COMPOSITION FOR TARGETING MEDULLARY THYROID CANCER

Abstract

Disclosed is a composition for targeting medullary thyroid cancer, the composition includes a diagnostic radionuclide-labeled ligand of the olfactory receptor OR51E2. The composition is internalized into parafollicular C cells by the olfactory receptor OR51E2 that is expressed on the parafollicular C cells of the thyroid gland and as such, can be advantageously used for diagnosing parafollicular C cell-derived medullary thyroid cancer and identifying whether or not parafollicular C cell-derived medullary thyroid cancer metastases are. In addition, a pharmaceutical composition includes an acetate-associated therapeutic radionuclide for treatment of medullary thyroid cancer can be used for treatment of medullary thyroid cancer because the composition is internalized into cancer cells through the binding of the acetate to the olfactory receptor OR51E2.

Claims

1. A composition for targeting medullary thyroid cancer, comprising a diagnostic radionuclide-labeled ligand of an olfactory receptor OR51E2.

2. The composition for targeting medullary thyroid cancer according to claim 1, wherein the medullary thyroid cancer is derived from parafollicular C-cells.

3. The composition for targeting medullary thyroid cancer according to claim 1, wherein the diagnostic radionuclide is selected from the group consisting of carbon-11 (C-11), nitrogen-13 (N-13), oxygen-15 (O-15), fluorine-18 (F-18), phosphorus-32 (P-32), copper-64 (Cu-64), gallium-67 (Ga-67), gallium-68 (Ga-68), rubidium-82 (Rb-82), zirconium-89 (Zr-89), technetium-99m (Tc-99m), indium-111 (In-111), iodine-123 (I-123), iodine-131 (I-131), xenon-133 (Xe-133), thallium-201 (Tl-201) and thorium-229 (Th-229).

4. The composition for targeting medullary thyroid cancer according to claim 1, wherein the ligand of the olfactory receptor OR51E2 is acetate, propionate, nonanoic acid, beta-ionone, azelaic acid, estriol, epitestosterone, 19-OH AD (19-hydroxyandrost-4-ene-3,17-dione), palmitic acid, androstenedione, D-alanyl-d-alanine, glycylglycine, kojibiose, urea, AFMK (N-acetyl-N-formyl-5-metoxykynurenamine), pelargonidin, hydroxypyruvic acid, bradykinin, 8-hydroxyguanine, imidazolone, 2-pyrrolidinone, 2-ketoglutaric acid, L-glyceric acid, glycine or 13-cis retinoic acid.

5. A method for diagnosing medullary thyroid cancer, comprising steps of: (a) administering the composition of claim 1 to an individual suspected of developing medullary thyroid cancer; (b) detecting a signal of the diagnostic radionuclide of the administered composition; and (c) comparing the detected signal of the diagnostic radionuclide with that of a normal individual.

6. The method for diagnosing medullary thyroid cancer according to claim 5, wherein the method for diagnosing medullary thyroid cancer further comprises a step of determining that the individual has developed medullary thyroid cancer when the detected signal in step (c) is higher than that of the normal individual.

7. A method for identifying metastasis of medullary thyroid cancer, comprising steps of: (a) administering the composition of claim 1 to an individual suffering from medullary thyroid cancer; (b) detecting a signal of the diagnostic radionuclide of the administered composition; and (c) identifying whether or not the signal of the detected diagnostic radionuclide appears in areas other than the thyroid gland.

8. A pharmaceutical composition for treating medullary thyroid cancer comprising acetate and a therapeutic radionuclide associated therewith as active ingredients.

9. The pharmaceutical composition for treating medullary thyroid cancer according to claim 8, wherein the therapeutic radionuclide is selected from the group consisting of carbon-11 (C-11), nitrogen-13 (N-13), oxygen-15 (O-15), fluorine-18 (F-18), phosphorus-32 (P-32), copper-64 (Cu-64), gallium-67 (Ga-67), gallium-68 (Ga-68), rubidium-82 (Rb-82), zirconium-89 (Zr-89)), technetium-99m (Tc-99m), indium-111 (In-111), iodine-123 (I-123), iodine-131 (I-131), xenon-133 (Xe-133), thallium-201 (Tl-201), thorium-229 (Th-229), yttrium-90 (Y-90), holmium-166 (Ho-166) and rhenium-188 (Re-188).

