KR101960040B1 - Use of human bitter taste receptor TAS2R10 for the diagnosis and treatment of cancer - Google Patents

Abstract

The present invention relates to the use of human bitter receptor TAS2R10 for the diagnosis and treatment of cancer. Specifically, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising an agent that promotes expression of TAS2R10 as an active ingredient, a screening method for cancer treatment using measurement of TAS2R10 expression level, and a method for measuring the expression level of TAS2R10 A biomarker composition and kit for the diagnosis or prognosis analysis of cancer as an active ingredient, and a method for providing information necessary for diagnosis or prognosis analysis of cancer using the expression level of TAS2R10.
The human bitter receptor TAS2R10 of the present invention effectively inhibits the cancer stem cell and self regenerating ability of cancer cells and inhibits tumor formation and inhibits the metastasis and infiltration of cancer cells and promotes the expression thereof, . In addition, the expression level of TAS2R10 of the present invention is changed according to development stage and degree of differentiation of cancer cells, and thus it can be effectively used for diagnosis and prognosis of cancer.

Description

Use of the human bitter taste receptor TAS2R10 for the diagnosis and treatment of cancer TAS2R10 for the diagnosis and treatment of cancer [

The present invention relates to the use of human bitter receptor TAS2R10 for the diagnosis and treatment of cancer.

Specifically, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising an agent that promotes expression of TAS2R10 as an active ingredient, a screening method for cancer treatment using measurement of TAS2R10 expression level, and a method for measuring the expression level of TAS2R10 A biomarker composition and kit for the diagnosis or prognosis analysis of cancer as an active ingredient, and a method for providing information necessary for diagnosis or prognosis analysis of cancer using the expression level of TAS2R10.

The sub-population of cancer cells has been demonstrated to represent representative features of cancer stem cells (CSC), including differentiation, self-renewal potential and tumorigenesis, (Clarke MF, et al. Cancer stem cells - perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res. 2006 ; 66 (19): 9339-9344). Thus, a cancer-targeting strategy targeting CSC is expected to be an effective treatment for malignant cancer.

In addition, many cancers are known to affect the mortality of cancer patients by metastasis of cancer cells, rather than primary tumors themselves. Cell invasion and migration in these cancer metastases are the two basic processes of cancer cell metastasis. Therefore, it is expected that the therapeutic strategy of inhibiting the cell infiltration and migration of cancer cells and inhibiting the metastasis of cancer cells will be an effective treatment for malignant cancer.

Neuroblastoma (NB) is a type of cancer that occurs in the early stages of an embryo or fetus and is derived from immature neurons. Most cases of NB occur in childhood and in children younger than 10 years of age. In fact, NB is the most commonly diagnosed type of cancer in infants under one year of age. The NB cell line is divided into three cell types based on the phenotype and gene expression pattern, such as nerve cell / neural endocrine precursor (N-type), substrate-adhesion / Schwannian (S-type) Respectively. Among them, type I cells represent the most immature and malignant population of NB cells, and these represent intermediate characteristics of N-type and S-type cells. In the gene expression profile of malignant NB, type I cells have been used as in vitro models for the potential therapeutic targets of NB, particularly for the study of CSC.

The taste is generally known as a flavor-dependent chemosensory system of taste buds located on the protuberance of the tongue. In addition, five basic flavors including sweet, umami, sour, salty and bitter taste are known to be detected by special sensory cells confined to the tongue. Among these cells, the mechanism of sweetness, umami and bitter taste has been found to involve small intermolecular interactions with certain types of G-protein-coupled receptors (GPCRs). GPCRs belong to the superfamily of transmembrane receptors that respond to a variety of extracellular stimuli such as neurotransmitters, light, taste and odor.

Human bitter receptors, referred to as TAS2R, represent about 25 chemosensory receptor groups that respond to bitter substances. Recent studies have demonstrated that TAS2R is also expressed in non-arousal tissues including the gastrointestinal tract, cardiovascular, lung, reproductive, immune and central nervous system tissues. These results suggest that TAS2R mediates the function of taste and the distinctive function. Studies on bitter receptors have hitherto been limited, but the detection of bitter taste is generally accepted to be related to a self-defense system that can protect itself from the ingestion of potentially harmful and dangerous toxins (Singh N, et al . Functional bitter taste receptors are expressed in brain cells. Biochem Biophys Res Commun. 2011; 406 (1): 146-151).

Although TAS2R is presumed to be related to various diseases such as asthma and gastrointestinal diseases related thereto based on the fact that TAS2R is also expressed in non-arousal tissue including gastrointestinal tract, cardiovascular, lung, genital, immune system and central nervous system tissues, The anti-amyloidogenic and anti-metastatic effects of TAS2R are not known, and the possibility of cancer treatment has not been elucidated.

Clarke MF, et al., Cancer Res. 2006; 66 (19): 9339-9344 Singh N, et al., Biochem Biophys Res Commun. 2011; 406 (1): 146-151

It is an object of the present invention to provide a diagnostic and therapeutic use of TAS2R10, a human bitter receptor, for cancer.

