JP2022096337A - Anti-cancer effect enhancer - Google Patents

Abstract

[Problem] To provide a technology for enhancing anti-cancer activity. [Solution] An anti-cancer activity enhancer containing at least one selected from the group consisting of TFF1 (Trefoil Factor Family 1) protein and a polynucleotide containing a TFF1 protein coding sequence. [Selected Figures] None

Description

The present invention relates to an anticancer action enhancer and the like.

In cancer treatment with anticancer drugs, anticancer drug sensitivity may become a problem. Antineoplastic susceptibility may be due, for example, to the nature of the individual patient's cancer, or to the resistance acquired by the cancer upon administration of the anticancer agent. Therefore, in anticancer drug treatment, the anticancer drug to be used may be selected after confirming the sensitivity of the anticancer drug by a test. However, the options for anticancer agents are limited, and it is assumed that anticancer agents that are expected to have high therapeutic effects cannot be selected. Therefore, there is a demand for a technique for improving the sensitivity to an anticancer drug, that is, enhancing the anticancer effect in chemotherapy.

The Trefoil Factor Family 1 (TFF1) is under research on gastric and pancreatic cancers. It has been reported that TFF1 can be used as a biomarker for pancreatic cancer (Patent Document 1). In addition, it has been clarified that TFF1 is abundantly produced mainly in the gastric mucosal epithelium, and it is said that gastric cancer develops in TFF1 knockout mice lacking the TFF1 gene, and it has a cancer-suppressing effect on gastric cancer. It is suggested that. However, on the other hand, it was suggested that TFF1 also has a cancer-suppressing effect on pancreatic cancer, but subsequent studies have shown that it survives between TFF1-producing pancreatic cancer mice and non-producing pancreatic cancer mice. It was found that there was no significant difference in the rates, and these results suggested that TFF1 alone could not provide effective treatment for cancer.

Special Table 2018-504609 Gazette

An object of the present invention is to provide a technique for enhancing an anticancer effect.

As a result of diligent research in view of the above problems, the present inventor is an anticancer activity enhancer containing at least one selected from the group consisting of TFF1 protein and a polynucleotide containing a TFF1 protein coding sequence. If so, we found that the above problems could be solved. The present inventor has completed the present invention as a result of further research based on this finding. The present invention includes the following aspects.

Item 1. An anticancer activity enhancer containing at least one selected from the group consisting of a TFF1 (Trefoil Factor Family 1) protein and a polynucleotide containing a TFF1 protein coding sequence.
Item 2. Item 2. The anticancer action enhancer according to Item 1, which contains the TFF1 protein.
Item 3. Item 2. The anticancer action enhancer according to Item 1 or 2, wherein the TFF1 protein is a chemically synthesized protein.
Item 4. Item 3. The anticancer action enhancer according to any one of Items 1 to 3, wherein the target cancer cells are gastrointestinal cancer cells.
Item 5. Item 6. The anticancer action enhancer according to any one of Items 1 to 5, which is used in combination with an anticancer agent.
Item 6. Item 6. The anticancer activity enhancer according to any one of Items 1 to 6, which is an agent for enhancing the anticancer activity of nucleic acid analogs.
Item 7. Item 6. The anticancer action enhancer according to any one of Items 1 to 7, which is a drug or a reagent.
Item 8. A composition for improving cancer, which comprises the anticancer action enhancer and the anticancer agent according to any one of Items 1 to 7.

According to the present invention, it is possible to provide a technique for enhancing an anticancer action and a cancer treatment technique using the technique.

The results of Test Example 1 are shown. A: Shows changes in the expression level of EMT markers due to TFF1 overexpression. B: Shows changes in cell number due to anticancer drug treatment when TFF1 is overexpressed. ** indicates p <0.01. C and D: Changes in the number of apoptotic cells due to anticancer drug treatment when TFF1 is overexpressed. C shows a histogram of flow cytometry, and D shows a graph of the number of apoptotic cells. The results of Test Example 2 are shown. A and B: Changes in the ratio of apoptotic cells due to chemical synthetic TFF1 treatment and anticancer drug treatment are shown. A shows a histogram of flow cytometry, and B shows a change in the apoptotic cell ratio. C: Changes in the amount of apoptosis markers due to chemical synthetic TFF1 treatment and anticancer drug treatment are shown. D: Shows changes in cell number due to chemical synthesis TFF1 treatment and anticancer drug treatment. ** indicates p <0.01. The results of Test Example 3 are shown. A, B, and C: Changes in EMT marker expression (A), changes in epithelial marker expression (B), and changes in Wnt target gene expression due to chemosynthetic TFF1 treatment and anticancer drug treatment (A) The results of analysis of C) at the mRNA level are shown. * Indicates p <0.05 and ** indicates p <0.01. D, E, and F: Changes in EMT marker expression (D), changes in epithelial marker expression (E), and Wnt target protein β-catenin due to chemosynthetic TFF1 treatment and anticancer drug treatment. The results of analysis of changes in phosphorylation status (F) at the protein level are shown. The results of Test Example 4 are shown. A: Shows the test schedule. B: A photograph of the appearance of the tumor 4 weeks after the start of the chemically synthesized TFF1 treatment and the anticancer drug treatment is shown. C: Shows changes in tumor volume since the start of chemically synthesized TFF1 treatment and anticancer drug treatment. From the plot on the left side of the graph of C, it is at the start of administration, 1 week after the start of administration, 2 weeks, 3 weeks, and 4 weeks. D: Shows the tumor volume 4 weeks after the start of chemically synthesized TFF1 treatment and anticancer drug treatment. * Indicates p <0.05 and ** indicates p <0.01. The results of Test Example 5 are shown. A to C: Changes in the expression level of cancer stem cell markers by chemically synthesized TFF1 treatment and anticancer drug treatment are shown. A shows the histogram of flow cytometry, B shows the result of Western blotting, and C shows the result of RT-PCR. ** indicates p <0.01. The results of Test Example 6 are shown. A: Shows the survival rate of KPC mice. B and C: Changes in the expression level of cancer stem cell markers by recombinant TFF1 treatment and anticancer drug treatment. B is an immunostaining image, and C shows the ratio of the number of individuals in the group in which the expression of the cancer stem cell marker is weak (less than 50% of the stained area) and the group in which the expression of the cancer stem cell marker is strong (stained area of 50% or more). The results of Test Example 7 are shown. It is the result of RT-PCR showing the change in the expression level of the cancer stem cell marker by the chemical synthesis TFF1 treatment and the anticancer drug treatment.

