KR101394828B1 - Use of GKN1 amino―terminal regulatory motif having anticancer activity - Google Patents

Abstract

The present invention relates to an anticancer composition comprising an amino-terminal regulatory motif of GKN1, and more particularly to an anticancer composition comprising an amino-terminal regulatory motif of GKN1, particularly an active region of a GKN1 gene having an activity of inhibiting the development of gastric cancer and inhibiting cancer cell metastasis will be. The GKN1 amino-terminal regulatory motif according to the present invention has an excellent activity of inhibiting the survival and proliferation of cancer cells, and has an effect of stopping cell cycle progression. Therefore, it can be effectively used as a composition for anticancer therapy and a screening method for anticancer agent.

Description

Use of an amino-terminal regulatory motif of GKN1 with anticancer activity {Use of GKN1 amino-terminal regulatory motif having anticancer activity}

The present invention relates to an anticancer composition comprising an amino-terminal regulatory motif of GKN1, and more particularly to an anticancer composition comprising an amino-terminal regulatory motif of GKN1, particularly an active region of a GKN1 gene having an activity of inhibiting the development of gastric cancer and inhibiting cancer cell metastasis will be.

In recent years, attempts have been made to discover therapeutic targets and to use them in the development of diagnostic and therapeutic agents by studying the function of genes related to the generation and treatment of cancer in the world. With the activation of genome research, human genetic DNA chip or proteome analysis studies have been actively conducted and a large number of genes related to cancer have been discovered, so a database of many genetic information has been established. However, Specific biological function and cancer relevance have not yet been studied or are uncertain. Therefore, based on these information, there is a continuing need for efforts to discover diagnostic and target genes related to actual cancer.

On the other hand, the gastrointestinal tract is always exposed to digestive food fragments, various harmful substances, and acidic pH stress, and thus, trauma is likely to occur. Endogenous molecules such as TFF1 (trefoil factor family 1) protect gastric mucosa from these harmful environments (Babyatsky, et al., 1996, Oral trefoil peptides protect against ethanol- and indomethacin-induced gastric injury in rats Gastroenterology 110, 489-497). Recently, gastrokinetic 1 (GKN1) has been isolated from gastric mucosal cells of various mammals including mice (Martin et al., 2003, A novel mitogenic protein that is highly expressed in cells of the gastric antrum mucosa. ... Gastrointest Liver Physiol 285, G332-G343), GKN1 is a novel autocrine / paracrine protein that is specifically expressed in the gastric mucosa (Martin et al, 2003;. . Xing et al, 2012, Gastrokine 1 induces senescence through p16 / Rb pathway activation in gastric cancer cells. Gut 61 , 43-52). Toback et al . Reported that GKN1 protects the antral mucosa and promotes restitution and proliferation after injury to help heal wounds (Toback et al., 2003, Peptide fragments of AMP-18, a novel secreted gastric antrum mucosal protein, are mitogenic and motogenic, Am. J. Physiol. Gastrointest. Liver Physiol. 285, G344-G353). GKN1 has also been shown to play an important role in gastric cancer cell migration by epithelial-mesenchymal transition (EMT), reactive oxygen species (ROS), and PI3K / Akt pathway (Yoon et al. , 2011).

Furthermore, the inventors have discovered that GKN1 induces inactivation of NF-kB and COX-2 and stimulates lymphocyte migration, which can inhibit gastric mucosal infiltration and cell proliferation and thus GKN1 is involved in gastric cancer development It is assumed that it plays an important role by regulating the cell cycle. This assumption was confirmed in a recent study that GKN1 inhibits cell growth through G2 / M arrest in SGC-7901 cells (Yan et al., 2011, Proteomics characterization of gastrokinetic 1-induced growth inhibition gastric cancer cells. Proteomics 11 , 3657-3664). Consistent with these results, Xing et al. Recently reported that GKN1 promotes senescence through p16 / Rb pathway activity in stomach cancer cells (Xing et al., 2012). However, the molecular mechanism of GKN1 that inhibits the cell cycle has not yet been elucidated.

In addition, the GKN1 protein is a member of the BRICHOS superfamily, which is a protein associated with dementia or the respiratory distress syndrome (Sanchez-Pulido et al., 2002, BRICHOS: a conserved domain in association with dementia, respiratory distress and cancer. Trends Biochem. Sci. 27, 329-332). The BRICHOS superfamily proteins have four distinct regions: hydrophobic region, linker, BRICHOS domain and C-terminus. The role of the BRICHOS domain is associated with post-translational processing of this protein, and the N-terminal hydrophobic region is reported to be part of the signal peptide (Sanchez-Pulido et al., 2002). However, there is not much research on the specific and clear function and role of each domain that constitutes GKN1.

