KR101285434B1 - Compositions for anticancer and cancer sensitization comprising RRP12 inhibitor - Google Patents

Abstract

The present invention relates to an anticancer and anticancer composition comprising an inhibitor of RRP12 (ribosomal RNA processing 12 homolog) gene or an inhibitor of RRP12 protein as an active ingredient, and a method for screening an anticancer or anticancer agent. In accordance with the present invention can be used in the targeted treatment of cancer against RRP12, by inhibiting the anticancer drug resistance of cancer cells to increase the anticancer effect of the anticancer drugs to inhibit the anticancer drug resistance of cancer cells to be used in the development of anticancer drugs and anticancer adjuvants that can be used for cancer treatment Can be.

Description

Compositions for anticancer and cancer sensitization comprising RRP12 inhibitors

The present invention relates to an anticancer and anticancer composition comprising an inhibitor of RRP12 (ribosomal RNA processing 12 homolog) gene or an inhibitor of RRP12 protein as an active ingredient, and a method for screening an anticancer or anticancer agent.

Cancer (malignant tumor) is the leading cause of mortality in modern society, and despite many studies to date, there is no groundbreaking treatment. Treatment of cancer with chemotherapy, such as anticancer drugs, has some effect, but a lot of research is required due to the various pathogenesis of cancer and the expression of anticancer drug resistance.

In recent decades, advances in diagnostic and therapeutic techniques have resulted in a limited positive outcome of improving cancer treatment and functional preservation for cancer treatment, but five-year survival rates range from 5 to 50% for many advanced cancers. These cancers can be characterized by aggressive invasion, lymph node metastasis, distant metastasis and the development of secondary cancers. In some cancers, survival rates have not changed much over the past 20 years despite various studies and treatments. Recently, many attempts have been made to increase the therapeutic effect through molecular biological approaches to these cancers, and researches on target therapies related to cancer proliferation, metastasis and cell death have been actively conducted.

Despite the development of many anticancer drugs to date, only a few cancers can be cured with only anticancer drugs. It decreases, but develops resistance to anticancer drugs during or after treatment. Therefore, for effective anticancer drug treatments, resistance to anticancer drugs, such as cancer cell resistance to anticancer drugs, must be overcome.

Meanwhile, the RRP12 (ribosomal RNA processing 12 homolog) gene is a gene at the position of 10q24.1 on chromosome 10 of human gene ID 23223 and is also called FLJ20231, KIAA0690 or DKFZp762P1116. The protein encoded by the RRP12 gene is known to influence the in vivo synthesis of the ribosome to help the nucleolar transport, but the exact function and mechanism of action is not yet known, especially the RRP12 protein related to cancer treatment The function is unknown.

In view of the above, the present inventors have studied intensively the functions of the RRP12 gene and RRP12 protein, and as a result, the inhibitory expression of the RRP12 gene in the tumor cells or the inhibitory activity of the RRP12 protein in the tumor cells have anticancer and anticancer effects The present invention has been completed by revealing the having.

It is an object of the present invention to provide an anticancer composition comprising an inhibitor of RRP12 gene expression or an inhibitor of RRP12 protein activity.

Another object of the present invention is to provide an anticancer sensitizing composition comprising an inhibitor of RRP12 gene or an inhibitor of RRP12 protein as an active ingredient.

Still another object of the present invention is to provide an anticancer composition comprising the anticancer sensitizing composition and an anticancer agent.

Still another object of the present invention is to provide a method for screening an anticancer agent or an anticancer agent.

In order to achieve the above object, the present invention provides an anticancer composition comprising, as an embodiment, an inhibitor of RRP12 gene expression or an inhibitor of RRP12 protein activity.

In the present invention, the RRP12 (ribosomal RNA processing 12 homolog) gene is a gene on human chromosome 10, and may have an mRNA sequence of SEQ ID NO: 1. The protein encoded by the RRP12 gene is known to help the phosphorus transport of ribosomes to affect the synthesis in vivo, but the exact function and mechanism of action is not known, it may have a sequence of SEQ ID NO: 2. The inventors have found an association with the tumor cells of the RRP12 protein, and particularly, the activity of the RRP12 protein was increased during doxorubicin treatment, and thus the RRP12 was designated as a doxorubicin activating protein (DXAP). In the following, DXAP has the same meaning as RRP12. In addition, the present inventors confirmed that RRP12 is an essential protein for the survival of tumor cells, and that the phosphorylation activity of RRP12 upon the administration of an anticancer agent is a survival mechanism for the anticancer agent of tumor cells. The present inventors newly identify the cancer-related properties of the RRP12 protein, and provide an anticancer or anticancer sensitizing composition including the RRP12 inhibitor as an active ingredient.

Inhibitors of the expression of the RRP12 gene or the inhibitor of the activity of the RRP12 protein included as an active ingredient in the anticancer composition of the present invention is not limited thereto, siRNA, antisense oligonucleotides, aptamers, antibodies and single chain variable region fragments specific to RRP12 And preferably siRNA specific to the RRP12 gene.

As used herein, the term "siRNA" is a nucleic acid molecule capable of mediating RNA interference or gene silencing, and thus, may be used as an efficient gene knockdown method or gene therapy method because it may inhibit expression of a target gene. do. siRNA was first discovered in plants, worms, fruit flies, and parasites, but recently, siRNAs have been developed and used in mammalian cell research.