1. 1. Definitions In the present specification, the expressions "contains" and "contains" include the concepts of "contains", "contains", "substantially consists" and "consists of only".

"Identity" of an amino acid sequence refers to the degree of coincidence of two or more contrastable amino acid sequences with each other. Therefore, the higher the match between two amino acid sequences, the higher the identity or similarity of those sequences. The level of amino acid sequence identity is determined, for example, using FASTA, a tool for sequence analysis, with default parameters. Alternatively, the algorithm BLAST by Karlin and Altschul (KarlinS, Altschul SF. “Methods for assessing the statistical significance of molecular sequence features by using general scoringschemes” Proc Natl Acad Sci USA. 87: 2264-2268 (1990), KarlinS, Altschul SF. It can be determined using “Applications and statistics for multiple high-scoring segments in molecular sequences.” Proc Natl Acad Sci USA. 90: 5873-7 (1993). A program called BLASTX based on such a BLAST algorithm has been developed. Specific methods for these analysis methods are known, and the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/) can be referred to. The "identity" of the base sequence is also defined according to the above.

As used herein, "conservative substitution" means that an amino acid residue is replaced with an amino acid residue having a similar side chain. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine is a conservative substitution. Other amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine and cysteine; alanine, valine, leucine, isoleucine, Amino acid residues with non-polar side chains such as proline, phenylalanine, methionine and tryptophan; amino acid residues with β-branched side chains such as threonine, valine and isoleucine; aromatic side chains such as tyrosine, phenylalanine, tryptophan and histidine Substitutions between amino acid residues are also conservative substitutions.

In the present specification, "nucleic acid" and "polynucleotide" are not particularly limited and include both natural and artificial ones. Specifically, in addition to DNA, RNA and the like, known chemical modifications may be applied as illustrated below. Substituting the phosphate residue (phosphate) of each nucleotide with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, or phosphorodithionate to prevent degradation by a hydrolyzing enzyme such as nuclease. Can be done. In addition, the hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide is converted into -OR (R is, for example, CH3 (2'-O-Me), CH2CH2OCH3 (2'-O-MOE), CH2CH2NHC (NH) NH2, It may be replaced with CH2CONHCH3, CH2CH2CN, etc.). Further, the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl. Can be mentioned. Further, examples thereof include those in which the phosphoric acid moiety and the hydroxyl moiety are modified with a biotin, an amino group, a lower alkylamine group, an acetyl group and the like, but the present invention is not limited thereto. Further, BNA (LNA) or the like in which the conformation of the sugar portion is fixed to N type by cross-linking the 2'oxygen and 4'carbon of the sugar part of the nucleotide can also be used.

2. Anticancer Action Enhancer, Composition for Ameliorating Cancer In one aspect of the present invention, at least one selected from the group consisting of a TFF1 protein and a polynucleotide containing a TFF1 protein coding sequence (“the present invention”. It relates to an anticancer activity enhancer (sometimes referred to as “the enhancer of the present invention” in the present specification) containing an active ingredient (which may also be referred to as “active ingredient”). Further, in one embodiment of the present invention, the composition for improving cancer containing the enhancer of the present invention and the anticancer agent (in the present specification, the present invention may be referred to as "the composition of the present invention". There is.). These will be described below.

2-1. TFF1 Protein In a preferred embodiment of the invention, the enhancer of the invention contains a TFF1 protein.

As the TFF1 protein, for example, Trefoil Factor Family 1 protein derived from various mammals such as human, monkey, mouse, rat, dog, cat, rabbit, pig, horse, cow, sheep, goat, and deer can be adopted. Among them, the TFF1 protein of the organism derived from the target cancer cell of the enhancer of the present invention and the composition of the present invention, or an organism species that is evolutionarily closer to the organism (for example, the organism species derived from the target cancer cell is human). For example, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, deer, etc.) are preferable.

