KR100818047B1 - -22 -22 Method of inducing cell death by inhibition of smooth muscle-22 alpha and anticancer drug containing smooth muscle-22 alpha inhibitor - Google Patents

Abstract

The present invention relates to a method for promoting the death of cancer cells by inhibiting the activity of smooth muscle-22α and an anticancer agent comprising an activity inhibitor of smooth muscle-22α as an active ingredient. Antisense nucleotides that complementarily bind to smooth muscle-22α mRNA, peptide mimetics that bind smooth muscle-22α protein, and an activity inhibitor selected from the group consisting of antibodies. The method of inhibiting the activity of smooth muscle-22α and the inhibitor of the present invention can be usefully used for anticancer treatment by promoting the cell killing effect of cancer cells.

Smooth muscle-22 alpha, an anticancer agent

Description

Method of inducing cell death by inhibition of smooth muscle-22 alpha and anticancer drug containing smooth muscle- by inhibiting the activity of smooth muscle-22α and promoting the death of cancer cells 22 alpha inhibitor}

1 is a plasmid map and sequence used for introducing sense and antisense SM-22α into lung cancer cell line A549 cells and hepatocarcinoma cell line HepG2 cells, A is a plasmid map, and B is a SM-22α sequence list,

2 is a diagram showing the results of SM-22α expression by introducing SM-22 sense nucleotides into lung cancer cell line A549 cells and hepatic cancer cell line HepG2 cells. (A) RT-PCR and Western blot method of lung cancer cell line A549 cells. A549 is a normal cell, E-pcDNA is an empty vector transformed cell, SM22-S is an SM-22α sense nucleotide vector transformed cell, and (B) RT-PCR of a hepatic cancer cell line HepG2 cell. And Western blots showing the results, HepG2 is normal cells, E-pcDNA is the empty vector transformed cells, SM22-S is SM-22α sense nucleotide vector transformed cells,

Figure 3 is a diagram showing the results of flow cytometry (FACS) investigation of the H ₂ O ₂ resistance of lung cancer cell line and liver cancer cell line when SM-22α overexpression, (A) is the result of measuring lung cancer cell line A549 cells by FACS, A549 / E-pcDNA is an empty vector transformed cell, SM22-Sense is an SM-22 sense nucleotide vector transformed cell, (B) is the result of measuring the liver cancer cell line HepG2 cells by FACS, and HepG2 / E-pcDNA is empty Vector transforming cell, SM22-sense is SM-22α sense nucleotide vector transforming cell,

Figure 4 shows the results of FACS investigation for gamma radiation resistance of lung cancer cell line and liver cancer cell line when SM-22α overexpression, (A) is the result of measuring lung cancer cell line A549 cells by FACS, A549 / E-pcDNA is empty Vector transformed cells, SM22-Sense is SM-22α sense nucleotide vector transformed cells, (B) is the result of measuring liver cancer cell line HepG2 cells by FACS, HepG2 / E-pcDNA is empty vector transformed cells, SM22- sense represents a SM-22α sense nucleotide vector transformed cell,

Figure 5 shows the results of FACS irradiation for luteolin (luteolin) lung cancer cell line and liver cancer cell line when SM-22α overexpression, (A) is the result of measuring the lung cancer cell line A549 cells by FACS, A549 / E-pcDNA Is an empty vector transformed cell, SM22-Sense represents an SM-22α sense nucleotide transformed cell, (B) is a result of measuring liver cancer cell line HepG2 by FACS, HepG2 / E-pcDNA is an empty vector transformed cell, SM22-sense represents SM-22α sense nucleotide vector transformed cells,

FIG. 6 is a diagram showing the results of using FACS for methyl metal sulfonate (MMS) of lung cancer cell line and liver cancer cell line upon SM-22α overexpression, and (A) measured lung cancer cell line A549 cells by FACS. As a result, A549 / E-pcDNA represents an empty vector transformed cell, SM22-Sense represents an SM-22α sense nucleotide vector transformed cell, (B) is a result of measuring liver cancer cell line HepG2 by FACS, and HepG2 / E- pcDNA represents an empty vector transformed cell, SM22-sense represents an SM-22α sense nucleotide vector transformed cell,

7 is a diagram showing the results of using FACS for serum-free medium of lung cancer cell line and liver cancer cell line upon SM-22α overexpression, (A) is a result of measuring lung cancer cell line A549 cells by FACS, and A549 / E- pcDNA represents the empty vector transformed cells, SM22-Sense represents the SM-22α sense nucleotide vector transformed cells, (B) is the result of measuring the liver cancer cell line HepG2 cells by FACS, HepG2 / E-pcDNA is empty vector transformed Cell, SM22-sense, represents a SM-22α sense nucleotide vector transformed cell,

8 is a diagram showing the results obtained by staining the cell death degree with Trypan blue when transforming a cancer cell line SM-22α antisense nucleotide vector, E-pcDNA is a co-vector transgenic cell, SM22-AS is SM-22α antisense nucleotide vector transformed cells, A549 represents lung cancer cell line, HT-29 represents colon cancer cell line, HT-1080 represents fibromyoma cell line, HepG2 represents liver cancer cell line,

9 is a diagram showing the results of investigation of cell death by FACS when transforming a cancer cell line with SM-22α antisense nucleotide vector, wherein E-pcDNA is an empty vector transformed cell, and SM22-AS is a SM-22α antisense nucleotide vector transformed. Cells, A549 represents lung cancer cell line, HT-29 represents colorectal cancer cell line, HT-1080 represents fibromyoma cell line, and HepG2 represents liver cancer cell line

The present invention relates to an anticancer agent comprising an antisense nucleotide of smooth muscke-22α (hereinafter referred to as "SM-22α") as an active ingredient, an expression vector comprising the nucleotide, and a transformant transformed thereto. .