Specifically, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising an agent that promotes expression of TAS2R10 as an active ingredient, a screening method for cancer treatment using measurement of TAS2R10 expression level, and a method for measuring the expression level of TAS2R10 A biomarker composition and kit for the diagnosis or prognosis analysis of cancer contained as an active ingredient, and a method for providing information necessary for diagnosis or prognosis analysis of cancer using the expression level of TAS2R10.

The present invention provides a pharmaceutical composition for preventing or treating cancer comprising as an active ingredient an agent for promoting the expression of the human bitter receptor TAS2R10.

The human bitter receptor TAS2R10 of the present invention is one of the five basic taste receptors of humans including sweet taste, umami, sour taste, salty taste and bitter taste. Among them, a receptor for a bitter taste substance, protein coupled receptors.

The present inventors have found that when TAS2R10 expression is promoted in cancer cells, cancer stem cell (CSC) and self-renewal capacity of cancer cells are suppressed, tumor formation is inhibited, and cancer cell metastasis and invasion invasion of the cancer is suppressed and the effect of preventing and curing cancer was first identified.

Specifically, in the example of the present invention, transfection of SK-N-BE (2) C (hereinafter referred to as "BE (2) C") with the recombinant plasmid pcDNA + TAS2R10 resulted in induction of TAS2R10 overexpression , And the expression of the representative cancer stem cell markers DLK1, CD133, Notch1 and Sox2 was significantly decreased, thereby confirming the inhibition of cancer stem cell by cancer cell promoting expression of TAS2R10 (FIGS. 4A and 4B).

In another embodiment of the present invention, BE (2) C is transfected with a recombinant plasmid pcDNA + TAS2R10 to induce overexpression of TAS2R10, followed by clonogenic assay and sphere formation assay As a result, both colony formation and spheroid formation of BE (2) C were significantly inhibited, thereby confirming inhibition of cancer cell self-renewal potential by promoting expression of TAS2R10 (FIGS. 5 and 6).

In another embodiment of the present invention, BE (2) C was transfected with a recombinant plasmid pcDNA + TAS2R10 to induce overexpression of TAS2R10 and then injected subcutaneously into a mouse model to observe the formation of tumor. As a result, It was confirmed that the tumor formation was inhibited to the level of about 80% as compared with the control (Table 1).

In another embodiment of the present invention, a wound-healing assay was performed using BE (2) C, which was transfected with pcDNA + TAS2R10 and induced overexpression of TAS2R10. As a result, Cell migration was performed at a rate as low as 30%, thereby confirming inhibition of cancer cell metastasis by promoting expression of TAS2R10 (FIG. 7).

In another embodiment of the present invention, transwell invasion assay was performed using BE (2) C in which TAS2R10 overexpression was induced by transfection with pcDNA + TAS2R10. As a result, Was significantly reduced (Fig. 8).

In another embodiment of the present invention, BE2C was transfected with recombinant plasmid pcDNA + TAS2R10 to induce overexpression of TAS2R10, followed by gelatin zympgraphy assay. As a result, It was confirmed that the expression of MMP-2 and MMP-9, which are known to decompose macromolecules of substrate (ECM) and promote the migration and invasion of cancer cells, is inhibited (FIG. 9).

In another embodiment of the present invention, qRT-PCR analysis and western blot analysis were performed using BE (2) C, which was transfected with pcDNA + TAS2R10 and induced overexpression of TAS2R10. As a result, It was confirmed that expression of HIF-la, which is known to induce metastasis, was significantly inhibited (FIG. 10).

The term " agent that promotes the expression of TAS2R10 " used in the present invention includes all agents that promote expression of TAS2R10, and specifically binds to a gene expression regulatory region of TAS2R10 to induce TAS2R10 expression promotion Carbohydrates, peptides, aptamers, antibodies, and the like.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier in addition to an agent which promotes the expression of TAS2R10 as an active ingredient.

The kind of carrier which can be used in the present invention is not particularly limited and any carrier commonly used in the technical field can be used. Examples of the carrier include, but are not limited to, saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, maltodextrin, glycerol, . These may be used alone or in combination of two or more.

In addition, the pharmaceutical composition of the present invention may be supplemented with other pharmaceutically acceptable additives such as excipients, diluents, antioxidants, buffers or bacteriostats, and may be used as fillers, extenders, wetting agents, disintegrants, , A binder, a lubricant, and the like may be additionally used.

In the pharmaceutical composition of the present invention, the agent for promoting the expression of TAS2R10 may be contained in an amount of 0.00001 wt% to 99.99 wt%, preferably 0.1 wt% to 90 wt%, based on the total weight of the pharmaceutical composition, More preferably 0.1% by weight to 70% by weight, and still more preferably 0.1% by weight to 50% by weight, based on the total weight of the pharmaceutical composition of the present invention. . If desired, it may also be included in the total content of the pharmaceutical composition.

The pharmaceutical composition of the present invention may be formulated into various formulations suitable for oral administration or parenteral administration.

Non-limiting examples of oral dosage forms using the pharmaceutical compositions of the present invention include troches, lozenges, tablets, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft Capsules, syrups, and elixirs.

In order to formulate the pharmaceutical composition of the present invention for oral administration, a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin; Excipients such as dicalcium phosphate and the like; Disintegrating agents such as corn starch or sweet potato starch; Magnesium stearate, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax, and the like. Sweetening agents, fragrances, syrups, and the like may also be used. Furthermore, in the case of capsules, in addition to the above-mentioned substances, liquid carriers such as fatty oils can be further used.

