KR20170005309A - Development of Dignosting Marker for Cisplatin-Resistant comprising GFRA1 as Active Ingredient and Method for Molecular Screening Cisplatin-Resistant Drug - Google Patents

Abstract

The present invention relates to a molecular screening method for developing a marker for diagnosing cisplatin resistance and prognosis of an osteosarcoma anti-cancer drug comprising GFRA1 as active component, and for developing cisplatin-resistant medicine and composition. In the present invention, a criteria for judging the prognosis of cisplatin treatment for a cancer patient is suggested, and a method for screening cisplatin adjurvants which can maximize the efficacy of cancer treatment by activating autophagy of cisplatin is provided.

Description

TECHNICAL FIELD The present invention relates to a method for the diagnosis of osteosarcoma anticancer cisplatin resistance and prognosis diagnosis using GFRA1 as an active ingredient, and a molecular screening technique for the development of a cisplatin resistant therapeutic agent and composition. The present invention also relates to a method for screening cisplatin-resistant GFRA1 as an active ingredient for Molecular Screening Cisplatin- Resistant Drug}

The present invention relates to a marker for the diagnosis and prognosis of ovarian cancer anticancer drug cisplatin resistant to GFRA1 as an active ingredient, and a molecular screening technique for developing a cisplatin resistant therapeutic agent and composition.

Osteosarcoma is the predominant form of early bone cancer. These osteosarcomas are usually malignant tumors that occur in childhood and adolescence. ¹ The use of chemotherapy with surgery increases the long-term survival rate of osteosarcoma patients by about 70%. ^2,3 Osteosarcoma is an anticancer drug commonly used as doxorubicin, cisplatin, and methotrexate. In particular, cisplatin is the most widely used platinum-based anticancer drug for solid tumors including osteosarcoma. ⁴ interacts with the nucleophilic N7 site of the purine base in DNA and induces DNA damage to inhibit tumor cell division and induce apoptosis or initiation of prognostic cell death. ⁵ These methods of treatment have high efficiency but are limited to acquired or intrinsic tolerance of cancer cells to anticancer agents. ⁶ Therefore, in order to develop more effective treatments for osteosarcoma, it is necessary to understand the molecular mechanisms that induce anticancer drug resistance.

Autophagy is an important process for the self digestion of cells to maintain homeostasis under metabolic stress and the recycling of unnecessary or ineffective cellular components. ^7,8 During the early stages of childhood, cell proteins, organelles and cytoplasm are isolated and surrounded by autophagosomes. Next, the canine fuses with the lysosome to form the autolysosome, and the isolated proteins and organs are degraded by the lysosomal hydrolase. ^9,10 Childhood plays an important role in cell death, such as apoptosis-deficient cells, an alternative cell death mechanism known as the prospective cell death type II, and can function as a tumor suppressor through this mechanism. ^11-13 Paradoxically, autophagy is known to be involved in cell survival mechanisms induced by environmental stresses such as poor nutrition, chemotherapy, radiation and hypoxia. ^11,12,14 Several studies have shown that the induction of cell viability for cell survival gives resistance to chemotherapy in some cancers. ^15-19 However, the relative contribution of cell survival as an apoptotic cell death or survival-promoting mechanism as a tumor suppressive mechanism of the child action on cancer chemotherapy has not been fully elucidated.

GNDF (Glycosylphosphatidylinositol-linked glial cell line-derived neurotrophic factor) receptor α or GFRα is a common receptor that recognizes the GNDF family of ligands including GDNF, neurturin, artemin and persephin coreceptor. ^20,21 Four types of GFRa were identified. Each GFR [alpha] recognizes its GDNF ligand specifically. The binding of GFRa to the ligand activates the tyrosine kinase RET and then activates Src, one of the Src family of cytoplasmic tyrosine kinases. ²⁰ GDNF / GFR [alpha] signaling has the potential of therapeutic targets for neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease, as it protects and promotes the survival of dopaminergic neurons to regulate the development and maintenance of the nervous system. ^22,23 GFRα1 specifically recognizes GDNF, which is involved in the regulation of neural cell survival and differentiation. Several studies have shown that the precise tumorigenic mechanism is not clear, but GFRα1 promotes metastasis and penetration, thus promoting human cancer, such as breast and pancreatic cancer. ^24-27

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have sought to understand the molecular mechanism of inducing anticancer drug resistance in patients with osteosarcoma, a rare cancer. As a result, it was confirmed that GFRα1 of osteosarcoma patients is involved in anticancer drug resistance and promotes the survival mechanism of cisplatin-induced autophagy. Thus, GFRα1 can diagnose susceptibility to cisplatin treatment in osteosarcoma patients The present invention has been completed.

Accordingly, an object of the present invention is to provide a method for determining susceptibility to cisplatin.

Another object of the present invention is to provide a kit for predicting susceptibility to cisplatin in a cancer patient.

It is still another object of the present invention to provide a screening method of cisplatin adjuvant.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a method for determining susceptibility to cisplatin comprising the steps of:

(a) separating a biological sample from a cancer patient;

(b) determining the expression level of the GFR alpha 1 gene or protein in the biological sample, wherein the degree of expression of the GFR alpha 1 gene or protein is down-regulated ) Is judged to be susceptible to cisplatin, and when the degree of expression of the GFR [alpha] l gene or protein is up-regulated from that of normal cells, it is determined that cisplatin resistance is present.

The present inventors have sought to understand the molecular mechanism of inducing anticancer drug resistance in patients with osteosarcoma, a rare cancer. As a result, it was confirmed that GFR [alpha] l of osteosarcoma patients is involved in anticancer drug resistance and promotes the survival mechanism of autophagy by cisplatin, so that GFR [alpha] l can diagnose susceptibility to cisplatin treatment in osteosarcoma patients.

Methods of cancer treatment are classified into surgical resection, chemotherapy, and radioactivetherapy. Chemotherapy, which is represented by chemotherapy, is one of the most important therapeutic methods for the treatment of tumors with surgery. Various chemotherapeutic agents have been developed to improve the therapeutic response rate and decrease the side effects. Despite the high therapeutic response rates of anticancer drugs, the inherent / inherent resistance of cancer cells to anticancer drugs has become a problem and understanding of the molecular mechanisms leading to anticancer drug resistance has emerged as an important task. Accordingly, the present inventors have identified GFR [alpha] l, a gene capable of predicting the susceptibility to cisplatin for solving such a problem.

The method for determining susceptibility to cisplatin of the present invention will be described step by step.

Step (a): Separation of biological samples

First, the biological sample is separated from the cancer patient.

According to one embodiment of the present invention, the cancer is selected from the group consisting of osteosarcoma, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, colon adenocarcinoma, prostate carcinoma, The present invention relates to a method for the treatment of endotheliosarcoma (for example, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, renal cell carcinoma, hepatocellular carcinoma, biliary cancer, fibrosarcoma, mucosal sarcoma, liposarcoma, chondrosarcoma, Lymphangioendotheliosarcoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, rhabdomyosarcoma, glandular carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, papillary adenocarcinoma, papillary adenocarcinoma, Cystadenocarcinoma, adenocarcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, squamous cell carcinoma, pineal gland carcinoma, angiomyoblastoma, cervical carcinoma , Ectopic glioma, meningioma, neuroblastoma, retinoblastoma, myeloma, lymphoma or leukemia.

According to another embodiment of the present invention, the cancer is osteosarcoma.

The present invention separates a biological sample from an osteosarcoma patient.

According to an embodiment of the present invention, the biological sample may be a tissue, a cell, a blood, a lymph, a bone marrow fluid, a saliva, a milk, a urine, a feces, an ocular fluid, a semen, a homogenate of a cerebrospinal fluid, Amniotic membrane fluid and cell tissue fluid.

According to another embodiment of the present invention, the biological sample is a tissue or a cell.

According to a particular embodiment of the invention, the biological sample is an osteosarcoma tissue or an osteosarcoma cell.

Step (b): Determination of expression of GFRα1 gene or protein

Next, whether or not the gene or protein of GFR? 1 (glial cell line-derived neurotrophic factor receptor? 1) of the biological sample is expressed is determined.

According to one embodiment of the present invention, expression of the GFRα1 gene or protein may be determined by hybridization, gene amplification, or immunoassay.

The hybridization method confirms the presence of GFR [alpha] l using a probe.

As used herein, the term " probe " refers to a linear oligomer of natural or modified monomer or linkages and includes deoxyribonucleotides and ribonucleotides and can specifically hybridize to a target nucleotide sequence, Present or artificially synthesized. The probe of the present invention is preferably a single strand, and is an oligodioxyribonucleotide.

In the microarray of the present invention, the probe is used as a hybridizable array element and immobilized on a substrate. Preferred gases include, for example, membranes, filters, chips, slides, wafers, fibers, magnetic beads or non-magnetic beads, gels, tubing, plates, polymers, microparticles and capillaries, as suitable rigid or semi-rigid supports. The hybridization array elements are arranged and immobilized on the substrate. Such immobilization is carried out by a chemical bonding method or a covalent bonding method such as UV. For example, the hybridization array element may be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and may also be bound by UV on a polylysine coating surface. In addition, the hybridization array element may be coupled to the gas through a linker (e.g., ethylene glycol oligomer and diamine).

On the other hand, the sample DNA to be applied to the microarray of the present invention can be labeled and hybridized with the array elements on the microarray. Hybridization conditions can be varied. The detection and analysis of the hybridization degree can be variously carried out according to the labeling substance.

The method for determining susceptibility to cisplatin of the present invention can be carried out based on hybridization. In this case, a probe having a sequence complementary to the sequence of SEQ ID NO: 5, which is a nucleotide sequence encoding GFRα1 of the present invention described above, is used.

Hybridization-based analysis using the probe hybridized to the nucleotide sequence encoding GFR [alpha] l of the present invention described above can be used to determine susceptibility to cisplatin.

The label of the probe may provide a signal to detect hybridization, which may be linked to an oligonucleotide. Suitable labels include fluorescent moieties (e.g., fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5 (Pharmacia)), chromophores, chemiluminescent moieties, magnetic particles, (P ³² and S ³⁵ ), mass labels, electron dense particles, enzymes (alkaline phosphatase or horseradish peroxidase), joins, substrates for enzymes, heavy metals such as gold and antibodies, streptavidin , Haptens with specific binding partners such as biotin, digoxigenin and chelating groups. Markers can be generated using a variety of methods routinely practiced in the art such as the nick translation method, the Multiprime DNA labeling systems booklet (Amersham, 1989) and the kaination method (Maxam & Gilbert, Methods in Enzymology , 65: 499 (1986)). The label provides signals that can be detected by fluorescence, radioactivity, colorimetry, weighing, X-ray diffraction or absorption, magnetism, enzymatic activity, mass analysis, binding affinity, hybridization high frequency, and nanocrystals.

