PHARMACEUTICAL COMPOSITION FOR TREATING THYROID CANCER COMPRISING TYROSINE KINASE ACTIVITY INHIBITOR AS ACTIVE INGREDIENT
A pharmaceutical composition containing a tyrosine kinase activity inhibitor as an active ingredient may increase the expression of sodium iodide symporter in thyroid cancer cells, thereby promoting iodine uptake and killing thyroid cancer cells. Thus, the composition may be useful for the treatment of thyroid cancer.
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The disclosure relates to a pharmaceutical composition for treating thyroid cancer containing a tyrosine kinase activity inhibitor as an active ingredient.
BACKGROUND ARTThyroid cancer is the most common tumor of the endocrine system, and examples thereof include papillary carcinoma, follicular carcinoma, Hurtle cell neoplasm, anaplastic carcinoma (undifferentiated thyroid cancer), medullary thyroid carcinoma, and the like. Among these, differentiated tumors such as thyroid papillary carcinoma and thyroid follicular carcinoma show a relatively good prognosis, but in the case in which these tumors infiltrated the surrounding tissues or metastasized to other organs, the survival rate of patients with these tumors decreases rapidly. Anaplastic carcinoma is a rare, highly malignant undifferentiated tumor which is characterized by aggressiveness, rapid progression, a poor prognosis, and high mortality. Unlike differentiated thyroid cancer, anaplastic carcinoma does not exhibit the characteristics of thyroid follicular cells, such as iodine uptake and thyroglobulin synthesis, and is resistant to radioactive iodine (RAI) therapy due to the reduced iodine uptake thereof.
Sodium iodide symporter (NIS) is an intrinsic transmembrane glycoprotein, and co-transports two sodium cations (Na2+) and one iodine anion (I) across the basolateral membrane of thyroid follicular cells. Anaplastic carcinoma shows the worst prognosis among thyroid carcinomas due to suppressed expression of the sodium iodide symporter therein and the reduced iodine uptake thereof.
DISCLOSURE Technical ProblemOne object of the present disclosure is to provide a composition for treating or preventing thyroid cancer and an anticancer adjuvant for thyroid cancer, which contain, as an active ingredient, a compound of the following Formula 1, a salt thereof or a solvate thereof:
One aspect of the present disclosure is directed to a composition for treating or preventing thyroid cancer containing, as an active ingredient, a compound of the following Formula 1, a salt thereof or a solvate thereof:
According to one embodiment of the present disclosure, the thyroid cancer may be anaplastic thyroid cancer or differentiated thyroid cancer.
According to one embodiment of the present disclosure, the compound of Formula 1 may increase the expression of sodium iodide symporter.
Another aspect of the present disclosure is directed to an anticancer adjuvant for thyroid cancer containing, as an active ingredient, a compound of the following Formula 1, a salt thereof or a solvate thereof:
According to one embodiment of the present disclosure, the compound of Formula 1 may increase the expression of sodium iodide symporter.
Advantageous EffectsThe use of the composition according to one embodiment of the present disclosure may increase the expression of sodium iodide symporter in thyroid cancer cells, thereby promoting iodine uptake and killing thyroid cancer cells. Thus, the composition may be useful for the treatment of thyroid cancer.
One aspect of the present disclosure is directed to a composition for treating or preventing thyroid cancer containing, as an active ingredient, a compound of the following Formula 1, a salt thereof or a solvate thereof:
According to one embodiment of the present disclosure, the compound of Formula 1 may be obtained by extraction and separation from natural products or produced by a conventional organic synthesis method, but is not limited thereto.
According to one embodiment of the present disclosure, the thyroid cancer may be undifferentiated thyroid cancer (anaplastic thyroid cancer) or differentiated thyroid cancer, but is not limited thereto. Unlike differentiated thyroid cancer, undifferentiated thyroid cancer has suppressed expression of the sodium iodine symptor and reduced iodine uptake.
According to one embodiment of the present disclosure, the compound of Formula 1 may increase the expression of sodium iodide symporter in thyroid cancer cells, thereby promoting the uptake of radioactive iodine and killing thyroid cancer cells. The compound of Formula 1 may also decrease colony-forming ability in cancer cell.
The pharmaceutical composition of the present disclosure may further contain pharmaceutically acceptable additives. As the pharmaceutically acceptable additives, there may be used starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, maltose, gum Arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, dextrose, sorbitol, and talc.
