KR20110101391A - Composition comprising expression or activity inhibitors of dkk3 for the prevention and treatment of cancer - Google Patents

Abstract

The present invention relates to a composition for the prevention and treatment of cancer containing the expression or activity inhibitor of DKK3 protein, and more particularly, after transfection of DKK3 siRNA in H460 non-small cell lung cancer, cancer cell death and intracellular activity Since it has an effect of increasing the oxygen level, it can be usefully used as a composition for the prevention and treatment of cancer or an anticancer adjuvant containing a DKK3 protein expression or activity inhibitor.

Description

Composition comprising expression or activity inhibitors of DKK3 for the prevention and treatment of cancer}

The present invention relates to a composition for the prevention and treatment of cancer containing a DKK3 expression or activity inhibitor.

Cancer is one of the greatest threats to human health, a disease that occurs when cells multiply through a series of mutations and proliferate and immortalize in an unlimited and unregulated manner. Cancer causes include environmental or external factors such as chemicals, viruses, bacteria, ionizing radiation, and internal factors such as congenital gene mutations (Klaunig & Kamendulis, Annu Rev Pharmacol Toxicol. , 44: 239-267). , 2004).

In the early stages of cancer, there are treatments such as surgery, radiation, and chemotherapy, but the side effects are also a big problem. In the case of terminal or metastatic cancers, life ends in a limited-time life without special treatment. In addition, various biochemical mechanisms related to cancer have been elucidated and treatments have been developed accordingly, but fundamental treatments for cancer have not been suggested.

Reactive oxygen species (ROS) are produced in mitochondria during respiration and are known to be produced by the action of specific enzymes (Kamata and Hirata, Cell Signal , 1999). Low levels of ROS regulate intracellular signaling and are known to play an important role in normal cell growth (Burdon, Free Radic. Biol. Med , 1995). It is also reported that ROS products are increased in cancer cells Cancer Res ., 1991); Burdon, 1995). Increased ROS during tumor progression is the expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and activator protein 1 (AP-1) transcription factors. Induction (Gupta et al., Carcinogenesis, 1999). Oxidative stress also causes DNA damage, leading to genome instability, which is known to affect cancer progression (Jackson and Loeb, Mutat. Res. , 2001). Thus, ROS are believed to play several roles in the development, progression and maintenance of cancer. Recent studies have reported that ROS occur in cells stimulated by growth factors such as epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) (Bae et al. , J. Biol. Chem. , 1997, 2000).

ROS plays a role in promoting cell growth under stress, but it is known to activate and regulate apoptosis in conditions under stress. Levels of ROS increase when cells are exposed to various stressors (eg, anticancer agents) (Jabs, Biochem. Pharmacol. , 1999). In addition, apoptosis signal-regulating kinase 1 (ASK1), C-Jun N-terminal kinase (JNK), and p38-related genes such as apoptosis-stimulating apoptosis-related genes It is known to induce death (Benhar et al., Mol. Cell. Biol., 2001). In addition, it is known to play an important role in inducing apoptosis through p53 (Polyak et al., Nature , 1997).

Dickkopf (DKK) genes present in mammals are known as a kind of extracellular signaling substances that regulate embryonic development and tissue development. Currently, four types of DKK genes are known, DKK1, DKK2, DKK3, and DKK4, and they have been actively studied as inhibitors of Wnt / β-catenin signaling.

Of the four types of DKK genes, DKK3 is known as a potential Wnt inhibitor and is generally low expressed in human cancer cells such as lung cancer cell lines and is recognized as a potential cancer suppressor gene. In many cancers, this low expression of DKK3 is known to be due to the methylation of CpG islands present in the promoter site of DKK3, and much of the research on DKK3 is focused on this methylation. However, even cancer cells are partially expressed because DKK3 is not completely suppressed according to their type. Especially, studies on completely suppressing the low expression of DKK3 and on the cause and signal transduction process of intracellular toxicity caused by inhibition of DKK3 expression are It is not happening.

Therefore, the present inventors transfected DKK3 siRNA in H460 non-small cell lung cancer still expressing DKK3, and completely inhibited the expression of DKK3, and apoptosis and intracellular activity in the transfected H460 non-small cell lung cancer. By confirming the effect of increasing oxygen levels, the present invention has been completed.

An object of the present invention is to provide a composition for the prevention and treatment of cancer containing inhibitors of the expression or activity of DKK3 protein.

In order to achieve the above object, the present invention provides a composition for preventing and treating cancer containing DKK3 protein expression or activity inhibitor.

The present invention also provides an anticancer adjuvant containing an inhibitor of expression or activity of DKK3 protein.

In addition, the present invention provides a method for screening a composition for preventing or treating cancer.

The DKK3 protein expression or activity inhibitor of the present invention inhibits the expression of DKK3, and after transfection of DKK3 siRNA into non-small cell lung cancer H460 cells, apoptosis is observed, and with increased expression of apoptosis-related proteins, Since ROS has an effect of increasing, it can be usefully used as a composition for preventing and treating cancer or anticancer adjuvant containing DKK3 protein expression or activity inhibitor.

