KR20170023335A

KR20170023335A - Pharmaceutical composition for treating cancer having radioresistant phenotype

Info

Publication number: KR20170023335A
Application number: KR1020150117824A
Authority: KR
Inventors: 황상구; 윤홍식; 김재성; 박종국; 송지영; 엄홍덕
Original assignee: 한국원자력의학원
Priority date: 2015-08-21
Filing date: 2015-08-21
Publication date: 2017-03-03
Also published as: KR101772447B1

Abstract

The present invention relates to a pharmaceutical composition for treating radiation-resistant cancer, which comprises an agent for inhibiting the expression or activity of a protein that increases resistance to radiation therapy or a gene encoding the protein, a pharmaceutical composition for treating the radiation- A radiation-tolerant cancer diagnostic kit comprising the diagnostic composition, and a method of providing information for diagnosis of radiation-tolerant cancer using the kit or the diagnostic composition. By using the pharmaceutical composition of the present invention, it is possible to treat resistant cancer while using the conventional radiation treatment method as it is, and it is possible to reduce the cost of developing cancer drug for resistant cancer as well as the burden of cancer treatment for patient, And can be widely used for effective and effective treatment.

Description

[0001] The present invention relates to a pharmaceutical composition for the treatment of radiation-resistant cancer,

The present invention relates to a pharmaceutical composition for treating radiation-resistant cancer, and more particularly, the present invention relates to a pharmaceutical composition for treating radiation-resistant cancer, which comprises a protein which increases the resistance to radiation therapy or an agent which inhibits the expression or activity of a gene A pharmaceutical composition for therapeutic use, a radiological cancer diagnostic composition comprising an agent capable of measuring the expression level of the protein or gene, a radiation-resistant cancer diagnostic kit comprising the diagnostic composition, and the radiation-resistant cancer using the diagnostic composition or the kit And a method of providing information for diagnosis.

Methods that are mainly used for cancer treatment include surgery, chemotherapy, and radiation therapy. Of these, surgery and radiation therapy are localized therapies that are effective only at the site of resection or irradiation, and chemotherapy is a systemic therapy that is effective for the whole body. Most cancers develop locally and cause systemic metastasis. Unless they are detected very early, there is already a fine systemic metastasis, which often leads to recurrence despite effective topical therapy. First, local therapy is used. The efficacy of radiation therapy used in such topical therapies varies depending on the nature of the cancer, the patient and the radiation being combined with other therapies. Cancers in which radiation therapy is widely practiced include head and neck cancer, laryngeal cancer, cervical cancer, parietal cancer, lung cancer, brain cancer, breast cancer, and colon cancer. Although these carcinomas may be the main targets of radiation therapy, strategies that improve treatment efficiency are needed because of poor response to radiation therapy. In addition, although the initial response is good due to good radiation therapy, recurrence frequently occurs, and it is important to take measures against recurrent cancer to improve the efficiency of radiation therapy. Although there is a difference in the sensitivity of radiation therapy to cancer cells, if it is possible to predict whether or not each individual shows resistance to radiation before radiation therapy, it can be a great help in establishing a more effective cancer treatment strategy.

Cancer cells are often known to alter cellular components as a way to protect them from chronic radiation damage. For example, growth regulatory proteins such as epidermal growth factor receptor (EGFR), phosphoinositide 3-kinase (PI3K) / Akt and RAS are activated or overexpressed in radiation-resistant tumors. The status / expression of p53 or Bcl-2 / -xl plays an important role in cell cycle and apoptosis and is associated with tumor radiation resistance, and cellular antioxidant proteins such as peroxiredoxin-II and Mn-SOD (manganese superoxide dismutase) It is involved in oxidative stress tolerance of cancer cells. In addition, several investigators have reported that radiation therapy can induce acquired radiation resistance and chemotherapeutic drug resistance by modulating proteins associated with P-glycoprotein and multiple drug resistance. Factors associated with radiation resistance that have been identified so far include cyclooxygenase-2 (Terakado N, Shintani S, Yano J, Chunnan L, Mihara M, Nakashiro K, et al., Oral Oncol 2004; 40: 383-9) (EGFR) (Milas L, Fanz, Andratschke NH, Ang KK., Int J Radiat Oncol Biol Phys 2004; 58 (3); 966-71), Cyclin D1 (Milas L, Akimoto T, Hunter NR (IGFBP-1 receptor), manganese superoxide dismutase (MGF-I), and manganese superoxide dismutase , And survivin. However, the mechanism of the disease has been partially elucidated, and there are few markers that can be used as useful predictors of radiation therapy results and increase the radiation sensitivity. If a gene marker providing resistance to radiation therapy can be identified, the level of expression of the gene marker can be measured to confirm the effectiveness of the radiation therapy, and the expression of the gene marker can be inhibited, It is expected that a radiation therapy strategy will be established.

Under these circumstances, the present inventors have made intensive researches to discover novel gene markers that provide resistance to radiation therapy. As a result, it has been found that a gene encoding four kinds of proteins (PAI-2, NOMO2, KLC4 or PLOD3) The present inventors have completed the present invention.

It is an object of the present invention to provide a pharmaceutical composition for treating radiation-resistant cancer, which comprises an agent that inhibits the expression or activity of a protein that increases resistance to radiation therapy or a gene that encodes the protein.

Another object of the present invention is to provide a radiation-tolerant cancer diagnostic composition comprising an agent capable of measuring the expression level of the protein or gene.

It is still another object of the present invention to provide a radiation-tolerant cancer diagnostic kit comprising the diagnostic composition.

It is still another object of the present invention to provide a method for providing information for diagnosing radiation-tolerant cancer using the diagnostic composition or the kit.

The present inventors prepared a mutant cell line showing radiation resistance by irradiating a known cancer cell line with a radiation to discover a novel gene marker that provides resistance to radiation therapy. As a result of examining the characteristics of the prepared mutant cell line, it was confirmed that it exhibited resistance to radiation, exhibited characteristics as cancer stem-like cells, and maintained the characteristics as cancer cells. As a result, eight kinds of proteins (FASN, VIM, GRP78, UQCRC1, PAI-2, NOMO2, KLC4 and PLOD3) were identified as a result of the protein whose expression level was increased in the mutant cell line as compared with the known cancer cell lines. Of these, four proteins (PAI-2, NOMO2, KLC4 or PLOD3) were found to have no known association with radiation performance. As a result of confirming the effect of suppressing the expression of the four kinds of proteins in the mutant cell line, apoptosis was induced both when the radiation was irradiated and when the radiation was not irradiated, and when the radiation was irradiated, the apoptosis progressed . Therefore, by measuring the expression levels of the above four kinds of proteins (PAI-2, NOMO2, KLC4 or PLOD3), it is possible to confirm whether or not the cancer cells expressing the gene have resistance to radiation, Of the present invention can be used as an active ingredient of a pharmaceutical composition for treating radiation-resistant cancer.