The composition of the present disclosure may be administered in various oral and parenteral formulations for actual clinical administration. For formulation, preparation may be performed by using a normally used diluents or excipients such as a filler, an extender, a binder, a wetting agent, a disintegrating agent, and a surfactant. Examples of solid formulations for oral administration include a tablet, a pill, power, a granule, a capsule, etc. The solid formulations may be prepared by mixing a herbal composition of the present invention having an increased content of oil-soluble polyphenol with at least one excipient such as starch, calcium carbonate, sucrose, lactose or gelatin. In addition to simple excipients, lubricants such as magnesium stearate or talc may also be used.
The composition of the present disclosure can be administered orally or parenterally in accordance with the desired method. When the composition is administered parenterally, the administration method is preferably selected from intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection and intrathoracic injection. The dose range of the composition varies depending on the patient's body weight, age, sex, health condition, diet, time of administration, administration method, excretion rate, and disease severity.
The composition according to the present disclosure may be administered in a pharmaceutically effective amount. In the present disclosure, “pharmaceutically effective amount” refers to an amount sufficient to treat diseases, at a reasonable benefit/risk ratio applicable to any medical treatment. The effective dosage level of the composition may be determined depending on factors, including the type and the severity of the disease, the activity of the drug, the patient's sensitivity to the drug, the time of administration, the route of administration, the excretion rate, the duration of treatment, and drugs used in combination with the composition, and other factors well known in the medical field. The composition of the present disclosure may be administered individually or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. The composition may be administered in a single or multiple dosage form. It is important to administer the composition in the minimum amount that can exhibit the maximum effect without causing side effects, in view of all the above-described factors, and this amount can be easily determined by a person skilled in the art.
Specifically, the effective dosage of the composition according to the present disclosure may vary depending on the patient's age, sex and body weight. In general, the composition may be administered daily or every other day or administered 1 to 3 times a day, at a dosage of 1 mg to 200 mg per kg of body weight, preferably 10 mg to 200 mg per kg of body weight. However, the dosage is not intended to limit the scope of the present disclosure in any way, because it may vary depending on the route of administration, the severity of the disease, the patient's sex, body weight, and age, etc.
Another aspect of the present disclosure is directed to an anticancer adjuvant for thyroid cancer containing, as an active ingredient, a compound of the following Formula 1, a salt thereof or a solvate thereof:
As used herein, the term ‘anticancer adjuvant’ refers to an agent capable of improving, enhancing or increasing the anticancer effect of an anticancer drug. For example, when an agent that exhibits anticancer activity in a concentration-dependent manner is used together with an anticancer drug at a level that does not exhibit any anticancer activity by itself, the agent may be used as an anticancer adjuvant capable of improving, enhancing or increasing the anticancer effect of the anticancer drug.
According to one embodiment of the present disclosure, since the compound of Formula 1 may promote the uptake of radioactive iodine by increasing the expression of sodium iodide symporter, it may be used as an anticancer adjuvant for the treatment of thyroid cancer, and may be co-administered with 131I in the treatment of thyroid cancer.
The anticancer adjuvant may be administered through any normal route, as long as it may reach a target tissue. The anticancer adjuvant of the present disclosure may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally or orally, depending on the intended use thereof, but is not limited thereto. In addition, the anticancer adjuvant may be administered using any device capable of delivering the active ingredient to a target cell.
Furthermore, the anticancer adjuvant may be administered alone before or after administration of an anticancer drug, or may be co-administered as an adjuvant for cancer therapy with an anticancer drug. When the anticancer adjuvant of the present disclosure is co-administered with an anticancer drug, it may be co-administered with the anticancer drug at a suitable ratio depending on the patient's condition, the dosage of the anticancer drug, and the administration duration of the anticancer drug. Specifically, the anticancer adjuvant may be administered in an amount equal to 0.01 to 10 times the total weight of the anticancer drug.
MODE FOR INVENTIONHereafter, one or more embodiments will be described in more detail with reference to examples. However, these examples are merely to illustrate one or more embodiments, and the scope of the present disclosure is not limited to these examples.
Experimental Methods1. Cell Culture
8505C cells, which are a human anaplastic thyroid cancer (hereinafter referred to as ATC) cell line, were cultured with RPMI-1640 medium (Hyclone) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Hyclone) in a humidified incubator at 37° C. under 5% CO2.