Non-limiting examples of the parenteral preparation using the pharmaceutical composition of the present invention include injections, suppositories, respiratory inhalation powders, aerosol preparations for spraying, ointments, powder for application, oils and creams.

In order to formulate the pharmaceutical composition of the present invention for parenteral administration, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations and external preparations may be used. Examples of the non-aqueous solutions and suspensions include propylene glycol, polyethylene Glycerol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like.

When the pharmaceutical composition of the present invention is formulated into an injection, the pharmaceutical composition of the present invention is mixed with a stabilizer or a buffer in water to prepare a solution or suspension, which is used for unit administration of an ampoule or a vial Can be formulated.

When the pharmaceutical composition of the present invention is formulated into an aerosol formulation, a propellant or the like may be added together with the additive such that the water-dispersed concentrate or the wet powder is dispersed.

When the pharmaceutical composition of the present invention is formulated into an ointment, a cream, a powder for application, an oil, an external preparation for skin or the like, an animal oil, a vegetable oil, a wax, a paraffin, a starch, a tracer, a cellulose derivative, a polyethylene glycol, , Silica, talc, zinc oxide or the like as a carrier.

The pharmaceutically effective amount and the effective dose of the pharmaceutical composition of the present invention can be varied depending on the formulation method, administration method, administration time and / or administration route of the pharmaceutical composition, and the kind of reaction to be achieved by administration of the pharmaceutical composition Including the age, weight, general health condition, degree and severity of the disease, sex, diet, excretion, drugs used simultaneously or simultaneously with the subject, and other compositional components, etc. May be varied according to factors well known in the art and in the pharmaceutical arts, and those of ordinary skill in the art can readily determine and prescribe dosages that are effective for the desired treatment.

The administration of the pharmaceutical composition of the present invention may be administered once a day or divided into several doses. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. Taking into consideration all of the above factors, in such an amount as to obtain the maximum effect in a minimal amount without side effects, which can be easily determined by those skilled in the art.

The route of administration and the mode of administration of the pharmaceutical composition of the present invention may be independent of each other, and may be arbitrarily selected depending on the route of administration and the mode of administration as long as the pharmaceutical composition can reach the desired site. The pharmaceutical composition may be administered orally or parenterally.

Examples of the parenteral administration method of the pharmaceutical composition of the present invention include intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration or subcutaneous administration, and the method of applying, spraying or inhalation the above composition But are not limited thereto.

The pharmaceutical composition of the present invention may be used alone, but may be used in combination with various cancer treatment methods such as radiotherapy and chemotherapy in order to increase the therapeutic efficiency.

The present invention provides a screening method for a cancer therapeutic agent comprising treating a sample expressing the human bitter receptor TAS2R10 with a candidate substance for cancer treatment and measuring the expression level of TAS2R10.

The present invention can be useful for screening cancer therapeutic agents by comparing changes in the expression level of TAS2R10 in the presence and absence of cancer candidate agents. Specifically, the cancer screening method of the present invention comprises measuring the expression level of TAS2R10 in a sample in the absence of a candidate for cancer treatment, measuring the expression level of TAS2R10 in the presence of a candidate for cancer treatment, As a result, when the expression level of TAS2R10 in the presence of the candidate substance is increased above the expression level in the absence of the candidate substance, the candidate substance can be predicted as a prophylactic or therapeutic agent for cancer.

The term " expression level of TAS2R10 " used in the present invention can be measured by measuring the expression level of the protein of TAS2R10 or the expression level of mRNA of TAS2R10.

Specifically, measuring the level of protein expression of TAS2R10 can be accomplished by Western blotting, ELISA, radioimmunoassay, radioimmunoassay, or immunoassay using, for example, antibodies, aptamers, antagonists or combinations thereof specifically binding to the TAS2R10 protein But are not limited to, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assays, complement fixation, FACS, protein chips, and the like.

In addition, measuring the level of expression of mRNA of TAS2R10 can be accomplished by using, for example, an antisense oligonucleotide that specifically binds to the mRNA of TAS2R10, a primer pair, a probe, or a combination thereof, But are not limited to, reverse transcription polymerase chain reaction, real-time reverse transcription polymerase chain reaction, RNase protection analysis, Northern blotting, DNA microarray chip, and the like.

The present invention provides a biomarker composition for the diagnosis or prognosis analysis of cancer comprising as an active ingredient an agent for measuring the expression level of the human bitter receptor TAS2R10.

The present invention also provides a kit for the diagnosis or prognosis of cancer comprising as an active ingredient an agent for measuring the expression level of the human bitter receptor TAS2R10.

The term " agent for measuring the expression level of TAS2R10 " as used in the present invention means any agent that can be used to measure the expression level of the protein of TAS2R10 or the expression level of mRNA of TAS2R10. Specifically, the agent for measuring the protein expression level of TAS2R10 may be, for example, an antibody, an aptamer, an antagonist or a combination thereof that specifically binds to a TAS2R10 protein, and may be a preparation for measuring the expression level of mRNA of TAS2R10 May be, for example, but are not limited to, an antisense oligonucleotide, a primer pair, a probe, or a combination thereof that specifically binds to the mRNA of TAS2R10.

