WO2021206105A1 - 細胞外小胞の分泌を抑制する細胞外小胞分泌抑制剤、およびその用途 - Google Patents
細胞外小胞の分泌を抑制する細胞外小胞分泌抑制剤、およびその用途 Download PDFInfo
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Definitions
- the present invention relates to an extracellular vesicle secretion inhibitor that suppresses the secretion of extracellular vesicles from cells, and its use.
- extracellular vesicles such as exosomes secreted from cells have been attracting attention.
- the extracellular vesicles contain nucleic acids and proteins such as microRNA (miRNA).
- miRNA microRNA
- the extracellular vesicles since these inclusions are transmitted from the cell secreting the extracellular vesicle to the receiving cell via the extracellular vesicle, it is considered to function as a communication tool between cells. ..
- the involvement of extracellular vesicles secreted from the primary cancer has been reported for cancer metastasis.
- the mechanism of extracellular vesicle secretion is elucidated, for example, by suppressing the secretion of the extracellular vesicle based on the mechanism, the effect of the secretion of the extracellular vesicle on the living body can be facilitated. It is also possible to analyze it. Further, in the case of a disease or the like caused by the secretion of the extracellular vesicles, the disease can be treated by suppressing the secretion of the extracellular vesicles based on the mechanism.
- an object of the present invention is to provide a new secretory inhibitor and secretory inhibitory method for the secretion of extracellular vesicles from cells.
- the present invention is an extracellular vesicle secretion inhibitor that suppresses the secretion of extracellular vesicles from cells, which comprises an inhibitor of the serine synthesis pathway.
- the method for suppressing secretion of the present invention is a method for suppressing the secretion of extracellular vesicles from cells, and is characterized by comprising a step of administering the extracellular vesicle secretion inhibitor of the present invention to an administration subject.
- the screening method of the present invention is a method for screening a candidate substance for an extracellular vesicle secretion inhibitor that suppresses the secretion of extracellular vesicles from cells. It is characterized by being selected as a candidate substance that suppresses the secretion of extracellular vesicles from cells.
- the present inventors have involved the serine synthesis pathway in the secretion of extracellular vesicles from cells, and by inhibiting the serine synthesis pathway, the secretion of extracellular vesicles from cells is suppressed. I found out what I could do. As described above, the mechanism of secretion of extracellular vesicles from cells has not been elucidated, and the present inventors have discovered it for the first time. According to the present invention, the secretion of extracellular vesicles from cells can be suppressed by inhibiting the serine synthesis pathway.
- the present invention can be said to be a very useful technique in the medical field, for example.
- FIG. 1 (A) is a graph showing the relative value of the EV amount by the ExoScreen method for the transformant (miR-891b) transfected with miR-891b and the transformant (NC) of the negative control.
- (B) is a graph showing the relative value of the EV amount by the NTA method.
- (A) is a graph showing the relative value of the EV amount by the ExoScreen method for the transformant (siPSAT1) transfected with siPSAT1 and the transformant (NC) of the negative control
- (B) Is a graph showing the relative value of the EV amount by the NTA method.
- FIG. 1 is a graph showing the relative value of the EV amount by the ExoScreen method for the transformant (miR-891b) transfected with miR-891b and the transformant (NC) of the negative control.
- (B) is a graph showing the relative value of the EV amount by the NTA method.
- (A) is a graph showing the relative value of the expression level of the PSAT1 gene with respect to the transformant (miR-891b) and the transformant (NC)
- (B) is a graph showing the relative value of the expression level of the PSAT1 gene. It is a graph showing a relative value
- (C) is a figure showing the relationship between 3'UTR of PSAT1 gene and miR-891b
- (D) is a transformation obtained by transfecting PSAT1 gene and miR-891b. It is a graph which shows the expression level of PSAT1 in a body.
- FIG. 4 is a graph showing relative values of the amount of secreted EV by the NTA method for a transformant (siPSAT1) obtained by transfecting various cancer cells with siPSAT1 and a transformant (NC) with a negative control.
- FIG. 5 is a graph showing the relative amount of CD63-positive EV in cells of a transformant (siPSAT1) obtained by transfecting cancer cells with siPSAT1 and a transformant (NC) of a negative control.
- FIG. 6 shows the results of culturing a transformant (siPSAT1) obtained by transfecting cancer cells with siPSAT1 and a negative control transformant (NC) in a serine-deficient medium or a serine-containing medium, and (A) is a result.
- the graph which shows the relative value of the secreted EV amount by the ExoScreen method (B) is the graph which shows the relative value of the secreted EV amount by the NTA method.
- FIG. 7 is a graph showing the relative amount of CD63-positive EV in cells.
- FIG. 8 is a graph showing the relative value of the amount of secreted EV by the NTA method for cancer cells coexisting with an inhibitor of serine synthesis.
- FIG. 9 is a graph showing relative values of the amount of secreted EV by the NTA method for a transformant (siPSAT1) obtained by transfecting various cancer cells with siPSAT1 and a transformant (NC) with a negative control.
- FIG. 10 shows the results of the breast cancer metastatic strain MDA-MB-231_Luc_D3H2LN
- (A) is a graph showing the relative value of the secreted EV amount by the NTA method
- (B) is a Western blot showing the expression of PSAT1 protein. It is a photograph of.
- FIG. 11 shows the results of mice transplanted with a breast cancer metastatic strain
- (A) is a graph of the tumor volume of the primary tumor (mammary gland) of the mouse
- (B) is the tumor weight of the primary lesion (mammary gland). It is a graph.
- FIG. 11 shows the results of mice transplanted with a breast cancer metastatic strain
- (A) is a graph of the tumor volume of the primary tumor (mammary gland) of the mouse
- (B) is the tumor weight of the primary lesion (mammary gland). It is a graph.
- FIG. 12 is a graph showing the results of lung cancer cells silenced with PSAT1 or PHGDH, (A) is the result of cell viability, and (B) is a graph showing the relative value of the amount of secreted EV by the NTA method.
- (C) is a graph showing a relative value of the amount of secreted EV by the ExoScreen method.
- FIG. 13 is a graph showing the results of colorectal cancer cells silenced with PSAT1 or PHGDH, (A) is the result of cell viability, and (B) is the relative value of the amount of secreted EV by the NTA method.
- (C) is a graph showing the relative value of the secreted EV amount by the ExoScreen method.
- FIG. 14 is a graph showing the amount of EV secreted per cell, (A) is the result of normal epithelial cells of the large intestine, and (B) is the result of normal epithelial cells of the lung.
- the extracellular vesicle (EV) secretion inhibitor of the present invention is hereinafter referred to as an EV secretion inhibitor.
- the EV secretion inhibitor of the present invention is, for example, an expression inhibitor that suppresses the expression of an enzyme protein in the serine synthesis pathway or a catalytic function inhibitor that suppresses the catalytic function of the enzyme protein in the serine synthesis pathway.
- the serine synthesis pathway is a synthetic pathway containing PSAT1.
- the inhibitor of the serine synthesis pathway is a PSAT1 protein expression inhibitor or a catalytic function inhibitor.
- the inhibitor of the serine synthesis pathway is a PHGDH protein expression inhibitor or a catalytic function inhibitor.
- the inhibitor of the serine synthesis pathway is a PSDH protein expression inhibitor or a catalytic function inhibitor.
- the cells are cancer cells.
- the cancer cells selected from the group consisting of colon cancer cells, lung cancer cells, melanoma cells, breast cancer cells, pancreatic cancer cells, and multiple myeloma cells. It is one.
- the cells are virus-infected cells.
- the inhibitor is a low molecular weight compound, a protein, or a peptide.
- the EV secretion inhibitor of the present invention is, for example, from a substance in which an expression inhibitor for the enzyme protein suppresses transcription from a gene encoding the enzyme protein, a substance that decomposes a transcribed transcript, and the transcript. At least one selected from the group consisting of substances that suppress the translation of proteins in the protein.
- the EV secretion inhibitor of the present invention is, for example, at least one nucleic acid substance in which the expression inhibitor is selected from the group consisting of miRNA, siRNA, antisense, and ribozyme.
- the EV secretion inhibitor of the present invention is, for example, an expression vector in which the expression inhibitor expresses the nucleic acid substance.
- the function-suppressing substance of the protein is an activity-inhibiting substance or an activity-neutralizing substance for the enzyme protein.
- the activity neutralizer is an antibody or antigen-binding fragment against the protein.
- the EV secretion inhibitor of the present invention is, for example, an expression vector in which the function inhibitor expresses the activity neutralizer.
- the cell is a cancer cell.
- the cancer cells were selected from the group consisting of colon cancer cells, lung cancer cells, melanoma cells, breast cancer cells, pancreatic cancer cells, and multiple myeloma cells. At least one.
- the cell is a virus-infected cell.
- the administration target is a human or non-human animal.
- the administration is performed in vivo or in vitro.
- the cell is a cancer cell.
- the cancer cells consist of colon cancer cells, lung cancer cells, melanoma cells, breast cancer cells, pancreatic cancer cells, and multiple myeloma cells. At least one selected from the group.