10. A method for treating medullary thyroid cancer comprising a step of administering the pharmaceutical composition for treating medullary thyroid cancer of claim 8 to an individual in need of treatment.

Description

DESCRIPTION OF DRAWINGS

[0040] FIG. 1 shows the results of staining normal thyroid cells and medullary thyroid cancer cells with an olfactory marker protein (OMP) and calcitonin.

[0041] FIG. 2 shows the results of staining medullary thyroid cancer cell lines MZ-CRC1 and TT, undifferentiated thyroid cancer cell lines FRO and SW1736, a papillary thyroid cancer cell line TPC-1, and a follicular thyroid cancer cell line FTC-133 with an olfactory marker protein antibody.

[0042] FIG. 3A shows the results of identifying the expression of OR51E1 (olfactory receptor 51E1) and OR51E2 in various thyroid cancer cell lines.

[0043] FIG. 3B shows the results of staining the thyroid medullary cancer cell lines MZ-CRC1 and TT with OR51E1 antibody.

[0044] FIG. 3C shows the results of staining the thyroid medullary cancer cell lines MZ-CRC1 and TT with OR51E2 antibody.

[0045] FIG. 4A shows the results of identifying the phosphorylation level of p42/44 after treating the parafollicular C-cell line with acetate, nonanoic acid, propionate and azelaic acid which are the olfactory receptor ligands.

[0046] FIG. 4B shows the results of identifying the phosphorylation level of CREB and p42/44 after treating the parafollicular C-cell line with acetate.

[0047] FIG. 4C shows the results of identifying the level of cAMP.

[0048] FIG. 5A shows the results of analyzing the degree of cell proliferation with bromodeoxyuridine after treating the parafollicular C-cell line with acetate.

[0049] FIG. 5B is the results of identifying the intracellular expression level of calcitonin.

[0050] FIG. 5C shows the results of identifying the release level of calcitonin by inhibiting the expression of OR51E2 with siRNA, followed by acetate treatment.

[0051] FIG. 6 shows the results of staining thyroid tissues of mice with the olfactory marker protein (OMP), calcitonin and OR51E2 antibody, respectively.

[0052] FIG. 7A shows the results of staining thyroid tissues of a transgenic mouse, in which the OR51E2 gene is replaced with a green fluorescent protein (GFP), with an antibody.

[0053] FIG. 7B shows the results of measuring the calcitonin level in serum after administration of acetate to a wild-type mouse and the transgenic mouse.

[0054] FIG. 8A shows the results of measuring the degree of positron emission after treating various thyroid cancer cell lines with .sup.11C-acetate.

[0055] FIG. 8B is the results of measuring CPM (counter per minute) after treating various thyroid cancer cell lines with .sup.11C-acetate.

[0056] FIG. 8C is the results of measuring CPM by introducing siOR51E2 into the medullary thyroid cancer cell line, followed by .sup.11C-acetate treatment.

[0057] FIG. 8D shows the results of measuring CPM after treating OR51E2 knockout mice with .sup.11C-acetate.

[0058] FIG. 9A shows the results of measuring CPM by first treating MZ-CRC-1 cell line, which is a medullary thyroid cancer cell line, with cold acetate, followed by competitive treatment with .sup.11C-acetate.

[0059] FIG. 9B shows the results of measuring CPM by first treating TT cell line, which is a medullary thyroid cancer cell line, with cold acetate, followed by competitive treatment with .sup.11C-acetate.

[0060] FIG. 9C shows the results of measuring CPM by first treating MZ-CRC-1 cell line, which is a medullary thyroid cancer cell line, with .sup.11C-acetate, followed by competitive treatment with cold acetate.

[0061] FIG. 9D shows the results of measuring CPM by first treating TT cell line, which is a medullary thyroid cancer cell line, with .sup.11C-acetate, followed by competitive treatment with cold acetate.

[0062] FIG. 10 shows the results of staining the cells with an OR51E2 antibody after treating the MZ-CRC-1 cell line with acetate.

[0063] FIG. 11A shows the results of performing positron emission tomography (PET) after administering .sup.11C-acetate to a nude mouse in which a tumor has been formed by administering the MZ-CRC-1 cell line.

[0064] FIG. 11B shows the results of measuring the calcitonin level in the serum of the nude mouse.

