JP6969778B2 - Treatment of cancer by suppressing the DNA repair pathway, which tends to be erroneous - Google Patents

Description

The present invention relates to a cancer cell growth inhibitor and a late-stage disorder reducing agent containing an SSA inhibitor that tends to target a DNA repair pathway, a concomitant agent containing an SSA inhibitor and a platinum preparation, and an SSA inhibitor and a platinum preparation and / Or related to a cancer treatment method using radiation.

Conventional drug-based cancer treatment methods are always accompanied by soaring medical costs due to expensive anti-cancer drugs, cost-to-life-prolonging effects, side effects and secondary carcinogenic problems, and sustainable anti-cancer drug development is worldwide. Even if you look at it, it is a difficult situation. Further, in view of such a situation, in order to enhance the therapeutic effect of existing anticancer agents, for example, combination therapy with other agents is being studied.

In Patent Document 1, (-) gallocatechin-3-gallate (hereinafter, may be abbreviated as GCG), (-) epigallocatechin-3-gallate (hereinafter, may be abbreviated as EGCG), (-). ) Catechin-3-gallate (hereinafter, may be abbreviated as CG), (-) Epicatechin-3-gallate (hereinafter, may be abbreviated as ECG), etc. Is described in a method of treating cancer in a subject, which comprises administering the subject in combination with an anticancer agent (eg, flaballocatechin, adriamycin, etoposide, taxol, cisplatin). Further, Patent Document 1 also describes a treatment method in which a chemical sensitizer and an anticancer agent are administered at the same time, and a treatment method in which the chemical sensitizer is administered before the anticancer agent.

Patent Document 2 describes a method for treating a proliferative disease in which administration of a composition containing epigallocatechin-3-gallate or the like is combined with radiation therapy, chemotherapy (for example, administration of cisplatin, etc.) and the like. There is.

However, the molecular biological mechanism of side effects of cancer treatment using anticancer drugs such as cisplatin and radiation, such as secondary carcinogenesis, has not been elucidated at present. Research has not progressed on drugs suitable for concomitant use with anticancer drugs for suppressing the occurrence of secondary carcinogenesis, and the dosage and administration for concomitant use, and there is still no effective solution.

Japanese Patent Publication No. 2007-524633 Special Table 2016-514135 Gazette

DNA is damaged in various types by cancer treatment using radiation or anticancer drugs. In order to cope with such damage, cells have various repair mechanisms. For example, in DNA double-strand breaks, which is one of DNA damage, homologous recombination is the repair mechanism. (Hereinafter, it may be abbreviated as HR)), non-homologous end recombination, alternative non-homologous end recombination, microhomology-mediated end-joining, break-induced replication. ), Single-strand annealing (hereinafter, may be abbreviated as SSA) and the like. Of these repair pathways, non-homologous end rebinding, alternative non-homologous end rebinding, microhomology-mediated end binding, DNA strand break-induced replication, and single-stranded annealing are repairs that are prone to DNA sequence errors. It is known as an error-prone repair pathway, which is prone to DNA errors.

Recently, various cancers in which SSA is activated are being discovered. To date, breast cancer and ovarian cancer with mutations in the BRCA1 and BRCA2 genes, and familial adenomatous polyposis due to APC gene mutations have been discovered as examples of special cancers in which SSA is easily activated.

In recent years, EGC has been identified as an error-prone repair pathway inhibitor, and from the results of structural analysis of the protein, this inhibitor EGC mediates van der Waals force at the functional site of Rad52, which is the main protein of the error-prone repair pathway. By making a special binding, the conventional EGC concentration range is 10 to 500 μM (Bigelow and Cardelli, Oncogene, 2006, 25, 1922-30; Sugihara et al., Free Radic Biol Med, 1999, 27, 1313-23. ) Was found to produce a Rad52 inhibitory effect at low concentrations around 1 μM (Hengel et al., Elife, 2016; 5: e14740).

However, molecular studies of radiation and anti-cancer agents, their repair mechanisms, and their association between radiation and anti-cancer agents-induced late effects, such as secondary carcinogenesis, and the SSA inhibitors. Studies on the low-concentration effect of the drug have not progressed, and there is almost no study at the individual level, especially using mice. The reason why research on the low concentration effect of the SSA inhibitor EGC has not progressed is that the anticancer effect of polyfer, which is a component of green tea, is the antioxidant effect of EGCG, which has the highest content in green tea, and the immune system transcription factor NFkB. Surh, Cancer chemoprevention with dietary phytochemicals. 2003, Nat Rev Cancer, 3, 768-780; Yang et al., Cancer prevention by tea: animal studies, molecular mechanisms and human relevance. 2009, Nat Rev Cancer, 9: 429-439). From such a background, EGCG has been intensively studied both at the cellular level and in animal experiments, and EGC having a lower content in green tea and lower physiological activity than EGCG is used, and it is orders of magnitude lower than before. It was difficult to come up with the idea of conducting experiments using cells at concentrations, especially animal experiments.

An object of the present invention is to amplify genetic instability due to accumulation of errors in DNA repair, DNA replication, and DNA recombination, and to cause long-term and side effects of malignant transformation of cancer such as onset of secondary carcinogenesis and metastasis. The purpose is to provide a new therapeutic means that can be suppressed in the absence of cancer.

The present inventor has focused on RECQL4 deficient cells, a tumor suppressor gene related to Rothmund-Thomson syndrome (Kitao et al., Nature Genet, 1999, 22, 82-84), and independently used human B lymphocytic leukemia cells Nalm-. When RECQL4 knock-in cells were established using 6 and phenotypic analysis was performed, it was clarified that RECQL4-deficient cells were highly sensitive to radiation and cisplatin (Kohzaki et al., Carcinogenesis, 2012, 33, 1203-1210). ..

As a result of diligent studies to solve the above problems based on the findings, first, RECQL4 deficient cancer HCT116 cells other than Nalm-6 and non-cancer RECQL4 deficient MCF10A cells were independently established, and the result was due to RECQL4 deficiency. We have found that high sensitivity to radiation and cisplatin is a cancer cell-specific and universal property.

Next, at the molecular level, what kind of DNA repair pathway the RECQL4-deficient cancer HCT116 cells, which are highly sensitive to radiation and cisplatin commonly used in anticancer treatment, survive and induce secondary carcinogenesis. The research was conducted with the aim of clarifying new weaknesses of cancer cells. As a result, we have found the mechanism and specific timing at which cancer cells activate SSA and try to survive after treatment with anticancer drugs and radiation, from the molecular level to the individual level. The present inventors have completed the present invention as a result of further studies based on these findings.