Figure 1 shows the methylation measurement of non-small cell lung cancer H460 cells and the transfection of DKK3 siRNA, and confirm the expression inhibition of DKK3:
Figure 1 A is a measure of the methylation of the DKK3 gene present in non-small cell lung cancer H460 cells;
FIG. 1B shows a transtranscription polymerase chain reaction of DKK3 after transfection of a negative control (NC) NC siRNA and a DKK3 siRNA that inhibits DKK3 expression in non-small cell lung cancer H460 cells. Figures confirmed using Polymerase Chain Reaction (RT-PCR);
H460: non-small cell lung cancer H460 cells;
H460 / NC: group that transfected H460 cells with NC siRNA as a control; And
H460 / SiDKK3: group transfected with DKK3 siRNA to H460 cells.
Figure 2 shows the inhibition of cell growth after DKK3 protein expression inhibition:
FIG. 2A is a diagram illustrating the morphology of cells after transfection of NC siRNA and DKK3 siRNA to H460 cells; FIG.
H460: non-small cell lung cancer H460 cells;
H460 / NC: group that transfected H460 cells with NC siRNA as a control;
H460 / SiDKK3: group transfected with DKK3 siRNA to H460 cells;
2B is a graph counting cells in the fraction of viable cells after transfection of NC siRNA, DKK3 siRNA to H460 cells;
●: non-small cell lung cancer H460 cells;
○: group transfected with NC siRNA as a control group to H460 cells;
■: group transfected with DKK3 siRNA to H460 cells;
2C is a graph confirming cell viability after transfecting NC siRNA and DKK3 siRNA to H460 cells;
●: non-small cell lung cancer H460 cells;
○: group transfected with NC siRNA as a control group to H460 cells;
■: group transfected with DKK3 siRNA to H460 cells;
Figure 2 D is a picture of measuring colony formation after transfection of NC siRNA, DKK3 siRNA to H460 cells;
H460: non-small cell lung cancer H460 cells;
H460 / NC: group transfected with NC siRNA as a control to H460 cells; and
H460 / SiDKK3: group transfected with DKK3 siRNA to H460 cells.
3 is a graph showing flow cytometry after transfection of NC siRNA, DKK3 siRNA to H460 cells:
H460: non-small cell lung cancer H460 cells;
H460 / NC: group transfected with NC siRNA as a control to H460 cells; and
H460 / SiDKK3: group transfected with DKK3 siRNA to H460 cells.
4 is a diagram showing the expression of apoptosis-related proteins and ROS levels after transfection of NC siRNA and DKK3 siRNA into H460 cells:
4A is a diagram showing the expression of apoptosis-related proteins after transfection of NC siRNA and DKK3 siRNA to H460 cells using Western blot;
H460: non-small cell lung cancer H460 cells;
H460 / NC: group that transfected H460 cells with NC siRNA as a control;
H460 / SiDKK3: group transfected with DKK3 siRNA to H460 cells;
4B is a graph confirming changes in ROS levels in H460 cells after transfection of NC siRNA and DKK3 siRNA to H460 cells;
H460: non-small cell lung cancer H460 cells;
H460 / NC: group transfected with NC siRNA as a control to H460 cells; and
H460 / SiDKK3: group transfected with DKK3 siRNA to H460 cells.

Hereinafter, the present invention will be described in detail.

The present invention provides compositions for the prevention and treatment of cancer containing DKK3 protein expression or activity inhibitors.

The DKK3 protein is preferably characterized by an amino acid sequence represented by SEQ ID NO: 1, but is not limited thereto.

Preferably, the expression inhibitor of the DKK3 protein is any one selected from the group consisting of antisense nucleotides, small interfering RNAs, and short hairpin RNAs, which complementarily bind to mRNA of the DKK3 gene. However, the present invention is not limited thereto.

 The activity inhibitor of the DKK3 protein is any one selected from the group consisting of compounds, peptides, peptide mimetics, aptamers and antibodies that specifically bind to DKK3 protein, but is not limited thereto.

The cancer is preferably any one selected from the group consisting of liver cancer, colon cancer, cervical cancer, kidney cancer, gastric cancer, prostate cancer, breast cancer, brain tumor, lung cancer, uterine cancer, colon cancer, bladder cancer, blood cancer and pancreatic cancer. More preferably, but not limited to colorectal cancer, lung cancer or colorectal cancer.

Antisense  Nucleotide

Antisense nucleotides, as defined in Watson-click base pairs, bind (hybridize) the complementary sequences of DNA, immature-mRNA or mature mRNA to disrupt the flow of genetic information as a protein in DNA. The nature of antisense nucleotides specific to the target sequence makes them exceptionally multifunctional. Since antisense nucleotides are long chains of monomeric units they can be easily synthesized for the target RNA sequence. Many recent studies have demonstrated the usefulness of antisense nucleotides as biochemical means for studying target proteins (Rothenberg et al. al ., J. Natl. Cancer Inst . , 81: 1539-1544, 1999). The use of antisense nucleotides can be considered as a new type of inhibitor because of recent advances in nucleotide synthesis and in the field of nucleotide synthesis that exhibit improved cell adsorption, target binding affinity and nuclease resistance.

