KR102532779B1 - Composition for preventing or treating cancer comprising DDIAS inhibitor and death receptor ligand - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising a DNA damage-induced apoptosis suppressor (DDIAS) inhibitor and a death receptor ligand as active ingredients.
The DDIAS inhibitor of the present invention effectively inhibits the degradation promotion of caspase-8 and the inhibition of DISC (death-inducing signaling complex) formation by the action of DDIAS, thereby inhibiting apoptosis in cancer cells by acting together with death receptor ligands. By exerting an excellent synergistic effect that promotes, it can be used in various ways as a composition for preventing and treating cancer, including lung cancer and liver cancer.

Description

Composition for preventing or treating cancer comprising a DDIAS inhibitor and a death receptor ligand

The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising a DNA damage-induced apoptosis suppressor (DDIAS) inhibitor and a death receptor ligand as active ingredients.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF protein family that promotes apoptosis in various types of cancer cells. TRAIL is known to induce apoptosis by binding to extrinsic receptors (DR) 4 and DR5 and recruiting the death-inducing signaling complex (DISC) in the extrinsic apoptotic pathway. The Fas-associated protein death domain (FADD) first interacts with the DR, then the procaspase via the death domain (DD) and the death effector domain (DED). Interacts with (procaspase)-8. Activation of caspase-8 through DISC formation is known to induce TRAIL-induced apoptosis by inducing cleavage and activation of downstream effector caspases such as caspase-3 and -7, but some cancer cells are dysregulated by TRAIL receptors. It is known that TRAIL becomes resistant as a result of deficiency of the DISC component.

Caspase-8 is a cellular protein known to be involved in TRAIL resistance and signal transduction. Reduction of caspase-8 expression due to mutations, epigenetic silencing and stability induces TRAIL resistance, which leads to It is known that TRAIL-induced apoptosis is impeded. Post-translational modifications of caspase-8, particularly phosphorylation of caspase-8, are known to reduce stability and activity. P90 ribosomal S6 kinase (RSK) 2 phosphorylates caspase-8 at T263 to induce ubiquitination and proteasome-related degradation, and Src phosphorylates caspase-8 at Y380 to inhibit its enzyme activity do. In addition, anti-cellular cFLIP (Fas-associated death domain-like interleukin-1β converting enzyme-like inhibitory protein), PEA-15 (phosphoprotein enriched in astrocytes-15), DJ-1 or mucin (MUC)-1 Apoptotic proteins are known to inhibit the activity of caspase-8, and these proteins are known to inhibit DR-induced apoptosis by blocking the movement of caspase-8 to the DISC.

DNA damage-induced apoptotic suppressor (DDIAS) is a target gene of nuclear factor, a transcription factor of activated T cell 1 (NFATc1), and is induced by epithermal growth factor (EGF) to produce β-catenin (catenin). ) is known to actively regulate cancer cell invasion by stabilizing it. DDIAS protects cancer cells from DNA-damaging reagents such as camptothecin and cisplatin, and nuclear DDIAS binds to DNA polymerase as a cofactor in the DNA polymerase-primase complex. While it is known to promote tumorigenesis in hepatocellular carcinoma (HCC), EGF-induced DDIAS is localized to the cytoplasm and is known to regulate β-catenin stability in HeLa cells.

Based on these previous studies, DDIAS is believed to have the potential to regulate cancer cell survival through various pathways (Korean Patent Registration No. 10-1168726, Korean Patent No. 10-1591278), but TRAIL, which induces apoptosis in cancer cells, The effect of DDIAS on the action of DDIAS has not been identified at all, and there has been no research on how to effectively prevent and treat cancer by demonstrating an excellent synergistic effect by combining a DDIAS inhibitor and a death receptor including TRAIL using this. Nothing happened.

Republic of Korea Patent No. 10-1168726 Republic of Korea Patent Registration No. 10-1591278

An object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising a DNA damage-induced apoptosis suppressor (DDIAS) inhibitor and a death receptor ligand as active ingredients.

The present invention provides a pharmaceutical composition for preventing or treating cancer comprising a DNA damage-induced apoptosis suppressor (DDIAS) inhibitor and a death receptor ligand as active ingredients.

The term "DDIAS" used in the present invention is a homologous protein of mouse Noxin and has been reported as the human Noxin gene hNoxin (GenBank accession numbers: NM_145018, C11orf82, FLJ25416, FLJ13936, UniProtKB/TrEMBL entry Q8IXT1). Recently, human Noxin was named a DNA damage-induced apoptotic suppressor (DDIAS) based on the fact that it inhibits apoptosis of DNA-damaged cells (Human Noxin is an anti-apoptotic protein in response). to DNA damage of A549 non-small cell lung carcinoma.Int J Cancer.2014 Jun 1;134(11):2595-2604).

In the present invention, DDIAS inhibitors may include all without particular limitation as long as they inhibit the expression of the DDIAS gene. For example, the DDIAS inhibitor may preferably be selected from the group consisting of siRNA, antisense oligonucleotide and shRNA that inhibit the expression of the DDIAS gene.

As used herein, the term "siRNA (small interfering RNA)" refers to a short RNA capable of mediating RNA interference (RNAi) or gene silencing, and generally has a size of about 20 nucleotides or less. have Since siRNA can suppress the expression of a target gene, it can be provided as an efficient gene knockdown method or gene therapy method.

In the present invention, siRNA may have a structure in which a sense RNA strand having a sequence homologous to the mRNA of a target gene and an antisense RNA strand having a sequence complementary thereto form a double strand, or a self-complementary sense and It may have a single-chain structure with an antisense strand.

The siRNA is not limited to complete pairing of double-stranded RNA parts paired with each other, but mismatch (corresponding bases are not complementary), bulge (no base corresponding to one chain) Unpaired parts may be included by the like. The siRNA end structure can be either a blunt end or a cohesive end structure, as long as the expression of the target gene can be inhibited by the RNA interference effect.

In the present invention, as the siRNA as a DDIAS inhibitor, only one type of siRNA may be used to selectively bind to only one target sequence position among mRNAs of DDIAS, or two or more types of siRNA may be used together to bind to one or more target sequence positions. may be

In the present invention, the siRNA against DDIAS may preferably be siRNA against a target sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 among the mRNA sequences of DDIAS. Specific target sequences to which siRNAs can be bound in DDIAS are shown in Table 1 below.

[Table 1]

In the present invention, the siRNA against DDIAS is more preferably a siRNA having a sense sequence of SEQ ID NO: 4 and an antisense sequence of SEQ ID NO: 5; siRNA having a sense sequence of SEQ ID NO: 6 and an antisense sequence of SEQ ID NO: 7; And it may be selected from the group consisting of siRNA having a sense sequence of SEQ ID NO: 8 and an antisense sequence of SEQ ID NO: 9. The specific sequences of siRNAs against DDIAS are shown in Table 2 below.

[Table 2]

As used herein, the term "antisense oligonucleotide" refers to DNA or RNA containing an oligonucleotide sequence complementary to a specific mRNA sequence, or a derivative thereof, and binds to a complementary sequence in mRNA to form a protein of mRNA. It acts to impede translation into .

In the present invention, as the antisense oligonucleotide as a DDIAS inhibitor, only one type of antisense oligonucleotide can be used to selectively bind to only one target sequence position of DDIAS mRNA, or two or more kinds can be used to bind to one or more target sequence positions. Antisense oligonucleotides may also be used together.