Accordingly, the present inventors have studied the effect of GKN1 on cell viability and proliferation, cell cycle, and epigenetic modification of cell cycle-related proteins, and confirmed the antitumor activity of GKN1. Especially, (Amino-terminal regulatory motif) and completed the present invention.

Korean Patent No. 10-1134675 Korean Patent Publication No. 10-2012-0078269

It is an object of the present invention to provide an anticancer composition comprising an amino-terminal regulatory motif of GKN1 (Gastrokine 1) protein.

It is another object of the present invention to provide a method for screening anticancer agents using the amino terminal regulatory motif of GKN1.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for anticancer chemotherapy comprising a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or a nucleic acid encoding the same, as an active ingredient.

In one embodiment of the present invention, the nucleic acid may be a nucleotide sequence of SEQ ID NO: 2.

In one embodiment of the present invention, the cancer may be a stomach cancer.

In one embodiment of the present invention, the nucleic acid may be contained in an expression vector.

The present invention also provides a method for producing a recombinant cell, comprising culturing a polypeptide of SEQ ID NO: 1 or a recombinant cell expressing the polypeptide and a candidate substance; And measuring the effect of the polypeptide of SEQ ID NO: 1 on the activity or the intracellular level of the anticancer agent.

In one embodiment of the present invention, when the candidate substance increases the activity of the polypeptide of SEQ ID NO: 1 or increases the expression level of the gene encoding the polypeptide in the cell, And judging it as an anticancer agent.

In one embodiment of the present invention, the activity or expression level of an intracellular gene in the polypeptide is determined by coimmunoprecipitation, radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunohistochemistry, Western blot (Western Blotting), and flow cytometry (FACS).

In one embodiment of the present invention, the cancer may be a stomach cancer.

The GKN1 amino-terminal regulatory motif according to the present invention has an excellent activity of inhibiting the survival and proliferation of cancer cells, and has an effect of stopping cell cycle progression. Therefore, it can be effectively used as a composition for anticancer therapy and a screening method for anticancer agent.

Figure 1 shows the anticancer activity of GKN1 through cell survival and proliferation inhibition. (A) In MTT assays, GKN1 -transfected AGS cells inhibited cell survival in a time-dependent manner, whereas shGKN1 -transfected HFE-145 cells increased cell survival in a time-dependent manner. (B) In the BrdU insertion assay, GKN1 - transfected AGS cells inhibited cell proliferation in a time-dependent manner, and shGKN1 -transfected HFE-145 cells increased cell proliferation in a time-dependent manner.
FIG. 2 is a diagram showing the structure of mutants used in wild-type GKN1 and the embodiment of the present invention. FIG.
Figure 3 shows the results of Western blot analysis showing the expression levels of the respective GKN1 plasmids and GAPDH. GAPDH was used as a loading control.
Figure 4 shows the results of transient transfection of pGKNl ^D13N , pGKNl ^{[Delta] 68-199} , pGKNl ^{[ Delta] 1-67} , ^165-199 and pGKNl ^{[Delta] 1-67} in AGS cells in a time dependent manner.
Figure 5 shows the results of transient transfection of pGKNl ^D13N , pGKNl ^{[Delta] 68-199} , pGKNl ^{[ Delta] 1-67} , ^165-199 and pGKNl ^{[Delta] 1-67} in AGS cells in a time dependent manner. On the other hand, in AGS cells, pGKN1 ^D13A And pGKN1 ^{[Delta] 1-164} did not affect cell viability and proliferation.
FIGS. 6 and 7 show the result of measuring the anticancer effect with the colony-forming assay in AGS cells. Wild-type GKN1 , pGKN1 ^D13N , pGKN1 ^{? 68-199} , pGKN1 ^{? 1-67, 68-199} and pGKN1 ^{? 1-67} significantly inhibited colony formation.
Figure 8 shows that 5-FU treatment in (A) AGS cells and (B) HFE-145 cells inhibits cell survival in a time- and dose-dependent manner.
9 shows that synergistic ^effects of wild-type GKNl , pGKNl ^D13N , pGKNl ^{? 68-199} , pGKNl ^{? 1-67} , ^165-199 , pGKNl ^{? 1-67} , and recombinant GKNl protein treatment on cell survival inhibition by 5-FU .
Figure 10 shows that wild-type GKNl and recombinant GKNl protein treatment synergize with G0 / G1 and G2 / M cell cycle arrest by 5-FU.
Figure 11 shows Western blot analysis results after treatment of AGS cells with GKNl transfection, 5-FU and recombinant GKNl protein. Synergy was observed in the increased expression of p53 and p21, but the expression of cyclin B and cyclin D was decreased.
Figures 12 and 13 show ^transcripts (SEQ ID ^{NOs: 1} , 2, 3, and 4) with the GKN wild type (Figure 12) and mutants (pGKN1 ^D13N , pGKN1 ^{? 68-199} , pGKN1 ^{? 1-67} , ^165-199 , and pGKN1 ^{? 1-67} ) G1 / G1 and G2 / M groups in the activated AGS cells.
14 is a Western blot analysis result of transfected AGS cells. The expression of p53 and p21 was slightly increased, and the downregulation of p16 recurrence and CDK4, cyclin D, E2F1, and cyclin A expression was observed. Knockdown of GKN1 in HFE-145 cells down-regulated the expression of p53, p16 and p21, but up-regulated the expression of cell cycle-modulating factors including CDK4 and cyclin A.
Figure 15 shows downregulation of GKN1 on G2 / M-based protein expression. In AGS cells, GKN1 down-regulated p-cdc2, PLK1, CDK2, cdc25a, cdc25c and cyclin B whereas GKN1 silencing in HFE-145 cells upregulated p-cdc2, PLK1 and CDK2 expression .
Figure 16 shows that when expressing ^{pGKN1 D13N} , pGKN1 ^{? 68-199} , pGKN1 ^{? 1-67} , ^165-199 , and pGKN1 ^{? 1-67} in transient transfection, CDK4, Cyclin D, Cyclin A, Cyclin E , And down-regulation of cyclin B. < RTI ID = 0.0 >

