WO2013035895A1 - Novel apoptosis regulator - Google Patents

Abstract

The present invention relates to a novel apoptosis regulator which comprises siRNA consisting of at least one nucleic acid sequence selected from the group consisting of nucleic acid sequences of SEQ ID NOs: 9-11, wherein the novel apoptosis regulator is useful for the treatment of diseases resulting from low levels of apoptosis, by targeting an apoptosis-inhibiting gene.

Description

Novel Apoptosis Regulators

The present invention relates to novel apoptosis regulators that promote apoptosis by inhibiting or inhibiting expression of apoptosis inhibiting genes.

Apoptosis refers to the destruction or suicide of cells in eukaryotic cells and is a basic intracellular process for maintaining homeostasis in individuals, including controlling the normal development of animals, or eliminating unnecessary or abnormal cells. to be. Apoptosis occurs in response to various external and internal stimuli, with the progression of apoptosis, such as cytoplasmic breakdown, blebbing of cell membranes, changes in the cytoskeleton, cell contraction, chromosome condensation and DNA fragmentation. Characteristic changes occur ⁸ .

Such apoptosis is a physiologically important phenomenon and is precisely regulated by in vivo complex mechanisms, and induction of inappropriate cell death, ie, inhibition or promotion of apoptosis due to abnormal regulation, is associated with many diseases.

In particular, excessive apoptosis can lead to the destruction of certain cells and thereby the loss of function in vivo, which can lead to central nervous system diseases such as a number of acute and chronic degenerative diseases (eg, Alzheimer's disease, Parkinson's disease, cerebral ischemia). / Stroke) ^{13, 20, 21, 18} , cardiovascular disease ²⁷ , and autoimmune diseases ^16,12 such as Graves' disease and type 2 diabetes.

On the other hand, a representative disease associated with abnormal inhibition of cell death is cancer. That is, the accumulation of tumor cells that could not be properly removed due to the underactivation of apoptosis is known to be closely related to the development of cancer ²² .

Therefore, it is possible to understand the mechanism of development of various diseases through the identification of factors and regulatory mechanisms that control apoptosis, which can be usefully used in the development of therapeutic / diagnostic agents for diseases related to apoptosis. In this regard, a number of apoptosis regulators which act at each stage leading to apoptosis are known to date.

Pathways leading to apoptosis can be classified into pathways through and / or not through apoptosis receptors. The former is apoptosis induced by the binding of a specific ligand to its receptor, and the receptors involved therein include Fas, tumor necrosis factor receptor 1 (TNFR), and TRAIL (TNF-related apoptosis-inducing ligand). The latter is apoptosis induced by stress, and stresses that can cause apoptosis include ultraviolet rays, heat shock, gamma irradiation, and hypoxia. Apoptosis caused by this stimulus is completed via factors acting in the downstream stages, a representative caspase protease. They are a group of proteins that act sequentially in the signaling pathway leading to cell death, and are involved in cell death by cleaving specific amino acid (aspartate) sites to activate a series of proteins.

Thus, various factors acting in this series of steps can be targeted to modulate apoptosis, for example, Walczak et al. ²⁴ use TRAIL protein as a cancer therapeutic agent as apoptosis promoting factor induced via cellular receptors. Started. Fulda et al. ⁴ also proposed the use of Fas-L as a target of cell receptor-induced apoptosis regulation.

Furthermore, p53 is a representative protein associated with induction of apoptosis, and it has been found that p53 is involved in the removal of these cells as mediators of apoptosis in cells showing abnormal cell growth by oncogenic genes such as Myc ^{. 23} . p53 is a representative tumor suppressor protein involved in repairing damaged DNA and cell cycle regulation. It acts as a transcriptional regulator with high affinity for a target gene in a target gene for regulation of expression of the target gene at the transcription level. It plays an important role in inhibiting the division of cells or selectively destroying abnormal cells with damaged DNA or abnormal division, thereby preventing their progression to cancer ¹⁵ .

WO 03/076647 discloses JADE genes, proteins that function as cell death and cell cycle regulators downstream of cell death, and methods for screening apoptosis control substances using the same.

Korean Patent Application Publication No. 2001-113088 discloses CIA proteins, genes and their use to interact with specific DNases activated by caspases to regulate apoptosis.

Korean Patent Application Publication No. 2002-40521 discloses an anticancer agent comprising microlactone using apoptosis.

However, in response to a variety of stimuli, an accurate understanding of the apoptosis mechanisms induced by complex steps interacting with each other, and on the basis of the There is a need for the development of additional factors involved in regulation.

Accordingly, the present inventors have disclosed a new gene KIAA0317, which inhibits cell death by inhibiting the activity of protease caspase, which is essential for cell death, in Korean Patent Application Publication No. 2008-80863.

(Patent Document 1) International Patent Publication WO 03/076647

(Patent Document 2) Korean Patent Application Publication 2001-113088

(Patent Document 3) Korean Patent Application Publication 2002-40521

(Patent Document 4) Korean Patent Application Publication 2008-80863

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An object of the present invention is to provide apoptosis control agent and a therapeutic agent for apoptosis-related diseases that can inhibit or inhibit the expression of genes that inhibit apoptosis.

To achieve the above object, the present invention provides an apoptosis control agent comprising an siRNA consisting of the nucleic acid sequence of SEQ ID NO: 9 to 11 and at least one nucleic acid sequence selected from the group consisting of the complementary sequence of the nucleic acid sequence.

The present invention also provides a shRNA vector expressively comprising an siRNA consisting of one or more nucleic acid sequences selected from the group consisting of the nucleic acid sequences of SEQ ID NOs: 9-11.

For example, the apoptosis modulator may inhibit or inhibit the expression of apoptosis inhibitory gene or protein, and the apoptosis inhibitory gene may be KIAA0317 gene.

For example, the apoptosis modulator may be useful for the treatment or prevention of cancer, but is not limited thereto.

In another embodiment, the apoptosis modulator may be used for the treatment of cancer, or as an adjuvant, and also increases anticancer agent sensitivity of cancer cells.

