JPWO2018221735A1 - New oligonucleotide - Google Patents

Abstract

An object of the present invention is to provide a novel nucleic acid having an anticancer action. Oligonucleotides comprising the nucleotide sequences shown in SEQ ID NOs: 1 to 6 have a growth inhibitory action on cancer cells and have no effect on normal cells, and are therefore effective as anticancer agents.

Description

  The present invention relates to an oligonucleotide having an anticancer action. The present invention also relates to a medicine and an anticancer agent using the oligonucleotide.

  In recent years, the survival rate of cancer patients has risen due to advances in cancer treatments and methods, but it remains the leading cause of death in Japan, and even today, more than 300,000 people suffer cancer every year. Is dead.

  Conventionally, an anticancer agent using a molecular targeting drug such as imatinib has been developed, and a high effect has been recognized in cancer treatment. However, in some cases, such a molecular-targeted drug causes a side effect due to the invasion of normal cells, which makes it impossible to continue administration, or the cancer cells acquire drug resistance, resulting in a marked decrease in the effect. It may happen.

  On the other hand, nucleic acid drugs have the advantage of high sequence specificity and the ability to target DNA and RNA that could not be targeted by molecularly targeted drugs, and therefore are expected to have high efficacy and safety, and can be applied in various disease fields. Is being considered. Also in the field of cancer diseases, nucleic acid drugs capable of inhibiting the functions of in-vivo molecules such as unique DNA, RNA, and proteins existing in cancer cells are drawing attention as next-generation anticancer agents.

  Conventionally, nucleic acids having anticancer activity have been vigorously studied (see, for example, Patent Documents 1 to 3), but there are still few candidate products that are considered to be clinically useful as anticancer agents. is the current situation. Against this background, there is a demand for the development of new nucleic acids having anticancer activity.

International Publication No. 2014/112144 JP, 2014-217311, A JP, 2010-31051, A

  An object of the present invention is to provide a novel nucleic acid having an anticancer action, and a medicine using the nucleic acid.

  The present inventors have conducted extensive studies to solve the above-mentioned problems, and as a result, oligonucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 1 to 6 have a growth inhibitory action on cancer cells, and moreover on normal cells. Found no effect. That is, it was found that the above-mentioned oligonucleotide has an excellent cancer treatment effect and safety, and is effective as an anticancer agent. The present invention has been completed by further studies based on such findings.

That is, the present invention provides the inventions of the following modes.
Item 1. An oligonucleotide consisting of the base sequence shown in any of SEQ ID NOs: 1 to 6 below.
5'-GGTTAGGG-3 '(SEQ ID NO: 1)
5'-GGTTAGGGT-3 '(SEQ ID NO: 2)
5'-GGTTAGGGTT-3 '(SEQ ID NO: 3)
5'-GGTTAGGGTTA-3 '(SEQ ID NO: 4)
5'-GGTTAGGGTTAG-3 '(SEQ ID NO: 5)
5'-GTTAGGGT-3 '(SEQ ID NO: 6)
Item 2. An anticancer agent comprising the oligonucleotide according to Item 1.
Item 3. A pharmaceutical composition comprising the oligonucleotide according to Item 1.
Item 4. Item 4. The pharmaceutical composition according to Item 3, further comprising a carrier for nucleic acid delivery.
Item 5. Item 5. The pharmaceutical composition according to Item 3 or 4, which is used for treating cancer or preventing cancer metastasis.
Item 6. Item 6. The pharmaceutical composition according to any one of Items 3 to 5, wherein the cancer is pancreatic cancer, lung cancer, osteosarcoma, or leukemia.
Item 7. Use of the oligonucleotide according to Item 1 for producing an anticancer agent.
Item 8. Item 8. The use according to Item 7, wherein the anticancer agent is used for treating cancer or preventing cancer metastasis.
Item 9. Item 9. The use according to Item 8, wherein the cancer is pancreatic cancer, lung cancer, osteosarcoma, or leukemia.
Item 10. A method for cancer treatment or metastasis prevention, comprising the step of administering an effective amount of the oligonucleotide according to Item 1 to a cancer patient or a person who is required to prevent cancer metastasis.
Item 11. Item 11. The method for treating or preventing metastasis of cancer according to Item 10, wherein the cancer is pancreatic cancer, lung cancer, osteosarcoma, or leukemia.

