KR101948247B1 - A composition for suppressing OCT4 and use thereof - Google Patents
A composition for suppressing OCT4 and use thereof Download PDFInfo
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Abstract
The present invention relates to a composition for inhibiting OCT4 (Octamer-Binding Transcription Factor 4) and its use. A composition for inhibiting the formation of a teratoma or a teratocarcinoma of a cell therapeutic agent for removing undifferentiated cells using the composition; A pharmaceutical composition for preventing or treating testicular cancer; A method for removing undifferentiated cells; Methods of inhibiting teratoma or teratocarcinoma formation; A method for producing a cell therapeutic agent; To a method of preventing or treating testicular cancer.
Description
The present invention relates to a composition for inhibiting OCT4 (Octamer-Binding Transcription Factor 4) and its use. A composition for inhibiting the formation of a teratoma or a teratocarcinoma of a cell therapeutic agent for removing undifferentiated cells using the composition; A pharmaceutical composition for preventing or treating testicular cancer; A method for removing undifferentiated cells; Methods of inhibiting teratoma or teratocarcinoma formation; A method for producing a cell therapeutic agent; To a method of preventing or treating testicular cancer.
Human induced pluripotent stem cells are pluripotency cells that can be differentiated into endoderm, mesoderm, and ectoderm that constitute the human body. They are cells that sustain self-renewal. They are regenerative medicine, Lt; RTI ID = 0.0 > cells. ≪ / RTI > In other words, induced pluripotent stem cells can differentiate into neurons, hepatocytes, blood cells, etc. that can treat damaged tissue. Induced pluripotent stem cells were produced by Yamanaka in 2006 by introducing four transcription factors (Oct4, Sox2, c-Myc and Klf4 transcription factors) into adult cells (Cell. 126 (4): 663-76) Various methods for the production of inducible pluripotent stem cells have been proposed. In general, OCT4 and SOX2 transcription factors are very important factors for the induction of stem cells in adult cells. Myc gene plays a role in improving the efficiency of stem cell production. In particular, the expression of OCT4 is important for maintaining the pluripotency of induced pluripotent stem cells.
In order to obtain cells differentiated from human induced pluripotent stem cells expressing OCT4, it is necessary to induce the differentiation of human induced pluripotent stem cells into specific therapeutic cells, and to remove the surface makers present in the cell membrane that specify the differentiated cells Using antibodies, Fluorescent Activated Cells Sorter (FACS) is used to obtain only the differentiated cells expressing the marker. However, there is a risk of damaging the therapeutic cell by the laser used when the flow cytometer separator is used.
Magnetic cell sorting (MACS) has been used to label cells using antibodies against markers present in the cell membrane to avoid damage to therapeutic cells. The Magnetic Separation Method (MACS) has the advantage of eliminating the risk of cell damage to the laser used when using a flow cell separator.
However, both FACS and MACS methods can not completely exclude the possibility that pluripotent pluripotent pluripotent stem cells are included in the therapeutic cell population. If there is a technical limitation in selecting only cells that have differentiated into 100% purity as therapeutic cells, serious safety problems may arise. Particularly, there is a possibility that undifferentiated cells expressing OCT4 are mixed in the differentiated cells derived from embryonic stem cells. Therefore, in the development of a cell therapy agent, there is a risk of tumor formation due to undifferentiated pluripotent stem cells called " teratoma " Problems are constantly emerging (Stem Cells. 2009, 27 (5): 1050-1056.).
Many interspecies studies have reported on studies of cell therapy by inoculating mouse stem cells into the brain of rats, in which case teratoma formation was inhibited. However, in allogeneic studies, in mouse embryonic stem cells, tumors were formed in all mice when only 500 stem cells were inoculated into mouse brain tissue (J. CEREBRAL BLOOD FLOW MET, 2003, 23: 780). Therefore, there is a desperate need to develop a technique for selectively removing undifferentiated cells expressing OCT4 in which there is a risk of teratoma without affecting the differentiated cells.
On the other hand, the onset of the tumor and the development of the embryo have similar characteristics. Embryonic carcinoma cells (ECC) refers to stem cells of teratocarcinoma, which are tumors of embryonic stem cells (ESC) from the inner cell mass of the embryo. Interestingly, these ECCs and ESCs have phenotypic plasticity and thus differentiate in certain circumstances. So far, important transcription factors involved in self-renewal and pluripotency of ECC and ESC have been reported (Jung et al. 2010, PLoS ONE 5, e10709; Young, 2011, Cell, 144, 940- 954.).
Under these circumstances, the present inventors have completed the present invention by confirming that the capturing derivative compounds can modulate the expression and activity of the stem cell transcription factor OCT4 to specifically / selectively remove OCT4 expressing cells.
One object of the present invention is to provide a composition for inhibiting OCT4 (Octamer-Binding Transcription Factor 4) comprising a compound represented by the formula (1) or a pharmaceutically acceptable salt thereof.
Another object of the present invention is to provide a compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof; Or for removing undifferentiated cells comprising said composition; Or a composition for inhibiting the formation of a teratoma or a teratocarcinoma of a cell therapy agent.
Another object of the present invention is to provide a compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof; Or a pharmaceutical composition for preventing or treating testicular cancer, which comprises the composition.
Another object of the present invention is to provide a compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof; Or treating the composition with a cell therapeutic agent; Or a method of inhibiting teratoma or teratocarcinoma formation.
Another object of the present invention is to provide a compound represented by the general formula (1) or a pharmaceutically acceptable salt thereof; Or a method for producing a cell therapeutic agent comprising the step of treating the composition with a cell therapeutic agent.
Another object of the present invention is to provide a compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof; Or a method for the prophylaxis or treatment of testicular cancer, comprising the step of administering the composition.
This will be described in detail as follows. On the other hand, each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention fall within the scope of the present invention. Further, the scope of the present invention is not limited by the detailed description described below.