[0065] FIG. 12 shows the results of performing PET after administering .sup.11C-acetate to a patient with medullary thyroid cancer.

MODE FOR INVENTION

[0066] Hereinafter, one or more embodiments will be described in more detail through examples. However, these examples are intended to illustratively explain one or more embodiments and the scope of the present invention is not limited to these examples.

Example 1: Expression Identification of Olfactory Marker Protein

[0067] 1-1. Thyroid Tissue and Thyroid Cancer Cell Line Staining

[0068] In order to identify whether an olfactory receptor is expressed in the thyroid gland, the expression of the olfactory marker protein (hereinafter, referred to as OMP) was identified. As a result of staining normal thyroid gland tissues and medullary thyroid cancer (MTC) cells with an OMP antibody (rabbit anti-OMP; Santa Cruz, Calif., USA), it was identified that the OMP was expressed in the thyroid gland, and it could be seen that the OMP was expressed only in parafollicular C-cells among cells constituting the thyroid gland (FIG. 1). Medullary thyroid cancer is a malignant thyroid tumor originating from parafollicular C-cells, where calcitonin is a marker protein for parafollicular C-cells.

[0069] 1-2. Thyroid Cancer Cell Line Staining

[0070] MZ-CRC1 and TT as medullary thyroid cancer cell lines, FRO and SW1736 as undifferentiated thyroid cancer cell lines, TPC-1 as a papillary thyroid cancer cell line, FTC-133 as a follicular thyroid cancer cell line were stained with an 01VIP antibody.

[0071] As a result, it could be identified that OMPs were highly expressed in MZ-CRC1 and TT cell lines as medullary thyroid cancer cell lines derived from parafollicular C-cells among various thyroid cancer cell lines (FIG. 2).

[0072] 1-3. Identification of Types of Olfactory Receptors

[0073] To identify the types of olfactory receptors expressed in thyroid cancer cell lines, RNA was isolated and then cDNA was synthesized to perform real-time polymerase chain reaction (RT-PCR) according to a method known in the art.

[0074] As a result, it could be identified that OR51E1 (olfactory receptor 51E1) and OR51E2 were highly expressed in MZ-CRC1 and TT cell lines as medullary thyroid cancer cell lines, but rarely expressed in other thyroid cancer cell lines (A in FIG. 3). In addition, as a result of staining the MZ-CRC1 and TT cell lines with OR51E1 and OR51E2 antibodies, respectively, it could be seen that the expression of the olfactory receptor almost coincided with the expression pattern of OMP (B and C in FIG. 3).

Example 2: Identification of Influences of Olfactory Receptor Ligands

[0075] 2-1. Treatment of Olfactory Receptor Ligands on Parafollicular C-Cells

[0076] Since OR51E1 and OR51E2 were highly expressed in the medullary thyroid cancer cell lines, MZ-CRC1 and TT cell lines as parafollicular C-cell lines were treated with acetate, nonanoic acid, propionate and azelaic acid as olfactory receptor ligands by 100 μM each to identify their influences.

[0077] As a result of the experiment, it could be seen that the level of phosphorylation of p42/44 MAPK (mitogen-activated protein kinase) was increased by acetate among various ligands (A in FIG. 4). In addition, the phosphorylation levels of p42/44 MAPK and CREB (cAMP response element-binding protein) increased in proportion to the acetate treatment concentration (B in FIG. 4), and as the phosphorylation level of CREB increased, the level of intracellular cAMP also increased. (C in FIG. 4).

[0078] Thereafter, the parafollicular C-cells were treated with acetate at each concentration and cultured, and then using a bromodeoxyuridine (BrdU) cell proliferation assay kit (Millipore, Mass.), the degree of BrdU binding was analyzed according to the manufacturer's protocol.

[0079] As a result, it could be seen that the cell proliferation was increased by identifying the increase of the BrdU binding level by the acetate treatment (A in FIG. 5). In addition, the intracellular expression level of calcitonin was increased by the acetate treatment (B in FIG. 5), and when the expression of OR51E2 was suppressed with siRNA, the release of calcitonin was not increased even with the acetate treatment (C in FIG. 5).

[0080] 2-2. Thyroid Tissue Staining in Mouse

[0081] The thyroid gland was isolated from an 8-week-old female C57BL/6 mouse to prepare tissue sections, and OR51E2, calcitonin, and OMP were stained with the respective antibodies. As a result, it could be seen that OR51E2, OMP, and calcitonin were co-localized in the thyroid tissue (FIG. 6).