In the present invention, the antisense oligonucleotide as a DDIAS inhibitor may preferably be antisense RNA.

As used herein, the term "antisense RNA" refers to a single-stranded RNA composed of an antisense sequence for a target gene, and binds to the target gene to stop expression at the stage of transcription, translation or splicing, thereby enabling the expression of the target gene by the target gene. It has been proposed to prevent expression of the encoded protein. It contains antisense RNA structurally modified to prevent rapid degradation by in vivo nucleases. A person skilled in the art can synthesize and modify the antisense RNA in a desired manner using methods known in the art (European Journal of Biochemistry. 2003;270:1628-1644. Methods Enzymol. 2000: 313, 3-45. reference).

In the present invention, the antisense RNA for DDIAS may be preferably selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9. The specific sequences of antisense RNAs against DDIAS are shown in Table 3 below.

[Table 3]

As used herein, the term "short hairpin RNA (shRNA)" refers to RNA having a short hairpin structure in which sense and antisense sequences of a siRNA target sequence are connected by a linker (loop sequence) of about 3 to 10 bases. It is a structure designed to overcome disadvantages such as high cost of biosynthesis of siRNA and short-term maintenance of RNA interference effect due to low cell transfection efficiency. shRNA can be expressed by introducing it into cells using adenovirus, lentivirus, plasmid expression vector system, etc., and such shRNA is converted into siRNA having a precise structure by siRNA processing enzyme (Dicer or Rnase Ⅲ) present in the cell. can be converted to induce silencing of target genes.

In the present invention, shRNA as a DDIAS inhibitor may use only one shRNA to selectively bind to only one target sequence position among mRNAs of DDIAS, or use two or more shRNAs together to bind to one or more target sequence positions. may be

In the present invention, the shRNA against DDIAS may preferably be encoded with a DNA sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. The specific sequences of shRNAs against DDIAS are shown in Table 4 below.

[Table 4]

In the present invention, a safe and efficient delivery system can be used to increase the efficiency of delivering a nucleic acid material such as siRNA, antisense oligonucleotide or shRNA into cells. For example, siRNA of the present invention, antisense RNA, or shRNA may be included in a composition along with a nucleic acid delivery system.

In the present invention, the nucleic acid delivery system can be largely divided into viral vectors and non-viral vectors. The most widely used is a viral vector, which has the advantage of high delivery efficiency and long duration. Among various viral vectors, retroviral vectors, adenoviral vectors, and adeno-associated viral vectors are mainly used. Nonviral vectors have advantages such as low toxicity and immune response, repeated administration, easy complex formation with ribonucleic acid, and easy mass production. In addition, there is an advantage in enabling long-term cell-selective nucleic acid delivery by conjugating a specific ligand to a diseased cell or tissue site with a non-viral vector. As non-viral vectors, various formulations such as liposomes, cationic polymers, micelles, emulsions, and nanoparticles can be used. A nucleic acid delivery system can significantly enhance the transport efficiency of a target nucleic acid within animal cells, and can deliver nucleic acids to any animal cell depending on the purpose of use of the nucleic acid to be delivered.

In the present invention, the nucleic acid delivery system may preferably be at least one selected from cationic liposomes and cationic polymers. Since nucleic acid delivery systems having formulations such as cationic liposomes and cationic polymers are positively charged, the positive charge of the nucleic acid delivery system and the anionic charge of nucleic acids such as siRNA result in electrostatic bonding by simple mixing to obtain a nucleic acid delivery system and nucleic acid. Can form liver complexes.

In the present invention, methods known in the art may be used to prepare expression constructs/vectors including siRNA, antisense oligonucleotide or shRNA. For example, U.S. Patent Publication Nos. 20040106567 and 20040086884 describe a number of expression constructs as well as delivery mechanisms including viral vectors, non-viral vectors, liposomal delivery vehicles, plasmid injection systems, artificial viral envelopes and polylysine conjugates. We are providing truckt/vector.

The specific preparation / acquisition method of siRNA, antisense oligonucleotide or shRNA used as a DDIAS inhibitor in the present invention may be performed in the same manner as that of Korean Patent Registration No. 10-1168726, which is a prior patent of the present applicant, but is limited thereto It is not.

In the present invention, the DDIAS inhibitor can effectively inhibit the degradation promotion of caspase-8 by the action of DDIAS.

Specifically, in an example of the present invention, expression levels of caspase-8 and FADD according to DDIAS knockdown were evaluated. As a result, the protein level of caspase-8 increased in cells with reduced DDIAS (FIG. 11a), but knockdown of DDIAS did not affect the mRNA level of caspase-8 (FIG. 11b). In addition, as a result of examining the half-life of caspase-8 protein in A549 cells treated with EGF and cycloheximide (CHX) under the reduced DDIAS, the half-life was stable compared to control cells (FIG. 11c). Through this, the inhibitor of DDIAS can achieve an effect of inhibiting destabilization of caspase-8 by suppressing the caspase-8 degradation promoting action of DDIAS.

In the present invention, the DDIAS inhibitor can effectively inhibit the formation of a death-inducing signaling complex (DISC) by the action of DDIAS.

As used herein, the term "death-inducing signaling complex (DISC)" is a multi-protein complex formed by members of the death receptor family of cell receptors that induce apoptosis. For example, DISC may be formed upon trimerization by binding the death receptor FasR to its ligand RasL, but is not limited thereto. The DISC may be composed of death receptors, FADD and caspase-8.

Specifically, in the examples of the present invention, whether DDIAS is involved in the formation of DISC in the extrinsic apoptosis pathway was analyzed. As a result, knockdown of DDIAS enhanced the movement of FADD and caspase-8 to the DISC in TRAIL-treated cells, but had no effect on the movement of DR5 to the DISC (FIG. 10b). In addition, DDIAS interacted with FADD, specifically with the N-terminal region of FADD (Figs. 10c and 10d), and FADD interacted with DDIAS containing the N-terminal region (Fig. 10e). In addition, when an excessive amount of DDIAS was added, the binding between FADD and caspase-8 was reduced (FIG. 10h). Through this, the DDIAS inhibitor can achieve the effect of promoting the formation of DISC by inhibiting the interaction between DDIAS and FADD and increasing the binding between FADD and caspase-8 by the interaction.

In the present invention, the death receptor ligand may be preferably selected from the group consisting of TRAIL (TNF-related apoptosis-inducing ligand), TRAs (TRAIL receptor agonists), TNF-α, and FasL (Fas ligand; CD95L, APO-1L) And, more preferably, it may be TRAIL.

As used herein, the term "death receptor ligand" refers to currently known death receptors such as TNF receptor-1 (DR1, CD120a, p55, p60), Fas (CD95, DR2, APO-1), DR3 (APO-3, LARD). , TRAMP, WSL1), DR4 (TRAILR1, APO02), DR5 (TRAILR2, KILLER, TRICK2) or DR6 (EDAR, NGFR) means a ligand capable of mediating an apoptosis signal.

As used herein, the term "TRAIL (TNF-related apoptosis-inducing ligand)" is a member of the TNF protein family that promotes cell death in various types of cancer cells, and binds to extrinsic receptors (DR) 4 and DR5 and induces extrinsic apoptosis. It is known to induce apoptosis by recruiting DISC in the pathway.