- the cell is a virus-infected cell.
- the extracellular vesicle (EV) secretion inhibitor (hereinafter referred to as EV secretion inhibitor) of the present invention is an agent that suppresses the secretion of extracellular vesicles from cells.
- the EV secretion inhibitor of the present invention is characterized by containing an inhibitor of the serine synthesis pathway as described above.
- the EV secretion inhibitor of the present invention is characterized by containing the inhibitor, and other configurations and conditions are not particularly limited. Further, as the EV secretion inhibitor of the present invention, the description of the method for suppressing EV secretion of the present invention, which will be described later, can be incorporated.
- the inhibitor may be a substance that suppresses the expression of the enzyme protein in the serine synthesis pathway, or may be a substance that suppresses the catalytic function of the enzyme protein in the serine synthesis pathway.
- the present invention is characterized in that the expression behavior of the serine synthesis pathway controls EV secretion, and by inhibiting the synthesis of serine, EV secretion from cells can be suppressed. Therefore, there are no restrictions on the type of substance that inhibits serine synthesis and the method of inhibition.
- the inhibitor only needs to be able to inhibit the serine synthesis pathway, and the form of inhibition is not particularly limited. That is, it may be inhibited by suppressing the expression of the enzyme protein in the serine synthesis pathway, or it may be inhibited by suppressing the catalytic function of the enzyme protein in the serine synthesis pathway.
- the inhibitor is, for example, a substance that suppresses the expression of the enzyme protein in the serine synthesis pathway
- the inhibitor is, for example, a substance that suppresses the catalytic function of the enzyme protein in the serine synthesis pathway.
- the EV secretion inhibitor of the present invention may contain, for example, the expression inhibitor, the catalytic function inhibitor, or both the former and the latter as the inhibitor of the active ingredient.
- the type of the inhibitor is not particularly limited, and examples thereof include low molecular weight compounds such as nucleic acid substances, proteins such as antibodies, and peptides such as antigen-binding fragments.
- the expression-suppressing substance suppresses any step of transcription or translation in the expression of the enzyme protein (hereinafter, also referred to as a target protein) from a gene encoding the enzyme protein (hereinafter, also referred to as a target gene). It may be a thing, and is not particularly limited.
- transcriptional repression include inhibition of transcription from DNA to pre-mRNA, inhibition of RNA processing for forming mature mRNA from pre-mRNA, degradation of pre-mRNA or mature mRNA.
- Examples of the suppression of translation include inhibition of translation from mature mRNA, inhibition of modification of translation products, and the like.
- the expression-suppressing substance is, for example, a nucleic acid substance (hereinafter, also referred to as a nucleic acid-type suppressing substance), may be in a form in which expression is suppressed as it is (first form), or in an in vivo, in vitro, or ex vivo environment. It may be in the form of a precursor (second form) in which the expression is suppressed underneath.
- a nucleic acid substance hereinafter, also referred to as a nucleic acid-type suppressing substance
- Examples of the expression-suppressing substance of the first form include antigene, antisense (antisense oligonucleotide), RNA interference (RNAi) substance, and ribozyme.
- RNAi substances include siRNA and miRNA.
- Antigene for example, inhibits transcription of mRNA
- antisense and miRNA for example, inhibits translation from mRNA
- siRNA and ribozyme for example, degrade mRNA.
- For these expression-suppressing substances for example, either the entire region or a partial region of the target gene may be used as the target region.
- antisense and miRNA can be designed to bind, for example, to the 3'UTR region of the mRNA transcribed from the target gene, and siRNA and ribozyme are, for example, one of the mRNA transcribed from the target gene. It can be designed to be perfectly complementary to the region of the part.
- the expression-suppressing substance of the first form can be obtained by, for example, a screening method as described later, or can be designed from the sequence of the target gene.
- the expression-suppressing substance of the first form may be, for example, single-stranded or double-stranded.
- the constituent unit of the expression-suppressing substance is not particularly limited, and examples thereof include a sugar, a base such as purine or pyrimidine, and phosphoric acid, and a deoxyribonucleotide skeleton or a ribonucleotide skeleton can be mentioned. It may be a non-nucleotide skeleton containing a base such as pyrrimidine or piperidine. These skeletons may be modified or unmodified. Further, the structural unit may be, for example, a natural type or an artificial non-natural type.
- the expression-suppressing substance may be formed from, for example, the same structural unit, or may be formed from two or more types of structural units.
- the expression-suppressing substance of the second form is the precursor, and specific examples thereof include a precursor expressing the expression-suppressing substance of the first form.
- the expression-suppressing substance of the first form can be expressed and function in an environment of in vivo, in vitro, or ex vivo.
- the precursor examples include a form containing the expression-suppressing substance of the first form and a linker.
- the precursor has a form in which both strands of siRNA are linked by the linker.
- the precursor is cleaved to remove the linker from the precursor, resulting in a double-stranded siRNA. Can be produced (expressed).
- shRNA that produces siRNA by cleavage can be exemplified.
- the precursor may be, for example, an expression vector into which the coding sequence of the expression-suppressing substance of the first form is inserted.
- the expression vector for example, the expression-suppressing substance of the first form can be expressed in an environment of in vivo, in vitro, or ex vivo.
- the coding sequence of a precursor such as the above-mentioned shRNA may be inserted into the expression vector.
- the type of the expression vector is not particularly limited, and examples thereof include a plasmid vector and a viral vector. Examples of the viral vector include an adenovirus vector and a Sendai viral vector.
- Examples of the catalytic function inhibitor include an activity inhibitor that inhibits the activity of the enzyme protein and an activity neutralizing substance that neutralizes the activity of the enzyme protein.
- the activity inhibitor is not particularly limited, and examples thereof include low molecular weight compounds.
- the active neutralizing substance examples include an antibody against the enzyme protein or an antigen-binding fragment (antigen-binding peptide) (hereinafter, also referred to as an antibody-type inhibitor).
- the antibody-type inhibitor is also referred to as a neutralizing antibody or a neutralizing antigen-binding fragment because the function of the enzyme protein can be suppressed by binding to the enzyme protein, for example.
- the antibody-type inhibitor can also be obtained by, for example, a screening method as described later.
- the antibody may be, for example, a monoclonal antibody or a polyclonal antibody, and the isotype thereof is not particularly limited, and examples thereof include IgG, IgM, and IgA.
- the antibody is administered to humans, for example, a fully human antibody, a humanized antibody, a chimeric antibody and the like are preferable.
- the antigen-binding fragment may be, for example, a fragment having a complementarity determining region (CDR) of the antibody, as long as it can recognize and bind to the target site of the target protein.
- CDR complementarity determining region
- Specific examples of the antigen-binding fragment include fragments of Fab, Fab', F (ab') and the like.
- the catalyst function-suppressing substance may be, for example, a first form that suppresses the catalytic function of the enzyme protein as it is, or a precursor that is in a state of suppressing expression in an in vivo, in vitro, or ex vivo environment.
- the second form of the above may be used.
- the catalyst function inhibitor of the first form is, for example, the antibody-type inhibitor as described above.
- examples of the precursor of the second form include an expression vector into which a coding sequence of a protein or peptide that suppresses the catalytic function of the enzyme protein is inserted.
- the type of the expression vector is not particularly limited, and examples thereof include a plasmid vector and a viral vector as described above.
- the catalyst function suppressing substance may be, for example, a substance that suppresses a state in which the enzyme protein has catalytic activity and does not lose its function as a catalyst, but can function. That is, as a specific example, an inhibitory substance such as reducing the substrate required for the enzyme protein to function or changing the substrate may be used. The reduction of the substrate may be, for example, suppression of substrate production or substrate degradation.
- the serine synthesis pathway examples include the synthesis pathway (I) represented by the following formula.
- the EV secretion inhibitor of the present invention preferably contains, for example, an inhibitor of the serine synthesis pathway (I) including PSAT1.
- PSAT1 an inhibitor of the serine synthesis pathway
- the present inventors have found that the expression behavior of the serine synthesis pathway regulates EV secretion. Specifically, in abnormal cells such as cancer cells, the amount of EV secreted is higher than that in normal cells due to excessive expression of genes in the serine synthesis pathway and proteins encoded by them. We obtained the finding that it will increase.
- the expression of the gene encoding the protein is suppressed (inhibited) or the function is suppressed (inhibited). It was confirmed that the increase in EV secretion can be suppressed in abnormal cells, and the present invention was established.
- the suppression of EV secretion in the present invention can be said to be, for example, suppression of an increase in EV secretion, and specifically, for example, suppression of an increase in EV secretion above a normal level.
- PHGDH is D-3-Phosphoglycerate Dehydogenase.
- the human PHGDH protein and the PHGDH gene encoding the same are registered under Gene ID: 26227 in a database (Genetic Testing Registry (GTR)).
- GTR Genetic Testing Registry
- the expression-suppressing substance for PHGDH can be set from, for example, the sequence of the PHGDH gene.
- an inhibitor such as NCT-503 or CBR-5884, or a neutralizing antibody such as PHGDH antibody can be used as the catalytic function inhibitor for PHGDH.