The present invention also provides a pharmaceutical composition comprising the apoptosis modulator of the present invention, useful for the treatment or prevention of a disease associated with the under activity of apoptosis. Preferably, the pharmaceutical composition may be used in combination with an anticancer agent.

Apoptosis regulators comprising an siRNA consisting of at least one nucleic acid sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NOs: 9 to 11 according to the present invention, by targeting a gene that inhibits apoptosis, It can be usefully used for the treatment of the resulting disease.

Figure 1 schematically shows the isolation process of cell lines resistant to apoptosis induced by p53, and the cloning method of apoptosis inhibiting gene therefrom.

FIG. 2 compares homology at the protein level for the KIAA0317 protein C terminal region and HECT regions derived from various proteins. FIG. 2A shows a comparison of the protein comprising the HECT region at the amino acid level with KIAA0317, and FIG. 2B shows the percent homology in the HECT region. * Indicates cysteine residues important for thioester bond formation in the previously identified HECT region.

Figure 3 shows the E3 ubiquitin ligase activity of KIAA0317 protein. Substitution of a cysteine residue in the HECT region with an alanine residue indicates loss of E3 ubiquitin ligase activity.

Figure 4a is a schematic of the primary structure of the KIAA0317 protein used in the inhibitory action analysis of apoptosis, Figure 4b shows the inhibitory action of p53, TNFα and STS (strausporine) induced apoptosis of KIAA0317 protein.

FIG. 5 shows that KIAA0317 protein inhibits the activity of protease caspase 9 and caspase 3, a protease that is activated by the apoptosis cascade.

FIG. 6 shows that upon apoptosis stimulation by KIAA0317 expression, ubiquitination of XIAP is inhibited and ubiquitination of XIAP antagonists smac / DIABLO, HtrA2, ATRS protein is increased.

7 shows that KIAA0317 protein ubiquitizes smac / DIABLO, HtrA2, ARTS protein in vitro.

8 is As a result of Western blocking the expression level of the protein in 10 lung cancer cell lines and 9 colorectal cancer cell lines using an antibody against KIAA0317, it is shown that KIAA0317 is overexpressed in 13 cell lines.

9 shows aspects of KIAA0317 expression in cancer and normal tissues from various organs.

10 is a measure of the degree of inhibition of the expression of KIAA0317 gene when a total of 11 siRNAs are introduced into the H1299 cancer cell line by RT-PCR method. Among the siRNAs tested, siRNAs 3, 5 and 6 showed the highest effect on the inhibition of KIAA0317 gene expression.

Figure 11 shows that the introduction of siRNA that inhibits the expression of the KIAA0317 gene into the H1299 cancer cell line, the effect of anticancer drugs such as doxorubicin and etoposide.

Figure 12 shows the formation of siRNA (SEQ ID NO: 9) expression vector that inhibits the expression of the KIAA0317 gene and introducing it into the H1299 cancer cell line, and then observed cancer formation in SCID mice. Cancer formation has been shown to be inhibited in cancer cells expressing the siRNA of KIAA0317.

Hereinafter, the present invention will be described in detail.

The present inventors screened a mouse-derived cDNA library using a Saos-2 cell line (FIG. 1) to isolate cDNAs of about 2.2 kb that confer strong resistance to apoptosis induced by p53, and sequenced. Was determined (SEQ ID NO: 5). As a result, 9-51 corresponds to a gene located on human chromosome 14 (14q34.3) (National Center for Biotechnology Information (NCBI, www.ncbi.nlm.nih.gov) name KIAA0317 (SEQ ID NO: 1)). Its mouse homologues are shown in SEQ ID NO: 2.

Results of homology analysis using a computer using a program called BLASTN or BLASTX (Altschul et al 1990; http://www.ncbi.nlm.nih.gov.) Developed based on BLAST (Karlin et al., 1993) KIAA0317 of the present invention was found to be similar to the sequences of GenBank AK077015 (mouse) and AB002315 (human), but its function is not known at all. However, as a result of homology analysis using the program, the C-terminal region of KIAA0317 was found to have a very low similarity with the HECT (Homology to E6-AP Carboxy Terminus) region (Fig. 2).

The HECT (Homology to E6-AP Carboxy Terminus) region is a conserved region consisting of about 350 amino acid residues, which is primarily an E3 ubiquitin ligase involved in the ubiquitination of proteins, such as E6-AP (Huibregtse et al., 1995), Nedd4 (Harvey and Kumar, 1999), SMURF2 (Lin et al., 2000), and PUP1 (Arendt and Hochstrasser, 1997) and the like (Huibregtse et al., 1995). In mammals, E3 ubiquitin ligase has been found to cover about 20 HECT regions to date, but their role varies greatly depending on the intracellular function involved in ubiquitination (You and Pickart, 2001). Therefore, even a protein containing an HECT region requires many experiments that are not obvious to those skilled in the art before elucidating specific functions thereof in the cell, and in particular, such as KIAA0317 of the present invention, E6-AP, hNedd4, hSMURF2, This is even more the case when both the HECT region and homology of hPUP1 and yRSP5 are very low, less than 35% (FIG. 2).

Accordingly, the present inventors have found that KIAA0317 can effectively inhibit cell death as an E3 ubiquitin ligase including the HECT region.

In addition, in the present invention, it was confirmed that the KIAA0317 protein had E3 ubiquitin ligase activity (FIG. 3), and in vivo, overexpression of the KIAA0317 gene was induced by p53, TNF-alpha, and staurosporine (STS). It was confirmed that can be significantly inhibited (about 50% or more, Figure 4). In addition, the expression of KIAA0317 in normal and cancerous tissues of cancer patients was investigated. As a result, 48%, 46%, 33%, 41% of various cancers, for example, stomach cancer, colon cancer, liver cancer, breast cancer, and lung cancer, respectively, And overexpression of KIAA0317 was observed in 42% (FIG. 6). Furthermore, inhibition of endogenous KIAA0317 protein expression by siRNA (Small Interfering RNA) not only induced apoptosis but also inhibited the growth of cancer cells and induced sensitivity to anticancer agents (FIGS. 7 and 8). These results demonstrate that the KIAA0317 gene can be a target molecule for treating diseases related to inhibition of apoptosis.

