WO2011105551A1 - Procédé de détection de cellules souches cancéreuses, et agent thérapeutique ou agent prévenant la récurrence de cancer - Google Patents

Procédé de détection de cellules souches cancéreuses, et agent thérapeutique ou agent prévenant la récurrence de cancer Download PDF

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WO2011105551A1
WO2011105551A1 PCT/JP2011/054287 JP2011054287W WO2011105551A1 WO 2011105551 A1 WO2011105551 A1 WO 2011105551A1 JP 2011054287 W JP2011054287 W JP 2011054287W WO 2011105551 A1 WO2011105551 A1 WO 2011105551A1
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cancer
cells
agent
recurrence
measuring
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Japanese (ja)
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正樹 森
直紹 原口
秀始 石井
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国立大学法人大阪大学
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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
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/351—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom not condensed with another ring
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • G01N33/57446—Specifically defined cancers of stomach or intestine

Definitions

  • the present invention relates to a method for detecting cancer stem cells and a drug for treating or preventing cancer recurrence.
  • Cancer cells have the property of being capable of self-proliferation and being able to wet to surrounding tissues or metastasize to distant tissues. However, not all of the cancer cells forming the cancer tissue have these characteristics, and there are very few cancer cells that develop cancer or advance cancer. It is known to be a cancer stem cell. Cancer stem cells exhibit undifferentiated surface characteristics like normal stem cells, have self-renewal ability and differentiation ability, and have the property of producing all cancer cells in various differentiation stages constituting cancer tissue. That is, cancer stem cells are thought to be responsible for generating the majority of cancer cells by differentiation while maintaining the same cells as themselves by self-replication in cancer tissues.
  • Cancer stem / progenitor cells are highly enriched in CD133 (+) CD44 (+) population in hepatocellular carcinoma.
  • Yang ZF et al. Significance of CD90 + cancer stem cells in human liver cancer. Cancer Cell. 2008; 13 (2): 153-166.
  • Yang ZF et al. Identification of local and circulating cancer stem cells in human liver cancer. Hepatology.2008; 47 (3): 919-928. Haraguchi N, et al. Characterization of a side population of cancer cells from human gastrointestinal system. Stem Cells. 2006; 24 (3): 506-513. Naka K, Muraguchi T, Hoshii T, Hirao A.
  • An object of the present invention is to provide a technique for accurately detecting cancer stem cells by newly identifying functional molecules involved in the control of cancer stem cells.
  • Another object of the present invention is to provide a technique for leading to DNA fragmentation and apoptosis for cancer stem cells and curing the cancer, that is, a radical therapeutic agent for cancer (cancer radical treatment) and a treatment method (cancer radical treatment method).
  • a further object of the present invention is to provide a technique for preventing recurrence of cancer by leading to DNA fragmentation and apoptosis of cancer stem cells and curing the cancer, that is, a cancer recurrence preventive agent and a recurrence preventive method. .
  • CD13 is involved in the control of the reactive oxgen species (ROS) removal pathway in cancer stem cells and maintains cancer stem cells. It was found that cancer stem cells are CD13-positive cells (CD13 + cells), and that cancer stem cells can be accurately detected by using CD13 as an index. Furthermore, by combining conventional cancer treatments such as chemotherapy and ionizing radiation with treatment with a compound having an action of inhibiting CD13 (hereinafter referred to as “CD13 inhibitor”), the ROS concentration is low and the cell cycle is reduced.
  • CD13 inhibitor a compound having an action of inhibiting CD13
  • cancer stem cells that have the characteristic of being in the dormant or late state and exhibit extremely high treatment resistance, and as a result, the therapeutic effect of cancer can be significantly improved In particular, it was found to be effective in the cure of cancer and prevention of recurrence.
  • a combination of a CD13 inhibitor and an anticancer agent or ionizing radiation treatment can fundamentally treat cancer and prevent cancer recurrence, in other words, a CD13 inhibitor and cancer therapy ( It means that combined use with anticancer drug administration or ionizing radiation treatment is effective as a cancer radical treatment method or a cancer recurrence prevention therapy.
  • CD13 + cells are suppressed in DNA double-strand damage or have activated double-strand damage repair ability (see FIG. 14), so a CD13 inhibitor and, for example, 5-fluorouracil, etc.
  • a CD13 inhibitor and, for example, 5-fluorouracil, etc. it is considered that a higher cancer curative effect or cancer recurrence preventing effect can be obtained by using in combination with an anticancer agent having a DNA synthesis inhibitory action.
  • an anthracycline anticancer agent doxorubicin having a very high ROS increase effect is used in combination to enhance the ROS excretion effect of the CD13 inhibitor, thereby increasing the ROS by the CD13 inhibitor. It is possible to damage cancer stem cells in the induced state with very high efficiency, and to lead to the cure of cancer.
  • the present invention has been completed by further studies based on such knowledge.
  • Cancer stem cell detection method (I-1). A method for detecting cancer stem cells, comprising measuring a cell expressing CD13 for a test cell derived from a cancer tissue or a tissue after cancer treatment. (I-2). The detection method according to (I-1), wherein the measurement of cells expressing CD13 is performed using an antibody capable of specifically binding to CD13. (I-3). The detection method according to (I-2), wherein the antibody is labeled with a labeling substance selected from a fluorescent substance and an enzyme. (I-4). The detection method according to any one of (I-1) to (I-3), which comprises a step of measuring cells expressing CD90 together with CD13. (I-5).
  • (II) Method of measuring the degree or risk of cancer symptoms (II-1).
  • a method of measuring the degree of cancer symptoms (cancer severity) or cancer risk of a subject (A) a step of measuring a cell expressing CD13 for a test cell derived from a cancer tissue of a subject or a tissue after cancer treatment, and (B) a CD13-expressing cell measured in the step (A) Determining the cancer severity or cancer risk according to the number of The above-mentioned measuring method characterized by including. (II-2).
  • step (B) when the number of CD13-expressing cells measured in the step (A) is large, the cancer severity and cancer risk are high, and when the number of CD13-expressing cells is small, the cancer severity and
  • (II-3) The measurement method according to (II-1) or (II-2), wherein the measurement of CD13-expressing cells is performed using an antibody capable of specifically binding to CD13.
  • II-4 The measurement method according to (II-3), wherein the antibody is labeled with a labeling substance selected from a fluorescent substance and an enzyme.
  • (II-8) The measurement method according to any one of (II-1) to (II-7), wherein measurement of cells expressing CD13 or CD90 is performed by a flow cytometry method. (II-9). The measurement method according to any one of (II-1) to (II-8), wherein the cancer severity or cancer risk is the probability that the cancer will recur.
  • (III) Method for measuring the therapeutic effect of cancer (III-1).
  • a method for measuring the therapeutic effect of cancer on a cancer patient (A) a step of measuring a cell expressing CD13 in a test cell derived from a tissue after cancer treatment of a cancer patient, and (b) the presence or absence of a CD13-expressing cell measured in the step (a) Depending on the step of determining the therapeutic effect of the cancer, The above-mentioned measuring method characterized by including. (III-2).
  • the step (b) is a step of determining that the cancer treatment effect is poor when CD13-expressing cells are detected in the step (a) and that the cancer treatment effect is good when CD13-expressing cells are not detected.
  • III-4 The measurement method according to (III-3), wherein the antibody is labeled with a labeling substance selected from a fluorescent substance and an enzyme.
  • III-5 The measuring method according to any one of (III-1) to (III-4), wherein the step is a step of measuring cells expressing CD90 together with CD13.
  • III-6 The measurement method according to (III-5), wherein the measurement of cells expressing CD90 is performed using an antibody capable of specifically binding to CD90.
  • III-7 The measurement method according to (III-6), wherein the antibody is labeled with a labeling substance selected from a fluorescent substance and an enzyme.
  • III-8 The measurement method according to any one of (III-1) to (III-7), wherein measurement of cells expressing CD13 or CD90 is performed by a flow cytometry method.
  • (IV) Method of measuring the risk of cancer recurrence (IV-1).
  • a method for measuring the risk of cancer recurrence after cancer treatment for a cancer patient (A ′) a step of measuring a cell expressing CD13 in a test cell derived from a tissue after cancer treatment of a cancer patient, and (b ′) CD13 expression measured in the step (a ′) Determining the cancer recurrence risk of the cancer patient according to the presence or absence of cells,
  • the above-mentioned measuring method characterized by including. (IV-2).