The nucleic acid sample to be analyzed can be prepared using mRNA obtained from various biosamples. The raw sample is preferably a cancer cell. Instead of the probe, the cDNA to be analyzed may be labeled and subjected to a hybridization reaction-based analysis.

When a probe is used, the probe is hybridized with the cDNA molecule. In the present invention, suitable hybridization conditions can be determined by a series of procedures by an optimization procedure. This procedure is performed by a person skilled in the art in a series of procedures to establish a protocol for use in the laboratory. Conditions such as, for example, temperature, concentration of components, hybridization and washing time, buffer components and their pH and ionic strength depend on various factors such as probe length and GC amount and target nucleotide sequence. The detailed conditions for hybridization are described in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); And MLM Anderson, Nucleic Acid Hybridization , Springer-Verlag New York Inc .; NY (1999). For example, high stringency conditions were hybridized at 65 ° C in 0.5 M NaHPO ₄ , 7% SDS (sodium dodecyl sulfate) and 1 mM EDTA, followed by addition of 0.1 x SSC / 0.1% SDS Lt; RTI ID = 0.0 > 68 C < / RTI > Alternatively, high stringency conditions means washing at < RTI ID = 0.0 > 48 C < / RTI > in 6 x SSC / 0.05% sodium pyrophosphate. Low stringency conditions mean, for example, washing in 0.2 x SSC / 0.1% SDS at 42 ° C.

After the hybridization reaction, a hybridization signal generated through the hybridization reaction is detected. The hybridization signal can be carried out in various ways depending on, for example, the type of label attached to the probe. For example, when a probe is labeled with an enzyme, the substrate of the enzyme can be reacted with the result of hybridization reaction to confirm hybridization. Combinations of enzymes / substrates that may be used include, but are not limited to, peroxidases (such as horseradish peroxidase) and chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis- Acetyl-3,7-dihydroxyphenox), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2) , 2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o -phenylenediamine (OPD) and naphthol / pyronin; (BCIP), nitroblue tetrazolium (NBT), naphthol-AS-B1-phosphate, and ECF substrate; alkaline phosphatase and bromochloroindoleyl phosphate; Glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS (phenzaine methosulfate). When the probe is labeled with gold particles, it can be detected by a silver staining method using silver nitrate. Therefore, when the method for determining susceptibility to cisplatin according to the present invention is carried out based on hybridization, specifically (i) hybridizing a probe having a sequence complementary to the nucleotide sequence encoding GFR [alpha] l of the present invention to a nucleic acid sample; (ii) detecting whether the hybridization reaction has occurred. By analyzing the intensity of the hybridization signal by the hybridization process, it is possible to judge whether the cisplatin is susceptible or not. That is, when the hybridization signal for the nucleotide sequence encoding GFRα1 of the present invention is down-regulated from the normal sample (for example, normal cells), the sample is judged to be cisplatin-susceptible, and the hybridization signal If it is up-regulated rather than normal, it is judged to be resistant to cisplatin.

According to a preferred embodiment of the present invention, the present invention can be embodied by a hybridization method. In this case, a probe that specifically binds to the nucleotide encoding GFR [alpha] l of the present invention is used.

The method for determining susceptibility to cisplatin of the present invention can be carried out by gene amplification.

The term " amplification " as used herein refers to a reaction that amplifies a nucleic acid molecule. A variety of amplification reactions have been reported in the art, including polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202 and 4,800,159), reverse-transcription polymerase chain reaction (RT-PCR) (Sambrook et al., Molecular Cloning. (LCR) (see, for example, A Laboratory Manual , 3rd Ed. Cold Spring Harbor Press (2001)), Miller, HI (WO 89/06700) and Davey, C. et al (EP 329,822) 17,18), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA, WO 88/10315) (SEQ ID NO: 1), self-sustained sequence replication (WO 90/06995), selective amplification of target polynucleotide sequences (US Patent No. 6,410,276), consensus sequence primed polymerase chain reaction CP-PCR), U.S. Patent No. 4,437,975), random plasmids (AP-PCR), U.S. Patent Nos. 5,413,909 and 5,861,245), nucleic acid sequence based amplification (NASBA), U.S. Patent No. 5,130,238, 5,409,818, 5,554,517, and 6,063,603), strand displacement amplification (21,22), and loop-mediated isothermal amplification. (LAMP) 23, but is not limited thereto. Other amplification methods that may be used are described in U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617 and U.S. Patent No. 09 / 854,317.

PCR is the most well-known nucleic acid amplification method, and many variations and applications thereof have been developed. For example, touchdown PCR, hot start PCR, nested PCR and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR. In addition, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction chain reaction (IPCR), vectorette PCR and thermal asymmetric interlaced PCR (TAIL-PCR) have been developed for specific applications. For more information on PCR, see McPherson, MJ, and Moller, SG PCR . BIOS Scientific Publishers, Springer-Verlag New York Berlin, Heidelberg, NY (2000), the teachings of which are incorporated herein by reference.

When the diagnostic method of the present invention is carried out using a primer, the gene amplification reaction is carried out to examine the degree of expression of the nucleotide sequence of GFRα1 of the present invention. Since the present invention analyzes the degree of expression of the nucleotide sequence of GFR alpha 1 of the present invention, the amount of mRNA of the GFR alpha 1 nucleotide sequence of the present invention is examined in a sample (for example, a cancer cell) to be analyzed and the nucleotide sequence of GFR alpha 1 of the present invention And the like.

Therefore, in principle, the present invention uses a mRNA in a sample as a template and performs a gene amplification reaction using a primer that binds to mRNA or cDNA.

To obtain mRNA, total RNA is isolated from the sample. The isolation of total RNA can be carried out according to conventional methods known in the art (see Sambrook, J. et al., Molecular Cloning , A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001); Tesniere , C. et al, Plant Mol Biol Rep, 9:..... 242 (1991); Ausubel, FM et al, Current Protocols in Molecular Biology, John Willey & Sons (1987); and Chomczynski, P. et al Anal. Biochem . 162: 156 (1987)). For example, TRIzol can be used to easily isolate total RNA in a cell. Next, cDNA is synthesized from the separated mRNA, and this cDNA is amplified. Since the total RNA of the present invention is isolated from a human sample, it has a poly-A tail at the end of the mRNA, and the cDNA can be easily synthesized using the oligo dT primer and the reverse transcriptase using such a sequence characteristic ( reference: PNAS USA, 85: 8998 ( 1988); Libert F, et al, Science, 244:.... 569 (1989); and Sambrook, J. et al, Molecular Cloning A Laboratory Manual, 3rd ed Cold Spring Harbor Press (2001)). Next, the synthesized cDNA is amplified through gene amplification reaction.

The primer used in the present invention is hybridized or annealed at one site of the template to form a double-stranded structure. Conditions suitable nucleic acid hybridization to form such double-stranded structure is Joseph Sambrook, such as, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, etc., Nucleic Acid Hybridization , A Practical Approach , IRL Press, Washington, DC (1985).

A variety of DNA polymerases can be used in the amplification of the present invention, including the " Clenow " fragment of E. coli DNA polymerase I, the thermostable DNA polymerase and the bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtainable from a variety of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis , Thermis flavus , Thermococcus literalis , and Pyrococcus furiosus (Pfu).

When performing the polymerization reaction, it is preferable to provide the reaction vessel with an excessive amount of the components necessary for the reaction. The excess amount of the components required for the amplification reaction means an amount such that the amplification reaction is not substantially restricted to the concentration of the component. To provide joinja, dATP, dCTP, dGTP and dTTP, such as Mg ^{+ 2} to the reaction mixtures to have a desired degree of amplification can be achieved is required. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, buffers make all enzymes close to optimal reaction conditions. Therefore, the amplification process of the present invention can be carried out in a single reaction without changing the conditions such as the addition of reactants.

In the present invention, annealing is carried out under stringent conditions that allow specific binding between the target nucleotide sequence and the primer. The stringent conditions for annealing are sequence-dependent and vary with environmental variables.

The thus amplified cDNA of the nucleotide sequence of GFR? 1 of the present invention is analyzed by a suitable method to examine the degree of expression of the nucleotide sequence of GFR? 1 of the present invention. For example, the result of amplification reaction described above is subjected to gel electrophoresis, and the resulting band is observed and analyzed to examine the degree of expression of the nucleotide sequence of GFRα1 of the present invention. Through the amplification reaction, when the expression of the nucleotide sequence of GFR [alpha] l of the present invention is down-regulated in normal cancer cells (normal cells) in cancer cells isolated from the in vitro, it is judged that cisplatin is susceptible and the expression is normal If the cell is up-regulated, it is judged to be cisplatin resistant.

Therefore, the method of determining susceptibility to cisplatin according to the present invention comprises: (i) performing an amplification reaction using a primer annealed to the nucleotide sequence of GFR? 1 of the present invention when performing the amplification reaction using cDNA; And (ii) analyzing the product of the amplification reaction to determine the degree of expression of the nucleotide sequence of GFR [alpha] l of the present invention.

According to a preferred embodiment of the present invention, the susceptibility determination method for cisplatin of the present invention can be carried out by gene amplification using primers of Sequence Listing 1 or Sequence Listing 4 as a primer.

The method of determining susceptibility to cisplatin of the present invention can be carried out by immunoassay.

The antibody used in the present invention is a polyclonal or monoclonal antibody, preferably a monoclonal antibody. Antibodies can be produced using methods commonly practiced in the art, such as the fusion method (Kohler and Milstein, European Journal of Immunology , 6: 511-519 (1976)), the recombinant DNA method (US Patent No. 4,816,56) 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)). General procedures for antibody preparation are described in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual , Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques , CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY , Wiley / Greene, NY, 1991, the disclosures of which are incorporated herein by reference. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing an immortalized cell line with an antibody-producing lymphocyte, and the techniques necessary for this process are well known and readily practicable by those skilled in the art. Polyclonal antibodies can be obtained by injecting a protein antigen into a suitable animal, collecting the antiserum from the animal, and then separating the antibody from the antiserum using a known affinity technique.

When the method of the present invention is carried out using an antibody, the present invention can be carried out according to a conventional immunoassay method and used to diagnose susceptibility to cisplatin.