It is not known at all that the four newly discovered proteins (PAI-2, NOMO2, KLC4 or PLOD3) in the present invention exhibit the property of imparting radiation resistance to cancer cells. Accordingly, the pharmaceutical composition provided by the present invention may be administered alone to the individual in which the cancer has been developed, but it is preferable that the pharmaceutical composition is administered together with the composition for radiotherapy or other composition for chemotherapy For example, doxorubicin, vinblastine, etc.).

In order to achieve the above-mentioned object, the present invention provides an agent for inhibiting the expression or activity of a protein selected from the group consisting of PAI-2, NOMO2, KLC4, PLOD3 and a combination thereof, And a pharmaceutical composition for treating radiation-resistant cancer.

The term "plasminogen activator inhibitor-2 " (PAI-2) of the present invention refers to an auxiliary regulatory factor for inactivating tPA and urokinase. Generally, a 60-kDa extracellular glycosylated form And 43-kDa in size. The PAI-2 is expressed in most cells, but is known to be expressed in large quantities, especially in monocytes / macrophage cells. The specific base sequence and protein information of the gene encoding PAI-2 are known in NCBI (GenBank: NM_001143818, NP_001137290, etc.).

The term "NOMO2 (NODAL modulator 2) " of the present invention means a part of a complex involved in the Nodal signal transduction pathway that is active during embryonic development. The specific nucleotide sequence and protein information of the gene encoding NOMO2 are known from NCBI (GenBank: NC_000016.10, NC_018927.2, NT-187260.1, etc.).

The term "KLC4 (Kinesin light chain 4)" of the present invention means a protein involved in Class I MHC mediated antigen processing and antigen presentation. The specific nucleotide sequence and protein information of the gene coding for KLC4 are known from NCBI (GenBank: NM_001289034, NP_001275963, etc.).

The term "PLOD3 (Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3)" of the present invention refers to a membrane-bound homozygous dimer-type enzyme located in the sister or region of RER. It is known to perform a role of catalyzing a misfiring reaction. The specific nucleotide sequence and protein information of the gene coding for PLOD3 are known from NCBI (GenBank: NM_001084, NP_001075, etc.).

The term "agent capable of inhibiting expression" of the present invention means a substance capable of inhibiting the production of a transcript or protein expressed and produced in a gene. Examples of the agent include a transcription factor that binds to a gene and inhibits at a transcription level; Interfering RNAs such as miRNA, siRNA, and shRNA that bind to the transcribed and synthesized transcript and degrade the transcript; An antibody capable of binding to an expressed protein, an aptamer, an antagonist, and the like.

The term "short interfering RNA " of the present invention means a double stranded RNA capable of inducing RNAi which inhibits the activity of a gene. In the present invention, the interfering RNA may be miRNA, siRNA, shRNA, or the like capable of inhibiting the expression of HRP-3. The interfering RNA may be any one selected from the group consisting of PAI-2, NOMO2, KLC4 or PLOD3 For example, siRNAs obtained by chemical synthesis or biochemical synthesis or in vivo synthesis, or double-stranded RNAs of 10 bases or more in which the double-stranded RNA of about 40 bases or more is degraded in the body can be used As one example, the siRNA of SEQ ID NO: 1 inhibiting the expression of PAI-2, the siRNA of SEQ ID NO: 2 inhibiting the expression of NOMO2, the siRNA of SEQ ID NO: 3 inhibiting the expression of KLC4, the sequence inhibiting the expression of PLOD3 SiRNA of No. 4, and the like.

The interference RNA is not limited to at least about 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% Or an RNA or double-stranded variant thereof containing a double-stranded portion may be used. The sequence portion having homology may be, but is not limited to, 15 nucleotides or more, 19 nucleotides or more, 20 nucleotides or more, or 21 nucleotides or more.

The term "antibody" of the present invention means a proteinaceous molecule capable of specifically binding to an antigenic site of a protein or peptide molecule. Such an antibody can be prepared by cloning each gene into an expression vector according to a conventional method, A protein encoded by the marker gene can be obtained and can be produced from the obtained protein by a conventional method. The form of the antibody is not particularly limited, and any polyclonal antibody, monoclonal antibody or antigen-binding antibody thereof may be included in the antibody of the present invention, and any immunoglobulin antibody may be included, Specific antibodies of the invention. In addition, the antibody comprises a functional fragment of an antibody molecule as well as a complete form having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule means a fragment having at least an antigen-binding function, and can be Fab, F (ab ') 2, F (ab') ₂ , Fv and the like.

In the present invention, the antibody may be an antibody capable of specifically binding to a PAI-2, NOMO2, KLC4 or PLOD3 protein. For example, the antibody may specifically bind PAI-2, NOMO2, KLC4 or PLOD3 protein A monoclonal antibody, or a portion thereof.

The term "aptamer " of the present invention means a nucleic acid molecule having a binding activity to a predetermined target molecule. The aptamer may be RNA, DNA, modified nucleic acid, or a mixture thereof. The aptamer may be in the form of a linear or cyclic form. Generally, the shorter the sequence of the nucleotide constituting the aptamer, the more the chemical synthesis and mass production It is known that it is easier to use, has excellent cost advantages, is easy to chemically modify, has excellent in-vivo stability, and has low toxicity.

For the purpose of the present invention, the aptamer can be interpreted as means for inhibiting the activity of the protein by binding to PAI-2, NOMO2, KLC4 or PLOD3 protein.

The term "antagonist " of the present invention means a molecule capable of directly or indirectly reducing a biological activity of a receptor, and when used with a ligand of a receptor, a molecule capable of reducing the action of the ligand But are not limited thereto.

For the purpose of the present invention, the antagonist includes any molecule that inhibits the activity of the PAI-2, NOMO2, KLC4 or PLOD3 proteins, and the antagonist specifically binds to PAI-2, NOMO2, KLC4 or PLOD3 But is not limited thereto.

The term "resistant cancer" of the present invention means cancer which shows extremely low sensitivity to radiation therapy and does not exhibit symptoms of amelioration, alleviation, alleviation or treatment of cancer by the above-mentioned treatment method. The resistant cancer may be resistant to certain radiation treatments from the outset and may not tolerate initially, but may result in long-term treatment resulting in a mutation of the gene in cancer cells that is no longer susceptible to the same level of radiation therapy It is possible.

In the present invention, the resistant cancer may be any cancer that is resistant to radiation therapy, specifically, but not limited to, radiation. Examples of such cancer include cancer such as radiation induced by overexpression of PAI-2, NOMO2, KLC4 or PLOD3 Lung cancer, cervical cancer, colon cancer, breast cancer, etc., which are resistant to treatment or chemotherapy, and the lung cancer may be non-small cell lung cancer, but is not limited thereto.