That is, the present invention is as follows.
[1] A cancer cell proliferation inhibitor containing an SSA inhibitor, which is used in combination with chemotherapy or radiation therapy using a platinum formulation, wherein the cancer cell proliferation inhibitor is the platinum formulation to a subject. A proliferation inhibitor that is administered after administration of SSA, or after irradiation with the radiation, before activation of SSA in cancer cells in the subject, or at the time of activation.
[2] The cancer cell growth inhibitor according to [1], wherein the SSA inhibitor is a Rad52 inhibitor.
[3] The Rad52 inhibitor is (-) epigallocatechin or 5-amino-1-((2R, 3R, 4S, 5R) -3,4-dihydroxy-5- (hydroxymethyl) -tetrahydrofuran-2. -Il) -1H-imidazole-4-carboxamide, the cancer cell growth inhibitor according to [2].
[4] The cancer cell growth inhibitor according to any one of [1] to [3], wherein the platinum preparation is selected from the group consisting of oxaliplatin, carboplatin, cisplatin, and nedaplatin.
[5] The above-mentioned one of [1] to [4], wherein the cancer cell growth inhibitor is administered within 2 days after the administration of the platinum preparation or the irradiation with the radiation. Cell growth inhibitor.
[6] The cancer cell proliferation inhibitor according to any one of [1] to [5], wherein the cancer cell is a cancer cell containing Rad52 factor and whose SSA activity is increased.
[7] The cancer cells containing the Rad52 factor and whose SSA activity is increased are selected from the group consisting of RECQL4-deficient cancer cells, BRCA1 and / or BRCA2-deficient cancer cells, and APC-deficient cancer cells [6]. ] The cancer cell proliferation inhibitor according to.
[8] A late disorder reducing agent caused by the chemotherapy or radiation therapy, which comprises an SSA inhibitor and is used in combination with chemotherapy or radiation therapy using a platinum preparation, wherein the late disorder reducing agent is: A late injury reducing agent administered after administration of the platinum preparation to a subject, or after irradiation with the radiation, before activation of SSA in cancer cells in the subject, or at the time of activation.
[9] The late disorder reducing agent according to [8], wherein the late disorder is a secondary carcinogen.
[10] The late disorder reducing agent according to [8] or [9], wherein the SSA inhibitor is a Rad52 inhibitor.
[11] The Rad52 inhibitor is (-) epigallocatechin or 5-amino-1-((2R, 3R, 4S, 5R) -3,4-dihydroxy-5- (hydroxymethyl) -tetrahydrofuran-2. -Il) -1H-imidazole-4-carboxamide, the late disorder reducing agent according to [10].
[12] The late disorder reducing agent according to any one of [8] to [11], wherein the platinum preparation is selected from the group consisting of oxaliplatin, carboplatin, cisplatin, and nedaplatin.
[13] The late disorder reducing agent according to any one of [8] to [12], wherein the late disorder reducing agent is administered within 2 days after the administration of the platinum preparation or the irradiation with the radiation. Agent.
[14] The late-stage disability reducing agent according to any one of [8] to [13], wherein the cancer cell is a cancer cell containing Rad52 factor and whose SSA activity is increased.
[15] The late-stage disorder reducing agent according to [14], wherein the cancer cells are selected from the group consisting of RECQL4-deficient cancer cells, BRCA1 and / or BRCA2-deficient cancer cells, and APC-deficient cancer cells.
[16] A concomitant drug consisting of a platinum preparation and an SSA inhibitor, wherein the SSA inhibitor activates SSA in cancer cells in the subject after administration of the platinum preparation to the subject. A cancer cell proliferation inhibitor administered before or at the time of activation.
[17] The cancer cell growth inhibitor according to [16], which is further used in combination with radiation therapy.
[18] A combination drug consisting of a platinum preparation and an SSA inhibitor, wherein the SSA inhibitor activates SSA in cancer cells in the subject after administration of the platinum preparation to the subject. A late disorder reducing agent caused by administration of the platinum preparation, which is administered before or at the time of activation.
[19] The late disorder reducing agent according to [18], wherein the late disorder is a secondary carcinogen.
[20] A method for treating cancer and reducing late effects caused by chemotherapy and / or radiation therapy, wherein the method requires the following:
(I) After administration of a therapeutically effective amount of platinum preparation and / or irradiation, and (ii) after administration of the platinum preparation and / or irradiation of radiation, SSA in cancer cells in the subject. A method comprising administering a therapeutically effective amount of an SSA inhibitor before or at the time of activation.
[21] The method according to [20], wherein the therapeutically effective amount of the SSA inhibitor is administered within 2 days after administration of the platinum preparation and / or after irradiation with radiation.
[22] The method according to [20] or [21], wherein the cancer cells are selected from the group consisting of RECQL4 deficient cancer cells, BRCA1 and / or BRCA2 deficient cancer cells, and APC deficient cancer cells. ..
[23] The method according to any one of [20] to [22], wherein the cancer is colorectal cancer, ovarian cancer or breast cancer.

According to the present invention, the SSA pathway specifically activated in cancer cells is selectively activated by administering an SSA inhibitor within a predetermined period after chemotherapy or radiotherapy by administration of a platinum preparation. Long-term and side effects such as suppression, induction of genetic instability by further DNA repair, DNA replication, and accumulation of errors in DNA recombination, and associated malignant transformation of cancer such as secondary carcinogenesis and metastasis. It can be effectively suppressed in the absence of it, and is expected to have a therapeutic effect on cancer.

FIG. 1 shows that RECQL4-deficient cells were established for colon cancer cells HCT116 and immortalized breast epithelial cells MCF10A, which are non-cancer cells, using Cas9-CRISPR technology (A and C). FIG. 1 also shows that RECQL4-deficient colorectal cancer cells are highly sensitive to radiation and cisplatin (B). Furthermore, FIG. 1 shows that RECQL4-deficient MCF10A non-cancer cells are not hypersensitive to radiation and cisplatin (D). FIG. 2 shows the results of a visual quantitative analysis of the behavior of DNA repair proteins after radiation and cisplatin treatment in HCT116 cells by fluorescent immunostaining (AF). Further, FIG. 2 shows that the DNA double-strand break recognition markers γH2AX and Rad51 decreased with the passage of time after irradiation (A and B). Furthermore, FIG. 2 shows that the DNA single-strand break recognition markers RPA and Rad52 increased with the passage of time after irradiation (C and D). FIG. 3 shows that Rad52 and RPA2 accumulated in the chromatin fraction over time, and this increase was significant in RECQL4-deficient cells (A and B). FIG. 4 shows the results of the GFP reporter assay, showing a significant increase in SSA activity in RECQL4-deficient cancer cells, but suppression of SSA activity by ectopic expression of the RecQL4 protein (A). ~ C). FIG. 5 shows the results of artificially suppressing SSA activity by siRNA and examining whether SSA activity is necessary for the survival of cancer cells (A and B). FIG. 5 also shows that RECQL4-deficient cancer cells increase SSA activity for survival (B). FIG. 6 shows the results of an experiment to suppress the growth of RECQL4-deficient cancer cells using epigallocatechin and the metabolism regulator AICAR (AF). In addition, FIG. 6 shows that both treatments of epigallocatechin and the metabolic regulator AICAR significantly suppressed RECQL4-deficient cancer cells in a concentration-dependent manner (A to C). Furthermore, FIG. 6 shows that salicylate, which is a direct AMPK activator, and 2-deoxy-D-glucose treatment, which is an indirect AMPK activator, do not show a specific inhibitory effect on RECQL4-deficient cancer cells. It shows that (DF). FIG. 7 shows the suppressive effect of cisplatin-induced genome instability by EGC treatment. FIG. 8 shows a xenograft experiment in which p53-deficient HCT116 cells, RECQL4-deficient HCT116 cells, and their wild-type cells are subcutaneously administered to BALB / cAJcl-nu / nu mice to examine their ability to grow cancer cells in vivo. The results are shown (A to C). FIG. 8 shows that the growth inhibitory effect of the AICAR treatment was greater in the RECQL4 deficient cells than in the p53-deficient HCT116 cells and the wild-type cells (A to C). FIG. 9 shows the results of oral administration of epigallocatechin to mice (A to C). FIG. 9 shows that the growth inhibitory effect of EGC was significantly observed in the RECQL4 deficient cancer cells as in the result of AICAR shown in FIG. 8 (A to C). FIG. 10 shows that a synergistic cancer cell inhibitory effect was obtained by simultaneous treatment of AICAR and cisplatin in RECQL4-deficient cancer cells (A). In addition, FIG. 10 shows cancer cells that are more effective than the treatment in the first 24 hours by treating with an SSA inhibitor at the timing when the SSA is delayedly activated 16 hours after the treatment with cisplatin. It shows that the inhibitory effect was obtained (B). FIG. 11 shows the synergistic effect of suppressing the growth of cancer cells by the combined use of cisplatin and EGC at the individual mouse level (A to E). FIG. 12 is a schematic diagram relating to the treatment of cancer by suppressing the DNA repair pathway, which tends to be erroneous.