- PSAT1 is Phosphoserine Transaminase 1.
- the human PSAT1 protein and the PSAT1 gene encoding it are registered in the database (GTR) under Gene ID: 29966.
- the expression-suppressing substance for PSAT1 can be set from, for example, the sequence of the PSAT1 gene, and as a specific example, miR-891b or the like can be used.
- a neutralizing antibody such as PSAT1 antibody can be used as the catalytic function inhibitory substance for PSAT1 antibody can be used.
- PPPH is Phosphoserine Phosphatase.
- the human PPPH protein and the PSDH gene encoding it are registered in the database (GTR) under Gene ID: 5723.
- the expression-suppressing substance for PPPH can be set from, for example, the sequence of the PPPH gene.
- a neutralizing antibody such as PPPH antibody can be used.
- the inhibitor of the serine synthesis pathway is, for example, a PSAT1 protein expression-suppressing substance and a catalytic function-suppressing substance, a PHGDH protein expression-suppressing substance and a catalytic function-suppressing substance, and a PSDH protein expression-suppressing substance and a catalytic function-suppressing substance. Any one of them may be included, any one of them may be included, and two or more of them may be included.
- the EV secretion inhibitor of the present invention may contain, for example, only the active ingredient, or may further contain other additive components.
- the additive component is not particularly limited, and examples thereof include the following components, preferably pharmacologically acceptable components.
- the additive component can be appropriately set according to, for example, the administration method of the EV secretion inhibitor, the administration target, the dosage form, and the like.
- the additive component examples include excipients.
- the excipient include an aqueous solvent, an alcohol solvent, a polyalcohol solvent, an oily solvent, a liquid medium such as a mixed solvent thereof (for example, an emulsifying solvent), lactose, starch and the like.
- the aqueous solvent include water, physiological saline, an isotonic solution such as sodium chloride, and examples of the oily solvent include soybean oil.
- the additive components include, for example, a binder such as starch paste; a disintegrant such as starch and carbonate; and a lubricant such as talc and wax.
- the additive component may include, for example, a DDS agent for delivering the active ingredient to a target site.
- the cells targeted for suppression of EV secretion are not particularly limited, and the target cells are those for which the effect of secretion or the effect of suppression of secretion is to be confirmed by suppressing the secretion of extracellular vesicles.
- the cell may be, for example, a normal cell or a cell that is abnormal with respect to the target item.
- the item of interest is not particularly limited as abnormal.
- the abnormal cell may be a cancer cell, or when the item is a virus infection, the abnormal cell. May be a virus-infected cell.
- cancer cells are preferable as the cells because the mechanism of cancer development, metastasis, treatment and the like can be analyzed.
- cancer cells of primary cancer that metastasize to other organs are preferable.
- the cancer cells are not particularly limited, and examples thereof include colon cancer cells, lung cancer cells, melanoma cells, breast cancer cells, pancreatic cancer cells, and multiple myeloma cells.
- a virus-infected cell is preferable as the cell because the mechanism of virus infection can be analyzed.
- the type of the virus is not particularly limited, and examples thereof include influenza virus and coronavirus.
- the extracellular vesicles are, for example, exosomes, microvesicles (microvesicles), extracellular vesicles, etc. secreted by the endocytosis pathway, and among them, for example, exosomes.
- Exosomes can generally be detected by marker molecules such as Alix, Tsg101, CD81, CD63, CD9, and flotilin.
- the method of using the EV secretion inhibitor of the present invention is not particularly limited, and for example, it may be added to a subject who wants to suppress the secretion of extracellular vesicles, and the addition method is not particularly limited, for example, in vivo. It may be in vitro or ex vivo.
- the target to which the EV secretion inhibitor of the present invention is added is, for example, a cell or tissue, a living body, or the like.
- the types of cells and tissues and the parts (organs) of a living body are not particularly limited, and examples thereof include large intestine, lung, skin, breast, mammary gland, mammary gland, pancreas, and bone marrow.
- the cells and tissues may be, for example, those isolated from a living body, cell lines or cultures thereof.
- the cells and tissues to be added may be, for example, derived from humans or non-human animals.
- the living body to be added may be, for example, a human or a non-human animal. Examples of the non-human animal include mammals such as mice, rats, rabbits, horses, sheep, cows, and camels.
- the EV secretion inhibitor corresponds to, for example, a target protein (the enzyme protein) or a target gene derived from the specific non-human animal relatively specifically.
- the inhibitor is, for example, an inhibitor that corresponds relatively specifically to the human-derived target protein or target gene. It is preferable to have.
- the EV secretion suppressing method of the present invention is a method of suppressing EV secretion from cells, and is characterized by including a step of administering the EV secretion suppressing agent of the present invention to an administration subject.
- the point of the present invention is to use the EV secretion inhibitor of the present invention, and other steps and conditions are not particularly limited.
- the description in the EV secretion suppressing agent of the present invention can be incorporated.
- EV secretion from the cell can be suppressed by adding and incubating the EV secretion inhibitor in the presence of a medium.
- the conditions of the incubation are not particularly limited, and for example, the medium, temperature, time, humidity and the like can be set according to the type of cells.
- the administration target is a tissue
- EV secretion from the cells constituting the tissue can be suppressed by adding and incubating the EV secretion inhibitor in the presence of a medium.
- the incubation conditions are not particularly limited, and for example, the medium, temperature, time, humidity, and the like can be set according to the type, size, and the like of the tissue.
- the administration method of the EV secretion inhibitor is not particularly limited, and examples thereof include parenteral administration, oral administration, and intravenous administration.
- the administration conditions are not particularly limited, and can be appropriately determined, for example, according to the type of living body, the type of organ to be administered, and the like.
- the administration site may be, for example, a target organ, that is, an organ having cells for which EV secretion is desired to be suppressed (also referred to as a target site), or the active ingredient of the EV secretion inhibitor up to the target site. It may be a site capable of delivering the inhibitor.
- a target organ that is, an organ having cells for which EV secretion is desired to be suppressed
- the administration site may be the large intestine of the target site or a site capable of delivering the inhibitor to the large intestine.
- the administration site may be the lung of the target site, a site capable of delivering the inhibitor to the lung, and the same applies to other organs.
- parenteral administration method examples include affected area injection, intravenous injection, subcutaneous injection, intradermal injection, drip injection, and transdermal administration.
- the form of the EV secretion inhibitor is not particularly limited, and as described above, it can be appropriately set according to the administration method and the like, and the above description can be incorporated.
- the dosage form is not particularly limited and can be appropriately determined depending on the administration method.
- it may be liquid, creamy, gel or the like, and can be prepared by mixing the medium and the inhibitor.
- the aqueous solvent is, for example, physiological saline, isotonic solution, etc.
- the oily solvent is, for example, soybean oil, etc.
- the emulsifying solvent is, for example, a mixed solution thereof.
- the parenteral administration agent may further contain, for example, alcohol, polyalcohol, surfactant and the like.
- the parenteral administration agent may contain a DDS agent for effectively delivering the inhibitor from a site other than the target site to the target site.
- the parenteral administration agent contains, for example, a DDS agent that specifically recognizes the cancer cells. But it may be.
- the dosage form of the oral administration is not particularly limited, and examples thereof include tablets, pills, granules, powders, capsules, and syrups.
- the orally administered agent may contain, for example, a diluent, an excipient, a carrier and the like.
- the oral administration agent may include, for example, a DDS agent for effectively delivering the inhibitor to the target site.
- the oral administration agent may contain, for example, a DDS agent that specifically recognizes the cancer cells. good.
- the administration conditions of the EV secretion inhibitor of the present invention can be appropriately determined according to, for example, age, body weight, type of organ to be administered, sex, and the like.
- the present inventors secrete extracellular vesicles from cancer cells of the primary cancer, and the transmission of information between cells via the extracellular vesicles causes cancer metastasis.
- the EV secretion inhibitor of the present invention as described above, by administration to a target site (cancer organ) of a patient suffering from cancer, secretion of extracellular vesicles from cancer cells at the target site is performed.
- the EV secretion inhibitor of the present invention can be used, for example, as a therapeutic agent for cancer.
- the treatment includes, for example, the meaning of so-called actions for alleviating the progression of cancer, curing cancer, etc., as well as preventive actions for preventing morbidity and recurrence of cancer. ..
- the EV secretion inhibitor of the present invention may be used, for example, for any one purpose, or may be used for two or more purposes.
- the administration condition of the EV secretion inhibitor of the present invention is, for example, in addition to the items exemplified above, further, the type of cancer (for example, colon cancer, lung cancer, melanoma, etc.). Breast cancer, pancreatic cancer, multiple myeloma, etc.), the degree of cancer progression, etc. can be determined as appropriate.
- the living body may be, for example, a subject suffering from the cancer from the viewpoint of treatment, a subject not suffering from the cancer from the viewpoint of prevention, or a subject whose presence or absence is unknown. ..
- the use of the present invention for suppressing EV secretion is not limited to such an example.
- EVs such as exosomes are involved in cell-cell transmission in the body.