When siRNA molecules are used in the present invention, a structure in which a sense strand (a sequence corresponding to the RRP12 mRNA sequence (SEQ ID NO: 1)) and an antisense strand (a sequence complementary to the RRP12 mRNA sequence) are positioned opposite to each other to form a double strand or It may have a single chain structure with self-complementary sense and antisense strands.

siRNAs are not limited to the complete pairing of double-stranded RNA pairs that pair with RNA, but include mismatches (corresponding bases are not complementary), expansion / bulge (bases corresponding to one chain). None) and the like may be included.

The siRNA terminal structure can be either blunt or cohesive, as long as the expression of the RRP12 gene can be suppressed by RNA interference (RNAi) effects. The cohesive end structure is possible for both 3'-end protrusion structures and 5'-end protrusion structures.

In the present invention, the siRNA molecule may have a total length of 10 to 50 bases, preferably 15 to 30 bases, more preferably 18 to 25 bases. In one embodiment of the present invention, the siRNA molecule may be formed of 19 bases using siDESIGN SOFTWARE. siRNA was prepared to check the expression level of the RRP12 gene. SiRNA of the present invention may be selected from the group consisting of SEQ ID NO: 5 to 10.

As used herein, the term “antisense oligonucleotide” refers to a DNA or RNA or derivative thereof containing a nucleic acid sequence complementary to a sequence of a particular mRNA, which binds to a complementary sequence in the mRNA to inhibit translation of the mRNA into a protein. It works. Antisense oligonucleotide sequence refers to a DNA or RNA sequence that is complementary to RRP12 mRNA and capable of binding to RRP12 mRNA, and refers to translation, translocation into the cytoplasm, maturation, or any other overall biological function of RRP12 mRNA. May inhibit essential activity. The length of the antisense oligonucleotide may be 6 to 100 bases, preferably 8 to 60 bases, more preferably 10 to 40 bases.

The antisense oligonucleotides may be modified at one or more bases, sugars or backbone positions to enhance efficacy. Oligonucleotide backbones can be modified with phosphorothioates, phosphoroesters, methyl phosphonates, short chain alkyls, cycloalkyls, short chain heteroatomics, heterocyclic intersaccharide linkages, and the like. In addition, antisense oligonucleotides may include one or more substituted sugar moieties. Antisense oligonucleotides may comprise modified bases. Modified bases include hypoxanthine, 6-methyladenine, 5-methyl pyrimidine (particularly 5-methylcytosine), 5-hydroxymethylcytosine (HMC), glycosyl HMC, gentobiosil HMC, 2-aminoadenine, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine, 2,6-diaminopurine Etc. In addition, the antisense oligonucleotides of the present invention may be chemically bound to one or more moieties or conjugates that enhance the activity and cellular adsorption of the antisense oligonucleotides. A cholesterol moiety, a cholesteryl moiety, a cholic acid, a thioether, a thiocholesterol, an aliphatic chain, a phospholipid, a polyamine, a polyethylene glycol chain, adamantane acetic acid, a palmityl moiety, octadecylamine, hexylamino- And liposoluble moieties such as Cole sterol moieties. Methods of preparing oligonucleotides comprising fat-soluble moieties are already well known in the art (US Pat. Nos. 5,138,045, 5,218,105 and 5,459,255). The modified oligonucleotides can increase stability to nucleases and increase the binding affinity of the antisense oligonucleotides with the target mRNA.

The antisense oligonucleotides may be synthesized in vitro in a conventional manner to be administered in vivo or to allow the antisense oligonucleotides to be synthesized in vivo. One example of synthesizing antisense oligonucleotides in vitro is using RNA polymerase I. One example of allowing antisense RNA to be synthesized in vivo is to allow the antisense RNA to be transcribed using a vector whose origin is a multi-cloning site (MCS) in the opposite direction. The antisense RNA is preferably such that the translation stop codon is present in the sequence so that it is not translated into the peptide sequence.

The design of antisense oligonucleotides that can be used in the present invention can be readily prepared according to methods known in the art with reference to the nucleotide sequence of the RRP12 gene.

As used herein, the term "aptamer" refers to a nucleic acid molecule having binding activity to a given target molecule. The aptamer may inhibit the activity of the polynucleotide or protein by binding to the RRP12 polynucleotide or protein. The aptamers of the invention may be RNA, DNA, modified nucleic acids or mixtures thereof, and may be in linear or cyclic form. The length of the aptamer of the present invention is not particularly limited and may be usually 15 to 200 nucleotides, but is, for example, 100 nucleotides or less, preferably 80 nucleotides or less, more preferably 60 nucleotides or less, even more preferably 45 It may be up to nucleotides. The length of the aptamers of the invention may also be, for example, at least 18, 20 or 25 nucleotides. Less total nucleotides make chemical synthesis and mass production easier and cost-effective. In addition, it is easy to chemical formula, high in vivo stability, low toxicity.