In the present specification, the abbreviations such as (poly) nucleotides are referred to as the IUPAC-IUB Communication on Biological Nomenclature, Eur. J. Biochem., 138: 9 (1984)], "base sequence or amino acid. Follow the "Guidelines for Preparation of Specifications, etc. Containing Sequences" (edited by the Japanese Patent Office) and the idiomatic symbols in the field.

1. 1. Definitions The definitions of terms herein are described below, but the following definitions do not limit the scope of the invention.

As used herein, the term "error-prone (DNA) repair pathway" refers to, among various DNA repair mechanisms possessed by cells, for example, non-homologous end reconnection, alternative non-homologous end reconnection, microhomology-mediated end binding, and DNA strand. It refers to a repair pathway mediated by cleavage-induced replication and single-stranded annealing (SSA), and more preferably a repair pathway mediated by single-stranded annealing (SSA).

Other terms used herein may be used in the meaning commonly used in the art, unless otherwise noted.

2. 2. Agents and Concomitant Agents of the Present Invention The present invention uses (1) a cancer cell growth inhibitor containing an SSA inhibitor, which is used in combination with chemotherapy or radiotherapy using a platinum preparation, and (2) a platinum preparation. Provided are late injury reducing agents resulting from the chemotherapy or radiation therapy, including SSA inhibitors, which are used in combination with chemotherapy or radiation therapy.
The cancer cell growth inhibitor and the late-stage disorder reducing agent of the present invention are used after administration of a platinum preparation to a subject or after irradiation with radiation, before activation of SSA in cancer cells in the subject, or at the time of activation. It is characterized by being administered.

The present invention also provides (3) a combination drug consisting of a platinum preparation and an SSA inhibitor, and the SSA inhibitor is used in cancer cells in the subject after administration of the platinum preparation to the subject. It is characterized by being administered before or during activation of SSA.
The concomitant drug of the present invention can be formulated into a single preparation by using a platinum preparation and an SSA inhibitor as a mixture, but it is preferable that the platinum preparation and the SSA inhibitor are formulated separately.
The concomitant agent of the present invention is suitable as a cancer cell growth inhibitor or a late disorder reducing agent caused by administration of a platinum preparation.

The "chemotherapy" of the present invention specifically means chemotherapy for cancer, and refers to general treatment of cancer with an anticancer drug.

The "platinum preparation" of the present invention refers to an anticancer agent classified into a platinum complex, and specific examples thereof include cisplatin, carboplatin, oxaliplatin, and nedaplatin, with preference given to cisplatin.

The "radiation therapy" of the present invention is a treatment for the purpose of irradiating a target malignant tumor portion with radiation to suppress the growth of cancer cells, and the radiation used for the treatment is X-ray. , Electron beam, γ ray, particle beam and the like. Further, in the radiation therapy of the present invention, a radiation dose close to the lethal dose of cancer cells may be irradiated, or a radiation dose less than the lethal dose may be irradiated, specifically, local irradiation or repeated irradiation. Including, for example, it may be in the range of 0.01 mGy to 100 Gy / day.

The "SSA inhibitor" of the present invention is used interchangeably with the "error-prone repair pathway inhibitor" described herein, attenuating or eliminating SSA activation and SSA-mediated DNA repair. Refers to an agent that can suppress. Specifically, for example, a Rad52 inhibitor is mentioned, and more specifically, for example, (-) epicatechin, (-) epigallocatechin (hereinafter, may be abbreviated as EGC), (-) epi. Catechin gallate, (-) Epigallocatechin gallate, 5-amino-1-((2R, 3R, 4S, 5R) -3,4-dihydroxy-5- (hydroxymethyl) -tetrahydrofuran-2-yl) -1H- Examples thereof include imidazole-4-carboxamide (hereinafter, may be abbreviated as AICAR). In the present invention, (-) epigallocatechin or 5-amino-1-((2R, 3R, 4S, 5R) -3,4-dihydroxy-5- (hydroxymethyl) -tetrahydrofuran-2-yl) -1H -Imidazole-4-carboxamide is preferred. Further, here, "activation of SSA" means that DNA repair by SSA is enhanced in cells by chemotherapy, radiotherapy, or the like.

The "object" of the present invention is a mammal including humans, and the mammals include, for example, rodents such as mice, rats, hamsters and guinea pigs, experimental animals such as rabbits, pigs, cows and goats. , Livestock such as horses, sheep and minks, pets such as dogs and cats, humans, monkeys, crab monkeys, red-tailed monkeys, marmosets, orchid utans, chimpanzees, primates such as gorillas and the like.

The "cancer" of the present invention refers to all malignant tumors. The types of malignant tumors to which the agents and concomitant agents of the present invention can be applied include, for example, pediatric brain tumors selected from the group consisting of stellate cell tumors, malignant medullary blastomas, embryonic cell tumors, cranial pharyngeal tumors and lining tumors; Adult brain tumors selected from the group consisting of glioma, glioma, medullary carcinoma, pituitary adenomas and neurosal carcinoma; maxillary sinus cancer, pharyngeal cancer (eg, nasopharyngeal cancer, mesopharyngeal cancer, hypopharyngeal cancer), laryngeal Head and neck cancer selected from the group consisting of cancer, oral cancer, lip cancer, tongue cancer and parotid adenocarcinoma; chest cancer and tumor selected from the group consisting of small cell lung cancer, non-small cell lung cancer, thoracic adenomas and mesotheloma Gastrointestinal cancer and tumor selected from the group consisting of esophageal cancer, liver cancer, primary liver cancer, bile sac cancer, bile duct cancer, gastric cancer, colon cancer, colon cancer, rectal cancer, anal cancer, pancreatic cancer and pancreatic endocrine tumor; Urinary and tumors selected from the group consisting of renal pelvis / urinary tract cancer, renal cell carcinoma, testicular tumor (also called testicle tumor), prostate cancer, bladder cancer, Wilms tumor and urinary tract epithelial cancer; Gynecologic cancers and tumors selected from the group consisting of cervical cancer, uterine body cancer, endometrial cancer, uterine sarcoma, villous cancer, vaginal cancer, breast cancer, ovarian cancer and ovarian germ cell tumor; soft sarcoma in adults and children Bone tumors selected from the group consisting of osteosarcoma and Ewing tumors; Endocrine tissue cancers and tumors selected from the group consisting of adrenal cortex cancer and thyroid cancer; Malignant lymphoma, non-Hodgkin lymphoma, Hodgkin's disease, multiple myeloma, Malignant lymphoma and leukemia selected from the group consisting of plasmacytoid tumor, acute myeloid leukemia, acute lymphocytic leukemia, adult T-cell leukemia lymphoma, chronic myeloid leukemia and chronic lymphocytic leukemia; or chronic myeloproliferative disease, malignant Examples include skin cancers and tumors selected from the group consisting of melanoma, spinous cell carcinoma, basal cell carcinoma and fungal cystitis.