As used herein, the term "TRAIL receptor agonists (TRAs)" may be a recombinant form of TRAIL, or a TRAIL-R1 or TRAIL-R2 specific antibody capable of binding to a TRAIL receptor and performing TRAIL-mediated death signaling. . Specifically, the recombinant TRAIL may include Dulanermin, and the TRAIL-R1 specific antibody may include Mapatumumab, a derivative thereof, or an antigen-binding fragment thereof, and the TRAIL-R1 specific antibody may include R2 specific antibodies include Drozitumumab, a derivative thereof or an antigen-binding fragment thereof; Conatumumab, a derivative thereof or an antigen-binding fragment thereof; Lexatumumab, a derivative thereof or an antigen-binding fragment thereof; Tigatuzumab, a derivative thereof or an antigen-binding fragment thereof; Alternatively, LBY-135, a derivative thereof, or an antigen-binding fragment thereof may be included, and any other type of agonist capable of performing TRAIL-mediated death signaling may be used without limitation.

As used herein, the term "TNF-α (tumor necrosis factor-α)" is a tumor necrosis factor that is activated from macrophages, natural killer cells (NK cells), some T cells, or some tumor cells. known to be produced.

As used herein, the term "FasL (Fas ligand; CD95L)" is a type 2 transmembrane protein belonging to the tumor necrosis factor (TNF) family, and is known to induce apoptosis by binding to its receptor. FasL/receptor interaction is known to play an important role in the regulation of the immune system and cancer progression.

In the present invention, even when TRAs, TNF-α or FasL are used as death receptor ligands, excellent anticancer effects are exhibited, but in particular, when TRAIL is used, the effect of effectively inhibiting inactivation of caspase-8 and inhibition of DISC formation is exhibited. DDIAS inhibitor and TRAIL, which have the effect of inducing apoptosis in cancer cells, work together to exert a more excellent synergistic effect, thereby significantly promoting apoptosis in cancer cells, showing a more excellent effect in preventing and treating cancer there is

In an embodiment of the present invention, drugs capable of enhancing cancer cell death induced by DDIAS knockdown were screened. Specifically, the growth inhibition ability of a total of 20 inhibitors, including doxorubicin, gefitinib, rapamycin, BAY 11-7082 and recombinant TRAIL, on A549 cells simultaneously treated with siRNA against DDIAS was experimented with. Compared to the control (scramble siRNA), TRAIL significantly inhibited the growth of A549, HCC15 and NCI-H1703 NSCLC cells transfected with DDIAS siRNA (Fig. 1a). Similarly, when DDIAS siRNA and TRAIL were treated in parallel, HepG2, SK-HEP-1 and Li-7 HCC cell proliferation was inhibited (Fig. 2a). In addition, the expressions of cleaved caspase-8, caspase-3 and PARP-1 (poly(ADP-ribose)polymerase-1) were up-regulated in a TRAIL dose-dependent manner (Figs. 1b and 2b). In A549 cells transfected with DDIAS siRNA, TRAIL strongly induced caspase-3/7 activity and apoptosis (Figures 1c and 1d). However, tyrosine kinase inhibitors (TKIs) such as gefitinib or sorafenib did not affect the growth inhibition of A549 or HepG2 cells due to DDIAS knockdown (FIG. 3). Death receptor ligands such as Fas ligand (FasL) or TNF-α significantly induced apoptosis and growth inhibition of HCC15 cells transfected with DDIAS siRNA (FIGS. 1e and 4). Through this, the death receptor ligand can act together with the DDIAS inhibitor to achieve a synergistic effect in the prevention and treatment of cancer.

As used in the present invention, the term "cancer" medically refers to a problem in the regulation function of normal division, differentiation or death of cells, abnormally proliferating, infiltrating surrounding tissues or organs to form a lump, and forming a lump. Any state that destroys or transforms a structure. Cancer is largely divided into primary cancer that exists in the site of origin and metastatic cancer that has spread to other parts of the body from the site of origin.

"Cancer", a disease to be prevented or treated according to the present invention, is interpreted to include all diseases as long as they fall within the category of cancer medically, and specific disease names are not particularly limited, but are preferably lung cancer, liver cancer, ovarian cancer, colorectal cancer, Pancreatic cancer, stomach cancer, breast cancer, cervical cancer, thyroid cancer, parathyroid cancer, brain tumor, prostate cancer, gallbladder cancer, blood cancer, bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, uterine cancer, rectal cancer, fallopian tube carcinoma, endometrial carcinoma, esophageal cancer , small intestine cancer, endocrine adenocarcinoma, adrenal cancer, and urethral cancer. In the present invention, the cancer may preferably be lung cancer, liver cancer, brain tumor, prostate cancer, pancreatic cancer or breast cancer that is resistant to TRAIL.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier in addition to containing the DDIAS inhibitor and the death receptor ligand as active ingredients.

The type of carrier that can be used in the present invention is not particularly limited, and any carrier commonly used in the art may be used. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, maltodextrin, glycerol, ethanol, and the like. can These may be used alone or in combination of two or more.

In addition, if necessary, the pharmaceutical composition of the present invention may be used by adding other pharmaceutically acceptable additives such as excipients, diluents, antioxidants, buffers or bacteriostatic agents, fillers, extenders, wetting agents, disintegrants, dispersants, surfactants , a binder or a lubricant may be additionally added and used.

In the pharmaceutical composition of the present invention, the DDIAS inhibitor and death receptor ligand may be included in an amount of 0.00001 wt% to 99.99 wt%, preferably 0.1 wt% to 90 wt%, based on the total weight of the pharmaceutical composition. Preferably, it may be included in 0.1% to 70% by weight, more preferably 0.1% to 50% by weight, but is not limited thereto, and may be variously changed depending on the condition of the subject to be administered, the type of specific disease, the degree of progression, etc. there is. If necessary, it may be included in the entire content of the pharmaceutical composition.

The pharmaceutical composition of the present invention may be formulated and used in various dosage forms suitable for oral or parenteral administration.

Non-limiting examples of formulations for oral administration using the pharmaceutical composition of the present invention include troches, lozenges, tablets, aqueous suspensions, oily suspensions, powdered preparations, granules, emulsions, hard capsules, and soft capsules, syrups or elixirs; and the like.

In order to formulate the pharmaceutical composition of the present invention for oral administration, a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin; excipients such as dicalcium phosphate and the like; disintegrants such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax may be used, and sweeteners, aromatics, syrups, and the like may also be used. Furthermore, in the case of capsules, a liquid carrier such as fatty oil may be additionally used in addition to the above-mentioned materials.

Non-limiting examples of parenteral preparations using the pharmaceutical composition of the present invention include injection solutions, suppositories, powders for respiratory inhalation, aerosols for sprays, ointments, powders for application, oils, creams, and the like.

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

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

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

When the pharmaceutical composition of the present invention is formulated into an ointment, cream, powder for application, oil, external skin preparation, etc., animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite , silica, talc, zinc oxide, etc. may be formulated using a carrier.

In another aspect, the present invention provides a method for preventing or treating cancer comprising administering to an animal a pharmaceutical composition comprising a DNA damage-induced apoptosis suppressor (DDIAS) inhibitor and a death receptor ligand as active ingredients to provide.

As used herein, the term "animal" may include mammals including humans, but is not limited thereto.