The aptamer of this invention can be manufactured by using SELEX method and its improvement method. The SELEX method is a method of selecting oligonucleotides that specifically bind to a target substance from a pool of oligonucleotides having about 10 to 14 different nucleotide sequences. The oligonucleotide used has a structure in which a random sequence of about 40 residues is sandwiched between primer sequences. This oligonucleotide pool is associated with a target substance to recover only the oligonucleotides bound to the target substance using a filter or the like. The recovered oligonucleotide is amplified by RT-PCR and used as a template for the next round. By repeating this operation about 10 times, an aptamer specifically binding to a target substance can be obtained. In the SELEX method, by increasing the number of rounds or using a competitive material, aptamers having a stronger binding strength to the target material can be concentrated and selected. Therefore, by adjusting the number of rounds of SELEX or changing the contention state, it is possible to obtain aptamers having different binding forces, aptamers having different binding forms, and aptamers having the same binding force or binding form but different base sequences. In addition, although the SELEX method includes the amplification process by PCR, by providing a mutation by using manganese ions in the process, it becomes possible to perform a SELEX richer in diversity.

In addition, aptamers can be obtained by using the Cell-SELEX technique for complex targets, ie, living cells and tissues, in addition to the conventional SELEX technique. The Cell-SELEX technique can be used to detect aptamers for disease cells even when surface marker targets are not known. It has the advantage of being able to develop. In addition, the Cell-SELEX technique has advantages over the conventional SELEX procedure because the isolated protein may not exhibit its original properties, since the target protein in the physiological state allows for a more functional approach in the selection process. have.

On the other hand, aptamers bind to the target substance by various binding modes such as ionic bonds using a negative charge of phosphate groups, hydrophobic bonds and hydrogen bonds using ribose, and hydrogen bonds or stacking bonds using nucleic acid bases. In particular, the ionic bond using the negative charge of the phosphate group present by the number of nucleotides strongly binds to the positive charge of lysine or arginine on the surface of the protein. For this reason, the nucleic acid base which is not related to direct binding with a target substance can be substituted. In particular, since the portion of the stem structure is already made of base pairs and is directed toward the inside of the double helix structure, the nucleic acid base is difficult to bind directly with the target substance. Therefore, even if the base pair is replaced with another base pair, the activity of the aptamer is often not reduced. Even in a structure in which a base pair is not formed, such as a loop structure, the base can be substituted when the nucleic acid base is not involved in direct binding to the target molecule. For example, at the 2 'position of the ribose, it may be a nucleotide in which a hydroxyl group is substituted with any atom or group. As such arbitrary atoms or groups, for example, hydrogen atom, fluorine atom or -O-alkyl group (e.g. -O-CH3), -O-acyl group (e.g. -O-CHO), amino group (e.g. -NH2) And nucleotides substituted with. Thus, aptamers retain their activity unless they substitute or delete functional groups involved in direct binding to the target molecule.

In addition, aptamers are easy to modify because of their chemical synthesis. By using the MFOLD program, aptamers predict the secondary structure, or predict the three-dimensional structure by X-ray analysis or NMR analysis, and can predict to what extent nucleotides can be substituted or deleted and where new nucleotides can be inserted. . The aptamer of the predicted new sequence can be easily chemically synthesized and can be confirmed by existing assay systems whether the aptamer is active.

As used herein, the term "antibody" refers to a substance that is produced by stimulation of an antigen in the immune system and specifically binds to a specific antigen to generate an antigen-antibody reaction, and in the present invention, specific to RRP12 protein in order to inhibit the activity of the RRP12 protein. Antibodies that bind can be used. In particular, phosphorylation inhibitory antibodies specific for threonine-glutamic acid-tyrosine (Thr-Glu-Tyr, TEY) moieties that are phosphorylated on RRP12 protein and are involved in protein activation can be used.

Antibodies specific for the RRP12 protein that may be used in the present invention may be polyclonal or monoclonal antibodies, preferably monoclonal antibodies. Antibodies specific for the RRP12 protein are commonly practiced in the art, such as fusion methods (Kohler and Milstein, European Journal of Immunology, 6: 511-519 (1976)), recombinant DNA methods (US Pat. No. 4,816,567). Or) phage antibody library methods (Clackson et al, Nature, 352: 624-628 (1991) and Marks et al, J. Mol. Biol., 222: 58, 1-597 (1991)). have. General procedures for antibody preparation can use methods known in the art, for example, the preparation of hybridoma cells producing monoclonal antibodies is achieved by fusing immortalized cell lines with antibody-producing lymphocytes, The techniques required for this process are well known to those skilled in the art and can be readily implemented. Polyclonal antibodies can be obtained by injecting the RRP12 protein antigen into a suitable animal, collecting the antiserum from the animal, and then separating the antibody from the antiserum using known affinity techniques.

In the present invention, the antibody may include a single chain variable region fragment (scFv). The single chain variable region fragment may be composed of "variable region (VL) -linker-heavy chain variable region (VH) of light chain". The linker means an amino acid sequence of a certain length that serves to artificially link the variable regions of the heavy and light chains.

As used herein, the term "anticancer" means inhibiting or preventing the growth of cancer. Inhibiting or preventing cancer growth is a concept that includes reducing cancer growth and cancer metastasis as compared to when not treated or treated. The metastasis refers to a process in which tumor (cancer) cells spread to distant parts of the body.

In the present invention, the term "cancer" refers to a state in which a problem arises in the regulation function of the cell itself, and abnormal cells that normally need to be killed proliferate and invade surrounding tissues and organs to form agglomerates and destroy or modify existing structures. It is used in the same sense as a malignant tumor.

In the present invention, the cancer may be selected from the group consisting of osteosarcoma, megacytoma, chondroma, synovial sarcoma, bladder cancer, gastric cancer, breast cancer, colon cancer, cervical cancer, prostate cancer and epidermal cancer, preferably osteosarcoma or synovial sarcoma And more preferably osteosarcoma. In addition, the cancer may include a cancer generated through the metastasis process.