2. Construction of pNIS-FL2-TurboFP635-pCMV-Rluc Recombinant Vector
A pNIS-FL2-TurboFP635-pCMV-Rluc recombinant vector expressing reporter genes driven by dual promoters was constructed by Cosmo Genetech Co., Ltd. (Republic of Korea) using a pNIS-FL2-TurboFP635 vector and a pcDNA3.1/Hygro(+) vector. Specifically, in order to screen a substance that increases the activity of NIS promoter, Firefly-luciferase2 (F-luc2) and TurboFP635 were included downstream (clockwise) of the NIS promoter. In order to count cells, CMV promoter and Renilla-luciferase (R-luc) were included upstream (counterclockwise) of the NIS promoter. The pNIS-FL2-TurboFP635 vector was provided from Dr. Abhijit De (ACTREC, India), and the pcDNA3.1/Hygro(+) vector was purchased from Invitrogen (USA).
3. Establishment of Stable Cell Line
The 8505C cell line was transfected with the pNIS-FL2-TurboFP635-pCMV-Rluc recombinant vector using the Fu-GENE HD solution according to the protocols of the manufacturer (Promega). The ratio of the recombinant vector to the Fu-GENE HD solution was 1:4, and the transfected cells were selected using 600 μg/ml of geneticin (AG Scientific). The selected cell line that stably expresses the dual-reporter gene system is referred to herein as ‘8505C-PNIS-PCMV’ cell line.
4. Screening of Substance that Increases Expression of Sodium Iodide Symporter
To examine transcriptional regulation of the NIS (sodium iodide symporter) promoter, the 8505C-PNIS-PCMV cells were treated with a tyrosine kinase inhibitor (hereinafter referred to as TKI) library for 24 hours. Next, D-luciferin (150 μg/ml) was added to the cells, and the activity of NIS promoter was measured by IVIS Lumina III (Perkin-Elmer, Wellesley, Mass., USA). To normalize the NIS promoter activity to cell number, 10 μg/ml of h-coelenterazine was added to the cells, and the activity of R-luc was measured. The activity of F-luc2 was divided by the R-luc activity. The TKI library used in the experiment was obtained from the Korea Chemical Bank (http://www.chembank.org/) in the Korea Research Institute of Chemical Technology (Republic of Korea). Among the TKI candidates, TKI-0266 was selected, purchased from Chemdiv (USA), dissolved in DMSO, and then stored at −80° C.
5. Protein Isolation and Western Blotting
8505C-PNIS-PCMV cells were treated with TKI-0266 and incubated for 24 hours, and then the cell pellet was washed twice with cold phosphate-buffered saline (hereinafter referred to as PBS). An RIPA buffer (Thermo Fisher Scientific, USA) containing a protease/phosphatase cocktail inhibitor kit (Thermo Fisher Scientific) was added to the cell pellet to lyse the cells. The lysed cells were gently vortexed three times at a time interval, and centrifuged at 13000×g at 4° C. to isolate a soluble protein. The isolated protein was quantified using a BCA protein assay kit (Thermo Fisher Scientific).
Membrane protein and cytosolic protein were isolated using Mem-PER™ plus kit (Thermo Fisher scientific) according to the manufacturer's protocols. Briefly, the collected cell pellet was washed with a cell wash solution and centrifuged at 300×g for 5 minutes, and this washing and centrifugation process was repeated two times. After centrifugation, the supernatant was discarded, and a permeabilization buffer was added to the cell pellet, followed by vortexing. Thereafter, the cell pellet was left to stand at 4° C. for 10 minutes with continuous stirring. After centrifugation, the supernatant containing cytosolic protein was collected, and a solubilization buffer was added to the remaining cell pellet which was then left to stand at 4° C. for 10 minutes with continuous stirring. After centrifugation again at 300×g for 5 minutes, the membrane protein was transferred into a fresh tube.
Equal amounts of proteins were separated by 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and then the proteins were transferred to PVDF membranes (Millipore, USA). The PVDF membrane was blocked with 3% BSA (bovine serum albumin) in TBS containing Tween-20 (TBS-T) for 1 hour, and incubated with primary antibodies in 0.5% BSA overnight at 4° C. The next day, the PVDF membrane was incubated with HRP-conjugated secondary antibodies at room temperature for 1 hour, and washed three times with TBS-T. The protein bands were visualized using ECL detection reagent (GE Healthcare Life Sciences, Italy) and the Fusion FX chemiluminescence analyzer system (Vilber lourmat, France).