Such immunoassays can be performed according to various quantitative or qualitative immunoassay protocols developed in the past. The immunoassay format may include, but is not limited to, radioimmunoassays, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or hardwood analysis, sandwich analysis, flow cytometry, But are not limited to, fluorescent staining and immunoaffinity purification. Methods of immunoassay or immunostaining are described in Enzyme Immunoassay , ET Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology , Vol. 1, Walker, JM ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.

For example, if the method of the present invention is carried out according to the method radioactive immunoassay, a radioactive isotope to the antibody labeling (e. G., C ^14, I ^125, P ³² and S ³⁵⁾ detects a GFRα1 of the invention Can be used.

When the method of the present invention is carried out by an ELISA method, a specific embodiment of the present invention comprises the steps of (i) coating the surface of a solid substrate with an unknown cell sample lysate to be analyzed; (Ii) reacting the cell lysate with an antibody to GFR [alpha] l as a primary antibody; (Iii) reacting the result of step (ii) with an enzyme-conjugated secondary antibody; And (iv) measuring the activity of the enzyme.

Suitable as said solid substrate are hydrocarbon polymers (e.g., polystyrene and polypropylene), glass, metal or gel, and most preferably microtiter plates.

The enzyme bound to the secondary antibody may include an enzyme catalyzing a chromogenic reaction, a fluorescence reaction, a luminescent reaction, or an infrared reaction, but is not limited thereto. For example, an alkaline phosphatase,? -Galactosidase, Radish peroxidase, luciferase, and cytochrome P ₄₅₀ . In the case where alkaline phosphatase is used as an enzyme binding to the secondary antibody, it is preferable to use, as a substrate, bromochloroindole phosphate (BCIP), nitroblue tetrazolium (NBT), naphthol-AS -Bl-phosphate and ECF (enhanced chemifluorescence) are used. When horseradish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (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 naphthol / pie Ronin, glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS a substrate such as phenzaine methosulfate may be used All.

When the method of the present invention is carried out in the Capture-ELISA mode, a specific embodiment of the present invention comprises: (i) coating an antibody against GFR [alpha] l of the present invention as a capturing antibody on the surface of a solid substrate; (Ii) reacting the capture antibody with a cell sample; (Iii) reacting the result of step (ii) with a detecting antibody which is labeled with a signal generating label and specifically reacts with MLN 51 protein; And (iv) measuring a signal originating from said label.

The detection antibody has a label that generates a detectable signal. The label may be a chemical (e.g., biotin), an enzyme (alkaline phosphatase,? -Galactosidase, horseradish peroxidase and cytochrome P ₄₅₀ ), a radioactive material (such as C ¹⁴ , I ¹²⁵ , P ³² And S ³⁵ ), fluorescent materials (e.g., fluorescein), luminescent materials, chemiluminescent materials, and fluorescence resonance energy transfer (FRET). Various labels and labeling methods are described in Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press,

In the ELISA method and the capture-ELISA method, measurement of the activity of the final enzyme or measurement of the signal can be performed according to various methods known in the art. This signal detection enables qualitative or quantitative analysis of GFR [alpha] 1 of the present invention. If biotin is used as a label, it can be easily detected by streptavidin. When luciferase is used, luciferin can easily detect a signal.

The susceptibility to cisplatin can be determined by analyzing the intensity of the final signal by the above-described immunoassay. That is, when the signal for GFR alpha 1 of the present invention is down-regulated in the sample, the signal is judged to be cisplatin-susceptible and the signal is judged to be resistant to cisplatin when the signal is up-regulated above the normal cell.

The method of the present invention may additionally include other components in addition to the above components. For example, if the method of the present invention is applied to a PCR amplification procedure, the kit of the present invention may optionally include reagents necessary for PCR amplification, such as buffers, DNA polymeraseThermus aquaticus (Taq),Thermus thermophilus (Tth),Thermus filiformis,Thermis flavus,Thermococcus literalis orPyrococcus furiosus (Thermostable DNA polymerase from Pfu), DNA polymerase joins and dNTPs.

The GFR? 1 of the present invention is a biomolecule that is low expressed in an object susceptible to cisplatin. Such low expression of GFR [alpha] l can be measured at the mRNA or protein level. As used herein, the term " low expression " means that the expression level of a nucleotide sequence to be a subject in a sample to be irradiated is lower than that of a normal sample. For example, expression analysis methods conventionally used in the art, such as RT-PCR or ELISA methods (see Sambrook, J. et al., Molecular Cloning , A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001) In the case of expression analysis, it means that the expression is analyzed to be low. For example, when analyzed according to the above-described assay method, when the GFR [alpha] 1 of the present invention is low expressed about 2 to 20 times as much as that of normal cells, it is judged as " low expression " .

According to another aspect of the present invention there is provided a kit for predicting susceptibility to cisplatin in a cancer patient comprising a nucleotide sequence encoding GFR [alpha] l, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide sequence, or an antibody specific for GFR [ .

According to one embodiment of the present invention, the nucleotide sequence is a nucleotide sequence represented by the genbank accession number NM_005264 (SEQ ID NO: 5).

The susceptibility prediction kit for cisplatin in the cancer patient is a hybridization kit, a gene amplification kit, or an immunoassay kit.

Since the kits of the present invention utilize the susceptibility determination method for cisplatin, the description common to both is omitted in order to avoid the excessive complexity of the present specification.

According to another aspect of the present invention, the present invention provides a screening method of cisplatin adjuvant that inhibits the resistance of cisplatin to cancer cells, comprising the steps of:

(a) contacting a candidate substance with a cell comprising GFR [alpha] l;

(b) determining the level of GFR [alpha] 1 expression in the cell, and determining that the GFR [alpha] l expression is down-regulated relative to the control, the cisplatin adjuvant.

The present invention demonstrates that GFR [alpha] l is involved in the anticancer drug resistance of osteosarcoma patients and promotes the survival mechanism of cisplatin-induced child action. Specifically, GFR [alpha] l overexpressed by cisplatin phosphorylates Src, and phosphorylated Src phosphorylates AMPK, initiating the child's action.

The screening method of the cisplatin adjuvant will be described step by step.

Step (a): contacting the cisplatin and the candidate substance

First, cisplatin and the candidate substance are contacted with cells having the somatic signaling pathway.

As used herein, the term " candidate substance " refers to an unknown substance used in screening to test whether it affects the inactivation of the child ' s signaling pathway. Such samples include, but are not limited to, chemicals, nucleotides, antisense-RNA, siRNA (small interference RNA) and natural extracts.

Step (b): Measurement of GFR? 1 expression level

Next, measurement of GFR [alpha] l expression level of the cell is judged as a cisplatin adjuvant when the GFR [alpha] l expression is down-regulated as compared with the control group treated with only cisplatin.

Expression of GFR [alpha] l may be carried out using the hybridization method, gene amplification method or immunoassay described above.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a method for determining susceptibility to cisplatin, a kit for predicting susceptibility to cisplatin in cancer patients, and a screening method for cisplatin adjuvant.

(b) The present invention provides a criterion for determining the cisplatin therapeutic prognosis in cancer patients.

(c) The present invention provides a method for screening cisplatin adjuvant capable of maximizing anticancer effect by activating autophagy of cisplatin.

(d) The present invention enables a patient-customized treatment by judging in advance whether or not the anticancer drug resistance is possible.