Since the RRP12 inhibitor of the present invention has not only an anticancer effect but also an effect of reducing the cancer cell's resistance to anticancer agents, the anticancer composition of the present invention is remarkably excellent in anticancer effects as compared to conventional anticancer agents.

As another aspect, the present invention provides an anticancer sensitizing composition comprising an inhibitor of RRP12 gene expression or an inhibitor of RRP12 protein activity.

As used herein, the term "anticancer sensitization" is used in the same sense as "chemical sensitization", and when the cancer is treated with a chemical anticancer agent, the chemical anticancer agent is more toxic to cancer cells than when there is no anticancer sensitizing composition. As it is strengthened or increased, it means that resistance to anticancer drugs, such as anticancer drug resistance, is reduced and the effects of the anticancer drugs are better exhibited. In the present invention, the anticancer sensitizing composition comprising the RRP12 inhibitor sensitizes cancer cells to the anticancer agent, thereby reducing the resistance of the cancer cells to the anticancer agent and increasing the effect of the anticancer agent.

The resistance to the anticancer agent refers to the fact that the drug administered at the time of administering the anticancer agent is insufficient in killing or killing the cancer cells, and the anticancer drug resistance is the most frequent cause. In general, when treating cancer patients using "cancer drug resistance" or "anticancer drug resistance", there is no treatment effect at the beginning of treatment or early cancer treatment effect, but the cancer treatment effect is lost in the continuous treatment process. it means. As described above, it is known that the effect is gradually decreased when the anticancer agent is continuously administered to the cancer patient, which is due to the appearance of cancer cells resistant to the anticancer agent in vivo, which makes the chemotherapy of cancer difficult. In the present invention, the anticancer sensitizing composition may be characterized by inhibiting the anticancer drug resistance.

In one embodiment of the present invention, when doxorubicin is administered with siRP specific to the RRP12 gene in osteosarcoma cell line (MG63) known to be resistant to anticancer agents, it was confirmed that proliferation of osteosarcoma cell line was significantly reduced, thereby to RRP12 gene. Specific siRNA was confirmed to increase the anticancer effect of doxorubicin by inhibiting the resistance of osteosarcoma cell line (MG63) to doxorubicin (Fig. 11).

Cancer that can obtain the anticancer sensitizing effect in the present invention may be selected from the group consisting of osteosarcoma, giant cell tumor, chondroma, synovial sarcoma, bladder cancer, gastric cancer, breast cancer, colon cancer, cervical cancer, prostate cancer and epidermal cancer, preferably Preferably it may be osteosarcoma or synovial sarcoma, more preferably osteosarcoma. In addition, the cancer may include a cancer generated through the metastasis process.

Inhibitors of the expression of the RRP12 gene or the inhibitor of the activity of the RRP12 protein included as an active ingredient in the anticancer sensitizing composition of the present invention, but is not limited thereto, siRNA, antisense oligonucleotides, aptamers, antibodies and single chain variable region fragments specific to RRP12 It may be, preferably siRNA specific to the RRP12 gene, the siRNA may be selected from the group consisting of SEQ ID NOs: 5 to 10. The siRNA, antisense oligonucleotides, aptamers, antibodies and single chain variable region fragments are as described above.

As another aspect, the present invention provides an anticancer composition comprising the anticancer sensitizing composition and an anticancer agent. Since the RRP12 inhibitor of the present invention reduces the resistance of cancer cells to anticancer drugs, the RRP12 inhibitor may be used as an adjuvant in combination with a conventional anticancer agent to enhance the effect of the anticancer agent.

In the present invention, the anticancer agent is a generic term for all drugs used to kill cancer cells, and most drugs block the replication, electron, and translation process of DNA of cancer cells. The kind of anticancer agent which can be used for the composition of this invention is not specifically limited. The anticancer agent may be selected under general principles to be considered in selecting an anticancer agent such as the type of cancer cells, the rate of absorption of the anticancer agent (the duration of treatment and the route of administration of the anticancer agent), the location of the tumor, and the size of the tumor. Specifically, the anticancer agent that can be used in the present invention is a DNA alkylating agent (DNA alkylating agent), methyllothamine (mechloethamine), chlorambucil (chlorambucil), phenylalanine (phenylalanine), mustard (mustard), cyclophosphamide (cyclophosphamide), ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin (streptozotocin), busulfan, thiotepa, cisplatin ) And carboplatin. In addition, the anticancer agent that can be used in the present invention is anti-cancer antibiotics (dactinomycin), doxorubicin (doxorubicin), daunorubicin (daunorubicin), idarubicin, mitosantron (mitoxantrone) ), Plicamycin, mitomycin and C bleomycin. In addition, the anticancer agent that can be used in the present invention is a plant alkaloid (vincristine), vinblastine (vinblastine), paclitaxel (paclitaxel), docetaxel (docetaxel), etoposide (tenposide), teniposide (teniposide) , Topotecan and iridotecan. Preferably the anticancer agent may be doxorubicin. However, anticancer agents that can be used in the present invention are not limited thereto.

As another aspect, the present invention provides a method for screening an anticancer agent or an anticancer agent. Specifically, (a) analyzing the expression of the RRP12 gene or activity of the RRP12 protein after treating the test substance; And (b) determining whether the expression of the RRP12 gene or the activity of the RRP12 protein after treatment with the test substance is inhibited compared to the expression of the RRP12 gene without treatment with the test substance or the activity of the RRP12 protein. A method for screening an anticancer agent or an anticancer agent is provided. The "inhibitor of expression of RRP12 gene" or the "activator of RRP12 protein" of the present invention can be obtained through the above screening method.