The present invention provides an apoptosis regulator comprising a KIAA0317 protein, a gene encoding the same, or a functional equivalent thereof as an active ingredient.

The KIAA0317 protein, the gene sequence encoding it, can be used from a variety of mammals, but is preferably of human origin, which is purified from natural sequence sources, present in all cell types of human and non-human mammal species. , Chemically synthesized, produced by DNA recombination techniques, or prepared by a combination of these and / or other methods.

Furthermore, the KIAA0317 protein of the present invention, and the gene encoding the same, include its functionally equivalent variants. Functionally equivalent variants for the purposes of the present invention refer to compounds that exhibit biological activity comparable to the naturally occurring sequences found. Considering the object of the present invention, functionally equivalent variants are meant to have the effect of the present invention including the activity of inhibiting apoptosis. Specifically, functionally equivalent variants include GenBank Accession Nos. AB002315 (Human) (SEQ ID NO: 1) and AK077015 (Mouse) (SEQ ID NO: 2), 29470 sequence and protein described in US Patent Publication 20050196754, for the gene BAA20775 But not limited to those derived from (mouse) and BAC36566 (human) sequences.

In addition, such functionally equivalent variants may be substituted, deleted, added, and / or inserted with the nucleotides constituting the gene sequence or the amino acid sequence constituting the protein sequence as long as they have the effect of the present invention including the activity of inhibiting apoptosis. Sequence variants.

Such protein sequence variations are well known to those skilled in the art, for example, site-directed mutagenesis, Kramer, W. & Fritz, HJ. Oligonucleotide-directed construction of mutagenesis via gapped duplux DNA. Enzymology, 154, 350-367, 1987) and the like. Polynucleotide sequence variations also occur naturally.

Such variations in sequence may or may not involve amino acid sequence variations that make up the protein. For example, there are degeneracy mutations in which protein sequence variations do not involve amino acid variations in polypeptides, and such degeneracy mutants are also included in the genes of the invention.

Genes encoding variants that are functionally equivalent to the KIAA0317 protein are known in the art, for example hybridization techniques (Southern, EM, Journal of Molecular Biology, 98, 503, 1975) or PCR methods (Saiki et al., Science). 230: 1350-1354, 1985; Saiki et al., Science, 239: 487-491, 1988). For example, those skilled in the art will be able to isolate a gene having high homology with the KIAA0317 gene by designing a primer that can hybridize specifically to the KIAA0317 gene from the above-mentioned KIAA0317 protein sequence.

The protein encoded by this isolated gene has high homology with the native KIAA0317 protein at the amino acid level. High homology refers to the identity of at least 50%, in particular at least 70%, more particularly at least 90% (eg, at least 95%) of sequences throughout an amino acid sequence.

The homology of amino acid sequences or nucleotide sequences is based on BLAST (Proc. Natl. Acad. Sci. USA, 90, 5873-5877, 1993), a program called BLASTN or BLASTX (Altschul et al, J. Mol. Biol. , 215, 403-410, 1990) and specific methods are known on the following website (http://www.ncbi.nlm.nih.gov.).

In addition, the KIAA0317 protein, and the gene encoding it, also include fragments having biological activities comparable to their natural sequences. As used herein, the term “fragment” refers to a sequence corresponding to a portion of a gene or protein, in the case of a gene, including physically, endonucleated, or chemically cleaved, and in the case of a protein, cleaved with protease. Or chemically cleaved ones. Furthermore, in the case of proteins, variants for altering the safety, shelf life, solubility, etc. of the KIAA0317 protein, or variants for altering interaction with other proteins that interact, are also included in the functionally equivalent variants.

The KIAA0317 gene of the present invention or a variant thereof functionally equivalent may be used by itself or in a vector for achieving the object of the present invention, and methods for introducing DNA into the vector are known in the art (Molecular Cloning: A Laboratory Manual, 3rd Ed., Sambrook and Russel, Cold Spring Harbor Laboratory Press, 2001; Current Protocols in Molecular Biology Ausubel, Brent, Kingston, More, Feidman, Smith and Stuhl eds, Greene Publ. Assoc., Wiley-Interscience, 1992 ). In one embodiment of the invention it was introduced and used as pCEV29 (Michieli et al., 1996; Yamanaka et al., 2001) vector for expression in eukaryotic cells.

Various vectors are known which can be expressed and / or amplified in cells, and otherwise introduced into the chromosome or present in the cell independently of the chromosome, and the vector is suitable for linear DNA, plasmid vector, or other purposes. Contains any vector. Furthermore, such vectors include chemical conjugate vectors, viruses, including ligands or nucleic acid binding moieties (eg, polylysine binding sites) to receptors on the cell surface (eg, those described in WO 93/04701). Vector (e.g., DNA or RNA virus), fusion protein moiety (e.g., antibody moiety that recognizes the antigen of the cell, Glutathione S-transferase for ease of isolation and detection) Or a fusion protein expression vector comprising a fluorescent protein moiety, and selection of a vector appropriate for the purpose will be apparent to those skilled in the art. For example, the KIAA0317 gene of the present invention may be introduced into an expression vector used in a gene therapy system or the like, such as an adenovirus vector, and then included in a virus particle as a carrier according to a known method.