The primary antibodies used in the experiment were as follows: NIS (Thermo Fisher Scientific; working dilution 1:2500), Phospho-p44/42 MAPK (p-ERK1/2) (Cell Signaling; dilution 1:2500), p44/42 MAPK (Total ERK1/2) (Cell Signaling; dilution 1:2500), Phospho-Akt (Cell Signaling; dilution 1:2500), Akt (Cell Signaling; dilution 1:2500), Thyroglobulin (Santa Cruz Biotechnology; dilution 1:2000), TSHR (Santa Cruz Biotechnology; dilution 1:2000), Thyroperoxidase (Santa Cruz Biotechnology; dilution 1:2000), TTF-1 (Santa Cruz Biotechnology; dilution 1:2000), Pax-8 (Santa Cruz Biotechnology; dilution 1:2000), Luciferase (Promega; dilution 1:5000), Renilla luciferase (Abcam; dilution 1:5000), GAPDH (Santa Cruz Biotechnology; dilution 1:5000) and R-actin (Santa Cruz Biotechnology; dilution 1:5000). HRP-conjugated secondary antibodies used in the experiment were anti-mouse (Cell Signaling) and anti-rabbit (Cell Signaling) antibodies.
6. In Vitro 125I Uptake Assay
8505C cells (1.25×105) were seeded and incubated in a 24-well plate for 24 hours, and then treated with TKI-0266 and further incubated for 24 hours. Thereafter, the medium was removed, and the cells were washed with bHBSS (Hank's balanced salt solution containing 0.5% BSA). The cells were incubated with 500 μl of bHBSS, 3.7 kBq carrier-free 125I (Perkin-Elmer), and 10 μmol/L sodium iodide (specific activity of 740 MBq/mmol) at 37° C. for 30 minutes. Thereafter, the cells were washed twice with cold bHBSS and lysed by adding 500 μL of 2% SDS solution thereto. Radioactivity was measured using a Cobra II gamma-counter (Canberra Packard, Canada). The 125I uptake values were normalized by comparing the amount of total protein determined by BCA protein assay kit.
7. 131I Clonogenic Assay
8505C cells (4×105) cells were seeded in a six-well plate, and then treated with TKI-0266 and incubated for 24 hours. Next, the medium was removed, and the cells were washed twice with bHBSS, and then incubated in the presence or absence of 50 ρCi/ml 131I (KIRAMS, Korea) containing 30 μM NaI for 7 hours at 37° C. After incubation, the cells were washed twice with bHBSS, re-seeded in a new six-well plate at a density of 1,000 cells/well, and then incubated for 7 days to induce colony formation. On day 7, the medium was removed and the cells were washed twice with PBS. A fixation buffer containing acetic acid:methanol at a ratio of 1:7 was added to the cells and the plate was left to stand at room temperature for 5 minutes to fix colonies. The fixed colonies were stained with 0.05% crystal violet for 1 hour and immersed in tap water to remove crystal violet. The colonies containing 50 or more cells were counted. Percentage inhibition was calculated as the number of colonies formed in the TKI-0266-treated group relative to the vehicle control.
8. Immunofluorescence Microscopy
8505C cells were seeded in a plate at a density of 2×105 cells and treated with the TKI inhibitor TKI-0266. After 24 hours, the cells were fixed with 4% paraformaldehyde for 30 minutes at room temperature and washed three times with PBS for 10 minutes each wash. Next, the cells were treated with PBS containing 0.7% Triton for 10 minutes at room temperature and quenched with PBS containing 50 mM NH4Cl for 10 minutes at room temperature. The cells were washed three times with PBS for 5 minutes each wash, and then blocked with 5% BSA-containing PBS and incubated with anti-NIS primary antibody (diluted at 1:50; Abcam, UK) overnight at 4° C. The next day, the cells were washed three times with PBS for 10 minutes each wash and incubated with Alexa-Fluor 488-conjugated secondary antibody (diluted at 1:300; Thermo Fisher Scientific) for 1 hour. After 1 hour, the cells were washed three times with PBS for 10 minutes for each wash, and mounted on a coverslip using a Vecta mounting medium (Vector Laboratories, USA) containing 4′,6-diamidino-2-phenylindole (DAPI). NIS staining was observed by confocal laser microscopy (Zeiss, LSM 5 exciter, Germany).