The pharmaceutical composition for the treatment of radiation-tolerant cancer of the present invention inhibits the expression or activity of PAI-2, NOMO2, KLC4 or PLOD3 protein and suppresses resistance to radiation. Therefore, Or by administering a conventional anticancer agent which does not exhibit any therapeutic effect due to the resistance caused by the PAI-2, NOMO2, KLC4 or PLOD3 protein and the pharmaceutical composition of the present invention, Radiation-resistant cancer can be treated. In particular, when the pharmaceutical composition of the present invention is administered in combination with a conventional anticancer agent, the anticancer activity of the conventional anticancer agent and the anticancer activity of the pharmaceutical composition of the present invention are overlapped with each other, thereby more effectively treating the cancer. Conventional anticancer agents that can be used at this time are not particularly limited as long as they are resistant to PAI-2, NOMO2, KLC4 or PLOD3 proteins, but preferably doxorubicin, vinblastine, taxol, ediposide, cisplatin, 5- FU, and IFOSPHASMIDE may be used alone or in combination.

According to one embodiment of the present invention, human H460 NSCLC cells were irradiated with radiation to produce mutants exhibiting resistance to radiation, and their characteristics were confirmed. As a result, they showed high cell viability (FIGS. 1A and 1B) (Fig. 1C), exhibited excellent cell proliferation rate (Fig. 1C), resistance to radiation treatment (Fig. 1D), low apoptosis rate by irradiation (Fig. 1E), Hsp90 and Her- (FIG. 1F). In addition, it was confirmed that the mutant exhibited characteristics as cancer stem-like cells (Figs. 2A to 2D), retained its characteristics as cancer cells (Figs. 3A to 3D), and inhibited cell senescence (Figs. 4A to 4C). As a result of proteomic analysis, a protein having an expression level 2 times or more higher than that of human H460 NSCLC cell was found to be 8 kinds of proteins (FASN, VIM, GRP78, UQCRC1, PAI- (PAI-2, NOMO2, KLC4, or PLOD3) is not known to be associated with the radiation-induced effects of HOMO2, KLC4, and PLOD3. (Table 1 and Table 2). As a result of examining the effect of inhibiting the expression of the four kinds of proteins on the radiation tolerance of cancer cells, cancer cells in which the expression of the four kinds of proteins were inhibited were observed not only in the case of irradiation, (FIG. 6A and FIG. 6B), it was confirmed that the expression level of truncated PARP, which is a cell apoptosis marker induced by irradiation, is rapidly increased (Fig. 6C).

Therefore, four proteins (PAI-2, NOMO2, KLC4, or PLOD3) that are not known to be related to radiation performance inhibit apoptosis induced by irradiation, thereby imparting tolerance to radiation Therefore, by measuring the expression levels of the above four kinds of proteins (PAI-2, NOMO2, KLC4 or PLOD3), it is possible to confirm whether or not the cancer cells expressing the gene have resistance to radiation, Of the present invention can be used as an active ingredient of a pharmaceutical composition for treating radiation-resistant cancer.

The pharmaceutical composition of the present invention may be prepared in the form of a pharmaceutical composition for the treatment of radiation-resistant cancer, further comprising an appropriate carrier, excipient or diluent conventionally used in the production of a pharmaceutical composition, (non-naturally occuring carrier). Specifically, the pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method . In the present invention, the carrier, excipient and diluent which may be contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use may include various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are simple diluents commonly used in suspension agents, solutions, emulsions and syrups have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The content of the formulation in the pharmaceutical composition according to an embodiment of the present invention is not particularly limited, but may be in the range of 0.0001 to 50% by weight, more preferably 0.01 to 10% by weight, based on the total weight of the final composition .

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount " of the present invention means a therapeutic or prophylactic treatment of a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention And the effective dose level refers to the level of the disease to be treated, the severity of the disease, the activity of the drug, the age, body weight, health, sex, sensitivity of the patient to the drug, Duration, duration of administration, factors involved in combination with or contemporaneously with the composition of the present invention, and other factors well known in the medical arts. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered singly or multiply. It is important to take into account all of the above factors and administer an amount that will achieve the maximum effect in the least amount without side effects.

The dosage of the pharmaceutical composition of the present invention can be determined by those skilled in the art in consideration of the purpose of use, the degree of addiction to the disease, the age, body weight, sex, history, or kind of the substance used as the active ingredient. For example, the pharmaceutical composition of the present invention can be administered to a mammal including a human at a dose of 10 to 100 mg / kg, more preferably 10 to 30 mg / kg, and the administration frequency of the composition of the present invention It may be administered once to three times a day, or may be administered several times in divided doses.

In another aspect, the present invention provides a method for treating a radiation-tolerant cancer comprising administering the pharmaceutical composition to a subject suffering from radiation-resistant cancer in a pharmaceutically effective amount. In this case, the pharmaceutical composition may be administered alone to the subject, or the pharmaceutical composition may be administered while being irradiated with radiation. In addition, the composition for chemotherapy other than the pharmaceutical composition of the present invention (for example, doxorubicin, Etc.) may be administered in combination.

The term "individual" of the present invention means all animals including human having the radiation-tolerant cancer. By administering the composition of the present invention to a subject, radiation resistant cancer can be treated.

The term "treatment" in the present invention means any action that improves or alleviates the radiation tolerance cancer by administering the pharmaceutical composition of the present invention.

The term "administration" of the present invention means the introduction of the pharmaceutical composition of the present invention to a subject by any appropriate method, and the administration route can be administered through various routes of oral or parenteral administration, have.

In the method of treating radiation-tolerant cancer of the present invention, the route of administration of the pharmaceutical composition may be administered through any conventional route so long as it can reach the target tissue. The pharmaceutical composition of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intracorporally, or rectally, as desired, though not particularly limited thereto. In addition, the composition may be administered by any device capable of transferring the active agent to the target cell.

The present invention provides, in yet another aspect, a pharmaceutical composition comprising an agent capable of measuring the level of a protein selected from the group consisting of PAI-2, NOMO2, KLC4, PLOD3 and combinations thereof or the expression level of a gene encoding said protein. A composition for diagnosing radiation-tolerant cancer is provided.

The term "agent capable of measuring the level of the protein or the expression level of the gene encoding the protein" of the present invention means an agent capable of measuring the level of the protein or the expression level of the gene encoding the protein.

In the present invention, the agent capable of measuring the level of the protein or the expression level of the gene encoding the protein is specifically a protein selected from the group consisting of PAI-2, NOMO2, KLC4, PLOD3, and combinations thereof An antibody capable of binding, an aptamer, an antagonist, and the like; A primer used for measuring the level of mRNA expressed from a gene encoding a protein selected from the group consisting of PAI-2, NOMO2, KLC4, PLOD3, and combinations thereof. At this time, the antibody, the aptamer and the antagonist are the same as described above.

The term "primer" of the present invention refers to a nucleic acid sequence having a short free 3 'hydroxyl group, capable of forming base pairs with a complementary template and having a starting point for template strand copying &Lt; / RTI > The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i.e., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. The PCR conditions, the lengths of the sense and antisense primers can be modified based on what is known in the art. The primers can also be modified, for example, by methylation, capping, substitution of nucleotides or modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, phosphoramidate , Carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). In order to more effectively recognize the PCR product amplified using the primer pair, the 5 'or 3' end of the primer can be labeled with a fluorescent material or the like. In this case, the fluorescent material to be used is not particularly limited, but may be selected from the group consisting of FAM (6-carboxyfluorescein), HEX (2 ', 4', 5 ', 7', - tetrachloro-6-carboxy-4,7-dichlorofluorescein) Etc. may be used.