  Since the oligonucleotide of the present invention can effectively inhibit the growth of cancer cells without inhibiting the growth of normal cells, it is useful as a nucleic acid drug having an excellent cancer therapeutic effect and safety.

FIG. 5 is a diagram showing the results of evaluation of the growth inhibitory effect on human pancreatic cancer cells of each oligonucleotide in Test Example 1. 8 is a diagram showing the results of evaluation of the growth inhibitory effect on human osteosarcoma cells of each oligonucleotide in Test Example 2. FIG. FIG. 9 is a diagram showing the results of evaluation of the growth inhibitory effect on human non-small cell lung cancer cells of each oligonucleotide in Test Example 3. FIG. 8 is a diagram showing the results of evaluation of the growth inhibitory effect on human chronic myelogenous leukemia cells of each oligonucleotide in Test Example 4. 8 is a diagram showing the results of evaluation of the growth inhibitory action on human dermal fibroblasts for each oligonucleotide in Test Example 5. FIG. 8 is a diagram showing the results of evaluation of the growth inhibitory effect on human pancreatic cancer cells of each oligonucleotide in Test Example 6. FIG. 8 is a diagram showing the results of evaluation of the growth inhibitory action on human pancreatic cancer cells for each oligonucleotide in Test Example 7. FIG. FIG. 9 is a diagram showing the results of evaluation of the growth inhibitory action on human skin fibroblasts for each oligonucleotide in Test Example 8. FIG. 11 is a diagram showing the results of analysis of protein expression differences in human pancreatic cancer cells for each oligonucleotide in Test Example 9. FIG. 9 is a diagram showing the results of evaluation of the antitumor effect against human pancreatic cancer cells in vivo for each oligonucleotide in Test Example 10.

1. Oligonucleotide The oligonucleotide of the present invention is characterized by comprising the nucleotide sequence shown in any of the following SEQ ID NOs: 1 to 6.
5'-GGTTAGGG-3 '(SEQ ID NO: 1)
5'-GGTTAGGGT-3 '(SEQ ID NO: 2)
5'-GGTTAGGGTT-3 '(SEQ ID NO: 3)
5'-GGTTAGGGTTA-3 '(SEQ ID NO: 4)
5'-GGTTAGGGTTAG-3 '(SEQ ID NO: 5)
5'-GTTAGGGT-3 '(SEQ ID NO: 6)

  The base sequences shown in SEQ ID NOS: 2 to 5 are the base sequences shown in SEQ ID NO: 1 with T, TT, TTA, and TTAG added to the 3'end side. Further, the base sequence shown in SEQ ID NO: 6 is obtained by deleting the guanine (G) at the 5'end in the base sequence shown in SEQ ID NO: 2.

  When the nucleotide sequence shown in SEQ ID NO: 1 is modified by addition, substitution, and / or deletion of nucleotides so that it does not correspond to the nucleotide sequence shown in any of SEQ ID NOs: 2 to 6, it is against cancer cells. There is a tendency that the growth-inhibiting effect is reduced or that a growth-inhibiting effect cytotoxin appears on normal cells. That is, the oligonucleotide of the present invention does not inhibit the growth of normal cells and specifically inhibits the growth of cancer cells based on having the specific base sequences shown in SEQ ID NOs: 1 to 6. Is possible.

  Among the oligonucleotides consisting of the base sequences shown in SEQ ID NOS: 1 to 6, from the viewpoint of providing further excellent anticancer action and safety, preferably from the base sequences shown in SEQ ID NOS: 2, 3, 4 and 6. The following oligonucleotides are listed.

  In the oligonucleotide of the present invention, the pentose having each base bonded thereto may be either D-ribose or deoxy-D-ribose, and these may be mixed. That is, the oligonucleotide of the present invention may be a DNA oligonucleotide, an RNA oligonucleotide, or a DNA / RNA chimera oligonucleotide. Preferred is a DNA oligonucleotide. When the oligonucleotide of the present invention is an RNA oligonucleotide, T (thymine) in the base sequences shown in SEQ ID NOs: 1 to U (uracil). Further, when the oligonucleotide of the present invention is a DNA / RNA chimeric oligonucleotide, when T (thymine) is present in the RNA portion in the base sequences shown in SEQ ID NOS: 1 to 6, the T (thymine) is converted to U (uracil). Become.