The amino acid sequences of TFF1 proteins from various species are known. Specifically, for example, the human TFF1 protein includes a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (NCBI Reference Sequence: NP_003216), and the mouse TFF1 protein includes a protein consisting of the amino acid sequence shown in SEQ ID NO: 2. (NCBI Reference Sequence: NP_033388) and the like. In addition, the TFF1 protein has an N-terminal signal sequence (for example, an amino acid sequence consisting of the 1st to 24th amino acids from the N-terminal in SEQ ID NO: 1 and an amino acid sequence consisting of the 1st to 21st amino acids from the N-terminal in SEQ ID NO: 2). It may be deleted (for example, a protein consisting of the amino acid sequence shown in SEQ ID NO: 3, a protein consisting of the amino acid sequence shown in SEQ ID NO: 4, etc.).

The TFF1 protein may have mutations such as amino acid substitutions, deletions, additions, and insertions as long as it has anticancer activity enhancing activity. Examples of the mutation include substitution, more preferably conservative substitution, from the viewpoint that the anticancer activity enhancing activity is less likely to be impaired.

Preferred specific examples of the TFF1 protein include the protein described in (a) below and the protein described in (b) below:
It consists of (a) a protein consisting of the amino acid sequence shown in any of SEQ ID NOs: 1 to 4, and (b) an amino acid sequence having 85% or more identity with the amino acid sequence shown in any of SEQ ID NOs: 1 to 4. In addition, at least one selected from the group consisting of proteins having anticancer activity enhancing activity can be mentioned.

In (b) above, the identity is preferably 90% or more, more preferably 93% or more, still more preferably 95% or more, still more preferably 97% or more, and particularly preferably 98% or more.

The anti-cancer effect enhancing activity can be determined according to or according to a known method. For example, after allowing both the test protein and the anticancer drug to act on cancer cells at a concentration and / or frequency that do not exert a cancer-suppressing effect on their own, the degree of cell proliferation and apoptosis. If the degree of cancer suppression is confirmed as a result, it can be determined that the test protein has anticancer activity enhancing activity. Specifically, the measurement can be performed according to the method described in Examples described later.

As an example of the protein described in (b) above, for example, one or more amino acids are substituted, deleted, added, or inserted into the amino acid sequence shown in any one of (b') SEQ ID NOs: 1 to 4. Examples thereof include a protein having an amino acid sequence and having an anticancer activity enhancing activity.

In the above (b'), the plurality is, for example, 2 to 8, preferably 2 to 6, more preferably 2 to 4, still more preferably 2 or 3, and particularly preferably. Is two.

The TFF1 protein may be chemically modified as long as it has anticancer activity enhancing activity.

The C-terminal of the TFF1 protein may be any of a carboxyl group (-COOH), a carboxylate (-COO- ⁾ , an amide (-CONH ₂ ) or an ester (-COOR).

Here, R in the ester is, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl; for example, a C _3-8 cycloalkyl group such as cyclopentyl, cyclohexyl; for example, phenyl. , C _6-12 aryl groups such as α-naphthyl; phenyl-C _1-2 alkyl groups such as benzyl, phenethyl; C _7- such as α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl. ₁₄ Alkyl group; Pivaloyloxymethyl group etc. are used.

The TFF1 protein may have a carboxyl group (or carboxylate) other than the C-terminal amidated or esterified. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used.

Furthermore, in TFF1 proteins, the amino group of the N-terminal amino acid residue is protected by a protective group (for example, a C _1-6 acyl group such as C _1-6 alkanoyl such as a formyl group or an acetyl group). , N-terminal glutamine residue that can be cleaved and produced in vivo is pyroglutamine oxidized, substituents on the side chain of amino acids in the molecule (eg-OH, -SH, amino group, imidazole group, indole group) , A guanidino group, etc.) is protected by a suitable protective group (eg, a C _1-6 acyl group such as a C _1-6 alkanoyl group such as a formyl group or an acetyl group), or a so-called sugar chain attached. Complex proteins such as glycoproteins are also included.

The TFF1 protein may be added with a known protein tag as long as it has an anticancer activity enhancing activity. Examples of the protein tag include a histidine tag, a FLAG tag, a GST tag and the like.

The TFF1 protein may be in the form of a pharmaceutically acceptable salt with an acid or base. The salt is not particularly limited as long as it is a pharmaceutically acceptable salt, and either an acidic salt or a basic salt can be adopted. Examples of acidic salts are inorganic acid salts such as hydrochlorides, hydrobromide, sulfates, nitrates, phosphates; acetates, propionates, tartrates, fumarates, maleates, apples. Organic acid salts such as acid salts, citrates, methane sulfonates and paratoluene sulfonates; amino acid salts such as asparagates and glutamates can be mentioned. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt.

The TFF1 protein may be in the form of a solvate. The solvent is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include water, ethanol, glycerol, acetic acid and the like.

The TFF1 protein can be obtained by a known method, for example, by chemical synthesis, purification from mammalian cells or tissues (eg, serum, etc.), purification from a transformant containing a polynucleotide encoding the TFF1 protein, and the like. .. In a preferred embodiment of the invention, the TFF1 protein is a chemically synthesized protein. When obtained by purification from a transformant, the transformant is not particularly limited as long as it is a cell capable of expressing the TFF1 protein from a polynucleotide containing a TFF1 protein coding sequence, and bacteria such as Escherichia coli, insect cells, etc. Various cells such as mammalian cells can be used.