The pharmaceutically effective amount and effective dose of the pharmaceutical composition of the present invention may vary depending on the formulation method, administration method, administration time and/or route of administration of the pharmaceutical composition, and the type of response to be achieved by administration of the pharmaceutical composition. and degree, type of subject to be administered, age, weight, general health condition, symptom or severity of disease, sex, diet, excretion, drugs used simultaneously or simultaneously with the subject, and other components of the composition, etc. It can be varied according to factors and similar factors well known in the medical field, and those skilled in the art can easily determine and prescribe an effective dosage for the desired treatment.

Administration of the pharmaceutical composition of the present invention may be administered once a day, or may be divided into several administrations. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. Considering all of the above factors, it can be administered in an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

The administration route and administration method of the pharmaceutical composition of the present invention may be independent, and may follow any route and administration method without particular limitation, as long as the pharmaceutical composition can reach the target site. The pharmaceutical composition may be administered orally or parenterally.

As a parenteral administration method of the pharmaceutical composition of the present invention, intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration, subcutaneous administration, or administration into cancer tissue may be used, and the composition may be applied to a diseased area or Nebulization and inhalation methods may also be used, but are not limited thereto.

The pharmaceutical composition of the present invention can exert excellent effects even when used alone, but can be used in combination with various methods such as hormone therapy and drug therapy to increase treatment efficiency.

The DDIAS (DNA damage-induced apoptosis suppressor) inhibitor of the present invention effectively inhibits the promotion of caspase-8 degradation and the inhibition of DISC (death-inducing signaling complex) formation by the action of DDIAS, ) It can be used in various ways as a composition for preventing and treating cancer, including lung cancer and liver cancer, by exerting an excellent synergistic effect of promoting apoptosis in cancer cells by acting together with a ligand.

In Figure 1 (a), NSCLC (A549, HCC15 and NCI-H1703) cells transfected with siRNA against DDIAS (siDDIAS) or scrambled siRNA (siScr) (10 nM) for 48 hours and treated with TRAIL for 24 hours It shows the results of cell growth analysis in . Cell growth percentage was determined by SRB assay. Data are presented as the mean ± SEM of three independent experiments. *P <0.05, **P <0.01, ***P <0.001. (b) shows the immunoblot analysis results for DDIAS expression and PARP, caspase-8 and -3 cleavage in NSCLC cells transfected with siDDIAS for 48 hours and treated with TRAIL for 4 hours. GAPDH was used as a loading control. (c) shows images of caspase-3/7 activation and annexin V staining in A549 cells transfected with siDDIAS for 48 hours and treated with TRAIL (100 ng/ml) for 18 hours. Scale bar represents 50 μm. (d) quantifies activation and apoptosis of caspase-3/7 in FIG. 1(c). (e) analyzes apoptosis based on annexin V staining in HCC15 cells transfected with siDDIAS for 48 hours and treated with FasL (100 ng/ml) or TNF-α (10 ng/ml) for 18 hours that showed the result. *P < 0.05, **P < 0.01.
In Figure 2 (a), HCC cells (HepG2, SK-HEP-1 and Li -7) shows the results of cell growth analysis. Cell growth percentage was determined by SRB assay. *P<0.05, **P<0.01, ***P<0.001 (Student's test). (b) shows the results of analysis of DDIAS expression and PARP, caspase-8 and -3 cleavage in HCC cells transfected with siDDIAS or siScr for 48 hours and treated with TRAIL for 4 hours.
In Figure 3 (a) shows the results of cell growth analysis in A549 cells transfected with siRNA against DDIAS (siDDIAS) or scrambled siRNA (10 nM) for 24 hours and treated with gefitinib for 48 hours. Cell growth percentage was determined by SRB assay. (b) shows the results of cell growth analysis in HepG2 cells transfected with siDDIAS or siScr (20 nM) for 24 hours and treated with sorafenib for 48 hours.
Figure 4 shows the results of cell growth analysis in HCC15 cells transfected with siRNA against DDIAS (siDDIAS) or scrambled siRNA (10 nM) for 48 hours and treated with TRAIL, FasL or TNF-α for 24 hours. Cell growth percentage was determined by SRB assay. *P<0.05, **P <0.01, ***P <0.001.
In Figure 5 (a) shows the results of cell growth analysis in NCI-H1703 and HepG2 cells transfected with Flag-DDIAS for 36 hours and treated with TRAIL for 24 hours. Cell growth percentage was determined by SRB assay. *P<0.05, **P<0.01. (b) is Flag-DDIAS expression and PARP, caspase-8 and -3 in NCI-H1703 and HepG2 cells transfected with Flag-DDIAS for 36 hours and treated with TRAIL (50 ng/ml) for 4 hours It shows the results of the analysis of the cleavage. (c) shows activation of caspase-3/7 in HepG2 cells transfected with Flag-DDIAS for 36 hours and treated with TRAIL (50 ng/ml) for 18 hours. Scale bar represents 50 μm. (d) is a quantification of apoptosis in FIG. 2(c). **P<0.01.
In Figure 6 (a) shows the protein array in HepG2 cells transfected with siRNA against DDIAS (siDDIAS) or scrambled siRNA (siScr) (20 nM) for 72 hours. (b) is HepG2 cells transfected with 20 nM siDDIAS for 48 hours, treated with TRAIL for 4 hours, and then p-p53 (S15), p-p53 (S392), DR4, DR5 and The results of immunoblot analysis for DDIAS levels are shown. GAPDH was used as a loading control. (c, d) were transfected with siDDIAS, siRNA against p53 (sip53), or siScr for 48 hours, and TRAIL (100 ng/ml) for 4 hours (immunoblotting) or 24 hours (cell growth assay) The results of cell growth analysis and immunoblot analysis for the expression of PARP, caspase-8, DDIAS, p53 and GAPDH (loading control) in the treated A549 cells are shown. (e, f) PARP, caspase-8 and -3 in NCI-H1299 cells transfected with siDDIAS for 48 hours and treated with TRAIL for 4 hours (immunoblotting) or 24 hours (cell growth assay). , Cell growth analysis and immunoblot analysis results for the expression of DDIAS and GAPDH are shown. *P<0.05, **P<0.01.
FIG. 7 shows the results of quantitative real-time PCR on the transcript levels of DDIAS, DR4, and DR5 in A549 cells transfected with siRNA against DDIAS (siDDIAS) or scrambled siRNA (10 nM) for 72 hours. **P<0.01.
In FIG. 8 (a), p-MKK4, p in A549 cells transfected with DDIAS siRNA (siDDIAS) or scrambled siRNA (siScr) for 48 hours and treated with TRAIL (100 ng/ml) for 4 hours. -The results of immunoblot analysis for the levels of MKK7, p-JNK, p-p38, p38 and GAPDH are shown. (b, c) are cells in A549 cells transfected with siDDIAS for 48 hours, pretreated with p38 MAPK and JNK inhibitors (SB203580 or SP600125, 10 μM), and then treated with TRAIL (100 ng/ml) for 24 hours It shows the results of the growth analysis. (d) is A549 treated with TRAIL (100 ng/ml) for 24 hours after transfection with siDDIAS and siRNA against caspase-8 (siCasp8) or siRNA against caspase-9 (siCasp9) for 48 hours. It shows the results of cell growth analysis in cells. **P<0.01. (e) is the level of PARP, caspase-8, -3, DDIAS and GAPDH in A549 cells transfected with siDDIAS, siCASP8 or siCASP9 for 48 hours and then treated with TRAIL (100 ng/ml) for 4 hours It shows the result of immunoblot analysis for. (f) is the expression of p-p53 (S15), p-JNK, p-p38 and GAPDH in A549 cells transfected with siDDIAS or siCASP8 for 48 hours and then treated with TRAIL (100 ng/ml) for 4 hours. It shows the result of immunoblot analysis for expression.
Figure 9 shows the expression of p-MKK4, p-MKK7, p-MKK4 in HepG2 cells transfected with siRNA against DDIAS (siDDIAS) or scrambled siRNA (siScr) for 48 hours and treated with TRAIL (10 ng/ml) for 4 hours. The results of immunoblotting analysis for the levels of JNK, p-p38, p38, DDIAS and GAPDH are shown.
In FIG. 10 (a), it was confirmed whether or not DISC was formed in HepG2 cells transfected with DDIAS siRNA (siDDIAS) or scrambled siRNA (siScr) for 60 minutes and treated with Flag-TRAIL for 30 minutes. Cell lysates were immunoprecipitated using anti-Flag agarose and immunoprecipitates were probed with the indicated antibodies. (b) shows endogenous binding between FADD and DDIAS, and shows the results of immunoprecipitation of HepG2 cell lysates with anti-DDIAS antibodies and immunoblotting with anti-FADD, caspase-8 and -DDIAS antibodies. (c) shows the binding of HA-FADD and Flag-DDIAS in 293T cells transfected with HA-FADD and Flag-DDIAS, and cell lysates were prepared using anti-Flag agarose or anti-HA beads. Immunoprecipitated and immunoprecipitates were probed with the indicated antibodies. (d, e) 293T cells were transfected with the indicated combinations of deletion mutants of Flag-DDIAS and HA-FADD, cell lysates were immunoprecipitated using anti-Flag agarose or anti-HA beads and , The results of probing the immunoprecipitate with anti-HA or -Flag antibodies are shown. (f) shows the results of immunoprecipitation of A549 cell lysates with anti-FADD antibodies and probing with anti-DDIAS, -caspase-8 and -FADD antibodies. (g) shows the results of immunoprecipitation of A549 cell lysates with anti-caspase-8 antibodies and probing of the immunoprecipitates with anti-DDIAS and -caspase-8 antibodies. (h) analyzes FADD/caspase-8 binding. NCI-H1299 cells were transfected with HA-FADD, Myc-caspase-8, and Flag-DDIAS for 36 hours, and cell lysates were treated with anti-HA. After immunoprecipitation with agarose, the results of probing the immunoprecipitate with anti-Myc, -Flag and -HA antibodies are shown.
In FIG. 11 (a, b), immunoblotting with antibodies directed against the expression of caspase-8, FADD and DDIAS in cells transfected with siRNA against DDIAS (siDDIAS) or scramble siRNA (siScr) for 60 hours was performed. It shows the result of lotting (a) and the result of performing quantitative real-time PCR (b). GAPDH was used as an internal control. (C) shows the expression of caspase-8 in cells transfected with siDDIAS for 48 hours and treated with EGF (100 ng/ml) and cycloheximide (CHX; 20 μM) for the indicated times. *P<0.05, **P<0.01. (d) shows the phosphorylation of RSK2 in cells transfected with siDDIAS for 60 hours and treated with EGF (100 ng/ml) for the indicated times, and the results of immunoblotting of cell lysates with the indicated antibodies it is shown (e) shows the ubiquitination of caspase-8 in cells transfected with siDDIAS for 60 hours and treated with EGF (100 ng/ml) and MG132 (10 μM). It shows the results of immunoprecipitation with the -8 antibody and immunoblotting with the indicated antibodies. (f) shows the expression of caspase-8 in cells co-transfected with DDIAS siRNA and Myc-RSK2 for 48 hours.
Figure 12 shows the DDIAS-mediated caspase-8 regulation model. Under normal conditions, DDIAS interacts with FADD and phosphorylates RSK2 to promote proteosome-associated degradation of caspase-8. In the presence of TRAIL, FADD dissociates from DDIAS to form DISC in the extrinsic apoptotic pathway. In the presence of DDIAS knockdown and TRAIL, caspase-8 is stabilized and DISC formation is promoted to induce TRAIL-mediated apoptosis.
Figure 13 shows brain tumor cells (T98G, U-87MG), pancreatic cancer cells (Panc1, Panc1, AsPC1), breast cancer cells (MCF7, Hs578T) and prostate cancer cells (DU145, PC3) cell growth analysis results are shown. Cell growth percentage was determined by SRB assay.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples.