  The oligonucleotide of the present invention may have various modifications generally applied to nucleic acids, if necessary, in order to impart resistance to nucleases and the like, as long as the effects of the present invention are not impaired. Examples of such modification include modification of sugar chain moiety such as 2′-O methylation; modification of base moiety; modification of phosphate moiety such as amination, lower alkyl amination, acetylation, phosphorothioate and the like. Be done. Modification of such a nucleic acid can be performed according to a known method.

  The oligonucleotide of the present invention can be obtained by a known production method. For example, the oligonucleotide of the present invention can be obtained by chemical synthesis, or can be obtained by amplification using the complementary strand of the oligonucleotide of the present invention as a template.

  Since the oligonucleotide of the present invention can effectively inhibit the growth of cancer cells without inhibiting the growth of normal cells, it can be preferably used as an anticancer agent. When the oligonucleotide of the present invention is used as an anticancer agent, the formulation, suitable cancer type, administration method, dose, etc. are as shown in the section of "2. Pharmaceutical composition" described later. ..

2. Pharmaceutical composition When the oligonucleotide is used as an anticancer agent, the oligonucleotide may be formulated as a pharmaceutical composition. That is, the pharmaceutical composition of the present invention is characterized by containing the above-mentioned oligonucleotide.

  The pharmaceutical composition of the present invention preferably further contains a carrier for nucleic acid delivery in order to efficiently deliver the oligonucleotide into cancer cells.

  The carrier for nucleic acid delivery is not particularly limited as long as it can be used for nucleic acid delivery and is pharmaceutically acceptable, and for example, lipofectamine, oligofectamine, lipofectin, atelocollagen, liposome, polyethyleneimine, polyamine. , Polybrene, exosome, nanoparticles (polyamino acid, metal, apatite, etc.), peptide, α-dextrin, β-dextrin, γ-dextrin, polyethylene glycol, cholesterol, vitamin A, vitamin E, folic acid, chitosan, water-soluble chitosan, etc. Is mentioned. These nucleic acid delivery carriers may be used alone or in combination of two or more.

  The mode of use of these nucleic acid delivery carriers may be appropriately set according to the type of the nucleic acid delivery carrier to be used, and for example, the nucleic acid delivery carrier is bound to the oligonucleotide by non-covalent bond such as hydrogen bond or hydrophobic bond. It may be used in a complexed form, or may be used by linking a carrier for nucleic acid delivery to the oligonucleotide by a covalent bond. When the carrier for nucleic acid delivery is covalently linked to the oligonucleotide, a known bifunctional linker may be used.

  In the pharmaceutical composition of the present invention, the compounding amount of the nucleic acid delivery carrier is set to an effective amount capable of delivering the oligonucleotide into cancer cells, depending on the type, dosage form, administration method, etc. of the nucleic acid delivery carrier used. Just set it.

  The dosage form of the pharmaceutical composition of the present invention is not particularly limited and may be appropriately set depending on the administration method and the like. Specific examples of the dosage form of the pharmaceutical composition of the present invention include solid preparations such as tablets, capsules, pills, powders, granules, suppositories, and patches; liquids, suspensions, emulsions, syrups, Liquid preparations such as injections can be mentioned. Among these dosage forms, a liquid preparation is preferable.

  Further, the pharmaceutical composition of the present invention is formulated by adding a pharmaceutically acceptable carrier or additive depending on its dosage form. For example, in the case of a solid preparation, it can be prepared using an excipient, a binder, a disintegrating agent, a lubricant, a solubilizing agent, an antioxidant, an isotonicity agent and the like. In the case of a liquid preparation, it can be prepared using physiological saline, a buffer solution or the like.

  Since the oligonucleotide can exert an anticancer effect by inhibiting the growth of cancer cells, the pharmaceutical composition of the present invention is used for treating cancer or preventing cancer metastasis.

  The cancer type to be treated or to prevent metastases is not particularly limited as long as it is a cancer to be treated with chemotherapy, but specifically, pancreatic cancer, lung cancer, osteosarcoma, colon cancer, colon cancer. , Gastric cancer, rectal cancer, liver cancer, breast cancer, bladder cancer, prostate cancer, cervical cancer, head and neck cancer, bile duct cancer, gallbladder cancer, oral cancer, melanoma, solid tumors such as brain tumors; leukemia, blood cancer such as malignant lymphoma Is mentioned. Among these cancer types, pancreatic cancer, lung cancer, osteosarcoma, and leukemia are preferable.