Smooth muscle cells, unlike fast skeletal muscle cells and slow skeletal muscle cells, can simultaneously express proliferation and only systematically restricted proteins. One such protein expressed exclusively in smooth muscle is the SM-22α, 22-KD protein. This protein is structurally similar to calponin, a thin filament myofibrillar regulatory protein in vertebrates, the Drosophila muscle protein mp20 (Lees-Miller, JP et al., J. Biol . Chem . 262: 2988 -2993, 1987). Therefore, SM-22α begins with the expression of the super SM-α actin and SM-myosin heavy chains during the differentiation of smooth muscle cells into vascular smooth muscle cells, and together with the production of calponin, vinculin, and tropomyosin. One of the important markers of differentiation expressed. SM-22α protein was first isolated from chicken gizzard smooth muscle cells (Lees-Miller, JP et al., Biochem . J. 244: 705-709, 1987; Lees-Miller, JP et al ., J. Biol . Chem. 262: 2988-2993, 1987), consisting of 201 amino acids, associated with calponin and known to be involved in the contraction of smooth muscle cells due to the interaction of tropomyosin with F-actin (Fu, Y. et. al ., J. Appl . Physiol . 89: 1985-1990, 2000; Kobayashi, R. et al ., Biochem . Biophys . Res . Commun . 198: 1275-1280, 1994; Nishida, W. et al ., Gene 130: 297-302, 1993). Human SM-22αcDNA was cloned as part of the study of nucleotides involved in replicative senescence and was found to be located on chromosome 11q23.2 (Camoretti-Mercado, B. et. al ., Genomics 49: 452-457, 1988). As a result of the various methods, it was observed that the fibroblasts were significantly increased in the old fibroblasts compared to the young fibroblasts.

SM-22 is an actin binding protein and is a monomeric spherical 22kDa basic protein consisting of at least three isoforms α, β, and γ. The most abundant α isoform, along with other differentiation marker proteins including α-actin, calponin and smooth muscle myosin, is expressed at particularly high levels in contractile mammalian smooth muscle cells (Owens, GK, Acta). physiol . Scand . 164: 623-635, 1998). Although the actin binding properties of SM-22α have been revealed (Fu, Y. et al ., J. Appl . Physiol . 89: 1985-1990, 2000), the function of SM-22α is still vague and fibroblasts (fibroblasts) It is known to be the same protein as the transgelin named by its inclusion in actin filaments (Shapland, C. et. al ., J. Cell Biol . 121: 1065-1073, 1993; Lawson, D. et al ., Cell Motil . Cytoskeleton 38: 250-257, 1997).

During development, SM-22α is detected first in all muscle systems in the early embryonic stage. It gradually decreases in the heart and segment as the embryonic process progresses, but is restricted to smooth muscle cells during late embryonic and postnatal development (Li, L. et. al ., Circ . Res . 78 (2): 188-195, 1996). Structurally, the SM-22α nucleotide consists of five exons and four introns, and the adjacent 5 'upstream DNA sequence is very well preserved from chicken to human (Li, L. et. al , Dev . Biol . 187: 311-321, 1997).