- Specific examples include transmission of an infected virus (for example, influenza virus, coronavirus, etc.) in the body. Therefore, according to the EV secretion inhibitor of the present invention, by suppressing the secretion of EV, for example, the transmission of a virus can be suppressed, and as a result, the spread of virus infection in the body can be inhibited.
- the EV whose secretion is suppressed by the EV secretion inhibitor of the present invention plays a role of transmitting information between cells by, for example, transferring an information substance in a certain cell to another cell. It is known. Therefore, the EV secretion inhibitor of the present invention can be said to be, for example, an intercellular signal transduction inhibitor, and the EV secretion inhibitor of the present invention can be said to be, for example, an intercellular signal transduction inhibitory method.
- the screening method of the present invention is a method for screening a candidate substance for an EV secretion inhibitor that suppresses the secretion of EV from cells, and an inhibitor that inhibits the serine synthesis pathway is selected from a test substance as an extracellular small substance from cells. It is characterized by being selected as a candidate substance that suppresses the secretion of vesicles.
- the type of the cell is not particularly limited and is as described above, and for example, cancer cells can be exemplified. As described above, the cancer cells include, for example, colon cancer cells, lung cancer cells, melanoma cells, breast cancer cells, pancreatic cancer cells, multiple myeloma cells and the like.
- the screening method of the present invention is, for example, a subject to an expression system expressing the enzyme protein (the target protein) from a gene encoding the enzyme protein (the target gene).
- the step of selecting the test substance, which is relatively lower than the above, as the candidate substance is included.
- the screening method of the present invention is, for example, a step of bringing the test substance into contact with the enzyme protein (the target protein), a step of detecting the catalytic activity of the enzyme protein. , And the step of selecting the test substance whose catalytic activity of the enzyme protein is relatively lower than that of the control system in which the test substance does not coexist is selected as the candidate substance.
- the screening method of the present invention is, for example, a step of contacting the test substance with the enzyme protein (the target protein), the target protein. It includes a step of detecting the binding between the substance and the test substance and a step of selecting the test substance bound to the target protein as the candidate substance.
- the present invention is the inhibitor for use in suppressing the secretion of extracellular vesicles from cells.
- the description of the EV secretion inhibitor of the present invention and the EV secretion suppression method of the present invention can be incorporated.
- Human colon adenocarcinoma cell line HCT116 (ATCC CCL-247) was used as the colon cancer cell, and human alveolar basal epithelial adenocarcinoma cell line A549 (ATCC CCL-185) was used as the lung cancer cell.
- Human melanoma cell line A375 (ATCC CRL-1619) as melanoma cells
- human breast cancer cell line MM231 (ATCC HTB-26) as breast cancer cells
- human pancreatic adenocarcinoma cell line Panc-1 (ATCC) as pancreatic cancer cells.
- CRL-1469 a human multiple myeloma cell line RPMI8226 (ATCC CCL-155) was used as the multiple myeloma cells.
- nucleic acid molecule As the nucleic acid molecule miRNA, miR-891b (also referred to as miR-891b mimic; SEQ ID NO: 1: GCACUUACCUGACAUGA) (product number 4446066, Ambion) was used, and as the nucleic acid molecule miRNA for negative control, miRNA MicNegive 4464058) (Ambion) was used. As the nucleic acid molecule siRNA, siPSAT1 (product number siGENOME SMART pool siRNA M-01398, Dharmacon) was used, and as the nucleic acid molecule siRNA for negative control, ALL STAR negative control siRNA (SI0365038) (both Qiagen) was used.
- siPSAT1 product number siGENOME SMART pool siRNA M-01398, Dharmacon
- RPMI complete medium containing 10% heat-inactivated fetal bovine serum (FBS) and antibiotic-antifungal solution (Gibco) added to RPMI 1640 medium (Gibco) was used.
- the medium for HCT116 is Maccoy5A complete medium supplemented with 10% heat-inactivated FBS and antibiotic-antifungal agent, and the medium for other cells is 10% heat-inactivated in DMEM medium (Gibco).
- DMEM complete medium supplemented with FBS and antibiotic-antifungal agents was used.
- the culture conditions were 37 ° C., 5% carbon dioxide, and 95% relative humidity (RH). Approximately 100,000 cells were seeded in 18 mL of the complete medium and incubated for 3-4 days using cells with less than 20 passages.
- the cultured cancer cells are washed with phosphate buffered saline (PBS) and the medium is replaced with advanced RPMI or advanced DMEM containing the antibiotic-antifungal agent and 2 mmol / L L-glutamine (Gibco). rice field.
- PBS phosphate buffered saline
- the prepared culture solution after the replacement was centrifuged at 2,000 ⁇ g for 10 minutes to remove cells, and the obtained supernatant was filtered through a 0.22 ⁇ m filter (Millipore).
- the obtained filtrate was centrifuged at 110,000 xg for 70 minutes to obtain EV-concentrated pellets.
- the pellet was washed with 11 mL of PBS, ultracentrifuged at 110,000 xg for 70 minutes, and recovered again.
- a biotinylated anti-human CD63 antibody and an AlphaLISA acceptor bead-binding anti-human CD63 antibody were used. Then, after incubating the plate at 37 ° C. for 1 hour, 25 ⁇ L of 80 ⁇ g / mL AlphaScreen streptavidin-coated donor beads was added, and the plate was further incubated at 37 ° C. for 30 minutes in the dark. Next, the detection device was set to an excitation wavelength of 680 nm and an emission detection wavelength of 615 nm, and the emission in the wells of the plate was measured.
- mouse monoclonal anti-human CD9 (clone 12A12) and CD63 antibody (clone 8A12) both Cosmo Bio
- Nanoparticle tracking tracking analysis EV analysis by NTA The recovered secretory EV was suspended in PBS, and a dilution system was prepared with PBS and analyzed by NanoSight particle tracking analysis (LM10, software Ver. 2.03). The particle tracking obtained at least five 60-second videos from each sample at camera level 14. The analysis settings were optimized and kept constant across the samples. The EV concentration was calculated as particles / cells in the culture medium, and a net EV secretion rate was obtained. Since the EV measurement result by the ExoScreen method showed a correlation with the EV measurement result by the NTA analysis, it was confirmed that the secreted EV amount can be measured by the ExoScreen method.
- Transient transfection assay In a 6-well plate, 2 mL of a suspension of cancer cells was seeded under the condition of 1.0 ⁇ 10 5 cells / well, incubated for 24 hours, and then 10 nmol / L of the desired nucleic acid molecule was added to the transfection reagent ( The nucleic acid molecule was transfected under the trade name DharmaFECT Transfection Reagent 1). As the nucleic acid molecule, the miRNA and the siRNA were used. After the 24 hour incubation, the medium was replaced with the advanced RPMI 1640 medium or advanced DMEM medium containing the antibiotic-antifungal agent and 2 mmol / L-glutamine (Gibco). Forty-eight hours after the replacement, total RNA was extracted and expression of the gene of interest was measured by qPCR.
- RNA extraction and qPCR analysis Total RNA was extracted from cultured cells using commercially available reagents (trade name QIAzol, trade name miRNeasy Mini Kit, Qiagen). The reverse transcription reaction was carried out using a commercially available kit (trade name: High-Capacity cDNA Reverse Transcription Kit, Applied Biosystems) and a random hexamer primer. Real-time PCR analysis was performed using a commercially available kit (trade name StepOne Plus, trade name TaqMan Universal PCR Master Mix, Thermo Fisher Scientific). mRNA expression was normalized with ⁇ -actin. As the probe for PSAT1, a TaqMan probe (Applied Biosystems) was used.
- Example A [Example A1] Target genes involved in the regulation of EV secretion in colorectal and lung cancers have been identified.
- FIG. 1 (A) is a graph showing the relative value of the secreted EV amount by the ExoScreen method, and (B) is the graph showing the relative value of the secreted EV amount by the NTA method.
- the transformants (miR-891b) of colon cancer cell HCT116 and lung cancer cell A549 transfected with miR-891b were transformed in both the CM and EV fractions.
- the amount of secreted EV was significantly suppressed from the body (NC).
- FIG. 1 (B) the same result was obtained by the NTA method. From these results, it was found that miR-891b suppresses EV secretion in colon cancer cells and lung cancer cells.
- the transformant (siPSAT1) of colon cancer cell HCT116 and lung cancer cell A549 transfected with siPSAT1 is a transformant (NC) in both the CM and EV fractions. Therefore, the amount of secreted EV was significantly suppressed. Further, as shown in FIG. 2 (B), the same result was obtained by the NTA method. As described above, EV secretion was suppressed by downregulation of the PSAT1 gene by siPSAT1, and it was found that the target gene of miR-891b is the PSAT1 gene, together with the result of (1) above.
- FIG. 3A is a graph showing a relative value of the expression level of the PSAT1 gene.
- the expression of PSAT1 protein was detected by Western blotting for the transformant (miR-891b) and the control transformant (NC) for the transformant (miR-891b).
- ⁇ -actin was also detected as a control.
- FIG. 3 (B) is a photograph of a Western blot showing the expression of PSAT1 protein.