  The administration method of the pharmaceutical composition of the present invention is not particularly limited and may be systemic administration or local administration, and may be appropriately set depending on the cancer type to be applied. Specific examples of systemic administration include intravascular (intraarterial or intravenous) administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, inhalation administration and the like. Intravascular administration includes continuous infusion as well as intravascular injection.

  The dose of the pharmaceutical composition of the present invention may be appropriately set within an effective range capable of exerting an anticancer effect, depending on the cancer type to be applied, the degree of symptoms, the sex of the patient, the age, etc. For example, the dose of the above oligonucleotide may be set to about 1 ng to 300 mg / kg (body weight), and the oligonucleotide may be administered 1 to 21 times / week.

  Further, the pharmaceutical composition of the present invention may optionally contain other anti-cancer agents in order to more effectively exhibit the anti-cancer effect, and also with other anti-cancer agents It may be administered in combination.

  The type of the other anticancer agent contained or used in combination with the pharmaceutical composition of the present invention is not particularly limited and may be appropriately set according to the cancer type to be applied, for example, antimetabolite Agents, alkylating agents, microtubule acting agents, anticancer antibiotics, topoisomerase inhibitors, platinum preparations and the like. Specifically, antimetabolites such as 5-fluorouracil, methotrexate, doxyfluridine, tegafur, 6-mercaptopurine, cytarabine, gemcitabine; clofosfamide, ifosfamide, thiotepa, carbocon, alkylating agents such as nimustine hydrochloride; docetaxel, paclitaxel, vincristine. , Vindesine, vinorelbine and other microtubule agents; anticancer antibiotics such as doxorubicin hydrochloride, mitomycin, amrubicin hydrochloride, pirarubicin hydrochloride, epirubicin hydrochloride, aclarubicin hydrochloride, mitoxantrone hydrochloride, bleomycin hydrochloride, peplomycin sulfate; irinotecan, nogitecan hydrochloride Topoisomerase inhibitors such as; and platinum preparations such as cisplatin, oxaliplatin, carboplatin, and nedaplatin.

  Hereinafter, the present invention will be described with reference to examples. However, the present invention is not construed as being limited to the following examples.

Production Example: Oligonucleotide synthesis Oligonucleotides (DNA molecules) having the nucleotide sequences shown in Table 1 were synthesized using a DNA / RNA synthesizer NTS M-MX-A20 (manufactured by Nippon Techno Service Co., Ltd.).

Test Example 1: Evaluation of growth inhibitory effect on human pancreatic cancer cells Human pancreatic cancer cell line (MIA PaCa-2) (purchased from JCRB Cell Bank, Institute for Medical Science, Health and Nutrition) 100 Units / ml penicillin, 100 μg / ml streptomycin , A cell solution suspended at 3.0 × 10 ⁴ cells / ml in Eagle's minimum essential medium (E-MEM) medium containing 0.1 mM non-essential amino acids and 10% fetal bovine serum was prepared. did. The cell solution was added to a 96-well plate at 100 μl / well and cultured overnight at 37 ° C. in a 5% CO ₂ atmosphere. Then, the oligonucleotides of Examples 1 and 6 and Comparative Examples 1 and 2 were introduced into the cells using a transfection reagent X-treme GENE HP Transfection reagent (manufactured by Roche) according to the attached protocol. Specifically, 10 μl of the mixed solution of the oligonucleotide and the transfection reagent was added to each well to make a total volume of 110 μl. The final concentration of the oligonucleotide in the well was 5.5 ng / μl. After transfection, the cells were cultured at 37 ° C for 48 hours. Then, 10 μl of Cell Counting Kit-8 (manufactured by Dojindo) was added, and the mixture was incubated at 37 ° C. for 1 hour, and then the absorbance was measured at wavelengths of 450 nm and 650 nm using a microplate reader (manufactured by Tecan). As a control, a test was conducted under the same conditions as above except that the oligonucleotide was not added (untreated). The survival rate of cells treated with each of the oligonucleotides was calculated based on the following formula, with the survival rate of cells treated with only the transfection reagent (untreated cells) as 100%.

  The obtained results are shown in FIG. The oligonucleotide of Comparative Example 1 did not have a growth inhibitory effect on human pancreatic cancer cells, and the oligonucleotide of Comparative Example 2 had a growth inhibitory effect on human pancreatic cancer cells. On the other hand, it was confirmed that the oligonucleotides of Examples 1 and 6 had a markedly higher growth inhibitory effect on human pancreatic cancer cells than Comparative Example 2 and had an excellent anticancer effect.