9. Establishment of Anaplastic Thyroid Cancer Xenograft Mouse Models
Female Balb/c nude mice, 5.5-week old with an average weight of 18.9±0.37 g (mean±standard deviation [SD]), were purchased (Hamamatsu, Shizuoka, Japan). The mice were acclimated under specific pathogen-free conditions for one week before starting the experiment, and were maintained at a temperature of 20 to 25° C. and a relative humidity of 40 to 70%. To establish anaplastic thyroid cancer xenograft mouse models, 8505C-PNIS-PCMV cells (5×106) were mixed with Matrigel (Corning) at a ratio of 1:1 and injected subcutaneously into the right flank region of each mouse.
10. In Vivo 99mTc-Pertechnetate Imaging
When the injected 8505C-PNIS-PCMV cells grew to a size of about 50 mm3, the mice were divided into two groups (n=5): a vehicle group; and a TKI-0266-administered group (50 mg/kg) injected with TKI-0266 by intraperitoneal injection every day for 5 days. To acquire gamma-camera images, 18.5 to 22.2 MBq 99mTc-pertechnetate was administered to all the mice by intravenous injection. Whole body pinhole gamma camera imaging using a 2-mm pinhole collimator was acquired by Infinia II gamma camera (GE Healthcare, USA). During the experiment, the mice were maintained under anesthesia with isoflurane (Forane, ChoongWae Co., Ltd., Korea). In order to estimate the count value, quantitative analysis was performed by quantifying the regions of interest (hereinafter referred to as ROI) in the mouse tumor region. Finally, in order to confirm the pertechnetate absorbed into the tumor, normalization was performed with a background activity ROI of the same size in the mouse head region.
11. Analysis by In Vivo Bioluminescence Imaging
In order to examine whether the expression of NIS would be increased by TKI-0266, mice for use in the experiment were divided into two groups (n=5): the vehicle group; and the TKI-0266-administered group (50 mg/kg). 50 mg/kg of TKI-0266 was administered intravenously to the established anaplastic thyroid cancer xenograft mice every day for 5 days. On day 0 (before administration of TKI-0266) and day 6 after the start of administration of TKI-0266, to measure the R-luc signal with IVIS Lumina III, h-coelenterazine (15 μg/ml) was administered to the mice by intravenous injection. Next, D-luciferin (150 μg/ml) was administered intraperitoneally to the mice, and the bioluminescence image (hereinafter referred to as BLI) was acquired.
In addition, in order to evaluate the effect of TKI-0266 administration on the treatment of thyroid cancer, mice for use in the experiment were divided into four groups (n=5) the vehicle group; the 131I-administered group (injected intraperitoneally with 1 mCi Na-131I on day 2 of the experiment; the TKI-0266-administered group (injected intraperitoneally with 50 mg/kg of TKI-0266 for 5 days); and the group co-administered with 131I and TKI-0266 (injected intraperitoneally with 50 mg/kg of TKI-0266 for 5 days, and injected intravenously with 1 mCi Na-131I on day 2; n=5). To measure the degree of tumor growth, h-coelenterazine (15 μg/ml) was administered to the mice by intravenous injection, and then the R-luc activity was monitored by BLI every 5 days.
12. Immunohistochemistry
At the end of the experiment, the mice were sacrificed, and the tumors were isolated from the mouse's right flank and fixed overnight with 4% formalin. The isolated tumors were embedded in paraffin, sectioned to a 4-μm thickness, and mounted on slides. The tumor sections on the slides were de-paraffinized and stained with hematoxylin & eosin. For immunohistochemistry, NIS (Thermo Fisher Scientific; diluted at 1:200) and cleaved caspase-3 (Cell Signaling; diluted at 1:200) antibodies were used.
13. Statistical Analysis
All experimental results are expressed as mean±SD. The results from two groups were statistically analyzed by Student's t-test using GraphPad Prism 5 software version 5.01 (GraphPad Software, Inc., USA). A P value of less than 0.05 was considered statistically significant. Error bars represent standard deviations (SD).