The present invention provides an anticancer composition containing an amino-terminal regulatory motif of GKN1 (Gastrokine 1) as an active ingredient and a method of screening an anticancer agent using the same.

The inventors of the present invention have investigated the anti-cancer effect of GKN1, a novel protein isolated from gastric mucosal cells of mammals, while studying for the development of a novel cancer therapeutic agent capable of inhibiting cancer development (Korean Patent Publication No. 10-2012 -0051258). GKN1 (Gastrokine 1), also known as AMP-18 (antrum mucosal protein-18) or CA11, is known to be expressed in gastric epithelia. In addition, GKN1 has been reported to protect the mucous membranes of the whole body and facilitate wound healing by facilitating recovery and proliferation after wounding (Toback. FG et al., Am J Physiol Gastrointest Liver Physiol 285 : G344-353, 2003), but the anti-cancer effect of GKN1 was not known. The present inventors confirmed that the expression of GKN1 gene is decreased in cancer cells unlike the normal cells, so that the GKN1 gene is presumed to be involved in the pathogenesis of cancer. Actually, it was firstly found that GKN1 has anticancer activity .

The inventors of the present invention have further studied based on the invention of the above patent publication which confirmed the anti-cancer effect of GKN1. As a result, the amino-terminal regulatory motif of GKN1 alone can exhibit a similar anti-cancer effect to that of GKN1 And completed the present invention.

GKN1 is composed of 199 amino acids and contains three conserved regions, the amino-terminal regulatory motif (1-67), the middle brichos domain (68-164), and the carboxy-terminal conserved region carboxy-terminal conserved domain (165-199) (Fig. 2). To investigate the antitumor activity of GKN1, the present inventors examined wild-type GKN1 and four types of internal-deletion mutations (pGKN1 ^{? 68-199} , pGKN1 ^{? 1-67} , 165-199, pGKN1 ^{? 1-164} and pGKN1 ^{[Delta] 1-67} ), and two mutants (pGKN1 ^D13N and pGKN1 ^D13A ), respectively (Fig. 2). Among these, pGKN1 ^{? 68-199} is a clone containing an amino-terminal regulatory motif.