- miR-891b perfectly matches the continuous 7-base region in the 3'UTR (SEQ ID NO: 2: UGGACUUAAUAAUGCAAGUGUGC is a partial region of 3'UTR) of human PSAT1 mRNA.
- SEQ ID NO: 2 UGGACUUAAUAAUGCAAGUGUGC is a partial region of 3'UTR
- SEQ ID NO: 4 TTCAACG
- the wild-type PSAT1 gene or mutant PSAT1 gene is subcloned into the plasmid vector psiCHECK2, and the recombinant vector is transfected into human fetal kidney cells (HEK293 cells) with miR-891b or negative control miRNA using Lipofectamine 3000. bottom. Forty-eight hours after transfection, the luciferase activity of the transformant was quantified with a plate reader according to the procedure of Dual-Luciferase Reporter Assay System.
- FIG. 3D is a graph showing the expression level of PSAT1 as a relative value of luciferase activity.
- transfection of miR-891b suppressed the expression of the PSAT1 gene and the expression of the PSAT1 protein. Furthermore, in the case of wild-type PSAT1 in which the sequence of the recognition region of miR-891b is a wild type, the expression level of PSAT1 was significantly reduced by transfection of miR-891b, but the sequence of the recognition region of miR-891b was mutated. By doing so, the expression level of PSAT1 did not decrease. From these facts, it was found that miR-891b recognized the 3'UTR of PSAT1 and cleaved PSAT1. That is, it was confirmed that the PSAT1 gene is a direct target of miR-891b.
- Example A2 It was confirmed that the suppression of EV secretion from various cancer cells by suppressing the expression of the PSAT1 gene.
- siRNA siPSAT1 was transfected and nanoparticle tracking, except that melanoma cell A375, breast cancer cell MM231, pancreatic cancer cell Panc-1, and multiple myeloma cell RPMI8226 were used.
- EV secretion was measured by follow-up analysis (NTA). These results are shown in FIG. FIG. 4 is a graph showing a relative value of the amount of secreted EV by the NTA method.
- the amount of secreted EV was significantly suppressed in the transformant (siPSAT1) transfected with siPSAT1 as compared with the transformant (NC) of the negative control. From these results, it was found that EV secretion of not only colon cancer cells and lung cancer cells but also various cancer cells can be suppressed by suppressing the expression of PSAT1. From this, it can be said that by suppressing the expression of PSAT1, it is possible to suppress EV secretion from various cancer cells, treat these cancers, and prevent cancer metastasis to other organs.
- siRNA siPSAT1 was transfected into colon cancer cell HCT116 and lung cancer cell A549, and EV biogenesis after PSAT1 silence was confirmed.
- CD63 and PSAT1 were confirmed by immunofluorescence for each transformant after silencing, accumulation of CD63, which is an EV marker, was confirmed in the cytoplasm after PSAT1 silencing.
- miR-891b was transfected, the accumulation of CD63 was confirmed. From this result, it was confirmed that by suppressing the expression of PSAT1, the production of EV itself is maintained, but the produced EV is accumulated in the cytoplasm and the secretion of EV from the cell to the outside is suppressed. ..
- the transformant after PSAT1 silence was co-immunostained with CD63, which is an EV marker, and EEA1, which is an early endosome marker, or Rab7, which is an late endosome marker.
- CD63 which is an EV marker
- EEA1 which is an early endosome marker
- Rab7 which is an late endosome marker.
- CD63 which is an EV marker
- EEA1 which is an early endosome marker
- Rab7 which is an late endosome marker
- Example B In Example A, it was confirmed that PSAT1 is involved in EV secretion, and that suppression of PSAT1 expression can suppress EV secretion. Since PSAT1 is an enzyme protein of the serine synthesis pathway, it was speculated that the serine synthesis pathway is involved in EV secretion, and that inhibition of the serine synthesis pathway, that is, inhibition of serine synthesis can suppress EV secretion. Therefore, in Example B, it was confirmed that EV secretion can be suppressed by inhibiting any step of the serine synthesis pathway regardless of PSAT1.
- Example B1 Since normal DMEM medium contains serine, a serine-deficient medium was used, and the effect of serine addition on EV secretion was confirmed.
- Serum-free selenium-deficient MEM medium containing 1 ⁇ insulin, transferrin, selenium solution (100 ⁇ ITS-G), 1 ⁇ MEM vitamin solution, and serine (4 mmol / L), except that it does not contain serine.
- Serum-free selenium-deficient MEM medium was used.
- Colorectal cancer cells HCT116 and lung cancer cells A549 were seeded in 96-well plates at 5 ⁇ 10 3 cells / well and in 6-well plates at 1.5 ⁇ 10 5 cells / well (Day 0). ), Medium (DMEM containing 10% FBS and 1 ⁇ antibiotic-antimycotic), incubated for 24 hours.
- each cell was transfected with siRNA (siPSAT1) or the ALL STAR negative control siRNA (NC) and incubated for an additional 24 hours.
- siRNA siPSAT1
- NC ALL STAR negative control siRNA
- the medium in the wells was replaced with the Serine-deficient MEM medium or the Serine-containing MEM medium, and after further incubation for 48 hours (Day 4), the 96-well plate was used in the ExoScreen method. The medium after culturing was collected from the well plate. Secreted EV was recovered from the recovered medium and NTA was performed.
- the amount of secreted EV by the ExoScreen method was determined as a relative value with the result of the transformant transfected with siRNA (siPSAT1) as 1, and the amount of secreted EV by NTA was the amount of transformed product transfected with siRNA (siPSAT1).
- the relative value was calculated with the amount of secreted EV as 1.
- FIG. 6 (A) is a graph showing the relative value of the secreted EV amount by the ExoScreen method, and (B) is the graph showing the relative value of the secreted EV amount by the NTA method.
- the negative control (NC) not silenced by PSAT1 showed almost the same results in the serine-deficient MEM medium and the serine-containing MEM medium.
- the amount of EV secreted was lower than that of the negative control transformant (NC).
- the EV secretion amount was significantly increased by the use of the serin-containing MEM medium (that is, the addition of serin) as compared with the result of using the serin-deficient MEM medium.
- the result was similar to that of Negative Control (NC). From these results, it was found that serine synthesis is involved in EV secretion, and that inhibition of serine synthesis can suppress EV secretion.
- Example B2 Inhibitors in the serine synthesis pathway were used to confirm the effect on EV secretion.
- NCT-503 an inhibitor of the enzyme protein (PHGDH) in the serine synthesis pathway, was used.
- the test was conducted as follows. Colorectal cancer cells HCT116 and lung cancer cells A549 were seeded in 6-well plates at 1.5 ⁇ 10 5 cells / well (Day 0), respectively, and the medium (DMEM with 10% FBS, 1x anti-anti medium). Incubated for 24 hours. After incubation (Day 1), the medium was removed, and then a serine-deficient medium and an inhibitor solution were added to the wells. The inhibitor solution was prepared by dissolving NCT-503 in DMSO, and the concentration of the inhibitor per well was 2.5 ⁇ mol / L. Then, after further incubation for 48 hours (Day 3), the medium in the well was collected and the cells in the well were counted. Negative control (NC) was carried out in the same manner except that DMSO was added instead of the inhibitory solution.
- DMEM fetal bovine serum
- the intracellular accumulated EV was analyzed as follows. That is, the collected cells were immunostained, the area of CD63 single positive was measured based on the fluorescence intensity, and the value divided by the number of nuclei was determined as the amount of EV accumulated in the cells. These results are shown in FIG. On the other hand, for secreted EV, EV was recovered from the recovered medium and NTA was performed. The amount of secreted EV by NTA was determined as a relative value with the amount of secreted EV of the negative control as 1. The result is shown in FIG.
- Example C Using various cancer cells, we confirmed the regulation of EV secretion by serine synthesis.
- PSAT1 silencing was used to inhibit the serine synthesis pathway, but as described above, it is not limited to PSAT1 silencing, and if serine synthesis is inhibited, EV secretion is similarly suppressed. It has been confirmed that it can be done.
- colon cancer cell lines HCT15, COLO201, COLO205, HT-29
- normal colon fibroblasts CCD-18co
- lung cancer cell lines A427, H1650, H2228) and normal lung epithelial cells
- ovarian cancer, breast cancer, melanoma, head and neck cancer, multiple myeloma, and pancreatic cancer also have PSAT1 in cancer cells rather than normal cells. It was confirmed that the expression level was significantly high.
- the expression level of PSAT1 in lung cancer patients showed a high correlation with the survival rate. Specifically, the higher the expression level, the lower the survival rate.
- FIG. 9 is a graph showing a relative value of the amount of secreted EV by the NTA method.
- Example D Using NCT-503, an inhibitor of the enzyme protein (PHGDH) in the serine synthesis pathway, reduction of tumor volume and suppression of EV secretion in vivo were confirmed.
- PEGDH enzyme protein
- MDA-MB-231_Luc_D3H2LN cell line (hereinafter referred to as D3H2LN) was used as a breast cancer metastatic strain.