FIGS. 1A through 1E show induction of GFRα1 expression by cisplatin in osteosarcoma cells. FIGS. 1A through 1C show immunoblot analysis of osteosarcoma cell lysates using antibodies specific for GFRα1 and β-actin. FIG. Figure 1a shows MG-63 and U-2 OS cells treated with doxorubicin (5 [mu] M), cisplatin (20 [mu] M) or methotrexate (1 mM) for 24 hours. Immunoblot analysis of GFR [alpha] l (left) and quantification (right) results of GFR [alpha] l expression after treatment of the chemotherapeutic agent. Figure 5b shows MG-63 and U-2 OS cells treated with various concentrations of cisplatin for 24 hours. Figure 1c shows MG-63 and U-2 OS cells treated with 20 [mu] M cisplatin and collected at designated times. Figures 1d and 1e show representative images (above) and quantitative analysis (below) of GFR [alpha] l mRNA expression as a result of quantitative real-time PCR of GFR [alpha] l mRNA expression after cisplatin treatment. Values are expressed as means ± SD (n = 3), ** means p <0.05 by t-test. Figure 1d shows MG-63 and U-2 OS cells treated with various concentrations of cisplatin for 24 hours. Fig. 1e shows MG-63 and U-2 OS cells treated with 20 [mu] M cisplatin and collected at designated times.
Figures 2a to 2h are results showing inhibition of cisplatin-induced osteosarcoma cell chemosensitivity by GFR [alpha] l. 2A shows the preparation of GFR [alpha] l-deficient osteosarcoma cell line using GFR [alpha] l shRNA. MG-63 and U-2 OS cells were transfected with either control or GFR [alpha] l shRNA. Control and GFRa1-deficient stable osteosarcoma cell lysates were immunoblot analyzed using antibodies specific for GFR [alpha] l and [beta] -actin. FIG. 2b shows the cell viability of GFRα1-deficient osteosarcoma cells after cisplatin treatment. Control or GFRa1-deficient cells were cultured and various concentrations of cisplatin were treated for 24 hours. Cell viability was measured using WST-1 assay. Values are expressed as means ± SD (n = 3), ** means p <0.05 by t-test. FIG. 2C shows the apoptotic response of GFRα1-deficient osteosarcoma cells after cisplatin treatment. Control and GFR [alpha] l-deficient cells were cultured and various concentrations of cisplatin were treated for 24 hours. Apoptotic cells (stomach) were stained with FITC-Annexin V staining and viable cells (bottom) were stained with PI staining. Values are expressed as means ± SD (n = 3), ** means p <0.05 by t-test. Figure 2D shows immunoblot analysis using antibodies specific for truncated PARP and truncated caspase-3, the apoptotic proteins of control and GFRa1-deficient cell lysates. Control and GFR [alpha] l-deficient MG-63 cells were treated with 20 [mu] M cisplatin for 24 hours and harvested. Figure 2e shows the relative caspase-3 activity of control and GFRa1-deficient MG-63 cells after cisplatin treatment. Control and GFRalpha1-deficient cells were cultured and cisplatin at various concentrations was treated for 24 hours with or without 50 [mu] M ZVAD-FMK. Values are expressed as means ± SD (n = 3), ** means p <0.05 by t-test. FIG. 2f shows the preparation of GFRα1-overexpressing osteosarcoma cells using the GFRα1 expression vector. MG-63 and U-2 OS cells were transfected with a control or human GFR [alpha] l expression vector. The immunoassay results of the control group using antibodies specific for GFR [alpha] l and [beta] -actin and the GFR [alpha] l-overexpressing stable osteosarcoma cell lysate are shown. Figure 2g shows the cell viability of GFRa1-overexpressing osteosarcoma cells after cisplatin treatment. Control and GFR [alpha] l-deficient cells were cultured and various concentrations of cisplatin were treated for 24 hours. Cell viability was measured using WST-1 assay. Values are expressed as means ± SD (n = 3), ** means p <0.05 by t-test. Figure 2h shows the apoptotic response of GFRa1-overexpressing osteosarcoma cells following cisplatin treatment. Control and GFR [alpha] l-deficient cells were cultured and various concentrations of cisplatin were treated for 24 hours. Cell viability was measured using WST-1 assay. Apoptotic cells (stomach) were stained with FITC-Annexin V staining and viable cells (bottom) were stained with PI staining. Values are expressed as means ± SD (n = 3), ** means p <0.05 by t-test.
Fig. 3 shows the improvement of anticancer drug resistance of cisplatin-induced osteosarcoma cells by GFR [alpha] l -induced child action. Figure 3a shows transient transfection of the control and GFR [alpha] l-deficient MG-63 cells with the mRFP-GFP tandem fluorescent-tagging LC3 (mRFP-GFP-LC3) vector and cisplatin (20 [mu] M) for 24 h. On the left, representative images of RFP-LC3 and GFP-LC3 points, the scale bar is 20 μm. Right, the number of yellow dots in the combined image, and the number of RFP-LC3. Values are expressed as means ± SD (n = 3), ** means p <0.05 by t-test. Figure 3b shows that the control and GFR [alpha] l-deficient MG-63 cells were treated with cisplatin (20 [mu] M) for 24 hours and stained with acridine orange. The representative image of cells stained with acridine orange stain is 20 μm in scale bar. The results of quantitative analysis of the number of AVOs are shown below. Values are expressed as means ± SD (n = 3), ** means p <0.05 by t-test. Figure 3c shows cefplatin (20 [mu] M) treated with control and GFR [alpha] l-deficient MG-63 cells for 24 hours and analyzed by TEM. The representative image of the child's action vacuole (yellow arrow) detected in the stomach and control MG-63 cells after treatment with cisplatin, the scale bar is 100 μm. The results of quantitative analysis of the number of child actinic vacuoles are shown below. Values are expressed as means ± SD (n = 3), ** means p <0.05 by t-test. Figure 3d shows immunoblot analysis using antibodies specific for GFR [alpha] l, LC3 and [beta] -actin of control and GFR [alpha] l-overexpressing osteosarcoma cell lysate. GFRα1-overexpressing MG-63 cells were treated with DMSO, 3-MA (10 μM) or Baf (100 nM). Figure 3E shows quantitative analysis of the number of yellow points and number of RFP-LC3 points in the control and GFRa1-overexpressing MG-63 cells. Control and GFRα1-overexpressing MG-63 cells were transiently transfected with the mRFP-GFP tandem fluorescent-tagged LC3 (mRFP-GFP-LC3) vector and cisplatin (20 μM) was treated for 24 hours. FIG. 3f shows that the control and GFRα1-overexpressing MG-63 were pretreated with 3-MA (10 μM) for 2 hours before the mRPF-GFR-LC3 transfection and reacted for 24 hours. FIG. 3g shows that the control and GFRα1-overexpressing MG-63 cells were treated with cisplatin (20 μM) for 24 hours and stained with acridine orange. The representative image of cells stained with acridine orange stain is 20 μm in scale bar. The results of quantitative analysis of the number of AVOs are shown below. Values are expressed as means ± SD (n = 3), ** means p <0.05 by t-test. FIG. 3h shows that the control and GFPα1-overexpressing MG-63 cells were treated with cisplatin (20 μM) for 24 hours and analyzed by TEM. The scale bar is a representative image of the child's action vacuole detected after cisplatin treatment on stomach, control and GFRα1-overexpressing MG-63 cells, and the scale bar is 100 μm. The results of quantitative analysis of the number of child actinic vacuoles are shown below. Values are expressed as means ± SD (n = 3), ** means p <0.05 by t-test. Figure 3i shows the cell viability of GFR [alpha] l-overexpressing osteosarcoma cells following cisplatin peri in the presence of the inhibition of the offspring. Control or GFRα1-overexpressing MG-63 cells were cultured with DMSO, 3-MA or Baf and cisplatin (20 μM) was treated for 24 hours. Values are expressed as means ± SD (n = 3), ** means p <0.05 by t-test. Figure 3j shows the cell viability of GFRa1-overexpressing osteosarcoma cells following cisplatin treatment in the presence of Beclin1 (20 nM) or HMGB1 (20 nM) siRNA. Control or GFRα1-overexpressing MG-63 cells were cultured with Beclin1 or HMGB1 siRNA for 48 hours and treated with cisplatin (20 μM) for 24 hours. Cell viability was measured using WST-1 assay. Values are expressed as means ± SD (n = 3), ** means p <0.05 by t-test. Figure 3k shows the effect of GFR [alpha] l deficiency or overexpression in the presence of cisplatin (20 [mu] M) in colony formation. The control (control shRNA), GFRα1-deficient (GFRα1 shRNA), control (mitotic vector) and GFRα1-overexpressing (GFRα1 expression vector) MG-63 cells were plated to the same number (1 × 10 ⁴ cells). All cell lines were treated with cisplatin (20 [mu] M) for 10 days. Colonies were visualized by staining with crystal violet. The left is the representative image of the colony formation and the right is the quantitative relationship of the number of colonies and the plating efficiency. Values are expressed as means ± SD (n = 3), ** means p <0.05 by t-test. Figure 31 shows the effect of the inhibitory action on colony formation of GFR [alpha] l-over-expressing cells in the presence of cisplatin (20 [mu] M). The control and GFR [alpha] l-overexpressing clone # 4 were plated and treated with 3-MA (10 [mu] M), Baf (100 nM) or CQ (30 [mu] g / ml), respectively. All cell lines were treated with cisplatin for 10 days. All plates were scanned with a scanner and the number of colonies was quantified using image-J software. On the left is a representative image of colonization. Right, the number of colonies and the survival fraction. Values are expressed as means ± SD (n = 3), ** means p <0.05 by t-test.
Figures 4A-4G show the regulation of GFR [alpha] l by the Src / AMPK signaling in osteosarcoma cells. FIGS. 4A and 4B are graphs showing osteosarcoma cell lysate using an antibody specific for GFRα1, p-Src, Src, p-AMPK, AMPK, p-mTOR, mTOR, p-p70 S6Kinase, Beclin 1, HMGB1, Lt; / RTI &gt; Figure 4a shows that MG-63 and cells were treated with cisplatin (20 [mu] M) for 24 hours. Figure 4b shows that cisplatin (20 [mu] M) was treated for 24 hours in the control and GFR [alpha] l-deficient MG-63 cells. Figures 4c to 4e show immunoblot analysis of osteosarcoma cell lysates using antibodies specific for GFR [alpha] l, p-Src, Src, p-AMPK, AMPK, LC3 and beta -actin. Figure 4c shows that cisplatin (20 [mu] M) was treated for 24 hours in the control and Src-deficient MG-63 cells. Figure 4d shows that MG-63 cells were cultured with or without PPl (5 [mu] M) and treated with cisplatin (20 [mu] M) for 24 hours. 4E shows that MG-63 cells were cultured under Compound C treatment or no treatment, and cisplatin (20 μM) was treated for 24 hours. Figure 4f shows the cell viability of MG-63 cells after treatment with cisplatin in the presence of PPl. MG-63 cells were cultured under DMSO or PPl (2, 5 [mu] M) and cisplatin (20 [mu] M) for 24 h. Cell viability was determined by WST-1 assay. Values are expressed as means ± SD (n = 3), ** means p <0.05 by t-test. Figure 4g shows the cell viability of MG-63 cells after cisplatin treatment in the presence of Compound C. MG-63 cells were cultured in DMSO or compound C (5, 10 μM) and treated with cisplatin (20 μM) for 24 h. Cell viability was determined by WST-1 assay. Values are expressed as means ± SD (n = 3), ** means p <0.05 by t-test.
Figures 5a to 5f show the Sikkim GFRα1- mediated autophagy This promotes vivo anticancer drug resistance, and tumor growth. Figure 5A shows that BALb / c nude mice (n = 40) received MG-63 transfected with either a control or GFR [alpha] l expression vector. Tumor volume was measured every 3-4 days. Values are expressed as mean ± standard deviation (n = 3). Figure 5b shows that BALb / c nude mice (n = 20) received MG-63 transfected with either control or GFR [alpha] l shRNA. Tumor volume was measured every 3-4 days. Values are expressed as mean ± standard deviation (n = 3). FIG. 5C shows that PBS, CQ, cisplatin or cisplatin + CQ were directly administered to tumors of mice to which MG-63 cells transfected with the GFRα1 expression vector were administered. Tumor volume was measured every 3-4 days. Values are expressed as mean ± standard deviation (n = 3). ** p &lt; 0.05 vs PBS, CQ or cisplatin-treated tumor. The left side of FIG. 5D shows representative images of HMGB1 immunofluorescence staining of tumor sections in which PBS, CQ, cisplatin or cisplatin + CQ were directly administered to mouse tumors to which MG-63 cells transfected with GFR? 1 expression vector were transfected. The right represents a quantitative analysis of the percentage of cytosolic HMGB1-positive cells in HMGB1-positive tumors. Values are expressed as means ± SD (n = 3), ** means p <0.05 by t-test. The left side of FIG. 5E is representative images of TUNEL-positive cells of tumor sections in which PBS, CQ, cisplatin or cisplatin + CQ were directly administered to mouse tumors to which MG-63 cells transfected with GFRα1 expression vector were administered. The right represents a quantitative analysis of the percentage of cytoplasmic TUNEL-positive cells in HMGB1-positive tumors. Values are expressed as means ± SD (n = 3), ** means p <0.05 by t-test. FIG. 5f shows the survival rate of mice in which cisplatin or cisplatin + CQ were directly administered to mouse tumors to which MG-63 cells transfected with GFRα1 expression vector were administered. Figure 5g shows a representative image of GFR [alpha] l immunohistochemical staining of human osteosarcoma patient sections before and after cisplatin treatment. Figure 5h shows a representative image of immunofluorescence staining of GFR [alpha] l and HMGBl of human osteosarcoma patient sections before / after cisplatin treatment.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Method

Human tissue

The sample of this study was supported by the National Center for Human Resources (http://koreabiobank.re.kr), Hwasun Hospital (Gwangju, Jeonnam, Korea), supported by the Ministry of Health and Welfare. All samples were obtained with an approved subject agreement from the Chonnam National University Clinical Examination Committee.