In the present invention, step (a) is a step of analyzing the expression of the RRP12 gene or the activity of the RRP12 protein after treating the test material, the assay can be analyzed in cells or in vitro, but is not limited thereto. RT-PCR (Reverse Transcription Polymerase Chain Reaction), Northern blotting, cDNA microarray hybridization reaction, in situ hybridization reaction, radioimmunoassay, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), western blotting And the like can be analyzed.

As used herein, the term "test substance" refers to an unknown substance used in screening to test whether the RRP12 gene expression or RRP12 protein activity is affected. The test substance may include, but is not limited to, chemicals, peptides and natural extracts. The test substance analyzed by the screening method of the present invention may be a single compound or a mixture of compounds, and may be obtained from a library of synthetic or natural compounds.

In the present invention, the step (b) is determined to be an anticancer agent or an anticancer agent when the expression of the RRP12 gene or the activity of the RRP12 protein is suppressed compared to the expression of the RRP12 gene or the activity of the RRP12 protein after treatment with the test substance. As a step, it can be determined as an anticancer agent or anticancer agent by measuring the down-regulation of the expression of RRP12 gene or RRP12 protein activity by the test substance.

The screening methods of the present invention can be carried out in a variety of ways, in particular in a high throughput manner according to various binding assays known in the art.

In the screening method of the present invention, the test substance or RRP12 protein may be labeled with a detectable label. For example, the detectable label may be a chemical label (eg biotin), an enzyme label (eg horseradish peroxidase, alkaline phosphatase, peroxidase, luciferase, β-galactosidase and β- Glucosidase), radiolabels (eg C14, I125, P32 and S35), fluorescent labels (eg coumarin, fluorescein, fluoresein Isothiocyanate (FITC), rhodamine 6G, rhodamine B, TAMRA (6 -carboxy-tetramethyl-rhodamine), Cy-3, Cy-5, Texas Red, Alexa Fluor, DAPI (4,6-diamidino-2-phenylindole), HEX, TET, Dabsyl and FAM), luminescent labels, chemiluminescence ( chemiluminescent label, fluorescence resonance energy transfer (FRET) label, or metal label (eg, gold and silver).

In the case of using a RRP12 protein or a test substance labeled with a detectable label, whether binding between the RRP12 protein and the test substance occurs can be analyzed by detecting a signal from the label. For example, when alkaline phosphatase is used as a label, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (naphthol-AS-B1-phosphate) Signal is detected using a chromogenic reaction substrate such as) and enhanced chemifluorescence (ECF). When hose radish peroxidase is used as a label, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium nitrate), resorupin benzyl ether, luminol, Amplex Red Reagent (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and substrates such as naphthol / pyronin to detect the signal.

Alternatively, binding of the test substance to the RRP12 protein may be assayed without labeling the interactants. For example, a microphysiometer can be used to analyze whether the test substance binds to the RRP12 protein. Microphysiometers are analytical tools that measure the rate at which cells acidify their environment using a light-addressable potentiometric sensor (LAPS). The change in acidification rate can be used as an indicator for binding between test substance and RRP12 protein (McConnell et al., Science 257: 19061912 (1992)).

The ability of the test substance to bind to RRP12 protein can be analyzed using real-time bimolecular interaction analysis (BIA) (Sjol and er & Urbaniczky, Anal. Chem., 63: 2338-2345 (1991), and Szabo et. al., Curr. Opin.Struct. Biol. 5: 699-705 (1995). BIA is a technique for analyzing specific interactions in real time, and can be performed without labeling of interactants (eg, BIAcore ™). Changes in surface plasmon resonance (SPR) can be used as indicators for real-time reactions between molecules.

By using the anti-cancer and anti-cancer sensitizing composition comprising an inhibitor of the expression of the RRP12 gene or an inhibitor of the activity of the RRP12 protein as an active ingredient, the anti-cancer agent may be used for the targeted treatment of cancer against RRP12, and the anticancer agent may be inhibited by inhibiting the anticancer drug resistance of the cancer cells. By increasing the anticancer effect, it can be used to develop anticancer agents and anticancer adjuvants that can be used to treat cancer.