Experimental Results1. Establishment of Anaplastic Thyroid Cancer Cells Containing pNIS-FL2-TurboFP635-pCMV-Rluc Recombinant Vector
To monitor the transcriptional regulation of NIS promoter expression in anaplastic thyroid cancer cells, an anaplastic thyroid cancer cell line containing a pNIS-FL2-TurboFP635-pCMV-Rluc recombinant vector (
Whether F-luc2, R-luc and TurboFP635 would be expressed in the 8505C-PNIS-PCMV cells was examined to verify whether the reporter genes would be driven. As a result, it could be confirmed that F-luc2, R-luc and TurboFP635 were stably expressed in the 8505C-PNIS-PCMV cells, not 8505C cells.
2. Screening of Substance that Increases NIS Expression
8505C-PNIS-PCMV cells were treated with TKI candidates for 24 hours, and then the BLI signal was examined, thereby excavating TKI-0266. It could be seen that when the cells were treated with TKI-0266 (hereinafter referred to as the TKI-0266-treated group), the BLI signal activity of F-luc2, which indicated the NIS promoter activity, 3.19-fold increased, and the BLI signal activity of R-luc, which indicates cell viability, was about 75% compared to that in the vehicle group. From the above experimental results, it can be seen that TKI-0266 can regulate the activity of NIS promoter.
3. Confirmation of the Increase in NIS Promoter Activity by TKI-0266
After 8505C-PNIS-PCMV cells were treated with TKI-0266, the activity of NIS promoter was measured depending on the treatment time and the treatment concentration.
As a result, it could be seen that as the treatment concentration of TKI-0266 increased, the BLI signal of F-luc2 increased, and the concentration of 12.5 μM showed the highest activity. On the other hand, it was confirmed that the BLI signal activity of R-luc did not significantly differ between the vehicle group and the TKI-0266-treated group. NIS promoter activity normalized by cell viability means a difference in F-luc2 and R-luc signal intensity.
4. Examination of Change in Protein Expression in 8505C-PNIS-PCMV Cells by TKI-0266
4-1. Examination of Change in NIS Expression
Whether the expression of endogenous NIS protein would be changed by TKI-0266 treatment was examined. As a result, it could be confirmed that the expression of endogenous NIS protein in the TKI-0266-treated group significantly increased, and the expression of NIS protein was the highest when the treatment concentration was 12.5 μM. The quantitative analysis of the band intensity on the Western blot also showed the same results.
In addition, as a result of examining intracellular regions expressing the NIS protein, it could be confirmed that expression of the NIS protein increased mainly in the cell membrane and that the expression of cytosolic NIS protein by TKI-0266 treatment was uptick.
4-2. Examination of Protein Involved in MAPK Signaling Pathway
The effect of TKI-0266 on the MAPK (mitogen-activated protein kinase) and PI3K-AKT signaling pathway related to the expression of thyroid-specific genes was evaluated.
As a result, it could be seen that the level of phosphorylated-AKT (hereinafter referred to as p-AKT) protein had little difference between the vehicle group and the TKI-0266-treated group. However, it could be confirmed that the level of phosphorylated ERK (hereinafter referred to as p-ERK) protein significantly decreased in the TKI-0266-treated group.
4-3. Examination of Changes in Expression of TPO, TSHR, Tg, TTF-1 and Pax-8
TPO (thyroid peroxidase), TSHR (thyroid stimulatine hormone receptor), Tg (thyroglobulin), TTF-1 (thyroid transcription factor-1) and Pax8 (paired box gene 8) are known to play an important role in thyroid follicular cells. Changes in the expression of the proteins in the 8505C-PNIS-PCMV cells were examined, and as a result, it could be confirmed that the expression of the proteins increased in the TKI-0266-treated group.
5. Evaluation of the Effect of TKI-0266 Treatment on Iodine and Radioiodine Uptake in Anaplastic Thyroid Cancer (ATC) Cells
Iodine uptake significantly increased in the TKI-0266-treated group compared to the vehicle group, and iodine uptake was completely blocked by treatment with the NIS inhibitor KCIO4. In addition, it could be confirmed that the colony-forming ability in the cells in the group co-treated with 131I and TKI-0266 significantly decreased compared to that in the vehicle group, the 131I-treated group and the TKI-0266-treated group. Further, it could be confirmed that the colony-forming ability of the cells in the TKI-0266 treated group decreased compared to that in the vehicle group.