According to an embodiment of the present invention, ectopic expression of GKN1 in AGS cells significantly reduced cell survival, proliferation and colony formation (Figs. 1, 4 to 7). On the other hand, it was confirmed that silencing of GKN1 expression in HFE-145 normal gastric mucosa increases cell viability and proliferation capacity (Fig. 1). MTT assays were also performed on AGS and HFE-145 cells to investigate the effect of GKN1 on the chemosensitivity of 5-FU. As expected, GKNl and 5-FU treatment showed synergistic effects of cell survival and proliferation inhibition through p53 and p21 expression (Figures 8-11). P53 and p21 activity by 5-FU anticancer drugs have already been reported in mouse L-TK cells and AGS cells (Sun et al., 1995, Identification of a novel p53 promoter element involved in genotoxic stress-inducible p53 gene expression. Mol. Cell. Biol. 15 , 4489-4496; Seo et al., 2011, Contribution of Epstein-Barr virus infection to chemoresistance of gastric carcinoma cells to 5-fluorouracil. Arch Pharm Res., 34, 635-643). In addition, the above-mentioned GKN1 antitumor activity required an amino-terminal regulatory motif of GKN1. These results show that GKN1 has an anticancer effect and that an amino terminal regulatory motif is required to exhibit anticancer activity.

The fact that the expression of GKN1 suppresses cell proliferation suggests that GKN1 can modulate cell cycle-regulating components. Therefore, we performed flowcytometry on AGS cells transfected with wild-type GKN1 to investigate the effect of GKN1 on the cell cycle and regulatory components of G1 / S and G2 / M groups. As a result, it was confirmed that the G0 / G1 group and the G2 / M group were simultaneously increased (FIG. 12). Similar patterns were confirmed in AGS cells transfected with pGKN1 ^D13N , pGKN1 ^{? 68-199} , pGKN1 ^{? 1-67} , ^165-199 , and pGKN1 ^{? 1-67} (Fig. 13). In G1 / S phase, negative cell cycle regulators such as p15, p16, p18, p19, p27 and p21 are key regulators that inhibit the cyclin D1 / CDK4, 6 or cyclin E / CDK2 complex and Reddy, 1995, Cell cycle control in mammalian cells: role of cyclins, cyclin dependent kinases (CDKs), growth suppressor genes and cyclin-dependent kinase inhibitors (CKIs), Oncogene 11 , 211-219). GKN1 expression in the G1 / S phase increased the expression of p53 and p21, selectively induced p16 re-expression, and inhibited CDK4, cyclin D1, E2F, and Cyclin A expression at the same time (Fig. 14). On the other hand, positive cell regulatory factors including CDK4 and cyclin A were up-regulated in GKN1 knockdown HFE-145 cells (Fig. 14). GKNl also inactivated G2 / M-group progression through modulation of cdc25, PLK1 and cyclin B expression in AGS cells (Figure 15). Also, when pGKNl ^D13N , pGKNl ^{? 68-199} , pGKNl ^{? 1-67} , ^165-199 , and pGKNl ^{? 1-67} were expressed by transient transfection, CDK4, Cyclin D, Cyclin A, Cyclin E, And down-regulation of cyclin B (Figure 16). In addition, when the expression of GKN1 was inhibited in HFE-145 cells, the expression of p-cdc2, PLK1 and CDK2 was upregulated (Fig. 15). These results are consistent with previous studies that overexpression of GKN1 causes cell cycle arrest in the G1 or G / M phase (Xing et al., 2012; Yan et al., 2011). Taken together, these results indicate that GKN1 is directly involved in the regulation of a series of proteins involved in cell cycle regulation, for which the amino terminal regulatory motif of GKN1 is essential.

Accordingly, the present invention can provide an anticancer composition comprising a GKN1 amino-terminal regulating motif or a nucleotide encoding the same. Preferably, the GKN1 amino-terminal regulatory motif may have the amino acid sequence of SEQ ID NO: 1, and the polynucleotide encoding the GKN1 amino-terminal regulatory motif may have the nucleotide sequence of SEQ ID NO: 2.

The GKN1 amino-terminal modulating motif according to the present invention may also preferably be a functional equivalent to a polypeptide having the amino acid sequence of SEQ ID NO: 1. The 'functional equivalent' means a polypeptide having at least 60%, preferably 70%, more preferably 80% or more sequence homology with the amino acid sequence of SEQ ID NO: 1 as a result of addition, substitution or deletion of amino acid Refers to a polypeptide exhibiting substantially the same activity as GKN1 of the present invention. Here, 'substantially homogenous activity' means the activity of GKN1 described above. Such functional equivalents may include, for example, amino acid sequence variants in which some of the amino acids of the GKN1 amino acid sequence according to the invention are substituted, deleted or added. Substitution of amino acids can be preferably conservative substitutions, and examples of conservative substitutions of amino acids present in nature are as follows: (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). Deletion of the amino acid may preferably be located at a site that is not directly involved in the activity of GKN1 of the present invention. The functional equivalents may also include polypeptide derivatives in which the chemical structure of the polypeptide is modified while maintaining the basic skeleton of GKN1 and its physiological activity. For example, the polypeptide of the present invention may include a fusion protein made by fusion with another protein while maintaining structural stability and physiological activity to change the stability, shelf-life, volatility, or solubility of the polypeptide of the present invention.