Transcripts and protein products of the nucleotides have been studied under four different names: SM-22α (Kim, S. et. al ., Mol . Cell . Biol . 17 (4): 2266-2278, 1997; Lees-Miller, JP et al ., Biochem . J. 244: 705-709, 1987; Lees-Miller, JP et al ., J. Biol. Chem . 262: 2988-2993, 1987; Li, L. et al ., Circ . Res . 78 (2): 188-195, 1996; Li, L. et al ., J. Cell Biol . 132 (5): 849-859, 1996; Moessler, H. et al., Development 122 (8): 2415-2425, 1996; Nishida, W. et al ., Biochem . Int . 23 (4): 663-668, 1991; Nishida, W. et al ., Gene 130 (2): 297-302, 1993; Osbourn, JK et al ., Gene 154 (2): 249-253, 1995; Shanahan, CM et al ., Circ. Res . 73 (1): 193-204, 1993; Shishibori, T. et al ., Biochem . Biophys . Res. Commun . 229: 225-230, 1996; Solway, J. et al ., J. Biol . Chem . 270 (22): 13460-13469, 1995); transgelin (Prinjha, RK et al ., Cell Motil . Cytoskel . 28 (3): 243-255, 1994; Shapland, C. et al ., J. Cell Biol . 121: 1065-1073, 1993; Shapland, C. et al ., J. Cell Biol . 107 (1): 153-161, 1988); -10 WS3 (Grigoriev, VG et al ., Exp . Gerontol . 31 (1/2): 145-157, 1996; Murano, S. et al ., Mol . Cell. Biol . 11 (8): 3905-3914, 1991; Thweatt, R. et al ., Biochem . Biophys . Res. Commun . 187: 1-7, 1992; Thweatt, R. et al ., Exper . Gerontol . 27 (4): 433-440, 1992); mouse p27 (Almendral, JM et al ., Exp . Cell Res . 181: 518-530, 1989; Santaren, JF et al ., Exp . Cell Res . 173: 341-348, 1987). One can confirm that these four are identical to SM-22α, and the cDNA sequence comparison is very similar. The physiological role of SM-22α is not yet clear, but its potential function has been studied under several different names. Transgelin binds to actin monomer in a 1: 6 molar ratio in vitro (Shapland, C. et. al ., J. Cell Biol . 121: 1065-1073, 1993; Shapland, C. et al ., J. Cell Biol . 107 (1): 153-161, 1988), crosslinks with actin filaments. In vivo, transgelin binds to actin stress fibers in adherent 3T3 fibroblasts, but its expression is markedly reduced in normal and transformed cells that grow unattached (Genini, M. et. al ., Int. J. Cancer 66 (4): 571-577, 1996; Shapland, C. et al ., J. Cell Biol . 107 (1): 153-161, 1988). SM-22α mRNA as WS3-10 is significantly increased in fibroblasts and aged primary human skin fibroblasts of Werner syndrome patients (Grigoriev, VG). et al ., Exp . Gerontol . 31 (1/2): 145-157, 1996; Murano, S. et al ., Mol . Cell . Biol . 11 (8): 3905-3914, 1991; Thweatt, R. et al., Biochem . Biophys . Res . Commun . 187: 1-7, 1992; Thweatt, R. et al ., Exper. Gerontol . 27 (4): 433-440, 1992). Mouse p27 mRNA is a cycloheximide treatment, which is significantly increased in serum-extracted 3T3 cells (Santaren, JF et al., Exp . Cell Res . 173: 341-348, 1987). Immunization confirmed that SM-22α is located in the actin stress fiber of cultured smooth muscle cells, but the longer 25-kDa form of this protein in bovine aorta is associated with the cell membrane in the presence of calcium (Shishibori, T. et. al ., Biochem . Biophys . Res . Commun . 229: 225-230, 1996). In contrast, the shorter 22-kDa form is present in the cytoplasm regardless of calcium concentration. Increased expression in senescent cells, decreased expression in transformed cells (tumor cells), and increased expression by serum exposure indicate that SM-22α / transgelin / WS3-10 / p27 can act through actin binding. Imply an important role in cell differentiation by stabilization of the backbone.

SM-22α expression is regulated by a serum response factor (SRF) that binds to CArG in its promoter (Solway, J. et. al ., J. Biol . Chem . 270: 13460-13469, 1995), which is reversely regulated by spherical actin (Mack, CP et. al ., J. Biol . Chem . 276: 341-347, 2001). Thus, the formation of actin cytoskeleton affects the expression of SM-22α, and factors that deactivate the polymerization of actin decrease SM-22α synthesis (Zeidan, A. et al., Am . J. Physiol . Cell Physiol . 284: C1387-C1396, 2003). The mechanical properties of smooth muscle are closely related to the expression of SM-22α. If the rat's portal vein is cultured without swelling, shortening of organ life leads to decreased contractility, reduced actin ratio, and reduced synthesis of SM-22α. (Zeidan, A. et al ., Am . J. Physiol . Cell Physiol. 284: C1387-C1396, 2003; Zeidan, A. et al ., Circ . Res . 87: 228-234, 2000). This may include structural changes in cytoskeletal proteins, including actin filaments. The function of SM-22α is not known yet, but it is thought to affect the structure and function of actin filament.

So far, SM-22α is a 22 kDa protein that is normally the most ubiquitous in smooth muscle tissue of mature vertebrates (Lees-Miller, JP et al., Biochem. J. 244: 705-709, 1987; Lees-Miller, JP et al ., J. Biol . Chem . 262: 2988-2993, 1987; Nishida, W. et al ., Biochem . Int . 23 (4): 663-668, 1991; Nishida, W. et al ., Gene 130 (2): 297-302, 1993; Shanahan, CM et al ., Circ . Res . 73 (1): 193-204, 1993), which are known to be one of the early markers of smooth muscle differentiation during embryogenesis and are thought to play an important role in the production of muscle tissue during early embryogenesis or differentiation. . SM-22α is abundantly expressed in smooth blood cells of adult blood vessels and intestines (Lawson, D. et. al ., Cell Motil . Cytoskeleton 38: 250-257, 1997; Li, L. et al ., Circ . Res . 78: 188-195, 1996), as well as high levels in various tissues such as the lung, uterus, vein and intestine. In various cancer cells, the expression level of SM-22α is low and the expression of aged cells is increased. Recent studies in yeast show that the increase of SM-22α induces mitochondrial instability, which causes reactive oxygen species to cause cell death. And decrease in life span (Li, L. et. al ., Circ . Res . 78 (2): 188-195, 1996).