KIAA0317 polynucleotides according to the invention and all variants described above can be prepared using known organic chemical methods for polypeptide synthesis, and can also be combined with each other in sequence synthesized into fragments to obtain the desired full length sequence. (The Peptides, Analysis, Synthesis, Biology, Vol. 1-9, Gross, Udenfriend and Meienhofer Ed. 1979-1987, Academic Press Inc.). In particular, the KIAA0317 polynucleotide, fragment thereof, or functionally equivalent variant thereof according to the present invention preferably uses genetic recombination techniques. The native KIAA0317 polynucleotide can be expressed in a suitable host cell to make a cell lysate, or KIAA0317 mRNA can be translated in vitro and purified by protein isolation methods known in the art, and can be purified by general genetic recombination techniques. And protein purification methods are described, for example, in Sambrook et al., Molecular Clonning: A Laboratory Mannual, Second Edition, Cold Spring Harbor Laboratory Press, 1989; Current Protocols in Molecular Biology, Ausubel et al Ed., Greene Publishing Associates and Wiley Interscience 1991). Such KIAA0317 polypeptides of the invention include, for example, those in the form of a fused protein or amino acid residue bound to a carrier for delivery or administration to a purified protein, water soluble protein, or target cell.

Intracellular expression of KIAA0317 of the present invention significantly inhibited apoptosis induced by p53, TNF-a, STS and the like (about 50% or more, Figure 4). Furthermore, inhibition of endogenous KIAA0317 protein expression with siRNA specific for KIAA0317 induced apoptosis. These results demonstrate that the KIAA0317 gene of the present invention has a novel apoptosis inhibitory function, and the KIAA0317 protein of the present invention, which functions to inhibit apoptosis, can be used for suppressing abnormally induced apoptosis. It may be useful as a therapeutic agent for a disease caused by.

Accordingly, the apoptosis modulator of the present invention is characterized in that it is for the treatment or prevention of a disease associated with excessive activity of apoptosis. Diseases associated with such hyperactivity of apoptosis are, but are not limited to, central nervous system diseases such as a number of acute and chronic degenerative diseases (eg, Alzheimer's disease, Parkinson's disease, cerebral ischemia / stroke) (Mochizuki et al., 1996; Smale et al., 1995; Thomas et al., 1995; Robertson et al., 2000), cardiovascular disease (Yue et al., 1999), and autoimmune diseases such as Graves' disease and type 2 diabetes ( O'Reilly et al., 1999; Magge et al., 1998).

The present invention is also characterized in that the KIAA0317 protein, the gene encoding it, or a variant functionally equivalent thereto is particularly associated with the HECT region of the KIAA0317 protein. In one embodiment of the present invention, it was demonstrated that only the HECT region of the C-terminal region of KIAA0317 can sufficiently inhibit apoptosis.

The present invention is based on the utility of KIAA0317 as an apoptosis inhibitor and the finding that KIAA0317 is overexpressed in a disease associated with underactivity of apoptosis, such as cancer cells (see FIG. 6), and in this respect the present invention relates to KIAA0317 gene or protein expression. It provides a pharmaceutical composition useful for the treatment or prevention of diseases associated with the under activity of apoptosis comprising an inhibitor as an active ingredient.

Diseases caused by the underactivation of apoptosis in the present invention include, but are not limited to, cancers in particular by inactivation of p53 (Vogelstein et al., 2000; Vousden et al., 2002). One embodiment of the present invention confirmed the overexpression of KIAA0317 in gastric cancer, liver cancer, colon cancer, and lung cancer, which means suppression of apoptosis in the cancer cells constituting the cancer tissue, apoptosis due to the loss of function of KIAA0317 Suggests that induction may be effective in the treatment of cancer.

As used herein, the term "inhibitor" is not particularly limited as long as it is a substance capable of inhibiting the expression of the KIAA0317 gene, the expression of the KIAA0317 protein, and / or the activity of the expressed protein, for example, in a synthetic or genetic recombination technique. Substances obtained by the above, substances derived from nature, or derivatives thereof include, for example, antibodies, antisense nucleotides, and the like. In one embodiment of the present invention, siRNA was used as an inhibitor to effectively inhibit KIAA0317's function as an apoptosis inhibitor.

siRNA is expressed in the process of development of the living body, or the defense mechanism of the body against the virus, and when bound to the target mRNA, induces the binding of the intracellular enzyme complex that recognizes it, and degrades the corresponding mRNA (Fire, 1998; Montgomery et al., 1998; Sharp PA,, 1999).

In mammals, siRNAs are typically 20 or 21mer double stranded RNA, consisting of 19 complementary sequences and a dimer of 3'-end non-complementary thymidine or uridine. Such double-stranded RNA has been shown to specifically inhibit the expression of the corresponding gene upon introduction into cells (Elbashir et al., 2001).

The siRNA according to the present invention may comprise two complementary nucleotide molecules or may comprise sense and antisense sequences in one molecule (transcript), for example, in the form of a hairpin. The siRNA may be at least about 10 nucleotides in length, the longest naturally occurring KIAA0317 transcript. In particular, the siRNA is about 100 nucleotides or less in length, most particularly 15 to about 25 nucleotides. In a preferred embodiment of the present invention, the function represented by SEQ ID NO: 3 or 4 as the sense strand and antisense strand constituting the double-stranded molecule, respectively, effectively inhibited the function of KIAA0317.

Preferably, the siRNA capable of inhibiting or inhibiting the function of the KIAA0317 as an apoptosis inhibitor of the present invention is an siRNA sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 9 to 11 or a complementary sequence of the sequences. to be. The sequences of SEQ ID NOs: 9 to 11 bind to the target mRNA and interfere with its translation, and when the sequences of SEQ ID NOs: 9 to 11 are sensed, the complementary sequences thereof may be viewed as antisense strands.

Complementary polynucleotide sequences can be hybridized under appropriate conditions to form several mismatched or mismatched double-stranded molecules or hairpin-structured double-stranded molecules. Given the object of the present invention, two sequences comprising up to 5 mismatches can be considered complementary.

Accordingly, the present invention provides an apoptosis regulator comprising an siRNA consisting of one or more nucleic acid sequences selected from the group consisting of the nucleic acid sequences of SEQ ID NOs: 9-18, preferably SEQ ID NOs: 9-11. Preferably, the nucleic acid sequence is an RNA sequence.

The apoptosis modulator may be a short hairpin RNA (shRNA) vector that expresses siRNA. The shRNA vector is designed to convert shRNA into siRNA in a cell, and plasmid vector or viral vector can be used. Such shRNA vectors are well known in the art and can be readily performed by those skilled in the art. In contrast to siRNAs, shRNA vectors may be beneficial in terms of long term stable gene expression inhibition by transfection in vivo.