The "cancer cells having increased SSA activity containing Rad52 factor" in the present invention are, for example, cancer cells having increased SSA activity containing Rad52 factor due to chemotherapy, radiotherapy, etc., and treatment thereof. Means cancer cells in which SSA activity containing Rad52 factor is increased irrelevantly, but is preferably cancer cells in which SSA activity including Rad52 factor is increased due to chemotherapy, radiotherapy, or the like. In addition, "cancer cells whose SSA activity including Rad52 factor is increased due to chemotherapy or radiotherapy" may or may not be activated before chemotherapy or radiotherapy. , If activated, means cancer cells whose activity is further increased after chemotherapy or radiation therapy. Examples of cancer cells containing Rad52 factor and increased SSA activity include, but are not limited to, RECQL4 deficient cancer cells, BRCA1 and / or BRCA2-deficient cancer cells, and APC-deficient cancer cells.

The "RECQL4 deficient cancer cell" of the present invention is a cancer cell having a mutation in the RECQL4 gene, and as a result, a cancer cell in which the CHECKCL4 protein does not have a normal function, particularly a cancer in which the helicase activity of RECQL4 is lost. Refers to a cell. Examples of RECQL4 deficient cancer include osteosarcoma, lymphoma, and skin cancer (Wang, Am J Med Genet, 2001, 102, 11-17; Siitonen, Eur J Hum Genet, 2009, 17). , 151-158), but not limited to these.

The "BRCA1 and / or BRCA2-deficient cancer cells" and "APC-deficient cancer cells" of the present invention are breast cancer and ovarian cancer cells due to BRCA1 gene and / or BRCA2 gene mutation, and familial colon adenomatosis due to APC gene mutation. Examples include, but are not limited to, cells.

The "late disorder caused by chemotherapy or radiation therapy" of the present invention means a health problem that occurs months to several decades after the end of treatment with chemotherapy or radiation therapy. For example, secondary carcinogenesis, metastasis, endocrine disorder, motor disorder and the like. In addition, "secondary carcinogenesis" is a type of cancer that is different from the original disease months to decades after treatment due to cell damage caused by anticancer drugs or radiation. Or to cause leukemia.

Regarding the timing of administration of the cancer cell growth inhibitor and the late-stage disorder reducing agent of the present invention, the agent may be administered together with the platinum preparation to the subject or at the time of irradiation with radiation, but the agent may be administered to the subject. It is preferable to administer after administration of the platinum preparation, after irradiation with radiation, before activation of SSA in cancer cells in the subject, or at the time of activation. Specifically, it is preferably within 2 days after administration of the platinum preparation to the subject or after irradiation with radiation, more preferably within 8 to 44 hours, further preferably within 16 to 40 hours, and particularly preferably within 20 to 36 hours. preferable.

Regarding the administration timing of the concomitant agent of the present invention, the platinum preparation in the concomitant agent and the SSA inhibitor may be simultaneously administered to the subject, but after the administration of the platinum preparation in the concomitant agent to the subject, the cancer cells in the subject. It is preferable to administer the SSA inhibitor in the concomitant agent before or at the time of activation of the SSA in the medium. Specifically, it is preferably within 2 days after administration of the platinum preparation to the subject or after irradiation with radiation, more preferably within 8 to 44 hours, further preferably within 16 to 40 hours, and particularly preferably within 20 to 36 hours. preferable.

As shown in Examples described later, the cancer cell growth inhibitor and the late-stage disorder reducing agent of the present invention can be administered to a subject at the above-mentioned predetermined time, and after the anticancer agent or radiation treatment, can be used. It specifically inhibits the mechanism by which cells activate SSA to survive, resulting in suppression of cancer cell growth, as well as DNA repair, DNA replication, and DNA repair induced by anticancer drugs and radiation treatment. Late disorders caused by genetic instability due to accumulation of errors in DNA recombination can also be reduced, and a long-term therapeutic effect on cancer can be expected without side effects.

As shown in Examples described later, the concomitant agent of the present invention can be obtained by administering the SSA inhibitor of the concomitant agent, in particular, within a predetermined period after administration of the platinum preparation of the concomitant agent to a subject. It specifically inhibits the mechanism by which cancer cells activate SSA to survive, resulting in suppression of cancer cell growth and the accumulation of errors in DNA replication induced by anticancer drugs and radiation treatment. Late disorders caused by genetic instability due to the disease can also be reduced, and a long-term, side-effect-free cancer therapeutic effect can be expected.

The conditions of use other than the administration time of the cancer cell growth inhibitor, the late disorder reducing agent, and the concomitant agent of the present invention are not particularly limited, and are, for example, depending on the administration target, administration route, degree of disease, and the like. , Administration form, administration method, dose and the like can be appropriately set, and specific examples thereof are as follows.

The cancer cell growth inhibitor, late injury reducing agent, and concomitant agent of the present invention are used by methods known per se, for example, using conventional mixing, granulation, coating, solubilization, freeze-drying, and the like. Can be manufactured.

In addition, the cancer cell growth inhibitor, the late disorder reducing agent, and the concomitant agent of the present invention may contain any pharmaceutically acceptable carrier, for example, various conventional organic or inorganic carrier substances, for example, solid. Examples thereof include excipients, lubricants, binders, disintegrants in pharmaceuticals, solvents in liquid formulations, solubilizing agents, suspending agents, tonicity agents, buffering agents, soothing agents and the like.

Further, the cancer cell growth inhibitor, the late disorder reducing agent, and the concomitant agent of the present invention are added with ordinary preservatives, antioxidants, colorants, sweeteners, adsorbents, wetting agents, etc., if necessary. An appropriate amount of the substance may be contained.

Examples of the excipient include lactose, sucrose, D-mannitol, starch, cornstarch, crystalline cellulose, light anhydrous silicic acid and the like.
Examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica and the like.
Examples of the binder include crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone, starch, gelatin, methyl cellulose, sodium carboxymethyl cellulose and the like.
Examples of the disintegrant include starch, carboxymethyl cellulose, calcium carboxymethyl cellulose, sodium carboxymethyl starch, L-hydroxypropyl cellulose and the like.
Examples of the solvent include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil, olive oil and the like.
Examples of the solubilizing agent include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like.
Examples of the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glycerin monostearate; for example, polyvinyl alcohol, polyvinylpyrrolidone, and the like. Examples thereof include hydrophilic polymers such as sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose and hydroxypropyl cellulose.
Examples of the tonicity agent include glucose, D-sorbitol, sodium chloride, glycerin, D-mannitol and the like.
Examples of the buffering agent include buffer solutions such as phosphates, acetates, carbonates and citrates.
Examples of the soothing agent include benzyl alcohol and the like.
Examples of the preservative include paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.
Examples of the antioxidant include sulfites, ascorbic acid, α-tocopherol and the like.