According to one aspect of the present invention, there is provided a composition for inhibiting OCT4 (Octamer-Binding Transcription Factor 4) comprising a compound represented by the following
In Formula 1,
Further, in one embodiment of the present invention,
R1 is H, or hydroxy;
R2 is H, or Ci- 4 alkoxy;
R3 is H, hydroxy, or C 1-4 alkoxy;
R4 is H, C 1-4 alkoxy, or halogen;
R5 is H;
R1 'is H, hydroxy or C 1-4 alkoxy;
R2 ' is H, or halogen;
R3 'is H, or C 1-4 alkoxy;
R4 ' is H;
R5 'may be a H, hydroxy, or C 1-4 alkoxy, but is not limited thereto.
In one embodiment of the present invention, R1, R2, R3, R4, R5, R1 ', R2', R3 ', R4' and R5 'are each independently selected from the group consisting of H, hydroxy, methoxy and bromo But are not limited thereto.
Further, in one embodiment of the present invention,
R1 is H, or hydroxy;
R2 is H, or methoxy;
R3 is H, hydroxy, or methoxy;
R4 is H, methoxy, or bromo;
R5 is H;
R1 'is H, hydroxy, or methoxy;
R2 ' is H, or bromo;
R3 ' is H, or methoxy;
R4 ' is H;
R5 'can be H, hydroxy, or methoxy, but is not limited thereto.
The compounds represented by Formula 1 according to the present invention include all pharmaceutically acceptable salts thereof as well as possible solvates and hydrates thereof which may be prepared therefrom and include all possible stereoisomers. The solvates, hydrates and stereoisomers of the compound represented by the formula (1) can be prepared from the compound represented by the formula (1) using conventional methods.
The compound represented by the formula (1) according to the present invention may be prepared in a crystalline form or in an amorphous form, and may be optionally hydrated or solvated when prepared in crystalline form. In the present invention, compounds containing various amounts of water as well as stoichiometric hydrates of the compound represented by the formula (1) may be included. Solvates of the compounds of formula (I) according to the present invention include both stoichiometric solvates and non-stoichiometric solvates.
The method for obtaining the compound represented by
The compound represented by the above-mentioned
In addition to the specific inhibitory effect on the OCT4 of the compound represented by the above formula (1), the effect of removing undifferentiated cells, the specific inhibitory effect on the teratoma or teratocarcinoma, and the preventive and therapeutic effect of testicular cancer, It is a new effect without bar.
In the present invention, the term "pharmaceutically acceptable salt" means a salt commonly used in the medical industry. Examples of the salt include inorganic ion salts such as calcium, potassium, sodium and magnesium, hydrochloric acid, nitric acid, phosphoric acid, There may be mentioned inorganic acid salts prepared from acid, iodic acid, perchloric acid, tartaric acid and sulfuric acid and the like, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, acetic acid, trifluoroacetic acid, , Acetic acid, carbonic acid, vanillic acid, lactic acid, glycolic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid , Hydroiodic acid, etc., sulfonic acid salts such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and nattalenesulfonic acid, glycine, N, lysine and the like, and amine salts prepared with trimethylamine, triethylamine, ammonia, pyridine, picoline and the like. However, the types of salts defined in the present invention are limited by the listed salts no.
In the present invention, 'Octamer-Binding Transcription Factor 4' (OCT4) It is a transcription factor also called POU5F1 (POU domain,
In the present invention, " OCT4 inhibition " means decreasing or inhibiting the expression and / or activity of OCT4. Since OCT4 inhibition inhibits cell growth and induces apoptosis, it can prevent or treat cell proliferative diseases and can selectively kill undifferentiated cells. As a result, compounds inhibiting OCT4 can be used as a therapeutic agent for cell therapy, Can be used as the compound. In addition, the compound inhibiting OCT4 can inhibit the generation of teratoma or teratocarcinoma due to the administration of a cell therapy agent, effectively kills cancer cells expressing OCT4, and can be used as an excellent chemotherapeutic agent.
Specifically, the compound represented by
The compound of
The compound of
The compound of
In the present invention, the compound of
The compound of formula 4 is a compound having the formula C 17 H 18 BrO 5 , the IUPAC designation is 5-bromo-2,2'-dihydroxy-4 ', 6'-dimethyl- 2'-dihydroxy-4 ', 6'-dimethoxychalcone).
The compound of Formula 4 is the compound of
The compound of
The compound of formula (5) is a compound of formula (1) wherein
The compound of
The compound of formula (6) is a compound of formula (1) wherein R1 is substituted by hydroxy and the remainder by hydrogen. In the present invention, the compound of formula (6) is designated as KRIBB59 (K59).
The inhibition of OCT4 may be, but is not limited to, in vitro, in vivo, or ex vivo.
In one specific example of the present invention, the compounds of the
The present invention relates to a compound of the above formula (1), a compound of any one of the above formulas (2) to (6), or a pharmaceutically acceptable salt thereof; Or a composition comprising the same. The present invention also provides a composition for removing undifferentiated cells. Specifically, the composition may be a composition for removing undifferentiated cells of a cell therapy agent, but is not limited thereto.
The present invention also relates to the aforementioned compound of
In the present invention, "cell therapy products" refers to living cells used for cell therapy, and more specifically refers to living cells used for autologous, allogenic, xenogenic, ) Means a drug that contains cells that have been proliferated and selected in vitro. The types of the cell therapy agents can be divided into somatic cell therapy agents and stem cell treatment agents depending on the degree of differentiation. The stem cell treatment agents can be divided into embryonic stem cell treatment agents, induced pluripotent stem cell treatment agents and adult stem cell therapy agents, It does not. In addition, the cell therapeutic agent may include, but is not limited to, undifferentiated cells.
The term " undifferentiated cell " as used herein refers to a cell that does not yet undergo differentiation and has room for differentiation, and includes embryonic stem cells, induced pluripotent stem cells, tumor cells, and cancer cells. More specifically, the undifferentiated cell may be an undifferentiated cell expressing OCT4.