statistical analysis

Data herein are presented as the mean±SEM of three independent experiments. Differences between groups were evaluated with two-tailed Student's t test followed by analysis of variance by Newman-Keuls test. P<0.05 was considered significant.

<Materials and methods>

Example 1: Preparation of Materials and Reagents

Recombinant human TRAIL and Flag-tagged TRAIL were obtained from Peprotech (Rocky Hill, NJ, USA) and Enzo Life Science (New York, NY, USA), respectively. Recombinant human FasL, TNF-α, EGF, Z-VAD-FMK, Z-IETD-FMK, Z-LEHD-FMK and Proteome Profiler Human Apoptosis array kits were obtained from R&D Systems (Minneapolis, MN, USA). ) was purchased. SB203580 and SP600125 were purchased from Millipore (Billerica, MA, USA). Sorafenib and gefitinib were obtained from Selleckchem (Houston, TX, USA). Cycloheximide, MG132 and DAPI were obtained from Sigma-Aldrich (St. Louis, MO, USA). siRNAs against DDIAS were obtained from ST pharm (Siheung, Korea), and siRNAs against p53, caspase-8 and caspase-9 were obtained from Bioneer (Daejeon, Korea). Antibodies used were: anti-Mc, -HA, -PARP and -p53 (all obtained from Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-caspase-3, -caspase-8, -caspase-9, -FADD, -phospho-(p-)MKK4, -pMKK7, -MKK4, -MKK7, -p-p38, anti-p- JNK, -p38, -JNK, -p-p53(S46), -p-p53(S392), -DR4, -DR5, -p-RSK2 (S227), -p-Src (Y416), -Src and - RSK2 (all obtained from Cell Signaling Technology, Beverly, MA, USA); anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (AbFrontier, Seoul, Korea); anti-Flag (Sigma-Aldrich); and anti-DDIAS (Atlas Antibodies, Stockholm, Sweden).

Example 2: DNA construct preparation

Human FADD cDNA (GenBank accession no. NM_003824) was obtained from Korea Human Gene Bank (Daejeon, Korea). DYK-tagged human caspase-8 was obtained from GeneScript (Piscataway, NJ, USA). Human RSK2 cDNA (GenBank accession no. NM_004586) was obtained from A549 cDNA. These clones were amplified by PCR and inserted into pcDNA3.1 with HA or Myc tags. Full-length and deletion mutants of Flag-DDIAS have already been reported (Won KJ et al. Cell Death Dis 2017; 8(1): e2554). A deletion mutant of FADD was subcloned into an HA-tagged fusion plasmid. All constructs were sequenced.