[0082] 2-3. OR51E2 Knockout Mice

[0083] C57/BL6 mice in which a green fluorescent protein (GFP) was inserted into the OR51E2 gene loci were purchased (Jackson Laboratory, USA) and reared under sterile conditions. Through a week of acclimatization, the mice were each sacrificed 30 minutes after injecting 100 μl of 99% acetate thereto to separate the thyroid gland and serum. The thyroid gland was stained with calcitonin and a GFP antibody, respectively, and the calcitonin concentration was measured in the separated serum.

[0084] As a result of thyroid gland staining, it could be identified that the calcitonin and GFP were located at the same site (A in FIG. 7), and it could be seen that in the case of wild-type mice, the level of calcitonin in the serum was increased by acetate, but in OR51E2 knockout mice, the level of calcitonin was maintained almost constant (B in FIG. 7).

Example 3: Identification of Acetate Influences on Olfactory Receptor OR51E2

[0085] 3-1. .sup.11C-Acetate Treatment

[0086] Thyroid cancer cell lines having different origins were treated with .sup.11C-acetate labeled with a radioactive isotope (20 μCi), and the degree of positron emission was identified. A control group was treated with F-18-fluorodeoxyglucose as the most commonly used radiopharmaceutical.

[0087] As a result, it was shown that the level of positron emission was high in the medullary thyroid cancer cell line, whereby it could be seen that acetate was bound to OR51E2 of the parafollicular C-cells (A in FIG. 8), and it was shown that the result of measuring CPM (counter per minute) also was the highest in the medullary thyroid cancer cell line (B in FIG. 8).

[0088] In the case of suppressing the expression of OR51E2 by treating the thyroid medullary cancer cell line with siOR51E2, CPM did not increase even with the .sup.11C-acetate treatment (C in FIG. 8), and in the case of OR51E2 knockout mice, CPM did not increase even with the .sup.11C-acetate treatment as compared with wild-type mice (D in FIG. 8).

[0089] 3-2. Acetate Binding Competition Experiment

[0090] MZ-CRC-1 and TT cell lines as medullary thyroid cancer cell lines were treated with 100 μM cold acetate (acetate labeled with no radioactive isotope), and then treated with .sup.11C-acetate at concentrations of 2, 5, 10, 20, 40 and 60 μCi to measure CPM. In addition, MZ-CRC-1 and TT cell lines were treated with .sup.11C-acetate (20 μCi), and then treated with cold acetate at concentrations of 0, 20, 40, 80, 150 and 300 μM to measure CPM.

[0091] In the case of the first cold acetate treatment and .sup.11C-acetate treatment, CPM increased as the treatment concentration increased (A and B in FIG. 9). Conversely, in the case of the .sup.11C-acetate treatment and then cold acetate treatment, CPM decreased as the treatment concentration increased (C and D in FIG. 9). This result means that cold acetate and .sup.11C-acetate bind to OR51E2 competitively with each other.

[0092] 3-3. Use of Acetate to Diagnose Thyroid Cancer

[0093] To identify whether the binding of acetate and OR51E2 can be utilized in the diagnosis of thyroid cancer, the MZ-CRC-1 cell line (parafollicular C-cells) was treated with 100 μM of acetate, and the cells were stained after 1, 5 and 30 minutes to identify OR51E2. As a result, it could be identified that OR51E2 migrated into the cells by the acetate treatment (FIG. 10).

[0094] Nude mice (BALB/c) were administered with the MZ-CRC-1 cell line (4×10.sup.6 cells/mouse) to induce tumorigenesis, and administered with .sup.11C-acetate, followed by performing positron emission tomography (PET). As a result, it could be seen that .sup.11C-acetate was accumulated in the tumor-producing sites by the MZ-CRC-1 cell line (A in FIG. 11), and it could be identified that the calcitonin concentration in the serum was increased by the administration of .sup.11C-acetate (B in FIG. 11).

[0095] Through the results so far, it was identified that the ligand of the olfactory receptor OR51E2 was acetate, and it was identified that since OR51E2 migrated into the cells by the binding of OR51E2 and acetate, acetate could be used for diagnosis of medullary thyroid cancer.