As the insect cells, for example, Sf cells, MG1 cells, High Five ^TM cells, BmN cells and the like are used. As the Sf cells, for example, Sf9 cells (ATCC CRL1711), Sf21 cells and the like are used. As the animal cells, for example, monkey COS-7 cells, monkey Vero cells, Chinese hamster cell CHO, mouse L cells, mouse AtT-20 cells, mouse myeloma cells, rat GH3 cells, human FL cells and the like are used.

The TFF1 protein can be a single protein or a combination of two or more proteins.

2-2. Polynucleotide containing the TFF1 protein coding sequence
The TFF1 protein coding sequence is not particularly limited as long as it is a polynucleotide consisting of a base sequence encoding a TFF1 protein.

The polynucleotide containing the TFF1 protein coding sequence is not particularly limited as long as it can express the expression of the TFF1 protein. Typically, the polynucleotide comprises a promoter sequence and a TFF1 protein coding sequence (and optionally a transcription termination signal sequence). The polynucleotide can also be in the form of an expression vector.

The promoter is not particularly limited, and examples thereof include a CMV promoter, an EF1 promoter, an SV40 promoter, an MSCV promoter, an hTERT promoter, a β-actin promoter, a CAG promoter and the like. In addition, various promoters that can be induced by a drug can also be used.

The above-mentioned polynucleotide may be another element (eg, multicloning site (MCS), drug resistance gene, replication origin, enhancer sequence, repressor sequence, insulator sequence, reporter protein (eg, fluorescent protein, etc.), if necessary). A coding sequence, a drug resistance gene coding sequence, etc. may be included.).

The expression vector is not particularly limited, and examples thereof include plasmid vectors such as animal cell expression plasmids; virus vectors such as retrovirus, lentivirus, adenovirus, adeno-related virus, herpesvirus, and Sendai virus.

The polynucleotide can be easily obtained according to a known genetic engineering technique. For example, it can be produced by utilizing PCR, restriction enzyme cleavage, DNA ligation technique, or the like.

2-3. Uses, Other Ingredients, etc. The active ingredient of the present invention is an anticancer effect enhancing activity, that is, an anticancer effect (for example, cell growth inhibitory effect, apoptosis induction) of a substance having an anticancer effect (anticancer agent, etc.). It has an activity to enhance the action, tumor volume reduction action, etc.). Therefore, the active ingredient of the present invention can be used as an anticancer activity enhancer (for example, in addition to pharmaceuticals and reagents, food compositions, oral compositions, health promoters, nutritional supplements (supplements, etc.), etc.). Furthermore, it can be used as a composition for improving cancer (for example, in addition to pharmaceuticals and reagents, food composition, oral composition, health promoting agent, nutritional supplement (supplement, etc.)) together with an anticancer agent. be. The active ingredient of the present invention can be applied to animals, humans, and various cells (for example, administration, ingestion, inoculation, treatment, etc.) as it is or in combination with conventional ingredients in various compositions.

In addition, the active ingredient of the present invention suppresses EMT (Epithelial-Mesenchymal Transition) induced by an anticancer drug, and activates (upwardly regulated) the Wnt pathway by an anticancer drug. It is also possible to suppress it, suppress stemness induced by an anticancer drug, and the like. Therefore, the active ingredient of the present invention, the enhancer of the present invention, and the like can be used for applications utilizing these activities.

The mode of use of the enhancer of the present invention and the composition of the present invention is not particularly limited, and an appropriate mode of use can be adopted depending on the type thereof. Depending on its use, the agent of the present invention can be used, for example, in vitro (for example, added to the medium of cultured cells) or in vivo (for example, administered to an animal). You can also.

The application of the enhancer of the present invention and the composition of the present invention is not particularly limited, but in mammals, for example, humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, etc. Examples include deer. An animal (eg, a patient) of interest in one aspect of the invention has cancer. Examples of cells to be applied include animal cells and the like. The type of cell to be applied is also not particularly limited, and for example, blood cells, hematopoietic stem cells / precursor cells, spp. Examples thereof include cells, muscle cells, epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, cancer cells and the like.

The cancers (cancer cells) targeted by the enhancer of the present invention and the composition of the present invention are not particularly limited, and include, for example, pancreatic cancer, kidney cancer, liver cancer, esophageal cancer, gastric cancer, and colon. Cancer, leukemia, lung cancer, prostate cancer, skin cancer, breast cancer, cervical cancer, etc. Among these, gastrointestinal cancers such as pancreatic cancer, kidney cancer, liver cancer, esophageal cancer, gastric cancer, and colon cancer are preferable, and pancreatic cancer is particularly preferable.

The enhancer of the present invention is preferably used in combination with an anticancer agent. The combination includes not only the case where it is applied at the same time but also the case where it is applied at intervals (for example, several minutes to several days (1 minute to 10 days, etc.)).

The anti-cancer agent used in combination with the enhancer of the present invention and the anti-cancer agent contained in the composition of the present invention are not particularly limited, and various anti-cancer agents can be used. Examples of the anticancer agent include antimetabolites, alkylating agents, microtube inhibitors, antibiotic anticancer agents, topoisomerase inhibitors, platinum preparations, molecular targeting drugs, hormonal agents, biological preparations and the like, which are preferable. Examples include antimetabolites (particularly preferably, among the antimetabolites, nucleic acid analogs).