Example 3: Cell culture and transfection

293T cells, NSCLC cells (A549, NCI-H1299 and NCI-H1703), HCC cells (HepG2, SKHEP-1 and Li-7), brain tumor cells (T98G, U-87MG), pancreatic cancer cells (Panc1, AsPC1), breast cancer Cells (MCF7, Hs578T) and prostate cancer cells (DU145, PC3) were cultured in each Dulbecco's modified form containing 10% fetal bovine serum, 50 U/ml penicillin, and 50 μg/ml streptomycin (Invitrogen, Carlsbad, CA, USA). Eagle's medium and Roswell Park Memorial Institute-1640 medium were cultured in an incubator at 37°C and 5% CO ₂ conditions. The cells were transfected with siRNA through electroporation (Neon transfection system, Invitrogen) according to the manufacturer's instructions. The target sequences used were as follows:

DDIAS, 5'-CAGAAGAGAUCUGCAUGUU-3' (SEQ ID NO: 8);

p53, 5'-CACUACAACUACAUGUGUA-3' (SEQ ID NO: 13);

Caspase-8, 5'-GCAAUCUGUCCUUCCUGAA-3' (SEQ ID NO: 14);

Caspase-9, 5'-CAGAUUCGCUCAUAGAUCA-3' (SEQ ID NO: 15); and

Scramble (control), 5′-CCUACGCCACCAAUUUCGU-3′ (SEQ ID NO: 16).

Example 4: Cell viability, cell death and caspase-3/7 assay

Cell viability was evaluated using a conventionally known sulforhodamine B (SRB) assay (Kim BK et al. Biochim Biophys Acta 2014; 1839(5): 364-73). The amount of SRB dye bound to the cell substrate was measured at a wavelength of 530 nm using a spectrophotometer. Apoptosis was analyzed using CellPlayer reagent-based annexin V and caspase-3/7 assays (Essen Bioscience, Ann Arbor, MI, USA). Cell images were taken at 2-hour intervals using a conventionally known IncuCyte Zoom system (Essen Bioscience) (Kim BK et al. Biochim Biophys Acta 2014; 1839(5): 364-73). Relative fluorescence was expressed by dividing green or red fluorescence by cell confluence. Images were used to confirm morphological changes due to transfection of siRNAs against DDIAS or TRAIL, or scrambled control siRNAs.

Example 5: Quantitative real-time PCR

Total RNA was extracted from cells using TRIzol reagent (Invitrogen), and 2 μg was reverse transcribed into cDNA using TOPscript RT DryMIX (dT18) (Enzynomics, Daejeon, Korea) according to the manufacturer's instructions. Quantitative real-time PCR was performed using a Rotor-Gene 6000 system (Corbett/Thermo Fisher Scientific, Waltham, MA, USA). Cycling conditions were performed at 95 °C for 5 minutes, 95 °C for 30 seconds, 55 °C for 30 seconds, and 72 °C for 30 seconds. The primers used are shown in Table 5 below.

[Table 5]

Primers for DR4 (P166162), DR5 (P195586), caspase-8 (P169836) and FADD (P162725) were obtained from Bioneer (Daejeon, Korea).

Example 6: immunoprecipitation ( Immunoprecipitation ) and immunoblotting ( immunoblotting ) analyze

Cells were cultured at 0.5 in 1 × phosphate-based saline (PBS) containing 1 mM Na3VO4, 1 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail (Roche, Basel, Switzerland). % Nonidet (N) P-40. The protein concentration in the lysate was measured with a bicinchoninic acid assay kit (Bio-Rad, Hercules, CA, USA). The lysate was incubated with FLAG M2 affinity gel, HA-agarose (Sigma-Aldrich) or Myc-agarose (Santa Cruz Biotechnology), and immunoblotting was performed with a specific antibody. Immune complexes were detected with an enhanced chemiluminescence kit (Millipore). For endogenous binding, lysates were incubated with 2 μg of control IgG or DDIAS, FADD, Caspase-8 antibodies overnight at 4°C, followed by incubation with IgG agarose for 1 hour at 4°C. Precipitates were immunoblotted with specific antibodies. For the protein array, HepG2 cells were transfected with DDIAS siRNA for 72 hours and then analyzed with a proteome profile array according to the manufacturer's protocol.

Example 7: DISC ( death - inducing signaling complex ) analyze

Cells were stimulated several times with 100 ng/ml Flag-TRAIL and 1 μg/ml ligand enhancer, then immediately washed in PBS and supplemented with 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.2% NP-40, 10% glycerol. and complete protease inhibitor cocktail (Roche) for 15 min on ice. The lysate was separated twice by centrifugation at 12,000×g for 10 minutes. The soluble fraction was pre-separated with 20 μl of Sepharose-6B (Sigma-Aldrich) at 4° C. for 2 hours, and then immunoprecipitated with 20 μl of anti-Flag-agarose (Sigma-Aldrich) at 4° C. . The beads were recovered by centrifugation and washed 4 times with 500 μl of lysis buffer. Sample buffer was added and beads were heated for 5 minutes before proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting.

<result>

<1> of cancer cells TRAIL -Judo apoptosis for sensitivity DDIAS Check the knockdown effect

The present inventors screened for drugs capable of enhancing cancer cell death induced by DDIAS knockdown by siRNA of DDIAS (siDDIAS). Growth inhibition of a total of 20 inhibitors including doxorubicin, gefitinib, rapamycin, BAY 11-7082 and recombinant TRAIL against A549 cells simultaneously treated with siRNA against DDIAS of SEQ ID NO: 8 abilities were tested. As a result, it was confirmed that TRAIL significantly inhibited the growth of A549, HCC15, and NCI-H1703 cells transfected with DDIAS siRNA, compared to the control group (scramble siRNA, siScr) (FIG. 1a). Similarly, HepG2, SK-HEP-1 and Li-7 HCC cell proliferation was inhibited when siDDIAS and TRAIL were treated in parallel (Fig. 2a). In addition, the expressions of cleaved caspase-8, caspase-3 and PARP-1 (poly(ADP-ribose)polymerase-1) were up-regulated in a TRAIL dose-dependent manner (Figs. 1b and 2b). Furthermore, TRAIL strongly induced caspase-3/7 activity and apoptosis in A549 cells transfected with siDDIAS (FIGS. 1c and 1d).

However, tyrosine kinase inhibitors (TKIs) such as gefitinib or sorafenib did not affect the growth inhibition of A549 or HepG2 cells due to DDIAS knockdown (FIG. 3). This suggests a specific function of DDIAS in TRAIL-induced apoptosis.

Death receptor ligands such as Fas ligand (FasL) or TNF-α significantly induced apoptosis and growth inhibition of HCC15 cells transfected with siDDIAS ( FIGS. 1E and 4 ). This shows that DDIAS plays a critical role in apoptosis induced by death signals.

Consistent with these observations, overexpression of DDIAS inhibited the increase in apoptosis and cleavage of caspase or PARP-1 in TRAIL-treated NCI-H1703 and HepG2 cells (Fig. 5a-d), suggesting that DDIAS is a death receptor ligand. It was confirmed that it inhibits apoptosis induced by

<2> Confirmation of Independence of p53 Signal in Increased TRAIL Sensitivity by DDIAS Knockdown

Protein array analysis was performed to elucidate the function of DDIAS in TRAIL-induced apoptosis. As a result, it was confirmed that p53 phosphorylation at S15 and S392 was increased in DDIAS-depleted cells (FIG. 6a). In addition, it was confirmed that p53 phosphorylation at S15 and S392 was significantly increased in HepG2 cells transfected with siDDIAS (FIG. 6b).

Previous studies have reported that p53 activation by chemotherapeutic agents induces DR4/5 expression in non-small cell lung cancer. We investigated whether this occurred in cells with reduced DDIAS. As a result, although p53 was activated, it was confirmed that the mRNA and protein levels of DR4/5 were not affected by DDIAS knockdown (FIGS. 6b and 7).

