質、m/z 12,440~12,560のピークとして検出されるタンパク質、m/z 17,100~17,270のピークとして検出されるタンパク質、m/z 18,290~18,470のピークとして検出されるタンパク質、m/z 24,660~24,750のピークとして検出されるタンパク質、m/z 35,980~36,290のピークとして検出されるタンパク質、m/z 9,100~9,200のピークとして検出されるタンパク質であることを見出した。
さらに、5-FU/L-OHP併用曝露後に、高感受性細胞と低感受性細胞において、細胞内発現レベルの異なる経時的変化が認められるピークを見出し、そのピークが、質量分析計において、本体もしくはそのフラグメントがm/z 5,300~5,400のピークとして検出されるタンパク質、m/z 6,130~6,230のピークとして検出されるタンパク質、m/z 7,000~7,080のピークとして検出されるタンパク質、m/z 7,840~7,920のピークとして検出されるタンパク質、m/z 8,920~9,000のピークとして検出されるタンパク質、m/z 12,440~12,560のピークとして検出されるタンパク質、m/z 17,100~17,270のピークとして検出されるタンパク質、m/z 18,290~18,470のピークとして検出されるタンパク質、m/z 24,660~24,750のピークとして検出されるタンパク質、m/z 35,980~36,290のピークとして検出されるタンパク質、m/z 9,100~9,200のピークとして検出されるタンパク質、カルシウム結合タンパク質S100A10であることを見出した。
また、高感受性細胞と低感受性細胞において、薬剤曝露前の細胞内発現レベルが異なるピークを見出し、そのピークが、質量分析計において、本体もしくはそのフラグメントがm/z 6,130~6,230のピークとして検出されるタンパク質、m/z 17,100~17,270のピークとして検出されるタンパク質、m/z 8,650~8,750のピークとして検出されるタンパク質、m/z 11,760~11,890のピークとして検出されるタンパク質であることを見出した。
かかる知見に基づき、さらに検討した結果、がん患者由来の生体試料中の当該タンパク質の濃度を測定すれば、当該がん患者のがんが、5-FU、L-OHP、或いは5-FU/L-OHPの併用療法に対して感受性を有するか否かを判定できること、また、これらの物質の発現変動を指標とすれば抗がん剤感受性亢進剤のスクリーニングが可能になること、さらに当該抗がん剤感受性亢進剤と感受性亢進の対象となる抗がん剤を併用すれば、当該抗がん剤の治療効果が飛躍的に向上することを見出し、本発明を完成した。
また、本発明は、カルシウム結合タンパク質S100A10(以下、タンパク質Fという)からなる、フルオロウラシル又はその塩、又はフルオロウラシル又はその塩とオキサリプラチン又はその塩との組み合わせからなる抗がん剤の感受性判定マーカーを提供するものである。
また、本発明は、検体中の上記のタンパク質A~Nを測定することを特徴とする抗がん剤感受性の判定方法を提供するものである。
また、本発明は、検体中の上記のタンパク質A~Nを測定するためのプロトコールを含むことを特徴とする抗がん剤感受性の判定方法を実施するためのキットを提供するものである。
さらに本発明は、上記のタンパク質A~Nの発現変動を指標とする抗がん剤感受性亢進剤のスクリーニング方法を提供するものである。
さらにまた本発明は、上記のスクリーニング方法により得られた抗がん剤感受性亢進剤を提供するものである。
さらに本発明は、上記の抗がん剤感受性亢進剤と、感受性亢進の対象となる抗がん剤とを組み合せてなるがん治療用組成物を提供するものである。
さらに本発明は、抗がん剤感受性を判定するための上記タンパク質A~Nを提供するものである。
さらに、また、このマーカーを用いれば、抗がん剤感受性を亢進させる薬剤がスクリーニングでき、その対象となった抗がん剤と抗がん剤感受性亢進剤とを併用すれば、がん治療効果が飛躍的に向上する。本発明の抗がん剤感受性判定マーカーの測定試薬は、抗がん剤感受性判定試薬として有用である。
なお、タンパク質F(カルシウム結合タンパク質S100A10)がL-OHPの抗がん剤感受性判定マーカーとして利用できることは、本発明者らによる先行出願(WO2009/96196号国際公開パンフレット)で既に明らかになっており、今回の実施例でも同様の結果が得られた。
また、投与後早期の段階において、タンパク質A~C、F、H~Kの濃度が所定の標準濃度より高いと判断される濃度を有する場合は、そのがんは対象とする抗がん剤に対して感受性ではないと判定できる。対象とする抗がん剤に対して感受性を有さない場合は、その薬効を期待することができず、このような薬効の期待できない抗がん剤の投与が続けられた場合、がんの進行、副作用の増大が危惧される。このように、本発明における抗がん剤感受性判定マーカーは、抗がん剤治療反応性の判定のみならず、薬効の期待できない抗がん剤の継続投与に伴うがんの進行や副作用の増大を回避するためのマーカーとしても使用できる。