The term "diagnosis" of the present invention is used to confirm the presence or characteristic of a pathological condition, and for the purpose of the present invention, not only to confirm the onset of radiation tolerant cancer, , Drug responsiveness, resistance, and the like.

In another aspect, the present invention provides a radiation-tolerant cancer diagnostic kit comprising the diagnostic composition.

The radiation-tolerant cancer diagnostic kit of the present invention may further comprise a level of a protein selected from the group consisting of PAI-2, NOMO2, KLC4, PLOD3 and combinations thereof capable of imparting radiation resistance to cancer or an expression level As well as one or more other component compositions, solutions, or devices suitable for the assay method.

As an example, the kit of the present invention may include a substrate, a suitable buffer solution, a secondary antibody labeled with a chromogenic or fluorescent substance, and a chromogenic substrate for immunological detection of the protein. The substrate contained in the kit may be a nitrocellulose membrane, a 96-well plate synthesized with a polyvinyl resin, a 96-well plate synthesized with a polystyrene resin, a slide glass made of glass, or the like, and the chromogenic enzyme may be peroxidase peroxidase, alkaline phosphatase and the like can be used. As the fluorescent material, FITC, RITC and the like can be used. The coloring substrate solution is ABTS (2,2'-azino-bis- 6-sulfonic acid), or OPD (o-phenylenediamine), TMB (tetramethylbenzidine) may be used. In addition, Western blotting, ELISA, otA (badioimmunoassay), radial immunodiffusion, Ouchteroni immunodiffusion, rocket immunoelectrophoresis, tissue But are not limited to, immunostaining, immunoprecipitation assays, complement fixation assays, FACS and protein chips.

As another example, the kit of the present invention may comprise an essential element for carrying out PCR for measuring the expression level of a gene encoding a protein selected from the group consisting of PAI-2, NOMO2, KLC4, PLOD3 and combinations thereof May be included. In addition to the primers for performing PCR, the kit can also be used in the form of test tubes, other appropriate containers, reaction buffers (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), various enzymes (such as Taq polymerase) , DEPC-water (DEPC-water), sterilized water, and the like. In addition, a primer pair specific to a gene used as a control group may be included.

According to another aspect of the present invention, there is provided a diagnostic composition or a kit for evaluating the expression level of a protein selected from the group consisting of PAI-2, NOMO2, KLC4, PLOD3, The method comprising the steps of: a) measuring the radiation dose of the radiation-tolerant cancer;

As one example, the method for providing information for the diagnosis of radiation-tolerant cancer of the present invention comprises the steps of (a) extracting PAI-2, NOMO2, KLC4, PLOD3 and combinations thereof from a sample isolated from a susceptible individual Measuring the level of the protein selected from the group; (b) measuring the level of a protein selected from the group consisting of PAI-2, NOMO2, KLC4, PLOD3 and combinations thereof from a sample isolated from a normal subject; And (c) comparing the levels of the proteins measured in steps (a) and (b). In the above method, when the level of the protein measured in step (a) is relatively high as compared with the level of the protein measured in step (b), it can be determined that the individual has developed radiation-tolerant cancer .

As another example, the method for providing information for the diagnosis of radiation-tolerant cancer of the present invention comprises the steps of (a) extracting PAI-2, NOMO2, KLC4, PLOD3 and combinations thereof from a sample isolated from a susceptible individual Measuring the mRNA level of a gene encoding a protein selected from the group; (b) measuring the mRNA level of a gene encoding a protein selected from the group consisting of PAI-2, NOMO2, KLC4, PLOD3 and combinations thereof from a sample isolated from a normal individual; And (c) comparing the levels of mRNA measured in steps (a) and (b). In the above method, when the level of the mRNA measured in the step (a) shows a relatively high value as compared with the level of the mRNA measured in the step (b), it can be determined that the individual has developed radiation resistant cancer .

The term "isolated sample " of the present invention means a protein isolated from the individual and comprising a protein selected from the group consisting of PAI-2, NOMO2, KLC4, PLOD3 and a combination thereof, or mRNA of a gene encoding the protein, In the present invention, the expression level of the protein or gene can be measured. The sample is not particularly limited, but may be, for example, a separated tissue or a separated cell.

By using the pharmaceutical composition of the present invention, it is possible to treat resistant cancer while using the conventional radiation treatment method as it is, and it is possible to reduce the cost of developing cancer drug for resistant cancer as well as the burden of cancer treatment for patient, And can be widely used for effective and effective treatment.

1A is a photograph showing cells surviving after treatment of H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells with radiation.
FIG. 1B is a graph showing the results of measuring cell viability after treating H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells with radiation.
1C is a graph showing the results of quantitative analysis of proliferated cells after culturing H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells for 6 days.
FIG. 1D is a graph showing the results of comparing susceptibility of H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells to radiation treatment.
FIG. 1E is a photograph showing the results of comparing the incidence of apoptosis induced by radiation treatment of H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells. Western blot analysis photograph showing the result, and bottom photograph showing DAPI staining result.
FIG. 1F is a graph showing the results of comparing mRNA levels of Hsp90 and Her-3 genes expressed in H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells.
2A is a graph showing the results of performing cancer stem-like cell surface marker analysis confirmed by flow cytometry in H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells.
FIG. 2B is a Western blot analysis image showing the results of comparing the levels of the stem cell marker proteins CD44, Notch1, Nanog, Oct4 or Sox2 in H460 cells, RR-Full cells, RR- # 2 cells and RR- to be.
FIG. 2C is a graph showing the results of comparing the number of searched spears and a photograph showing the morphology of H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells cultured under sphere culture conditions.
FIG. 2D is a photomicrograph showing the result of comparing the morphological changes of the sphere with the passage of time after treating the sphere of RR- # 2 cells with serum.
3A is a photograph and a graph showing the results of analysis of invasiveness of H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells.
FIG. 3B is a photograph and a graph showing the results of analyzing the mobility of H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells.
FIG. 3C is a photograph showing the results of wound healing analysis on H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells.
FIG. 3D is a graph showing a photograph showing colonies obtained by culturing H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells, and a result of analyzing the number of colonies.
The left side of FIG. 4A is a photomicrograph showing the morphology of H460 cells, RR-Full cells and RR- # 2 cells cultured for 48 hours while irradiating 0-6 Gy of radiation. In the center and right side of FIG. . The results are shown in the photographs and the graphs showing the results of comparing the changes in cell senescence level by SA-β-gal staining.
4B is a Western blot analysis showing the results of comparing the expression levels of aging-related marker proteins (p16, p21, p53 and pp53) expressed in H460 cells, RR-Full cells and RR- It is a photograph.
FIG. 4c is a graph showing the results of classification of H460 cells and RR- # 2 cells cultured with irradiation, according to the cell cycle.
FIG. 5A shows the results of 2-D PAGE analysis of H460 cells, RR-Full cells and RR- # 2 cells, showing that the expression levels in RR-Full and RR- , Which is a significant increase in the number of proteins.
FIG. 5B is an enlarged view of the enlarged portion of the protein having the increased expression level in FIG. 5A.
FIG. 5c is a Western blot analysis image showing the results of comparing the expression levels of PAI-2, NOMO2, KLC4 or PLOD3 in H460 cells and RR- # 2 cells.
FIG. 6A is a photomicrograph showing the results of comparing changes in cell morphology after culturing with irradiation of radiation-resistant cells in which the expression of PAI-2, NOMO2, KLC4 or PLOD3 was inhibited.
FIG. 6B is a graph showing the results of comparing cell death rates measured by flow cytometry after culturing with irradiation of radiation-resistant cells in which the expression of PAI-2, NOMO2, KLC4 or PLOD3 was inhibited.
FIG. 6C is a graph showing changes in the expression levels of PAI-2, NOMO2, KLC4 or PLOD3 in the RR- # 2 cells cultured without or with 10 Gy of each siRNA, Western blot analysis showing the expression level of the protein inhibited by each siRNA and the expression level of cleaved PARP.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Preparation and characterization of radiation-resistant cells