Antimetabolites include, for example, enocitabine, carmofur, capecitabine, tegafur, tegafur uracil, tegafur gimeracil oteracil potassium, gemcitabine, cytarabine, cytarabine ocphosphat, nerarabine, fluorouracil, fludarabine, pemetrexet. Examples include cladribine, doxifludine, hydroxycarbamide, mercaptopurine and the like.

Examples of the alkylating agent include cyclophosphamide, iphosphamide, nitrosourea, dacarbazine, temozolomide, nimustine, busulfan, melphalan, procarbazine, and ranimustine.

Examples of the microtubule inhibitor include alkaloid-based anticancer agents such as vincristine and taxane-based anticancer agents such as docetaxel and paclitaxel.

Examples of the antibiotic anticancer agent include mitomycin C, doxorubicin, epirubicin, daunorubicin, bleomycin, actinomycin D, acralubicin, idarubicin, pirarubicin, peplomycin, mitoxantrone, amrubicin, and dinostatinstimalamar.

Examples of the topoisomerase inhibitor include CPT-11 having a topoisomerase I inhibitory action, irinotecan, nogitecan, and etoposide and sobzoxane having a topoisomerase II inhibitory action.

Examples of the platinum preparation include cisplatin, nedaplatin, oxaliplatin, carboplatin and the like.

Examples of hormonal agents include dexamethasone, finasteride, tamoxifen, astrosol, exemestane, ethinyl estradiol, chlormaginone, goseleline, bicalutamide, flutamide, bredonizolone, leuprorelin, retrozol, estramustine, toremifene, fosfestrol, and mittan. Examples thereof include methyltestosterone, medroxyprogesterone, and mepitiostane.

Bioforms include, for example, interferon α, β and γ, interleukin 2, ubenimex, dried BCG and the like.

Molecular-targeted drugs include, for example, rituximab, alemtuzumab, trastuzumab, cetuximab, panitzummab, imatinib, dasatinib, nirotinib, gemtuzumab, elrotinib, temsirolimus, bebashizumab, vEGF trap , Ibritumomab tiuxetan, Tamibarotene, tretinoin and the like. In addition to the molecular-targeted drugs specified here, human epithelial growth factor receptor 2 inhibitor, epithelial growth factor receptor inhibitor, Bcr-Abl tyrosine kinase inhibitor, epithelial growth factor tyrosine kinase inhibitor, mTOR inhibition Agents, inhibitors targeting angiogenesis such as vascular endothelial growth factor receptor 2 inhibitors (α-VEGFR-2 antibody), various tyrosine kinase inhibitors such as MAP kinase inhibitors, inhibitors targeting cytokines, Molecular-targeted drugs such as proteasome inhibitors, antibody-anticancer drug formulations, etc. can also be included. These inhibitors also include antibodies.

The enhancer of the present invention and the composition of the present invention may further contain other components, if necessary. The other ingredients are not particularly limited as long as they are ingredients that can be blended in, for example, pharmaceuticals, food compositions, oral compositions, health-promoting agents, dietary supplements (supplements, etc.), but are not particularly limited, for example, bases. , Carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents and the like. Pharmaceutically acceptable carriers and additives include, for example, excipients such as sucrose and starch; binders such as cellulose and methylcellulose; disintegrants such as starch and carboxymethylcellulose; Agents; Aroma agents such as citric acid and menthol; Preservatives such as sodium benzoate and sodium hydrogen sulfite; Stabilizers such as citric acid and sodium citrate; Suspension agents such as methyl cellulose and polyvinylpyrrolid; Surface active agents and the like Dispersants; diluents such as water and physiological saline; base waxes and the like, but are not limited thereto. The form of the enhancer of the present invention and the composition of the present invention is not particularly limited, and can take a form usually used in each use depending on the use.

As a form, if the use is pharmaceutical, any dosage form, for example, tablets (including medially disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly-like drops, etc.), rounds, granules, etc. Oral preparation forms such as fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, and syrups), jelly, and injections (for example, drip injections (eg, drip injections). For example, intravenous drip injectables, etc.), intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections), external preparations (eg, ointments, poultices, lotions), suppository inhalants, ophthalmic agents, etc. Parenteral pharmaceutical forms such as ophthalmic ointments, nasal drops, ear drops, and liposomes can be taken.

The route of administration of the enhancer of the present invention and the composition of the present invention is not particularly limited as long as the desired effect can be obtained, and is enteral administration such as oral administration, tube feeding, enema administration; intravenous administration, and transintestinal administration. Examples thereof include parenteral administration such as arterial administration, intramuscular administration, intracardiac administration, subcutaneous administration, intradermal administration, and intraperitoneal administration.

As for the form, when the use is a health enhancer, a nutritional supplement (supplement, etc.), for example, a tablet (including an orally disintegrating tablet, a chewable tablet, an effervescent tablet, a troche agent, a jelly-like drop agent, etc.), Pharmaceutical form suitable for oral ingestion such as pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions and syrups), jelly (including drinks, suspensions and syrups) Oral formulation), nasal drops, inhalants, anal suppositories, inserts, enema, jelly, injections, patches, lotions, creams, etc. Form).