The present inventors have not shown that overexpression of SM-22α in cancer cells has been shown to be resistant to various stresses including methyl methanesulfonate (MMS), which does not promote cell growth or promote cell aging but rather directly damage DNA. After confirming that, on the contrary, it was confirmed that low expression of SM-22α in cancer cell lines induced cell death.

It is an object of the present invention to provide a method for promoting the death of cancer cells by inhibiting the activity of smooth muscle-22α.

In order to achieve the above object, the present invention provides a method for promoting the death of cancer cells by inhibiting the activity of smooth muscle-22α (hereinafter referred to as "SM-22α").

The present invention also provides an anticancer agent comprising an activity inhibitor of SM-22α as an active ingredient.

In addition, an SM-22α antisense nucleotide expression vector is provided.

In addition, the present invention provides an anticancer agent comprising the SM-22α antisense nucleotide expression vector as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for promoting the death of cancer cells by inhibiting the activity of smooth muscle-22α (hereinafter referred to as "SM-22α").

The inventors of the present invention transformed SM-22α sense nucleotide expression vectors into lung cancer cell line A549 cells and hepatic cancer cell line HepG2 cells to transform various stressors, namely H ₂ O ₂ , gamma radiation, luteolin, an anticancer agent, methyl methane, a genotoxic reagent. The degree of apoptosis of cancer cells in sulfonate and serum free media was measured by flow cytometry (FACS). As a result, it was confirmed that cell death was suppressed when the stress agent was treated in lung cancer cell lines and liver cancer cell lines overexpressing SM-22α (see FIGS. 3 to 7). Based on the above results, the SM-22α antisense nucleotide was cloned to suppress the expression of SM-22α in cancer cells and transformed into lung cancer cell line A549 cells, liver cancer cell line HepG2, colon cancer cell line HT-29 cells, and fibromyoma cell line HT-1080 cells. It was. As a result of microscopic observation of the transformed cells, cell death was promoted (see FIG. 8), and FACS measured about 27 times in lung cancer cell line, about 20 times in liver cancer cell line, and about in colorectal cancer cell line. Cell death increased by 85-fold and about 44-fold in fibromyoma cell lines (see FIG. 9 and Table 1). Therefore, inhibiting the expression of SM-22α can effectively inhibit the cell death of cancer cells.

The method for promoting cell death may be performed using an activity inhibitor selected from the group consisting of antisense nucleotides complementarily binding to mRNA of smooth muscle-22α, peptide mimetics binding to proteins, and antibodies.

Antisense  Nucleotide

Antisense nucleotides are currently recognized as therapeutic agents with promise in the treatment of many human diseases. Nucleotides, as defined in Watson-click base pairs, bind (hybridize) the complementary sequences of DNA, immature-mRNA or mature mRNA to disrupt the flow of genetic information as proteins in DNA. The nature of antisense nucleotides to make them specific for the target sequence makes them exceptionally multifunctional. Since antisense nucleotides are long chains of monomeric units they can be easily synthesized for the target RNA sequence. Many recent studies have demonstrated the utility of antisense nucleotides as biochemical means for studying target proteins (Rothenberg et al., J. Natl . Cancer Inst . , 81: 1539-1544, 1999). The use of antisense nucleotides can be considered as a novel form of therapeutic agent because of recent advances in the field of nucleotide synthesis that exhibit oligonucleotide chemistry and improved cell adsorption, target binding affinity and nuclease resistance. For example, the use of antisense oligonucleotides targeted to cmyb has been used to completely remove myeloid leukemia cells from bone marrow derived from myeloid leukemia patients (Gewirtz and Calabreta, US Pat. No. 5,098,890). Antisense nucleotides are known to have in vivo human efficacy of treating cytomegalovirus retinitis infections. In the embodiment of the present invention, by producing the SM-22α antisense nucleotide described in SEQ ID NO: 9 to investigate the cell killing effect, cell death was effectively promoted (see FIGS. 8, 9 and Table 1). SM-22α antisense nucleotides according to the present invention are provided to specifically hybridize with RNA derived from a gene encoding SM-22α, and nucleotides having sufficient identity and number to be effective such specific hybridization are within the scope of the present invention. And a nucleotide having a nucleotide sequence set forth in SEQ ID NO: 9.

As a substance that inhibits the function of the SM-22α protein, peptides, antibodies, peptide mimetics and the like that bind to the protein may be used.

Peptide mimetics ( Peptide Minetics )

Mimetics (eg, peptides or non-peptidic agents) that inhibit the binding domain of SM-22α can be prepared to inhibit the activity of SM-22α.

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

The present invention provides an anticancer agent comprising the SM-22α activity inhibitor as an active ingredient. The anticancer agent of the present invention is an anticancer agent comprising an antisense nucleotide complementary to the mRNA of SM-22α, a peptide that binds to the SM-22α protein, a peptide mimetics, and an active inhibitor selected from the group consisting of antibodies. It is characterized by.

The anticancer agent of the present invention targets cancers consisting of lung cancer, colorectal cancer, myeloma, and liver cancer.