  • step (b ′) when CD13-expressing cells are detected in the step (a ′), the cancer patient is determined to have a high risk of cancer recurrence, and when CD13-expressing cells are not detected,
  • IV-3 The measurement method according to (IV-1) or (IV-2), wherein measurement of CD13-expressing cells is performed using an antibody capable of specifically binding to CD13.
  • IV-4 The measurement method according to (IV-3), wherein the antibody is labeled with a labeling substance selected from a fluorescent substance and an enzyme.
  • V Cancer stem cell detection reagent and detection kit
  • a cancer stem cell detection reagent comprising a substance that specifically binds to CD13 (hereinafter referred to as “CD13-binding substance”) as an active ingredient.
  • V-2 At least one selected from the group consisting of an antibody (hereinafter referred to as “anti-CD13 antibody”), a microRNA, an RNA aptamer, and a dominant negative mutant, wherein the CD13 binding substance can specifically bind to CD13.
  • V-3 A cancer stem cell detection kit comprising a CD13 binding substance as a cancer stem cell detection reagent.
  • V-4 A cancer stem cell detection kit comprising a CD13 binding substance as a cancer stem cell detection reagent.
  • CD90-binding substance a substance that specifically binds to CD90
  • a cancer therapeutic agent comprising a CD13 inhibitor and an anticancer agent.
  • the cancer therapeutic agent according to (VI-2) wherein the CD13 inhibitor is at least one selected from the group consisting of a CD13 neutralizing antibody and Ubenimex.
  • Any of (VI-1) to (VI-6), wherein the cancer to be treated is a solid cancer expressing CD13, preferably liver cancer, lung cancer and gastrointestinal cancer, more preferably liver cancer and colon cancer
  • the anticancer agent as described in. (VI-8).
  • a pharmaceutical kit for treating cancer comprising a first agent containing a CD13 inhibitor and a second agent containing an anticancer agent in separate packaging forms. (VI-9).
  • Any of (VI-7) to (VI-11), wherein the cancer to be treated is a solid cancer expressing CD13, preferably liver cancer, lung cancer and gastrointestinal cancer, more preferably liver cancer and colon cancer
  • the kit according to 1. (VI-13).
  • a step of administering a CD13 inhibitor and an anticancer agent preferably a DNA synthesis inhibitor or an anticancer agent having an ROS-raising action
  • a CD13 inhibitor administration treatment and an ionizing radiation treatment to a cancer patient
  • a radical cure for cancer preferably a radical cure for cancer.
  • the CD13 inhibitor is at least one selected from the group consisting of a CD13 neutralizing antibody and Ubenimex.
  • the anticancer agent is a DNA synthesis inhibitor or an anticancer agent having an ROS increasing action.
  • a combination of a CD13 inhibitor and an anticancer agent preferably a combination of a CD13 inhibitor and a DNA synthesis inhibitor or an anticancer agent having an ROS-elevating action
  • a combination of a CD13 inhibitor and an anticancer agent preferably a combination of a CD13 inhibitor and a DNA synthesis inhibitor or an anticancer agent having an ROS-raising action, which is used for radically treating cancer.
  • VI-18 A combination of a CD13 inhibitor and an anticancer agent, preferably a combination of a CD13 inhibitor and a DNA synthesis inhibitor or an anticancer agent having an ROS-raising action, which is used for radically treating cancer.
  • a combination preparation comprising a combination of a CD13 inhibitor and an anticancer agent (preferably a DNA synthesis inhibitor or an anticancer agent having an ROS-raising action) containing a CD13 inhibitor and an anticancer agent (preferably a DNA synthesis inhibitor or an anticancer agent having an ROS-raising action).
  • a combination according to (VI-17) which is a pharmaceutical kit containing a CD13 inhibitor and an anticancer agent (preferably a DNA synthesis inhibitor or an anticancer agent having a ROS-elevating action) in a separate packaging form.
  • a cancer recurrence preventive agent comprising a CD13 inhibitor and an anticancer agent.
  • a cancer recurrence preventive agent comprising a CD13 inhibitor and an anticancer agent.
  • the cancer recurrence preventive agent according to (VII-2) wherein the CD13 inhibitor is at least one selected from the group consisting of a CD13 neutralizing antibody and Ubenimex.
  • VI-4 Cancer recurrence preventive agent, cancer recurrence prevention pharmaceutical kit and cancer recurrence prevention method.
  • the target cancer is a solid cancer expressing CD13, preferably liver cancer, lung cancer or gastrointestinal cancer, more preferably liver cancer or colon cancer, any of (VII-1) to (VII-5) For preventing cancer recurrence. (VII-7).
  • a pharmaceutical kit for preventing cancer recurrence comprising a first agent containing a CD13 inhibitor and a second agent containing an anticancer agent.
  • an anticancer agent preferably a DNA synthesis inhibitor or an anticancer agent having an ROS-raising action
  • the CD13 inhibitor is at least one selected from the group consisting of a CD13 neutralizing antibody and Ubenimex.
  • the anticancer agent is a DNA synthesis inhibitor or an anticancer agent having an ROS increasing action.
  • a combination of a CD13 inhibitor and an anticancer agent preferably a combination of a CD13 inhibitor and a DNA synthesis inhibitor or an anticancer agent having an ROS-raising action
  • a combination of a CD13 inhibitor and an anticancer agent preferably a combination of a CD13 inhibitor and a DNA synthesis inhibitor or an anticancer agent having an ROS-raising action, used for preventing cancer recurrence.
  • VII-16 A combination of a CD13 inhibitor and an anticancer agent, preferably a combination of a CD13 inhibitor and a DNA synthesis inhibitor or an anticancer agent having an ROS-raising action, used for preventing cancer recurrence.
  • a combination preparation comprising a combination of a CD13 inhibitor and an anticancer agent (preferably a DNA synthesis inhibitor or an anticancer agent having an ROS-raising action) containing a CD13 inhibitor and an anticancer agent (preferably a DNA synthesis inhibitor or an anticancer agent having an ROS-raising action).
  • a combination described in (VII-15) which is a pharmaceutical kit containing a CD13 inhibitor and an anticancer agent (preferably a DNA synthesis inhibitor or an anticancer agent having a ROS-elevating action) in a separate packaging form.
  • cancer stem cells can be accurately detected. Therefore, the present invention can contribute to the improvement of cancer treatment technology, and is an innovative that is extremely useful for predicting the degree of cancer symptoms such as cancer occurrence, progression, metastasis, and recurrence or the risk of cancer. Can be technology.
  • cancer therapeutic agent or cancer treatment pharmaceutical kit of the present invention by using a CD13 inhibitor in combination with an anticancer agent, preferably a DNA synthesis inhibitor, not only cancer cells but also cancer stem cells are eliminated, Since cancer can be suppressed, it is also possible to provide a cancer root treatment method and a cancer root treatment method that exhibit an unprecedented excellent therapeutic effect. Further, according to the cancer recurrence preventive agent or the cancer recurrence prevention pharmaceutical kit of the present invention, cancer recurrence is prevented by eradicating cancer cells or cancer stem cells survived by conventional cancer therapy, and thus the survival rate of cancer patients. Can be significantly improved.
  • A It is a flowchart which shows the operation procedure which performed "1. Confirmation that CD13 is a marker candidate of a cancer stem cell” in an Example.
  • B It is a figure which shows the expression intensity
  • C and D shows the results of analyzing the expression of CD13, CD133, and CD90 in hepatitis infection negative cell line (HuH7) and hepatitis infection positive cell line (PLC / PRF / 5: described as “PLC” in the figure)
  • FIG. It is a figure which shows the result of the term of "1. Confirmation that CD13 is a cancer stem cell marker candidate" in an Example.
  • A The results of examining the expression status of CD13 and CD31 in HuH7, Hep3B, and PLC / PRF / 5 are shown.
  • B shows the relationship between CD13 and Hu fraction in HuH7. It can be seen that CD31 + cells are mainly present in the G2 / M / SP fraction. It is a figure which shows the result of the term of "2. Confirmation that CD13 is a marker of the hepatocellular carcinoma (HCC) cell in a dormant stage" in an Example.
  • A The result of examining the relationship between the expression of CD13 of HuH7 and PLC (PLC / PRF / 5) and the cell cycle of HuH7 and PLC (PLC / PRF / 5).
  • FIG. 4 is a view showing the results in the section “3. Confirmation that CD13 is specifically expressed in moderately to poorly differentiated colorectal cancer cells” in Examples.