In one embodiment of the present invention, it was confirmed that when codon 13 of GNK1 is aspartic acid or asparagine, it also exhibits an anticancer effect like full length GNK1.

The polynucleotide encoding the GKN1 (Gastrokine 1) amino-terminal regulatory motif may be introduced into an expression vector such as a plasmid or a viral vector by a known method, and then the expression vector may be transduced by various methods known in the art ) Or transfection into the target cell in an expression form.

The plasmid expression vector is an FDA approved gene transfer method that can be used for human, and is a method of directly delivering plasmid DNA to human cells (Nabel, EG et al., Science, 249: 1285-1288, 1990) Has the advantage that it can be homogeneously purified, unlike the viral vector. As a plasmid expression vector that can be used in the present invention, mammalian expression plasmids known in the art can be used. In one embodiment of the present invention, a recombinant expression vector was prepared using pcDNA3.1 (Invitrogen) plasmid.

A plasmid expression vector containing a nucleic acid according to the present invention may be prepared by a method known in the art such as, but not limited to, transient transfection, microinjection, transduction, , Cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection ), Electroporation, gene gun, and other known methods for introducing DNA into cells (Wu et al., J. Bio. Chem., 267: 963-967, 1992; Wu et al., Bio. Chem., 263: 14621-14624, 1988). Preferably, transient transfection methods can be used.

In addition, the vector capable of expressing the GKN1 amino-terminal regulating motif may be administered into cells, tissues or the body by a known method, for example, topically, parenterally, orally, nasally, , Subcutaneously, or by any other suitable means. In particular, the vector may be injected directly into a target cancer or tumor cell in an amount effective to treat the tumor cells of the target tissue. In particular, in the case of a cancer or tumor in the body cavity such as the eye, gastrointestinal tract, genitourinary tract, lung and bronchial system, the pharmaceutical composition of the present invention is administered to a hollow organ affected by cancer or tumor, May be injected directly using a needle, catheter or other type of transport tube.

Meanwhile, since the GKN1 amino-terminal regulatory motif of the present invention inhibits the proliferation of cancer cells, promotes apoptosis, and inhibits the metastasis and invasion of cancer cells, the present invention can provide a method for screening anticancer agents using the gene have.

That is, the method of screening an anticancer agent according to the present invention comprises: (a) culturing a GKN1 amino-terminal regulating motif or a recombinant cell expressing the motif and a candidate substance; And (b) determining the effect on the activity or intracellular level of the GKN1 amino-terminal modulating motif.

The intracellular level increase of the GKN1 amino-terminal regulatory motif means that the expression of the gene encoding the GKN1 amino-terminal regulatory motif is increased or the degradation of the GKN1 amino-terminal regulatory motif is inhibited, thereby increasing the concentration of the GKN1 amino-terminal regulatory motif It says. The GKN1 amino-terminal regulatory motif gene expression includes transcription and translation of a gene encoding the GKN1 amino-terminal regulatory motif. Therefore, when the candidate substance increases expression of the GKN1 amino-terminal regulatory motif gene and the protein level in the cell, the candidate substance can be judged as a substance having anticancer activity.

In addition, various methods known in the art can be used to measure the activity and intracellular level of the GKN1 amino-terminal regulating motif, including, but not limited to, coimmunoprecipitation, enzyme immunoassay an enzyme-linked immunosorbent assay, radioimmunoassay (RIA), immunohistochemistry, Western blotting and flow cytometry (FACS).

In addition, the screening method based on GKN1 of the present invention can be applied to high throughput screening (HTS). HTS is a method of simultaneously testing a plurality of candidate substances and simultaneously or almost simultaneously screening biological activities of a plurality of candidate substances. As a specific embodiment, the cell line can be cultured in a 96-well microtiter plate or a 192-well microtiter plate, followed by treatment with a plurality of candidate substances, and the degree of expression of GKN1 can be measured by an immunochemical method. The wells typically require assay volumes ranging from 50 to 500, and in addition to plates, a number of instruments, instruments, pipettes, robots, and plate washers may be used to adapt the 96-well format to a wide range of uniform and non- And plate readers are commercially available.