As used herein, the term " diagnosis " generally refers to identifying the presence or characteristic of a pathologic condition, and " prognosis " generally refers to prediction of disease progression and recovery.

The kit of the present invention may further comprise instructions for describing optimal reaction performance conditions. The instructions may be in the form of, for example, a brochure or flyer-shaped brochure, a label attached to the kit, a description of the package surface including the kit, and may include information disclosed or provided via an electronic medium such as the Internet .

The kit of the present invention can be, but not limited to, an RT-PCR kit, a kit for DNA analysis (e.g., a DNA chip) or a kit for protein analysis (e.g., a protein chip).

Specifically, the kit of the present invention may be a kit containing essential elements necessary for performing RT-PCR. For example, RT-PCR kits can be used to screen primer pairs for TAS2R10 genes, test tubes or other appropriate containers, reaction buffers (varying in pH and magnesium concentration), deoxynucleotides (dNTPs), Taq polymerase, Enzymes such as reverse transcriptase, DNase, RNAse inhibitors, DEPC-water, sterile water, and the like. It may also contain a primer pair specific for the gene used as a quantitative control.

In addition, specifically, the kit of the present invention may be a kit containing essential elements necessary for performing DNA analysis. The DNA assay kit (DNA chip kit) is typically a flat solid support plate, typically with a gridded array of nucleic acid species attached to a glass surface that is no larger than a microscope slide, , Which means a hybridization reaction between a nucleic acid on a DNA chip and a complementary nucleic acid contained in a solution treated on the surface of the chip to enable massive parallel analysis.

In addition, specifically, the kit of the present invention may be a kit containing essential elements necessary for performing protein analysis. The protein assay kit (protein chip kit) may be a kit in which one or more antibodies against the marker are arranged at predetermined positions on the substrate and immobilized at a high density. The method of using a protein chip can detect the presence or the expression level of the protein by separating the protein from the sample and hybridizing the separated protein with the protein chip to form an antigen-antibody complex and reading the protein.

The kit of the present invention comprises an antibody that specifically binds to a marker component, a secondary antibody conjugate conjugated with a marker that develops upon reaction with the substrate, a chromogenic substrate solution that reacts with the label, a wash solution and an enzyme reaction Stop solution, and the like, and may be made from a number of separate packaging or compartments, including the reagent components used.

The marker of the secondary antibody conjugate is preferably a conventional coloring agent that undergoes a chromogenic reaction and is preferably selected from the group consisting of HRP (horseradish peroxidase), alkaline phosphatase, colloid gold, poly L-lysine-fluorescein fluorescein such as isothiocyanate and rhodamine-B-isothiocyanate, and dye may be used.

The present invention provides a method for providing information necessary for the diagnosis or prognosis analysis of cancer comprising the step of measuring the expression level of the human bitter receptor TAS2R10 in a sample separated from a subject suspected of having cancer.

The present invention can be used to provide information necessary for diagnosis or prognosis analysis of cancer by measuring the expression level of TAS2R10 in a sample isolated from a suspected individual. Specifically, the information providing method of the present invention measures the expression level of TAS2R10 in a sample isolated from a subject suspected of having cancer, and the expression level of TAS2R10 in a normal control group is measured. As a result, If the expression level of TAS2R10 in the sample is increased than in the normal control, it can be predicted as cancer.

In the present invention, the term " individual " may be a mammal including, but not limited to, a human being who can develop or develop cancer.

In the present invention, the term " a sample isolated from a suspected cancer " means all samples capable of measuring the expression moisture thereof, including proteins or genes of TAS2R10, isolated from suspected cancerous individuals, But are not limited to, tissue, cells, whole blood, serum, plasma, saliva, cerebrospinal fluid, urine, etc., separated from an individual.

The human bitter receptor TAS2R10 of the present invention effectively inhibits the cancer stem cell and self regenerating ability of cancer cells and inhibits tumor formation and inhibits the metastasis and infiltration of cancer cells and promotes the expression thereof, .

In addition, the expression level of TAS2R10 of the present invention is changed according to development stage and degree of differentiation of cancer cells, and thus it can be effectively used for diagnosis and prognosis of cancer.