For example, the apoptosis modulator may inhibit or inhibit the expression of apoptosis inhibitory gene or protein, and the apoptosis inhibitory gene may be KIAA0317 gene.

For example, the apoptosis modulator may be useful for the treatment or prevention of cancer, in particular, gastric cancer, liver cancer, colon cancer, lung cancer and the like, but is not limited thereto.

In another embodiment, the apoptosis modulator may be used for the treatment of cancer, or as an adjuvant, and also increases anticancer agent sensitivity of cancer cells.

The present invention also provides a pharmaceutical composition comprising the apoptosis modulator of the present invention, useful for the treatment or prevention of a disease associated with the under activity of apoptosis. Preferably, the pharmaceutical composition can be used in combination with known anticancer agents such as but not limited to cisplatin, doxorubicin, etoposide and the like. The combination may be simultaneous administration, sequential administration in any order or sequential administration at regular time intervals.

In addition, the present invention expresses siRNA, or siRNA consisting of one or more nucleic acid sequences selected from the group consisting of the nucleic acid sequence of SEQ ID NO: 9 to 18, preferably SEQ ID NO: 9 to 11 and the complementary sequence of the nucleic acid sequence It provides an anticancer agent characterized by using a combination of an expression construct and an anticancer agent possibly included. The inhibitor is characterized by increasing the sensitivity of cancer cells to anticancer agents.

Apoptosis modulators according to the present invention, and pharmaceutical compositions useful for the treatment or prevention of diseases associated with underactivity of apoptosis, can be administered in a suitable formulation together with carriers, diluents or excipients known in the art. Apoptosis modulators or pharmaceutical compositions of the invention can be administered via a general route as long as they can reach the desired tissue, and may be administered parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) according to the desired method. Or oral administration, especially parenteral administration, more particularly intravenous injection, the dosage form depends on the method of administration chosen.

Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and one or more of these components, as necessary. And other conventional additives such as buffers and bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions, and the like, and may act specifically on target organs. Target organ specific antibodies or other ligands may be used in combination with the carriers so as to be used. Furthermore, it may be suitably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA). have.

In addition, other pharmaceutical delivery systems such as liposomes and emulsions well known in the art may be employed. Certain organic solvents such as dimethylsulfoxide may also be employed.

The dosage will vary depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and severity of the disease. For example, therapeutically effective dosages can be initially determined using in vitro assays through cell culture, and performed in animal models to determine IC ₅₀ (in vitro) in which concentration ranges of blood KIAA0317 were determined in cell culture. In assays, drug treatments, the concentration of the test compound at a lethal dose relative to 50% of the cultured cells) can be determined. Those skilled in the art will be able to determine the amount effective for treatment without undue experimentation, and this information can be used to more accurately determine the dosage useful in humans.

The pharmaceutical composition of the present invention may be provided alone or in combination with drugs such as surgery, hormonal therapy, and anticancer agents for the treatment of cancer.

EXAMPLE

Hereinafter, examples are provided to help understand the present invention. However, the following examples are merely provided to more easily understand the present invention, and the present invention is not limited to the following examples.

General methods of molecular genetics and genetic engineering of the present invention are described in Molecular Cloning: A Laboratory Manual, (Sambrook et. Al., Cold Spring Harbor); Gene Transfer Vectors for Mammalian Cells (Miller & Calos ed.); And Current Protocols in Molecular Biology (F.M. Ausubel et. Al, ed., Wiley & Sons). The reagents, cloning vectors, and genetic kits mentioned in the specification of the present invention are commercially available such as Gibco / BRL, Bio-Rad, Stratagene, Invitrogen, ClonTech and Sigma-Aldrich Co. You can buy it at Cell culture methods are generally described in Culture of Animal Cells: A Manual of Basic Technique (R.I. Freshney ed, Wiley &Sons); It is described in the current edition of General Techniques of Cell Culture (M.A. Harrison & I.F.Rae, Cambridge Univ. Press). Materials and reagents necessary for cell culture are available from commercially available companies such as Gibco / BRL, Nalgene-Nunc International, Sigma Chemical Co. And ICN Biochemicals.

Example 1. Cloning of Genes That Inhibit Apoptosis

1-1 Cell Culture and Transfection

The cells were treated at 37 ° C. in DMEM medium (Dulbecco's Modified Eagle's Medium, Hycolon) containing 10% fetal bovine serum (Hyclone), glucose, penicillin (100 units / ml) and streptomycin (100 μg / ml). Incubated in a 5% CO ₂ environment. Transfection was performed basically using lipofectamine (Invitrogen, USA) as recommended by the manufacturer. In addition, reagents and instruments necessary for cell culture were purchased from Invitrogen.

Cloning of genes that inhibit 1-2 apoptosis

A mouse testicular cDNA library was introduced into Saos-2 cells (American Type Culture Collection, ATCC; Cat. No. HTB-85) derived from human osteosarcoma in which apoptosis was induced by p53. The cDNA library extracts and purifies mRNA from Trizol (Invitrogen) according to the manufacturer's recommendation from mouse testes, and synthesizes cDNA using Superscipt II (Invitrogen) as a template, and also uses the pCEV29 expression vector ( Michieli et al., 1996; Yamanaka et al., 2001) were cloned into the Sfi I restriction enzyme site. In the pCEV29 expression vector, the cloned cDNA was expressed under the MuMLV-LTR (Moloney murine leukemia virus long terminal repeat) promoter to express a neomycin-resistant gene resistant to G418.

After 1 × 10 ⁶ Saos-2 cells were placed in a plate of 100 mm in diameter and incubated for 2 days in 10 ml of culture solution (DMEM + 10% FBS), the prepared 2 μg cDNA library was introduced into the cells by CaPO ₄ , followed by 500 Incubated by adding μg / ml of G418 to the culture. After about two weeks, G418 addition was discontinued after the cDNA-non-injection was removed, with the cells forming G418 resistant colonies containing approximately 100-2,000 cells. Then, when each colony grew to about 1 mm in diameter, they were separated, and each colony was separated into two wells of two 24-well plates and cultured.