Cell culture

Human osteosarcoma cell line MG-63 and U-2 OS, human embryonic kidney cell line 293T, human fibrosarcoma cell line HT-1080, human pancreatic cancer cell line MIA PaCa-2 and anamus embryo fibroblast cell line NIH / , Rockville, Md.). The cell lines were DMEM (Dulbecco's Modified Eagle's Medium; MG-63, 293T, MIA PaCa-2 and NIH / 3T3) containing 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin / streptomycin. , EMEM (Eagle's Minimum Essential Medium; HT-1080) or McCoy's 5a medium (U-2 OS). The cells were cultured under a humidified atmosphere of 5% CO ₂ at 37 ° C.

Production of stable cell line

The human pLenti / GFR [alpha] l expression vector and control vector were purchased from GeneCopoeia (Rockville, Md.). 293T cells were used for viral packaging, and titration of whole lentiviral vectors was performed using Invitrogen Gateway System and ViraPower Lentiviral Expression System. The presence of GFR [alpha] l was confirmed by PCR and the correct insertion of the clone was confirmed by sequencing. HT-1080 cells, lentivirus was transfected, expressed and titrated. The packed virus was concentrated by centrifugation (20,000 x g, 2 hours, 4 &lt; 0 &gt; C) using Centric filter (Millipore, Billerica, MA). The produced GFRα1 over-expressing MG-63, U-2 OS or NIG / 3T3 was infected with a lentivirus containing the pLenti / cotrol or pLenti / GFRα1 vector. The cells were then cultured for 4-5 weeks in a culture medium containing 400 [mu] g / ml G418. GFR [alpha] l -specific shRNAs and control shRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.). Specific shRNAs or control shRNAs were transfected into MG-63 and U-2 OS cells using Lipofectamine 2000 (Life Technologies, Grand Island, NY) and 100 μg / ml puromycin Lt; RTI ID = 0.0 &gt; GFRa &lt; / RTI &gt; 1 knockdown cell line.

Preparation of cisplatin-resistant cell lines

Cisplatin-resistant (CIS ^R ) cell lines were prepared by continuously exposing cisplatin-1 (1 μM to 1 mM cisplatin to an MG-63 parental cell line with an IC50 value for 24 hours according to an initial dose-response study of cisplatin Respectively. Initially, 1 [mu] M cisplatin was treated for 72 hours in the CIS ^R cell line. The medium was removed and cells were allowed to recover for 72 hours. Then, cisplatin (1 to 50 [mu] M) was gradually exposed at a low concentration to a high concentration to prepare a resistant cell line. These generators were run for about 6 months, and the cells produced were maintained for two months in the presence of new IC50 concentrations of cisplatin.

Reagents and Antibodies

3-methyladenine (3-MA), and chloroquine diphosphate salt (CQ-Diamineplatinum (II) dichloride), DMSO (dimethyl sulfoxide), DMF (N, N-dimethylformamide), Bafilomycin A1, Were purchased from Sigma-Aldrich, MO. Methotrexate and doxorubicin were purchased from Santa Cruz Biotechnology. Anti-phospho-mTOR, anti-mTOR, anti-c-Src, anti-caspase-3 and anti-beta-actin were purchased from Santa Cruz Technology and anti-HMGB1 was purchased from Abcam ), Anti-phospho-Src, anti-Bechlin 1, anti-phospho-AMPK, anti-AMPK, anti-LC3B and anti-phospho-p70 S6 kinase were purchased from Cell Signaling Technology (Beverly, MA) .

Quantitative real-time-PCR (qPCR)

Total RNA was isolated from cells using the RNeasy system (Qiagen, Hilden, Germany) according to the manufacturer's protocol. CDNA was prepared from 30 [mu] l reaction containing total RNA (3 [mu] g), reverse transcriptase and oligo dT primer (Promega, Madison, Wis.). Real-time amplification of GFRα1 and NFκB p50 cDNA was performed using the LightCycler 96 Instrument (Roche, Indianapolis, IN) using the SYBR Master Mix FastStart Essential DNA Green Master (Roche). A set of gene-specific human primers of 18S rRNA, GFRα1 and NFκB p50 was designed using Primer Express software v3.0.1 (Applied Biosystems, Grand Island, NY). The primers used for qPCR synthesized are as follows:

p18S rRNA sense primer 5'-GAGGATGAGGTGGAACGTGT-3 'and the antisense primer 5'-TCTTCAGTCGCTCCAGGTCT-3' (166 bp site amplification); 5'-TCCAATGTGTCGGGCAATAC-3 'and antisense primer (SEQ ID No. 2) 5'-GGAGGAGCAGCCATTGATTT-3' (106 bp site amplification); qGFRα1 sense primer (Sequence Listing 1st sequence); qNFkB p50 sense primer 5'-AAGCACAAAAAGGCAGCACT-3 'and antisense primer 5'-TGCCAATGAGATGTTGTCGT-3' (197 bp site amplification). After one cycle of 95 ° C for 10 seconds, it was amplified for 48 cycles under conditions of 95 ° C for 5 seconds and an annealing step of 60 ° C for 20 seconds. Melting curve analysis was performed to determine the exact size of the amplicon. A negative control without cDNA was run every PCR to assess the specificity of the reaction. Analysis of the results was performed using LightCycler 96 software 1.1 (Roche).

Semi-quantitative RT-PCR

Gene-specific human primer sets of GFR [alpha] l, GDNF and GAPDH were designed using Primer Express software v3.0.1 (Applied Biosystems, Grand Island, NY). The primers used for qPCR synthesized are as follows:

5'-CTTCTGTGCCTGTAAATTTGCA-3 '(387 bp site amplification) of GFRα1 sense primer (SEQ ID NO: 3) 5'-TGTCAGCAGCTGTCTAAAGG-3' and an antisense primer (Sequence Listing 4 sequence); GDNF sense primer 5'-CCAACCCAGAGAATTCCAGA-3 'and antisense primer 5'-AGCCGCTGCAGTACCTAAAA-3' (150 bp site amplification); GAPDH sense primer 5'-TGACCACAGTCCATGCCATC-3 'and antisense primer 5'-TTACTCCTTGGAGGCCATGT-3' (494 bp site amplification). Denaturation step; 94 캜 - 3 min, amplification step; (94 ° C.-30 seconds) - (58 ° C.-30 seconds) - (72 ° C.-30 seconds) 28 amplification cycles and elongation steps; The PCR reaction was optimized at 72 ° C for 10 minutes. The amplified product was electrophoresed on a 1.5% agarose gel and visualized by EtBr (Ethidium bromide) staining.

siRNA (small interfering RNA) -based experiments

Human GFRα1 siRNA, Beclin 1 siRNA, HMGB1 siRNA, AMPK siRNA, c-Src siRNA, NFκB p50 siRNA, APE siRNA and negative control siRNA were purchased from Santa Cruz Biotechnology. The cell line was transiently transfected with siRNA using Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer's manual.

Cell viability assay

WST-1 cell viability assay reagents were purchased from Roche. The same number of cells were seeded in triplicate in 48-well plates and cultured in growth medium containing 10% FBS. Cells were treated with PBS, GDNF (50 ng / ml), GDNF (50 ng / ml) + cisplatin (20 μM) or cisplatin (20 μM) in the presence of serum for 24 hours. Prior to treatment with PBS, GDNF, GDNF + cisplatin or cisplatin, cells were previously treated with inhibitors (3-MA, PPl, Compound C, Baf and CQ) and incubated for 1 hour. The WST-1 reagent was then treated to cells at a defined time and incubated at 37 [deg.] C for 2 hours. After WST-1 treatment, the absorbance was measured to determine cell viability. The spectrophotometer absorbance of the sample was measured at 450 nm using an Ultra Multifunctional Microplate Reader (Tecan, Durham, NC). Experiments were performed at least three independent cultures.

Apoptosis analysis

Suspension or trypsin-isolated cells were harvested and washed once with ice cold PBS and analyzed with FITC Annexin V Apoptosis Detection Kit (BD Biosciences, San Diego, Calif.). Apoptotic cells were analyzed with Cell Quest software (Becton Dickinson, Franklin Lakes, NJ) and FACS Calibur flow cytometry analyzer. Results were expressed as the mean of three measurements of at least 10,000 cells per assay. The sub-G1 population was counted as apoptotic cells. Caspase 3 activity was analyzed by the Colorimetric CaspACETM assay system (Promega, Madison, Wis.) According to the manufacturer &apos; s instructions. The degree of tissue apoptosis was evaluated by TUNEL (terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling) analysis of ApopTag Plus Peroxidase In Situ (Millipore, Billerica, MA).

FACS analysis

The cells were harvested by trypsinization, washed with PBS, fixed with 70% ethanol, washed with PBS, and reacted with 0.02 mg / ml propidium iodide containing RNase. Cells were analyzed using a FACS Calibur flow cytometer and Cell Quest software.

TUNEL analysis

The tissue apoptosis index was determined by TUNEL analysis. The sections were stained with ApopTag Plus Peroxidase In Situ. The sections were incubated overnight at 60 ° C. The sections were deparaffinized with xylene for one hour and then rehydrated with continuous reducing alcohols (100, 95, 90, 70 and 50%). Slides were treated with 20 [mu] g / ml Proteinase K (Invitrogen, Camarillo, CA) and allowed to react for 15 minutes. Each step was washed with PBS. Treatment of 3% H ₂ O ₂ inhibited endogenous peroxidase activity. After washing with PBS, the sections were incubated with equilibrium buffer for 10-15 minutes at 37 ° C for 1 hour with TdT (Terminal Deoxynucleotidyl Transferase) enzyme [77 μl reaction buffer + 33 μl TdT enzyme mixture (1 μl Td enzyme) . Slides treated with stop / wash buffer (1:10) for 10 minutes at room temperature were incubated with anti-digoxigenin conjugate for 30 minutes. After washing three times for 5 minutes with PBS, the sections were stained with DAB to detect TUNEL-positive cells. Next, the cells were stained contrasted with methyl green. The results were analyzed and quantified with Image-J software (National Institutes of Health, Bethesda, MD).