In addition, the anti-cancer composition according to the present invention can be used as a pharmaceutical composition capable of preventing and treating cancer, and the pharmaceutical composition can further include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" as used herein refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like when administered to humans. Pharmaceutically acceptable carriers include, for example, carriers for oral administration such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like, water for parenteral administration such as water, suitable oils, saline, aqueous glucose and glycols And may further contain stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995). The pharmaceutical composition according to the present invention, together with a pharmaceutically acceptable carrier as described above, may be formulated into a suitable form according to a method known in the art. That is, the pharmaceutical composition of the present invention can be prepared in various parenteral or oral administration forms according to known methods, and isotonic aqueous solutions or suspensions are preferred for injection formulations typical of parenteral administration formulations. The injectable formulations may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be formulated for injection by dissolving in saline or buffer. Formulations for oral administration also include, but are not limited to, powders, granules, tablets, pills, and capsules.

The pharmaceutical composition formulated as described above may be administered in an effective amount through various routes including oral, transdermal, subcutaneous, intravenous, or muscular. The term " effective amount " as used herein refers to an amount that shows a preventive or therapeutic effect when administered to a patient. The dosage of the pharmaceutical composition according to the present invention can be appropriately selected depending on the route of administration, subject to be administered, age, gender, individual difference, and disease state. Preferably, the pharmaceutical composition of the present invention may contain the active ingredient in an amount of 1 to 10000 / kg body weight / day, more preferably 10 to 1000 / kg body weight / day, May be repeatedly administered several times a day at an effective dose of < RTI ID = 0.0 > The anticancer composition of the present invention may also be administered in combination with a known compound having an effect of preventing, ameliorating or treating cancer.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

< Example  1>

Cell culture and transfection ( transfection )

AGS gastric cancer cell line and HFE-145 normal gastric mucosa cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and Hassan (Washington, DC, USA). Both cell lines were cultured in RPMI-1640 medium (Lonza, Basel, Switzerland) supplemented with 10% heat-inactivated fetal bovine serum at 37 ° C (5% CO ₂ ). GKNl cDNA and shGKNl were cloned into the pcDNA3.1 expression vector (Invitrogen, Carlsbad, CA, USA). AGS and HFE-145 cells were transfected with expression plasmids (2 ug total DNA) in 60 mm-diameter dishes. Lipofectamine Plus transfection reagent (Invitrogen) was used according to the manufacturer's protocol.

< Example  2>

Plasmid production and GKN1 of Transfection

GKN1 is composed of 199 amino acids and contains three conserved regions, an amino-terminal regulatory motif (1-67), a central brichos domain (68-164) and a carboxy-terminal conservation domain carboxy-terminal conserved domain (165-199) (Fig. 2). The present inventors produced the following four kinds of deletion-formed plasmids. pGKN1 ^{△ 68-199} are the amino-terminus include, pGKN1 ^{△ 1-67} are each the brichos and carboxyl terminal-to-terminal control motifs, pGKN1 ^{△ 1-67,165-199} is a brichos domain, pGKN1 ^{△ 1-164} is carboxy . Post-translational modification of asparagine (D) to asparagine (N) at codon 13 of GKN1 has been reported in H. pylori- infected gastric mucosa (Nardone et al., 2007) Two mutations with mutations in codons were also generated using Quick Change Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). All plasmids were identified by sequencing.

The primer sequences used for the cloning of GKN1 and the PCR of the specific region are shown in Table 1 below.

All cDNAs were subcloned into pcDNA 3.1 (Invitrogen) and transient transfection was performed in AGS cells with 7 constructs containing wild type GKNl and 6 mutations. Lipofectamine (Invitrogen) was used according to the manufacturer's instructions. The effects of stomach mutations on cell survival, proliferation and colony formation were then observed in shGKN1 transfected AGS and HFE-145 cells.

In addition, to see if GKN1 contributes to the chemosensitivity of 5-FU (5-fluorouracil), each construct containing 6 wild-type and 6 mutant GKN1 , recombinant GKN1 protein, and 5-FU And MTT assays were performed in AGS and HFE-145 cells at 24 and 48 hours, respectively.