When the cells became about 50% lush, adenovirus expressing p53 (Jung et al., 2004) was infected with cells of one of the two wells, and the cultures were replaced after about 3 hours. After 3 days, apoptosis due to p53 expression was observed. One week later, cells were fixed and stained with Kimsa (Sigma USA) solution and cell growth was compared in p53 virus-infected wells and uninfected wells.

Colonies of wells in which apoptosis was not actively induced by p53 expression were separated and grown on 60 mm diameter plates. After p53 virus was again infected with the cells thus removed, the cells were re-checked for resistance to apoptosis induced by p53 in the same manner as described above, and cDNA introduced therefrom was isolated from the cells with confirmed apoptosis.

First, chromosomal DNA was extracted from p53-resistant cells according to a known method. To isolate the pCEV29 expression vector introduced into the chromosome, the chromosomal DNA (50 μg) was digested with only one restriction enzyme XhoI or NotI present in the vector, and then the DNA was purified by a known phenol extraction method. After purification, 1.2 μg of DNA was bound to T4 ligase (T4 ligase) at 16 ° C. for 24 hours, and then purified by phenol extraction. The concentration of bound DNA was confirmed by agarose gel electrophoresis, and the DNA was introduced into the E. coli DH10B cell line (Gibco-BRL, USA) by electroshock method (Gibco-BRL, USA) according to the manufacturer's recommendations. . Since the pCEV29 expression vector contains an ampicillin (ampicillin, Sigma, USA) resistance gene, resistant colonies were selected from the ampicillin containing medium. A total of 300 to 500 colonies were collected by washing the whole plate with LB medium (Luria-Bertani medium, Difco), adding Ampicillin (100 μg / ml) and incubating at 37 ° C. overnight, and then collecting the cells the next day. Plasmid DNA was isolated by a known alkaline-lysing method.

After the isolated DNA was introduced into E. coli DH10B cells, 4-5 plates were prepared to grow 50-100 colonies per plate. Single colonies were again taken from the plate and inoculated into LB medium, and then single plasmid DNA was isolated by a known alkaline-lysing method. In order to identify the cDNA inserted into the pCEV29 expression vector, primers composed of nucleotide sequences which are present near each of 5 'and 3' of the inserted gene of the vector (forward: 5'-CGACTGGAGCACGAGGACACTGA-3 '; reverse: 5'-CATCAAAAATAGCCAAAAGG -3 '), and using the pCEV29 expression vector extracted from the isolated p53 induced apoptosis resistant Saos-2 cell line as a template, 30 cycles of 94 ° C 1min, 48 ° C 1min, 72 ° C 10min as one cycle PCR reaction was performed.

In this method, 2.2 kb genes were isolated from 8 p53 induced apoptosis resistant Saos-2 clones, and sequenced using the sequencing kit (Applied Biosystmes, USA) as recommended by the manufacturer, and the BLASTN program. (Altschul et al, 1990; homology analysis revealed that the sequences constitute part of GenBank AK077015 (mouse) and AB002315 (human), including the putative HECT region, which was specifically identified as an E3-ubiquitin ligase. The function is unknown.

The gene was named KIAA0317 (Resistance to apoptosis induction).

Example 2. Determination of Ubiquitination Activity of KIAA0317

In order to confirm the function of KIAA0317 protein, it was observed whether KIAA0317 forms a ubiquitin and thioester complex in the presence of the ubiquitin-active enzymes E1 and E2.

The sequence encoding the HECT region (KIAA0317-H) of human-derived KIAA0317 or the HECT (KIAA0317-Hc / a) region in which the 720th cysteine residue of the HECT region was substituted with an alanine residue was transferred to pGEX5X-3 (Pharmacia, USA). Cloning as recommended, GST-KIAA0317-H and GST-KIAA0317-Hc / a vectors fused with Glutathione S trnasferase (GST), respectively, were constructed and expressed and purified in E. coli (BL21). The protein was reacted with ubiquitin and ATP at 25 ° C. for 90 minutes in the presence of E1 (Calbiocem, USA) and E2 (Calbiocem, USA). After the reaction, the mixture was washed with buffer to remove excess ubiquitin, followed by 12% SDS (Sodium Dodecy Sulfate) -polyacrylamide gel electrophoresis (PAGE) to transfer to nylon membrane, Anti-ubiquitin (Santa Cruze Biotechnology USA) and anti-GST antibody (Pharmingen, USA) as primary antibody, and anti-goat-horseradish peroxidase-fusion antibody and Chemiluminescence Detect Kit (Amersham, UK) as secondary antibody Western blot was performed using known methods and as recommended by the manufacturer.

As shown in FIG. 3, due to the high molecular weight, the KIAA0317 protein combined with ubiquitin can be separated by the difference between the unbound and the electrophoretic migration distance. The KIAA0317 protein of the present invention formed a complex with ubiquitin in the presence of E1 and E2, and the formation of this complex resulted in a mutation of a reducing agent (DTT) and a cysteine residue to an arine (KIAA0317-) that interfered with thioester binding with ubiquitin. Hc / a). E3-ubiquitin ligase, which is involved in the ubiquitination of target proteins in cells, is commonly known to bind ubiquitin and thioesters in the presence of E1 and E2. Thus, the above results demonstrate that the KIAA0317 protein of the present invention, in particular the HECT region, functions as an E3-ubiquitinase.