The dosage forms of the cancer cell growth inhibitor, the late disorder reducing agent, and the concomitant agent of the present invention include liquids, tablets, rounds, drinking liquids, powders, suspending agents, emulsions, granules, extracts, and fine granules. Agents, syrups, soaking agents, decoctions, eye drops, troches, paps, liniments, lotions, eye ointments, plasters, capsules (including soft capsules), suppositories, enema, injections (liquids, liquids, Suspensions, etc.), patches, ointments, jellies, pasta, inhalants, creams, sprays, nasal drops, aerosols, sustained-release preparations (eg, sustained-release microcapsules), fast Examples thereof include a release-release preparation, which is preferably a sustained-release preparation capable of sustaining the efficacy for a long time at the timing when the inhibitory effect on SSA activation can be most expected.

The cancer cell growth inhibitor, the late-stage disorder reducing agent, and the concomitant agent of the present invention are safely administered by a method according to various forms when used. For example, in the case of an external preparation, it is directly sprayed, affixed or applied to a required site such as skin or mucous membrane, and in the case of tablets, rounds, drinking solutions, suspensions, emulsions, granules and capsules, it is oral. It is administered intravenously, intramuscularly, intradermally, subcutaneously, intra-articularly, intraperitoneally or intratumorally in the case of injections, and intrarectally in the case of suppositories.

When the platinum preparation and the SSA inhibitor are separately formulated for the concomitant agent of the present invention, each preparation may be administered by the same method, or each preparation may be administered by a different method. ..

The content of the SSA inhibitor contained in the cancer cell growth inhibitor and the late-stage disorder reducing agent of the present invention is about 0.01 to 100% by weight, preferably about 0.1, based on the total amount of the usual preparation. It is about 50% by weight.

Further, regarding the concomitant agent of the present invention, the content of the platinum preparation and the SSA inhibitor in the concomitant agent is about 0.01 to 100% by weight, preferably about 0.1, respectively, with respect to the entire usual preparation. It is about 50% by weight.

The content of additives such as a pharmaceutically acceptable carrier that can be contained in the cancer cell growth inhibitor, the late disorder reducing agent, and the concomitant agent of the present invention varies depending on the form of the preparation, but is usually the whole preparation. On the other hand, it is about 1 to 99.99% by weight, preferably about 10 to 90% by weight.

The doses of the cancer cell growth inhibitor, the late-stage disorder reducing agent, and the concomitant agent of the present invention are the activity and type of the active ingredient, the administration mode (for example, oral and parenteral), the degree of the disease, and the animal to be administered. It can be appropriately selected depending on the species, drug acceptability to be administered, body weight, age and the like.

For example, the dose of the cancer cell growth inhibitor and the late disorder reducing agent of the present invention is such that the SSA inhibitor contained in the agent (-) epigallocatechin is used per day for an adult (body weight 60 kg). It is usually about 5 mg to 1000 mg, preferably about 50 mg to 100 mg, and for example, 5-amino-1-((2R, 3R, 4S, 5R) -3,4-dihydroxy-5- (hydroxymethyl) -tetrahydrofuran. -2-yl) -1H-imidazole-4-carboxamide is usually about 20 mg to 20 g, preferably about 200 mg to 2000 mg.

Further, for example, the dose of the concomitant drug of the present invention is usually about 1 mg to 1000 mg, preferably about 10 mg to 100 mg of the platinum preparation in the concomitant drug, for example, cisplatin, per day for an adult (body weight 60 kg). Yes, and the SSA inhibitor in the combination, eg, (-) epigallocatechin, is usually on the order of 5 mg to 1000 mg, preferably on the order of 50 mg to 100 mg, and also, for example, 5-amino-1-((2R). , 3R, 4S, 5R) -3,4-dihydroxy-5- (hydroxymethyl) -tetrahydrofuran-2-yl) -1H-imidazole-4-carboxamide is usually about 20 mg to 20 g, preferably 200 mg to 2000 mg. Degree.

The amount of the above-mentioned platinum preparation and SSA inhibitor can be administered once to three times a day. In addition, when cisplatin treatment or irradiation is performed multiple times, it is preferable to administer the SSA inhibitor at the dosage and administration described in the present specification each time.

Although the present invention has been described above by taking an agent and a concomitant agent as an example, it may also be used in the case of a method for treating the cancer of the present invention and reducing side effects caused by chemotherapy and / or radiotherapy. , The above is used as it is.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

(Materials and methods)
Example 1: Cell culture 10% of fetal bovine serum (FBS) (Sigma, Cat. No. 172012) obtained by heat-inactivating HCT116 cells at 56 ° C. for 30 minutes, and 100 U / ml penicillin + 0. In a Dulvecco's modified Eagle's medium (Wako, Cat. No. 043-30085) culture medium containing 1% of 1 mg / ml streptomycin (Wako, Cat. No. 168-23191). The cells were cultured using a humidified 37 ° C. incubator.

Horse serum (Bioconcept, Cat. No. 205F00I), 1 μM dexamethasone (Sigma, Cat. No. D8893), 5 μg / ml insulin (Sigma), in which MCF10A cell culture was heat-inactivated by treatment at 56 ° C. for 30 minutes. , Cat. No. I9278), human EGF 10 ng / ml (Sigma, Cat. No. E9644), and 100 U / ml penicillin + 0.1 mg / ml streptomycin (Wako, 168-23191) in 1% Dalveco modified eagle medium. -F12 (DMEM / F12) (Wako, Cat. No. 048-29785) was cultured in a humidified 37 ° C. incubator using a humidified 37 ° C. incubator (Costantino et al., Science, 2014, 343, 88-91).

Example 2: Vector construction A RECQL4 knock-in vector was constructed by introducing a stop codon at the 493th amino acid site of the RecQL4 protein (Kohzaki et al., Carcinogenesis, 2012, 33, 1203-1210). A full-length RecQL4 cDNA was inserted into the EcoRI site of the pApuro vector to prepare a RecQL4 protein expression vector (Takata et al., EMBO J, 1994, 13, 1341-1349). Regarding the expression of RecQL4 protein, transfection with Amaxa nucleofector 2b (Lonza, Nucleofector kit V, Cat. No. VCA-1003; program, D-032), X-tremeGENE HP DNA (Roche, Cat. No. 6366244001), And Lipofectamine LTX with PLUS Regent (Thermo Fisher Scientific, Cat. No. 15338100) was used for transient expression, and the expression level was quantified by the Western transfection method.

Example 3: Knock-in cell establishment by Cas9-CRISPR technology Using Cas9-CRISPR technology (Cong et al., Science, 2013, 339, 819-823), sgRNA1 targeting the RECQL4 gene exon6 and sgRNA2 targeting exon9. A primer pair for fabrication was used.

Amaxa nucleofector for ^{1 × 10 6} HCT116 cells using a total of 2 g of DNA of 0.5 g Cas9 sgRNA1, 0.5 g Cas9 sgRNA2 and 1 g EQUL4 knock-in construct linearized with restriction enzyme PvuI. Transfection with 2b was performed (Lonza, Nucleofector kit V, Cat. No. VCA-1003; program, D-032). After 24 hours, a single clone was established by culturing 6 g / ml or 10 g / ml concentration of Blasticidin (Bsr; Wako, Cat. No. 029-18701) medium on a 96-well plate. After a few weeks, drug-resistant single colonies were amplified and knock-in cells were established by screening.

Example 4: Colonization method The susceptibility to drugs and radiation was determined by the colonization method. HCT116 cells were seeded and adhered to plates containing 3 ml (6 hole plate), 1 ml (12 hole plate), and 0.5 ml (24 hole plate) culture medium. The next day, cisplatin / CDDP / cis-diaminedichloro-platinum (II); CAS 15663-27-1 (10-20 g / ml) (Wako, Cat No. 033-20091) was added to the adhered cells, 2 After culturing in a 37 ° C. incubator for hours and washing with PBS, normal medium was added. Irradiation was ^{performed using a 137} Cs Gammacelll 40 Exactor (0.7 Gy / min; MDS Nordion). In each case, after culturing for about 2 weeks, the colonies were fixed with methanol and then stained with Giemsa, and the number of colonies was counted.