Example 1-1: Preparation of radiation-resistant cells

Human H460 NSCLC cells were cultured in a Roswell Park Memorial Institute medium 1640 (Gibco-BRL, Rockville, Md.) Containing 10% FBS, 50 ug / ml streptomycin and 50 units / ml penicillin.

The cultured cells were cultured for 2 weeks at a dose of 2 Gy for 20 weeks using a ¹³⁷ cesium-ray source (Atomic Energy of Canada Ltd., Canada). The colonies formed after the culture were stained with trypan blue , And from which RR-Full (radioresistance phenotype of irradiated cell population) cells, which are resistant to radiation treatment, were selected. The selected RR-Full cells were treated with 6 Gy radiation and subcultured to select RR- # 2 cells and RR- # 5 cells, and their characteristics were compared with H460.

Example 1-2: Characterization of radiation-resistant cells

Example 1-2-1: Comparison of cell viability by radiation treatment

The H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells prepared in Example 1-1 were treated with 0, 4 or 6 Gy radiation and cultured for 14 days, To compare cell viability (Figs. 1A and 1B).

FIG. 1A is a photograph showing cells surviving after irradiation with radiation to H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells, FIG. 1B shows H460 cells, RR- RR- &num; 2 cells and RR- &num; 5 cells after radiation treatment. As shown in FIGS. 1A and 1B, it was confirmed that RR-Full cells, RR- # 2 cells and RR- # 5 cells showed a higher cell survival rate after radiation treatment than H460 cells.

Example 1-2-2: Cell proliferation rate compare

The H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells prepared in Example 1-1 were inoculated into a 60-mm culture dish at a density of 3 X 10 ⁵ cells and cultured for 6 days After that, the number of cells was quantitatively analyzed (Fig. 1C).

1C is a graph showing the results of quantitative analysis of proliferated cells after culturing H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells for 6 days. As shown in FIG. 1C, it was confirmed that RR-Full cells, RR- # 2 cells and RR- # 5 cells were superior to H460 cells in cell proliferation rate.

Example 1-2-3: Comparison of susceptibility to radiation treatment

The H460 cells, RR-Full cells, RR- # 2 cells, and RR- # 5 cells prepared in Example 1-1 were treated with 0 or 10 Gy radiation and cultured for 48 hours. Washed, and treated with PBS containing 0.1% triton X-100 for 5 minutes, and stained with 5 / / ml of PI (propidium iodide) at room temperature for 15 minutes to compare cell death rates (Fig. 1d) .

FIG. 1D is a graph showing the results of comparing susceptibility of H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells to radiation treatment. As shown in FIG. 1d, H460 cells showed relatively high sensitivity to radiation treatment, but RR-Full cells, RR- # 2 cells and RR- # 5 cells showed relatively low sensitivity to radiation treatment, In particular, it was confirmed that RR- # 2 cells exhibited the lowest sensitivity to radiation treatment.

Example 1-2-4: Radiation treatment Apoptotic change

The H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells prepared in Example 1-1 were treated with 0 or 10 Gy radiation and cultured for 48 hours, , DAPI staining was performed to compare nuclei changes, and extracts of each cell were obtained. Western blot analysis using the PARP specific antibody cleaved from each of the above extracts was performed to determine the apoptosis (Fig. 1E). At this time, β-actin was used as an internal control group.

FIG. 1E is a photograph showing the results of comparing the incidence of apoptosis induced by radiation treatment of H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells. Western blot analysis photograph showing the result, and bottom photograph showing DAPI staining result. As shown in FIG. 1E, the levels of PARP cleaved by radiation treatment of H460 cells were significantly increased, but the levels of PARP cleaved even after the irradiation of RR-Full cells, RR- # 2 cells and RR- Was not significantly changed.

Example 1-2-5: Changes in transcription level of radiation-resistant factors by radiation treatment

In order to confirm the radiation resistant phenotype, the transcription levels of Hsp90 and Her-3 genes, which are known as tumor radiation resistance factors, were evaluated in the H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells (Fig. 1F).

FIG. 1F is a graph showing the results of comparing mRNA levels of Hsp90 and Her-3 genes expressed in H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells. As shown in FIG. 1F, the transcription levels of Hsp90 and Her-3 genes, which are known as radiation resistance factors, were low in H460 cells. However, Hsp90 and Her-3 genes in RR-Full, RR- And the highest level in RR- # 2 cells.

The above results indicate that RR-Full cells, RR- # 2 cells and RR- # 5 cells obtained by irradiating human H460 NSCLC cells with radiation are radiation-tolerant cells.

Example 1-3: Cancer Stemlike cell( CSCs ) As Character analysis

To determine whether the three types of radiation-tolerant cells produced exhibited the characteristics as cancer stem-like cells (CSCs).

Example 1-3-1: Surface marker analysis

H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells prepared in Example 1-1 were washed with PBS, treated with PBS containing 0.1% triton X-100 for 5 minutes, Respectively. Subsequently, the cells were washed three times with PBS, and the anti-CD133 antibody or the anti-CD44 antibody was treated for 30 minutes, and then the secondary antibody bound to FITC was treated for 20 minutes to react. Finally, the cells were washed twice with PBS and flow cytometry was performed (Fig. 2a).

2A is a graph showing the results of performing cancer stem-like cell surface marker analysis confirmed by flow cytometry in H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells. As shown in FIG. 2A, CD133 expression was expressed in a low ratio (0.1-0.3%) in all cell lines, but the expression ratios of CD44 in RR-Full, RR- # 2 and RR- % And 95%, respectively, whereas it was 82% in H460 cells.