1 shows the results of Western blotting using phosphorylation (Thr218 / Tyr220) detection antibody upon doxorubicin administration in synovial sarcoma (HSSYII).
2 to 4 show the results of confirming the expression of the RRP12 gene by performing RT-PCR in normal cells and various tumor cells. Glyceraldehyde triphosphate dehydrogenase (GAPDH) was used as a control.
Figure 5 shows the results of confirming the phosphorylation activity of RRP12 protein by Western blotting when doxorubicin administered in normal cells (hDF) and synovial sarcoma (HSSYII). Beta-actin (β-actin) was used as a control.
Figure 6 shows the results of confirming the phosphorylation activity of RRP12 protein by Western blotting when doxorubicin administered in osteosarcoma cell lines (MG63, SaOS2, U2OS). Beta-actin (β-actin) was used as a control.
Figure 7 shows the results of confirming the activity of the RRP12 protein by performing cell-immunochemical staining when doxorubicin administration in osteosarcoma cell line (MG63). DAPI staining was performed to confirm the location of the nucleus.
Figure 8 shows the results of confirming the expression level of the RRP12 gene by RT-PCR when the siRNA having the sequence of SEQ ID NOS: 5 to 10 in the osteosarcoma cell line (MG63).
9 shows the results of confirming the inhibition of phosphorylated RRP12 protein activity using siRNA specific to RRP12 gene in osteosarcoma cell line (MG63) (ne: control siRNA administration, siDXAP: siRNA administration specific to RRP12 gene).
Figure 10 shows the results of confirming the proliferation of osteosarcoma cell lines by MTT assay when inhibiting RRP12 using siRNA specific to the RRP12 gene in normal cells (hDF) and osteosarcoma cell line (MG63) (ne: control siRNA administration , DXAP: administration of siRNA specific to the RRP12 gene).
Figure 11 shows the results obtained by MTT assay for the proliferation of osteosarcoma cell line when doxorubicin is administered with siRNA specific to the RRP12 gene in osteosarcoma cell line (MG63) (ne: control siRNA administration, siDXAP: specific to RRP12 gene SiRNA administration).
12 shows the results of summarizing the functions of RRP12.
FIG. 13 shows the result of performing flowcytometric analysis to confirm cell death of osteosarcoma cell lines when RRP12 is suppressed using siRNA specific to RRP12 gene in osteosarcoma cell line (MG63).
FIG. 14 shows Western blotting when inhibiting RRP12 using siRNA specific to the RRP12 gene in osteosarcoma cell line (MG63) and caspase-3 and poly (ADP-ribose) polymerase (Poly ADP-ribose) polymerase (PARP) is confirmed whether the activity of the results (ne: control siRNA administration, siDXAP: siRNA administration specific to the RRP12 gene).

Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. However, the following examples are only for illustrating the present invention, but the scope of the present invention is not limited by these examples.

Cell preparation

10% fetal bovine serum such as HSSYII, a synovial sarcoma cell (obtained from Seoul National University Hospital), fibroblasts derived from humans (fibroblast, hDF, Samsung Medical Center, organ transplant center), and osteosarcoma cell lines (MG63, SaOS2, U2OS) Incubated in DMEM (Dulbecco's modified Eagle's medium) filled with 50 U / ml penicillin and 50 ug / ml streptomycin.

Example  One: RRP1  Isolation of protein

Western blotting was performed using phosphorylation (Thr218 / Tyr220) detection antibody upon doxorubicin administration in synovial sarcoma (HSSYII). Cell lysates were prepared from the cultured synovial sarcoma cells, and 30 ug of cell lysates were electrophoresed in 10% polyacrylamide gel electrophoresis (SDS-PAGE), followed by polyvinylidene difluoride (PVDF) membrane ( bio-rad) and incubated with blocking solution (TBST with 5% bovine serum albumin) at room temperature for 1 hour. After the incubation, the cells were further incubated for 2 hours in a blocking solution containing phosphorylation (Thr218 / Tyr220) detection antibody (Cell signaling, USA) and β-actin (Santa Cruz Biotechnology) primary antibody (rabbit polyclonal antibody). The membrane was washed with water, and then incubated for 1 hour in a Tris-buffered Saline with tween-20 (TBST) solution containing a secondary antibody (1: 5,000) conjugated with a horseradish peroxidase (Santa Cruz Biotechnology). The membrane was washed again with water, then developed with an enhanced chemiluminescence reagent (Intron Biotechnology) and the results were described. A protein was always found to be constantly active at the 140 kd position (FIG. 1). All proteins found at 140 kd were collected and subjected to mass spectrometry (Gel MS) to find 13 candidate proteins (Table 1). Among them, one protein having an amino acid sequence recognized by the phosphorylation (Thr218 / Tyr220) detecting antibody, that is, threonine-glutamic acid-tyrosine (Thr-Glu-Tyr, TEY) was isolated. The protein was a protein called RRP12 or KIAA0690 of SEQ ID NO: 2, which was phosphorylated upon doxorubicin administration, but its function was still unknown.

Example  2: in various tumor cells RRP12  Confirm gene expression

In order to confirm whether the gene of the RRP12 protein isolated in Example 1 is overexpressed in various tumor cells, RT-PCR was performed to confirm the expression of the RRP12 gene in various tumor cells. Normal cells (hDF, HNSC), Epidermal cancer (A431), Osteosarcoma (SaOs2, U2OS, MG63), Giant cell tumor (giant tumor), Synovial sarcoma (HSSYII), Bladder cancer (T24), Gastric cancer (MKN1), Breast cancer (MCF7, sKBR3), colorectal cancer (HCT116), cervical cancer (HeLa), prostate cancer (PC3) and chondrroma cells from cells using the easy-blue ™ total RNA extraction kit (intron biotechbology) according to the manual provided It was. cDNA was synthesized according to the manual provided using cDNA synthesis (superscript III reverse transcriptase, invitrogen). PCR was performed using RRP12 primer F-GACGCCCATGGAAGAAGAGGC (SEQ ID NO: 3) and R-GCAGCGAAGTACTCAGTCTCC (SEQ ID NO: 4).

As a result, it was confirmed that the expression level of the RRP12 gene is very low in normal cells, while the expression level is significantly high in tumor cells (FIGS. 2 to 4). These results indicate that RRP12 gene is overexpressed in various tumor cells and is associated with survival or proliferation of tumor cells.