- This cell line is a modified strain of the parent breast cancer cell MDA-MB-231, and it has been confirmed that the amount of exosomes secreted is significantly higher than that of the parent strain and the expression level of PSAT1 is high. That is, with respect to the parent strain and the D3H2LN, the EV secretion amount was measured by NTA analysis and the expression of PSAT1 was measured by Western blotting in the same manner as in Example A1. The result is shown in FIG. In FIG.
- (A) is a graph showing a relative value of the amount of secreted EV by the NTA method
- (B) is a photograph of a Western blot showing the expression of PSAT1 protein. As shown in FIG. 10, it has been confirmed that D3H2LN has a significantly higher amount of exosome secretion and a significantly higher expression level of PSAT1 than the parent strain.
- mice immunodeficient mice C.I.
- a B-17 scid mouse female, 6 weeks old was used.
- the D3H2LN was suspended in PBS to prepare a 1 ⁇ 10 6 cell / 100 ⁇ L cell suspension.
- 100 ⁇ L of the cell suspension was injected subcutaneously into the mammary gland of the mouse and transplanted.
- NCT-503 solution solvent PBS
- the volume of the tumor in the primary lesion was measured weekly using IVIS-Spectrum (Sumisho Pharma International Co., Ltd.). Specifically, it was calculated by a calculation formula (minor axis ⁇ minor axis ⁇ major axis ⁇ 0.5) using the major axis and the minor axis of the tumor.
- the body weight of the mouse was measured every day. On the 35th day (Day 35), the mice were sacrificed, the mammary gland, which was the primary lesion, and the lung, which was the metastatic lesion, were excised, and the weight of the primary lesion was measured.
- (A) is a graph (calculated value) of the tumor volume of the primary tumor (mammary gland) of the mouse on Day 21 and Day 35
- (B) is the tumor weight of the primary tumor (mammary gland) of the mouse of Day 35. It is a graph (measured value).
- the volume of the tumor of the primary lesion was significantly reduced over time in the administration group to which the inhibitor was administered as compared with the non-administration group.
- the weight of the tumor in the primary lesion was significantly reduced in the administration group to which the inhibitor was administered as compared with the non-administration group.
- the lungs in the administration group tended to have fewer metastatic lesions than the lungs in the non-administration group.
- Example E Regarding PHGDH, which is an enzyme protein of the serine synthesis pathway, PSAT1 whose EV secretion suppression has been confirmed in the above examples is used as a positive control, and the expression of each gene (PHGDH gene and PSAT1 gene) is suppressed from cancer cells. Suppression of EV secretion was confirmed. Unless otherwise specified, the method was performed in the same manner as in Example B1.
- siPHGDH product number M-9518-01-0010, Dharmacon
- siPSAT1 was used in the same manner as in Example A.
- Lung cancer cells A549 and colon cancer cells HCT116 and were seeded on a 96-well plate at 5 ⁇ 10 3 cells / well, respectively, and on a 6-well plate at 1.5 ⁇ 10 5 cells / well, respectively.
- DMEM complete medium in which 10% heat-inactivated FBS and an antibiotic-antifungal agent were added to DMEM medium (Gibco) was used.
- siRNA siPSAT1 or siPHGDH
- ALL STAR negative control siRNA was incubated for an additional 24 hours.
- the medium in the wells is replaced with advanced DMEM medium, and after further incubation for 48 hours (Day 4), the 96-well plate is used for the ExoScreen method, and the cultured medium is recovered from the 6-well plate. bottom. Secreted EV was recovered from the recovered medium and NTA was performed. In addition, the number of cells of Day 4 cultured in the 6-well plate was counted, and the survival rate was calculated with the number of cells of the negative control as a relative value of 1.
- FIG. 12 shows the result of lung cancer cell A549
- FIG. 13 shows the result of colon cancer cell HCT116.
- (A) is a result of cell viability
- (B) is a graph showing a relative value of the amount of secreted EV by the NTA method.
- the silenced transformants showed little change in survival rate compared to the unsilencing control. .. Also, as shown in FIGS. 12 (B) and 12 (C), the silenced transformant had reduced EV secretion as compared to the control.
- Example F In Example B2, it was confirmed that EV secretion in cancer cells can be suppressed by using the inhibitor NCT-503 that inhibits PHGDH in the serine synthesis pathway.
- NCT-503 did not suppress EV secretion due to cell death of cells, but suppressed EV secretion peculiar to abnormal cells such as cancer cells by inhibiting the serine synthesis pathway. The supplementary data of this is shown.
- NCT-503 was added so as to be 2.5 ⁇ mol / L with respect to the medium of the well. Therefore, for each normal epithelial cell of lung (HBEC) and colon (HCoEpiC), the medium without NCT-503 added (DMSO added) or a predetermined concentration (0.156225 to 2.5 ⁇ mol / L) is adjusted. The cells were cultured using the NCT-503-added medium supplemented with NCT-503, and the cell viability and the amount of EV secreted by ExoScreen were confirmed.
- the test was conducted as follows.
- the normal epithelial cells were seeded on a 96-well plate at 5000 cells / well (Day 0) and incubated in medium for 24 hours (Day 1).
- As the medium for the large intestine cells CoEpiCM 1x anti-anti medium was used, and as the medium for the lung cells, BEBM 1x anti-anti medium was used.
- the inhibitor solution NCT-503 / DMSO
- An additional 48 hours of incubation was performed (Day 3). Then, the medium in the well was collected, and the cells in the well were counted.
- FIG. 14 is a graph showing the amount of EV secreted per cell, (A) is the result of normal epithelial cells of the large intestine, and (B) is the result of normal epithelial cells of the lung.
- A is the result of normal epithelial cells of the large intestine
- B is the result of normal epithelial cells of the lung.
- the EV secretion amount of the NCT-503-added medium is relative to the EV secretion amount of the DMSO-added medium without NCT (0M).
- the relative value was 0.98 for normal epithelial cells in the colon and 1.17 for normal epithelial cells in the lung, showing almost no difference. That is, the addition of NCT-503 did not affect EV secretion in normal epithelial cells.
- Example B2 by adding 2.5 ⁇ mol / L NCT-503 to the cancer cells, it was compared with the control in which only DMSO, which is the solvent of NCT-503, was added.
- the secretion of extracellular vesicles from cells can be suppressed by inhibiting the serine synthesis pathway. Therefore, by suppressing the secretion according to the present invention, it becomes possible to analyze the effect of the secretion of extracellular vesicles or the suppression of the secretion on the living body. Therefore, the present invention can be said to be a very useful technique in the medical field, for example.