タンパク質A~C、G~Kについて対象とする抗がん剤がL-OHPである場合、また、タンパク質A~C、F、H~Kについて対象とする抗がん剤が5-FU/L-OHPの併用である場合も上記と同様に抗がん剤に対する感受性を判定することができる。
また、投与後早期の段階において、タンパク質D、Mの濃度が所定の標準濃度より高いと判断される濃度を有する場合は、そのがんは対象とする抗がん剤に対して感受性ではないと判定できる。対象とする抗がん剤に対して感受性を有さない場合は、その薬効を期待することができず、このような薬効の期待できない抗がん剤の投与が続けられた場合、がんの進行、副作用の増大が危惧される。このように、本発明における抗がん剤感受性判定マーカーは、抗がん剤治療反応性の判定のみならず、薬効の期待できない抗がん剤の継続投与に伴うがんの進行や副作用の増大を回避するためのマーカーとしても使用できる。
タンパク質D、Mについて、対象とする抗がん剤がL-OHPである場合、また、対象とする抗がん剤が5-FU/L-OHPの併用である場合も上記と同様に抗がん剤に対する感受性を判定することができる。
また、投与後早期の段階において、タンパク質E、Gの濃度が所定の標準濃度より低いと判断される濃度を有する場合は、そのがんは対象とする抗がん剤に対して感受性ではないと判定できる。対象とする抗がん剤に対して感受性を有さない場合は、その薬効を期待することができず、このような薬効の期待できない抗がん剤の投与が続けられた場合、がんの進行、副作用の増大が危惧される。このように、本発明における抗がん剤感受性判定マーカーは、抗がん剤治療反応性の判定のみならず、薬効の期待できない抗がん剤の継続投与に伴うがんの進行や副作用の増大を回避するためのマーカーとしても使用できる。
また、これらのタンパク質の濃度が所定の標準濃度より高いと判断される濃度を有する場合は、そのがんは対象とする抗がん剤に対して感受性を有すると判定できるため、治療効果を期待できる患者を積極的に選出するためのマーカーとしても使用できる。
ここで用いられる対象となる抗がん剤としては、前記と同様であり、オキサリプラチン、シクロフォスファミド(cyclophosphamide)、イフォスファミド(ifosfamide)、チオテパ(thiotepa)、メルファラン(melphalan)、ブスルファン(busulfan)、ニムスチン(nimustine)、ラニムスチン(ranimustine)、ダカルバジン(dacarbazine)、プロカルバジン(procarbazine)、テモゾロミド(temozolomide)、シスプラチン(cisplatin)、カルボプラチン(carboplatin)、ネダプラチン(nedaplatin)、メトトレキサート(methotrexate)、ペメトレキセド(pemetrexed)、フルオロウラシル(fluorouracil)、テガフール/ウラシル(tegaful・uracil)、ドキシフルリジン(doxifluridine)、テガフール/ギメラシル/オテラシル(tegaful・gimeracil・oteracil)、カペシタビン(capecitabine)、シタラビン(cytarabine)、エノシタビン(enocitabine)、ゲムシタビン(gemcitabine)、6-メルカプトプリン(6-mercaptopurine)、フルダラビン(fuludarabin)、ペントスタチン(pentostatin)、クラドリビン(cladribine)、ヒドロキシウレア(hydroxyurea)、ドキソルビシン(doxorubicin)、エピルビシン(epirubicin)、ダウノルビシン(daunorubicin)、イダルビシン(idarubicine)、ピラルビシン(pirarubicin)、ミトキサントロン(mitoxantrone)、アムルビシン(amurubicin)、アクチノマイシンD(actinomycin D)、ブレオマイシン(bleomycine)、ペプレオマイシン(pepleomycin)、マイトマイシンC(mytomycin