Autophagy analysis

The intracellular endogenous or exogenous LC3-Ⅱ was monitored for the formation of the child's follicle with LC3B antibody or mRFP-GFP-LC3 plasmid (Samsung Medical Center, Sungkyunkwan University School of Medicine, Professor Lee Myung-shik). After mRFP-GFP-LC3 was transfected into the cell line, the degree of protein expression of LC3-Ⅱ was detected by Western blotting. Acidic Vesicular Organelles (AVO) were stained with acridine orange according to a known method. ⁴⁹ acridine orange (Polysciences, Warrington, Pa.) Was added to the final concentration of 0.5 mg / ml for 15 minutes. All fluorescence images, including AVO and LC3 points (puncta), are confocal images using an LSM510 laser scanning microscope (Carl Zeiss, Germany). Results were quantified and analyzed using Image-J software. LC3-labeling points were defined as mean cytofluorimetric &gt; 1.5 SD light points. At least 3 independent experiments were conducted to analyze the minimum 40 (AVO) or 20 (LC3) sections. ₁₅

Transmission electron microscope

2% para-formaldehyde and fix the cell 2% glutaraldehyde with 0.1 M phosphate buffer (pH 7.4) containing were fixed (postfix) and then for 2 hours in 1% OsO _4. Cells were hydrated by increasing alcohol concentration (30, 50, 70, 90 and 100%), infiltrated with LR white resin and embedded LR white resin. The hardened block was cut to a thickness of 60-nm and stained with uranyl acetate and lead citrate. Samples were observed with a transmission electron microscope (Hitachi H-7600; Hitachi, Tokyo, Japan). 10 sites of the images were selected and the child's follicles were quantified as described above.

Colony-Forming Analysis and Crystal Violet Dyeing

MG-63 and MG-63 resistant MG-63-CIS ^R cells, pLenti / GFRα1-, pLenti / empty vector-, shGFRα1- and control shRNA-transfected MG-63 cells were cultured in DMEM supplemented with 10% Lt; / RTI &gt; For colony-forming analysis, the same number of cells of each individual clone were plated in 12-well plates. After 24 hours, the cells were reacted with cisplatin and cisplatin + antifungal agents, respectively. The medium was changed every 3 days under the same conditions. Ten days later, colonies were visualized. Cells were washed with PBS, fixed with 4% PFA for 5 min and washed again with PBS. Immobilized cells were stained with 0.05% crystal violet for 1 hour and then washed with distilled water. The stained colonies were scanned with an appson scanner (GT9700F, Tokyo, Japan) and counted with Image-Pro Plus 5.1 software (Media Cybernetics, Bethesda, MD). The experiment was carried out three times.

Focus formation analysis

NIH / 3T3 cells (1 x ¹⁰⁶ ) were plated in a 60-mm dish and incubated overnight to form a monolayer. The following day, NIH / 3T3 cells (1 x 10 ³ ) transfected with an expression vector containing GFRα1 were plated on control NIH / 3T3 cells. The medium was changed every 2 days and the focus was quantitated 7-10 days later. Cells transfected with empty vectors were used as negative control. All cells were stained with crystal violet (0.5% in 20% ethanol).

Immune blotting

Cells were washed with PBS and lysed with RIPA buffer (Pierce, Rockford, IL). Protein content was determined with a die-binding microassay (Bio-Rad, Hercules, CA) and 10-50 [mu] g protein per lane was electrophoresed on a 4-12% SDS polyacrylamide gel Respectively. Protein was blotted on a high bond ECL membrane (Hybond ECL membrane, Amersham Pharmacia Bioech, Piscataway, NJ). Two protein ladders (GenDEPOT, Barker, TX) were used to determine the molecular weight of the protein. The blotted protein was detected using a chemiluminescence detection system (iNtRON, Seoul, Korea). Results were quantified and analyzed with Image-J software.

Immunocytochemistry

Osteosarcoma cells (2 × 10 ⁴ ) were seeded in a 60 μ-dish ^{35 mm high} (ibidi, GmbH, Am Klopferspitz, Germany). The next day, the cells were fixed with 4% PFA (paraformaldehyde) for 20 minutes. Washed with PBS and then reacted with 0.04% Triton X-100. After washing with PBS, the cells were reacted with 0.03% BSA for 10 minutes at room temperature. The antibodies were then reacted overnight at 4 ° C and washed with PBS for 1 hour. Alexa Fluor 488- or 647-conjugated secondary antibodies were reacted at 4 ° C for 1 hour and washed with PBS for 1 hour. Washed with PBS, mounted with VECTASHIELD (Vector Laboratories, Burlingame, Calif.) And sealed with clear manicure. Images were observed with a confocal microscope. LC3 point or APE was quantified and analyzed using Image-J. As in the previous analysis, there was no difference between the two observations according to biomarker expression pattern and score.

Immunohistochemistry

Tissue paraffin blocks from each donor were cut and arranged in 3 mm tissue biopsies. The sections (5 탆 thick) were deparaffinized and stained with H & E (Hematoxylin & Eosin). After antigen retrieval with 10 mM sodium citrate (pH 6.0), the sections were incubated with rabbit anti-HMGB1 and mouse anti-GGFR1 antibodies for 24 hours. The sections were reacted with biotin-conjugated secondary antibodies and the antibody label was visualized using the VECTASTAIN ABC system (Vector Laboratories). For immunofluorescence, patient tissues and mouse tumors were reacted with Alexa Fluor 488- and 647-conjugated secondary antibodies (Invitrogen) and contrasted with DAPI (Sigma-Aldrich) for nuclear staining. Immunofluorescence was detected with a confocal microscope. In patient tissue studies, tissues of all metastatic cancer patients showed strong immunoreactivity of GFR [alpha] l or HMGB1 between 4 weeks and 10 weeks after chemotherapy with cisplatin (within 4 weeks after the chemotherapy was administered / 1, weak, 2, moderate, 3, strong) and percentage of positive cells (0, non; 1, weak) , 5%, 1, 6-25%, 2, 26-50%, 3, 50-75%, 4, and 76% Thus, the indicated plus (+) indicates positive immunoreactivity and the minus (-) indicates negative immunoreactivity (not detected). In the preceding analysis, there was no difference between the biomarker expression patterns and the two observations according to the scores scored in the analysis section. The results were quantified and analyzed using Image-J.

Tumor formation in nude mice

The mice used in this study were purchased from Orient Bio (Sungnam, Korea) with 6 week old magnetic BALb / c nude mice. The mice were raised in pathogen-free organs according to animal welfare regulations and standard protocols. All protocols of this study were approved by Chonnam National University Laboratory Animals Committee. MG-63 cells transfected with control shRNA, GFR alpha 1 shRNA, plenti / GFR alpha 1 expressing viral vectors, or plenti / control viral vectors were harvested and suspended in PBS. Next, MG-63 cells (1 × 10 ⁶ ) were subcutaneously administered to the right side of BALb / c nude mice. Tumor size was measured with a caliper every 3-4 days. 31 days after subcutaneous administration, PBS, CQ (60 mg / kg), cisplatin (3 mg / kg) and cisplatin + CQ peritoneally were administered once a week to mice having GFRα1 expressing tumor cells. Tumor size was measured with a caliper every 4 days. Tumor weight was calculated by measuring the tumor size in mm with a caliper and using the long ellipsoid [(L × W ² ) / 2] (L is a long measure) formula.

Statistical analysis

Survival results were analyzed by Kaplan-Meier and log-rank tests of GraphPad Prism v5.04 (GraphPad Software, La Jolla CA). Plating Efficiency (PE,%) = (number of counted colonies / number of plated cells) x 100. Survival Fraction (SF,%) = (PE of cisplatin-treated clone / PE of untreated control clone). All values are expressed as mean ± standard deviation. Statistical analysis was performed using a two-tailed Student's t-test. ** p <0.05 means statistical significance.

Experiment result

Induction of GFRα1 expression in osteosarcoma cells by cisplatin

GFR [alpha] 1 expression was observed in malignant human cancers, and GFR [alpha] l plays a role in regulating tumor cell metastasis and penetration, indicating that GFR [alpha] l is involved in cancer progression and metastasis. ^25-27 To investigate whether GFRα1 is involved in osteosarcoma resistance, we examined the effects of doxorubicin, cisplatin and methotrexate on the expression of GFRα1 in well-known osteosarcoma cells MG-63 and U-2 OS. Cisplatin significantly increased the expression of GFR [alpha] l in both types of cells, whereas doxorubicin and methotrexate showed a slight effect on the expression of GFR [alpha] l (Fig. 1a). The effect of cisplatin on the expression of GFR [alpha] l was concentration and time dependent (Fig. 1b and 1c). Cisplatin also increased GFR [alpha] l mRNA expression in both types of cells in a concentration- and time-dependent fashion (Fig. 1b and 1c), indicating that GFR [alpha] l expression was induced in the transcriptional and translational stages of osteosarcoma cells by cisplatin. To investigate the effect of GFRα1 expression on the efficacy of chemotherapeutic agents, GFRα1 was knocked down with GFRα1-specific siRNAs in MG-63 and U-2 OS cells and treated with each of the three drugs. Treatment with cisplatin significantly reduced cell proliferation in GFRα1-deficient MG-63 and U-2 OS cells compared to the control, but doxorubicin and methotrexate showed little effect on cell proliferation of GFRα1-deficient cells. Considering that GDNF is a major ligand of GFR [alpha] l, the effect of cisplatin on the expression of GDNF was also investigated. In osteosarcoma cells, 10 and 20 μM cisplatin treatment increased GDNF mRNA expression. However, GNDF did not affect the proliferation of MG-63 cells. These results suggest that GFR [alpha] l inhibits cisplatin-induced apoptosis and that this effect is independent of the GFR [alpha] l ligand GDNF.