Peptide mimetics ( Peptide Minetics )

Peptide Minetics are peptides or non-peptides that inhibit the binding domain of DKK3 protein and inhibit the activity of DKK3 protein. Major residues of non-hydrolyzable peptide analogs include β-turn dipeptide cores (Nagai et. al . Tetrahedron Lett 26: 647, 1985), keto-methylene pseudopeptides (Ewenson et al . J Med Chem 29: 295, 1986; And Ewenson et al . in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, IL, 1985), Azepine (Huffman et al . in Peptides: Chemistry and Biology, GR Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), benzodiazepines (Freidinger et al . in Peptides; Chemistry and Biology, GR Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), β-aminoalcohol (Gordon et al . Biochem Biophys Res Commun 126: 419 1985) and substituted gamma lactam ring (Garvey et al . in Peptides: Chemistry and Biology, GR Marshell ed., ESCOM Publisher: Leiden, Netherlands, 1988).

siRNA  molecule

The sense RNA and the antisense RNA form a double stranded RNA molecule, wherein the sense RNA is preferably an siRNA molecule comprising a nucleic acid sequence identical to the target sequence of a continuous nucleotide of some of the DKK3 mRNA. The siRNA molecule is preferably composed of a sense sequence consisting of 10 to 30 bases selected from the base sequence of the DKK3 gene and an antisense sequence complementarily binding to the sense sequence, but is not limited thereto. Any double-stranded RNA molecule having a sense sequence capable of complementarily binding to a nucleotide sequence can be used. Most preferably, the antisense sequence has a sequence complementary to the sense sequence.

Antibodies

The anti-DKK3 antibody can be prepared by the injection of anti-DKK3 or commercially available. In addition, the antibodies include polyclonal antibodies, monoclonal antibodies, fragments capable of binding epitopes, and the like.

Polyclonal antibodies can be produced by conventional methods of injecting the DKK3 into an animal and collecting blood from the animal to obtain serum containing the antibody. Such polyclonal antibodies can be purified by any method known in the art and can be made from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, cattle, dogs and the like.

Monoclonal antibodies can be prepared using any technique that provides for the production of antibody molecules through the culture of continuous cell lines. Such techniques include, but are not limited to, hybridoma technology, human B-cell hybridoma technology, and EBV-hybridoma technology (Kohler G et. al ., Nature 256: 495-497, 1975; Kozbor D et al . , J Immunol Methods 81: 31-42, 1985; Cote RJ et al. Proc Natl Acad Sci 80: 2026-2030, 1983; And Cole SP et al ., Mol Cell Biol 62: 109-120, 1984).

In addition, antibody fragments containing specific binding sites for the DKK3 can be prepared. For example, but not limited to, F (ab ') 2 fragments can be prepared by digesting antibody molecules with pepsin, and Fab fragments can be prepared by reducing the disulfide bridges of F (ab') 2 fragments. Alternatively, the Fab expression library can be made smaller to quickly and easily identify monoclonal Fab fragments with the desired specificity (Huse WD et. al . , Science 254: 1275-1281, 1989).

The antibody can be bound to a solid substrate to facilitate subsequent steps such as washing or separation of the complex. Solid substrates include synthetic resins, nitrocellulose, glass substrates, metal substrates, glass fibers, microspheres and microbeads. In addition, the synthetic resins include polyester, polyvinyl chloride, polystyrene, polypropylene, PVDF and nylon.

Aptamers  ( Aptamer )

Aptamers are single-stranded nucleic acids (DNA, RNA or modified nucleic acids) which, by themselves, have a stable tertiary structure and are capable of binding to target molecules with high affinity and specificity. Aptamers have been developed since the first development of the aptamer excavation technology called SELEX (Systematic Evolution of Ligands by Exponential enrichment) (Ellington, AD and Szostak, JW., Nature 346: 818-822, 1990). Many aptamers have been unearthed that can bind to various target molecules. Aptamers are often compared to single antibodies because of their inherent high affinity (usually pM levels) and their specificity to bind to target molecules, particularly as a "chemical antibody" and thus have a high potential as an alternative antibody.

In one experimental example of the present invention, when examining the degree of methylation of the DKK3 gene present in H460 cells, an average of 44% methylation was observed. After transfecting the DKK3 siRNA to H460 cells, the expression of DKK3 was decreased. Completely inhibited (see FIG. 1).

In one experimental example of the present invention, after transfecting DKK3 siRNA to H460 cells, the cell death was observed. As a result, the cells did not normally attach to the culture plate and did not grow, compared with the control NC siRNA. The number of cells suspended in the supernatant of the medium increased (see A and B in FIG. 2). In addition, as a result of measuring the cell viability using the MTT assay, it was confirmed that the survival rate of cells in the group transfected with DKK3 siRNA is significantly reduced (see FIG. 2C). In addition, after transfecting the DKK3 siRNA to H460 cells, colony forming assay confirmed that the cells were not formed normally (see FIG. 2D).

In one experimental example of the present invention, after transfecting DKK3 siRNA to H460 cells and performing flow cytometry, the H460 cell group and the group transfected with DKK3 siRNA than the group transfected with NC siRNA to H460 cells. Apoptosis occurred, and after 3 days of transfection, it was confirmed that the difference between each group was greatest (see FIG. 3).

In one experimental example of the present invention, after transfecting DKK3 siRNA to H460 cells and comparing the expression of apoptosis related proteins, Cyclin D, Cycliln E, p53, p21 and Bax ( It was observed that Bax) expression was significantly increased as compared with the H460 cell group and the NC siRNA transfected group to H460 cells (see FIG. 4A). In addition, after transfecting the DKK3 siRNA to H460 cells, the intracellular ROS was measured, and it was confirmed that the ROS level was increased compared to the H460 cell group and the NC siRNA transfected group to H460 cells (FIG. 4). B).

Therefore, after transfection of DKK3 siRNA in H460 non-small cell lung cancer line, expression of DKK3 was completely inhibited and had an effect of increasing cancer cell death and intracellular ROS levels, and thus containing a DKK3 protein expression or activity inhibitor. It can be usefully used as a composition for the prevention and treatment of cancer.

The composition may contain one or more active ingredients exhibiting the same or similar function in addition to the expression or activity inhibitor of DKK3.