As a form, when the use is a food composition, liquid, gel or solid foods such as juice, soft drink, tea, soup, soy milk, salad oil, dressing, yogurt, jelly, pudding, sprinkle, milk powder for childcare. , Cake mixes, powdered or liquid dairy products, breads, cookies and the like.

In terms of form, when the application is an oral composition, for example, a liquid (solution, emulsion, suspension, etc.), a semi-solid (gel, cream, paste, etc.), a solid (tablet, particulate agent, capsule, etc.) Arbitrary forms such as film agents, kneaded products, molten solids, waxy solids, elastic solids, etc., more specifically dentifrices (kneaded teeth, liquid teeth, liquid teeth, powdered teeth, etc.), mouthwashes, Examples include coatings, patches, mouth fresheners, foods (eg, chewing gums, tablets, candy, gummy, films, troches, etc.).

The content of the active ingredient of the present invention in the enhancer of the present invention and the composition of the present invention depends on the type, use, mode of use, application target, state of application target, etc. of the active ingredient, and is limited. However, it can be, for example, 0.0001 to 100% by weight, preferably 0.001 to 50% by weight.

The amount of the enhancer of the present invention and the composition of the present invention applied (for example, administration, ingestion, inoculation, etc.) expresses the desired effect (anticancer effect enhancing activity, or an anticancer effect enhanced thereby). The amount of the active ingredient is not particularly limited as long as it is effective, and the weight of the active ingredient is generally 0.01 to 1000 mg / kg body weight per day. The above dose can be administered once a day or in multiple doses (2 to 3 times), and the dose may be adjusted according to the patient's age, pathological condition, and symptoms.

Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.

Materials and Methods Each experiment in the test examples described below used the materials described below and was performed according to the methods described below, unless otherwise noted.

Materials and methods 1. Human Samples Human pancreatic samples were obtained from patients who underwent pancreatic resection for invasive pancreatic liver cancer (n = 46) at Nagoya University Hospital in accordance with the guidelines of the clinical trial review committee of Nagoya University Hospital. ..

Materials and methods 2. Histology and immunohistochemical specimens were fixed overnight in 10% formalin / phosphate buffered saline. Histological analysis was performed on 4 μm paraffin-embedded sections. Immunohistochemical staining was performed using the antibodies shown in Table 1. The sections were incubated overnight with each primary antibody at 4 ° C and 1 hour at room temperature with the secondary antibody. Proteins were visualized using the EnVision detection system (Dako Japan). Slides were counterstained with hematoxylin. Approximately 10 randomly selected non-overlapping visual fields (x200 microscopic visual fields) of stained sections were imaged and evaluated using Keyence BZ-X800 (Keyence). The analysis was performed using ImageJ software published by the National Institutes of Health (NIH).

Materials and methods 3. Cell culture Human pancreatic cancer cell lines (panc1, MiaPaca2, KLM1, KP4, PK8, PK9, PK45h, and PK59) were obtained from the Cell Medicine Center, Institute of Aging Medicine, Tohoku University, Japan. All cell lines were cultured in RPMI 1640 medium (Invitrogen Life Technologies) containing 10% heat-inactivated fetal bovine serum (Equitech-Bio Inc) in a moist atmosphere containing 5% CO ₂ at 37 ° C.

Materials and methods 4. Transfection of TFF1 overexpressing plasmid According to the manufacturer's protocol, the TFF1 overexpressing plasmid or control plasmid was transfected into a pancreatic cancer cell line using the CUY21EDIT ver 2.10 electroporation system (BEX). After transfection, cancer cell lines were incubated for 48 hours and analyzed. In the gemcitabine treatment setting, cells were transfected, incubated for 24 hours, then gemcitabine treated (1 μM) and analyzed.

Materials and methods 5. Cell treatment with TFF1 protein Human recombinant TFF1 (hrTFF1) was purchased from Peprotech. Synthetic human TFF1 (sTFF1) is synthesized by the solid phase peptide synthesis method (Fmoc method), the obtained crude product is purified by reverse phase liquid chromatography, and DMSO is used as an oxidant in a phosphate buffer solution. Obtained by refolding. Human pancreatic cancer cell lines were seeded in 6-well plates and treated with gemcitabine (final concentration 1 μM) or 5-fluorouracil (final concentration 100 μM) and / or TFF1 (hrTFF1 or sTFF1, final concentration 100 ng / ml). After 36 hours, it was analyzed.

Materials and methods 6. Cell proliferation assay Cell proliferation was assessed using the WST-1 Cell Proliferation Assay System (Cell Proliferation Reagent WST-1; Roche). Cancer cell lines were transfected with the TFF1-plasmid and seeded in 96-well plates (5 × 10 ³ cells per well) or seeded and then treated with gemcitabine and / or sTFF1. After 72 hours of incubation, the medium was replaced with fresh medium containing 10 μl WST-1 reagent. Relative cell numbers were calculated by measuring the absorbance of each well at 450 nm.

Materials and methods 7. Western blotting
Proteins were extracted from cultured cells using Laemmli sample buffer. The protein was subjected to 10% or 15% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane (Immobilon; Millipore). Membranes were incubated with the primary antibodies shown in Table 2. Horseradish peroxidase (HRP) -bound anti-rabbit IgG (1: 1,000, Cell Signaling Technology) and HRP-bound anti-mouse IgG (1: 1,000, Cell Signaling Technology) were used as secondary antibodies. Protein expression was detected using the Pierce Western Blot Substrate (Thermo).