Example 5: Western blotting method Using the conventional Western blotting method (Laemmli, Nature, 1970, 227, 680-5), the following antibody: mouse anti-α-Actinin (1/2000, Millipore, clone AT6 / 172) , Cat. No. 05-384), mouse anti-β-Tubulin (1/2000, Wako, Cat. No. 014-25041), rabbit anti-Rad51 (1/2000, Bioacademia, Cat. No. 70-001) ), Mouse anti-Rad51 (1/1000, GeneTex, 14B4, Cat. No. GTX70230), mouse anti-phospho-Histone H2A. X (Ser139) (1/2000, Millipore, clone JBW301, Cat. No. 05-636), rabbit anti-Rad52 (1/1000, SantaCruz, H-300, Cat. No. sc-8350), rabbit anti- Rad52 (1/1000, Abcam, Cat. No. ab103067), rabbit anti-RPA32 (1/1000, GeneTex, Cat. No. GTX70258), rabbit anti-RecQL4 (1/1000, Novus, Cat. No. 25470002) , Rabbit anti-Histone H3 (1/2000, Cell Signaling, Cat. No. 9715) was used to quantify the amount of each protein.

Example 6: Fluorescent immunostaining method Wild-type and RECQL4-deficient HCT116 cells were seeded on a 12-well plate containing a cover glass (Matsunami Glass, Cat. No. C015001), and cultured and adhered for 2 days or more. Irradiate the adhered cells with 2%, 8 and 20 hours later with 2% sucrose (Wako, Cat. No. 193-00025) and 3% paraformaldehyde (Wako, Cat. No. 160-16061). The solution was prepared with PBS and fixed at room temperature for 15 minutes. Treat with 0.5% Triton-X100 (Wako, Cat. No. 160-24751) PBS solution for 5 minutes at room temperature to allow the cell membrane to permeate and for at least 30 minutes 1% BSA (Roche, Fraction V, Cat. No. . 10735078001) Blocking treatment was performed with the containing PBS solution. The primary antibodies used in the fluorescent immunostaining method are as follows: rabbit anti-Rad51 (1/1000, Bioacademia, Cat. No. 70-001), mouse anti-Rad51 (1/500, GeneTex, 14B4, Cat. No. GTX70230), mouse anti-fluor-Histone H2A.X (Ser139) (1/1000, Millipore, clone JBW301, Cat. No. 05-636), rabbit antibody-Rad52 (1/500, SantaCruz, H-300, Cat) . No. sc-8350), rabbit antibody-Rad52 (1/500, Abcam, Cat. No. ab103067), rabbit antibody-RPA32 (1/500, GeneTex, Cat. No. GTX70258). After reacting these primary antibodies at room temperature for 1 hour, they were washed 3 times in 5 minutes with PBS containing 0.05% Tween 20 (MP Biomedicals, Cat. No. 103168) (hereinafter, may be abbreviated as PBST). bottom. Next, Alexa Fluor 594-conjuged goat anti-mouse IgG (1/2000, Thermo Fisher, Cat. No. A11037) and Alexa Fluor 488-conjuged goat anti-mouse IgG (1/2000, Thermo Fisher, Cat. No.). After reacting with the secondary antibody of A11001) at room temperature for 45 minutes, it was washed twice with PBST for 5 minutes. Finally, it was stained with DAPI (Dojindo, Cat. No. 342-07431), washed once with PBST for 5 minutes, and then encapsulated with Fluoromount-G (Southern Biotech, Cat. No. 0100-01). The prepared sample was observed with a Zeiss AxioObsaver fluorescence microscope, and the foci of each protein was counted and analyzed.

Example 7: Concentration of chromatin fraction A method for concentrating chromatin fraction was performed without cross-linking with formaldehyde (Mendez and Stillman, MCB, 2000, 20, 8602-12; Petermann et al., Mol Cell, 2010. , 37, 492-502). 2 × 10 ⁷ HCT116 cells were collected and containing the cells in a hypotonic buffer (10 mM HEPES (pH 7) (Wako, Cat. No. 340-08233), 50 mM NaCl (Wako, Cat. No. 191-01665). ), 0.3M sucrose, 0.5% Triton X-100, protease inhibitor cocktail (Roche, Cat. No. 05892791001)) was treated on ice for 10 minutes and centrifuged at 1500 g for 5 minutes to remove cellular proteins. ..

Next, nuclear buffer (10 mM HEPES (pH 7), 200 mM NaCl, 1 mM EDTA (Wako, Cat. No. 311-90075), 0.5% NP-40 (Wako, Cat. No. 145-09701), protease. The inhibitor cocktail) was treated on ice for 10 minutes and centrifuged at 13000 g for 2 minutes to remove the nuclear soluble fraction protein. Add lysis buffer (10 mM HEPES (pH 7), 500 mM NaCl, 1 mM EDTA, 1% NP-40, protease inhibitor cocktail) to the pellet, mix well, and then sonicate with low amplitude (level 3) (TAITEC, VP). -15S) was performed 3 times in 10 seconds, and after centrifugation at 13000 g for 30 seconds, the supernatant was transferred to a new Eppendorf tube, and the amount of protein was measured by a DC protein assay (BioRad, Cat. No. 5000116JA) based on the Lowry method. Measured and 40 μg or less of protein was used for western blotting.

Example 8: DNA double-strand break repair assay using GFP reporter An hprtSAGFP vector was used to measure single-strand annealing, which is an error-prone repair pathway (Addgene, plasmid No. 41594). KpnI / SacI linearized hprtSAGFP vector was introduced at Amaxa into wild-type and RECQL4-deficient HCT116 cells (Stark et al., MCB, 2004, 24, 9305-9316), and 24 hours later, puromycin (Wako) in 96-well plates. , Cat. No. 160-23151) was cultured in a medium containing the cells to obtain several clones that stably retained the vector. Then, clones expressing I-SceI and becoming GFP positive were established with the pCBASceI vector (Addgene, plasmid No. 26477), and the repair efficiency was quantified using these several clones.

Example 9: siRNA Treatment and Rad52 Inhibitor Treatment AllStars Negative Control siRNA (Qiagen, Cat. No. SI03650318) was used as a control. The sequence information of siRNA of Rad52 is siRNA # 1; 5'-GGAGUGACUCAAGAAUUAATT-3'(SEQ ID NO: 5) and siRNA # 2; 5'-GGCCCAGAAUACAUAAGUATT-3'(SEQ ID NO: 6). Equal amounts were mixed and introduced into cells by HiPerFect Transfection Reagent (Qiagen, Cat. No. 301704) and used in the experiment 36 to 48 hours later. Rad52 inhibitors are (-) epigallocatechin (EGC); CAS 970-74-1 (Tokyo Chemical Industry, Cat. No. E1084) and 5-amino-1-((2R, 3R, 4S, 5R)). -3,4-dihydroxy-5- (hydroxymethyl) -tetra-2-yl) -1H-imidazole-4-carboxamide (AICAR); CAS 2627-69-2 (Wako, Cat. No. 011-22533) Using. AMPK activators other than AICAR are salicylate as a direct activator; CAS 54-21-7 (Wako, Cat. No. 191-03142) as an indirect activator, and 2-deoxy-D-glucose as an indirect activator. Hereinafter, it may be abbreviated as 2DG); CAS 154-17-6 (Wako, Cat. No. 040-06481) was used. A PBS solution was prepared for each drug, stored at -30 ° C after dispensing, and thawed at the time of use to be used as an inhibitor solution under the same conditions.