In the present invention, 'teratoma' refers to a teratoma composed of various cells and tissues such as skin cells, muscle cells, nerve cells, etc., and a problem that teratoma may be generated due to undifferentiated cells contained in a cell therapy agent have.
In the present invention, 'teratocarcinoma' means an embryonal carcinoma in which embryonal carcinoma and teratoma coexist, and also called teratocarcinoma.
The removal of the undifferentiated cells, inhibition of teratoma or teratocarcinoma formation may be accomplished in vitro, in vivo, or ex vivo, but is not limited thereto.
The compound represented by the formula (1) or the compound represented by any one of the formulas (2) to (6) of the present invention can inhibit the expression or activity of the OCT4 protein to remove undifferentiated cells contained in the cell therapeutic agent, Which can inhibit teratoma or teratocarcinoma.
An embodiment of the present invention relates to a method for treating a cell treatment agent before administration to a subject and / or treating a composition comprising the same and / or a composition comprising the compound of the present invention To remove undifferentiated cells of the cell therapy agent or to inhibit the formation of the teratoma or teratocarcinoma.
In a specific example of the present invention, it was confirmed that the expression of OCT4 protein level was observed only in undifferentiated cells such as inducible pluripotent stem cells and not observed in neural stem cells (FIG. 8) But also selectively induced pluripotent stem cells.
Therefore, the compound of the present invention specifically inhibits OCT4-expressing cells, and thus can be used not only for the removal of undifferentiated cells of cell therapy agents, but also for suppressing the formation of teratoma or teratocarcinoma of cell therapy agents .
Yet another aspect of the present invention relates to a method for preventing or treating cancer, comprising the step of administering to a patient a composition comprising the compound of
Specific examples of the cancer include, but are not limited to, liver cancer, colon cancer, breast cancer, stomach cancer, lung cancer, pancreatic cancer, thyroid cancer, uterine cancer, prostate cancer or testicular cancer.
The term " prophylactic, " as used herein, refers to any act that inhibits or delays the onset of cancer, specifically testicular cancer, by administration of a composition comprising a compound of the invention or a pharmaceutically acceptable salt thereof.
The term " treatment " as used herein refers to any action in which administration of a composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof improves or alleviates the symptoms of the disease.
The compound represented by the formula (1) or the compound represented by any one of the formulas (2) to (6) of the present invention specifically inhibits the cancer cell expressing OCT4 by inhibiting the expression or activity of the OCT4 protein, .
The pharmaceutical composition of the present invention may further include, but is not limited to, an anticancer agent (for example, an anticancer agent for testicular cancer and the like). The anticancer agent may be selected depending on the purpose of prevention or treatment.
In a specific embodiment of the present invention, the compound of the present invention is treated with a testicular cancer cell line to confirm the cancer cell apoptosis inducing effect of the compound of the present invention. As a result, (FIGS. 5B and 5C), it was confirmed that cancer cells can be specifically inhibited (FIG. 6A) without affecting normal cells, and thus can be useful for prevention and treatment of cancer .
In the present invention, the "pharmaceutical composition" may further comprise suitable carriers, excipients or diluents conventionally used in the production of pharmaceutical compositions. Specifically, the pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method .
In the present invention, the carrier, excipient and diluent which may be contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.
Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, Sucrose, lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.
Liquid preparations for oral use may include various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are simple diluents commonly used in suspension, liquid solutions, emulsions and syrups have.
Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.
Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.
The amount of the compound or pharmaceutically acceptable salt thereof contained in the pharmaceutical composition according to an embodiment of the present invention is not particularly limited, but is preferably 0.0001 to 50% by weight, more preferably 0.01 to 50% by weight, 5% by weight.
The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. The term " pharmaceutically effective amount " in the present invention means a therapeutically effective amount for treating or preventing a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention And the effective dose level refers to the amount of the effective amount of the composition of the present invention, and the effective dose level is determined depending on the severity of the disease, the activity of the drug, the age, body weight, health, sex, The duration of the treatment, the factors including the drugs used in combination or concurrently with the composition of the present invention used, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered singly or multiply. It is important to take into account all of the above factors and administer an amount that will achieve the maximum effect in the least amount without side effects.
The dosage of the pharmaceutical composition of the present invention can be administered, for example, such that the composition containing the compound is administered to an individual at 10 to 1,000 mg / kg, more specifically, 10 to 600 mg / kg for a day, The dosage of the composition of the present invention is not particularly limited, but may be administered once a day or divided into several doses.
In another aspect of the present invention, the present invention provides a pharmaceutical composition comprising the compound of
The term "compound of
The compounds or compositions of the present invention may be administered to a cell therapy agent in vitro, in vivo, or ex vivo.
An embodiment of the present invention relates to a method for treating a cell treatment agent before administration to a subject and / or treating a composition comprising the same and / or a composition comprising the compound of the present invention To remove undifferentiated cells of the cell therapy agent or to inhibit the formation of the teratoma or teratocarcinoma.
According to another aspect of the present invention, there is provided a pharmaceutical preparation for preventing or treating teratoma or teratocarcinoma, comprising a compound of
The term "compound of
The removal of the undifferentiated cells, inhibition of teratoma or teratocarcinoma formation may be accomplished in vitro, in vivo, or ex vivo, but is not limited thereto.
In a specific example of the present invention, it was confirmed that the expression of OCT4 protein level was observed only in undifferentiated cells such as inducible pluripotent stem cells and not observed in neural stem cells (FIG. 8) But also selectively induced pluripotent stem cells.
Therefore, the compound of the present invention specifically inhibits OCT4-expressing cells, and thus can be used not only for the removal of undifferentiated cells of cell therapy agents, but also for suppressing the formation of teratoma or teratocarcinoma of cell therapy agents .