  • CD13 + CD90 ⁇ cells Dormant Cancer stem Cells: Dormant CSCs
  • Activated CSCs Activated CSCs
  • CD13 ⁇ CD90 + cells Progesitors of Cancer Cells
  • CD90 + cells are also associated with a decrease in intracellular ROS due to an increase in antioxidant capacity. Therefore, conventional cancer treatments (genotoxic chemotherapy and radiation It shows resistance to radiation therapy. It is a figure which shows the result of the section of "5.
  • CD13 + cell shows resistance to cancer chemotherapy and radiotherapy" in an Example.
  • a and B shows that CD13 + cells are resistant to an anticancer drug (an anticancer drug having a ROS-raising action: doxorubicin hydrochloride (DXR)).
  • C Indicates that CD13 + cells are resistant to irradiation.
  • “RT 4G 24h” means 24 hours after irradiation (4 Gray)
  • “RT 4G 48h” means 48 hours after irradiation (4 Gray). It is a figure which shows the result of the term of "6. Confirmation that CD13 is selectively expressing in the cell which shows treatment tolerance" in an Example.
  • A In order to identify the expression of CD13 in clinically obtained HCC cells, the results of digesting an HCC sample and analyzing the hematopoietic CD45 (Lin / CD45) negative fraction by multicolor flow cytometry are shown.
  • B shows the result of confirming the expression of CD13 in a fresh cryosurgical specimen.
  • “After TAE” means a sample subjected to hepatic artery embolization therapy
  • “Non-TAE” and “Not treatment” mean samples not subjected to hepatic artery embolization therapy.
  • FIG. 8B in the left end and middle column images, CD13 is stained red and DAPI is blue. It is a figure which shows the result of the term of "7.
  • CD13 Ab means a CD13 neutralizing antibody.
  • Ube means Ubenimex. It is a figure which shows the result of the term of "7. Confirmation that CD13 inhibition leads to a cell apoptosis” in an Example.
  • DOX is synonymous with DXR. It is a figure which shows the result of the term of "8. Confirmation that CD13 inhibition induces tumor regression" in an Example.
  • Ube means Ubenimex. In the image of A in FIG.
  • RT 4Gy means a group irradiated with radiation
  • R4Gy with Tempol means a group irradiated after tempol treatment.
  • the red numbers in the figure are the percentage of ⁇ -H2AX in CD13 + cells (PLC / PRF / 5: CD13 + CD90 ⁇ cells, HuH7: CD13 + CD133 + cells), and the blue numbers are CD13 ⁇ cells
  • the ratio (%) of ⁇ -H2AX in (PLC / PRF / 5: CD13 ⁇ CD90 + cells, HuH7: CD13 ⁇ CD133 + cells) is shown.
  • the method for detecting a cancer stem cell of the present invention is characterized by measuring a cell expressing CD13 in a test cell derived from a cancer tissue of a cancer patient or a tissue after cancer treatment. .
  • the detection method of the present invention will be described in detail.
  • cancer stem cell means a cell that has self-replicating ability and maintains an undifferentiated state and can produce cancer cells by differentiation.
  • the “test cell” is a cell that is a target of determination of whether or not it expresses CD13, that is, whether or not it is a cancer stem cell.
  • the test cell may be a single cell isolated from cancer tissue of a cancer patient or tissue after cancer treatment, or may be a group of cells isolated from cancer tissue or tissue after cancer treatment. .
  • the cancer tissue from which the test cell is derived is not particularly limited, and examples thereof include liver cancer, colorectal cancer, esophageal cancer, gastric cancer, bile duct cancer (bile duct includes intrahepatic bile duct and extrahepatic bile duct), Gallbladder cancer (the left two or three are collectively called the biliary tract), pancreatic cancer (the pancreas is divided into duct and secretory gland tissue, the latter includes endocrine and exocrine systems), duodenal cancer, colon cancer (colon Includes ascending, transverse, descending, sigmoid colon, rectum), breast cancer, and solid tumor tissues such as brain tumors; and hematopoietic tumors (blood cancers) such as leukemia, malignant lymphoma, and multiple myeloma Hematopoietic tissue involved is exemplified. Since the present invention is suitable for detection of cancer stem cells contained in liver cancer, among these cancer tissues, examples of preferable cancer
  • the tissue after cancer treatment from which the test cells are derived is a tissue after the above cancer treatment, and the cancer treatment treatment is performed by at least one selected from surgical therapy, chemotherapy, and radiation therapy. Any organization after being applied may be used.
  • a cancer tissue is excised by surgical therapy, a tissue near the excision site can be targeted.
  • test cells to be detected in the detection method of the present invention may be those extracted from the cancer tissue of the cancer patient, or the tissue after cancer treatment of the cancer patient, or the cancer tissue of the cancer patient. Or the thing of the state which exists in the tissue after cancer treatment of a cancer patient may be sufficient. In the latter case, the target tissue may be in the body of the cancer patient, but is preferably in a state of being removed from the body of the cancer patient.
  • cancer patients targeted by the present invention are mammals including humans. Humans are preferred, but rodents such as mice, rats, and guinea pigs; and laboratory animals such as rabbits, cats, dogs, and monkeys can also be used.
  • CD13 is a molecule present on the surface of cancer stem cells and is involved in the control of the ROS elimination pathway, reducing ROS-induced DNA damage that occurs after genotoxic chemoradiation stress, and apoptosis.
  • Non-patent document 10 Haraguchi ⁇ N, Ishii H, Mimori K, Tanaka F, Ohkuma M, Kim HM, Akita H, Takiuchi D, Hatano H, Nagano H , Barnard GF, Doki Y, Mori M.
  • CD13 is a therapeutic target in human liver cancer stem cells. J Clin Invest. 2010; 120 (9): 3326-3339.).
  • CD13-binding substance a substance that specifically binds to CD13
  • a test cell a substance that specifically binds to CD13
  • the presence or absence of binding of the substance to the test cell is measured.
  • cells expressing CD13 can be measured. That is, for the test cell, a cell expressing CD13 can be detected using the binding of the CD13 binding substance as an index.
  • the CD13 binding substance is not particularly limited as long as it can specifically bind to CD13.
  • an antibody capable of specifically binding to CD13 hereinafter referred to as “anti-CD13 antibody”.
  • an anti-CD13 antibody is exemplified as a preferable one.
  • the CD13 binding substance is labeled with a fluorescent substance such as FITC or a labeling substance such as an enzyme from the viewpoint of easy detection.
  • Measurement of a test cell bound with a CD13 binding substance can be performed by a method known in the art. For example, if the CD13 binding substance is labeled with a fluorescent substance, the cells to which the substance is bound can be measured qualitatively and quantitatively by using flow cytometry.
  • test cells expressing CD90 can also be performed by contacting a test cell with a substance that specifically binds to CD90 (hereinafter referred to as “CD90-binding substance”), as in the case of CD13. it can.
  • CD90-binding substance a substance that specifically binds to CD90
  • the CD90-binding substance is not particularly limited as long as it can specifically bind to CD90.
  • an antibody that can specifically bind to CD90 hereinafter referred to as “anti-CD90 antibody”
  • examples include microRNA, RNA aptamer, and dominant negative mutant.
  • An anti-CD90 antibody is preferred.
  • the CD90 binding substance is desirably labeled with a fluorescent substance such as FITC or a labeling substance such as an enzyme, as in the case of the measurement of CD13.
  • a fluorescent substance such as FITC
  • a labeling substance such as an enzyme
  • the method for measuring a test cell bound with a CD90-binding substance can also be performed by a method known in the art, such as flow cytometry, as in the case of CD13.
  • test cells expressing CD13 or the test cells expressing CD13 and CD90 thus measured are detected and identified as cancer stem cells.
  • the cancer stem cells detected by the method of the present invention can be isolated, if necessary, and used for screening for a therapeutic drug for cancer or for evaluating the efficacy of a therapeutic drug for cancer.
  • cancer stem cells detected by the method of the present invention are used to advance drug discovery specifically for clones in the cell cycle stationary phase or drug-resistant clones, thereby acting only on resistant cancer cells against cancer treatment. Therefore, it is possible to develop a cancer therapeutic agent that has no side effects.
  • the development of drugs that prevent cancer recurrence or cancer recurrence, and the development of drugs whose drug efficacy spectrum is specialized for cancer stem cells Is also expected.
  • the presence of cancer stem cells in cancer tissue is used to determine the degree of cancer symptoms (cancer severity) and the risk of cancer progression, recurrence or metastasis
  • the detection method of the present invention can be used as a method for measuring cancer severity or cancer risk.