C)、アクラルビシン(aclarubicin)、ジノスタチン(zinostatin)、ビンクリスチン(vincristine)、ビンデシン(vindesine)、ビンブラスチン(vinblastine)、ビノレルビン(vinorelbine)、パクリタキセル(paclitaxel)、ドセタキセル(docetaxel)、イリノテカン(irinotecan)、イリノテカン活性代謝物(SN-38)、ノギテカン(nogitecan、topotecan)、エトポシド(etoposide)、プレドニゾロン(prednisolone)、デキサメタゾン(dexamethasone)、タモキシフェン(tamoxifen)、トレミフェン(toremifene)、メドロキシプロゲステロン(medroxyprogesterone)、アナストロゾール(anastrozole)、エキセメスタン(exemestane)、レトロゾール(letrozole)、リツキシマブ(rituximab)、イマチニブ(imatinib)、ゲフィチニブ(gefitinib)、ゲムツズマブ・オゾガマイシン(gemtuzumab ozogamicin)、ボルテゾミブ(bortezomib)、エルロチニブ(erlotinib)、セツキシマブ(cetuximab)、ベバシズマブ(bevacizumab)、スニチニブ(sunitinib)、ソラフェニブ(sorafenib)、ダサチニブ(dasatinib)、パニツムマブ(panitumumab)、アスパラギナーゼ(asparaginase)、トレチノイン(tretinoin)、三酸化ヒ素(arsenic trioxide)、又はそれらの塩、又はそれらの活性代謝物等が挙げられる。このうちフッ化ピリミジン系抗がん剤、白金錯体系抗がん剤が好ましく、特にフルオロウラシル、オキサリプラチン又はそれらの塩が好ましい。さらに、フルオロウラシル又はその塩と、オキサリプラチン又はその塩との併用剤を好適に利用することができる。
(1)方法
(a)使用細胞
ヒト大腸癌細胞株2種類(HCT-116、DLD-1)は、ECACCより入手した。培養は、φ100mm/Tissue Culture Dish(IWAKI)、培地(D-MEM、2mM Glutamine、10% Fetal Bovine Serum)、37℃、5%CO2の条件下にて行なった。
フルオロウラシル(5-FU)はSigma社より購入し、オキサリプラチン(L-OHP)原末は、株式会社ヤクルト本社より入手した。
2種類の大腸癌細胞株HCT116、DLD-1を用い、薬剤曝露24時間後と48時間後の細胞生存率をMTS assay(CellTiter96TMAQueous One Solution Cell Proliferation Assay、Promega)にて評価した。薬剤曝露条件は5-FU単剤としてControl(0μM)、0.001μM、0.01μM、0.1μM、1μM、3μM、10μM、30μM、100μM、1000μM、10000μMの11条件、L-OHP単剤としてControl(0μM)、0.001μM、0.01μM、0.1μM、0.3μM、1μM、3μM、10μM、30μM、100μM、1000μMの11条件を用いた。併用としては5-FUの2μM、10μM、30μM、100μMの4濃度それぞれに、L-OHP の0.01μM、0.1μM、0.3μM、1μM、3μM、10μM、30μM、100μM、1000μMの9濃度を加えた36条件、及び各併用条件と同濃度の5-FU単剤として4条件とControl(0μM)を用いた。感受性の評価は、1回の試験においてそれぞれの細胞株、薬剤曝露時間、薬剤曝露条件について3サンプルずつ測定し、異なる継代数の細胞にて3回実施した。
解析は、MTS assayの結果から算出した生存率をもとに行った。併用効果の有無の判定は、併用時における生存率(viability)と、併用時に用いたのと同濃度での各薬剤の単剤曝露時における生存率とを比較し、双方から有意に生存率が低下していれば併用効果があると判定した。
前記(c)の結果にもとづき、曝露実験に用いる薬剤濃度は、5-FU+L-OHPの併用として2μM+1μM、2μM+10μM、100μM+1μM、100μM+10μMの4種類、これら併用曝露で用いるのと同じ濃度の各薬剤単独曝露として5-FUは2μM、100μM、L-OHPは1μM、10μM、及びこれらに薬剤フリーのControlを加えた全9条件で曝露実験を行った。薬剤曝露時間は 曝露直前(0時間)、4時間、12時間、24時間、48時間の5通りとし、曝露終了時点における細胞数を計測し、細胞内タンパク質を抽出した。
dishより培地を除き氷冷PBSで3回洗浄後、ラバーポリスマンにて剥がしてかき集め、細胞懸濁液を1.5mLマイクロチューブに移した。4℃にて1,200×g、10分間遠心して細胞を集め、上清を除いた後に、細胞数1000万個あたり200μLの細胞溶解バッファー(9mol/L Urea、2% CHAPS、1mM DTT、プロテアーゼインヒビターカクテル(Sigma))を加え、氷冷下で超音波破砕処理を行なった後、4℃にて16,000×g、20分間遠心し、上清を液体窒素にて急速凍結後、分析するまで-80℃にて保存した。上清の一部を用いてタンパク質定量(DC Protein Assay Kit、Bio-Rad)を行なった。