In another aspect of the present invention, the present invention provides a pharmaceutical composition comprising the compound of
The term "compound of
The compound of the present invention or the composition for inhibiting OCT4 can kill undifferentiated cells remaining in a cell therapeutic agent through inhibition of the expression or activity of OCT4 protein and inhibit the generation of teratoma or teratocarcinoma, Can be produced.
In another aspect of the present invention, the present invention provides a pharmaceutical composition comprising the compound of
More specifically, the present invention provides a method of preventing or treating testicular cancer, comprising the step of administering the composition to a subject.
The compound of formula (1), the compound of any one of the above formulas (2) to (6), the pharmaceutically acceptable salt and the cancer are as described above.
As used herein, the term " individual " may refer to any animal, including a human, who has, or is likely to develop, cancer, specifically, testicular cancer. The animal may be, but is not limited to, a mammal such as a cow, a horse, a sheep, a pig, a goat, a camel, a nutrient, a dog, a cat,
The preventive or therapeutic method of the present invention may specifically include the step of administering the composition in a pharmaceutically effective amount to a subject who has developed or is at risk of developing cancer, specifically testicular cancer.
In a specific embodiment of the present invention, the compound of the present invention is treated with a testicular cancer cell line to confirm the cancer cell apoptosis inducing effect of the compound of the present invention. As a result, (FIGS. 5B and 5C), it was confirmed that cancer cells can be specifically inhibited (FIG. 6A) without affecting normal cells, and thus can be useful for prevention and treatment of cancer .
The compound of the present invention inhibits the expression or activity of OCT4 protein, thereby killing the undifferentiated cells remaining in the cell treatment agent, inhibiting the generation of teratoma or teratocarcinoma, and preventing or treating testicular cancer And can be usefully utilized as a pharmaceutical composition.
1 is a Western blot analysis image showing whether or not the expression of OCT4 is inhibited in NCCIT testicular cancer cells treated with 2 μM, 5 μM and 10 μM of the compounds K53, K55, K56, K58 and K59, respectively. OCT4 expression quantification was performed using LAS-2000 image analysis instrument.
FIG. 2 is a Western blot analysis image of an OCT4 protein expressed by treating compound K53 with NCCIT testicular cancer cells at different concentrations, using an OCT4-specific antibody. FIG.
FIG. 3 is a graph showing changes in mRNA and protein levels of genes NANOG and USP44, which are induced by OCT4 expression when compound K53 is treated to NCCIT testicular cancer cells at different concentrations.
FIG. 4 is a graph showing the inhibition of OCT4 expression and the degree of cleavage of PARP, a cell death marker, by treating NCCIT testicular cancer cells with 20 μM concentration of compound K53 at different times.
5A is a graph showing a change in body weight of nude mouse regression model by intraperitoneal administration of compound K53 (30 mg / kg).
FIG. 5B is a graph showing the tumor size change of the nude mouse regression model by the intraperitoneal administration of the compound K53 according to the administration period.
5c is an image of tumor of nude mouse regression model by intraperitoneal administration of compound K53.
6A is a graph showing the number of cells induced when compound K53 was treated with two testicular cancer cell lines (NCCIT, Tera-1) and two normal cell lines (HFF, MCF10A).
FIG. 6B is a Western blot analysis image showing the degree of expression of OCT4 in two testicular cancer cell lines (NCCIT, Tera-1) and two normal cell lines (HFF, MCF10A) using an OCT4-specific antibody.
FIG. 7 shows the results of affinity chromatography of K53-Biotin (KRIBB53-Biotin) synthesized by the specific binding of OCT4 protein to the K53 compound.
8 is an image of western blot analysis in which OCT4 expression of human induced pluripotent stem cells (iPSC) and neural stem cells (NSC) differentiated therefrom is analyzed using an OCT4 specific antibody. Induced pluripotent stem cells express OCT4, but the expression of OCT4 was inhibited in the neural stem cells differentiated therefrom.
FIG. 9A is a micrograph showing the cell morphology observed by treating human induced pluripotent stem cells (iPSC) and neural stem cells (NSC) with compounds K53, K55, K56, K58 or K59 at a concentration of 5 μM for 24 hours.
FIG. 9B is a graph showing survival cell numbers of human induced pluripotent stem cells (iPSC) and neural stem cells (NSC) treated with the compound K53, K55, K56, K58 or K59 at a concentration of 5 μM for 24 hours.
10 is a graph showing the number of surviving cells after treating NCCIT testicular cancer cells with the compounds K53, K55, K56, K58 and K59 at a concentration of 10 μM and 30 μM for 48 hours, respectively.
Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.
< Example 1 > The compounds K53, K55, K56, K58 and K59 OCT4 Identification of the activity inhibition effect
To confirm the inhibitory effect of the compounds K53, K55, K56, K58, and K59 on OCT4 activity, the following dual luciferase method was performed.
The OCT4-binding ORE (OCT4 Response Element) was prepared by the PCR method and inserted into the pTA-Luc plasmid (manufactured by Clontech) at the Korea Research Institute of Bioscience and Biotechnology to produce Firefly luciferase, [PORE-TA-Luc], which is a plasmid in which the expression of [pORE-TA-Luc] is increased. Regardless of the activity of OCT4, the [pRL-TK] vector, a plasmid that constantly expresses Renilla luciferase, was purchased from Promega. [pORE-TA-Luc] and [pRL-TK] plasmids were simultaneously infected with a testicular cancer cell line (NCCIT, purchased from ATCC) using X-tremeGENE (Roche, USA).
Plasmid-introduced testicular cancer cells were removed from the cell culture dish using 0.05% trypsin-EDTA and 20,000 cells were inoculated in each well of a 96-well assay plate. K56, K58, and K59 were cultured in DMSO (Sigma Chemical Co.) in a medium containing 10% FBS for 18 hours in a 37 ° C incubator containing 5% 1000 μM was added at a concentration of 10 μM and cultured in a 37 ° C. incubator for 24 hours. A separate substrate, Beetle luciferin (Promega) and coelenterazine (Promega), specific for Firefly luciferase and Renilla luciferase, were added to each well in an amount of 25 μl.