  • the present invention also provides a method for measuring cancer seriousness or cancer risk using the above-mentioned “cancer stem cell detection method”.
  • the method includes the following steps (A) and (B): (A) a step of measuring cells expressing CD13 (CD13-expressing cells) for test cells derived from cancer tissues of cancer patients or tissues after cancer treatment; (B) A step of determining the cancer severity or cancer risk of the cancer patient according to the number of CD13-expressing cells detected in the step (A).
  • step (A) means both qualitative analysis for measuring the presence or absence of CD13-expressing cells in a test cell and quantitative analysis for measuring the abundance thereof.
  • the CD13-expressing cells measured in step (A) correspond to cancer stem cells.
  • the step (B) corresponds to a step of predicting cancer severity or cancer risk using the number of cancer stem cells as an index.
  • the CD13 binding property may be labeled with a labeling substance such as a fluorescent substance or an enzyme. It can be carried out by bringing a substance into contact with a test cell and measuring the presence or absence of binding of the substance to the test cell.
  • a labeling substance such as a fluorescent substance or an enzyme. It can be carried out by bringing a substance into contact with a test cell and measuring the presence or absence of binding of the substance to the test cell.
  • CD13-expressing cells that is, cancer stem cells
  • Detection of CD13-expressing cells using the binding of the CD13-binding substance as an index can be performed by a method known in the art as described above.
  • CD13-binding substance when labeled with a fluorescent substance is labeled with a fluorescent substance can be performed by using flow cytometry.
  • the number (amount) of CD13-expressing cells (cancer stem cells) can be measured simultaneously based on the level of fluorescence intensity, so CD13-expressing cells (cancer stem cells) can be qualitatively and quantitatively determined. Can be measured.
  • the more CD13-expressing cells (cancer stem cells) measured in the step (A) using the detection method of the present invention the more severe the cancer symptoms, It is determined that the cancer risk is high.
  • the smaller the CD13-expressing cells (cancer stem cells) measured in step (A) using the detection method of the present invention the lighter the cancer symptoms and the lower the risk of cancer.
  • CD90 expression in addition, it is preferable to measure the expression of CD90 in addition to the expression of CD13. In this way, the accuracy of detection of cancer stem cells can be further increased, and the cancer severity or cancer risk can be increased. It can be determined more accurately.
  • CD90 expression can be measured in the same manner as CD13 expression.
  • Method for Measuring Cancer Treatment Effect or Risk of Cancer Recurrence Furthermore, the presence of cancer stem cells in tissues after cancer treatment can be used as an evaluation standard for cancer treatment effect and can be used to predict cancer recurrence. it can. Therefore, the “1. Method for detecting cancer stem cells” of the present invention can also be used as a method for measuring the therapeutic effect of cancer and a method for measuring the risk of cancer recurrence.
  • the present invention also provides a method for measuring the effect of cancer treatment or the risk of cancer recurrence using the above-described “method for detecting cancer stem cells”.
  • the method includes the following steps (a) and (b): (A) measuring CD13-expressing cells (CD13-expressing cells) for test cells derived from tissues after cancer treatment of cancer patients; (B) A step of determining a cancer therapeutic effect or cancer recurrence risk for the cancer patient according to the presence or absence of the CD13-expressing cells measured in the step (a).
  • measuring CD13-expressing cells means both qualitative analysis for measuring the presence or absence of CD13-expressing cells in a test cell and quantitative analysis for measuring the abundance thereof.
  • the CD13-expressing cells measured in step (a) are cancer stem cells as described above, and step (b) determines the cancer therapeutic effect or cancer recurrence risk for cancer patients using the presence or absence of the cancer stem cells as an index. It corresponds to the process to do.
  • the step (a) is a CD13 binding substance which may be labeled with a fluorescent substance or a labeling substance such as an enzyme, as described in the section of “1.
  • Detection method of cancer stem cells can be carried out by contacting the test cells with the test cells and measuring the presence or absence of binding of the substance to the test cells.
  • CD13-expressing cells that is, cancer stem cells
  • Detection of CD13-expressing cells using the binding of the CD13-binding substance as an index can be performed by a method known in the art as described above.
  • CD13-binding substance when labeled with a fluorescent substance is labeled with a fluorescent substance can be performed by using flow cytometry.
  • the number of CD13-expressing cells can also be measured simultaneously by the level of fluorescence intensity, so CD13-expressing cells (cancer stem cells) can be measured qualitatively and quantitatively. it can.
  • step (b) CD13-expressing cells (cancer stem cells) were detected from the tissue after cancer treatment in step (a) using the “method for detecting cancer stem cells” of the present invention.
  • cancer stem cells high risk of cancer recurrence.
  • cancer treatment effect is good and the possibility of cancer recurrence is low (the risk of cancer recurrence is low).
  • CD90 expression can be measured in the same manner as CD13 expression.
  • Cancer Stem Cell Detection Reagent and Detection Kit The present invention is intended to perform the “cancer stem cell detection method” described in 1 above, and to qualitatively or alternatively detect CD13 expressing cells (cancer stem cells) in the measurement methods described in 2 and 3 above.
  • a reagent for detecting cancer stem cells, which is used for quantitative measurement, is also provided.
  • the detection reagent of the present invention includes a substance that specifically binds to CD13 (CD13-binding substance).
  • the present invention is for performing the “cancer stem cell detection method” described in 1 above, and for qualitatively or quantitatively measuring CD13-expressing cells (cancer stem cells) in the measurement methods described in 2 and 3 above.
  • a kit for detecting cancer stem cells used in the above is characterized by containing a CD13 binding substance as a detection reagent for cancer stem cells.
  • the detection kit of the present invention may further contain a substance that specifically binds to CD90 (CD90-binding substance) as a detection reagent for cancer stem cells.
  • the detection kit of the present invention further includes “reagent for detecting cancer stem cells” described in 1 above, or other reagents and instruments required for performing the measurement methods described in 2 and 3 above.
  • the detection kit of the present invention may include a procedure manual for performing the above detection method.
  • CD13 binding substance and the CD90 binding substance used in the cancer stem cell detection reagent and detection kit of the present invention are as described in “1. Detection method of cancer stem cells” above.
  • the “cancer stem cell detection method” is a method for measuring the degree of cancer symptoms (cancer severity) or cancer risk, or a method for measuring cancer therapeutic effect or cancer recurrence risk. Therefore, the detection reagent and detection kit of the present invention can be used as a diagnostic agent and a diagnostic kit for measuring the degree of cancer symptoms (cancer severity) or cancer risk, and further the therapeutic effect of cancer or cancer. It can also be used as a diagnostic agent and a diagnostic kit for measuring the risk of recurrence.
  • the present invention further provides a therapeutic agent for cancer and a preventive agent for cancer recurrence , which further comprises a CD13 inhibitor and an anticancer agent.
  • a therapeutic agent for cancer and a preventive agent for cancer recurrence which further comprises a CD13 inhibitor and an anticancer agent.
  • the cancer therapeutic agent and cancer recurrence preventing agent of the present invention will be described in detail.
  • the cancer therapeutic agent of the present invention can be used for the purpose of curing cancer, and in this sense, it can also be referred to as a cancer curative.
  • the cancer therapeutic agent of this invention can prevent the recurrence of cancer as a result of curing cancer.
  • the CD13 inhibitor used in the cancer therapeutic agent and cancer recurrence preventive agent of the present invention is pharmaceutically acceptable and can inhibit the function of CD13, that is, can inhibit the control function of the ROS removal pathway.
  • CD13 inhibitors include CD13 neutralizing antibody, ubenimex (bestatin), aminopeptidase N (APN / CD13) inhibitor 24F (Novel aminopeptidase N (APN / CD13) inhibitor 24F can suppress invasion of hepatocellular carcinoma cells as . Biosci Trends. 2010 2010 Apr; 4 (2): 56-60).
  • CD13 inhibitors may be used alone or in combination of two or more.
  • These CD13 inhibitors are commercially available (for example, as for the CD13 neutralizing antibody, “mouse monoclonal anti-human CD13 antibody (clone WM15)” is available from Gene Tex, and Ubenimex is available from Nippon Kayaku). For this reason, although a commercial item may be used for a CD13 inhibitor, it may be manufactured and used by a known method.