Reduction of cisplatin efficacy by GFRα1 expression in osteosarcoma cells

To investigate the role of GFRα1 in cisplatin-induced apoptosis, stable GFRα1-deficient MG-63 and U-2 OS cell lines were prepared using GFRα1-specific shRNAs. The knockdown of GFR [alpha] l expression in osteosarcoma cell lines reduced GFR [alpha] l protein (Fig. 2a). Like the siRNA-mediated knockdown of GFR [alpha] l, the stable knockdown of GFR [alpha] l significantly reduced the proliferation of cisplatin-treated osteosarcoma cells in a concentration-dependent manner (Fig. In addition, knockdown of GFR [alpha] l significantly decreased cell viability and significantly increased cisplatin-induced apoptosis. This means that GFRa1-deficient cells are more sensitive to cisplatin treatment (Fig. 2C). The western blot results using truncated PARP and truncated caspase-3 apoptosis markers showed a significant increase in truncated PARP and truncated caspase-3 after cisplatin treatment in GFRα1-deficient MG-63 cells (FIG. 2d ). Consistently, caspase-3 activity was significantly increased in GFRα1-deficient MG-63 cells, which increased more dramatically in GFRα1-deficient MG-63 cells compared to cisplatin treated control cells (FIG. 2e). The addition of the pancispase inhibitor ZVAD-FMK reversed this effect, which means that the deficiency of GFR [alpha] l, when cisplatin is present, further stimulates apoptosis. In addition, MG-63 and U-2 OS cell lines were transiently transfected with the human GFR [alpha] l expression vector to stably overexpress human GFR [alpha] l (Fig. 2f). Overexpression of GFR [alpha] l did not increase cell proliferation of the cisplatin-treated osteosarcoma cell line as compared to the control group of the concentration-response experiment (Fig. 2g). However, additional analysis using FACS indicated that overexpression of GFR [alpha] l significantly reduced cisplatin-induced apoptosis, consistent with increased cell viability in both cell lines (Fig. 2h). Collectively, these results indicate that GFR [alpha] l decreases the sensitivity of osteosarcoma cells to cisplatin-induced apoptosis.

To cisplatin  by GFR initiation of α1

Childhood is a mechanism that can promote resistance to anticancer therapy that induces apoptosis and apoptosis. Therefore, we examined the role of the childhood in relation to GFR [alpha] l expression in cisplatin-induced apoptosis of osteosarcoma cells. We investigated whether GFRα1 deficiency regulates the formation of light chain 3 (LC3) points in the pups that are used as markers for child action. Fluorescence image analysis of LC3 point formation using ²⁸ mRFP-GFP-LC3 reporter (a RFP-GFP tandem fluorescence-tagging LC3 vector) showed that LC3 point formation in MG-63 cells transfected with cisplatin- , But no change in GFR alpha 1-deficient intracellular point formation (FIG. 3A). In particular, cisplatin treatment increased the formation of RFP / GFP-positive yellow spots and RFP-positive red spots (autolysosome) in control cells, and the number of RFP / GFP- Was larger than the number of positive red dots (Fig. 3A). Acridine orange staining of GFRα1-deficient MG-63 cells showed that GFRα1 knockdown did not increase the accumulation of AVO (Acidic Vesicular Organelle) -positive cells by cisplatin treatment, whereas cisplatin treatment of control cells induced AVO-positive cells (Fig. 3B). These results were also observed in GFRα1-deficient U-2 OS cells. In addition, ultrastructural analysis using TEM showed that the number of subcellular vacuoles per cell in the control cells after cisplatin treatment was significantly increased, but that GFR [alpha] l deficiency did not affect intracellular vacuoles after the same treatment (Fig. 3c ). This implies that cisplatin induces a malfunction in osteosarcoma and GFR [alpha] l is required for such a childhood reaction.

In order to investigate the effect of GFRα1 in cisplatin-induced child action and its effect on cisplatin resistance development, the conversion of LC3-Ⅰ into LC3-Ⅱ during child cell formation was investigated. ^9,11,12,29 Western blot analysis showed an increase in the over-expression GFRα1- MG-63 cells LC3-Ⅱ protein expression compared to the control (Fig. 3d), which means that the over-expression of GFRα1 initiate autophagy do. The addition of 3-MA (3-methyladenine), an inhibitor of early childhood action, reduced LC3-Ⅱ expression, while bafilomycin 1, a late-stage inhibitor of child action, increased LC3-Ⅱ expression (Fig. 3d) . Consistent with these results, LC3 point formation after cisplatin treatment was significantly increased in GFRα1-overexpressing MG-63 and U-2 OS cells compared to the control (FIG. 3e). The number of RFP / GFP-positive yellow dots in cisplatin-treated control cells was greater than that of RFP-positive red dots, but the number of RFP-positive red dots in GFRα1-overexpressing cells was much higher than the number of RFP / GFP- (Fig. 3E), indicating that GFR [alpha] l significantly increases sperm action. It should be noted that the basal LC3 point formation without cisplatin treatment is higher in GFR [alpha] l-over-expressing cells compared to control cells (Fig. 3e). 3-MA treatment inhibited point formation in control cells and GFRa1-overexpressing cells (Figure 3f). Addition of GDNF did not have a significant effect on control cells and GFR [alpha] l-over-expressing cells. Similar to these results, cisplatin-treated or untreated osteosarcoma cells GFR [alpha] l-overexpression increased AVO-positive cells compared to the control (Fig. 3g). GFR [alpha] l-overexpressing cells showed an increase in the number of child-acting vacuoles per treated or untreated cells as compared to control cells (Fig. 3h). In a known study, APE1 (apurine and apyrimidine endonuclease 1) upregulated GFRα1 expression in pancreatic cancer cells to increase GDNF reactivity ₂₇ , and APE1 was investigated for the role of GFRα1-mediated child action in osteosarcoma cells after cisplatin treatment . Also, the knockdown of APE1 does not decrease LC3 point formation following cisplatin treatment, which means that APE1 is not involved in this mechanism. RET, a downstream substrate of the GDNF / GFRα1 complex, was examined for the involvement of GFRα1-mediated child action after cisplatin treatment of osteosarcoma cells, and Western blotting confirmed that RET expression was not detected in MG-63 osteosarcoma cells .

Cell proliferation of GFRα1 overexpressing cells was significantly higher in the treated / untreated cisplatin than in the control cells. This is associated with increased suppression of GFRα1-overexpressing cells and suppression of the effects of addition of 3-MA (FIG. 3i). The increase in cell proliferation observed in GFR [alpha] l-over-expressing cells is inhibited by siRNA-mediated autophagic protein Beclin1 or HMGB1 silencing, suggesting that GFR [alpha] l -mediated action after cisplatin treatment contributes to increased cell proliferation Backed. To further demonstrate the role of GFR [alpha] l in cisplatin-mediated childhood and cell proliferation, control cells, GFR [alpha] l-deficient cell lines or GFR [alpha] l-over-expressing cell lines were cultured under cisplatin treatment and colonies counted. The GFRa1 over-expressing cell line increased the number of colonies during the 7 days of the reaction and showed a high plating efficiency, while the control and GFRa1 deficient cell lines showed no colonies (Fig. 3k). Treatment of GFRα1-overexpressing MG-63 clone # 4 and 3-MA, Baf or chloroquine (CQ), which exhibits high resistance to cisplatin, effectively reduces the number of colonies and viability by GFRα1 mediated formation, (Fig. 3i). MG-63 cells were treated with cisplatin for 10 days to produce three cisplatin-resistant MG63 clones. All cisplatin-resistant cell lines showed a greater number of colonies compared to control MG-63 cells. In addition, GFRα1 mRNA expression was significantly increased in all three cell lines compared to the control group.

The transcription factor NFκB p50 can bind to the promoter of the GFRα1 gene, thereby upregulating GFRα1 mRNA expression. ²⁷ is to be commensurate with the result, NFκB p50 mRNA expression is also cisplatin as compared to the control group were significantly increased in the resistant cell line. The reduction of GFRα1 mRNA expression in MG-63 cells by NFκB p50 knockdown using siRNA suggests the possibility of NFκB p50 activation of cisplatin to stimulate GFRα1 expression. Studies using NIH3T3 cells indicate that GFR [alpha] l is capable of transforming. Overexpression of GFR [alpha] l in NIH3T3 cells induced cell transformation by showing focus formation. No transfection was induced with NIH3T3 transfected with the control vector. Collectively, these findings support the important role of GFR [alpha] l in the regulation of cognition by cisplatin, which promotes osteosarcoma cell survival and anticancer drug resistance.

Src / AMPK  Through signaling GFR Childhood regulation of α1

Src is activated by GFRα1 / RET signaling. Activation of Src by GFRα1 in ²⁰ RET-deficient cells implies that GFRα1 can activate Src signaling in a RET-independent manner. ^{20,30 In} addition to its tumorigenic function in a variety of cancers, Src has not yet been elucidated, but is involved in obtaining child behavior, especially chemical resistance. ^31,32 Src-mediated AMPK activation is associated with the regulation of child behavior. ^33,34 AMPK-mediated child action is associated with cisplatin-induced chemical resistance. ³⁵

After cisplatin treatment, cisplatin-induced GFR [alpha] 1 expression in MG-63 cells increased phosphorylated Src and phosphorylated AMPK and, when AMPK was activated, reduced the downstream kinases phosphorylated mTOR and phosphorylated S6K associated with AMPK signaling (Fig. 4A). Upregulation of Src / AMPK signaling by GFR [alpha] l increased the expression of Beclin 1, HMGB1 and LC3-II (Fig. 4A). In contrast, in GFRα1-deficient cells, phosphorylated Src and phosphorylated AMPK were reduced and phosphorylated mTOR and phosphorylated S6K were increased compared to the control. GFR [alpha] l-deficient cells showed a decrease in the expression of the pancreatic-related protein as compared to the control (Fig. 4b). Inhibition of Src phosphorylation by siRNA or the selective inhibitor PPl reduced AMPK phosphorylation and LC3-II expression (Fig. 4c and 4d). The inhibition of AMPK phosphorylation by the compound C inhibiting the selective inhibition also reduced LC3-II expression without affecting Src phosphorylation (Fig. 4E), confirming that AMPK is a downstream kinase of Src. Inhibition of Src or AMPK activation by a screening inhibitor after cisplatin treatment of MG-63 cells reduces cell viability as compared to the control (Figs. 4f and 4g). These results indicate that induction of GFR [alpha] l by initiating cisplatin treatment of osteosarcoma initiates Src activation, thereby activating AMPK signaling and inducing initiation of the child's action.

Inhibition of GFRα1-Induced Childhood Tumor Growth Promotion

Using a mouse xenograft model was examined for the influence of GFRα1 vivo tumorigenesis. First, MG-63 cells or GFRα1-overexpressing MG-63 cells (MG-63 / GFRα1) were transplanted subcutaneously to the right side of nude maas. On day 5 after the administration, tumors developed in MG-63 / GFRα1 transplanted mice and became -90 31 size after 31 days, whereas MG-63 transplanted mice developed tumors 17 days after administration and the tumor size was -10 ㎣ (Fig. 5A). Next, GFRα1-deficient MG-63 (MG-63 / GFRα1 shRNA) was transplanted to investigate the effect of GFRα1 deficiency on tumor formation. Tumors were formed in mice transplanted with MG-63, but no tumors were formed in mice transplanted with MG-63 / GFR alpha 1 shRNA after 31 days of administration (FIG. 5B).