Example  3: in tumor cells upon administration of anticancer agent RRP12  Protein activity measurement

3-1. Synovial sarcoma ( HSSY In II) RRP12  Protein activity measurement

The activity of RRP12 protein was measured by western blotting when doxorubicin was administered in normal cells (hDF) and synovial sarcoma (HSSYII). Cell lysates were prepared from normal and synovial sarcoma cells, 30 ug of cell lysates were electrophoresed on 10% SDS-PAGE, then transferred to a polyvinylidene difluoride (PVDF) membrane (bio-rad). Incubated with blocking solution (TBST with 5% bovine serum albumin) at room temperature for 1 hour. After the culture, a block containing phosphorylation (Thr218 / Tyr220) detection antibody (Cell signaling, USA), RRP12 detection antibody (Novus, USA) and β-actin (Santa Cruz Biotechnology) primary antibody (rabbit polyclonal antibody) Further incubation for 2 hours in solution. After washing the membrane, the membrane was incubated for 1 hour in a Tris-buffered Saline with tween-20 (TBST) solution containing a secondary antibody (1: 5,000) conjugated with a horseradish peroxidase (SaRP Cruz Biotechnology). The membrane was washed again with water, then developed with an enhanced chemiluminescence reagent (Intron Biotechnology) and the results were described. Phosphorylation of RRP12 protein was observed in normal cells and synovial sarcoma without doxorubicin and 50 nM of doxorubicin. The phosphorylation activity of RRP12 protein was observed only when doxorubicin was administered in synovial sarcoma. 5).

3-2. In osteosarcoma RRP12  Protein activity measurement

The activity of RRP12 protein was measured upon doxorubicin administration in three different osteosarcoma cell lines (MG63, SaOS2, U2OS). The activity of phosphorylated RRP12 protein was measured in the osteosarcoma cell line without doxorubicin and with 50 nM of doxorubicin. After culturing three osteosarcoma cell lines and collecting the cells 24 hours after dosing rubicin 50nM administration, the protein activity was measured using the western blotting method shown in Example 3-1. As a result, the activity of RRP12 protein was not observed when doxorubicin was not administered, whereas the phosphorylation activity of RRP12 protein was observed when doxorubicin was administered (FIG. 6).

Cell immunohistochemical staining was performed 24 hours after doxorubicin 50nM administration in osteosarcoma cell line (MG63). The osteosarcoma cell line was fixed with 5% paraformaldehyde for 10 minutes, permeabilized with a buffer containing 0.1% Triton X-100, and then the primary antibody p-RRP12 (Cell). signaling, USA) and anti-RRP12 antibody (Novus, USA) treatment and then a secondary antibody (Alexa Fluor 488- or Alexa Fluor 546-conjugated anti-rabbit, mouse, goat or chicken antibody). Images were obtained by fluorescence microscopy or confocal microscopy. The location of the nucleus was confirmed by DAPI staining (blue fluorescence) and the results were analyzed by staining (green fluorescence) of the activated RRP12 protein. When doxorubicin was not administered, RRP12 protein was observed around the nucleus, but when doxorubicin was administered, RRP12 protein was activated and entered into the nucleus (FIG. 7).

These results indicate that there is a mechanism that causes the phosphorylation activity of RRP12 by the anticancer agent doxorubicin in the synovial sarcoma and osteosarcoma cell lines, which are tumor cells. It could be predicted to be associated with survival mechanisms for palpation or anticancer drugs.

Example  4: siRNA Using RRP12  Inhibition of gene expression

4-1. siRNA Making and siRNA Using RRP12  Inhibit gene expression

In order to prepare expression inhibitor of the RRP12 gene, siRNA specific to the RRP12 gene was constructed using siDESIGN SOFTWARE. Six candidate siRNAs having the sequences of SEQ ID NOs: 5-10 were synthesized (Table 2). Three days after transfection of the six candidate siRNAs into the osteosarcoma cell line (MG63), the expression level of the mRNA was confirmed using the RT-PCR method shown in Example 2, and then all six siRNAs were transduced. In the group it was confirmed that the expression of the RRP12 gene was reduced (Fig. 8).

These results indicate that the siRNA specific for the RRP12 gene produced above effectively inhibits the expression of the RRP12 gene. In particular, the siRNA having the sequences of SEQ ID NOs: 5, 7, and 10 has more significant effects, and thus, The experiment was performed using siRNA.

4-2. siRNA Phosphorylation RRP12  Inhibition of protein

It was confirmed whether phosphorylated RRP12 protein was inhibited by siRNA specific to RRP12 gene. Western blotting was performed on phosphorylated RRP12 protein by doxorubicin in osteosarcoma cell line (MG63). 120 pmol of siRNA specific to the RRP12 gene and 50 nM of doxorubicin after 2 days, the cells were collected after 24 hours, and the protein activity was obtained using the western blotting method shown in Example 3-1. Measured.

As a result, no phosphorylated RRP12 protein was detected when doxorubicin was not administered. Phosphorylated RRP12 protein was detected when doxorubicin was administered, but the amount of phosphorylated RRP12 protein was remarkable when siRNA specific to RRP12 gene was treated. It was observed that the decrease was very fast (Fig. 9).

These results indicate that the phosphorylated RRP12 protein is significantly inhibited by anticancer agents in tumor cells by siRNAs specific for the RRP12 gene.