The luminescence intensity due to the decomposition of the substrate was measured using a GloMax TM 96 Microplate Luminometer (Promega), and the measured value was calculated from the following formula (1) The measured firefly luciferase activity, which proportionally reflects the activity, is characterized by the nonspecific cytotoxicity and transfection that occurs in each experimental group using the activity of Renilla luciferase expressed by a constantly expressing promoter, The deviation of the efficiency was corrected).
[Equation 1]
Active inhibition (%) = 100 x [1-
(RLU: relative luminescence unit)
[PORE-TA-Luc] + [pRL-TK] + [DMSO], and the sample RLU represents [pORE-TA-Luc] + [pRL-TK] + [compound 1] , [PORE-TA-Luc] is a firefly luciferase expression plasmid having a promoter regulated by OCT4, and [pRL-TK] means a plasmid always constantly expressing Renilla luciferase .
As a result, as shown in Table 1, it was confirmed that the compounds K53, K55, K56, and K59 inhibited the activity of luciferase by 50% or more, thereby inhibiting OCT4 activity. In particular, K56 and K59 exhibited almost 100% active inhibition, indicating that the OCT4 activity inhibiting effect is more excellent.
< Example 2> Compounds of the compounds K53, K55, K56, K58 and K59 OCT4 Confirming expression suppression effect
In addition to the OCT4 activity inhibitory effect, the compounds K53, K55, K56, K58, and K59 inhibited OCT4 gene expression.
First, NCCIT cells were inoculated in a 6-well plate at 2.5 × 10 5 cells. After 18 hours, the compounds K53, K55, K56, K58 and K59 were treated with 2 μM, 5 μM and 10 μM, respectively, for 24 hours. The treated cells were lysed with RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM sodium vanadate, 0.5% sodium deoxycholate, 0.05% sodium deoxy sulfate). The cell lysate was centrifuged at 15,000 g to recover the supernatant, and the concentration of OCT4 and GAPDH protein in the recovered cell lysate was measured using Bradford reagent (Bio-Rad protein assay, USA).
After 15 μg of the cell lysate was separated by 10% SDS-PAGE (SDS-polyacrylamide gel electrophoresis), proteins were electrophoresed on a PVDF membrane (Millipore, USA) and TBST (50 mM Tris-HCl, 6, 150 mM NaCl, 0.1% tween 20). The cells were reacted with OCT4 antibody (Cell signaling, USA) and GAPDH antibody (Santacruz, USA) for 2 hours and reacted with HRP-conjugated secondary antibody (Jackson Immunolab, USA) for 30 minutes. Then, chemiluminescence POD reagent (Roche, Germany) were used to detect OCT4 and GAPDH expression levels.
As a result, as shown in FIG. 1, it was confirmed that the compounds K53, K55, K56, K58 and K59 showed an activity of inhibiting the expression of OCT4 in a concentration-dependent manner.
< Example 3> Concentration of Compound K53 OCT4 Confirming expression suppression effect
Next, it was tried to specifically confirm whether compound K53 inhibits OCT4 expression in a concentration-dependent manner.
First, NCCIT cells were inoculated in a 6-well plate at 2.5 × 10 5 cells. After 18 hours, the compound K53 was treated at a concentration of 3 μM, 5 μM, 10 μM, 20 μM and 30 μM for 12 hours. The treated cells were lysed with RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM sodium vanadate, 0.5% sodium deoxycholate, 0.05% sodium deoxy sulfate). The cell lysate was centrifuged at 15,000 g, and the supernatant was collected. The concentration of OCT4 and GAPDH protein in the recovered cell lysate was measured using a Bradford reagent (Bio-Rad protein assay, USA).
After 15 μg of the cell lysate was separated by 10% SDS-PAGE (SDS-polyacrylamide gel electrophoresis), proteins were electrophoresed on PVDF membrane (Millipore, USA) and TBST (50 mM Tris- 6, 150 mM NaCl, 0.1% tween 20). After incubation with OCT4 antibody (Cell signaling, USA) and GAPDH antibody (Santacruz, USA) for 2 hours and reaction with HRP conjugated secondary antibody (Jackson Immunolab, USA) for 30 minutes, chemiluminescence POD reagent ) Were used to detect OCT4 and GAPDH expression levels.
As a result, as shown in FIG. 2, it was confirmed that the compound K53 was able to inhibit the expression of OCT4 in a concentration-dependent manner.
< Example 4> of compound K53, OCT4 Downstream Gene NANOG And USP44 Confirming expression suppression effect
NANOG, which is induced by OCT4 transcription factors, plays an important role in stem cell maintenance. Therefore, the change in mRNA expression level of NANOG and USP44 induced by OCT4 when compound K53 was treated was measured by the following experimental method, and the inhibitory effect of compound K53 on NANOG and USP44 mRNA expression was confirmed.
The 6-well plate was inoculated with 2.5 x 10 < 5 > cells of NCCIT cells. After 18 hours, the compound K53 was treated at a concentration of 3 μM, 5 μM, 10 μM, 20 μM and 30 μM for 12 hours. The treated cells were lysed with RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM sodium vanadate, 0.5% sodium deoxycholate, 0.05% sodium deoxy sulfate). The cell lysate was centrifuged at 15,000 g to recover the supernatant, and the protein concentration of the recovered cell lysate was measured using a Bradford reagent (Bio-Rad protein assay, USA).
After 15 μg of the cell lysate was separated by 10% SDS-PAGE (SDS-polyacrylamide gel electrophoresis), proteins were electrophoresed on PVDF membrane (Millipore, USA) and TBST (50 mM Tris- 6, 150 mM NaCl, 0.1% tween 20). (Jackson Immunolab, USA) for 2 min. The reaction was then incubated with NANOG antibody (R & D systems, USA), USP44 antibody (Santacruz, USA) and GAPDH antibody (Santacruz, , and chemiluminescence POD reagent (Roche, Germany).