細胞溶解バッファー(プロテアーゼインヒビターを除く)にて2.5mg/mLに調製し、さらに、pH4.5の希釈/洗浄バッファー(50mM sodium acetate buffer)(以下、バッファー)にて0.5mg/mLに調製したサンプル100μLを、同バッファーにて前処理した陽イオン交換チップアレイ(CM10、Bio-Rad)のスポットにapplyし、1時間インキュベートして反応させた後、バッファーにて3回洗浄、milliQ水にて2回リンスした。風乾後、energy absorbing molecule(EAM:50% ACN/0.5% TFA溶液によるsinapinic acidの飽和溶液)1.0μLを、0.5μLずつ2回に分けて各スポットにapplyし、スポット表面が乾いた後、プロテインチップアレイの分析を行なった。
タンパク質発現解析は、表面エンハンス型レーザー脱離イオン化飛行時間型質量分析計(surface-enhanced laser desorption/ionization time-of-flight mass spectrometry:SELDI-TOF MS)にて行なった。分析機器としては、ProteinChipTM Reader(Model PCS4000 Personal Edition、Bio-Rad)を用い、Mass Range 0~70,000 Daltons、Focus Mass 8,000 Dalton、Energy 3000もしくは4000 nJ、1サンプルあたり合計265shots の条件にて分析を行なった。signal-to-noise ratio(S/N比)2以上のピークの抽出とタンパク質発現比較解析は、CiphergenExpressTM Data Manager 3.0を用いて行なった。
SELDI-TOF MSによる分析の結果、S/N比2以上の条件で各サンプルにつき88~143個のタンパク質ピークを抽出した。まず、CiphergenExpressTM Data Manager 3.0によりピーククラスターを作成した。次に、各条件において、薬剤曝露後に経時的に有意にピーク強度が変化しているピークと、各曝露時間(4、12、24、48時間)において、薬剤曝露条件によってピーク強度が有意に変化しているピークをピックアップした。さらに、両者に共通して検出されるピーク、すなわち曝露時間による発現変化と薬剤条件による発現変化のいずれをも認めるピークを絞り込んだ。
(a)HCT-116及びDLD-1における薬剤感受性の評価
100μMの5-FUにて48時間曝露後の細胞生存率は、HCT116で約24%、DLD-1で約49%となり、DLD-1がHCT116に比べ5-FU低感受性であることが確認された。1μMのL-OHPにて48時間曝露後の細胞生存率は、HCT116で約37%、DLD-1で約82%となり、DLD-1がHCT116に比べL-OHP低感受性であることが確認された。5-FU/L-OHP併用については、5-FU 2μM+L-OHP 1μMの24時間曝露後、及び5-FU 100μM+L-OHP 10μMの48時間曝露後において、HCT116では各々の単剤曝露時よりも併用時において有意に低い細胞生存率を示し併用効果が認められたが、DLD1では有意な併用効果は認められなかった。したがって、HCT116は5-FU/L-OHP併用に対する高感受性細胞、DLD-1は低感受性細胞であることが確認された。(図1)。
SELDI-TOF MSを用いたプロテオーム解析手法により、5-FU曝露、あるいはL-OHP曝露、あるいは5-FU/L-OHP併用曝露に伴う細胞内タンパク質の変動を網羅的に解析した。(1)方法で示した手法により解析した結果、各薬剤曝露後に特徴的な変動を示す以下のタンパク質を見出した。
5-FU曝露後の細胞内タンパク質の経時的変化について解析した結果、
(1)5-FU曝露後の細胞内レベルが、DLD-1でのみ上昇、あるいはHCT116と比較してDLD-1でより大きな上昇が認められたピーク(図2、図3、図4(ア)、図7(ア)、図9、図10、図11(ア)、図12)
・m/z 5,300~5,400(タンパク質A)
・m/z 6,130~6,230(タンパク質B)
・m/z 7,000~7,080(タンパク質C)
・m/z 11,020~11,120(タンパク質F)
・m/z 17,100~17,270(タンパク質H)
・m/z 18,290~18,470(タンパク質I)
・m/z 24,660~24,750(タンパク質J)
・m/z 35,980~36,290(タンパク質K)
(2)5-FU曝露後、HCT116で細胞内レベルの低下が認められたピーク(図5(ウ)、図14)
・m/z 7,840~7,920(タンパク質D)
・m/z 9,100~9,200(タンパク質M)
L-OHP曝露後の細胞内タンパク質の経時的変化について解析した結果、