The composition can be administered orally or parenterally during clinical administration and intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dural injection, cerebrovascular injection or intrathoracic injection during parenteral administration. And can be used in the form of general pharmaceutical formulations.

The composition can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers.

The daily dosage of the composition is about 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, preferably administered once or several times a day, but the weight, age, sex, health, diet of the patient The range varies depending on the time of administration, the method of administration, the rate of excretion and the severity of the disease.

The composition of the present invention may be administered in various parenteral formulations during actual clinical administration, when formulated using a diluent or excipient such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used. do. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The present invention also provides an anticancer adjuvant containing an inhibitor of expression or activity of DKK3 protein.

The DKK3 protein preferably has an amino acid sequence as set forth in SEQ ID NO: 1, but is not limited thereto.

The inhibitor of expression of the DKK3 protein is DKK3 It is preferably one selected from the group consisting of antisense nucleotides, short interfering RNAs, and short hairpin RNAs, which complementarily bind to mRNA of a gene, but are not limited thereto.

The activity inhibitor of the DKK3 protein is preferably any one selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, and antibodies that complementarily bind to DKK3 protein, but is not limited thereto.

The cancer is preferably any one selected from the group consisting of liver cancer, colon cancer, cervical cancer, kidney cancer, gastric cancer, prostate cancer, breast cancer, brain tumor, lung cancer, uterine cancer, colon cancer, bladder cancer, blood cancer and pancreatic cancer. More preferably, but not limited to colorectal cancer, lung cancer or colorectal cancer.

In one experimental example of the present invention, after transfecting DKK3 siRNA to H460 cells, the cells did not grow and adhere to the culture plate normally, and the number of cells suspended in the supernatant of the medium increased (see FIGS. 2A and 2B). In addition, it was confirmed that the cell viability is greatly reduced (see FIG. 2C and FIG. 3). In addition, after transfection of DKK3 siRNA to H460 cells, it was confirmed that colony formation was not normal (see FIG. 2D). In addition, after transfection of DKK3 siRNA to H460 cells, expression of apoptosis-related proteins increased (see FIG. 4). In addition, after transfecting the DKK3 siRNA to H460 cells, the ROS levels were measured in the cells. As a result, the ROS level was increased compared to the H460 cell group and the NC siRNA transfected group to H460 cells (FIG. 4). Reference).

Therefore, after transfection of DKK3 siRNA in H460 non-small cell lung cancer, expression of DKK3 was completely inhibited and had an effect of increasing the death of cancer cells and intracellular ROS levels, and thus containing a DKK3 protein expression or activity inhibitor. It can be usefully used as an anticancer adjuvant.

In addition, the present invention provides a method for screening a composition for preventing or treating cancer.

The method for screening a composition for preventing or treating cancer of the present invention is preferably, but not limited to, a method comprising the following steps:

1) treating a test substance to cancer cells as an experimental group;

2) measuring the expression level of DKK3 protein in the treated cells of step 1); And

3) selecting the test substance whose DKK3 protein expression level in step 2) is reduced compared to the control group.

In the screening method for the composition for preventing or treating cancer of the present invention, the expression level of DKK3 protein in step 2) is reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay (ELISA). , Immunohistochemistry, Western blotting and flow cytometry (FACS) is preferably measured by any one selected from the group consisting of, but not limited to.

Hereinafter, the present invention will be described in detail by the experimental and production examples. However, the following Experimental Examples and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Experimental Examples and Preparation Examples.

Experimental Example 1 Measurement of Methylation of H460 Cells and Inhibition of DKK3 Development

<1-1> Culture of H460 Non-Small Cell Lung Cancer Cell Line

H460 non-small cell lung cancer cell line (American Type Culture Collection, USA) was cultured in 10% Fetal Laboratories and 100 U / ml penicillin in RPMI-1640 medium (Hyclone Laboratories, USA). ) And 100 μg / ml streptomycin were cultured by mixing. Cells were maintained at 37 ° C. incubator at 5% CO ₂ , and the cells were planted in 6-well plates with 1 × 10 ⁵ cells and cultured.

<1-2> Methylation of CpG Island

EZ DNA Methylation-Gold Kit (Zymo Research, USA) to obtain DNA from cells to confirm methylation of DKK3, and then to make Bisulfite-modified genomic DNA (gDNA) Was used.

To perform the bisulfite reaction, 400 ng of genomic DNA (gDNA) and 130 μl CT conversion reagent are placed in a sample tube and reacted in a thermal cycler (MJ Research, USA). The reaction conditions are 10 minutes at 98 ℃, 2 hours 30 minutes at 64 ℃ and then stored at 4 ℃. The reaction DNA was purified using EZ DNA Methylation-Gold kit (Zymo Research). The purified sample is placed in a Zymo-Spin IC colum (column) containing 600 μl M-binding buffer and shaken. Centrifuge the Zymo-Spin IC column for 30 seconds, add 200 μl M-wash buffer to the column, centrifuge for 30 seconds, and add 200 μl M-desulphonation buffer to the column for 15 minutes at room temperature (20 ~ 30 ℃). Allow it to react for 20 minutes. After the reaction was completed, the column was centrifuged for 30 seconds, and finally, 200 μl M-wash buffer was added and centrifuged for 30 seconds. To completely remove the buffer, centrifugation was performed once more. The resulting gDNA was obtained by dissolving 20 μl of M-elution buffer in a column. GDNA was stored at -20 ° C prior to use in experiments.