The composition of the present invention comprises 0.0001 to 50% by weight of the active ingredient based on the total weight of the composition.

The composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions in addition to the active ingredients.

The composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. 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 needed. 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 preferably 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.

Nucleotides or nucleic acids used in the present invention can be prepared for the purpose of oral, topical, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal and the like. Preferably, the nucleic acid or vector is used in injectable form. Thus, in particular, the area to be treated may be mixed with any pharmaceutically acceptable vehicle for injectable compositions for direct infusion. The composition may comprise, in particular, an isotonic sterile solution or a lyophilized composition which allows for the composition of an injectable solution upon addition of sterile water or suitable physiological saline solution. Direct injection of nucleic acid into a patient's tumor is advantageous because it allows the treatment efficiency to be focused on the infected tissue. The dosage of nucleic acid used can be adjusted by various parameters, in particular by gene, vector, mode of administration used, disease in question or alternatively desired duration of treatment. In addition, the range varies depending on the weight, age, sex, health status, diet, administration time, administration method, excretion rate and severity of the patient. The daily dosage is about 0.01 to 12.5 mg / kg, preferably 1.25 to 2.5 mg / kg, preferably administered once to several times a day.

The present invention provides a smooth muscle-22α antisense nucleotide expression vector.

SM-22α antisense nucleotides contained in the expression vector is preferably a nucleotide sequence shown in SEQ ID NO: 9. Specifically, genes were amplified using the primers set forth in SEQ ID NOs: 1 and 2, and inserted into a pCDNA3.1 (Invitrogen, USA) vector. The expression vector is not particularly limited, but it is preferable to use a conventional plasmid vector or a viral vector, and a viral vector such as an adenovirus, an adeno-associated virus, a retrovirus including a lenti virus, and the like may be used. have.

In addition, the present invention provides an anticancer agent comprising the anti-nucleotide expression vector of SM-22α as an active ingredient.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the present invention, but the content of the present invention is not limited to the following embodiments.

< Example  1> SM -22α sense ( sense ) Preparation of Vector Expressing Nucleotide

SM-22α nucleotides of A549 cells, a human lung cancer cell line, were identified by the primers set forth in SEQ ID NO: 1, including the Hind III restriction enzyme sequence (5'-agcttaagcttgacatggccaacaag-3 ') and BamH I restriction enzymes, for the production of sense SM-22α. Sense SM 22α was amplified by PCR (Polymerase chain reaction) using a primer (SEQ ID NO: 2'-gcggatcctctccgctctaactg-3 ') comprising a sequence. PCR conditions are as follows. After 5 minutes of initial denaturation at 95 ° C, denaturation reaction at 95 ° C for 60 seconds, primer binding reaction at 59 ° C for 45 seconds, and length extension reaction at 72 ° C for 45 seconds, and this process was repeated 50 times. It was. The amplified sense SM-22α nucleotide was inserted into the Hind III / BamH I restriction enzyme site of the expression vector pcDNA3.1 (Invitrogen, USA), and the expression vector thus prepared was named "pcDNA / SM-22-S" ( See FIG. 1).

< Example  2> pcDNA Preparation of Transgenic Cells Transduced with SM / 22-S Expression Vector

Human lung cancer cell line A549 cells and liver cancer cell line HepG2 cells in 1 × 10 ^{6 in} RPMI 1640 medium (Gibco-BRL, USA) containing 100 units / ml penicillin (Sigma, USA), 10% fetal calf serum (Hyclone, USA) Cells were inoculated at a concentration of 5 ml and then cultured in a 37 ° C., 5% CO ₂ incubator. 4 μg of the pcDNA / SM-22-S expression vector and the E-pcDNA (empty vector) expression vector prepared in Example 1, respectively, using lung cancer cell line A549 cells and liver cancer cell line HepG2 cells using Lipofectamine 2000 (Invitrogen, USA). Was transduced. Cells transduced with the sense SM-22α nucleotides were cultured for one week in a medium containing 500 μg / ml G418 (DUCHEFA, Netherlands), followed by SM- among clones derived from each cell grown in a 96-well plate. The clones with the highest expression of 22α were selected with 500 μg / ml G418.

< Example  3> Reverse transcription  Polymerase chain reaction reverse transcriptase - polymerase chain  reaction: RT-PCR) analysis

The culture medium was removed from the transgenic animal cells prepared in Example 2, and 1 ml of TRIzol reagent (Invitrogen, USA) was added to the culture medium, and total RNA was isolated according to the method provided by Invitrogen. Transcription polymerase chain reaction was performed using 10 mM dNTP mixture, 5 × RT buffer, 0.5 μg / μL oligo dT, 4 μg total RNA heat treated at 70 ° C. for 4 minutes, 200 U / μL M-MLV RTase (Invitrogen, USA). Into each tube was added sterile water treated with 0.02% DEPC (diethyl pyrocarbonate) to adjust the final 20 μl volume, and then reacted at 45 ℃ for 30 minutes. Thereafter, the mixture was heated again at 95 ° C. for 5 minutes and then quenched in ice to inactivate M-MLV Rtase.