  • anticancer agents used in the cancer therapeutic agent and cancer recurrence preventive agent of the present invention include conventionally known alkylating agents (mustard drugs, nitroureas), antimetabolites (folic acid antimetabolites, pyrimidine antimetabolites, Purine antimetabolite, hydroxycarbamide), antitumor antibiotics (anthracycline drugs, others, mitomycin C, etc.), platinum preparations, topoisomerase inhibitors (topoisomerase I inhibitors, topoisomerase II inhibitors), etc. it can.
  • alkylating agents muscle drugs, nitroureas
  • antimetabolites folic acid antimetabolites, pyrimidine antimetabolites, Purine antimetabolite, hydroxycarbamide
  • antitumor antibiotics anthracycline drugs, others, mitomycin C, etc.
  • platinum preparations platinum preparations
  • topoisomerase inhibitors topoisomerase I inhibitors, topoisomerase II inhibitors
  • anticancer agents having an activity of inhibiting DNA synthesis in the present invention, such anticancer agents are collectively referred to as “DNA synthesis inhibitors”) and anticancer agents having an activity of increasing ROS.
  • DNA synthesis inhibitors collectively referred to as “DNA synthesis inhibitors”
  • anticancer agents having an activity of increasing ROS may be used individually by 1 type, and may be used in combination of 2 or more type.
  • DNA synthesis inhibitors include alkylating agents such as ifosfamide, melphalan, nimustine hydrochloride, ranimustine, and procarbazine hydrochloride; 5-fluorouracil (5-FU), 5-FU prodrugs (eg, doxyfluridine, tegafur, carmofur) , 5'-doxyfluridine prodrugs (eg capecitabine), cytarabine, cytarabine prodrugs (eg cytarabine ocphosphate phosphate hydrate), pyrimidine antagonists such as enocitabine, gemcitabine hydrochloride; mercaptopurine hydrate, fludarabine phosphate Purine antimetabolites such as esters and cladribine, and other antimetabolites such as levofolinate calcium and hydroxycarbamide; doxorubicin hydrochloride, daunorubicin hydrochloride, pirarubicin, and mitoxantron
  • the above pyrimidine antagonists such as 5-fluorouracil (5-FU) and prodrugs thereof are preferable, and 5-fluorouracil is more preferable.
  • These DNA synthesis inhibitors may be used individually by 1 type, and may be used in combination of 2 or more type.
  • an anticancer agent having a ROS-raising action is an anthracycline anticancer agent belonging to an antitumor antibiotic.
  • anthracycline anticancer agent include doxorubicin, daunorubicin, pirarubicin, epirubicin, idarubicin, aclarubicin, amrubicin, mitoxantrone, or a pharmaceutically acceptable salt thereof.
  • These anticancer agents may be used alone or in combination of two or more. Moreover, you may use together with the said DNA synthesis inhibitor.
  • the content of the CD13 inhibitor and the anticancer agent depends on the type of cancer to be treated, the degree of symptoms, A therapeutically effective amount according to the age, sex, etc. of the cancer patient is sufficient. Although it cannot be defined uniformly, for example, it is desirable that the content satisfy the following range as a dose per adult.
  • Dose of CD13 inhibitor per adult Usually 10 to 600 mg, preferably 15 to 90 mg, more preferably 30 to 60 mg ⁇
  • Dose of adult anticancer drug (preferably DNA synthesis inhibitor) per adult Usually 100 to 3000 mg, preferably 200 to 1000 mg, more preferably 250 to 750 mg.
  • the ratio of the CD13 inhibitor to the anticancer agent is appropriately determined within the range satisfying the therapeutically effective amount.
  • the ratio of the anticancer agent (preferably a DNA synthesis inhibitor or an anticancer agent having an ROS-raising action) to 1 part by weight of the CD13 inhibitor is usually 1 to 100 parts by weight, preferably 2 to 50 parts by weight, more preferably Examples are 4 to 30 parts by weight.
  • the cancer therapeutic agent and cancer recurrence preventive agent of the present invention include pharmaceutically acceptable carriers and additives in addition to a CD13 inhibitor and an anticancer agent (preferably a DNA synthesis inhibitor or an anticancer agent having an ROS-raising action). May be.
  • examples of such carriers and bases include excipients, extenders, binders, disintegrants, surfactants, lubricants, solubilizers, plasticizers, pH adjusters, buffers, chelating agents, Preservatives, antioxidants, solvents, disintegrants and the like can be mentioned.
  • the dosage forms of the cancer therapeutic agent and cancer recurrence preventing agent of the present invention are not particularly limited, and may be appropriately set according to the administration form.
  • Specific examples of the dosage form of the cancer therapeutic agent and cancer recurrence preventing agent of the present invention include tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules and the like.
  • the dosage form of the cancer therapeutic agent and cancer recurrence preventing agent of the present invention may be appropriately set according to the type of cancer to be treated, and may be oral administration such as buccal administration, sublingual administration, In addition, parenteral administration such as intravenous administration, intramuscular administration, subcutaneous administration, pulmonary administration, and rectal administration may be used.
  • the cancer to be treated by the cancer therapeutic agent and the cancer recurrence preventing agent of the present invention is not particularly limited as long as it is a cancer tissue expressing CD13.
  • cancer include gastrointestinal cancers such as esophageal cancer, gastric cancer, duodenal cancer, colorectal cancer, and colon cancer (the large intestine includes the ascending, transverse, descending, sigmoid colon, and rectum); liver cancer; lung cancer Breast cancer; brain tumor; bile duct cancer (the bile duct includes the intrahepatic and extrahepatic bile ducts); gallbladder cancer (the left two or three are collectively referred to as the biliary tract); pancreatic cancer (the pancreas is the duct and secretory tissue) The latter includes solid cancers such as the endocrine system and exocrine system); and hematopoietic tumors (blood cancers) such as leukemia, malignant lymphoma, and multiple myeloma
  • the cancer therapeutic agent of the present invention can be applied as a first-choice therapy to cancer patients suffering from the above cancer. That is, cancer treatment (radical treatment of cancer) can be performed using the cancer therapeutic agent before performing surgical treatment such as cancer tissue resection surgery or radiation treatment on the cancer patient.
  • cancer therapeutic agent of the present invention is also applied to cancer patients who are resistant to cancer therapy, and the cancer does not disappear by other cancer chemotherapy, surgical treatment such as cancer tissue resection surgery, or radiation treatment. be able to.
  • the cancer recurrence-preventing agent of the present invention is used for cancer patients who have achieved a temporary cancer therapeutic effect by other cancer chemotherapy, surgical treatment such as cancer tissue resection surgery, or radiation treatment. It can be applied for the purpose of preventing.
  • a pharmaceutical kit for treating cancer or preventing cancer recurrence provides a second agent comprising a first agent containing a CD13 inhibitor and an anticancer agent (preferably a DNA synthesis inhibitor or an anticancer agent having a ROS-elevating action).
  • an anticancer agent preferably a DNA synthesis inhibitor or an anticancer agent having a ROS-elevating action.
  • pharmaceutical kit both are collectively referred to as “pharmaceutical kit”.
  • the type, the content in the first agent, and the dose are as shown in the above “5. Cancer therapeutic agent and cancer recurrence preventive agent”. is there.
  • the anticancer agent preferably a DNA synthesis inhibitor or an anticancer agent having an ROS-raising action
  • the type, the content in the second agent, and the dosage are also described above. As described in "5. Cancer therapeutic agent and cancer recurrence preventive agent”.
  • first agent and the second agent may further contain a pharmaceutically acceptable carrier or additive, and examples of such carriers and additives are also described in “5. And the same as those used in the “cancer recurrence preventive agent”. Further, the formulation forms of the first agent and the second agent are not particularly limited, and specific examples thereof are the same as those in the case of “5. Cancer therapeutic agent and cancer recurrence preventing agent” above.
  • the cancer to be treated by the pharmaceutical kit of the present invention is the same as the cancer to which the above-mentioned “5. Cancer therapeutic agent and cancer recurrence preventing agent” is applied.
  • the pharmaceutical kit of the present invention may include an administration manual showing these administration methods and the like.
  • the present invention provides a radical cancer treatment method and a cancer recurrence prevention method (hereinafter, these methods are collectively referred to as "cancer radical cure and recurrence suppression therapy").
  • the cancer curative and recurrence-suppressing therapy of the present invention includes (1) a step of administering a CD13 inhibitor and an anticancer agent to a cancer patient, or (2) a CD13 inhibitor administration treatment and ionizing radiation irradiation for a cancer patient. It has the process of performing treatment, It is characterized by the above-mentioned.