On the other hand, phosphorylation of p53 at S15 and S392 was increased in DDIAS-reduced/TRAIL-treated A549 cells (Fig. 6b). As a result of examining whether p53 signaling is involved in TRAIL sensitivity in DDIAS-reduced cells, the present inventors confirmed that DDIAS knockdown increased apoptosis in A549 cells regardless of p53 knockdown (FIGS. 6c and 6d). In addition, it was confirmed that knockdown of DDIAS in TRAIL-treated NCI-H1299 (p53-null) cells increased cell death as well as cleavage of PARP-1 and caspase ( FIGS. 6e and 6f ).

These results indicate that DDIAS regulates TRAIL sensitivity regardless of p53 status, confirming that changes in p53 and DR are not related to DDIAS knockdown/TRAIL-induced apoptosis.

<3> Confirmation of necessity of caspase-8 activation in TRAIL sensitivity induced by DDIAS knockdown

Immunoblot was performed to confirm the effects of p38 MAPK, JNK, MKK4, and caspase-8 on TRAIL-induced apoptosis.

As a result, phosphorylation of MKK4 (mitogen-activated protein kinase kinase 4) as well as p38 MAPK and JNK known to mediate TRAIL-induced apoptosis were strongly increased in DDIAS knockdown/TRAIL-treated A549 and HepG2 cells. (FIGS. 8a and 9). However, it was confirmed that treatment with SB203580 or SP600125, which are p38 MAPK and JNK inhibitors, respectively, did not affect the apoptotic effect induced by DDIAS knockdown/TRAIL-treatment in A549 cells (FIG. 8b). This suggests that p38 MAPK and JNK activation are not involved in cell death induced by DDIAS knockdown/TRAIL-treatment. On the other hand, when treated with the pan-caspase inhibitor Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone), cell death by DDIAS knockdown/TRAIL-treatment It was confirmed that this hardly occurred (FIG. 8b).

In addition, treatment with the caspase-8 inhibitor Z-IETD-FMK (Z-Ile-Glu(OMe)-Thr-Asp(OMe)fluoromethylketone) inhibited apoptosis induced by DDIAS knockdown/TRAIL, whereas When treated with the caspase-9 inhibitor ZLEHD-FMK (Z-Leu-Glu(O-Me)-His-Asp(O-Me)), it was confirmed that there was no apoptosis inhibitory effect (FIG. 8c). Reduction of caspase-8, but not caspase-9, confirmed that DDIAS-reduced cells were able to overcome TRAIL-induced apoptosis as a result of reduced caspase and PARP-1 cleavage ( FIGS. 8D and 8E ). Furthermore, phosphorylation of p53, p38 MAPK and JNK by DDIAS knockdown/TRAIL treatment was not induced at all by inhibition of caspase-8 (FIG. 8f).

These results show that DDIAS knockdown promotes TRAIL-mediated apoptosis regardless of the intrinsic mitochondrial pathway, and it was found that activation of caspase-8 is required for TRAIL sensitization induced by DDIAS knockdown.

<4> Confirmation of DISC generation inhibition effect of DDIAS through interaction with FADD

Whether DDIAS is involved in the formation of DISC in the extrinsic apoptotic pathway was analyzed. As a result, it was confirmed that DISC was formed by DR5, FADD, and caspase-8 within 30 minutes when Flag-TRAIL, not DDIAS, was treated (FIG. 10a). In addition, it was confirmed that knockdown of DDIAS enhances the movement of FADD and caspase-8 to the DISC in cells treated with TRAIL (FIG. 10a). However, DDIAS knockdown had no effect on the movement of DR5 to the DISC (Fig. 10a).

Furthermore, in a co-immunoprecipitation experiment using an anti-DDIAS antibody, whether DDIAS interacts with FADD or caspase-8 was investigated, and as a result, endogenous DDIAS, not endogenous caspase-8, It was confirmed that FADD and co-immunoprecipitation were performed (FIG. 10b).

To further validate the interaction between DDIAS and FADD, reciprocal immunoprecipitation experiments were performed in HEK293T cells in the absence of TRAIL. As a result, HA-FADD was detected when Flag-DDIAS was immunoprecipitated with an anti-Flag antibody, and the interaction between DDIAS and FADD was determined by reverse co-immunoprecipitation using an anti-HA antibody. It was confirmed through (FIG. 10c).

To identify the region of FADD that interacts with DDIAS, a HA-FADD deletion construct containing either DED (amino acids 1-80) or DD (amino acids 81-208) was generated (Fig. 10d). As a result of the experiment, it was confirmed that DDIAS interacted with the FADD N-terminal region including DED, but did not interact with the C-terminal region including DD (FIG. 10d). In addition, it was confirmed that HA-FADD was co-immunoprecipitated with Flag-DDIAS including F (amino acids 1-998) and N (amino acids 1-400) (FIG. 10e). In particular, in the absence of TRAIL, the two proteins FADD and DDIAS could physically interact under normal conditions, but the binding between FADD and DDIAS was reduced when treated with TRAIL (FIG. 10f). In contrast, it was confirmed that DDIAS did not bind to caspase-8 regardless of TRAIL treatment or normal conditions (Fig. 10g).

Based on the observation that the N-terminal DED of procaspase-8 binds to the N-terminal DED of FADD, a competitive binding assay was performed to determine whether DDIAS affects the interaction between FADD and caspase-8. was performed. Specifically, experiments were performed after cells were transfected with fixed amounts of Myc-caspase-8 and HA-FADD and increasing amounts of Flag-DDIAS. As a result, it was confirmed that in the immunoprecipitate containing anti-HA agarose bound to Myc-caspase-8, the binding between FADD and caspase-8 was reduced by adding a larger amount of DDIAS (FIG. 10h). . These results suggest that DDIAS inhibits DISC formation through interaction with FADD.

<5> Confirmation of the effect of increasing ubiquitination of DDIAS and promoting caspase-8 degradation

Expression levels of caspase-8 and FADD following DDIAS knockdown were analyzed. As a result, it was confirmed that the protein level of caspase-8 increased in DDIAS-depleted cells, but the mRNA level of caspase-8 was not affected (FIG. 11b). In addition, as a result of examining the half-life of caspase-8 protein in A549 cells treated with EGF and cycloheximide (CHX) under the reduction of DDIAS, it was confirmed that the half-life was stable compared to control cells (siScr) (Fig. 11c). .

It has been previously reported that RSK2 phosphorylates caspase-8 to promote ubiquitination and proteasomal degradation. Accordingly, the present inventors investigated the effect of reducing DDIAS on the activation of Src or RSK2 in the presence of EGF in A549 cells. As a result, it was confirmed that DDIAS knockdown completely blocked RSK2 phosphorylation at S227 in the presence of EGF, but did not affect Src activation (FIG. 11d). In addition, it was confirmed that EGF treatment induced caspase-8 ubiquitination in the presence of the proteasome inhibitor MG132, but not at all by DDIAS knockdown (FIG. 11e). This means that proteasome-associated degradation of caspase-8 was induced by DDIAS-mediated phosphorylation of RSK at S227.

Furthermore, it was confirmed that overexpression of RSK2 inhibits DDIAS knockdown-mediated stabilization of caspase-8 (FIG. 11f). These results suggest that DDIAS promotes phosphorylation of RSK2 at S227, induces ubiquitination of caspase-8 against proteasome-related degradation, and protects cells from apoptosis.