1 μl of the cDNA synthesized by the above procedure was added to ₁₀ × buffer (without MgCl ₂ ), 2.5 mM daNTP mixture, 10 × MgCl ₂ , Tag DNA polymerase (Invitrogen, USA) and SM-22α sense primer (5), respectively. '-AGCTTAAGCTTGACATGGCCAACAAG-3') and SM-22α antisense primer (5'-GCGGATCCTCTCCGCTCTAACTG-3 ') as described in SEQ ID NO: 6 and β-actin sense primer (5'-GTGGGGCGCCCCAGGCACCA-3') as described in SEQ ID NO: 7 And β-actin antisense primer (5'-CTCCTTAATGTCACGCACGATTTC-3 ') as set forth in SEQ ID NO: 8. PCR conditions are as follows. After 5 minutes of initial denaturation at 94 ° C, a denaturation reaction at 94 ° C for 60 seconds, a primer binding reaction at 59 ° C for 60 seconds, a length extension reaction at 45 ° C for 45 seconds, and the process repeated 45 times. Then, length extension reaction was performed at 72 ° C. for 5 minutes. The changes observed through electrophoresis on 1% agarose gel were observed.

As a result of the above example, the expression level of SM-22α was observed in A549 cells, which are lung cancer cell lines, and HepG2 cells, which are liver cancer cell lines. As a result of transforming E-pcDNA vector and SM-22α cloned vector into cells, the transformed E-pcDNA vector showed similar expression to that of the control group, but when SM-22α expression vector was transformed, It was confirmed that expression was increased (see FIG. 2).

< Example  4> Weston Blot ( Western blot ) analysis

The transgenic animal cells prepared in Example 2 were collected 1 × 10 ⁷ cells and washed twice with PBS, followed by protease inhibitor cocktail (2 mM ARBSF, 1 mM EDTA, 130 μM Bestatin, 1 μM leupeptin, 14 μM E-). 64, 0.3 μM Aprotinin) cells were pulverized in a sonicator for 15-30 seconds, and then the supernatant was collected by centrifugation at 12,000 rpm and 4 ° C for 1 hour. Proteins were quantified by Lowry Method, loaded onto 12% acrylamide gel, separated by electrophoresis and transferred to nitrocellulose membrane. React with a primary antibody (Goat polyclonal anti-SM-22α antibody, abcam, UK) on a protein-transferred nitrocellulose membrane and then react with a secondary antibody (antigoat-IgG-HRP, Sigma, USA) to the ECL kit (Amersham Biosciences , UK) for 1 minute and exposed to Hyperfilm-MP (Amersham Biosciences, UK).

As a result of the above Example, it was confirmed that the protein expression shows the same aspect as the result of Example 3 (see Fig. 2).

< Example  5> SM -22α over stress Cancer cell line  Resistance evaluation

< Example  5-1> SM -22α overexpression Hydrogen Peroxide ( Hydrogen peroxide , H ₂ O ₂ For) Cancer cell line  Resistance evaluation

Lung cancer cell line A549 cells and liver cancer cell line HepG2 cells were each dispensed into ⁶ × well plates at 1 × 10 ⁶ cells and RPMI 1640 containing 100 units / ml penicillin (Sigma, USA), 10% fetal calf serum (Hyclone, USA) After incubation for 24 hours in a 37 ° C., 5% CO ₂ incubator using medium (Gibco-BRL, USA), H ₂ O ₂ was treated for 24 hours to 1.2 mM. Thereafter, 500 µl of cooled 70% ethanol was treated to fix cells at -20 ° C for 30 minutes, ethanol was removed by centrifugation at 3,000 rpm, and the fixed cells were collected. Collected cells were washed with PBS and propidium iodide (PI) staining reagent (PBS, pH 7.4, 0.1% triton X-100, 0.1 mM EDTA, 0.05 mg / ml RNase A (50 units / mg)). , 500 μl of 50 μg / ml propidium iodide) was added and stained at 4 ° C. for 1 hour, followed by flow cytometry (FACS).

As a result, in the lung cancer cell line A549 cells, no cell death occurred in the untreated cells. When H ₂ O ₂ was treated, apoptosis occurred in the cells transformed with the E-pcDNA vector (empty vector), but A549 cells transformed with the SM-22α expression vector suppressed cell death. (See Figure 3A). In addition, hepatic cancer cell line HepG2 cells were also inhibited by H ₂ O ₂ treatment due to the overexpression of SM-22α as in the lung cancer cell line (see FIG. 3B). This suggests that cancer cell lines acquire resistance to H ₂ O ₂ due to SM-22α overexpression.

< Example 5 -2> SM -22α over gamma radiation Cancer cell line  Resistance evaluation

Lung cancer cell line A549 cells and liver cancer cell line HepG2 cells were each dispensed into ⁶ × well plates at 1 × 10 ⁶ cells and RPMI 1640 containing 100 units / ml penicillin (Sigma, USA), 10% fetal calf serum (Hyclone, USA) After incubation for 24 hours in a 37 ° C., 5% CO ₂ incubator using medium (Gibco-BRL, USA), gamma radiation was treated. As a source, ⁶⁰ Co was used, and lung cancer cell line A549 cells were treated with 20 Gy (Gy) and incubated for 72 hours and measured by FACS as in Example 5-1, and liver cancer cell line HepG2 cells were treated with 10 gray for 24 hours. It was cultured and measured by FACS as in Example 5-1.