(1)L-OHP曝露後の細胞内レベルが、DLD-1でのみ上昇、あるいはHCT116と比較してDLD-1でより大きな上昇が認められたピーク(図2、図3、図4(イ)、図7(イ)、図8(ア)、図9、図10、図11(ウ)、図12)
・m/z 5,300~5,400(タンパク質A)
・m/z 6,130~6,230(タンパク質B)
・m/z 7,000~7,080(タンパク質C)
・m/z 11,020~11,120(タンパク質F)
・m/z 12,440~12,560(タンパク質G)
・m/z 17,100~17,270(タンパク質H)
・m/z 18,290~18,470(タンパク質I)
・m/z 24,660~24,750(タンパク質J)
・m/z 35,980~36,290(タンパク質K)
(2)L-OHP曝露後、HCT116で細胞内レベルの低下を認めたピーク(図5(ア)、図14)
・m/z 7,840~7,920(タンパク質D)
・m/z 9,100~9,200(タンパク質M)
5-FU/L-OHP併用曝露後の細胞内タンパク質の経時的変化について解析した結果、
(1)5-FU/L-OHP曝露後の細胞内レベルが、DLD-1でのみ上昇、あるいはHCT116と比較してDLD-1でより大きな上昇が認められたピーク(図2、図3、図4(ウ)、図7(ウ)、図9、図10、図11(イ)、図12)
・m/z 5,300~5,400(タンパク質A)
・m/z 6,130~6,230(タンパク質B)
・m/z 7,000~7,080(タンパク質C)
・m/z 11,020~11,120(タンパク質F)
・m/z 17,100~17,270(タンパク質H)
・m/z 18,290~18,470(タンパク質I)
・m/z 24,660~24,750(タンパク質J)
・m/z 35,980~36,290(タンパク質K)
(2)5-FU/L-OHP併用曝露後、HCT116で細胞内レベルの上昇が認められたピーク(図6、図8(イ))
・m/z 8,920~9,000(タンパク質E)
・m/z 12,440~12,560(タンパク質G)
(3)5-FU/L-OHP併用曝露後、HCT116で細胞内レベルの低下が認められたピーク(図5(イ)、図14)
・m/z 7,840~7,920(タンパク質D)
・m/z 9,100~9,200(タンパク質M)
薬剤曝露前(非曝露時)の細胞内レベルが、DLD-1と比較してHCT116で有意に高値を示したピーク
・m/z 6,130~6,230(タンパク質B)
・m/z 8,650~8,750(タンパク質L)
・m/z 11,760~11,890(タンパク質N)
・m/z 17,100~17,270(タンパク質H)
タンパク質Bにおける薬剤曝露前の細胞内レベルは、SELDI-TOF MS分析によるピーク強度(μA)(平均値±S.D.、n=27)として、HCT116では55.4±9.2、DLD-1では28.9±6.4、同様にタンパク質Lでは、HCT116
88.2±2.2、DLD-1 32.1±1.3、タンパク質Nでは、HCT116
85.1±9.3、DLD-1 50.6±6.0、タンパク質Hでは、HCT116
15.5±2.1、DLD-1 3.86±1.34であり、いずれもHCT116における薬剤曝露前(非曝露時)細胞内レベルはDLD-1よりも有意に高値を示した。
実施例1において見出されたすべてのピークに関して、分子量既知の標準物質として、Insulin oxidized B chain(bovine)(m/z 3496.94+1H)、Insulin(bovine)(m/z 5733.51+1H)、Cytochrome c (equine)(m/z 12360.96+1H)、Apomyoglobin(equine)(m/z 16952.27+1H)、Aldorase(reabbit muscle)(m/z 39212.28+1H)を用い、各目的ピークを挟む2種類の標準物質の分子量2点を用いた分子量補正(internal calibration)によりm/zの確認を行った。その結果、実施例1において見出されたピークは、それぞれ、m/z 5,300~5,400(タンパク質A)、m/z 6,130~6,230(タンパク質B)、m/z 7,000~7,080(タンパク質C)、m/z 7,840~7,920(タンパク質D)、m/z 8,920~9,000(タンパク質E)、m/z 11,020~11,120(タンパク質F)、m/z 12,440~12,560(タンパク質G)、m/z 17,100~17,270(タンパク質H)、m/z 18,290~18,470(タンパク質I)、m/z 24,660~24,750(タンパク質J)、m/z 35,980~36,290(タンパク質K)、m/z 8,650~8,750(タンパク質L)、m/z 9,100~9,200(タンパク質M)、m/z 11,760~11,890(タンパク質N)の間に検出されることを確認した。