&Lt; Example 3 >

Effect of GKN1 on cell survival, proliferation and colony formation

<3-1> Measurement of cell viability, proliferation and colony formation

For cell survival and growth analysis, MTT assays were performed at 24, 48, and 72 hours after transient transfection of each construct. Absorbance was measured at 540 nm using a spectrophotometer, and cell viability was expressed relative to the control (mock) (empty vector + lipofectamine).

For cell proliferation measurements, BrdU insertion assays were performed at 24, 48, and 72 hours after transient transfection of each construct. BrdU cell proliferation assay kit (Millipore, Billerica, MA, USA) was used according to the protocol of the manufacturer. Absorbance was measured at 450 nm using a spectrophotometer, and the cell proliferative capacity was expressed as a relative value to the control (mock).

In order to measure the proliferative ability of single cells in vitro, a plate clonogenic assay was performed. 1 X 10 ³ AGS cells transfected with each of the preparations were inoculated into 6-well plates and cultured in RPMI-1640 for 2 weeks to allow colonies to form. Colonies were fixed in 1% formaldehyde, stained with 0.5% crystal violet solution, and counted using a colony count program.

A Student t-test was performed to investigate the effect of GKN1 on cell survival and proliferation. Data were expressed as mean ± standard deviation of at least 3 replicates. A P value of less than 0.05 was considered statistically significant.

<3-2> GKN1 Effect on cell survival, proliferation and colony formation

The effects of GKN1 on cell growth and proliferation were confirmed by performing MTT, BrdU insertion and colony formation assays in AGS and HFE-145 cells. As a result, a significant time-dependent inhibitory effect on cell growth and proliferation was observed in wild-type GKN1 -transfected AGS cells (FIGS. 1A and B), consistent with previous studies (Yoon et al., 2011) . In contrast, in the expression GKN1 HFE-145 cells, expression control GKN1 (silencing) by shGKN1 the time of cell growth and proliferation showed dependent increase (Fig. 1A and B). To find specific regions of GKN1 that regulate cell proliferation, we first identified four internal-deletion mutations of GKN1, pGKN1 ^{? 68-199} , pGKN1 ^{? 1-67} , 165-199, pGKN1 ^{? 1- 164} and pGKN1 ^{? 1-67} were prepared (Fig. 2). In addition, one nucleotide at the 13 codon of GKNl was changed to create two mutant GKNl plasmids, pGKNl ^D13N and pGKNl ^D13A (Fig. 2). Expression of wild-type GKNl, the two mutants, and four deletion plasmids was confirmed by Western blot analysis from AGS cell lysates (Figure 3). Cell survival and proliferative capacity were also analyzed in AGS cells transfected with four kinds of erase plasmids and two mutant plasmids. As shown in Figures 4 and 5, the expression of the GKN1 wild-type, pGKNl ^D13N , pGKNl ^{[Delta] 68-199} , pGKNl ^{[ Delta] 1-67} , ^165-199 , and pGKNl ^{[Delta] 1-67} plasmids significantly enhanced cell survival and proliferation in AGS cells Respectively. However, the mutant plasmid pGKNl ^D13A and pGKNl ^{[Delta] 1-164} containing only the carboxy terminus had minimal effect (Figures 4 and 5).

In the colony forming assay, the expression of GKN1 wild-type, pGKN1 ^D13N , pGKN1 ^{? 68-199} , pGKN1 ^{? 1-67} , ^165-199 , and pGKN1 ^{? 1-67} were compared with control cells (mock) transfected with empty vector only Significantly reduced the number and size of colonies surviving AGS gastric cancer cell lines (Figs. 6 and 7).

In addition, in order to examine the effect of GKN1 on the anticancer sensitivity of 5-FU, 24 hours and 48 hours after co-treatment of 5-FU, wild-type or mutant GKN1 and recombinant GKN1 in AGS and HFE- Respectively. 5-FU treatment reduced time and dose-dependent cell viability in both cell lines (Figure 8). As expected, the wild-type and pGKN1 ^D13N, pGKN1 ^{68-199 △, △} pGKN1 ^{1-67,165-199,} mutations including pGKN1 ^{△ 1-67} GKN1, and AGS cells treated with the recombinant protein is GKN1 hours and administered in combination with 5-FU Lt; RTI ID = 0.0 &gt; (FIG. 9). &Lt; / RTI &gt; This means that GKN1 increases cytotoxicity of 5-FU. However, the two mutations, pGKNl ^D13A and pGKNl ^{[Delta] 1-164,} had only a minor effect on cell survival (Fig. 9). The effect of GKN1 on the anticancer drug sensitivity of 5-FU in cell cycle progression was confirmed by flow cytometry. Interestingly, treatment with 5-FU alone did not affect the cell cycle, but AGS cells treated with 5-FU and wild-type GKN1 or recombinant GKN1 protein increased the number of sub-G0 cells and increased G0 / G1 And G2 / M group progression (FIG. 10). In addition, GKN1 overexpression showed synergistic expression of p53 and p21, and decreased expression of cyclin B and D in AGS cells (Fig. 11).