Example 1-3-2: Marker Analysis of protein expression level

Each of the lysates from the H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells prepared in Example 1-1, Western blot analysis using antibodies against Oct4, Sox2 or < RTI ID = 0.0 > ss-catenin < / RTI > At this time, β-actin was used as an internal control group.

FIG. 2B is a Western blot analysis image showing the results of comparing the levels of the stem cell marker proteins CD44, Notch1, Nanog, Oct4 or Sox2 in H460 cells, RR-Full cells, RR- # 2 cells and RR- to be. As shown in FIG. 2B, it was confirmed that CD44 and Notch1 were specifically expressed in RR-Full, RR- # 2 and RR- # 5 cells compared with H460 cells. In addition, the pluripotency-related transcription factors Nanog, Oct4 and Sox2, as well as the β-catenin, an adjuvant regulator, were also relatively high in RR-Full, RR- # 2 and RR- # 5 cells compared to H460 cells Respectively.

Therefore, it was analyzed that RR-Full cells, RR- # 2 cells and RR- # 5 cells would exhibit characteristics as cancer stem cells.

Example 1-3-3: Spear Formation analysis

The H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells prepared in Example 1-1 were suspended in 20 ng / ml EGF, 20 ng / ml bFGF and B27 serum- The culture was incubated for 5 days, and the culture conditions were compared immediately after and 5 days after culturing. The number of spears obtained after culturing was compared (FIG. 2C).

FIG. 2C is a graph showing the results of comparing the number of searched spears and a photograph showing the morphology of H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells cultured under sphere culture conditions. As shown in FIG. 2C, the number of spores was significantly increased in RR-Full cells, RR- # 2 cells and RR- # 5 cells compared to H460 cells.

Meanwhile, sera were treated on the sphere of RR- # 2 cells which form the greatest number of spores in the RR-Full cells, RR- # 2 cells and RR- # 5 cells, and cultured at 0, 12, 24 and 24 hours Was photographed by an optical microscope (Fig. 2D).

FIG. 2D is a photomicrograph showing the result of comparing the morphological changes of the sphere with the passage of time after treating the sphere of RR- # 2 cells with serum. As shown in FIG. 2D, it was confirmed that RR- # 2 cells significantly changed with time, similar to tumor cells.

Example 1-4: Characterization of cancer cells

Example 1-4-1: Invasiveness and mobility analysis

The H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells prepared in Example 1-1 were inoculated into BD Bio coat Matrigel Invasion chambers pretreated with 10 mg / ml GF, The invasiveness and mobility of each cell was analyzed (Figures 3a and 3b).

3A and 3B are photographs and graphs showing the results of analysis of invasiveness of H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells. And cell and RR- # 5 cells. As shown in FIGS. 3A and 3B, it was confirmed that RR-Full cells, RR- # 2 cells and RR- # 5 cells showed higher invasiveness and mobility than H460 cells. In the case of invasiveness, RR-Full cells, RR- # 2 cells and RR- # 5 cells showed 1.7 times, 2.6 times, and 2.2 times, respectively, as compared with H460 cells. , RR- # 2 cells and RR- # 5 cells were 1.8, 2.9 and 2.7 times, respectively.

Example 1-4-2: Mobility analysis through wound healing analysis

The H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells prepared in Example 1-1 were cultured in a 60-mm culture container until saturation, Scratched with a pipette tip. Then, the cells were washed twice with PBS, fresh medium was supplied, and the cells were cultured for 48 hours. Immediately after the culture, 24 hours and 48 hours after the incubation, the cells were photographed by reversed-phase microscopy (FIG.

FIG. 3C is a photograph showing the results of wound healing analysis on H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells. As shown in FIG. 3C, relatively good cell mobility was exhibited in RR-Full cells, RR- # 2 cells and RR- # 5 cells as compared with H460 cells, and thus it was confirmed that the activity of effectively treating wounds was exhibited.

Example 1-4-3: Growth rate analysis

The H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells prepared in Example 1-1 were cultured, and the colonies formed were stained with trypan blue and counted to compare their proliferation rates 3d).

FIG. 3D is a graph showing a photograph showing colonies obtained by culturing H460 cells, RR-Full cells, RR- # 2 cells and RR- # 5 cells, and a result of analyzing the number of colonies. As shown in FIG. 3D, cell growth was promoted in RR-Full cells, RR- # 2 cells and RR- # 5 cells as compared with H460 cells, and the proliferation rate was higher in RR-Full cells, RR- RR- &num; 5 cells were 1.72, 2.5 and 2.2 times, respectively.

Example 1-5: Cell aging assay

Example 1-5-1: Analysis of shape change

The H460 cells, RR-Full cells and RR- # 2 cells prepared in Example 1-1 were cultured for 48 hours while irradiating 0-6 Gy of radiation, and the morphology of the cells was photographed by a microscope (FIG. 4A left side).

The left side of FIG. 4A is a photomicrograph showing the morphology of H460 cells, RR-Full cells and RR- # 2 cells cultured for 48 hours while irradiating 0-6 Gy of radiation. As shown in the left side of FIG. 4A, H460 cells were changed to broadly spread the cell shape as the radiation treatment level increased. However, RR-Full cells and RR- # 2 cells showed morphological changes Respectively.

Example 1-5-2: Analysis of SA-β-gal staining

The H460 cells, RR-Full cells and RR- # 2 cells cultured in the irradiation of radiation in Example 1-5-1 were washed twice with PBS and fixed with 3.7% formalin for 10 minutes. The cells were then washed twice with PBS and then stained with SA-β-gal dye solution (1 mg / ml 5-bromo-4-chloro-3-indolyl β-D-galactoside, 40 mM citric acid / sodium phosphate buffer, pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl and 2 mM MgCl ₂ ) at 37 ° C for 16 hours, and the result was observed under a microscope and the number of stained cells was counted Center and right of FIG. 4A).

4A are photographs and graphs showing the results of SA-β-gal staining comparing the changes in cell senescence level with irradiation. As shown in the center and right side of FIG. 4A, when H460 cells, RR-Full cells and RR- # 2 cells were treated with radiation, cell senescence was promoted with increasing levels of radiation, -Full cells and RR- &num; 2 cells, the level of cellular senescence after irradiation was drastically increased. For example, the proportion of SA-β-gal positive cells in RR-Full cells and RR- # 2 cells treated with 6 Gy of radiation was 32% and 23%, respectively, while H460 cells treated with 6 Gy of radiation The ratio of SA-β-gal positive cells was about 52%.

Example 1-5-3: Aging marker analysis

The H460 cells, RR-Full cells and RR- # 2 cells prepared in Example 1-1 were cultured for 48 hours while irradiated with 6 Gy of radiation, and the aging-related marker proteins (p16, p21, p53 And phosphorylated p53 (pp53)) were compared by Western blot analysis (Fig. 4B).