実施例1において見出されたすべてのピークに関して、その性質をさらに詳細に調べるために、pHの変化に伴うピーク強度の変化について検討を行った。
(a)検討に用いたプロテインチップアレイとバッファー条件
陽イオン交換チップアレイ(CM10、Bio-Rad)に対して、pH3.0(50mM Glycine-HCl buffer)、pH3.5(50mM sodium acetate buffer)、pH4.0(50mM sodium acetate buffer)、pH4.5(50mM sodium acetate buffer)、pH5.0(50mM sodium acetate buffer)、pH5.5(50mM sodium acetate buffer)、pH6.0(50mM phosphate buffer)、pH6.5(50mM phosphate buffer)、pH7.0(50mM phosphate buffer)、pH7.5(50mM phosphate buffer)、pH8.0(50mM Tris-HCl buffer)、pH8.5(50mM Tris-HCl buffer)、pH9.0(50mM Glycine-NaOH buffer)、pH9.5(50mM Glycine-NaOH buffer)、pH10.0(50mM Glycine-NaOH buffer)の15種類のバッファーを用いた。
CM10チップアレイを用いた分析のためのサンプル調製及びプロテインチップの作製は、(a)の各バッファーを用いて実施例1の(1)方法前記(f)に準じて実施した。
CM10チップアレイを用いた分析において、ピーク強度の低下を認めるpHがそのタンパク質のイオン化が失われる等電点(pI)付近と判断できる。その結果、実施例1で検出したピークの等電点(pI)は、
・m/z 7,000~7,080(タンパク質C)ではpH3.5~6.5、
・m/z 8,920~9,000(タンパク質E)ではpH4.0~7.0、
・m/z 11,020~11,120(タンパク質F)ではpH7.0~8.0、
・m/z 12,440~12,560(タンパク質G)ではpH4.5~7.5
・m/z 17,100~17,270(タンパク質H)ではpH5.0~8.0、
・m/z 18,290~18,470(タンパク質I)ではpH6.5~9.5、
・m/z 24,660~24,750(タンパク質J)ではpH3.5~6.5、
・m/z 35,980~36,290(タンパク質K)ではpH3.0~6.5
・m/z 8,650~8,750(タンパク質L)ではpH4.5~7.0
・m/z 9,100~9,200(タンパク質M)ではpH4.5~7.5
・m/z 11,760~11,890(タンパク質N)ではpH4.5~7.5
がpI付近に相当すると推定された。m/z 5,300~5,400(タンパク質A)、m/z 6,130~6,230(タンパク質B)、m/z 7,840~7,920(タンパク質D)では、ピーク強度が低下するpHが明確ではなかった。
実施例1で見出されたピークについて、実施例2及び3の情報にもとづき、The ExPASy proteomics serverのTagIdent tool(http://au.expasy.org/tools/tagident.html)を用いてデータベース検索を行った。その結果は以下のとおりであった。
実施例4のデータベース検索の結果、m/z 11,020~11,120(タンパク質F)では1種類のタンパク質(Protein S100-A10)のみがヒットしたことから、HCT116及びDLD-1における発現を確認した。
1)方法
HCT116及びDLD-1について、実施例1と同様の方法により細胞内タンパク質を抽出後、100V定電圧にてSDS-PAGEを行なった。泳動後、PVDF膜にタンパク質をブロットし、ブロッキングを行なった後、抗S100A10モノクローナル抗体(Purified Mouse Anti-Annexin II light chain monoclonal antibody、BD Transduction Laboratories)を用いて一次抗体反応を行なった。アルカリホスファターゼ標識抗マウスIgG抗体と二次抗体反応をさせた後、反応基質としてCDP-StarTM chemiluminescent substrateを添加して発光させ、ルミノ・イメージアナライザー(LAS-4000mini、FUJIFILM)により検出した。
実施例1において、m/z 11,020~11,120(タンパク質F)のHCT116、及びDLD-1におけるS100A10の発現は、抗S100A10モノクローナル抗体を用いたウエスタンブロット法により確認された(図13)。