<Example 4>

Effect of GKN1 on cell cycle

<4-1> Flow cytometry analysis of cell cycle

For cell cycle analysis, AGS cells from each experimental group were collected, digested with trypsin, fixed with cold 70% ethanol, and stained with PI (propidium iodide) for 45 minutes in the dark before analysis. Percentages of cells in different phases of the cell cycle were determined using a FACS Calibur Flow Cytometer equipped with CellQuest 3.0 software (BD Biosciences, Heidelberg, Germany). The experiment was repeated three times.

<4-2> Expression of Cell Cycle Regulator

To determine whether GKN1 is involved in cell cycle regulation, wild-type and mutant GKN1 Expression of G0 / G1 protein including p53, p21, p16, p15, CDK4, cyclin D1 and E2F in AGS cells 24 hours after transfection and HFE-145 cells 24 hours after shGKNl transfection , cdc2, cyclin B, cdc25A, cdc25C, aurora A, and Plk1, respectively. Cell lysates were separated on a 10% polyacrylamide gel and transferred to a Hybond PVDF membrane (Amersham Pharmacia Biotech, Piscataway, NJ, USA). After blocking, the membrane is incubated with antibodies against G0 / G1 proteins (including p53, p21, p16, p15, CDK4, cyclin D1 and E2F) and G2 / M group proteins (cdc2, cyclin B , cdc25A, cdc25C, aurora A, and Plk1). Protein bands were detected using an enhanced chemiluminescence reagent (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

<4-3> G0 / G1 and G2 / M arrest effects of GKN1

In addition, the effect of GKN1 on cell cycle progression was confirmed by flow cytometry. The ability of GKN1 to cause a replicative arrest was measured by flow cytometry analysis at 24 and 48 hours after transfection with wild type GKNl , 4 deletion plasmids, and 2 mutant GKNl . As expected, wild type GKN1, pGKN1 ^D13N, pGKN1 ^{68-199 △, △} pGKN1 ^{1-67,165-199,} and pGKN1 ^{1 -67} is conducted to increase the number of sub-G0 cells, cell cycle G0 / G1 and G2 / M group And a moderate effect was observed. However, pGKN1 ^D13A And pGKN1 ^{[Delta] 1-164} did not show any effect on cell cycle progression (FIGS. 12 and 13).

Expression of G0 / G1 and G2 / M-related proteins was examined to find potential molecular pathways involved in cell cycle arrest by GKN1. By transfection and the lysates shGKN1 for the AGS cells to the Specifications GKN1 was performed Western blot analysis of lysates from transfected with HFE-145 cells. During G0 / G1 arrest of AGS cells, the expression of GKN1 activates upregulates the expression of p53 and p21, causing re-expression of p16 and downregulating the expression of Cdk4, cyclin D, E2F1, and cyclin A (Fig. 14). In shGKNl transfected HFE-145 cells, silencing of GKN1 down-regulated the expression of p16 and p21, but upregulated the expression of positive cell cycle regulators including CDK4 and cyclin A (Figure 14) . During G2 / M arrest, GKN1 down-regulated the expression of p-cdc2, PLK1, CDK2, cdc25a, cdc25c and cyclin B in AGS cells whereas GKN1 expression in HFE-145 cells decreased expression of p-cdc2, PLK1 and cdk2 (Fig. 15). In AGS cells, pGKN1 ^D13N , pGKN1 ^{? 68-199} , pGKN1 ^{? 1-67} , ^165-199 , and pGKN1 ^{? 1-67} downregulated the expression of CDK4, cyclin D and cyclin B (FIG. 16) Is able to regulate the G1 / S and G2 / M groups of the cell cycle.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Attach an electronic file to a sequence list

Claims (8)

1. A pharmaceutical composition for anti-cancer comprising a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or a nucleic acid encoding the same, as an active ingredient. The method according to claim 1,
Wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 2. The method according to claim 1,
Wherein the cancer is gastric cancer. The method according to claim 1,
Wherein the nucleic acid is contained in an expression vector. delete delete delete delete

Images