Next, to investigate whether the MG-63 / GFRα1-treated mice were improved tumor formation by GFRα1-mediated action, tumors of MG-63 / GFRα1 treated cells were treated with PBS (control), CQ, cisplatin or cisplatin + CQ Lt; / RTI &gt; Compared with PBS-treated tumors, tumors treated with CQ or cisplatin decreased in size, and tumors treated with cisplatin and CQ decreased in size (FIG. 5C). These results indicate that treatment of cisplatin and CQ reduces tumor progression, whereas treatment of cisplatin or CQ alone allows tumors to survive (Fig. 5C). Cisplatin-treated tumor cells from MG-63 / GFR &agr; 1 transgenic mice exhibited a significant increase in HMGB1 expression in the cytoplasm, indicating increased activity. Simultaneous treatment of cisplatin with CQ reduced HMGB1 located in the cytoplasm (Fig. 5d). When CQ treated with cisplatin, the cisplatin-induced apoptosis in GFR [alpha] l -induced tumors was significantly increased (Fig. 5e) as compared to CQ- or cisplatin-treated GFR [alpha] l- expressing tumors. Furthermore, the survival rate of MG-63 / GFRα1 transplanted mice treated with CQ and cisplatin was significantly increased compared to mice treated with cisplatin only (Fig. 5f), indicating that inhibition of the childhood action of GFRα1-mediated protection of cisplatin-induced apoptosis . Taken together, these results indicate that GFRα1 enables the child action in response to cisplatin vivo and this autophagy reaction to promote the survival of osteosarcoma tumor GFRα1- mediated autophagy is important for the development of cisplatin resistance of osteosarcoma.

The contribution of clinical pathological GFRα1-mediated child action in cisplatin-resistant osteosarcoma patients

The complete surgical testing tissue samples obtained from 27 people osteosarcoma patients around the secondary and / or adjuvant therapy with resection were investigated GFRα1- vivo mediated autophagy. Among the cases, nine samples of tissue samples showed DAPI nuclear staining and survival of the bovine. For GFRα1 and HMGB1 immunostaining, osteosarcoma tissues were processed and analyzed before and after treatment with cisplatin from nine samples showing resistance to chemotherapy. Four samples showed GFR [alpha] l-positive expression, and only four GFR [alpha] l-positive samples showed positive HMGB1 expression (Fig. 5g, 5h and Table 1). Four tissue samples were obtained from patients receiving chemotherapy containing cisplatin for 4 to 15 weeks. In addition, the tumors obtained from the four osteosarcoma patients metastasized to the lungs (Table 1). The tissues of patients treated within 4 weeks did not express GFRα1 and HMGB1 (Table 1). Collectively, these studies suggest that GFR [alpha] l plays an important role in cisplatin-mediated anticancer drug resistance and metastasis.

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While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Development of Dignosting Marker for Cisplatin-Resistant          comprising GFRA1 as Active Ingredient and Method for Molecular          Screening Cisplatin-Resistant Drug <130> PN130555 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> qGFRa1 sense primer <400> 1 tccaatgtgt cgggcaatac 20 <210> 2 <211> 20 <212> DNA <213> qGFRa1 antisense primer <400> 2 ggaggagcag ccattgattt 20 <210> 3 <211> 20 <212> DNA <213> GFRa1 sense primer <400> 3 tgtcagcagc tgtctaaagg 20 <210> 4 <211> 22 <212> DNA <213> GFRa1 antisense primer <400> 4 cttctgtgcc tgtaaatttg ca 22 <210> 5 <211> 1398 <212> RNA <213> Homo sapiens GDNF family receptor alpha 1 (GFRA1), transcript variant 1 mRNA <400> 5 atgttcctgg cgaccctgta cttcgcgctg ccgctcttgg acttgctcct gtcggccgaa 60 gtgagcggcg gagaccgcct ggattgcgtg aaagccagtg atcagtgcct gaaggagcag 120 agctgcagca ccaagtaccg cacgctaagg cagtgcgtgg cgggcaagga gaccaacttc 180 agcctggcat ccggcctgga ggccaaggat gagtgccgca gcgccatgga ggccctgaag 240 cgaagtcgc tctacaactg ccgctgcaag cggggtatga agaaggagaa gaactgcctg 300 cgcatttact ggagcatgta ccagagcctg cagggaaatg atctgctgga ggattcccca 360 tatgaaccag ttaacagcag attgtcagat atattccggg tggtcccatt catatcagat 420 gtttttcagc aagtggagca cattcccaaa gggaacaact gcctggatgc agcgaaggcc 480 tgcaacctcg acgacatttg caagaagtac aggtcggcgt acatcacccc gtgcaccacc 540 agcgtgtcca acgatgtctg caaccgccgc aagtgccaca aggccctccg gcagttcttt 600 gacaaggtcc cggccaagca cagctacgga atgctcttct gctcctgccg ggacatcgcc 660 tgcacagagc ggaggcgaca gaccatcgtg cctgtgtgct cctatgaaga gagggagaag 720 cccaactgtt tgaatttgca ggactcctgc aagacgaatt acatctgcag atctcgcctt 780 gcggattttt ttaccaactg ccagccagag tcaaggtctg tcagcagctg tctaaaggaa 840 aactacgctg actgcctcct cgcctactcg gggcttattg gcacagtcat gacccccaac 900 tacatagact ccagtagcct cagtgtggcc ccatggtgtg actgcagcaa cagtgggaac 960 gacctagaag agtgcttgaa atttttgaat ttcttcaagg acaatacatg tcttaaaaat 1020 gcaattcaag cctttggcaa tggctccgat gtgaccgtgt ggcagccagc cttcccagta 1080 cagaccacca ctgccactac caccactgcc ctccgggtta agaacaagcc cctggggcca 1140 gcagggtctg agaatgaaat tcccactcat gttttgccac cgtgtgcaaa tttacaggca 1200 cagaagctga aatccaatgt gtcgggcaat acacacctct gtatttccaa tggtaattat 1260 gaaaaagaag gtctcggtgc ttccagccac ataaccacaa aatcaatggc tgctcctcca 1320 agctgtggtc tgagcccact gctggtcctg gtggtaaccg ctctgtccac cctattatct 1380 ttaacagaaa catcatag 1398

Claims (11)

A method for determining susceptibility to cisplatin comprising the steps of:
(a) separating a biological sample from a cancer patient;
(b) determining the expression level of the GFR alpha 1 gene or protein in the biological sample, wherein the degree of expression of the GFR alpha 1 gene or protein is down-regulated ) Is judged to be susceptible to cisplatin, and when the degree of expression of the GFR [alpha] l gene or protein is up-regulated from that of normal cells, it is determined that cisplatin resistance is present.
The method of claim 1, wherein the cancer is selected from the group consisting of osteosarcoma, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, large intestine, prostate carcinoma, colon cancer, breast cancer, Endotheliosarcoma, endotheliosarcoma, endometrioid adenocarcinoma, endometrial adenocarcinoma, adenocarcinoma, renal cell carcinoma, hepatocellular carcinoma, biliary cancer, fibrosarcoma, mucinous sarcoma, liposarcoma, chondrosarcoma, Sarcomas, lymphomas, lymphangioendotheliosarcoma, rhabdomyosarcoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, rhabdomyosarcoma, glandular carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, papillary adenocarcinoma, adenocarcinoma, A tumor of the thyroid gland, a tumor of the thyroid, a bronchial carcinoma, a choriocarcinoma, a testicular tumor, an embryonal carcinoma, a Wilms tumor, an astrocytoma, Kaposi sarcoma, a hematoblastoma, a craniopharyngioma, cell Species, meningioma, neuroblastoma, retinoblastoma, myeloma, characterized in that the lymphoma or leukemia.
8. The method of claim 1, wherein the cancer is osteosarcoma.
The method according to claim 1, wherein the step (b) is performed using a hybridization method, a gene amplification method, or an immunoassay method.
A kit for predicting susceptibility to cisplatin in a cancer patient, comprising a nucleotide sequence encoding GFR [alpha] l, a sequence complementary to the nucleotide sequence, a fragment of the nucleotide sequence, or an antibody specific for GFR [alpha] l.
6. The kit for predicting susceptibility to cisplatin according to claim 5, wherein the nucleotide sequence is a nucleotide sequence represented by the genbank accession number NM_5264.
6. The kit for predicting susceptibility to cisplatin according to claim 5, wherein the kit is a hybridization kit, a gene amplification kit or an immunoassay kit.
6. The method of claim 5, wherein the cancer is selected from the group consisting of osteosarcoma, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, colorectal adenocarcinoma, prostate carcinoma, colon cancer, breast cancer, Endotheliosarcoma, endotheliosarcoma, endometrioid adenocarcinoma, endometrial adenocarcinoma, adenocarcinoma, renal cell carcinoma, hepatocellular carcinoma, biliary cancer, fibrosarcoma, mucinous sarcoma, liposarcoma, chondrosarcoma, Sarcomas, lymphomas, lymphangioendotheliosarcoma, rhabdomyosarcoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, rhabdomyosarcoma, glandular carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, papillary adenocarcinoma, adenocarcinoma, A tumor of the thyroid gland, a tumor of the thyroid, a bronchial carcinoma, a choriocarcinoma, a testicular tumor, an embryonal carcinoma, a Wilms tumor, an astrocytoma, Kaposi sarcoma, a hematoblastoma, a craniopharyngioma, cell Kind, meningioma, neuroblastoma, retinoblastoma, myeloma, lymphoma or kit for predicting susceptibility to cisplatin in patients with cancer, wherein the leukemia.
6. The kit for predicting susceptibility to cisplatin according to claim 5, wherein the cancer is osteosarcoma.
6. The kit for predicting susceptibility to cisplatin according to claim 5, wherein the nucleotide sequence encoding GFR [alpha] l is highly expressed from a cisplatin-resistant patient.
A screening method of cisplatin auxiliaries for inhibiting resistance to cisplatin in cancer cells comprising the steps of:
(a) contacting a cell comprising GFR [alpha] l with a cisplatin and a candidate agent;
(b) determining the level of GFR [alpha] 1 expression in the cell, and determining that the GFR [alpha] l expression is down-regulated relative to the control, the cisplatin adjuvant.

Images