FIG. 1 is a graph showing the expression levels of TAS2R10 in animal tissues and cancer cells, wherein (a) is a graph showing the expression levels of TAS2R10 in tongue and liver tissues of a mouse, (b) (2) the expression level of TAS2R10 in C and SY5Y cells.
FIG. 2 is a graph of calcium migration analysis. FIG. 2 (a) is a graph of calcium mobilization analysis in HEK293 cells transfected with pcDNA + TAS2R10, and (b) is a graph of calcium migration analysis in BE (2) C cells. In the drawing lm1 is Ca ²⁺ - and fluorescence data in combination with Fura-2 dye, lm2 is Ca ²⁺ - a fluorescence data of the unbound dye.
FIG. 3 is a graph showing the expression levels of neurite outgrowth and neural differentiation-related markers of BE (2) C cells, wherein (a) shows the results of treatment of BE (2) C cells with all-trans retinoic acid (b) is a graph showing neurite outgrowth and neural differentiation-associated marker levels when BE (2) C cells were transfected with pcDNA + TAS2R10.
FIG. 4 is a graph showing the expression levels of CSC markers in BE (2) C cells. FIG. 4 (a) shows the levels of CSC marker expression when RA cells were treated with BE (B) is a graph showing CSC marker expression levels when BE (2) C cells were transfected with pcDNA + TAS2R10, (c) is a graph showing BE (2) C cells Lt; RTI ID = 0.0 &gt; CSC < / RTI &gt; marker and TAS2R10.
FIG. 5 is a photograph and a graph showing the results of colony formation analysis of BE (2) C cells transfected with pcDNA + TAS2R10.
6 is a photograph and a graph showing the result of spherical analysis of BE (2) C cells transfected with pcDNA + TAS2R10.
7 is a photograph and a graph showing the results of analysis of wound healing of BE (2) C cells transfected with pcDNA + TAS2R10.
8 is a photograph and a graph showing transwell infiltration analysis results of BE (2) C cells transfected with pcDNA + TAS2R10.
FIG. 9 is a photograph and a graph showing gelatin zymography analysis results of BE (2) C cells transfected with pcDNA + TAS2R10, wherein (a) is a photograph of the activity of MMP-2, And MMP-9 gene expression levels.
FIG. 10 is a photograph and a graph showing Western blot analysis for confirming the expression level of HIF-1a in BE (2) C cells transfected with pcDNA + TAS2R10, wherein (a) shows the expression level of HIF- (B) is a graph showing the expression levels of VEGF and GLUT1, downstream genes of HIF-1a.
(Significance sign of the graph of the present invention means: * p <0.05, ** p <0.01, *** p <0.001)

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

Statistical analysis

All experimental results of the examples were expressed as mean ± standard error (SEM) from at least three independent experiments and statistical significance analysis was performed using GraphPad PRISM (GraphPad Software, San Diego, Calif., USA). Data were compared between the two groups using the unpaired two-tailed t- test. Pearson's Chi-suqare test was performed to compare tumor incidence. P values less than 0.05 were considered statistically significant.

Example  1: Cell culture

SK-N-BE (2) C (BE (2) C) and SH-SY5Y (SY5Y), two human neuroblastoma (NB) cell lines, were obtained from the American Type Culture Collection (ATCC, Rockville, Respectively. Human embryonic kidney cell line, HEK293, was obtained from Dr. Park Tae-hyun of Seoul National University.

BE (2) C cells and SY5Y cells were cultured in a 1: 1 mixed medium of MEM (Minimum Essential Medium) and Ham's F-12 (Welgene, Daegu, Korea). HEK293 cells were cultured in a 1: 1 mixed medium of MEM and high-glucose DMEM (Dulbecco's Modified Eagle's Medium; Welgene).

All media were supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT, USA), 1% penicillin (100 U / mL) and 100 μg / mL streptomycin (Invitrogen, Carlsbad, CA, USA). To induce hypoxic conditions, 1 mM sodium pyruvate (Junsei Chemical, Tokyo, Japan) and 25 mM HEPES (Biosesang, Seongnam, Korea) were added to a pH 7.4 standard growth medium. Cell culture was maintained in a 5% CO ₂ and 37 ° C incubator.

All-trans retinoic acid (all- trans -retinoic acid, RA) and cobalt chloride (II) hexahydrate (CoCl _{2 ·} 6H ₂ O) was purchased from Sigma-Aldrich (St. Louis, MO , USA). RA (1 μM) was used to induce neural differentiation of NB cells and was made to be hypoxic at 21% O ₂ using CoCl _{2 ·} 6H ₂ O (100 μM). The reagents were added to the cell culture medium immediately before each assay was performed under dark illumination. Denatonium benzoate (Sigma-Aldrich) was used to activate TAS2R10 in a calcium mobilization assay.

Example  2: TAS2R10  Preparation of plasmid vectors and transfection

pcDNA3 + TAS2R10 was obtained from Dr. Park Tae-hyun of Seoul National University. Briefly, cDNA PCR products for TAS2R10 were digested with EcoRI and XhoI as restriction enzymes for TAS2R10 (Takara Bio, Mountain View, CA, USA). The FLAG-tagged pcDNA3 vector was digested with EcoRI and XhoI as restriction enzymes for TAS2R10.

The digested PCR products were separated by electrophoresis and extracted and purified from agarose gels for binding to pcDNA3 vector. These recombinant plasmids were amplified by transforming dh5-α competent cells. Plasmid DNA was recovered from a portion of the colonies for sequencing after transformed cells were picked in solid LB medium supplemented with ampicillin (Amresco, Solon, OH, USA) (BD Bioscience, San Jose, Calif., USA) .

BE (2) C cells were plated into 6-well plates and transfected with pcDNA3 (empty vector; EV) and pcDNA3 + TAS2R10 recombinant plasmids for 48 hours until cell density reached 70% to 80%.

Example  3: Quantitative real time PCR  analysis

PCR analysis performed in the examples of the present invention was performed in the following manner.