In order to measure the methylation, it was used for sequencing (pyrosequencing) Pyro. Using the prepared gDNA as a template A polymerase chain reaction (PCR) was performed. 5 μl of 10 × Taq buffer in 5 ng gDNA, 5 U of Hot / Start Taq polymerase (Enzynomics, Korea), 4 μl of each dNTP (2.5 mM) mixture, 10 pmol / primer-S (SEQ ID NO: 2: 5′-GGA GGG) GTT AGG GTT TGA AT-3 ') 2, 10 pmol / biotinylated-primer-As (SEQ ID NO: 3: 5'-biotin-TCR AAC ACA AAC TAA ACT CTA CTC CT-3') 2 The remaining volume was filled with distilled water. PCR conditions were 15 minutes at 95 ℃, 45 cycles were reacted for 40 seconds at 95 ℃, 40 seconds at 55 ℃ 40 seconds at 72 ℃, and finally reacted for 10 minutes at 72 ℃. 5 μl of PCR product was identified using ethidium bromide (EtBr) staining on a 3% agarose gel. 20-25 μl of the PCR product was prepared using multichannel pipettes according to the method described in PSQ 96 sample preparation guide using streptavidin Sepharose HP beads (Amersham Biosciences, Sweden). It was made from single-strand DNA (ssDNA).

According to the method described in the PSQ 96 sample preparation guide, first, 40 μl of 1 × annealing buffer (composition: 20 mM Tris, 2 mM magnesium acetate) and 100 pmole / 0.12 μl of a mixture of 5 μl of sequencing primer (5′-GGG GTT TGG GAG TTA TT-3 ′) was dispensed at 40 μl per well. 37 μl 2 × binding buffer and 3 μl streptavidin sepharose beads were added to a new 96 well PCR plate. 40 μl of the biotin-labeled PCR product was added to the streptavidin mixture and shaken for 5 minutes. The probe in the Vacuum prep workstation device was washed once with distilled water using a vacuum pump to capture a mixture of the biotin-labeled PCR product and streptavidin beads. The probe combined with the bead mixture was washed once with 70% ethanol, sequentially denatured solution (composition: 0.2M sodium hydroxide), washing buffer [composition: 10 mM Tris, pH 7.6] and tertiary distilled water. The bead mixture adhering to the probe was transferred to the PSQ 96 well plate containing 1 × annealing buffer by stirring. The bead mixture and the PSQ 96 well plate with 1 × annealing buffer were heated at 80 ° C. for 2 minutes.

Pyro Gold reagents kit (Biotage, Charlottesville, VA, USA) was used, and 15 pmol of each sequencing primer (SEQ ID NO: 2, 3, 4) was added to analyze single-stranded DNA using the PyroMark ID system. Pipette the substrate (S), enzyme (E), dATP (A), dCTP (C), dGTP (G) and dTTP (T) included in the Pyro Gold reagent kit into the appropriate area of the cartridge. Was put on. The prepared PSQ 96 well plates and cartridges were placed in a PyroMark ID instrument, and pyrosequencing was performed by running Pyro-Q-CpG software. Subsequently, methylation of CpG was calculated on average by methylation of the fifth or sixth CpG in pyro sequencing to confirm the degree of methylation.

As a result, as shown in FIG. 1A, 164, 175, 181 and 193 CpG sites (DKK3 121 to 240nt, y: CpG site, SEQ ID NO: 5: tyggggatag agtttaggtg agttggggtt tgggagttat tag of nucleotides 121 to 240 of DKK3) y gtagag gatt y gggtt y ggttttttt gg y gagggtt ttagtattat aggtgaggag tagagtttag tttgtgttyg) and methylation was observed, on average, 44% of the expression was confirmed (A in Fig. 1).

<1-3> Confirmation of Reduced DKK3 Expression After DKK3 siRNA Transfection

After one day of non-small cell lung cancer H460 cells were cultured, DKK3 siRNA (small intefering RNA) (SEQ ID NO: 6: 5'-AGC UGC UGC UAA AGC AUC AUC AGA A-3 ') (Invitrogen, USA) and its control siRNA ( NC siRNA) (Bioneer Co., Korea) 100 nM was transfected with Lipofectamine RNAi MAX reagent (Invitrogen). 2 days after transfection Later, RNA was extracted from the cells using an RNA extraction kit (Qiagen, USA).

Complementary DNAs (complementary DNAs, cDNAs) were synthesized using PCR premix kit (Intron Biotechnology, Korea) to perform reverse transcription polymerase chain reaction using the extracted RNA. DKK3 forward primer (SEQ ID NO: 7: 5'-GTT GAG GAA CTG ATG GAG GAC A-3 '), DKK3 reverse primer (SEQ ID NO: 8: 5'-TTG CAC ACA TAC ACC AGG CTG T-) 3 '), actin forward primer (SEQ ID NO: 9: 5'-ATG TGC AAG GCC CGC TTC G-3') and actin reverse primer (SEQ ID NO: 10: 5'-TTA ATG TCA CGC ACG ATT TCC-3 ') Polymerase chain reaction was performed. In order to amplify the GK-rich DKK3 template, i-GC capture solution (Intron Biotechnology, Korea) was used in addition to the PCR mixture. After PCR reaction of DKK3 for 30 seconds at 94 ℃, 40 cycles were reacted for 30 seconds at 94 ℃, 30 seconds at 61 ℃, and 1 minute at 72 ℃, and finally 5 minutes at 72 ℃. The amplified PCR product was confirmed by electrophoresis on 1% agarose gel.