4-3. siRNA Using RRP12  Confirmation of tumor cell proliferation upon inhibition of gene expression

In the osteosarcoma cell line (MG63), the inhibition of RRP12 using siRNA specific to the RRP12 gene was confirmed. In the normal fibroblast (hDF) and osteosarcoma cell lines (MG63), control siRNA and siRP specific to the RRP12 gene were administered, respectively, after 2 days, MTT assay was performed to measure the cell proliferation of the osteosarcoma cell line (MG63). It was. Normal fibroblasts and osteosarcoma cell lines were treated with 50 ug / ml of MTT solution, respectively, and incubated at 37 ° C. for 1 hour. After the incubation, the MTT solution was discarded, 200ul DMSO (dimethyl sulfoxide) was added to dissolve the insoluble substance (formazan crystal), and the O.D. (optical density) was measured at 595 nm using a spectrophotometer. O.D. of cells infected with RRP12 siRNA against cells infected with control siRNA. The ratio of the values was calculated and the results are described. All statistical treatments used the Student's T test and were considered significant when the p-value was 0.05 or less.

As a result, it was observed that the cell proliferation is reduced in the group administered siRNA specific to the RRP12 gene, in particular the cell proliferation is significantly reduced in osteosarcoma cell line (Fig. 10). These results indicate that the administration of RRP12 gene-specific siRNA in osteosarcoma cell lines inhibits RRP12 gene expression and inhibits cell proliferation. Since RRP12 gene is essential for the proliferation of osteosarcoma cell lines, anti-cancer effects are observed when RRP12 gene is inhibited. Means.

In addition, when doxorubicin was administered in combination with siRNA specific to RRP12 gene in osteosarcoma cell line (MG63), proliferation of osteosarcoma cell line was confirmed. In the osteosarcoma cell line, control siRNA and siRP specific to the RRP12 gene were respectively administered, and the proliferation of cells in the doxorubicin-administered group and the non-dosed group was compared by the MTT assay.

As a result, the proliferation of osteosarcoma cell line was decreased in the group administered with siRNA specific to the RRP12 gene than the group administered with doxorubicin, and the cell proliferation was further increased in the group administered with siRNA specific for the doxorubicin and RRP12 gene. It was confirmed that the marked reduction (Fig. 11).

These results indicate that RRP12 inhibitors have better anticancer effects than doxorubicin, and when administered with doxorubicin, an anticancer agent in osteosarcoma cell lines, siRNAs specific for RRP12 gene increase the anticancer effect of doxorubicin and inhibit cell proliferation. The result of summarizing the function of RRP12 based on the result of the above embodiment is shown in FIG.

4-4. siRNA Using RRP12  Of tumor cells upon inhibition of gene expression Cell death  Confirm

In the osteosarcoma cell line (MG63), cell death was observed when the RRP12 was inhibited by siRNA specific to the RRP12 gene. In the osteosarcoma cell line, control siRNA and siRP specific to the RRP12 gene were respectively administered, and flow cytometry analysis was performed to confirm whether or not cell death occurred in the doxorubicin-administered group and the non-dosed group. The activity of caspase-3 and poly (ADP-ribose) polymerase (PARP), enzymes activated during cell death, was analyzed by Western blotting (Fig. 13 to 14).

As a result, when a siRNA specific to the RRP12 gene was administered to the osteosarcoma cell line (MG63), cell death occurred. These results indicate that RRP12 is an essential protein for the survival of osteosarcoma cell lines. It was determined that the survival mechanism of tumor cells for the anticancer agent.

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Claims (12)

An anticancer composition comprising siRNA, antisense oligonucleotide or aptamer specific to RRP12 (ribosomal RNA processing 12 homolog) gene, or an aptamer, antibody or single chain variable region fragment specific to RRP12 protein as an active ingredient.
delete The composition of claim 1, wherein the siRNA is selected from the group consisting of SEQ ID NOs: 5-10.
The composition of claim 1, wherein the cancer is selected from the group consisting of osteosarcoma, megacytoma, chondroma, synovial sarcoma, bladder cancer, gastric cancer, breast cancer, colon cancer, cervical cancer, prostate cancer and epidermal cancer.
An anticancer sensitizing composition comprising an siRNA, antisense oligonucleotide or aptamer specific to a ribosomal RNA processing 12 homolog (RPRP12) gene, or an aptamer, antibody or single chain variable region fragment specific to a RRP12 protein.
6. The composition of claim 5, wherein the anticancer sensitizing composition inhibits anticancer drug resistance.
delete The composition of claim 5, wherein the siRNA is selected from the group consisting of SEQ ID NOs: 5-10.
6. The composition of claim 5, wherein the cancer is selected from the group consisting of osteosarcoma, megacytoma, chondroma, synovial sarcoma, bladder cancer, gastric cancer, breast cancer, colon cancer, cervical cancer, prostate cancer, and epidermal cancer.
An anticancer composition comprising the composition of any one of claims 5, 6, and 8-9 and an anticancer agent.
The anticancer composition according to claim 10, wherein the anticancer agent is doxorubicin.
(a) analyzing the expression of the RRP12 gene or the activity of the RRP12 protein after treating the test substance; And
(b) judging the test substance as an anticancer or anticancer agent if the expression of the RRP12 gene or the activity of the RRP12 protein after treatment with the test substance is inhibited compared to the expression of the RRP12 gene without the test substance or the activity of the RRP12 protein. Comprising, an anticancer agent or anticancer agent screening method.

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