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Abstract
Description
本発明の細胞外小胞(EV)分泌抑制剤(以下、EV分泌抑制剤という)は、細胞からの細胞外小胞の分泌を抑制する剤である。本発明のEV分泌抑制剤は、前述のように、セリン合成経路の阻害剤を含むことを特徴とする。本発明のEV分泌抑制剤は、前記阻害剤を含むことが特徴であって、その他の構成および条件は、特に制限されない。また、本発明のEV分泌抑制剤は、後述する本発明のEV分泌の抑制方法等の記載を援用できる。
本発明のEV分泌抑制方法は、細胞からのEVの分泌を抑制する方法であり、前記本発明のEV分泌抑制剤を、投与対象に投与する工程を含むことを特徴とする。本発明は、前記本発明のEV分泌抑制剤を使用することがポイントであって、その他の工程および条件は、特に制限されない。前記本発明のEV分泌抑制方法に関しては、前記本発明のEV分泌抑制剤における記載を援用できる。
本発明のスクリーニング方法は、細胞からのEVの分泌を抑制するEV分泌抑制剤の候補物質のスクリーニング方法であり、被検物質から、セリン合成経路を阻害する阻害物質を、細胞からの細胞外小胞の分泌を抑制する候補物質として選択することを特徴とする。前記細胞の種類は、特に制限されず、前述のとおりであり、例えば、がん細胞が例示できる。前記がん細胞は、前述のように、例えば、大腸がん細胞、肺がん細胞、メラノーマ細胞、乳がん細胞、膵臓がん細胞、および多発性骨髄腫細胞等があげられる。
本発明は、細胞からの細胞外小胞の分泌抑制に使用するための前記阻害剤である。前記抑制物質は、前記本発明のEV分泌抑制剤および前記本発明のEV分泌抑制方法の記載を援用できる。
大腸がん細胞として、ヒト結腸腺がん細胞株HCT116(ATCC CCL-247)、肺がん細胞として、ヒト肺胞基底上皮腺がん細胞株A549(ATCC CCL-185)を使用した。メラノーマ細胞として、ヒトメラノーマ細胞株A375(ATCC CRL-1619)、乳がん細胞として、ヒト乳癌細胞株MM231(ATCC HTB-26)、膵臓がん細胞として、ヒト膵臓腺がん細胞株Panc-1(ATCC CRL-1469)、多発性骨髄腫細胞として、ヒト多発性骨髄腫細胞株RPMI8226(ATCC CCL-155)を使用した。
核酸分子miRNAとして、miR-891b(miR-891b mimicともいう。配列番号1:GCAACUUACCUGAGUCAUUGA)(製品番号 4464066 、Ambion社)を使用し、ネガティブコントロール用の核酸分子miRNAとして、miRNA Mimic Negative Control#1(4464058)(Ambion)を使用した。核酸分子siRNAとして、siPSAT1(製品番号 siGENOME SMART pool siRNA M-010398、Dharmacon社)を使用し、ネガティブコントロール用の核酸分子siRNAとして、ALL STARネガティブコントロールsiRNA(SI03650318)(いずれもQiagen)を使用した。
MM231用の培地は、RPMI 1640培地(Gibco)に10%熱不活化ウシ胎児血清(FBS)および抗生物質-抗真菌溶液(Gibco)を添加したRPMI完全培地を使用した。HCT116用の培地は、Maccoy5Aに10%熱不活化FBSおよび抗生剤-抗真菌薬を添加したMaccoy5A完全培地を使用し、その他の細胞用の培地は、DMEM培地(Gibco)に10%熱不活化FBSおよび抗生剤-抗真菌薬を添加したDMEM完全培地を使用した。培養条件は、37℃、5%二酸化炭素、95%相対湿度(RH)とした。約100,000個の細胞を前記完全培地18mLに播種し、3~4日間インキュベートし、継代数20未満の細胞を使用した。
培養したがん細胞を、リン酸緩衝生理食塩水(PBS)で洗浄し、培地を、前記抗生物質-抗真菌薬と2mmol/L L-グルタミン(Gibco)とを含むadvanced RPMIまたはadvanced DMEMに置き換えた。置換後の調整培養液を2,000×gで10分間遠心分離して細胞を除去し、得られた上清を0.22μmフィルター(Millipore)でろ過した。得られたろ液を110,000×gで70分間遠心分離し、EVが濃縮されたペレットを得た。前記ペレットを11mLのPBSで洗浄し、さらに110,000×gで70分間超遠心分離し、再回収した。
EVの検出には、AlphaScreenストレプトアビジン被覆ドナービーズ(6760002)、AlphaLISA非結合アクセプタービーズ(6062011)、およびAlphaLISAユニバーサルバッファ(AL001F)から構成されるAlphaLISA試薬(Perkin Elmer)、96ウェルハーフエリアホワイトプレート(6005560、Perkin Elmer)、および検出装置EnSpire Alpha 2300 Mutilabelプレートリーダー(Perkin Elmer)を使用した。具体的には、前記プレートの各ウェルに、EV 5μLまたはCM(細胞の培養上清)10μLと、前記バッファで調製した5nmol/Lビオチン化抗体 10μLと、50μg/mL AlphaLISAアクセプタービーズ結合抗体 10μLとを添加した。CD9/CD9二重陽性EVの検出には、ビオチン化抗ヒトCD9抗体とAlphaLISAアクセプタービーズ結合抗ヒトCD9抗体とを使用し、CD9/CD63二重陽性EVの検出には、ビオチン化抗ヒトCD9抗体とAlphaLISAアクセプタービーズ結合抗ヒトCD63抗体とを使用した。また、CD63/CD63二重陽性EVの検出には、ビオチン化抗ヒトCD63抗体とAlphaLISAアクセプタービーズ結合抗ヒトCD63抗体とを使用した。そして、前記プレートを37℃で1時間インキュベートした後、80μg/mL AlphaScreenストレプトアビジン被覆ドナービーズ 25μLを添加し、さらに37℃で30分間、暗所でインキュベートした。つぎに、前記検出装置を励起波長680nmおよび発光検出波長615 nmに設定し、前記プレートのウェルにおける発光を測定した。抗体は、いずれも市販品として、マウスモノクローナル抗ヒトCD9(クローン12A12)およびCD63抗体(クローン8A12)(いずれもCosmo Bio)を使用した。
回収した分泌EVをPBSに懸濁し、さらにPBSで希釈系を調製し、NanoSight粒子トラッキング分析(LM10、ソフトウェアVer. 2.03)で分析した。前記粒子トラッキングは、カメラレベル14で各サンプルから少なくとも5つの60秒ビデオを得た。分析設定は、最適化し、サンプル間で一定に維持させた。EV濃度は、培養液の粒子/細胞として算出し、正味のEV分泌率が得られた。前記ExoScreen法によるEVの測定結果は、NTA分析によるEVの測定結果と相関関係を示したことから、ExoScreen法により分泌EV量を測定できることが確認できた。
6ウェルプレートに、1.0×105細胞/ウェルの条件でがん細胞の懸濁液2mLを播種し、24時間インキュベートした後、目的の核酸分子10nmol/Lを添加し、トランスフェクション試薬(商品名DharmaFECT Transfection Reagent 1)により前記核酸分子のトランスフェクションを行った。前記核酸分子は、前記miRNA、前記siRNAを使用した。前記24時間のインキュベート後、培地を、前記抗生物質-抗真菌薬と2mmol/L-グルタミン(Gibco)とを含む前記advanced RPMI 1640培地またはadvanced DMEM培地に置き換えた。置換の48時間後、トータルRNAを抽出し、目的の遺伝子の発現をqPCRで測定した。
トータルRNAは、市販の試薬(商品名QIAzol、商品名miRNeasy Mini Kit、Qiagen)を用いて培養細胞から抽出した。逆転写反応は、市販キット(商品名High-Capacity cDNA Reverse Transcription Kit、Applied Biosystems)およびランダムヘキサマープライマーを用いて行った。リアルタイムPCR分析は、市販キット(商品名StepOne Plus、商品名TaqMan Universal PCR MasterMix、Thermo Fisher Scientific)を用いて行った。mRNAの発現は、β-アクチンで正規化した。PSAT1用のプローブは、TaqManプローブ(Applied Biosystems)を使用した。
* P <0.05、** P <0.01。
[実施例A1]
大腸がんおよび肺がんにおけるEV分泌の調節に関与するターゲット遺伝子を同定した。
大腸がん細胞HCT116および肺がん細胞A549に、それぞれmiR-891bをトランスフェクションして、その形質転換体(miR-891b)の培養上清(CM)および前記形質転換体(miR-891b)から分泌されたEVを含むEV画分を回収し、前記ExoScreen法により分泌EV量を強度シグナルとして測定した。また、各形質転換体について、NTA分析により、分泌EV量を確認した。ネガティブコントロールとして、miR-891b miRNA Mimic Negative Control#1をトランスフェクションしたA549の形質転換体(NC)についても、同様に分泌EV量を測定した。そして、形質転換体(NC)の分泌EV量を1として、形質転換体(miR-891b)の分泌EVの相対値(n=3)を求めた。
前記(1)において、miR-891bのトランスフェクションによってEV分泌が抑制されたことから、発明者らは、さらなる鋭意研究によって、miR-891bにより発現が抑制されるターゲット遺伝子としてPSAT1遺伝子を見出した。そこで、PSAT1遺伝子の発現を抑制するsiRNA(siPSAT1)を大腸がん細胞HCT116および肺がん細胞A549にトランスフェクションして、ExoScreen法およびNTA法により分泌EV量を確認した。また、ネガティブコントロールとして、ALL STARネガティブコントロールsiRNAをトランスフェクションして、同様の確認を行った。これらの結果を図2に示す。図2において、(A)は、ExoScreen法による分泌EV量の相対値を示すグラフであり、(B)は、NTA法による分泌EV量の相対値を示すグラフである。
前記(2)におけるPSAT1遺伝子について、miR-891bの直接のターゲット遺伝子であることの確認を行った。
PSAT1遺伝子の発現抑制による、各種がん細胞からのEV分泌の抑制を確認した。
前記実施例A1(2)と同様に、大腸がん細胞HCT116および肺がん細胞A549にsiRNA(siPSAT1)をトランスフェクションし、PSAT1サイレンシング後のEVバイオジェネシスを確認した。サイレンシング後の各形質転換体について、免疫蛍光によりCD63およびPSAT1を確認したところ、PSAT1サイレンシング後の細胞質において、EVのマーカであるCD63の蓄積が確認された。また、miR-891bをトランスフェクションした場合も、同様に、CD63の蓄積が確認できた。この結果から、PSAT1の発現を抑制することによって、EVの産生自体は維持されるが、産生されたEVは細胞質内に蓄積され、細胞から外部へのEV分泌が抑制されることを確認できた。
前記実施例Aにおいて、EV分泌にPSAT1が関与しており、PSAT1の発現抑制により、EV分泌を抑制できることが確認された。PSAT1は、セリン合成経路の酵素タンパク質であるため、EV分泌にセリン合成経路が関与しており、セリン合成経路の阻害、すなわちセリン合成の阻害によってEV分泌を抑制できることが推測された。そこで、実施例Bにおいて、PSAT1にかかわらず、セリン合成経路についていずれかの工程を阻害することで、EV分泌を抑制できることを確認した。