4B is a Western blot analysis showing the results of comparing the expression levels of aging-related marker proteins (p16, p21, p53 and pp53) expressed in H460 cells, RR-Full cells and RR- It is a photograph. As shown in FIG. 4B, the expression levels of aging-related marker proteins (p16, p21, p53, and pp53) in H460 cells were higher than those of RR-Full cells and RR- # 2 cells. In particular, the expression level of p53 and activated p53 (pp53) was significantly increased in H460 cells compared to RR-Full cells and RR- # 2 cells.

Example 1-5-4: Cell cycle analysis

H460 cells and RR- # 2 cells prepared in Example 1-1 were cultured for 48 hours while irradiated with 6 Gy of radiation. After 0, 12, 24 or 48 hours, 70% ethanol was added and fixed at 4 DEG C for 2 hours. The fixed cells were treated with 100 mg / ml ribonuclease for 1 hour and stained with 5 μg / ml PI (propidium iodide) for 15 minutes at room temperature. Flow cytometry was performed on stained cells, and the results were analyzed by the Modfit cell cycle program (Verity Software House Inc., USA) to classify the cells in the G1, S, and G2 / M groups, respectively 4c).

FIG. 4c is a graph showing the results of classification of H460 cells and RR- # 2 cells cultured with irradiation, according to the cell cycle. As shown in FIG. 4C, G2 / M cell cycle arrest was induced after irradiation in H460 cells, and the ratio thereof was 56% at 12 hours treatment and 45% at 24 hours treatment, whereas G2 / It was confirmed that the ratio of M cell cycle arrest was 37% at 12 hours treatment and only 27% at 24 hours treatment time.

The results of FIGS. 1 to 4 indicate that RR-Full cells, RR- # 2 cells and RR- # 5 cells obtained by irradiating H460 cells with radiation show not only radiation resistance but also similar characteristics to cancer stem cells Respectively.

Example 2: Proteome ( Proteomic ) analysis

Example 2-1: 2-D PAGE analysis

2-D PAGE analysis was performed on the H460 cells, RR-Full cells and RR- # 2 cells prepared in Example 1-1 to detect proteins whose expression patterns were changed in each cell. Approximately, 2-D SDS-PAGE was performed on the extracts obtained from each of the above cells and expressed in RR-Full cells and RR- # 2 cells compared to H460 cells using silver salt and Imagemaster ™ 2D platinum software (Fig. 5A and 5B, Table 1). &Lt; tb > < TABLE >

FIG. 5A shows the results of 2-D PAGE analysis of H460 cells, RR-Full cells and RR- # 2 cells, showing that the expression levels in RR-Full and RR- FIG. 5B is a magnified photograph showing an enlarged portion of the protein having the increased expression level. FIG.

In RR-Full cells and RR- # 2 cells compared to H460 cells, the expression levels of proteins number designation Fold change SD Swiss-Prot Assession no. Mw (kDa) p / Coverage
MASCOT Score One
2
3
4
5
6
7
8 FASN
VIM
GRP78
UQCRC1
PAI2
NOMO2
KLC4
PLOD3 (+) 2.6
(+) 4.4
(+) 8.1
(+) 2.4
(+) 2.6
(+) 6.7
(+) 6
(+) 3.1 ± 0.71
± 0.27
± 0.26
± 1.25
± 0.33
± 0.78
± 1.32
± 1.13 P49327
P08670
P11021
P31930
P05120
Q5JPE7
Q9NSK0
O60568 275850
53676
72402
53297
46851
135077
70965
85302 5.99
5.06
5.07
5.94
5.46
5.51
5.95
5.69 12
59
40
53
38
30
20
43 101
237
235
203
124
146
74
292

Fold change indicates the ratio of the spot volume in H460 cells to the average volume of spot and RR-Full / RR- # 2 cells in spot volume, (+) indicating increased protein expression level in RR-Full / RR- # 2 cells .

Coverage represents the proportion of peptides matched in the total protein.

The Mascot Score represents the significance of the values obtained from the Mascot search engine.

As shown in FIGS. 5A and 5B and Table 1, eight proteins were detected in the RR-Full cells and RR- # 2 cells, which express more than two-fold higher expression levels than H460 cells.

Example 2-2: MALDI - TOF analysis

The 8 proteins detected in Example 2-1 were identified by MALDI-TOF mass spectrometry and MASCOT score (Table 2).

Identification of eight proteins that induce radiation resistance protein Expression level Radioactive resistance FASN
VIM
GRP78
UQCRC1
PAI2
NOMO2
KLC4
PLOD3 ↑ 2.6
↑ 4.4
↑ 8.1
↑ 2.4
↑ 2.6
↑ 6.7
↑ 6.0
↑ 3.1 Radiation resistance
Radiation resistance
Radiation resistance
Radiation reactivity
Unknown
Unknown
Unknown
Unknown

As shown in Table 2, the eight kinds of proteins include four kinds of proteins (PAI-2, FASN, VIM, GRP78 and UQCRC1) NOMO2, KLC4 and PLOD3).

Of the eight proteins, the association of the four proteins (PAI-2, NOMO2, KLC4 and PLOD3) with radiation performance has not been known at all and has been identified for the first time through the present invention.

Example 2-3: Western Blot analysis

The H460 cells and RR- # 2 cells prepared in Example 1-1 were tested for the presence of four kinds of proteins (PAI-2 , NOMO2, KLC4 or PLOD3) (Fig. 5C). At this time, HRP3 and? -Actin were used as internal control groups.

FIG. 5c is a Western blot analysis image showing the results of comparing the expression levels of PAI-2, NOMO2, KLC4 or PLOD3 in H460 cells and RR- # 2 cells. As shown in FIG. 5C, the PAI-2, NOMO2, KLC4, or PLOD3 proteins were all expressed at higher levels in RR- # 2 cells than H460 cells.

Example 3: PAI -2, NOMO2 , KLC4 or PLOD3 Of radiation tolerance

Example 3-1: PAI -2, NOMO2 , KLC4 or PLOD3 Of radiation-resistant cells

First, siRNA capable of inhibiting the expression of four kinds of proteins (PAI-2, NOMO2, KLC4 or PLOD3) which are not known to be associated with radiation-induced performance among the eight kinds of proteins identified in Example 2-2, Synthesized as follows:

siPAI-2: 5'-CAC UCU UUG CCC UCA AUU UUU-3 '(SEQ ID NO: 1)

siNOMO2: 5'-GCA GAU UAA UCA AUU UGA UUU-3 '(SEQ ID NO: 2)

siKLC4: 5'-CCA GAA UAA GUA UAA GGA AUU-3 '(SEQ ID NO: 3)

siPLOD3: 5'-GGA AGU ACA AGG AUG AUG AUU-3 '(SEQ ID NO: 4)

FBS using Lipofectamine RNAiMAX reagent ^® using, by the introduction of each of the synthesized siRNA in RR- # 2 cells prepared in Example 1-1 with each 50 nM separately four kinds of proteins (PAI-2, NOMO2, KLC4 Or PLOD3) expression was inhibited.