  • the administration of the CD13 inhibitor and the anticancer agent to the cancer patient may be performed simultaneously, that is, in parallel, or may be performed at different administration timings, or a drug withdrawal period may be provided in the middle. good.
  • the type, content, added carrier and additive, dose, formulation form, and administration route are as described in “5. Cancer therapeutic agent and cancer recurrence preventive agent” above.
  • the anticancer agent is preferably a DNA synthesis inhibitor or an anticancer agent having an ROS increasing action.
  • the formulation forms and administration routes of the CD13 inhibitor and anticancer agent may be the same or different.
  • administration of a CD13 inhibitor to a cancer patient may be performed simultaneously with the ionizing radiation irradiation treatment for the patient, that is, in parallel, or at a different time, and may be suspended during the course. A period or a rest period may be provided.
  • the type, content, carrier and additive to be added, dosage, formulation form, and administration route are as described in “5. Cancer therapeutic agent and cancer recurrence preventing agent” above.
  • the treatment conditions in the ionizing radiation irradiation treatment can be appropriately selected according to a standard method according to the type of cancer of the cancer patient, the seriousness such as the size and progression of the cancer, the age, sex and weight of the patient.
  • the cancer patient that is the subject of the cancer cure and the recurrence-suppressing therapy of the present invention is a cancer patient suffering from a cancer to which the above-mentioned “5. cancer therapeutic agent and cancer recurrence preventive agent” is applied, preferably Solid cancer patients, more preferably liver cancer, lung cancer, or gastrointestinal cancer patients, particularly preferably liver cancer or colon cancer patients.
  • Cancer radical therapy can be performed on such cancer patients before surgical treatment such as cancer tissue resection surgery or radiation treatment, and other cancer chemotherapy, surgery such as cancer tissue resection surgery, etc.
  • the present invention can also be applied to cancer patients who are resistant to cancer therapy and whose cancer has not disappeared even by treatment or irradiation treatment.
  • the method for preventing cancer recurrence according to the present invention provides cancer recurrence to a cancer patient who has achieved a temporary cancer therapeutic effect by other cancer chemotherapy, surgical treatment such as cancer tissue resection surgery, or radiation treatment. It is applied for the purpose of preventing.
  • ⁇ Experiment method> Cell culture human hepatoma cells HuH7 and PLC / PRF5 (obtained from Tohoku University Institute of Aging Medicine, Medical Cell Resource Center) in RPMI 1640 medium (Invitrogen) containing 10% FBS (fetal bovine serum; Equitech-Bio) Culture was performed. The culture was performed in an atmosphere of 37 ° C. and 5% CO 2 .
  • the PCR primers used for amplification are as follows.
  • GCLM 5'-TTGTGTGATGCCACCAGATTT-3 '(SEQ ID NO: 1) and 5'-TTCACAATGACCGAATACCG-3' (SEQ ID NO: 2)
  • GAPDH 5′-TTGGTATCGTGGAAGGACTCA-3 ′ (SEQ ID NO: 3) and 5′-TGTCATCATATTTGGCAGGTTT-3 ′ (SEQ ID NO: 4).
  • doxorubicin was added to the culture (0.01, 0.05 and 0.1 ⁇ g / ml). 72 hours after the addition of the cancer chemotherapeutic agent, live cells were measured in the same manner as in the analysis of cell proliferation.
  • DMEM / F-12 serum-free medium (Invitrogen) consists of 2 mM L-glutamine, 1% sodium pyruvate (Invitrogen), 1% MEM non-essential amino acids (Invitrogen), 1% insulin-transferrin-selenium-X supplement (Invitrogen) , 1 ⁇ M dexamethasone (Wako), 200 ⁇ M L-ascorbic acid 2-phosphate (Sigma), 10 mM nicotinamide (Wako), 100 ⁇ g / ml penicillin G, and 100 U / ml streptomycin, 20 ng / ml epidermal growth factor And 10 ng / ml fibroblast growth factor 2 (PeproTech). Cell passage was performed every 3 days.
  • a fragment with an immunohistochemical thickness of 4 ⁇ m was obtained with a cryostat and fixed with 4% paraformaldehyde for 15 minutes. After 1 hour blocking, the fragments were incubated with primary antibody overnight at 4 ° C. in a humidified chamber.
  • Primary antibodies include anti-human CD13 mouse monoclonal antibody (clone WM15, dilution 1:50; Santa Cruz Biotechnology), anti-human carbonic anhydrase IX (CA9) rabbit polyclonal antibody (dilution 1: 1000; Novus Biologicals), anti-human CD90 rabbit monoclonal antibody (dilution 1: 1000; Epitomics) and anti-human Ki-67 rabbit polyclonal antibody (dilution 1: 100; Santa Cruz Biotechnology) were used.
  • tumor tissue live cancer tissue samples were obtained from Osaka University.
  • the tumor tissue was cut into small pieces of 2 mm or less, further chopped with a sterile scalpel, and then washed twice with DMEM / 10% FBS.
  • the tumor tissue slices were then placed in DMEM / 10% FBS containing 2 mg / ml collagenase A (Roche Diagnostics) solution. Incubation was performed with stirring at 7 ° C. until the tumor tissue slices were completely digested.
  • Cells were collected by passing through a 40 ⁇ m nylon mesh, washed twice, removed by Ficoll (GE Healthcare) concentration gradient centrifugation, cell fragments and cell debris were removed, and staining for flow cytometry was performed.
  • DXR-R Doxorubicin resistant HuH7 cells were established by sequential treatment with 1 ⁇ g / ml doxorubicin (DXR) and selection of resistant clones. Cell apoptosis was measured using Propidium Iodide (PI) and APC-Annexin V (BD Pharmingen) together with Apoptosis Detection Kit (Bio Vision).
  • PI Propidium Iodide
  • APC-Annexin V BD Pharmingen
  • mice In vivo analysis 1 x 10 5 cells of HuH7 and PLC / PRF / 5 were injected into NOD / SCID mice under anesthesia to produce xenografted model mice. In the cell injection step, cells were resuspended in a mixture containing medium and Matrigel (BD Biosciences) in a 1: 1 ratio. In mice xenografted with HuH7 cells, 5-FU (30 mg / kg; intraperitoneal administration) as a DNA synthesis inhibitor or Ubenimex (20 mg / kg; oral administration) as a CD13 inhibitor was administered for 3 days. The next day, mice were dissected and tumors removed for immunochemical histological analysis.
  • 5-FU (30 mg / kg, intraperitoneal administration for 5 days and discontinuation of drug administration for 2 days, 2 courses, 14 days
  • Ubenimex (20 mg / kg, 14-day gavage)
  • Ubenimex and 5-FU (a combination of 2 courses of 30 mg / kg of 5-FU and 20 mg / kg of Ubenimex for 14 days), respectively.
  • Tumor size and relative tumor volume were calculated according to the following formula.
  • mice After 14 days of 5-FU treatment, the remaining tumor was excised, shredded to a size of 2 mm, and subcutaneously administered with Matrigel to the second NOD / SCID. Mice were treated with ubenimex (20 mg / kg) the next day 7 days after transplantation. Tumor growth was observed for 3 weeks. In order to statistically analyze the results, each experiment was performed with 4 or more mice.
  • ROS analysis To examine intracellular ROS levels, cells were loaded into 10 ⁇ M 2 ′, 7′-dichlorofluorescein diacetate (DCF-DA) at 37 ° C. for 30 minutes. ROS was activated when treated with 100 ⁇ M H 2 O 2 at 37 ° C. for 120 minutes.
  • DCF-DA 7′-dichlorofluorescein diacetate
  • CD13 inhibition To examine the effect of CD13 inhibition on ROS levels, cells were pretreated for 4 hours at 37 ° C. with 5 ⁇ g / ml CD13 neutralizing antibody (mouse monoclonal anti-human CD13 antibody, clone WM15) or 25 ⁇ g / ml Ubenimex. Stained with DCF-DA.
  • mitochondrial ROS detection cells were loaded into 5 ⁇ M MitoSOX (Molecular Probes) at 37 ° C. for 20 minutes.
  • Non-patent Document 7 In order to identify specific cell surface markers related to the SP cell fraction, the gene expression profile data of the SP fraction and the non-SP fraction were used by microarray analysis (Non-patent Document 7). In SP cells, from the list of 268 genes that are up-regulated more than twice, using UniProtKB database (http://www.uniprot.org/) to potentially encode cell surface proteins 56 genes were selected. Furthermore, the surface marker highly expressed by SP fraction was identified using the antibody (commercial item) couple
  • CD13 and CD31 were identified.