As a result, it was confirmed that cell death occurred in cells transformed with E-pcDNA vector in the lung cancer cell line during gamma radiation treatment, whereas cell death transformed in cells transformed with SM-22α expression vector was inhibited (FIG. 4A). Reference). Hepatic cancer cell line HepG3 cells were also found to inhibit apoptosis in cells transformed with SM-22α expression vector in the same manner as lung cancer cell lines upon gamma radiation treatment (see FIG. 4B). This suggests that cancer cell lines have increased resistance to gamma radiation due to SM-22α overexpression.

< Example  5-3> SM Luteolin at -22α overexpression Luteolin For) Cancer cell line  Resistance evaluation

Lung cancer cell line A549 cells and liver cancer cell line HepG2 cells were each dispensed into ⁶ × well plates at 1 × 10 ⁶ cells and RPMI 1640 containing 100 units / ml penicillin (Sigma, USA), 10% fetal calf serum (Hyclone, USA) After incubation for 24 hours in a 37 ° C., 5% CO ₂ incubator using medium (Gibco-BRL, USA), the anticancer drug luteolin was treated. Lung cancer cell line A549 cells were treated with 0.2 mM luteolin and cultured for 72 hours, and measured by FACS in the same manner as in Example 5-1. Hepatic cancer cell line HepG2 cells were treated with luteolin 0.5 mM and cultured for 48 hours. It was measured by FACS in the same manner as -1.

As a result of constant Example, no cell death was observed in the group not treated with luteolin. In the case of lung cancer cell line A549 cells, significant cell death was observed in cells transformed with E-pcDNA vector upon luteolin treatment, but cell death was inhibited in cells transformed with SM-22α expression vector (see FIG. 5A). Hepatocarcinoma cell line HepG2 cells were also observed to reduce cell death upon luteolin treatment in cells transformed with SM-22α expression vector in the same way as lung cancer cell lines (see FIG. 5B). This suggests that cancer cell lines acquire resistance to luteolin due to SM-22α overexpression.

< Example  5-4> SM -22α overexpression methyl  Methanesulfonade ( Methyl methanesulfonate , MMS For) Cancer cell line  Resistance evaluation

Lung cancer cell line A549 cells and liver cancer cell line HepG2 cells were each dispensed into ⁶ × well plates at 1 × 10 ⁶ cells and RPMI 1640 containing 100 units / ml penicillin (Sigma, USA), 10% fetal calf serum (Hyclone, USA) After incubation for 24 hours in a 37 ° C., 5% CO ₂ incubator using medium (Gibco-BRL, USA), methyl methanesulfonate (MMS), a direct-acting genotoxic agent, is used. Was treated. Lung cancer cell line A549 cells were treated with MMS 1 mM and cultured for 48 hours, and measured by FACS in the same manner as in Example 5-1. Hepatocarcinoma cell line HepG2 cells treated with MMS 0.5 mM and cultured for 48 hours, Example 5-1 In the same manner as measured by FACS.

As a result of constant Example, no cell death was observed in the group not treated with MMS. In the lung cancer cell line A549 cells, significant cell death was observed in cells transformed with the E-pcDNA vector upon MMS treatment, but cell death was inhibited in cells transformed with the SM-22α expression vector (see FIG. 6A). Hepatocarcinoma cell line HepG2 cells were also observed to reduce apoptosis in cells transformed with SM-22α expression vector in the same manner as lung cancer cell line upon MMS treatment (see FIG. 5B). This suggests that cancer cell lines acquire resistance to MMS due to SM-22α overexpression.

< Example  5-5> SM -22α overexpression Serum free  For badges Cancer cell line  Resistance evaluation

Lung cancer cell line A549 cells and liver cancer cell line HepG2 cells were each dispensed into ⁶ × well plates at 1 × 10 ⁶ cells and RPMI 1640 containing 100 units / ml penicillin (Sigma, USA), 10% fetal calf serum (Hyclone, USA) After culturing for 24 hours in a 37 ° C., 5% CO ₂ incubator using medium (Gibco-BRL, USA), the medium was replaced with serum medium for the control group and serum-free medium for the experimental group. Lung cancer cell line A549 cells were solid in serum-free medium and cultured for 4 days, and measured by FACS in the same manner as in Example 5-1, and liver cancer cell line HepG2 cells were replaced with serum-free medium and cultured for 10 days, Example 5-1. In the same manner as measured by FACS.

As a result of constant Example, no cell death was observed in the cells cultured with the existing serum medium. In the case of lung cancer cell line A549 cells, significant cell death was observed in cells transformed with E-pcDNA vector when cultured in serum-free medium, but cell death was inhibited in cells transformed with SM-22α expression vector (see FIG. 6A). ). When the liver cancer cell line HepG2 cells were also replaced with serum-free medium, it was observed that cell death was reduced in cells transformed with the SM-22α expression vector in the same manner as the lung cancer cell lines (see FIG. 5B). This suggests that cancer cell lines acquire resistance to H ₂ O ₂ due to SM-22α overexpression.