Test Example 2: Evaluation of growth inhibitory effect on human osteosarcoma cells (1) Using human osteosarcoma cell line (Saos-2) (purchased from RIKEN BioResource Center), (2) 5.0 × 10 ⁴ The cell solution suspended so that the number of cells / ml was 100 μl / well was added to a 96-well plate, (3) 100 Units / ml penicillin, 100 μg / ml streptomycin, and 10% fetal bovine serum were added to the medium. Was used, and (4) each oligonucleotide of Examples 1, 6 and Comparative Example 1 was used to give a final oligonucleotide concentration in the well of transfection of 9.1 ng / μl. Other than the above, tests were performed under the same conditions as in Test Example 1 above, and the cell viability when treated with each oligonucleotide was determined.

  The obtained results are shown in FIG. As a result, it was confirmed that the oligonucleotides of Examples 1 and 6 had an excellent growth inhibitory action on human osteosarcoma cells.

Test Example 3: Evaluation of growth inhibitory effect on human non-small cell lung cancer cells (1) Use of human non-small cell lung cancer cell line (A549) (purchased from JCRB Cell Bank, Institute for Pharmaceutical Sciences, Health and Nutrition) ( 2) The cell solution suspended at 5.0 × 10 ⁴ cells / ml was added to a 96-well plate at 100 μl / well, and (3) each of Example 1 and Comparative Example 1. Except that the final concentration of the oligonucleotide in the well at the time of transfection was set to 9.1 ng / μl using the oligonucleotide, the test was carried out under the same conditions as in Test Example 1 above, and the oligonucleotide was treated with each oligonucleotide. Cell viability was determined.

  The obtained results are shown in FIG. As a result, the oligonucleotide of Comparative Example 1 showed no growth inhibitory effect on human non-small cell lung cancer cells, but the oligonucleotide of Example 1 showed a growth inhibitory effect on human non-small cell lung cancer cells. Became clear.

Test Example 4: Evaluation of growth inhibitory action on human chronic myelogenous leukemia cells (1) Using human chronic myelogenous leukemia cell line (K562) (purchased from RIKEN BioResource Center), (2) 5.0 × Cell suspension suspended at 10 ⁴ cells / ml was added to a 96-well plate at 100 μl / well, (3) 100 Units / ml penicillin, 100 μg / ml streptomycin, and 10% bovine in the medium. Using RPMI-1640 containing fetal serum and (4) using each oligonucleotide of Example 1 and Comparative Example 1, the final concentration of oligonucleotide in the well at the time of transfection was 9.1 ng / μl. Other than the above, tests were performed under the same conditions as in Test Example 1 above, and the cell viability when treated with each oligonucleotide was determined.

  The obtained results are shown in FIG. As a result, it was confirmed that even in the case of human chronic myelogenous leukemia cells, the oligonucleotide of Example 1 has a growth inhibitory action.

Test Example 5: Evaluation of growth inhibitory effect on human dermal fibroblasts (normal cells) (1) Using human dermal fibroblast cell line (NHDF) (purchased from Takara Bio Inc.), (2) 2.5 The cell solution suspended at 10 ⁴ cells / ml was added to a 96-well plate at 100 μl / well, (3) 100 Units / ml penicillin, 100 μg / ml streptomycin, and 10% were added to the medium. Using RPMI-1640 containing fetal bovine serum and (4) using each oligonucleotide of Example 1 and Comparative Example 1, the final concentration of oligonucleotide in the well at the time of transfection was 4.5 ng / μl. The test was performed under the same conditions as in Test Example 1 above except that the cell viability when treated with each oligonucleotide was determined.

The obtained results are shown in FIG. As a result, in the oligonucleotides of Comparative Example 1 in which the growth inhibitory action on cancer cells was not observed in Test Examples 1 to 4, the growth inhibitory action on human skin fibroblasts (normal cells) was not observed. On the other hand, the oligonucleotide of Example 1 also showed no growth inhibitory effect on human skin fibroblasts, and was confirmed to have no adverse effect on normal cells. From the above results, it became clear that the oligonucleotide of the present invention can specifically inhibit the growth of cancer cells without inhibiting the growth of normal cells.

Test Example 6: Evaluation of growth inhibitory action on human pancreatic cancer cells A test was conducted under the same conditions as in Test Example 1 above except that the oligonucleotides of Examples 3 and 4 were used, and each oligonucleotide was treated. Cell viability was determined.