Example 10: Examination of the effect of EGC treatment on cisplatin-induced genomic deletion In order to eliminate genomic heterogeneity between HCT116 cells, cells were seeded so that one cell would fit into one hole using a 96-well plate. , and cultured for 3 weeks increases the single cells to 1x10 ^6, using this cell as a control genome. The control cells were treated with or without cisplatin treatment, and the cells were seeded using a 96-well plate so that one cell could enter one well, and cultured in EGC-containing medium or normal medium for 2 weeks. The EGC-containing medium at 2 weeks was replaced with regular medium, increased to 1x10 ⁶ one cell and cultured in total 3 weeks, using these cells as target genome, were compared analysis of the control genome (FIG. 7A). Array CGH (comparative genomic hybridization; array) is used with Affilix's CytoScan (registered trademark) HD Array (Tokushima University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Comprehensive Research Support Center), and Affilix's analysis software CytoScan (registered trademark) HD. Comparative analysis by the monochromatic method was performed using the Trademark Analysis Suite (ChAS) (FIG. 7B).

Example 11: Xenograft experiment using BALB / cAJcl-nu / nu mice A mouse experiment was performed based on an animal experiment plan approval application (AE15-016) approved by the Institute of Industrial Medicine. Logarithmically grown 3 × 10 ⁶ wild-type, RECQL4 deficient, and p53-deficient HCT116 cells were collected by centrifugation, washed with PBS, and the cell pellet was sufficiently suspended on ice with 100 μl PBS. An equal amount of 100 μl Matrigel® (Corning, Cat. No. 356234) was added and suspended well on ice to prepare a 1: 1 cell solution for administration. A total of two dosing cell solutions placed on ice were subcutaneously administered to the left and right flanks of 6-8 week old female BALB / cAJcl-nu / nu mice (Buzzai et al., Cancer Res, 2007, 67, 6745-52). Mice were weighed once a week and cancer volume (mm ³ ) was measured twice a week. The cancer capacity (mm ^{3 ) was calculated using the formula of d 2} × D / 2 (≈6 / π) with d as the minimum diameter and D as the maximum diameter.

(result)
B lymphocytes such as Nalm-6 are well known to be highly sensitive to radiation. Therefore, in order to confirm that the radiation and cisplatin hypersensitivity of RECQL4-deficient cells are a common phenotype in cancer cells, colon cancer cells HCT116 and immortalized breast epithelial cells MCF10A, which are non-cancer cells, are used. In contrast, RECQL4-deficient cells were established using Cas9-CRISPR technology (FIGS. 1A and C). As a result, it was confirmed that RECQL4-deficient colorectal cancer cells were also highly sensitive to cisplatin (Fig. 1B), but interestingly, RECQL4-deficient MCF10A non-cancer cells did not show high sensitivity (Fig. 1D). From these results, it was found that deficiency of RECQL4 function in cancer cells makes them highly sensitive to radiation and cisplatin.

Next, in order to clarify the molecular mechanism of radiation and cisplatin hypersensitivity, the behavior of DNA repair proteins after radiation and cisplatin treatment in HCT116 cells was visually quantitatively analyzed by fluorescent immunostaining. Rad51 (Shinohara et al., Cell, 1992, 69, 457-470), which is a major factor in homologous recombination repair (HR), which is one of the DNA double-strand break repair pathways, is a DNA double-strand break recognition marker. Similar to γH2AX (Rogakou et al., JBC, 1998, 273, 5858-68), it decreased over time after irradiation (FIGS. 2A and B). On the other hand, RPA, which is a DNA single-strand break recognition marker (Raderschall et al., PNAS, 1999, 96, 1921-1926), increased with the passage of time after irradiation (Fig. 2C). RPA is a major factor in single-strand annealing repair (SSA), which is one of the DNA double-strand break repair pathways, and the behavior of Rad52, another major factor in SSA, was also examined (Shinohara and Ogawa, Nature, 1998). , 391, 404-407), and showed the same behavior as RPA (Fig. 2D). Accumulation of RPA and Rad52 proteins was significantly increased in RECQL4-deficient cells and suppressed by ectopic expression of RecQL4 protein (FIGS. 2E and F). From this result, it was found that in RECQL4 deficient cells, the SSA repair pathway is significantly activated by DNA damage caused by radiation or cisplatin treatment.

It was found that double-strand breaks in DNA and the chromatin region that folds DNA are closely related, the heterochromatin region that is tightly folded is difficult to cut, and the euchromatin region that is loosely folded is easy to cut. (Goodarzi et al., Mol Cell, 2008, 31, 167-77). Therefore, the accumulation of SSA repair factor in the DNA-damaged site was analyzed biochemically by concentrating the chromatin fraction. There was a transient increase and decrease in Rad51 and γH2AX, as correlated with the results in FIG. 2, but Rad52 and RPA2 accumulated in the chromatin fraction over time, and this increase was remarkable in RECQL4-deficient cells. There were (Figs. 3A and B). Since the method of cross-linking protein and chromatin with formaldehyde is not used, the binding between SSA factor and chromatin is considered to be relatively strong. From the above results, it was found that in RECQL4 deficient cancer cells, DNA repair SSA factor is delayedly accumulated in chromatin after DNA damage due to irradiation.

Quantitative SSA activity measurements were performed using the GFP reporter assay to determine SSA activation in RECQL4-deficient cancer cells (Stark et al., MCB, 2004, 24, 9305-9316). A significant increase in SSA activity was observed in RECQL4-deficient cancer cells, but since SSA activity was suppressed by ectopic expression of RecQL4 protein (FIGS. 4A-C), RecQL4 protein was found in cancer cells. It was found that it plays a role in suppressing the disease.

Since we found that SSA activity was increased in RECQL4-deficient cancer cells, we artificially suppressed SSA activity by siRNA in order to investigate whether SSA activity is necessary for the survival of cancer cells (siRNA). FIG. 5A). As a result, it was found that the number of RECQL4-deficient cancer cells was significantly reduced by suppressing the expression of Rad52 protein. Moreover, since the phenotype is restored when the RECQL4 protein is ectopically expressed, it was found that the RECQL4-deficient cancer cells increase the SSA activity for survival (FIG. 5B).

Next, in order to confirm the results in FIG. 5, epigallocatechin (Hengel et al., ELife, 2016; 5: e14740), which is a component of green tea recently identified as a Rad52 inhibitor, and the metabolic regulator AICAR were used. (Sullivan et al., PLoS One, 2016, 11, e0147230). As a result, it was found that both drug treatments similarly significantly suppressed RECQL4-deficient cancer cells in a concentration-dependent manner (FIGS. 6A to 6C). AICAR, known as an AMPK activator (Sullivan et al., FEBS Lett, 1994, 353, 33-6), is a direct AMPK activator, salicylate, or an indirect AMPK activator, 2DG treatment. (Vincent et al., Oncogene, 2015, 34, 3627-39) did not show a specific inhibitory effect on RECQL4-deficient cancer cells (FIGS. 6E and F). From these results, it was clarified that the inhibitory effect on RECQL4-deficient cancer cells is a specific inhibitory effect targeting Rad52.