Example 3-2: PAI -2, NOMO2 , KLC4 or PLOD3 Analysis of Radiation-Induced Morphological Changes of Radiation-Resistant Cells with Inhibited Expression

Each of the transformants prepared in Example 3-1, the negative control into which nonspecific siRNA was introduced, and the positive control group into which siRNA (siHRP-3) capable of inhibiting the expression of HRP-3 were introduced were added with 0 or 10 Gy After culturing for 24 hours while irradiating with radiation, changes in the morphology of the cells were analyzed by an optical microscope (Fig. 6A).

FIG. 6A is a photomicrograph showing the results of comparing changes in cell morphology after culturing with irradiation of radiation-resistant cells in which the expression of PAI-2, NOMO2, KLC4 or PLOD3 was inhibited. As shown in FIG. 6A, the cells of the negative control group and the positive control group did not show any change in the cell shape after the irradiation, but the radiation-resistant cells in which the expression of PAI-2, NOMO2, KLC4 or PLOD3 was suppressed Of course, it was confirmed that apoptosis was induced even when radiation was not irradiated. In particular, it was confirmed that the progress of apoptosis accelerated when irradiated.

Example 3-3: Flow cell Through analysis Cell death rate analysis

Flow cytometry was performed on each cell obtained in Example 3-2, and the mortality rate of each cell was measured and compared (FIG. 6B). At this time, the negative control group and the positive control group were set the same as in Example 3-2.

FIG. 6B is a graph showing the results of comparing cell death rates measured by flow cytometry after culturing with irradiation of radiation-resistant cells in which the expression of PAI-2, NOMO2, KLC4 or PLOD3 was inhibited. As shown in FIG. 6B, the radiation-resistant cells in which the expression of PAI-2, NOMO2, KLC4 or PLOD3 was inhibited showed a higher apoptosis rate than the cells in the negative control and positive control irrespective of irradiation, It was confirmed that the cell death rate was further increased.

Example 3-4: Western Blot Through analysis Apoptosis Marker Protein (truncated PARP ) Expression levels

The extracts of RR- # 2 cells cultured with or without irradiation of 10 Gy of each of the siRNAs synthesized in Example 3-1 or not, Western blot analysis was performed using antibodies of the inhibited protein and antibodies of the cleaved PARP (Figure 6c). At this time, β-actin was used as an internal control group.

FIG. 6C is a graph showing changes in the expression levels of PAI-2, NOMO2, KLC4 or PLOD3 in the RR- # 2 cells cultured without or with 10 Gy of each siRNA, Western blot analysis showing the expression level of the protein inhibited by each siRNA and the expression level of cleaved PARP. As shown in FIG. 6C, the expression of each protein is inhibited by the introduction of siRNA regardless of irradiation, the expression of each protein is inhibited, and when the radiation is irradiated, the expression level of cleaved PARP is rapidly increased Respectively.

(PAI-2, NOMO2, KLC4, or PLOD3), which is not known to have an association with the performance in radiation, inhibits apoptosis induced by irradiation, To the cells.

Thus, by measuring the expression levels of four kinds of proteins (PAI-2, NOMO2, KLC4 or PLOD3) for which the correlation between the performance in radiation is first confirmed through the present invention, the cancer cells expressing the gene are resistant to radiation The present invention can be used as an active ingredient of a pharmaceutical composition for the treatment of radiation tolerance cancer.

<110> KOREA INSTITUTE OF RADIOLOGICAL & MEDICAL SCIENCES <120> Pharmaceutical composition for treating cancer having 라오르 레스 비 <130> KPA150540-KR <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siPAI-2 <400> 1 cacucuuugc ccucaauuuu u 21 <210> 2 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siNOMO2 <400> 2 gcagauuaau caauuugauu u 21 <210> 3 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siKLC4 <400> 3 ccagaauaag uauaaggaau u 21 <210> 4 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siPLOD3 <400> 4 ggaaguacaa ggaugaugau u 21

Claims

(2) is selected from the group consisting of PAI-2 (Plasminogen activator inhibitor-2), NOMO2 (NODAL modulator 2), KLC4 (Kinesin light chain 4), PLOD3 (Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3) A pharmaceutical composition for treating radiation-resistant cancer, which comprises an agent for inhibiting expression or activity of a protein or a gene encoding the protein.

The method according to claim 1,
Wherein the agent is an antibody, an aptamer, or an antagonist that inhibits the activity of a protein.

The method according to claim 1,
Wherein the agent is a miRNA, siRNA or shRNA that inhibits expression or activity of the gene.

The method according to claim 1,
The above agent is an siRNA of SEQ ID NO: 1 inhibiting the expression of PAI-2, an siRNA of SEQ ID NO: 2 inhibiting the expression of NOMO2, siRNA of SEQ ID NO: 3 inhibiting the expression of KLC4, Of siRNA. &Lt; / RTI >

The method according to claim 1,
Wherein said radiation resistant cancer is lung cancer, cervical cancer, colon cancer or breast cancer which is resistant to radiation therapy.

The method according to claim 1,
Wherein said radiation resistant cancer is non-small cell lung cancer that is resistant to radiation therapy.

The method according to claim 1,
Wherein said pharmaceutical composition is administered in combination with radiation upon treatment of radiation resistant cancer.

The method according to claim 1,
Wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient or diluent.

Wherein the composition is capable of measuring the level of a protein selected from the group consisting of PAI-2, NOMO2, KLC4, PLOD3, and combinations thereof, or an expression level of a gene encoding the protein.

10. The method of claim 9,
Wherein the agent is an antibody, an aptamer or an antagonist capable of specifically binding to the protein.

10. The method of claim 9,
Wherein the agent is a primer used for measuring the level of mRNA expressed from the gene.

A diagnostic kit for a radiation-tolerant cancer comprising the diagnostic composition of claim 9.

The diagnostic composition of claim 9 or the diagnostic kit of claim 12 can be used to measure the level of a protein selected from the group consisting of PAI-2, NOMO2, KLC4, PLOD3, and combinations thereof or the expression level of a gene encoding the protein Wherein the method comprises the steps of:

14. The method of claim 13,
The method comprising: (a) measuring the level of a protein selected from the group consisting of PAI-2, NOMO2, KLC4, PLOD3, and combinations thereof from a sample isolated from an individual suspected of developing radiation resistant cancer; (b) measuring the level of a protein selected from the group consisting of PAI-2, NOMO2, KLC4, PLOD3 and combinations thereof from a sample isolated from a normal subject; And (c) comparing the levels of the proteins measured in steps (a) and (b).

14. The method of claim 13,
The method comprises the steps of (a) measuring the mRNA level of a gene encoding a protein selected from the group consisting of PAI-2, NOMO2, KLC4, PLOD3 and combinations thereof from a sample isolated from an individual suspected of having a radiation- step; (b) measuring the mRNA level of a gene encoding a protein selected from the group consisting of PAI-2, NOMO2, KLC4, PLOD3 and combinations thereof from a sample isolated from a normal individual; And (c) comparing the levels of mRNA measured in steps (a) and (b).