As a result, after transfecting the DKK3 siRNA to H460 cells, it was confirmed that the expression of DKK3 is completely reduced (Fig. 1B).

Experimental Example 2 Inhibition of Cell Growth

<2-1> Inhibition of Growth of H460 Cells by Introduction of DKK3 siRNA

After transfecting the H460 cells with DKK3 siRNA and its control NC siRNA by the method described above, the cells were used two days later. Total cells, cells suspended in the supernatant of the medium, and cells attached to the bottom of the culture plate were observed using a fluorescence microscope (Leica Microsystems, USA). Images observed with a fluorescence microscope were taken using a digital camera (Cannon Power Shot S45).

As a result, floating cells which were not observed in the group transfected with H460 cells and the control NC siRNA did not adhere well to the culture plate after the transfection of DKK3 siRNA, and the floating cells increased (FIG. 2). A).

<2-2> Cell viability measurement

In order to measure the viability of the cells, the survival fraction of the cells was analyzed. After trypsin treatment for H460 cells, the number of cells was counted directly. In addition, MTT assay (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) was performed to determine cell viability. H460 cells were planted 1000-3000 cells in 96-well plate at three-fold per experimental group. After 24, 48 and 72 hours have elapsed, 100 μl of MTT solution is placed in the wells and the plate is allowed to react at 37 ° C. for 4 hours. The medium was removed, dimethyl sulfoxide (DMSO) was added to the wells to dissolve the MTT crystals, and the absorbance was measured at a GLOMAX 96 microplate luminometer (Promega) 570 nm wavelength. . The measured values were analyzed using a Microplate manager (Promega, USA).

As a result, the cell number was reduced by about 70% in the group transfected with DKK3 siRNA, compared to H460 in which DKK3 was expressed. In addition, it was confirmed that the growth of cells was significantly reduced in the group transfected with the DKK3 siRNA group compared with the group transfected with the H460 cell group and the control group si siRNA. This could be attributed to the increase in suspended cells observed in the morphology of these cells (B and C in FIG. 2).

<2-3> colony formation measurement

Colony forming assays were performed to confirm cell growth patterns. 24 hours after transfection of DKK3 siRNA into H460 cells, the cells were trypsinized and harvested, and then seeded at 5 × 10 ³ in a 35 mm culture dish. After incubation for 7 days, the cells were stained with 0.5% crystal violet and then photographed.

As a result, in the group transfected with DKK3 siRNA, it was confirmed that the generation of colonies was significantly lower than the group transfected with H460 cells and NC siRNA as a control group (FIG. 2D).

Experimental Example 3 Measurement of Cytotoxicity Induced by DKK3 Expression Inhibition

In order to measure the cytotoxic effects induced by transfection of DKK3 siRNA, H460 cells were stained with propidium iodide (PI), and flow cytometry was used. After H460 cells were fixed for 30 minutes at 4 ° C. using 70% ethanol in a dark place, ethanol was washed with phosphate-buffered saline (PBS), and then the cells were treated with 50 μl / ml of iodide propidium. Was stained. Stained DNA was measured using FACScan (EPICS XL; Beckman Coulter Counter, USA). At least 1 × 10 ⁴ cells were counted in each group, and changes in each cell were measured using Phoenix Multicycler software (Phoenix Flow System, USA).

As a result, the first day after transfection showed a similar pattern in the H460 cell group and the NC siRNA group, and cell death started from the second day. Apoptosis was less than 4% in the H460 and NC siRNA groups, whereas apoptosis in the DKK3 siRNA group was significantly different from the H460 and NC siRNA groups from day 2. The largest difference was seen on day 3 (FIG. 3). In other words, it was confirmed that cell death was induced by DKK3 expression inhibition.

Experimental Example 4 Apoptosis Protein Expression and ROS Measurement after DKK3 Expression Inhibition

<4-1> Expression of Apoptosis Proteins after Inhibition of DKK3 Expression

In order to determine the apoptosis pathway observed in H460 cells, the expression level of the apoptosis pathway related protein was confirmed using Western blot. Protein concentration was measured using a Lowry kit (Bio-Rad Laboratories, USA). The same amount of protein was separated on 10-12% SDS-SDS-PAGE (sodium dodecyl sulfate polyacrylamide) and then transferred to a nitrocellulose membrane (Hybond ECL; Amersham Pharmacia, USA). The protein-transported nitrocellulose membrane was prevented from nonspecific binding with blocking buffer in which 10% non-fat milk was dissolved in PBST (0.1% Tween in PBS) solution for 2 hours at room temperature. The membrane was then reacted with each antibody for 1 hour. Antibodies used were p21 ^{WAF1 / Cip1} (p21) ( ^Abcam , USA), beta-actin (β-actin) (Cell Signaling, USA), cyclin E (Cell Signaling, USA), phospho-Akt, pAkt) (Cell Signaling, USA), Cyclin D1 (Santa Cruz, USA), Cdk2 (Santa Cruz, USA), (Cyclin-dependent kinase 2, Cdk) (Santa Cruz, USA), PTEN (phosphatase and tensin homolog) ( Santa Cruz, USA), p53 (Santa Cruz, USA) and Bax (Santa Cruz, USA) were used diluted 1: 1,000. Thereafter, after reacting with a secondary antibody equipped with horseradish peroxidase (HRP), the result was obtained by luminescence using Westzol chemiluminescence dye kit (Intron Biotechnology).