Claims (35)
- 質量分析計において、本体もしくはそのフラグメントがm/z 5,300~5,400のピークとして検出されるタンパク質、m/z 6,130~6,230のピークとして検出されるタンパク質、m/z 7,000~7,080のピークとして検出されるタンパク質、m/z 7,840~7,920のピークとして検出されるタンパク質、m/z 8,920~9,000のピークとして検出されるタンパク質、m/z 12,440~12,560のピークとして検出されるタンパク質、m/z 17,100~17,270のピークとして検出されるタンパク質、m/z 18,290~18,470のピークとして検出されるタンパク質、m/z 24,660~24,750のピークとして検出されるタンパク質、m/z 35,980~36,290のピークとして検出されるタンパク質、m/z 8,650~8,750のピークとして検出されるタンパク質、m/z 9,100~9,200のピークとして検出されるタンパク質、及びm/z 11,760~11,890のピークとして検出されるタンパク質から選ばれるタンパク質からなる抗がん剤感受性判定マーカー。
- 抗がん剤が、フッ化ピリミジン系抗がん剤及び白金錯体系抗がん剤から選ばれる抗がん剤である請求項1記載の抗がん剤感受性判定マーカー。
- 抗がん剤が、フルオロウラシル、オキサリプラチン及びそれらの塩から選ばれるものである請求項1又は2記載の抗がん剤感受性判定マーカー。
- 抗がん剤が、フルオロウラシル又はその塩とオキサリプラチン又はその塩との組み合わせである請求項1又は2記載の抗がん剤感受性判定マーカー。
- 検体中の請求項1~4のいずれか1項記載のタンパク質を測定することを特徴とする抗がん剤感受性の判定方法。
- 検体が、がんを有する被験者由来の生体試料である請求項5記載の判定方法。
- 検体が、抗がん剤を投与された、がんを有する被験者由来の生体試料である請求項5又は6記載の判定方法。
- 抗がん剤が、フッ化ピリミジン系抗がん剤及び白金錯体系抗がん剤から選ばれる抗がん剤である請求項5~7のいずれか1項記載の判定方法。
- 抗がん剤が、フルオロウラシル、オキサリプラチン及びそれらの塩から選ばれるものである請求項5~8のいずれか1項記載の判定方法。
- 抗がん剤が、フルオロウラシル又はその塩とオキサリプラチン又はその塩との組み合わせである請求項5~8のいずれか1項記載の判定方法。
- 検体中の請求項1~4のいずれか1項記載のタンパク質を測定するためのプロトコールを含むことを特徴とする請求項5~10のいずれか1項記載の判定方法を実施するためのキット。
- 検体が、がんを有する被験者由来の生体試料である請求項11記載のキット。
- 検体が、抗がん剤を投与された、がんを有する被験者由来の生体試料である請求項11又は12記載のキット。
- 抗がん剤が、フッ化ピリミジン系抗がん剤及び白金錯体系抗がん剤から選ばれる抗がん剤である請求項11~13のいずれか1項記載のキット。
- 抗がん剤が、フルオロウラシル、オキサリプラチン及びそれらの塩から選ばれるものである請求項11~14のいずれか1項記載のキット。
- 抗がん剤が、フルオロウラシル又はその塩とオキサリプラチン又はその塩との組み合わせである請求項11~14のいずれか1項記載のキット。
- 請求項1~4のいずれか1項記載のタンパク質の発現変動を指標とする抗がん剤感受性亢進剤のスクリーニング方法。
- 抗がん剤が、フッ化ピリミジン系抗がん剤及び白金錯体系抗がん剤から選ばれる抗がん剤である請求項17記載のスクリーニング方法。
- 抗がん剤が、フルオロウラシル、オキサリプラチン及びそれらの塩から選ばれるものである請求項17又は18記載のスクリーニング方法。
- 抗がん剤が、フルオロウラシル又はその塩とオキサリプラチン又はその塩との組み合わせである請求項17又は18記載のスクリーニング方法。
- 請求項17~20のいずれか1項記載の方法により得られた抗がん剤感受性亢進剤。
- 請求項21記載の抗がん剤感受性亢進剤と、感受性亢進の対象となる抗がん剤とを組み合せてなるがん治療用組成物。
- 抗がん剤が、フッ化ピリミジン系抗がん剤及び白金錯体系抗がん剤から選ばれる抗がん剤である請求項22記載のがん治療用組成物。
- 抗がん剤が、フルオロウラシル、オキサリプラチン及びそれらの塩から選ばれるものである請求項22又は23記載のがん治療用組成物。
- 抗がん剤が、フルオロウラシル又はその塩とオキサリプラチン又はその塩との組み合わせである請求項22又は23記載のがん治療用組成物。
- カルシウム結合タンパク質S100A10からなる、フルオロウラシル又はその塩、又はフルオロウラシル又はその塩とオキサリプラチン又はその塩との組み合わせからなる抗がん剤の感受性判定マーカー。