Finally, epigallocatekin selectively suppresses the SSA pathway, leading to the induction of genetic instability by further DNA repair, DNA replication, and accumulation of DNA recombination errors, and associated secondary carcinogenesis and metastasis. Experiments were conducted to support the suppression of malignant transformation of cancer. The experimental results are shown in FIG. As shown in the results of FIG. 7, it was revealed that the epigallocatechin treatment suppressed the genomic deletion induced by the cisplatin treatment.

Through the above studies, it was found by analysis at the molecular and cellular level that inhibition of SSA in the error-prone DNA repair pathway is effective in suppressing RECQL4-deficient cancer cells.

Next, in order to demonstrate the SSA inhibitory effect at the individual level, wild-type and RECQL4 deficient HCT116 cells are subcutaneously administered to BALB / cAJcl-nu / nu mice to examine their ability to grow cancer cells in vivo. A xenograft experiment was performed. Since AICAR treatment has been shown to suppress the proliferation of p53-deficient HCT116 cells (Buzzai et al., Cancer Res, 2007, 67, 6745-52), p53-deficient HCT116 cells were used as controls. As reported, p53-deficient cells were suppressed in proliferation by AICAR treatment compared to wild-type cells (FIGS. 8A-C). Interestingly, RECQL4-deficient cells had a greater growth-suppressing effect by AICAR treatment than p53-deficient cells (FIGS. 8A-C).

Another SSA inhibitor, EGC, has a smaller biological effect than EGCG (epigallocatechin gallate), and EGCG has been intensively studied (for example, "epigallocatechin gallate" was searched for in PubMed. In the case of 4524 hits, but when searching for "epigallocatechin NOT gallate", only 305 hits), there are almost no studies of EGC administration to mice. However, considering that it is easy to clinically apply to humans, we decided to try oral administration of EGC to mice. Since studies on EGC administration to animals have only been reported in mice with intraperitoneal administration (ip) of 20 mg / kg (Wang et al, PLoS One, 2013; e56631), a lower concentration of 15 mg / kg orally (po) was given. The inhibitory effect of SSA was examined in mice. As a result, a significant growth inhibitory effect of EGC was observed in RECQL4 deficient cancer cells as in AICAR (FIGS. 9A to 9C).

Since the effect of suppressing SSA on the growth of cancer cells was confirmed at the individual mouse level, next, by inhibiting SSA activated after treatment with cisplatin, which is an anticancer drug, in a timely manner, it is effective and synergistic. We tested the hypothesis that it might have a cancer cell-suppressing effect. RECQL4-deficient cancer cells are also susceptible to treatment with AICAR and cisplatin alone (FIGS. 1B and 6B), but simultaneous treatment of the two confirmed a synergistic cancer cell inhibitory effect (FIG. 10A). Furthermore, by treating the SSA inhibitor at the timing of delayed activation of SSA after 24 hours, an effective cancer cell inhibitory effect was found as compared with the initial 24-hour treatment (FIG. 10B).

Finally, the synergistic effect of suppressing the growth of cancer cells at the cellular level was also examined at the individual mouse level. The administration method of oral administration of EGC was selected the day after administration of the anticancer drug cisplatin. As a result, EGC alone did not show an inhibitory effect on RECQL4 deficient cancer cells, because the administration method this time had a lower EGC administration frequency than that shown in FIG. 9, and the administration interval was longer. It is thought to be caused. The cancer cell-suppressing effect of cisplatin alone and the synergistic cancer cell-suppressing effect of cisplatin and EGC were observed (FIGS. 11A to 11E). From these results, it was confirmed that by inhibiting SSA activated by treatment with the anticancer drug cisplatin, the synergistic growth inhibitory effect of RECQL4-deficient cancer cells was confirmed even at the mouse individual level.

(Conclusion)
From the above results, special cancer cells in which SSA is easily activated in order to survive after anticancer drug treatment or irradiation, specifically, breast cancer and ovarian cancer having mutations in the BRCA1 gene and BRCA2 gene, for example. Anticancer drug treatment by treating cancer cells and cells with familial colon adenomatosis due to APC gene mutation with an SSA inhibitor in a delayed and timely manner after treatment with an anticancer drug or after irradiation. It has been clarified from the molecular level to the individual level that side effects caused by ovarian irradiation can be reduced and the growth of cancer cells can be synergistically suppressed.

Special cancer cells in which SSA is easily activated after anticancer drug treatment or irradiation for self-survival, specifically, breast cancer or ovarian cancer cells having mutations in the BRCA1 and BRCA2 genes, for example. In addition, by treating cells with familial colon adenomatosis due to APC gene mutation with an SSA inhibitor in a timely manner after anticancer drug treatment or irradiation, it can be used for anticancer drug treatment and irradiation. Since it is possible to synergistically suppress the growth of cancer cells while reducing the side effects caused by it, it is useful in that it can be a novel and useful therapeutic means for cancer.
In addition, even though it is a special cancer, the number of these cancer patients in the world is thousands every year even if 1% of colorectal cancer is considered to be due to APC mutation (Kinzler and Vogelstein, Cell, 1996, 87, 159-70), considering the current situation in which more than 1 million people are diagnosed with breast cancer each year and more than 400,000 die (Parkin et al., CA Cancer J Clin. 2005, 55). , 74-108), the number of cancer patients for which the therapeutic means is effective exists in the tens of thousands, and has sufficient industrial potential.

Although some of the specific embodiments of the present invention have been described in detail above, the specific embodiments shown by those skilled in the art will be modified in various ways to the extent that they do not substantially deviate from the teachings and advantages of the present invention. It is possible to make changes. Accordingly, all such amendments and modifications are within the spirit and scope of the invention claimed in the claims described below.

Claims (4)

A cancer cell proliferation inhibitor containing an SSA inhibitor, which is used in combination with chemotherapy or radiotherapy using a platinum preparation, wherein the cancer cells are RECQL4-deficient cancer cells, BRCA1 and / or BRCA2 deficient. At least one type of cancer cell selected from the group consisting of cancer cells and APC-deficient cancer cells, wherein the SSA inhibitor is (-) epigallocatekin or 5-amino-1-((2R). , 3R, 4S, 5R) -3,4-dihydroxy-5- (hydroxymethyl) -tetra-2-yl) -1H-imidazole-4-carboxamide, and the cancer cell proliferation inhibitor is applied to the subject. A proliferation inhibitor that is administered within 8 to 44 hours after administration of the platinum preparation or irradiation with the radiation. The cancer cell growth inhibitor according to claim 1, wherein the platinum preparation is selected from the group consisting of oxaliplatin, carboplatin, cisplatin, and nedaplatin. A late injury reducing agent resulting from the chemotherapy or radiation therapy, comprising an SSA inhibitor, which is used in combination with chemotherapy or radiation therapy using a platinum preparation, wherein the SSA inhibitor is (-) epi. Galocatechin or 5-amino-1-((2R, 3R, 4S, 5R) -3,4-dihydroxy-5- (hydroxymethyl) -tetrahex-2-yl) -1H-imidazole-4-carboxamide. A late disorder reducing agent, wherein the late disorder reducing agent is administered within 8 to 44 hours after administration of the platinum preparation to a subject or after irradiation with the radiation. The late disorder reducing agent according to claim 3 , wherein the late disorder is a secondary carcinogen.

Images