As a result, after suppressing the expression of DKK3, the expression of cyclin D1 and cyclin E was increased compared to the H460 cells and NC siRNA group, whereas no difference in expression was observed in Cdk2 and CdK4. In addition, there was no difference in the expression level of PTEN and pAkt between the group in which DKK siRNA was introduced, the group in which H460 cells and NC siRNA were introduced. However, specific expression levels of p53 and p21 were significantly increased in the group introduced with DKK3 siRNA. These results confirm that DKK3 siRNA was introduced into H460 cells, thereby suppressing expression and experiencing oxidative stress. It was observed that the expression of Bax was significantly increased in the group in which the DKK siRNA was introduced. Since Bax is transferred to the nucleus after the apoptosis starts, it was confirmed that the apoptosis occurred through this increase (FIG. 4A). .

<4-2> ROS Level Measurement after DKK3 Expression Suppression

Since the p53 transduction circuit can be initiated due to an increase in ROS, to determine whether the increased p53 identified by Western blot is due to a change in ROS inside H460 cells after inhibition of DKK3 protein expression, the value of ROS is determined. Measured.

After transfecting H460 cells with NC siRNA and DKK3 siRNA, the cells for 14 hours, 20 hours and 24 hours were trypsinized and then walked. After about 1 × 10 ⁶ H460 cells were walked by trypsin treatment, washed with PBS, suspended, and then dichlorofluorescein diacetate (DCFH-DA) was added to a final concentration of 10 uM. DCFH-DA is a nonpolar compound that is fused intracellularly and then converted into a membrane-impermeable nonfluorescent polar derivative (DCFH) by the cell's esterase. Intracellularly trapped DCFH is known to be rapidly oxidized to fluorescent 2 ', 7'-diclorofluorescein (DCF) by hydrogen peroxide in the cell. Therefore, after DCFH-DA was reacted to the cells in a dark place for 30 minutes at 37 ° C., ROS levels were measured by flow cytometry.

As a result, it was observed that the level of ROS was significantly increased after DKK3 protein expression inhibition, and the difference between H460 cells, NC siRNA and DKK3 siRNA groups was the largest after 2 hours of transfection of DKK3 siRNA (FIG. 4). B). In other words, it was confirmed that DKK3 plays a role in maintaining intracellular ROS levels.

The preparation examples for the compositions of the present invention are given below.

Preparation Example 1 Preparation of Pharmaceutical Formulation

<1-1> Preparation of powder

2 g of DKK3 expression or activity inhibitor

1 g lactose

The above components were mixed and packed in airtight bags to prepare powders.

<1-2> Preparation of Tablet

100 mg of DKK3 protein expression or activity inhibitor

Corn starch 100 mg

100 mg of milk

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

<1-3> Preparation of Capsule

100 mg of DKK3 protein expression or activity inhibitor

Corn starch 100 mg

100 mg of milk

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

&Lt; 1-4 >

Inhibitor of expression or activity of DKK3 protein 1 g

Lactose 1.5 g

Glycerin 1 g

0.5 g of xylitol

After mixing the above components, it was prepared to be 4 g per ring according to a conventional method.

<1-5> Preparation of granules

150 mg of DKK3 protein expression or activity inhibitor

Soybean Extract 50mg

Glucose 200 mg

600 mg of starch

After mixing the above components, 100 mg of 30% ethanol was added and dried at 60 ° C. to form granules, and then filled in fabric.

As described above, since the expression of DKK3 is completely suppressed in non-small cell lung cancer lines, cancer cell death and intracellular reactive oxygen levels are increased, thus preventing the cancer containing DKK3 protein expression or activity inhibitors and It can be usefully used as a therapeutic composition or as an anticancer adjuvant.