<6> Confirmation of the effects of TRAIL treatment and DDIAS knockdown in various cancer cells

The present inventors confirmed whether the simultaneous treatment of TRAIL and siDDIAS exhibits a cell proliferation inhibitory effect in various cancer cells. Specifically, the cell proliferation inhibitory effect on brain tumor cells T98G and U-87MG, pancreatic cancer cells Panc1 and AsPC1, breast cancer cells MCF7 and Hs578T, and prostate cancer cells DU145 and PC3 was confirmed. As a result, compared to the control group (scramble siRNA, siScr), it was confirmed that the cells transfected with DDIAS siRNA were significantly inhibited in cell proliferation when TRAIL was treated (FIG. 13). These results suggest that DDIAS inhibitors and death receptor ligands can exhibit cytostatic effects on various cancers.

In this specification, the detailed description of the contents that can be sufficiently recognized and inferred by those skilled in the art of the present invention is omitted, and the technical spirit or essential configuration of the present invention is changed in addition to the specific examples described in this specification. More diverse modifications are possible within the range not specified. Accordingly, the present invention may be practiced in a manner different from that specifically described and exemplified herein, which can be understood by those skilled in the art.

<110> KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY <120> Composition for preventing or treating cancer comprising DDIAS inhibitors and death receptor ligands <130> P17-080-KRI <160> 20 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> RNA <213> artificial sequence <220> <223> DDIAS_880-898 <400> 1 gattcatgga gccttgttt 19 <210> 2 <211> 19 <212> RNA <213> artificial sequence <220> <223> DDIAS_2673-2691 <400> 2 ctatcatttc cctgatcaa 19 <210> 3 <211> 19 <212> RNA <213> artificial sequence <220> <223> DDIAS_1153-1171 <400> 3 cagaagagat ctgcatgtt 19 <210> 4 <211> 19 <212> RNA <213> artificial sequence <220> <223> DDIAS siRNA (1)_sense <400> 4 gauucaugga gccuuguuu 19 <210> 5 <211> 19 <212> RNA <213> artificial sequence <220> <223> DDIAS siRNA(1)_anti sense <400> 5 aaacaaggcu ccaugaauc 19 <210> 6 <211> 19 <212> RNA <213> artificial sequence <220> <223> DDIAS siRNA (2)_sense <400> 6 cuaucauuuc ccugaucaa 19 <210> 7 <211> 19 <212> RNA <213> artificial sequence <220> <223> DDIAS siRNA(2)_anti sense <400> 7 uugaucaggg aaaugauag 19 <210> 8 <211> 19 <212> RNA <213> artificial sequence <220> <223> DDIAS siRNA (3)_sense <400> 8 cagaagagau cugcauguu 19 <210> 9 <211> 19 <212> RNA <213> artificial sequence <220> <223> DDIAS siRNA(3)_anti sense <400> 9 aacaugcaga ucucuucug 19 <210> 10 <211> 44 <212> RNA <213> artificial sequence <220> <223> DDIAS shRNA (1) <400> 10 gattcatgga gccttgtttg agctcaaaca aggctccatg aatc 44 <210> 11 <211> 44 <212> RNA <213> artificial sequence <220> <223> DDIAS shRNA (2) <400> 11 ctatcatttc cctgatcaag agctcttgat cagggaaatg atag 44 <210> 12 <211> 44 <212> RNA <213> artificial sequence <220> <223> DDIAS shRNA (3) <400> 12 cagaagat ctgcatgttg agctcaacat gcagatctct tctg 44 <210> 13 <211> 19 <212> RNA <213> artificial sequence <220> <223> p53_target <400> 13 cacuacaacu acaugugua 19 <210> 14 <211> 19 <212> RNA <213> artificial sequence <220> <223> caspase-8_target <400> 14 gcaaucuguc cuuccugaa 19 <210> 15 <211> 19 <212> RNA <213> artificial sequence <220> <223> caspase-9_target <400> 15 caguaucgcu cauagauca 19 <210> 16 <211> 19 <212> RNA <213> artificial sequence <220> <223> scramble <400> 16 ccuacgccac caauuucgu 19 <210> 17 <211> 20 <212> DNA <213> artificial sequence <220> <223> DDIAS primer_F <400> 17 cttgcagcag ttgttacgaa 20 <210> 18 <211> 20 <212> DNA <213> artificial sequence <220> <223> DDIAS primer_R <400> 18 gtgaccaagc acttcgagtt 20 <210> 19 <211> 20 <212> DNA <213> artificial sequence <220> <223> GAPDH primer_F <400> 19 tcatgaccac agtccatgcc 20 <210> 20 <211> 20 <212> DNA <213> artificial sequence <220> <223> GAPDH primer_R <400> 20 tccaccaccc tgttgctgta 20

Claims (11)

A pharmaceutical composition for preventing or treating cancer comprising a DNA damage-induced apoptosis suppressor (DDIAS) inhibitor and a death receptor ligand as active ingredients,
The DDIAS inhibitor is selected from the group consisting of siRNA, antisense oligonucleotide and shRNA that inhibit the expression of the DDIAS gene,
The death receptor ligand is selected from the group consisting of TRAIL (TNF-related apoptosis-inducing ligand), TRAs (TRAIL receptor agonists), TNF-α and FasL (Fas ligand; CD95L),
The TRAIL receptor agonists (TRAs) are selected from the group consisting of recombinant TRAIL, TRAIL-R1-specific antibodies and TRAIL-R2-specific antibodies that can bind to TRAIL receptors and perform TRAIL-mediated death signaling, pharmaceutical composition. delete The method of claim 1, wherein the siRNA,
siRNA having a sense sequence of SEQ ID NO: 4 and an antisense sequence of SEQ ID NO: 5;
siRNA having a sense sequence of SEQ ID NO: 6 and an antisense sequence of SEQ ID NO: 7; and
A pharmaceutical composition selected from the group consisting of siRNA having a sense sequence of SEQ ID NO: 8 and an antisense sequence of SEQ ID NO: 9. The pharmaceutical composition according to claim 1, wherein the antisense oligonucleotide is antisense RNA, and the antisense RNA has a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9. The pharmaceutical composition according to claim 1, wherein the shRNA is encoded with a DNA sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12. delete The pharmaceutical composition according to claim 1, wherein the death receptor ligand is TRAIL or TRAs. The pharmaceutical composition according to claim 1, wherein the DDIAS inhibitor inhibits inactivation of caspase-8. The pharmaceutical composition according to claim 1, wherein the DDIAS inhibitor promotes the formation of a death-inducing signaling complex (DISC). The method of claim 1, wherein the cancer is lung cancer, liver cancer, ovarian cancer, colon cancer, pancreatic cancer, stomach cancer, breast cancer, cervical cancer, thyroid cancer, parathyroid cancer, brain tumor, prostate cancer, gallbladder cancer, blood cancer, bladder cancer, kidney cancer, melanoma A pharmaceutical composition selected from the group consisting of colon cancer, bone cancer, skin cancer, uterine cancer, rectal cancer, fallopian tube carcinoma, endometrial carcinoma, esophageal cancer, small intestine cancer, endocrine cancer, adrenal cancer and urethral cancer. The pharmaceutical composition according to claim 10, wherein the cancer is lung cancer, liver cancer, pancreatic cancer, prostate cancer, breast cancer or brain tumor.

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