Example 3. Inhibition of Apoptosis of KIAA0317

To analyze the apoptosis inhibitory function of KIAA0317, the full-length KIAA0317 cDNA cloned in Example 1 (9-51), the KIAA0317 cDNA derived from humans, the KIAA0317-HECT region, and the 720th cysteine residue of the respective HECT region were alanine An expression vector pCDNA6-v5 comprising a KIAA0317-HECT region substituted with residues was introduced into Saos-2 cells to prepare a cell line stably expressing the KIAA0317 protein or its HECT region according to the method described in Example 1. Each cell was added to ⁴ × 10 ^{4 in} a 24-well plate and 1 ml of cell culture was added. After 1 day, TNF-alpha (0.1 ng /) containing adenovirus (Jung et al., 2004) (250 multiplicity of infection) expressing p53 in each well; 50 μg / ml of cyclohexamide ml); and adding strosporin (25 nM) to each well, replacing the cultures after 2 hours for adenovirus, and rest the cells in each well at the time of 24 hours in each container. After aggregating the cells, the cells were stained with 0.4% trypan blue (Sigma) to quantify cell death by measuring the number of killed cells under a microscope. BclXL, known to inhibit, was used (Jung et al., 2004).

As shown in FIG. 4B, cells expressing KIAA0317 and KIAA0317-H (amino acid sequences 455 to 823) 9-51 (amino acid sequences 551 to 823) of FIG. 4A are known to have various signals, p53, All positive controls induced by TNF-alpha and storosporin inhibited cell death to approximately the same level as bclXL, previously known as apoptosis inhibitor.

This apoptosis inhibitory effect was lost in KIAA0317 (KIAA0317 ca, KIAA0317-Hca), where the 720th cysteine residue in the HECT region was replaced with an alanine residue, indicating that the HECT region and the amino acid residue play an important role in the inhibition of apoptosis. Suggest. In addition, the HECT region of KIAA0317 alone proves to have an effect of inhibiting cell death.

Example 4 Inhibition of Protease Caspase-9 and Caspase-3 by KIAA0317

TNF-alpha (0.1 ng / ml) was added to cells expressing the normal and mutant KIAA0317 gene or Bcl-xL, a known apoptosis inhibitory gene used in Example 3, and then caspase activity was measured after 24 hours. .

Caspase activity was determined by DEVD (D: aspartic acid, E: glutamic acid, V: valine) conjugated to 7-amino-4-trifluoromethyl coumarin (AFC), and LEHD (L: leucine, H: histidine) -AFC Was analyzed as per the manufacturer's recommendations using a fluorescence assay kit (R & D Systems, USA) as substrates for caspases 3 and 9, respectively. The fluorescence signal was measured using a microplate fluorescent reader at an excitation wavelength of 400 nm and an emission wavelength of 505 nm.

As shown in FIG. 5, the activity of caspases 9 and 3 was increased by TNF-alpha as evidence of apoptosis. However, in the cells expressing KIAA0317, the activity of the caspase was maintained despite TNF-alpha treatment. Could be suppressed. Inhibition of this activity does not appear in the case of KIAA0317c / a in which a specific amino acid in the HECT region is substituted, indicating that the caspase inhibitory activity is due to KIAA0317.

Example 5 Reduced Ubiquitination of XIAP and Increased Ubiquitinylation of Smac / DIABLO, HtrA2 Proteins Promoting XIAP Ubiquitinization by KIAA0317

The normal KIAA0317 gene used in Example 3 was expressed in H1299 cancer cell line, followed by administration of eposide. After a predetermined time elapsed, the protein was extracted from the cells and analyzed by Western blot. Ubiquitination of XIAP protein was increased after eposide treatment in control HCT1116 cells, but XIAP ubiquitination was significantly inhibited in HCT116 cells expressing KIAA0317 gene. Upon cell death, ubiquitination of Smac / DIABLO, ARTS, HtrA2, etc., which is known to promote ubiquitination of XIAP, was markedly increased in HCT116 cells expressing KIAA0317. Therefore, it was found that KIAA0317 promotes ubiquitination of Smac / DIABLO, ARTS, HtrA2 and the like that promotes ubiquitination of XIAP, and inhibits ubiquitination of XIAP (FIG. 6).

Example 6 Ubiquitination of Smac / DIABLO, HtrA2 Proteins with KIAA0317 Protein in In Vitro Ubiquitination Experimental Conditions

KIAA0317 protein and Smac / DIABLO, ARTS or HtrA2 protein were added to the ubiquitin reaction solution under in vitro conditions, followed by one hour reaction at 30 ° C., and the protein was analyzed by Western blot. As a result of the Westin blot using ubiquitin and antibodies of each protein, it was found that all three proteins were ubiquitinated by KIAA0317 protein (FIG. 7).

Example 7. Overexpression of KIAA0317 in Cancer Cell Lines and Cancer Tissues

Since overexpression of KIAA0317 is associated with suppression of apoptosis, the expression patterns of KIAA0317 were investigated in cancer, a representative disease in which inhibition of apoptosis was observed.

Lung cancer and colorectal cancer cell lines were examined using Westinblock for the expression level of the protein using an antibody against KIAA0317. FIG. 8 shows that KIAA0317 is overexpressed in 13 cell lines among 19 cell lines.

In tissue microarrays (US Biomax, Inc, USA) containing samples from normal and cancerous tissues of the stomach, colon and liver, the Tyramide Signal Amplification Kit (NEN Life Science) as an immunochemistry and signal amplification agent was Analysis was performed using. In this experiment, 61, 124, 57, 184 and 59 samples were used for gastric cancer, colorectal cancer, liver cancer, lung cancer and breast cancer and their respective normal tissues.

Briefly, first, the KIAA0317 contained in the tissue was exposed by heating in a microwave in citric acid buffer at pH 6.0. Antibodies to KIAA0317 include the specification of the KIAA0317 protein sequence. Antibodies prepared by using a peptide covering the site as an epitope were used. The antibody was bound to tissue samples and then developed with diaminobenzidine (Sigma, USA) and counterstained with hematoxylin A. Non-immune rabbit serum was used as a negative control. The results were read independently by three pathologists and categorized the response to KIAA0317 antibodies in four steps as follows: (1) negative, 0-5%; (2) low, 5-30%; (3) medium, 30-50%; (4) high, 50% or more.

Since KIAA0317 expression was read as negative or low in normal tissues, it was considered overexpression to show moderate and high reactivity in the present results.