RNA was isolated using TRizol reagent (Invitrogen) and the first strand cDNA was synthesized by reverse transcription using RevertAid reverse transcriptase (Thermo Scientific, Waltham, Mass., USA). cDNA samples were combined with 2X SYBR Green PCR Master Mix (Qiagen) and quantitative real time PCR was performed using Rotor-Gene Q (Qiagen, Hilden, Germany) under the following conditions: start at 95 ° C (5 min) , Denaturation at 95 캜 (15 sec), annealing at 60 캜 and elongation (10 sec).

The primer sequences used in PCR are shown in Table 1 below, and GAPDH was measured as an internal control to generalize all qRT-PCR data.

Example  4: Western Blat  analysis

Western blot analysis performed in embodiments of the present invention was performed in the following manner.

Western blot analysis was performed in a manner known per se (Lim JY et al., Biochem Biophys Res Commun. 2014; 450 (4): 1475-1480). Briefly, cell protein extracts were collected in RIPA lysis buffer and an equal volume of each extract sample was separated in 8% or 10% sodium dodecyl sulfate (SDS) polyacrylamide gels. After transferring the sample to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA), it was resuspended in 5% bovine serum albumin (BSA) or skimmed milk in Tris- buffered saline / Tween 20 Lt; / RTI &gt;

The blocked membrane was labeled with a Flag (DYKDDDK) tag, a phospho-ERK, total ERK (Cell Signaling, Danvers, MA, USA),? -Tubulin III (Sigma-Aldrich), HIF- USA) and α-tubulin (Sigma-Aldrich) at 4 ° C overnight. Membranes were washed several times and then anti-mouse or anti-rabbit secondary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) was added as appropriate. One hour later, an ECL detection system was used to visualize the bound antibody.

Example  5: Calcium migration analysis TAS2R10's  Functional expression confirmation

Example  5-1: Experiments on tongue and liver tissue of animals

Mouse tissue experiments were performed using 5-week-old male C57BL / 6 mice (Central Lab Animal Inc, Seoul, Korea). Mice were isolated by extracting mouse tongue and liver tissue. Expression levels of mouse TAS2R108 (human TAS2R10) mRNA were analyzed in both tongue and liver tissues and GAPDH was used as a loading control. In addition, mRNA expression levels of TAS2R10 in BE (2) C cells and SY5Y cells, which were two NB cell lines cultured in Example 1, were analyzed.

As a result of the experiment, it was confirmed that the expression level of TAS2R10 was much higher in tongue tissues than in liver tissues (Fig. 1A). In addition, TAS2R10 was expressed at high levels in two NB cell lines, BE (2) C and SY5Y. Especially, TAS2R10 was shown to be a more malignant and less differentiated type I cell line BE (2) significantly lower levels in C cells (FIG. 1b). From these results, it was confirmed that the expression level of TAS2R10 is dependent on the differentiation state of the cells.

Example  5-2: Calcium migration analysis Calcium mobilization assay )

In order to evaluate the functional expression of TAS2R10 ectopic expression in HEK293 cells and endogenous expression of TAS2R10 in BE (2) C cells, calcium migration assay was performed as follows.

Fura-2 QBT ^TM calcium kit (Molecular Devices, Sunnyvale, Calif., USA) was used according to the manufacturer's instructions. Briefly, HEK293 cells (8 × 10 ⁴ ) and BE (2) C cells (5 × 10 ⁴ ) were dispensed into 96-well clear bottom black plates (Corning Inc., Corning, NY, USA). The next day, the HEK293 cells and BE (2) C cells were transfected with pcDNA + TAS2R10 and maintained for 48 hours. In order to measure the activation of each receptor in each cell line, denatonium benzoate, a common agent of bitter taste receptors, was used. Activation of the receptor was measured immediately after the application of the denatonium benzoate, Respectively. Signals were measured using a Flex Station III fluorescent plate reader (Molecular Devices) at 510 nm after stimulation at 340 nm for Ca ²⁺ -bonded Fura-2 dye and 380 nm for Ca ²⁺ -bonded dye.

As a result of the experiment, calcium migration was detected in BE (2) C cells and TAS2R10 transfected HEK293 cells (Figs. 2a and 2b). The degree of calcium mobilization varied, but the migration pattern was similar at the time of TAS2R10 activation and release of calcium into the cells. Based on these results, it was confirmed that TAS2R10 expressed exogenously and endogenously was functionally expressed.

Example  6: induction of neural differentiation and Cancer stem cell  For inhibition TAS2R10's  efficacy

Example  6-1: Neuronal differentiation

To test whether expression of TAS2R10 is affected by neuronal differentiation, BE (2) C cells were treated with RA (1 μM) for 5 days. As a result of treatment, neurite outgrowth was observed and it was confirmed that the expression of beta -tubulin III and phospho-ERK, which are neural differentiation-related markers, was upregulated (FIG. Similarly, after overexpression of TAS2R10 in BE (2) C cells, neurite outgrowth was observed and expression levels of p-tubulin III and phospho-ERK1 / 2 were significantly increased (Fig. 3B). It was confirmed that overexpression of TAS2R10 induces neuronal differentiation.

Example  6-2: Cancer stem cell Marker  Expression

In order to examine the effect of TAS2R10 on the expression of representative cancer stem cell (CSC) markers, the expression of DLK1, CD133, Notch1 and Sox2 was significantly downregulated in BE (2) C cells treated with RA While the expression level of TAS2R10 was confirmed to be up-regulated (Fig. 4A). In addition, when the TAS2R10 was overexpressed in BE (2) C cells, several representative stem cell markers were also significantly down regulated (Fig. 4B). This demonstrates that overexpression of TAS2R10 inhibits the expression of CSC markers.