  The obtained results are shown in FIG. Based on the results of FIG. 6, as in Examples 1 and 6, each of the oligonucleotides of Examples 3 and 4 was observed to have a growth inhibitory effect on human pancreatic cancer cells. It was confirmed that the example has an excellent anti-cancer effect.

Test Example 7: Evaluation of Growth Inhibitory Effect on Human Pancreatic Cancer Cells Except that each oligonucleotide of Example 1 and Comparative Examples 3 to 6 was used, a test was performed under the same conditions as in Test Example 1 above, and each oligonucleotide was used. The cell viability upon treatment was determined.

The obtained results are shown in FIG. 7. From the results of FIG. 7, the oligonucleotide of Comparative Example 3 which is a complementary strand of Example 1 and the oligonucleotides of Comparative Examples 4 to 6 in which Example 1 is substituted by several bases have a reduced ability to inhibit the growth of human pancreatic cancer cells. It became clear.
From the results of the above Test Examples 1 to 7, it was determined that the base sequence shown in SEQ ID NO: 1 does not correspond to the base sequence shown in any of SEQ ID NOs: 2 to 6 due to nucleotide addition, substitution, and / or deletion. When modified, it was confirmed that the growth inhibitory effect on cancer cells was reduced.

Test Example 8: Evaluation of Growth Inhibitory Effect on Human Skin Fibroblasts (Normal Cells) A test was conducted under the same conditions as in Test Example 5 except that each oligonucleotide of Examples 3, 4, and 6 was used, The cell viability when treated with each oligonucleotide was determined.

  The obtained results are shown in FIG. From the results shown in FIG. 8, with each of the oligonucleotides of Examples 3, 4, and 6 as in Example 1, no growth inhibitory effect on human dermal fibroblasts (normal cells) was observed, which adversely affected normal cells. It was confirmed that the result did not reach.

Test Example 9: Analysis of differences in protein expression in human pancreatic cancer cells Human pancreatic cancer cell line (MIA PaCa-2) (purchased from JCRB Cell Bank, Institute of Medical Science, Health and Nutrition) 100 Units / ml penicillin, 100 μg / ml streptomycin , A cell solution suspended at 5.7 × 10 ⁵ cells / ml in Eagle's minimum essential medium (E-MEM) medium containing 0.1 mM non-essential amino acid and 10% fetal bovine serum was prepared. did. The cell solution was added to a 48-well plate at 270 μl / well and cultured overnight at 37 ° C. in a 5% CO ₂ atmosphere. Then, the oligonucleotides of Example 1 and Comparative Example 3 were introduced into the cells using a transfection reagent X-treme GENE HP Transfection reagent (manufactured by Roche) according to the attached protocol. Specifically, 30 μl of the mixed solution of the oligonucleotide and the transfection reagent was added to each well to make a total volume of 300 μl, and the final concentration of the oligonucleotide in the well was 3.3 ng / μl. After transfection, the cells were cultured at 37 ° C for 24 hours. As a reference, the same operation as described above was performed using only the transfection reagent. Then, the cells were washed twice with 300 μl of PBS, and the cells were lysed with 300 μl of Lysis Buffer containing 10 μg / ml Aprotinin, 10 μg / ml Leupeptin, and 10 μg / ml Pepstatin A. Centrifugation was performed at 14000 × g for 5 minutes, and the supernatant was collected to obtain a cell extract. Using this cell extract, a membrane was prepared according to the attached protocol using a Proteome Profiler Apoptosis Array Kit (manufactured by R & D SYSTEMS), and measured using GE ImageQuant LAS4000mini (manufactured by GE Life Science), and intracellular protein was measured. Expression difference analysis was performed.

  The obtained results are shown in FIG. From this result, when comparing the results of Comparative Example 3 with the transfection reagent alone, there was almost no difference except for Catalase, but in Example 1, the expression of Heme Oxygenase-2 (HO-2) was enhanced in addition to Catalase. Was. Based on these results, when the oligonucleotide of Comparative Example 3 was introduced into cells, stress was applied in the cells and active oxygen was produced, but the active oxygen was decomposed and detoxified by Catalase, resulting in cell death. It is considered that it was not induced. On the other hand, by introducing the oligonucleotide of Example 1 into cells, active oxygen was decomposed by Catalase as in Comparative Example 3, but heme was decomposed by HO-2, which resulted in divalent iron. It is possible that free radicals were released and active oxygen was produced. Therefore, the oligonucleotide of Example 1 induces cell death by causing an increase in active oxygen in cancer cells and making it impossible to maintain the balance between the expression level of Catalase and the active oxygen content, and as a result, It was suggested to inhibit proliferation.