RNA was collected from TRIzol (Invitrogen). Approximately 2 μg of the obtained RNA was collected and the complementary DNA was synthesized using RevertAid first strand cDNA synthesis kit (Fermentas). The amount of mRNA expression of NANOG was confirmed by real - time reverse transcriptase chain reaction (PCR) using the fluorescence intensity of IQ ™ SYBR green supermix (BioRad) supplemented with this complementary DNA template.
As can be seen from FIG. 3, when the compound K53 was treated for 12 hours, the amount of NANOG mRNA expression was decreased as the concentration of the K53 compound was increased. In addition, it was confirmed that the amount of mRNA expression of USP44 was also decreased as the treatment concentration of compound K53 was increased.
< Example 5> Confirmation of induction of cancer cell death by compound K53
Poly (ADP-Ribose) polymerase (hereinafter referred to as PARP) is a marker that can induce apoptosis and is a typical intracellular repair system that enables the maintenance of gene stability against external stimuli. When cell death occurs, PARP is cleaved and cleavage of such PARP is considered to be a typical signal of apoptosis.
Therefore, we intend to confirm the cell apoptosis inducing activity and the teratoma inhibitory effect of compound K53 using PARP.
Specifically, a 60 mm cell culture dish was inoculated with 3 × 10 5 NCCIT cells, and the compound K53 was treated at a concentration of 20 μM and cultured for the indicated time. To analyze the cleavage of PARP by compound K53, the treated cells were dissolved in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM sodium vanadate, 0.5% sodium deoxycholate, 0.05% sodium deoxy sulfate) . The cell lysate was centrifuged at 15,000 g to recover the supernatant, and the protein concentration of the recovered cell lysate was measured using a Bradford reagent (Bio-Rad protein assay, USA).
After 30 μg of the cell lysate was separated by 7.5% SDS-PAGE (SDS-polyacrylamide gel electrophoresis), proteins were electrophoresed on a PVDF membrane (Millipore, USA) and TBST (50 mM Tris-HCl, 6, 150 mM NaCl, 0.1% tween 20). The cells were incubated with PARP antibody (Cell signaling, USA), GAPDH antibody (Santacruz, USA) for 2 hours and reacted with secondary antibody conjugated with HRP (Jackson Immunolab, USA) for 1 hour and then chemiluminescence POD reagent ). ≪ / RTI > The results are shown in Fig.
As can be seen from FIG. 4, it can be seen that the cleaved PARP form is observed in proportion to the treatment time of the compound K53. Therefore, it can be seen that the compound K53 induces cell death of NCCIT expressing OCT4, exhibits excellent anticancer effect, and can effectively kill undifferentiated cells or teratomas.
< Example 6> Nude mouse initiation model confirmed the side effects and anti-cancer effects of peritoneal administration of compound K53
A mixture of 5 × 10 6 cells in NCCIT Matrigel Matrix (BD biosciences) 100 μl were transferred to NOD.CB17-Prkdc scid Il2rg tm1Wjl / SzJmice (6 weeks old). One day after the inoculation, the mice were randomly divided into two groups (n = 6) in groups of 6, and the compound K53 of the present invention was added to 10% DMA (N, N-dimethylacetamide), 10% Cremophore and 80% distilled water / v / v), and compound K53 was intraperitoneally administered at a daily dose of 30 mg / kg for 50 days.
No specific symptoms were observed during the test period, and results of mouse weight change, tumor volume change, and tumor histology of the last day were as shown in Figs. 5A, 5B and 5C, respectively.
6-1) Observation of weight change
As shown in FIG. 5A, the results of the last day (day 51) showed no weight loss in the compound K53 (30 mg / kg) group as compared with the solvent control group. Therefore, it was confirmed that the compound K53 is a safe substance which does not cause side effects in vivo.
6-2) Observation of tumor volume change
As shown in FIG. 5B, the tumor size change by daily repeated intraperitoneal administration of compound K53 in the initiation model was observed for 51 days to confirm the anticancer effect. On the last day, the tumor volume was measured to be 387.8 ± 66.2 mm 3 in the solvent-treated group and 90.6 ± 26.2 mm 3 in the case of the compound K53, which was about 1/4 of the volume of the control group.
In addition, the results of FIG. 5c were calculated to show an excellent inhibition rate of 77% (p < 0.001) as a result of calculating tumor growth inhibition rate (%) through the following equation (2).
&Quot; (2) "
Tumor growth inhibition rate (%) = [1- (Test group of the t / vehicle control group t)] x 100
(Where t = Vt - Vo , Vt is measurement of the tumor volume)
6-3) Visual confirmation of tumor size
5c, which is a photograph showing the tissue of the vehicle control group and the compound K53 (30 mg / kg) administration group on the last day of the experiment (day 51).
As a result, it was confirmed that the tumor size extracted from the compound K53-treated group was remarkably small, and the compound K53 showed excellent anti-cancer effect, undifferentiated cell inhibitory effect and teratoma inhibitory effect.
< Example 7> of compound K53, OCT4 Expression Testicular cancer Identification of cell-specific growth inhibition
In order to confirm whether compound K53 selectively inhibited testicular cancer cell lines, the proliferation of testicular cancer cells NCCIT and Tera-1 was analyzed.
Specifically, 7,000 NCCIT and Tera-1 human testicular cancer cells were administered to each well of a 96-well plate and the cells were cultured in a medium containing 10% FBS in a 37 ° C incubator containing 5% carbon dioxide. After 24 hours, the medium was replaced with a medium containing the control (0.1% DMSO) or the compound K53 at 10 μM and 30 μM (the compound dissolved in DMSO was diluted with the medium) and then cultured for 72 hours. The number of cells was measured using a hematocytometer after mixing 1: 1 of the triplan blue and cells, and the results are shown in FIG. 6A.