通常のDMEM培地にはセリンが含まれているため、セリン欠乏培地を使用し、セリン添加によるEV分泌への影響を確認した。
セリン合成経路における阻害剤を使用して、EV分泌への影響を確認した。
様々ながん細胞を用いて、セリン合成によるEV分泌の制御を確認した。なお、本実施例において、セリン合成経路の阻害としてPSAT1サイレンシングを利用したが、前述のように、PSAT1のサイレンシングには限定されず、セリン合成の阻害であれば、同様にEV分泌を抑制できることは確認済みである。
セリン合成経路における酵素タンパク質(PHGDH)の阻害剤NCT-503を使用して、in vivoにおける腫瘍体積の減少、EV分泌の抑制等を確認した。
セリン合成経路の酵素タンパク質であるPHGDHについて、上述の実施例でEV分泌抑制が確認されているPSAT1をポジティブコントロールとして、それぞれの遺伝子(PHGDH遺伝子およびPSAT1遺伝子)の発現抑制による、がん細胞からのEV分泌の抑制を確認した。特に示さない限りは、前記実施例B1の方法と同様に行った。
前記実施例B2において、セリン合成経路のPHGDHを阻害する阻害剤NCT-503を用いて、がん細胞におけるEV分泌を抑制できることが確認された。ここで、NCT-503の添加によって、細胞が細胞死したことによりEV分泌が抑制されたのではなく、がん細胞等の非正常細胞に特有のEV分泌がセリン合成経路の阻害によって抑制されたことの補足データを示す。
Claims (26)
- セリン合成経路の阻害剤を含むことを特徴とする細胞からの細胞外小胞の分泌を抑制する細胞外小胞分泌抑制剤。
- 前記阻害剤が、セリン合成経路における酵素タンパク質の発現を抑制する発現抑制物質またはセリン合成経路における酵素タンパク質の触媒機能を抑制する触媒機能抑制物質である、請求項1に記載の細胞外小胞分泌抑制剤。
- 前記セリン合成経路が、PSAT1を含む合成経路である、請求項1または2に記載の細胞外小胞分泌抑制剤。
- 前記セリン合成経路の阻害剤が、PSAT1タンパク質の発現抑制物質または触媒機能抑制物質である、請求項1から3のいずれか一項に記載の細胞外小胞分泌抑制剤。
- 前記セリン合成経路の阻害剤が、PHGDHタンパク質の発現抑制物質または触媒機能抑制物質である、請求項1から3のいずれか一項に記載の細胞外小胞分泌抑制剤。
- 前記セリン合成経路の阻害剤が、PSPHタンパク質の発現抑制物質または触媒機能抑制物質である、請求項1から3のいずれか一項に記載の細胞外小胞分泌抑制剤。
- 前記細胞が、がん細胞である、請求項1から6のいずれか一項に記載の細胞外小胞分泌抑制剤。
- 前記がん細胞が、大腸がん細胞、肺がん細胞、メラノーマ細胞、乳がん細胞、膵臓がん細胞、および多発性骨髄腫細胞からなる群から選択された少なくとも一つである、請求項7に記載の細胞外小胞分泌抑制剤。
- 前記細胞が、ウイルス感染細胞である、請求項1から6のいずれか一項に記載の細胞外小胞分布抑制剤。
- 前記阻害剤が、低分子化合物、タンパク質、またはペプチドである、請求項1から9のいずれか一項に記載の細胞外小胞分泌抑制剤。
- 前記酵素タンパク質に対する発現抑制物質が、前記酵素タンパク質をコードする遺伝子からの転写を抑制する物質、転写された転写産物を分解する物質、および前記転写物からのタンパク質の翻訳を抑制する物質からなる群から選択された少なくとも一つである、請求項2から10のいずれか一項に記載の細胞外小胞分泌抑制剤。
- 前記発現抑制物質が、miRNA、siRNA、アンチセンス、およびリボザイムからなる群から選択された少なくとも一つの核酸物質である、請求項11に記載の細胞外小胞分泌抑制剤。
- 前記発現抑制物質が、前記核酸物質を発現する発現ベクターである、請求項12に記載の細胞外小胞分泌抑制剤。
- 前記タンパク質の機能抑制物質が、前記酵素タンパク質に対する活性阻害物質または活性中和物質である、請求項2から13のいずれか一項に記載の細胞外小胞分泌抑制剤。
- 前記活性中和物が、前記タンパク質に対する抗体または抗原結合断片である、請求項14に記載の細胞外小胞分泌抑制剤。
- 前記機能抑制物質が、前記活性中和物を発現する発現ベクターである、請求項15に記載の細胞外小胞分泌抑制剤。
- 請求項1から16のいずれか一項に記載の細胞からの細胞外小胞の分泌を抑制する細胞外小胞分泌抑制剤を、投与対象に投与する工程を含むことを特徴とする細胞からの細胞外小胞分泌の抑制方法。
- 前記細胞が、がん細胞である、請求項17に記載の抑制方法。
- 前記がん細胞が、大腸がん細胞、肺がん細胞、メラノーマ細胞、乳がん細胞、膵臓がん細胞、および多発性骨髄腫細胞からなる群から選択された少なくとも一つである、請求項18に記載の抑制方法。
- 前記細胞が、ウイルス感染細胞である、請求項17に記載の抑制方法。
- 前記投与対象が、ヒトまたは非ヒト動物である、請求項17から20のいずれか一項に記載の抑制方法。
- 前記投与が、in vivoまたはin vitroで行われる、請求項17から21のいずれか一項に記載の抑制方法。
- 被検物質から、セリン合成経路を阻害する阻害物質を、細胞からの細胞外小胞の分泌を抑制する候補物質として選択することを特徴とする細胞からの細胞外小胞の分泌を抑制する細胞外小胞分泌抑制剤の候補物質のスクリーニング方法。
- 前記細胞が、がん細胞である、請求項23に記載のスクリーニング方法。
- 前記がん細胞が、大腸がん細胞、肺がん細胞、メラノーマ細胞、乳がん細胞、膵臓がん細胞、および多発性骨髄腫細胞からなる群から選択された少なくとも一つである、請求項24に記載のスクリーニング方法。
- 前記細胞が、ウイルス感染細胞である、請求項23に記載のスクリーニング方法。
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EP21784748.2A EP4134097A1 (en) | 2020-04-07 | 2021-04-07 | Extracellular vesicle secretion inhibitor for inhibiting extracellular vesicle secretion, and use of the same |
CN202180026707.8A CN115867315A (zh) | 2020-04-07 | 2021-04-07 | 用于抑制细胞外囊泡的分泌的细胞外囊泡分泌抑制剂及其用途 |
US17/995,550 US20230151368A1 (en) | 2020-04-07 | 2021-04-07 | Extracellular vesicle secretion reducing agent for reducing extracellular vesicle secretion, and use of the same |
KR1020227032794A KR20220144399A (ko) | 2020-04-07 | 2021-04-07 | 세포외 소포의 분비를 억제하는 세포외 소포 분비 억제제, 및 그 용도 |
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Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20140045915A1 (en) * | 2010-08-31 | 2014-02-13 | The General Hospital Corporation | Cancer-related biological materials in microvesicles |
WO2017043370A1 (ja) * | 2015-09-09 | 2017-03-16 | 塩野義製薬株式会社 | エクソソーム分泌阻害剤 |
JP2018521038A (ja) * | 2015-06-30 | 2018-08-02 | アモーレパシフィック コーポレーション | 皮膚美白用組成物及び皮膚美白効能物質をスクリーニングする方法 |
US20190071400A1 (en) * | 2016-03-09 | 2019-03-07 | Raze Therapeutics, Inc. | 3-phosphoglycerate dehydrogenase inhibitors and uses thereof |
WO2019106126A1 (en) * | 2017-12-01 | 2019-06-06 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Mdm2 modulators for the diagnosis and treatment of liposarcoma |
JP2020069392A (ja) | 2018-10-30 | 2020-05-07 | 株式会社クラレ | 係合組立キットおよび係合組立体 |
JP2021022825A (ja) | 2019-07-26 | 2021-02-18 | アイシン精機株式会社 | 周辺監視装置 |
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- 2021-04-07 WO PCT/JP2021/014693 patent/WO2021206105A1/ja unknown
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Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20140045915A1 (en) * | 2010-08-31 | 2014-02-13 | The General Hospital Corporation | Cancer-related biological materials in microvesicles |
JP2018521038A (ja) * | 2015-06-30 | 2018-08-02 | アモーレパシフィック コーポレーション | 皮膚美白用組成物及び皮膚美白効能物質をスクリーニングする方法 |
WO2017043370A1 (ja) * | 2015-09-09 | 2017-03-16 | 塩野義製薬株式会社 | エクソソーム分泌阻害剤 |
US20190071400A1 (en) * | 2016-03-09 | 2019-03-07 | Raze Therapeutics, Inc. | 3-phosphoglycerate dehydrogenase inhibitors and uses thereof |
WO2019106126A1 (en) * | 2017-12-01 | 2019-06-06 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Mdm2 modulators for the diagnosis and treatment of liposarcoma |
JP2020069392A (ja) | 2018-10-30 | 2020-05-07 | 株式会社クラレ | 係合組立キットおよび係合組立体 |
JP2021022825A (ja) | 2019-07-26 | 2021-02-18 | アイシン精機株式会社 | 周辺監視装置 |
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EP4134097A1 (en) | 2023-02-15 |
US20230151368A1 (en) | 2023-05-18 |
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