Example  6-3: Sphere formation analysis ( Sphere formation assay )

BE (2) C cells (3x10 ⁴ / well) were inoculated on a cell culture plate previously coated with 1.2% poly-HEMA (Sigma-Aldrich) 2% B-27 (Invitrogen), 1% BSA (Rdtech Technology) and 2% penicillin-streptomycin (Invitrogen) (DMEM) and F-12 (Welgene). The following day, the cells were transfected with each plasmid prepared in Example 2. After 48 hours, the cells were further cultured for 10 days to 14 days, and the number of spheres containing 50 or more cells was counted and photographed (Nikon Instruments Co. Ltd., Tokyo, Japan).

As a result of the above experiment, it was confirmed that gene expression of TAS2R10 was significantly down-regulated in the formed sperm (Fig. 4C). This demonstrates that overexpression of TAS2R10 inhibits the expression of CSC markers.

Example  7: To inhibit the self-renewal ability of cancer cells TAS2R10's  efficacy

Self-renewal capacity is one of the characteristics of CSC and is known to exhibit physiological effects of cancer stem cells. In order to measure the self regenerating ability of cancer cells according to the overexpression of TAS2R10, spherical shape analysis was performed in the same manner as in Example 6-3, and colony formation analysis was performed by the following method.

Clonogenic assays were performed in a manner known per se (Lee HA et al., Oncol Rep. 2013; 30 (4): 1869-1877). Briefly, BE (2) C cells (800 cells / well) were plated on 6-well plates. On the next day, the cells were transfected with pcDNA (empty vector; EV) and pcDNA + TAS2R10 according to the manufacturer's instructions. After 48 hours, transfected cells were maintained for additional 7 days to 10 days. The colonies were then stained with crystal violet (Sigma-Aldrich). Plating efficiency (PE,%) was calculated as follows: number of stained colonies / number of cells divided x 100%.

As a result of the above experiment, it was confirmed that colony formation (Fig. 5) and sphere formation (Fig. 6) were significantly inhibited. This demonstrates that TAS2R10 plays an important role in inhibiting the self-renewal potential of cancer cells and eliminating cancer stem-like cells.

Example  8: In the xenotransplantation model Tumor formation  For inhibition TAS2R10's  efficacy

BE (2) C cells were transfected with pcDNA3 (empty vector; EV) and pcDNA3 + TAS2R10 plasmid. After 48 hours, the medium was replaced with growth medium containing G418 (Invitrogen). Two weeks later, one cell line that was stably established was selected and subcutaneously injected (4x10 ⁵ cells / injection) into the side of immunocompetent, 5-week-old male BALB / c nude mice (Central Lab Animal Inc, Seoul, Korea).

After tumors were established, tumor size was monitored twice a week using a digital caliper. Tumor volume was calculated as length (mm) x width ² (mm ² ) x 0.5. Animal protection and experimental protocols for this experiment were carried out with the approval of Ewha Womans University Animal Care and Use Committee (IACUC approval number: 16-046).

As a result of the above experiment, tumors were generated in 10 out of 10 EV control mice at the time of sacrifice of mice, while 7 out of 10 TAS2R10 mice developed tumors, as shown in the following Table 1. Thus, it was confirmed that the overexpression of TAS2R10 inhibited the tumor development to 30% as compared with the control EV group.

[Table 1]

(In the data in Table 1, comparison of tumor incidence among the experimental groups was performed using the chi-square test (P <0.05) .The final tumor volume and weight were expressed as mean ± SEM. Unpaired two-tailed t- test was performed for comparison.

Example  9: For cancer cell metastasis and invasion inhibition TAS2R10's  efficacy

Example  9-1: Wound healing analysis Wound - healing assay )

To investigate cell migration, wound healing assays were performed in a manner known per se (Park S et al., Biosci Biotechnol Biochem. 2013; 77 (4): 722-728). Briefly, BE (2) C cells were grown in 6-well plates as monolayers until the density reached 80% to 90%. Then, each monolayer was scratched with a sterile 200 μL pipette tip to form 4 to 6 lines of about 1 mm wide each. The dislodged cells were washed away with fresh medium, and the remaining cells were transfected with pcDNA3 (empty vector; EV) and pcDNA3 + TAS2R10. After 24 hours and 48 hours, cell migration was photographed and the distance traveled by the cells was measured.

As a result of the above experiment, it was confirmed that cell migration was performed at a slow rate of about 30% in all TAS2R10-transfected BE (2) C cells as compared with the EV control (Fig. 7).

Example  9-2: Transwell  Infiltration analysis Transwell invasion assay )

(Park S et al., Biosci Biotechnol Biochem. 2013; 77 (4): 1977), the extent of cellular invasion was examined by transwell infiltration analysis using a 24 well Matrigel infiltration chamber (Becton Dickinson, Bedford, Mass., USA) 722-728). Dispensing a brief, 3x10 ⁵ BE (2) C cells, the rehydration of the upper well (rehydration) containing serum-free medium, and the lower chamber is filled with a growth medium containing 5% FBS. Cells that were then dispensed into the upper wells were then transfected with each plasmid prepared in Example 2. After 48 hours, the cells that did not pass through the membrane pores of the upper well were carefully removed with a swab and the cells located in the lower well were fixed with methanol and stained with crystal violet (Sigma-Aldrich). The number of infiltrated cells was determined in five fields randomly selected for each well.