  • the expression analysis results of CD13 were 1.64 ⁇ 0.45 (relative value) in the SP fraction and 0.51 ⁇ 0.03 (relative value) in the non-SP fraction (P ⁇ 0.01) (B in FIG. 1).
  • CD31 expression was high in the G2 / M / SP fraction (Fig. 2B) and was not common for hepatoma cells (HuH7, PLC / PRF / 5 and Hep3B). (B in FIG. 1).
  • Non-patent Document 11 Ma S, et al. Identification and characterization of tumorigenic liver cancer stem / progenitor cells. Gastroenterology. 2007; 132 (7): 2542-2556
  • Non-Patent Document 12 Zhu Z, et al. Cancer stem / progenitor cells are highly enriched in CD133 (+) CD44 (+) population in hepatocellular carcinoma.
  • Non-Patent Document 13 Yang ZF, et al. Significance of CD90 + cancer stem cells in human liver cancer. Cancer Cell. 2008; 13 (2): 153-166.
  • Patent Document 14 Yang ZF, et al. Identification of local and circulating cancer stem cells in human liver cancer. Hepatology. 2008; 47 (3): 919-928.).
  • CD133 expression was detected in HuH7 but not in PLC / PRF / 5.
  • CD90 expression was detected in PLC / PRF / 5 but not in HuH7.
  • the expression of CD13 was confirmed in any of Hep3B, HuH7, and PLC / PRF / 5 (C in FIG. 1 and A in FIG. 2). In particular, in HuH7, many CD13 + cells were observed in the fraction (CD13 + CD133 + ) overexpressing CD133.
  • Multi-color analysis with Hoechst staining revealed clear localization of CD13 + cells in the SP fractions of HuH7 and PLC / PRF / 5.
  • the CD13 ⁇ CD133 + and CD13 ⁇ CD90 + fractions were localized in the G1 to G2 fractions, but not in the SP fraction (D in FIG. 1).
  • CD13 is a universal marker candidate closely related to the hepatoma cell SP fraction.
  • no single marker showing a stronger association with the SP fraction than CD13 was found.
  • CD13 is a marker of potentially dormant hepatocellular carcinoma (HCC) cells Considering hematopoiesis and leukemic stem cells are in G0 phase, dormant or poorly proliferating cancer cells are resistant to anticancer drugs It is thought to be related to resistance and cancer recurrence. Therefore, the identification and characterization of cancer cell populations that are dormant or have a low proliferative ability are very important in examining resistance to anticancer drugs and cancer recurrence. CD13 expression of HuH7 and PLC / PRF / 5 and the relationship between the cell cycle of HuH7 and PLC / PRF / 5 were examined.
  • CD13 + fractions were present in the G1 / G0 phase, and CD13 was strongly expressed.
  • the apparent population was localized in the G0 phase.
  • the cell cycle was analyzed using a DNA binding stain (Hoechst blue: Hoechst 33342) and an RNA binding stain (Pyronin Y).
  • the population of CD133 + cells in HuH7 and the fraction of CD90 + cells in PLC / PRF / 5 were distributed in G1 / G0 and G2 / M phases.
  • a relationship between the SP fraction and the G0 cell cycle was also observed, and the SP fraction was clearly localized in the G0 phase even in the absence of reserpine (ABC transporter blocker) (A in FIG. 3).
  • CD13 + CD133 + fractions showed cell fragmentation and apoptotic changes during culture.
  • CD13 + CD90 ⁇ population was mainly in the G0 / G1 phase, and the CD13 + CD90 + population was clearly in the S–G2 / M phase.
  • CD13 ⁇ CD90 + cells were present in all cell cycles, but clearly had a higher proportion in the G2 / M and S phases than the CD13 + CD90 ⁇ population (D in FIG. 3).
  • CD13 + cells are also used in colorectal cancer, which is a type of digestive cancer. It can be seen that it is a cancer stem cell positioned higher in the differentiation hierarchy and a cell resistant to an anticancer agent.
  • CD13 + cells form spheres and create a CD90 + phenotype
  • the formation of spheres is a common feature of stem cells.
  • expression of CD13 in spheres derived from HuH7, PLC / PRF / 5 and clinically obtained HCC was examined.
  • CD13 expression is both HuH7 (67.0%, 33.5-fold increase in the sphere versus control 2.0%) and PLC / PRF / 5 (83.8%, 5.51-fold increase in the sphere versus control 15.2%). It increased (A in FIG. 5). There was no significant difference in the expression of CD133 in HuH7.
  • CD90 expression was decreased in the sphere (2.5% in the sphere, a 14.28-fold increase in the sphere compared to 35.7% in the control).
  • CD13 expression was more immature stem-like than CD133 and CD90, suggesting a dormant population.
  • the sphere obtained from the clinically obtained HCC sample was localized in the CD13 + CD90 ⁇ CD133 ⁇ fraction as in the case of PLC / PRF / 5 (FIG. 5B).
  • the time course of CD13 and CD90 expression in PLC / PRF / 5 was examined.
  • the CD13 + CD90 ⁇ fraction was isolated from the PLC / PRF / 5 sphere and cultured in a serum-containing medium, the CD13 + CD90 + fraction was observed after 96 hours (C in FIG. 5).
  • the isolated CD13 ⁇ CD90 ⁇ fraction induced cell death within a few days and was unable to maintain viability.
  • the isolated CD13 ⁇ CD90 ⁇ fraction rapidly produced a CD13 + CD90 ⁇ fraction within 24 hours (FIG. 5C).
  • CD13 + CD133 + , CD13 ⁇ CD133 + and CD13 ⁇ CD133 ⁇ were confirmed in HuH7.
  • the CD13 + CD133 + fraction was more resistant to DXR than the CD13 ⁇ CD133 + and CD13 ⁇ CD133 ⁇ fractions (FIG. 7B).
  • the CD13 ⁇ CD133 ⁇ fraction showed a high drug resistance, although the cell growth rate was slow in proliferation tests and cell fate studies (FIGS. 7A and C).
  • PLC / PRF / 5 the CD13 ⁇ CD90 + fraction shifted to the CD13 + fraction and the CD90 + fraction decreased (A in FIG. 7).
  • HuH7 and PLC / PRF / 5 cells were irradiated with radiation, and cell surface markers expressed in the cells remaining after irradiation were analyzed. After 24 hours of irradiation, the remaining cells were localized in the CD13 + fraction in HuH7 and in the CD13 + CD90 - fraction in PLC / PRF / 5. After 48 hours of irradiation, the remaining cells started to grow, and CD13 ⁇ CD133 + cells were created with HuH7, and CD13 + CD90 + cells were created with PLC / PRF / 5 (C in FIG. 7). These results corroborate the examination results of changes with time (C in FIG. 6), and show that CD13 + cells exist as a core fraction of the cellular hierarchy.
  • the CD13 - CD90 + fraction was higher in the relapsed sample after hepatic artery embolization than in the sample without hepatic artery embolization (40 in the sample without hepatic artery embolization). Compared to ⁇ 18%, 12 ⁇ 5%: 3.3-fold increase in the sample of recurrence after hepatic artery embolization therapy) (A in FIG. 8). In all 12 clinically obtained HCC samples, the expression pattern was very close to PLC / RLF / 5. Here, the percentage of cells indicates the percentage (%) of cells surviving after mechanical and enzymatic digestion. Most hepatocellular carcinoma cells retain liver cell function, accumulate fat and glycogen, and produce bilirubin. Hepatocyte cancer cells are relatively large compared to other cancer cells and are more susceptible to damage by mechanical and enzymatic digestion.
  • CD13 expression was confirmed in fresh cryosurgical specimens. In cases after hepatic artery embolization, CD13 + HCC cells were present along fibrous capsules forming cell clusters. In cases where hepatic artery embolization was not performed, CD13 + HCC cells usually formed small cell clusters inside the cancer lesion (FIG. 8B). CD13 was expressed on the cell surface in the HCC case. In normal liver samples, CD13 was expressed in sinusoids and bile ducts, clearly different from CD13 expression in HCC samples.
  • HCC recurrence after hepatic artery embolization usually occurs in fibrotic capsules, and drug-resistant viable HCC (chemo-resistant viable HCC) is mainly present around fibrotic capsules, so immunity in samples after hepatic artery embolization Histochemical findings fully support clinical experience.
  • drug-resistant viable HCC chemo-resistant viable HCC
  • CD13 + CD90 + fraction was mainly present in the G2 / M / S phase (A in FIG. 3), this fraction increased after treatment with DXR, which is a DNA synthesis inhibitor.