As a result, as shown in FIG. 6 (a), when the K53 compound was treated at 30 μM, the proliferation of NCCIT and Tera-1 was inhibited by 88.69% and 70.4%, respectively. On the other hand, normal cell lines HFF and MCF10A treated with 30 μM of K53 did not inhibit proliferation.
Therefore, it can be seen that the compound K53 specifically suppresses only cancer cells and teratomas without affecting normal cells.
On the other hand, in order to confirm that the induction activity of the compound K53 is due to the inhibition of OCT4 activity, the expression level of OCT4 in the NCCIT, Tera-1, HFF and MCF10A cell lines was analyzed.
Specifically, NCCIT, Tera-1, HFF and MCF10A cells were inoculated in a 60 mm cell culture dish at 3 × 10 5 cells and cultured. Cells were lysed with RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM sodium vanadate, 0.5% sodium deoxycholate, 0.05% sodium deoxy sulfate). The cell lysate was centrifuged at 15,000 g to recover the supernatant, and the protein concentration of the recovered cell lysate was measured using a Bradford reagent (Bio-Rad protein assay, USA).
After 30 μg of the cell lysate was separated by 7.5% SDS-PAGE (SDS-polyacrylamide gel electrophoresis), proteins were electrophoresed on a PVDF membrane (Millipore, USA) and TBST (50 mM Tris-HCl, 6, 150 mM NaCl, 0.1% tween 20). The cells were reacted with OCT4 antibody (Cell signaling, USA) and Actin antibody (Santa Cruz Biotechnology, USA) for 2 hours. HRP conjugated secondary antibody (Jackson Immunolab, USA) was reacted for 1 hour and chemiluminescence POD reagent Roche, Germany). The results are shown in FIG. 6B.
As a result, as shown in FIG. 6B, it was confirmed that the compound K53 specifically inhibited OCT4-expressing testicular cancer cells and did not affect normal cells other than cancer cells.
< Example 8> OCT4 Confirmation of binding of compound K53 to the molecular target
In order to analyze more specifically the activity of K53 through OCT4 inhibition, we examined whether K53 binds to OCT4.
First, KRIBB53-biotin was synthesized by dissolving N-biotinylcaproic acid, EDC and DMAP in a DMF solvent and reacting K53 (KRIBB53) compound. The KRIBB53-biotin was then purified using HPLC.
The NCCIT lysate was prepared using homogenizer buffer (60 mM β-glycerophosphate, 25 mM MOPS (pH 7.2), 15 mM EGTA, 1 mM DTT, 1 mM phenyl phosphate, 100 μM benzamidine). After the KRIBB53-biotin was treated with 20 μM, the K53 compound was treated at 0, 50 μM, 100 μM, 200 μM and 500 μM concentrations in the solution. Then, NeutrAvidin-Agarose resin was added. The cell lysates not bound to KRIBB53-biotin were diluted with bead buffer (50 mM Tris (pH 7.4), 5 mM NaF, 250 mM NaCl, 5 mM EDTA, 5 mM EGTA, 0.1% Nonidet P-40, 10 μg / ml of leupeptin, aprotinin, and soybean trypsin inhibitor, and 100 μM benzamidine), followed by treatment with SDS-sample buffer to recover binding proteins. The recovered proteins were separated by using 10% SDS-PAGE (SDS-polyacrylamide gel electrophoresis), proteins were electrophoresed on a PVDF membrane (Millipore, USA), TBST (50 mM Tris-HCl, pH 7.6, 150 mM NaCl , 0.1% tween 20) containing 5% skim milk. The cells were incubated with OCT4 antibody (Cell signaling, USA) for 2 hours, reacted with HRP-conjugated secondary antibody (Jackson Immunolab, USA) for 1 hour and detected with chemiluminescence POD reagent (Roche, Germany). The results are shown in Fig.
As a result, as shown in FIG. 7, the binding of OCT4 to KRIBB53-biotin and the treatment of K53 (KRIBB53) showed a competitive decrease. Thus, it can be seen that K53 has an activity of directly binding to OCT4.
< Example 9> Induced pluripotent stem cells And In neural stem cells OCT4 Expression analysis
Western blotting was performed to confirm the expression of OCT4 in undifferentiated pluripotent stem cells and neural stem cells.
Derived neural stem cell CRL2097 NSC differentiated from induced pluripotent stem cell CRL2097 iPSC produced by reprogramming from human-derived fibroblast (CRL2097) purchased from ATCC was used. The differentiation into neural stem cells was based on Jun Takahashi's paper based on dual-smad inhibition. (Doi, Daisuke, et al., "Isolation of human induced pluripotent stem cell-derived dopaminergic progenitors by cell sorting for successful transplantation." Stem cell reports 2.3 (2014): 337-350.)
The pellet was loosened in RIPA (sigma # R0278-50ML) buffer and placed on ice for 1 hour to allow the protein to dissolve. After centrifugation at 16000g at 4 ° C for 20 minutes using a centrifuge, proteins were quantified by Bradford (Biorad # 500-0006) only after obtaining supernatant. 20 μg of which was loaded onto polyacrylamide gel (Biorad # 456-1083) and transferred to PVDF membrane (Biorad # 162-0177). After blocking for 1 hour at room temperature using 5% low fat dry milk (Biorad # 170-6404), OCT3 / 4 antibody (Santa Cruz # sc9081) was treated at a ratio of 1: 4000. The antibody reaction was carried out at 4 ° C for 16 hours. Then, anti-rabbit-HRP antibody (Santa Cruz # sc2004) was treated at a ratio of 1: 10000 for 1 hour at room temperature. Beta-actin antibody (Sigma # A5441) for internal control was treated at a 1: 5000 ratio.