- 検体中の請求項26記載のカルシウム結合タンパク質S100A10を測定することを特徴とする抗がん剤感受性の判定方法。
- 検体が、がんを有する被験者由来の生体試料である請求項27記載の判定方法。
- 検体が、抗がん剤を投与された、がんを有する被験者由来の生体試料である請求項27又は28記載の判定方法。
- 検体中の請求項26記載のカルシウム結合タンパク質S100A10を測定するためのプロトコールを含むことを特徴とする請求項27~29のいずれか1項記載の判定方法を実施するためのキット。
- 検体が、がんを有する被験者由来の生体試料である請求項30記載のキット。
- 検体が、抗がん剤を投与された、がんを有する被験者由来の生体試料である請求項30又は31記載のキット。
- 請求項26記載のカルシウム結合タンパク質S100A10の発現変動を指標とする抗がん剤感受性亢進剤のスクリーニング方法。
- 請求項33記載の方法により得られた抗がん剤感受性亢進剤。
- 請求項34記載の抗がん剤感受性亢進剤と、感受性亢進の対象となる抗がん剤とを組み合せてなるがん治療用組成物。
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CN201080049208.2A CN102597779B (zh) | 2009-10-30 | 2010-10-29 | 抗癌剂感受性的判定方法 |
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EP3550306A1 (en) | 2014-08-26 | 2019-10-09 | Keio University | Anti-cancer agent sensitivity-determining marker |
WO2020067228A1 (ja) * | 2018-09-28 | 2020-04-02 | 学校法人慶應義塾 | 併用抗がん剤の感受性の判定マーカー |
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EP3575799B1 (en) | 2011-03-24 | 2020-06-24 | Keio University | Marker for determination of sensitivity to anticancer agent |
AU2015279712B2 (en) * | 2014-06-26 | 2021-03-25 | Yale University | Compositions and methods to regulate renalase in the treatment of diseases and disorders |
US20200191772A1 (en) * | 2017-03-31 | 2020-06-18 | Keio University | Combined anticancer agent sensitivity determination marker |
CN108187053B (zh) * | 2018-03-07 | 2021-07-09 | 南京脑科医院 | 基因krtap20-1的应用 |
CN111638261B (zh) * | 2020-04-17 | 2023-04-07 | 融智生物科技(青岛)有限公司 | 一种计算设备、存储介质和地中海贫血筛查装置及系统 |
CN111812220A (zh) * | 2020-05-29 | 2020-10-23 | 南京品生医学检验实验室有限公司 | 一种检测血浆中抗肿瘤药物浓度的方法 |
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EP2495569B1 (en) | 2016-06-15 |
EP3081941A1 (en) | 2016-10-19 |
US8765713B2 (en) | 2014-07-01 |
EP2495569A4 (en) | 2013-03-13 |
CN102597779A (zh) | 2012-07-18 |
JPWO2011052749A1 (ja) | 2013-03-21 |
EP3081941B1 (en) | 2018-06-27 |
CN105699638A (zh) | 2016-06-22 |
US20120214831A1 (en) | 2012-08-23 |
EP2495569A1 (en) | 2012-09-05 |
CN102597779B (zh) | 2016-03-23 |
CN105699638B (zh) | 2018-05-22 |
JP5548694B2 (ja) | 2014-07-16 |
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