As can be seen in Figure 9, compared with normal tissue, overexpression of KIAA0317 in cancer tissues was observed, 48%, 46%, 33 in the gastric, colon, liver, lung and breast cancer tissue samples examined, respectively. Overexpression of KIAA0317 was observed in%, 42% and 41% of tissues.

These results suggest that inactivation of KIAA0317 in cancer tissues may be effective in the treatment of cancer.

Example 8. Construction and activity of novel siRNAs that regulate expression of apoptotic inhibitory genes

8-1. siRNA production

siRNA nucleic acid sequences were designed using the Ambion website (www. ambion.com/techlib/misc/siRNA_finder.html). In summary, starting with the AUG sequence of KIAA0317 cDNA, scanning in the 3 'direction to yield the AA sequence, this sequence and then 19 nucleic acid sequences were selected as potential siRNA target sites, and the 5' and 3 'untranslated sites and translations. The sequence of initiation site (within 75 base) was excluded from the target. The selected sequence was compared with the sequence of a known database, and the homology with the coding sequence of another gene was excluded. The target sequence of KIAA0317 used in this example is 5'-AATTGGTCCCTGAGAACCTTT-3 ', and the siRNA-forming sequence is shown in Table 1 below.

Table 1 siRNA number Sequence (5 '->3') SEQ ID NO: One uaugauuugggccaggaaagagcgg SEQ ID NO: 12 2 aaucccaggagacuuugcaagacc SEQ ID NO: 13 3 uacacgugcggcaagcucaaagag SEQ ID NO: 9 4 gacaucagugugucugacudtd SEQ ID NO: 14 5 cacugauccuaacacacaudtd SEQ ID NO: 10 6 cucagucuuucgcagucaudtd SEQ ID NO: 11 7 gcccaattccaacgtagtaa SEQ ID NO: 15 8 cagcgagagcttcggcaggt SEQ ID NO: 16 9 tcggctggccagtcaagtga SEQ ID NO: 17 10 cagaacatgggccctcgttt SEQ ID NO: 18

Scrambled siRNA was purchased from PROLIGO as a control siRNA that did not affect KIAA0317 gene expression. For intracellular expression of the siRNA, Knockott RNAi System (BD-Clontech, USA) BD was used as recommended by the manufacturer. In summary, an oligonucleotide comprising the sense sequence (Table 1) was synthesized according to the manufacturer's recommendation and cloned into pSIREN-Retro Q (BD-Clontech, USA) to express the siRNA targeting KIAA0317 (pSIREN). -KIAA0317si) was prepared and used for transfection of cells.

8-2. RNA interference analysis

293 cells were seeded in each well of a 24-well plate at a concentration of 3 x 10 ⁴ cells and incubated overnight, and then the cells were subjected to KIAA0317-Ready at 40 / well using Lipofectamine 2000 (Invitrogene) as recommended by the manufacturer. Treated with pmole concentration for 24 hours. After treatment, 48 hours later, RNA was extracted from the cells and the amount of KIAA0317 (Rani) mRNA was measured by RT-PCR method. As a result, gene expression interference by siRNA 3, 5, 6 appeared to be excellent (Fig. 10).

Example 9. Induction of apoptosis of siRNA and increased sensitivity to anticancer agents

In each siRNA treated cell (colon cancer cell line H1299), doxorubicin (ALZA Pharmaceuticals, USA), a known anticancer agent, and etoposide (Bristol-Myers Squibb Company) were treated in the same manner as described in Example 8-2. , USA) were treated at concentrations of 2 μM and 5 μM, respectively, to investigate apoptosis effects.

As shown in Figure 11, it can be seen that the increase in apoptosis effect by each anticancer agent during siRNA treatment.

These results suggest that not only siRNA but also can be effectively used for anticancer treatment when used in combination with known anticancer agents.

Example 10. Inhibitory Effect of siRNA on Cancer Growth in Cancer Cells

Experiments were performed to confirm siRNA tumor interference effects in mice. M12 was injected with H1299 cells expressing H1299 or siRNA (siRNA 3, SEQ ID NO: 9), and the extent of tumor formation was confirmed by size measurement. As a result, control mice (A) injected with only H1299 cells normally formed tumors, but H1299 cells (B) expressing siRNA (siRNA 3, SEQ ID NO: 9) showed almost no increase in tumor volume (FIG. 12). ). This proves that the expression of siRNA can effectively inhibit tumor formation.

Claims (11)

An apoptosis modulator comprising an siRNA consisting of one or more nucleic acid sequences selected from the group consisting of the nucleic acid sequences of SEQ ID NOs: 9-11. According to claim 1, wherein said apoptosis regulators Apoptosis modulator, characterized in that to inhibit or inhibit the expression of a gene or protein that inhibits apoptosis. The apoptosis regulator according to claim 2, wherein the apoptosis inhibitory gene is KIAA0317 gene. The apoptosis modulator according to claim 1, wherein the apoptosis modulator is useful for the treatment or prevention of cancer. According to claim 1, wherein the cancer cell death regulator, characterized in that the cancer, liver cancer, colon cancer or lung cancer. The apoptosis modulator according to claim 1, wherein the apoptosis modulator increases anticancer sensitivity of cancer cells. A shRNA vector expressively comprising an siRNA consisting of at least one nucleic acid sequence selected from the group consisting of the nucleic acid sequences of SEQ ID NOs: 9-11. A pharmaceutical composition comprising the apoptosis modulator according to any one of claims 1 to 7, which is useful for the treatment or prevention of a disease associated with the under activity of apoptosis. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition is used in combination with an anticancer agent. Use of an siRNA consisting of one or more nucleic acid sequences selected from the group consisting of the nucleic acid sequences of SEQ ID NOs: 9-11 for the treatment or prevention of diseases associated with underactivity of apoptosis. Related to the underactivity of apoptosis, comprising administering a siRNA consisting of one or more nucleic acid sequences selected from the group consisting of the nucleic acid sequences of SEQ ID NOs: 9-11 to a patient with a disease associated with the under activity of apoptosis Method of treatment of patients with the disease.

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