6. Examination of Change in NIS Expression in Anaplastic Thyroid Cancer Cells by TKI-0266 Treatment
The degree of NIS enhancement by TKI-0266 treatment was examined. As a result, it could be seen that the expression of endogenous NIS expression in the TKI-0266-treated group was robust, but that in the vehicle group was weak.
7. Examination of Change in Iodine Uptake in Anaplastic Thyroid Cancer Xenograft Mouse Models by TKI-0266 Administration
Based on the in vitro experiment results, whether the increase in NIS expression by TKI-0266 administration would have an effect on the radioisotope uptake in cancer cells was examined.
TKI-0266 was administered to anaplastic thyroid cancer xenograft mice constructed by transplanting 8505C cells (5×106), and 18.5 to 22.2 MBq 99mTc-pertechnetate was administered to the mice, followed by pinhole gamma camera imaging. As a result, it could be seen that in the case of the TKI-0266-administered group, 99mTc-pertechnetate accumulation in the cancer cells significantly increased and the iodine uptake of the thyroid cancer cells was enhanced by TKI-0266 administration.
In particular, it could be confirmed that when 50 mg/kg of TKI-0266 was administered, the radioisotope uptake was the best compared to that in the vehicle group.
8. Examination of Change in Luciferase Activity in Anaplastic Thyroid Cancer Xenograft Mice by TKI-0266 Administration
D-luciferin was administered to anaplastic thyroid cancer xenograft mice, and then the activity thereof was monitored. As a result, it could be seen that in the TKI-0266-administered group, the activity of F-luc2 significantly increased, meaning that the expression of NIS increased. In addition, it could be confirmed that in the vehicle group, the activity of F-luc2 was uptick because of tumor growth.
9. Examination of Tumor Growth Inhibitory Effect of Single Administration of TKI-0266 and Co-Administration of 131I and TKI-0266 in Anaplastic Thyroid Cancer Xenograft Mice
It could be confirmed that the vehicle group and the group administered with 131I alone showed a continuous increase in the tumor size on days 6 and 11, and the group administered with TKI-0266 alone also showed an increase in the tumor size on days 6 and 11. However, it could be confirmed that the group administered with TKI-0266 alone showed a slow tumor growth rate compared to the vehicle group and the group administered with 131I alone, indicating that the tumor growth inhibitory effect appeared in the group administered with TKI-0266 alone.
In addition, in the group co-administered with 131I and TKI-0266, the R-luc signal intensity decreased on day 6 after administration, and then was restored on day 11 after administration. This suggests that the group co-administered with 131I and TKI-0266 showed the best tumor growth inhibitory effect among the four experimental groups. However, it could be confirmed that all the experimental groups showed no significant change in the mouse body weight during the experiment.
10. Confirmation of Increased Expression of NIS and Cleaved Caspase-3 in Anaplastic Thyroid Cancer Xenograft Mice by Administration of TKI-0266
It could be seen that the expression of NIS-specific protein was high in the group administered with TIK-0266, and the expression of cleaved caspase-3 indicating cell death was the highest in the group co-administered with 131I and TKI-0266.
Those skilled in the art to which the present disclosure pertains will appreciate that the present disclosure may be embodied in modified forms without departing from the essential characteristics of the present disclosure. Therefore, the disclosed embodiments should be considered as being illustrative in all respects, rather than restrictive. The scope of the present disclosure should be defined by the appended claims rather than the above description, and all differences within the scope equivalent to the claims should be interpreted as falling within the scope of the present disclosure.
Claims
1. A composition for treating or preventing thyroid cancer containing, as an active ingredient, a compound of the following Formula 1, a salt thereof or a solvate thereof:
2. The composition of claim 1, wherein the thyroid cancer is anaplastic thyroid cancer or differentiated thyroid cancer.
3. The composition of claim 1, wherein the compound of Formula 1 increases expression of sodium iodide symporter.
4. An anticancer adjuvant for thyroid cancer containing, as an active ingredient, a compound of the following Formula 1, a salt thereof or a solvate thereof:
5. The anticancer adjuvant of claim 4, wherein the compound of Formula 1 increases expression of sodium iodide symporter.
Type: Application
Filed: Oct 19, 2018
Publication Date: Aug 6, 2020
Applicant: Kyungpook National University Industry-Academic Cooperation Foundation (Daegu)
Inventors: Byeong Cheol AHN (Daegu), Ji Min OH (Daegu)
Application Number: 16/757,486