< Example  6> SM -22α Antisense ( antisense ) Preparation of Vector Expressing Nucleotide

SM-22α nucleotides of A549 cells, a human lung cancer cell line, were identified by the primer (5'-ttggatccgacatggccaacaag-3 ') and Hind III restriction enzyme sequence as set forth in SEQ ID NO: 3, including the BamH I restriction enzyme sequence, for the production of antisense SM-22α. Antisense SM-22α was amplified by PCR using a primer (5′-gccctaagctttctaactgatgatct-3 ′) as described in SEQ ID NO: 4 comprising. PCR conditions are as follows. After the initial denaturation process at 95 ℃ for 5 minutes, the denaturation reaction for 60 seconds at 95 ℃, primer binding reaction for 45 seconds at 60 ℃, length extension reaction for 45 seconds at 72 ℃ and repeat this process 50 times It was. The amplified sense SM-22α nucleotide has a sequence set forth in SEQ ID NO: 9, and was inserted into the Hind III / BamH I restriction enzyme site of the expression vector pcDNA3.1 (Invitrogen, USA), and the expression vector thus prepared was "pcDNA / SM-22-AS "(see Figure 1).

< Example  7> pcDNA / SM-22- AS  Preparation of Transgenic Cells Transduced with Expression Vectors

Human lung cancer cell line A549 cells, liver cancer cell line HepG2 cells, cell line HT-29 cells, and fibromyoma cell line HT-1080 cells comprising 100 units / ml penicillin (Sigma, USA), 10% fetal bovine serum (Hyclone, USA) RPMI 1640 medium (Gibco-BRL, USA) was inoculated at a concentration of 1 × 10 ⁶ cells / 5 ml and then incubated in a 37 ° C., 5% CO ₂ incubator. 4 μg of each of the pcDNA / SM-22-AS expression vector and the E-pcDNA (empty vector) expression vector prepared in Example 6 were transduced into the four cancer cell lines using Lipofectamine 2000 (Invitrogen, USA). It was. Transduction method was performed in the same manner as in Example 2. Thereafter, the cells were fixed and trypan blue staining was performed under an optical microscope (Leica, Germany). As a result, the cells transformed with the SM-22α antisense nucleotide expression vector promoted cell death as compared with the cells transformed with the E-pcDNA vector (see FIG. 8).

< Example  8> SM Investigation of Apoptosis in Cancer Cell Lines by Inhibiting -22α Expression

Human lung cancer cell line A549 cells, liver cancer cell line HepG2 cells, colon cancer cell line HT-29 cells, and fibromyoma cell line HT-1080 cells, which are cancer cell lines transformed with SM-22α antisense nucleotide expression vector, were cultured for 10 days. It was measured by FACS in the same manner as -1.

As a result, cell death was not observed in the cell line transformed with the E-pcDNA vector, but cell death transformed with the SM-22α antisense nucleotide expression vector was confirmed to occur effectively (Fig. 9 and See Table 1).

FACS analysis of cancer cell line transformed with SM-22α antisense nucleotide expression vector Transformation of E-pcDNA Expression Vector SM-22 Antisense Nucleotide Expression Vector Transformation Lung Cancer Cell Line A549 Cells 3.27 ± 0.95 88.57 ± 2.41 Liver cancer cell line HepG2 cells 3.93 ± 0.50 77.80 ± 4.08 Colon cancer cell line HT-29 cells 1.17 ± 0.33 99.03 ± 2.90 Fibromyoma Cell Line HT-1080 Cells 2.07 ± 0.87 90.10 ± 2.59

As described above, it was confirmed that increasing the expression of SM-22α increases resistance to various stress agents, and inhibiting the expression of SM-22α promotes cell death. Therefore, an anticancer agent containing an inhibitor of SM-22α as an active ingredient can be usefully used to promote cell death of cancer cells.

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Claims (10)

A method of inhibiting the activity of smooth muscle-22α to promote cancer cell death in mammals except humans. The method of claim 1, wherein the inhibition of smooth muscle-22α activity is performed by using an antisense nucleotide or antibody that complementarily binds to smooth muscle-22α mRNA. . An anticancer agent comprising an antisense nucleotide or antibody complementarily binding to mRNA of smooth muscle-22α as an active ingredient. delete The anticancer agent according to claim 3, wherein the antisense nucleotide has a nucleotide sequence set forth in SEQ ID NO: 9. 4. The anticancer agent according to claim 3, wherein the cancer is selected from the group consisting of lung cancer, colorectal cancer, myeloma, and liver cancer. Smooth muscle-22α antisense nucleotide expression vector. 8. The expression vector of claim 7, wherein the expression vector is a plasmid vector or a viral vector. 9. The viral vector of claim 8, wherein the viral vector is a retrovirus comprising adenovirus, adeno-associated virus, lenti virus. An anticancer agent comprising the expression vector of any one of claims 7 to 9 as an active ingredient.

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