Materials and methods 8. Real-time PCR
RNA was extracted from the cell line using QIAcube (Qiagen) according to the manufacturer's protocol. CDNA was synthesized using a high volume cDNA reverse transcription kit (Applied Biosystems). Real-time PCR assays were performed using the 7300 Fast Real-Time PCR System (Applied Biosystems). Each reaction was performed with a 10 μl mixture containing TaqMan Universal PCR Master Mix (Applied Biosystems). TaqMan probes and primers were obtained from Applied Biosystems (shown in Table 3). In each assessment, the relative expression of the gene of interest was normalized to that of the 18S internal control.

Materials and methods 9. Apoptosis Assay Apoptosis analysis was performed using Muse Annexin V and Dead Cell Kit (Millipore) according to the manufacturer's protocol. Pancreatic cells were seeded on 6-well plates and apoptosis rates were analyzed after 48 hours of incubation with gemcitabine and / or TFF1.

Materials and methods 10. Flow cytometric cells were harvested, rinsed twice with PBS and incubated with antibody against CD133 (BD Pharmingen, clone: W6B3C1) and Hoechst 33342 (Invitrogen) for 1 hour at room temperature. After washing, cells were resuspended in PBS. 7-AAD (BD bioscience) was added before the flow cytometric analysis. 7AAD-negative and Hoechst-positive cells were gated and analyzed by flow cytometry (FACS Canto 2, BD).

Materials and methods 11. animal
Pdx1-Cre (stock number: 014647), LSL-p53 ^R172H (stock number: 008652), and LSL-KRAS ^G12D (stock number: 008179) mice were purchased from Jackson Laboratory (Bar Harbor). TFF1-KO (gene name: Tff1tm1a (EUCOMM) Wtsi) mice were purchased from IMPC (International Mouse Phenotyping Consortium). Mice of Pdx1-Cre, LSL-p53 ^R172H , and LSL-KRAS ^G12D mice were mated with TFF1-KO mice to generate mice of the following genotypes: LSL-KRAS ^G12D ; LSL-p53 ^R172H ; TFF1-KO (KPC). / TFF1KO). Recombinant mouse TFF1 (mrTFF1) was purchased from Peprotech (Cranberry, NJ). Gemcitabine (100 mg / kg, intraperitoneal injection) and mrTFF1 (1 μg / body, subcutaneous injection) were started at the age of 2 months. Mice were collected when they were moribund.

Materials and methods 12. Xenograft mouse model Male BALB / c nude mice (8 weeks old, body weight 20-25 g) were purchased from Japan SLC. Panc1 cells (1 × 10 ⁷ ) were inoculated into the thighs of each mouse. After 10 days, mice were randomly divided into 3 groups (5 mice per group) and treated with either PBS, gemcitabine (40 mg / kg) or sTFF1 (1 μg / body) twice weekly for 4 weeks. Tumor growth was assessed by measuring volume (mm ³ ). The volume was calculated by the following formula: (L × W ² ) / 2. Here, L is the major axis of the tumor and W is the minor axis of the tumor.

Materials and methods 13. Statistical analysis All data are shown as mean ± SD. Differences were tested for significance by analysis of variance. Mouse survival was calculated using the Kaplan-Meier method, and differences in survival curves were compared using the Logrank test. All statistical analyzes were performed using the Statistical Package for the Social Sciences (SPSS) ver. 24. A P-value <0.05 was considered statistically significant.

Test example 1. Analysis of the effect of TFF1 overexpression on EMT (Epithelial-Mesenchymal Transition) and anticancer drug susceptibility First, the expression levels of EMT markers (snail and slug) in panc 1 cells transfected with the TFF1 overexpression plasmid were examined. It was analyzed by blotting. The results are shown in Figure 1A. From FIG. 1A, it was found that the expression level of the EMT marker decreased due to the overexpression of TFF1.

Next, panc 1 cells transfected with the TFF1 overexpressing plasmid were treated with an anticancer drug (gemcitabine) and subjected to a cell proliferation assay. The results are shown in Figure 1B. From FIG. 1B, when TFF1 was overexpressed, cell proliferation suppression by anticancer drug treatment was enhanced.

Subsequently, panc 1 cells transfected with the TFF1 overexpressing plasmid were treated with an anticancer drug (gemcitabine) and subjected to an apoptosis assay. The results are shown in FIGS. 1C and 1D. From FIGS. 1C and 1D, when TFF1 was overexpressed, apoptosis by anticancer drug treatment was promoted.

Test example 2. Analysis of the effect of chemically synthesized TFF1 on anticancer drug-induced apoptosis Human pancreatic cancer cells are treated with the anticancer drug (gemcitabine) alone or with both the anticancer drug and the chemically synthesized TFF1 (sTFF1). An apoptosis assay was performed. The results are shown in Figures 2A and 2B. From FIGS. 2A and 2B, it was found that the proportion of apoptotic cells was higher when treated with both the anticancer drug and the chemically synthesized TFF1.