  • DXR ATP-binding Cassette
  • CD13 is presumed to be responsible for cytoprotection against anticancer drugs (resistance to anticancer drugs).
  • DXR is also known as an ABC transporter-dependent anticancer agent.
  • a DXR resistant HuH7 clone was established that was able to survive 90% of cells with 0.5 ⁇ g / ml DXR (FIG. 10). Note that 99% of the HuH7 parental cells can be killed by the above concentration of DXR.
  • CD13 Inhibition of CD13 was able to suppress cell proliferation to 50% or less in the DXR resistant HuH7 clone (C in FIG. 9). This finding can inhibit the multidrug resistance of cancer cells by inhibiting CD13, resulting in a reduction in the number of cancer cells that can remain after treatment with conventional anticancer drugs by inhibiting CD13 It suggests that you can.
  • CD13 + HuH7 cells have high tumorigenicity.
  • the CD13 + CD133 + fraction formed tumors from 100 cells
  • the CD13 ⁇ CD133 + fraction formed tumors from 1,000 cells
  • the CD13 ⁇ CD133 ⁇ fraction failed to form tumors even from 5,000 cells. .
  • CD13 + cells generated by treatment can be made fresh from the remaining CD90 + cells.
  • cancer stem cells which are CD13 + cells
  • a combination of a CD13 inhibitor such as Ubenimex and an anticancer agent such as 5-FU an anticancer agent based on DNA synthesis inhibition. It was confirmed that it induces apoptosis of cancer stem cells. From this, it is possible to eliminate not only cancer cells but also cancer stem cells that are resistant to cancer treatment by using a combination of a CD13 inhibitor and an anticancer agent, preferably an anticancer agent based on an inhibitory action on DNA synthesis. Treatment is expected to be possible.
  • cancer stem cells which are CD13 + cells
  • a combination of a CD13 inhibitor and an anticancer agent preferably an anticancer agent based on DNA synthesis inhibitory action
  • an anticancer agent preferably an anticancer agent based on DNA synthesis inhibitory action
  • CD13 + confirmed 10 of that cell contains a low level of ROS. Confirmation that DNA strand double strand damage is kept low in CD13 + cells
  • Dormant stem cells that are capable of self-renewal usually have low levels of ROS because intracellular ROS is low, or low ROS is beneficial for cell survival even if ROS levels are not reached immediately It has been reported that the operating principle of vital functions is shifted in the direction of maintaining the above, and that dysregulation of ROS levels impairs the function of stem cells (Non-patent Document 8).
  • Intracellular ROS was measured by 2 ', 7'-dichlorofluorescein diacetate (DCF-DA) staining.
  • DCF-DA 7'-dichlorofluorescein diacetate
  • the CD13 + fraction had a lower ROS concentration than the CD133 strong expression fraction and the CD90 + fraction.
  • ROS was clearly lower in the CD13 + fraction than in the CD13 ⁇ fraction.
  • Treatment with a CD13 neutralizing antibody or a CD13 inhibitor such as Ubenimex significantly increased the ROS concentration in CD13 + cells to the same extent as the ROS concentration in the CD13 ⁇ fraction (FIG. 12A).
  • the CD13 + CD90 ⁇ fraction showed a lower ROS concentration than the CD13 ⁇ CD90 + and CD13 + CD90 + fraction (FIG. 12B).
  • the CD13 + fraction contained MitoSOX, another ROS indicator (FIG. 12C).
  • GCLM a gene capable of encoding glutamine-cysteine ligase
  • Non-patent Document 9 It is known that cell death after cytotoxic chemotherapy and ionizing radiation is partly caused by free radicals (Non-patent Document 9). In this study, a low ROS concentration was shown in the CD13 + cell population, so it was verified whether the CD13 + fraction caused DNA damage. For this verification, purified CD13 + CD90 ⁇ , CD13 + CD90 + , CD13 ⁇ CD90 + , and CD13 ⁇ CD90 ⁇ HCC cells were irradiated and subjected to an alkaline comet assay. There was no significant difference in the extent of DNA damage in cells that were not irradiated after ionizing radiation, but when ionizing radiation was applied, DNA in CD13 + CD90 - cells was higher than in the other three fractions.
  • CD13 is specifically expressed on the surface of cancer stem cells, especially solid stem cancer stem cells of human liver cancer and gastrointestinal cancer (colon cancer), and is involved in the regulation of ROS elimination pathway Thus, it has an essential function for maintaining cancer stem cells.
  • CD13 is a specific marker for cancer stem cells, particularly cancer stem cells of solid cancers such as liver cancer and gastrointestinal cancer (colon cancer).
  • cancer stem cells expressing CD13 are in the cell cycle quiescence, ROS concentration is suppressed low, and DNA double-strand damage is suppressed or double. Since the ability to repair chain damage was activated, it was confirmed that the solid cancer stem cells were extremely resistant to conventional treatments such as chemotherapy and ionizing radiation.
  • problems in cancer treatment such as conventional chemotherapy and ionizing radiation (incurable, recurrence, metastasis) may be caused by CD13 + cells.
  • problems in conventional cancer treatment can be solved, cancer can be cured, and cancer recurrence and metastasis can be prevented.
  • hypoxia the tissue falls into acidosis, and the effects of anticancer drugs and anticancer treatment with radiation are diminished.
  • Hypoxia is considered to cause cancer cells with higher malignancy by further causing genetic changes in tumor cells.
  • cell division is likely to stop and the number of stationary cells increases.
  • Such quiescent cells have high resistance to cancer treatment by anticancer agents and radiation.
  • Hypoxia also occurs when tumor angiogenesis does not catch up with tumor growth (in part because the tumor itself moves its surrounding environment in a direction that favors cancer). It is also known that the anticancer agent itself does not reach the target site at such sites.
  • the cancer stem cells which are CD13 + cells are in a hypoxic state (ROS concentration is suppressed to a low level), they have high resistance to cancer treatment by anticancer agents and radiation irradiation.
  • the present invention uses a combination of a CD13 inhibitor such as a CD13 neutralizing antibody or ubenimex and a cancer treatment with an anticancer agent or radiation to induce apoptosis in cancer stem cells remaining in the conventional therapy. It is effective as a treatment method for cancer cure (cancer radical treatment method) and as a treatment method for preventing cancer recurrence (cancer recurrence prevention therapy).
  • CD13 + cells have DNA double strand damage suppressed or DNA double strand damage repair ability is activated. From this, it is possible to improve sensitivity to anticancer agents by increasing ROS by treating with CD13 inhibitors, and preferably by using anticancer agents having an inhibitory action on DNA synthesis such as 5-fluorouracil as anticancer agents, DNA fragmentation and apoptosis can be guided, and as a result, cancer can be cured and cancer recurrence can be prevented.
  • anticancer agents having an inhibitory action on DNA synthesis such as 5-fluorouracil as anticancer agents
  • SEQ ID NOs: 1 and 2 are the nucleotide sequences of PCR primers used for the amplification of the glutamine-cysteine ligase (GCLM) gene, and SEQ ID NOs: 1 and 2 are glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene sequences. It means the base sequence of PCR primer used for amplification.
  • GCLM glutamine-cysteine ligase
  • GPDH glyceraldehyde-3-phosphate dehydrogenase

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Abstract

L'invention porte sur une molécule fonctionnelle impliquée dans la régulation de cellules souches cancéreuses, laquelle molécule est nouvellement identifiée. Ainsi l'invention décrit : une technique pour détecter de manière exacte des cellules souches cancéreuses ; et une technique pour induire l'apoptose de cellules souches cancéreuses pour traiter complètement le cancer. CD13 est impliqué dans la régulation d'une voie d'élimination d'espèces oxygénées actives dans une cellule souche cancéreuse, et est donc une molécule essentielle pour l'entretien des cellules souches cancéreuses. Ainsi, des cellules souches cancéreuses peuvent être détectées de manière exacte en utilisant CD13 comme indicateur. L'invention porte aussi sur le traitement radical du cancer (particulièrement le cancer du foie et le cancer gastro-intestinal) qui peut être obtenu en utilisant un inhibiteur de CD13 et un agent anticancéreux ou une radiothérapie de manière combinée.
PCT/JP2011/054287 2010-02-26 2011-02-25 Procédé de détection de cellules souches cancéreuses, et agent thérapeutique ou agent prévenant la récurrence de cancer WO2011105551A1 (fr)

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