<110> Korea Atomic Energy Research Institute <120> Composition comprising expression or activity inhibitors of DKK3          for the prevention and treatment of cancer <130> 10p-02-15 <160> 10 <170> KopatentIn 1.71 <210> 1 <211> 350 <212> PRT <213> Homo sapiens <400> 1 Met Gln Arg Leu Gly Ala Thr Leu Leu Cys Leu Leu Leu Ala Ala Ala   1 5 10 15 Val Pro Thr Ala Pro Ala Pro Ala Pro Thr Ala Thr Ser Ala Pro Val              20 25 30 Lys Pro Gly Pro Ala Leu Ser Tyr Pro Gln Glu Glu Ala Thr Leu Asn          35 40 45 Glu Met Phe Arg Glu Val Glu Glu Leu Met Glu Asp Thr Gln His Lys      50 55 60 Leu Arg Ser Ala Val Glu Glu Met Glu Ala Glu Glu Ala Ala Ala Lys  65 70 75 80 Ala Ser Ser Glu Val Asn Leu Ala Asn Leu Pro Pro Ser Tyr His Asn                  85 90 95 Glu Thr Asn Thr Asp Thr Lys Val Gly Asn Asn Thr Ile His Val His             100 105 110 Arg Glu Ile His Lys Ile Thr Asn Asn Gln Thr Gly Gln Met Val Phe         115 120 125 Ser Glu Thr Val Ile Thr Ser Val Gly Asp Glu Glu Gly Arg Arg Ser     130 135 140 His Glu Cys Ile Ile Asp Glu Asp Cys Gly Pro Ser Met Tyr Cys Gln 145 150 155 160 Phe Ala Ser Phe Gln Tyr Thr Cys Gln Pro Cys Arg Gly Gln Arg Met                 165 170 175 Leu Cys Thr Arg Asp Ser Glu Cys Cys Gly Asp Gln Leu Cys Val Trp             180 185 190 Gly His Cys Thr Lys Met Ala Thr Arg Gly Ser Asn Gly Thr Ile Cys         195 200 205 Asp Asn Gln Arg Asp Cys Gln Pro Gly Leu Cys Cys Ala Phe Gln Arg     210 215 220 Gly Leu Leu Phe Pro Val Cys Thr Pro Leu Pro Val Glu Gly Glu Leu 225 230 235 240 Cys His Asp Pro Ala Ser Arg Leu Leu Asp Leu Ile Thr Trp Glu Leu                 245 250 255 Glu Pro Asp Gly Ala Leu Asp Arg Cys Pro Cys Ala Ser Gly Leu Leu             260 265 270 Cys Gln Pro His Ser His Ser Leu Val Tyr Val Cys Lys Pro Thr Phe         275 280 285 Val Gly Ser Arg Asp Gln Asp Gly Glu Ile Leu Leu Pro Arg Glu Val     290 295 300 Pro Asp Glu Tyr Glu Val Gly Ser Phe Met Glu Glu Val Arg Gln Glu 305 310 315 320 Leu Glu Asp Leu Glu Arg Ser Leu Thr Glu Glu Met Ala Leu Gly Glu                 325 330 335 Pro Ala Ala Ala Ala Ala Ala Leu Leu Gly Gly Glu Glu Ile             340 345 350 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DKK3 Forward primer <400> 2 ggaggggtta gggtttgaat 20 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> DKK3 Reverse primer <400> 3 tcraacacaa actaaactct actcct 26 <210> 4 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Sequencing primer <400> 4 ggggtttggg agttatt 17 <210> 5 <211> 120 <212> DNA <213> Homo sapiens <400> 5 tyggggatag agtttaggtg agttggggtt tgggagttat tagygtagag gattygggtt 60 yggttttttt ggygagggtt ttagtattat aggtgaggag tagagtttag tttgtgttyg 120                                                                          120 <210> 6 <211> 25 <212> RNA <213> Homo sapiens <400> 6 agcugcugcu aaagcaucau cagaa 25 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> DKK3 Forward primer for PCR <400> 7 gttgaggaac tgatggagga c 21 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> DKK3 Reverse primer for PCR <400> 8 ttgcacacat acaccaggct gt 22 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Actin Forward primer for PCR <400> 9 atgtgcaagg cccgcttcg 19 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Actin Reverse primer for PCR <400> 10 ttaatgtcac gcacgatttc c 21

Claims (14)

A composition for preventing and treating cancer containing DKK3 (Dickkopf 3) protein expression or activity inhibitors.
2. The composition for preventing and treating cancer according to claim 1, wherein the DKK3 protein has an amino acid sequence set forth in SEQ ID NO: 1.
The method of claim 1, wherein the inhibitor of expression of the DKK3 protein is any one selected from the group consisting of antisense nucleotides, short interfering RNAs, and short hairpin RNAs, which complementarily bind to mRNA of the DKK3 gene. Composition for the prevention or treatment of cancer, characterized in that.
The composition for preventing or treating cancer according to claim 3, wherein the siRNA of the DKK3 gene has a nucleotide sequence as set forth in SEQ ID NO: 6.
The method of claim 1, wherein the activity inhibitor of the DKK3 protein is any one selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, and antibodies that bind complementarily to the DKK3 protein. Composition.
According to claim 1, wherein the cancer is any one selected from the group consisting of liver cancer, colon cancer, cervical cancer, kidney cancer, stomach cancer, prostate cancer, breast cancer, brain tumor, lung cancer, uterine cancer, colon cancer, bladder cancer, blood cancer and pancreatic cancer A composition for the prevention or treatment of cancer, characterized in that.
An anticancer adjuvant containing an inhibitor of expression or activity of DKK3 protein.
7. The composition for preventing and treating cancer according to claim 6, wherein the DKK3 protein has an amino acid sequence set forth in SEQ ID NO: 1.
The method of claim 6, wherein the inhibitor of expression of the DKK3 protein is any one selected from the group consisting of antisense nucleotides, short interfering RNAs, and short hairpin RNAs that complementarily bind to the mRNA of the DKK3 gene. Anticancer adjuvant, characterized in that.
10. The anticancer adjuvant of claim 9, wherein the siRNA of the DKK3 gene has a nucleotide sequence set forth in SEQ ID NO: 6.
7. The anticancer adjuvant of claim 6, wherein the activity inhibitor of the DKK3 protein is any one selected from the group consisting of compounds, peptides, peptide mimetics, aptamers and antibodies that complementarily bind to the DKK3 protein.
According to claim 6, wherein the cancer is any one selected from the group consisting of liver cancer, colon cancer, cervical cancer, kidney cancer, stomach cancer, prostate cancer, breast cancer, brain tumor, lung cancer, uterine cancer, colon cancer, bladder cancer, blood cancer and pancreatic cancer Anticancer adjuvant characterized by the above-mentioned.
1) treating a test substance to cancer cells as an experimental group;
2) measuring the expression level of DKK3 protein in the treated cells of step 1); And
3) A method for screening a composition for preventing or treating cancer, comprising selecting a test substance whose amount of DKK3 protein expression in step 2) is reduced compared to a control.
The method of claim 11, wherein the expression level of the DKK3 protein in step 2) is reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay (ELISA), immunohistochemistry, Western blotting. And flow cytometry (FACS). The method for screening a composition for preventing or treating cancer, characterized in that the measurement by any one selected from the group consisting of.

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