Test Example 10: Evaluation of antitumor effect on human pancreatic cancer cells by animal experiment 2.1 × 10 ⁶ human pancreatic cancer cell lines (MIA PaCa-2) were subcutaneously administered to the right leg of mice, and the tumor size was 200 mm. ^{After 3} was reached, the oligonucleotides of Example 1 and Comparative Example 1 were used to prepare a drug according to the attached protocol using a transfection reagent in vivo jet PEI transfection reagent (manufactured by Polyplus), and directly administered to the tumor. Specifically, 100 μl of a mixed solution of the above-mentioned oligonucleotide and transfection reagent was administered to each mouse. The dose of each oligonucleotide was set to 0.5 mg / kg and 2.0 mg / kg in Example 1 and 2.0 mg / kg in Comparative Example 1. In addition, drug administration was performed every other day for a total of 3 times, followed by a 2-day rest period as 1 cycle (1 week), for a total of 7 cycles (7 weeks). Tumor volume was measured over time every week during the test period. The tumor volume was obtained by measuring the major axis and minor axis of the tumor with a caliper, and using the approximate expression V = (W ² × L) × 0.4 (V: volume, W: minor axis, L: major axis), the tumor volume (mm ³ ). In addition, for comparison, a group to which only the same amount of PBS was added instead of the drug (PBS group), a group to which only the same amount of transfection reagent was administered (Vehicle group), and an existing anticancer agent were used. Gemcitabine hydrochloride was administered (the same number of cycles as the above, with 80 mg / kg per dose being administered twice a week (once every 3 to 4 days) as one cycle (one week)). The same test was conducted for the positive control group). In the test, four mice were used for each group, and a multiple comparison test was performed using the Bonferroni method.

  The obtained results are shown in FIG. In the PBS group, Vehicle group, and Comparative Example 1, it was confirmed that the tumor continued to grow over the course of the test days. On the other hand, in Example 1, it was confirmed that the growth of the tumor was suppressed, and that the growth of the tumor was suppressed even when the dose was particularly small (0.5 mg / kg). Furthermore, in Example 1, not only is there a significant difference from the Vehicle group when the dose is high (2.0 mg / kg), but it is also comparable to the existing anticancer drug gemcitabine hydrochloride (positive control group). There was an antitumor effect. Therefore, it was confirmed that Example 1 has an excellent antitumor effect.

Claims (11)

An oligonucleotide consisting of the base sequence shown in any of SEQ ID NOs: 1 to 6 below.
5'-GGTTAGGG-3 '(SEQ ID NO: 1)
5'-GGTTAGGGT-3 '(SEQ ID NO: 2)
5'-GGTTAGGGTT-3 '(SEQ ID NO: 3)
5'-GGTTAGGGTTA-3 '(SEQ ID NO: 4)
5'-GGTTAGGGTTAG-3 '(SEQ ID NO: 5)
5'-GTTAGGGT-3 '(SEQ ID NO: 6)   An anticancer agent comprising the oligonucleotide according to claim 1.   A pharmaceutical composition comprising the oligonucleotide of claim 1.   The pharmaceutical composition according to claim 3, further comprising a carrier for nucleic acid delivery.   The pharmaceutical composition according to claim 3 or 4, which is used for treating cancer or preventing cancer metastasis.   The pharmaceutical composition according to claim 5, wherein the cancer is pancreatic cancer, lung cancer, osteosarcoma, or leukemia.   Use of the oligonucleotide according to claim 1 for producing an anticancer agent.   The use according to claim 7, wherein the anticancer agent is used for treating cancer or preventing cancer metastasis.   The use according to claim 8, wherein the cancer is pancreatic cancer, lung cancer, osteosarcoma, or leukemia.   A method for treating cancer or preventing metastasis, comprising the step of administering an effective amount of the oligonucleotide according to claim 1 to a cancer patient or a person who is required to prevent cancer metastasis.   The method for treating cancer or preventing metastasis according to claim 10, wherein the cancer is pancreatic cancer, lung cancer, osteosarcoma, or leukemia.