Human pancreatic cancer cells are then treated with chemically synthesized TFF1 alone, anticancer agent alone, or both anticancer agent and chemically synthesized TFF1 to produce apoptosis markers (cleavage (active) caspases 3 and 7). The amount was analyzed by western blotting. The results are shown in Figure 2C. From Fig. 2C, it was found that the apoptotic marker was not increased by the chemically synthesized TFF1 alone, but the apoptosis marker was increased by the combined use of the anticancer drug and the chemically synthesized TFF1 as compared with the anticancer drug alone. ..

Subsequently, human pancreatic cancer cells were treated with chemically synthesized TFF1 alone, anticancer drug alone, or both anticancer drug and chemically synthesized TFF1 to perform a cell proliferation assay. The results are shown in Figure 2D. From Fig. 2D, it was found that the chemosynthetic TFF1 alone did not suppress cell proliferation, but the combined use of the anticancer drug and the chemosynthetic TFF1 suppressed cell proliferation more than the anticancer drug alone. ..

Test example 3. Analysis of the effects of chemically synthesized TFF1 on the EMT and Wnt pathways induced by anticancer agents Human pancreatic cancer cells are treated with chemically synthesized TFF1 alone, anticancer agents alone, or both anticancer agents and chemically synthesized TFF1. Then, the expression levels of EMT markers, epithelial markers, and Wnt target genes were analyzed by RT-PCR. The results are shown in FIGS. 3A, 3B, and 3C. From FIGS. 3A, 3B, and 3C, it was found that the anticancer drug induces EMT and the Wnt pathway is upregulated, while the chemically synthesized TFF1 treatment suppresses these changes. Similar results were also obtained in protein-level analyzes (Fig. 3D, Fig. 3E, and Fig. 3F).

Test example 4. Analysis of the effect of chemically synthesized TFF1 on anticancer drug susceptibility According to the schedule shown in Fig. 4A, mice were implanted with human pancreatic cancer cells, and then the anticancer drug and chemically synthesized TFF1 were administered for a certain period of time. During the test schedule, the tumor diameter was measured from the body surface, and after the test schedule was completed, the tumor was removed, and the appearance was observed and the tumor volume was measured. The results are shown in FIGS. 4B, 4C, and 4D. From FIGS. 4B, 4C, and 4D, it was found that the administration of the anticancer drug and the chemically synthesized TFF1 can further suppress the tumor growth.

Test example 5. Analysis of the effect of chemically synthesized TFF1 on stemness induced by anticancer drugs 1
Human pancreatic cancer cells are treated with chemically synthesized TFF1 alone, anticancer drug alone, or both anticancer drug and chemically synthesized TFF1 to express cancer stem cell markers (CD133, NANOG, and SOX2). Analyzed by metric, western blotting, and RT-PCR. The results are shown in FIGS. 5A, 5B, and 5C. From FIGS. 5A, 5B, and 5C, it was found that the cancer stem cell marker whose expression is increased by the anticancer drug is suppressed by chemically synthesized TFF1.

Test example 6. Analysis of the effect of recombinant TFF1 on anticancer drug sensitivity
Survival was measured in KPC mice by administration of the anticancer drug alone or both the anticancer drug and chemically synthesized TFF1. The results are shown in Figure 6A. From FIG. 6A, it was found that the survival rate was further increased by the administration of the anticancer drug and the chemically synthesized TFF1.

On the other hand, the pancreatic cancer tissue in the above test was immunostained with an antibody against a cancer stem cell marker (CD133). From the immunostained image, the group was divided into a group in which the expression of CD133 was weak (less than 50% of the stained area) and a group in which the expression of CD133 was strong (stained area of 50% or more), and the number of individuals in each group was measured. The results are shown in FIGS. 6B and 6C. From FIGS. 6B and 6C, it was found that the cancer stem cell marker whose expression is increased by the anticancer drug is suppressed by recombinant TFF1.

Test example 7. Analysis of the effect of chemically synthesized TFF1 on stemness induced by anticancer drugs 2
Expression of cancer stem cell markers (CD133, NANOG, and SOX2) by treating human colorectal cancer cells (DLD1) with chemically synthesized TFF1 alone, anticancer agents alone, or both anticancer agents and chemically synthesized TFF1. Was analyzed by RT-PCR. The results are shown in Figure 7. From FIG. 7, it was found that the cancer stem cell marker whose expression is increased by the anticancer drug is suppressed by the chemically synthesized TFF1.

Claims (8)

An anticancer activity enhancer containing at least one selected from the group consisting of a TFF1 (Trefoil Factor Family 1) protein and a polynucleotide containing a TFF1 protein coding sequence. The anticancer action enhancer according to claim 1, which contains the TFF1 protein. The anticancer action enhancer according to claim 1 or 2, wherein the TFF1 protein is a chemically synthesized protein. The anticancer action enhancer according to any one of claims 1 to 3, wherein the target cancer cell is a gastrointestinal cancer cell. The anticancer action enhancer according to any one of claims 1 to 5, which is used in combination with an anticancer agent. The anticancer activity enhancer according to any one of claims 1 to 6, which is an agent for enhancing the anticancer activity of nucleic acid analogs. The anticancer action enhancer according to any one of claims 1 to 7, which is a pharmaceutical or a reagent. A composition for improving cancer, which comprises the anticancer action enhancer according to any one of claims 1 to 7 and the anticancer agent.

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