As a result of the above experiment, it was confirmed that the invasive potential of TAS2R10 was significantly reduced as compared with the EV control (Fig. 8). Thus, it was confirmed that the overexpression of TAS2R10 suppresses the ability of BE (2) C cells to migrate by inhibiting the migration and invasion ability of cancer cells.

Example  9-3: Gelatin Zymography  analysis( Gelatin zymography assay )

MMP is known to degrade ECM macromolecules to promote the migration and invasion of cancer cells. In order to analyze the enzymatic activity of MMP-2, gelatin zymography analysis was performed according to a conventionally known method (Park S et al., Biosci Biotechnol Biochem. 2013; 77 (4): 722-728). Briefly, BE (2) C cells were grown in serum-free medium and transfected with each plasmid prepared in Example 2. After 48 hours, each medium was collected and separated on a 10% SDS-PAGE gel containing 0.1 mg / mL gelatin. Electrophoresis was performed at 4 캜.

The gel was then washed with 2.5% Triton X-100 denaturing buffer to remove SDS and incubated in a reaction buffer containing 50 mM Tris-Cl (pH 7.5), 10 mM CaCl ₂ , 15 mM NaCl, and 0.2 mM ZnCl ₂ &Lt; / RTI &gt; overnight. The gel was stained with 0.5% Coomassie Brilliant Blue R-250 (Amresco, Solon, OH, USA) for 1 hour at room temperature with a shaker, and then washed with a desalting solution [MeOH: H ₂ O: acetic acid (40:50:10) ] Until the transparent band of MMP-2 was visible.

As a result of the above experiment, it was confirmed that not only the activity of MMP-2 but also the expression of MMP-2 and MMP-9 were decreased according to overexpression of TAS2R10 (FIGS. 9A and 9B). Thus, it was confirmed that overexpression of TAS2R10 inhibits the metastatic potential by inhibiting MMP activity and expression.

Example  10: Under hypoxic conditions HIF - 1α  Regulation of expression TAS2R10's  efficacy

It is known that the possibility of metastasis of tumor cells in hypoxic condition is increased. HIF-1α is a transcription factor known to modulate and mediate tumor metastasis by regulating downstream genes such as VEGF and GLUT1. QRT-PCR analysis and Western blot analysis were performed in the same manner as in Examples 3 and 4 to confirm that TAS2R10 can regulate the expression of HIF-1 alpha and related genes.

As a result of the above experiment, HIF-1α expression was strongly detected after induction of hypoxic state by CoCl ₂ , but was not detected in normal oxygen state. It was also found that overexpression of TAS2R10 significantly downregulated HIF-1α expression compared to EV control under hypoxic conditions induced by CoCl ₂ (FIG. 10A). Furthermore, the expression of downstream genes of HIF-1α is affected in such a way that VEGF and GLUT1 are up-regulated under hypoxic conditions induced by CoCl ₂ and downstream genes of HIF-1α are down-regulated upon overexpression of TAS2R10 (Fig. 10B). This demonstrates that TAS2R10 contributes to the regulation of the expression of hypoxic-related genes.

It is to be understood that both the foregoing general description and the following detailed description of the present invention are exemplary and explanatory and are intended to provide further explanation of the invention as claimed. More variations are possible within a range that does not. Accordingly, the present invention may be embodied in other forms without departing from the spirit or scope of the inventive concept as defined by the appended claims and their equivalents.

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Claims (15)

delete delete delete delete delete delete A method for screening a cancer therapeutic agent comprising treating a sample expressing the human bitter receptor TAS2R10 with a candidate for cancer treatment and measuring the expression level of TAS2R10. 8. The method of claim 7, wherein the measurement of the expression level measures the protein expression level of TAS2R10 or the mRNA expression level of TAS2R10. 8. The method of claim 7, further comprising the step of screening candidates with increased levels of TAS2R10 expression compared to before treatment of the candidate material with the sample. A biomarker composition for the diagnosis or prognosis analysis of cancer comprising an agent for measuring the expression level of the human bitter receptor TAS2R10 as an active ingredient. 11. The biomarker composition of claim 10, wherein the expression level of TAS2R10 is an expression level of a protein of TAS2R10 or an expression level of mRNA of TAS2R10. 12. The method according to claim 11, wherein the agent for measuring the expression level of the TAS2R10 protein is any one selected from the group consisting of an antibody, an aptamer, an antagonist and a combination thereof, and the agent for measuring the expression level of the TAS2R10 mRNA An antisense oligonucleotide, a primer pair, a probe, and combinations thereof. A kit for the diagnosis or prognosis of cancer comprising an agent for measuring the expression level of the human bitter receptor TAS2R10. And measuring the level of expression of the human bitter receptor TAS2R10 in a sample isolated from a subject suspected of having a cancer. 15. The method of claim 14, further comprising comparing the measured expression level of TAS2R10 to a normal control.

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