As a result, as shown in FIG. 8, OCT4 (size: 45 kDa) expression was observed only in induced pluripotent stem cells at the protein level and not in neural stem cells.
Therefore, it can be seen that the compound K53 inhibiting OCT4 affects undifferentiated cells such as inducible pluripotent stem cells or teratoma cells, but has no effect on neural stem cells.
< Example 10> Compounds of the compounds K53, K55, K56, K58 and K59 Induced pluripotent stem cells Specific growth inhibitory effect
When the K53, K55, K56, K58, and K59 compounds were treated, the cell counting was carried out as follows to confirm the growth inhibitory effect on the induced pluripotent stem cells.
Induced pluripotent stem cell CRL2097 iPSC cultured using TeSR-E8 (stemcell # 05940) medium was separated into single cells using Accutase (Millipore # SCR005) and seeded at 5 × 10 4 / well (12) . Two days later, the drug was treated at a time when cells were stably growing. In the case of neural stem cells, the cells were seeded at 5 × 10 5 / well (24) and treated for 11 days. To confirm the significance, each condition was repeated with n = 3. DMSO (control), compound K53 or K59 was treated with 5 [mu] M each for 24 hours, and then cell counting was carried out. Treffan blue and cells were mixed 1: 1, then Countess ™ AutomatedCellCounter (Invitrogen) was used and the number of viable cells was compared under each condition treated with the drug. Data were analyzed using T - test to compare the conditions of drug treatment with DMSO.
As a result, as shown in FIGS. 9A and 9B, in the case of induced pluripotent stem cell CRL2097 iPSC expressing OCT4, apoptosis was observed under conditions of treatment with K53, K55, K56, K58 and K59, Respectively. On the other hand, in the case of neural stem cell CRL2097 NSC not expressing OCT4, no significant difference was observed between K53, K55, K56, K58 and K59, and the number of cells was not decreased.
Therefore, the compounds K53, K55, K56, K58, and K59 exhibit a specific killing effect on cancer cells, OCT4 expressing cancer cells, undifferentiated cells, or teratoma cells, and thus exhibit excellent anticancer effects and inhibit undifferentiated cells or teratoma cell formation As shown in FIG.
< Example 11> Compounds of the compounds K53, K55, K56, K58 and K59 OCT4 Specific growth inhibition effect on expression testicular cancer cells
In order to confirm whether compounds K53, K55, K56, K58, and K59 specifically inhibited the growth of OCT4 expressing testicular cancer cell line NCCIT, the proliferation of testicular cancer cells NCCIT was analyzed.
Specifically, 2x10 5 NCCIT human testicular cancer cells were administered to each well of a 6-well plate and the cells were cultured in a medium containing 10% FBS in a 37 ° C incubator containing 5% carbon dioxide. After 24 hours, the medium was replaced with a control (0.1% DMSO) or a medium containing the compounds K53, K55, K56, K58 and K59 at 10 μM or 30 μM (the compound dissolved in DMSO was diluted with the medium) and then cultured for 48 hours. The number of cells was measured using a hematocytometer after mixing 1: 1 of the trypan blue and the cells, and the results are shown in FIG.
As shown in FIG. 10, when 10 μM of K53, K55, K56, K58 and K59 compounds were treated, the proliferation of NCCIT was inhibited by 27, 42, 87, 31 and 59%, respectively. When 30 μM of K53, K55, K56, K58 and K59 compounds were treated, the proliferation of NCCIT was inhibited by 59, 97, 100, 96 and 99%, respectively.
Therefore, the compounds K53, K55, K56, K58, and K59 inhibited proliferation of NCCIT cells expressing OCT4, and showed inhibitory effects on the death of undifferentiated cells, formation of teratoma and teratocarcinoma And can also have the preventive and therapeutic effects of testicular cancer.
From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all aspects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.
Claims (11)
[Chemical Formula 1]
In Formula 1,
R1 is H, or hydroxy;
R2 is H, or Ci- 4 alkoxy;
R3 is H, hydroxy, or C 1-4 alkoxy;
R4 is H, C 1-4 alkoxy, or halogen;
R5 is H;
R1 'is H, hydroxy or C 1-4 alkoxy;
R2 ' is H, or halogen;
R3 'is H, or C 1-4 alkoxy;
R4 ' is H;
R5 'is H, hydroxy, or C 1-4 alkoxy.
R1 is H, or hydroxy;
R2 is H, or methoxy;
R3 is H, hydroxy, or methoxy;
R4 is H, methoxy, or bromo;
R5 is H;
R1 'is H, hydroxy, or methoxy;
R2 ' is H, or bromo;
R3 ' is H, or methoxy;
R4 ' is H;
R5 ' is H, hydroxy, or methoxy.
(2)
(3)
[Chemical Formula 4]
[Chemical Formula 5]
[Chemical Formula 6]
[Chemical Formula 1]
In Formula 1,
R1 is H, or hydroxy;
R2 is H, or Ci- 4 alkoxy;
R3 is H, hydroxy, or C 1-4 alkoxy;
R4 is H, C 1-4 alkoxy, or halogen;
R5 is H;
R1 'is H, hydroxy or C 1-4 alkoxy;
R2 ' is H, or halogen;
R3 'is H, or C 1-4 alkoxy;
R4 ' is H;
R5 'is H, hydroxy, or C 1-4 alkoxy.
R1 is H, or hydroxy;
R2 is H, or methoxy;
R3 is H, hydroxy, or methoxy;
R4 is H, methoxy, or bromo;
R5 is H;
R1 'is H, hydroxy, or methoxy;
R2 ' is H, or bromo;
R3 ' is H, or methoxy;
R4 ' is H;
R5 ' is H, hydroxy, or methoxy.
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| 2-Hydroxychalcone and xanthohumol inhibit invasion of triple negative breast cancer cells, Chemico-Biological Interactions, 203, 565-572 (2013.) 1부.* |
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