KR101118880B1 - Method for screening inhibitors of liver cancer - Google Patents

Abstract

The present application relates to a screening system of a substance that interferes with the interaction between HBx, which is a protein of hepatitis B virus, and an APC protein. HBx binds to the host cell's APC protein and activates signaling pathways that cause liver cancer. Therefore, the interference between the proteins can effectively inhibit liver cancer.

Hepatitis B Virus, HBx, APC

Description

Method for screening inhibitors of liver cancer

The present invention relates to the field of cancer inhibitor screening.

An estimated 350 million people worldwide are infected with Hepatitis B Virus (HBV), of which 5 to 10% suffer from chronic hepatitis B for life, with chronic hepatitis B (Hepatocellular Carcinoma, HCC) (Ahn, SH, YN Park, et al. (2005). "Long-term clinical and histological outcomes in patients with spontaneous hepatitis B surface antigen seroclearance." J Hepatol 42 (2): 188-94 Montalto, G., M. Cervello, et al. (2002). "Epidemiology, risk factors, and natural history of hepatocellular carcinoma." Ann NY Acad Sci 963: 13-20). Hepatocellular carcinoma is the fourth most incidence cancer in Korea (National Cancer Information Center, 2003-2005). The main treatments for liver cancer include liver resection, hepatic artery embolization, chemotherapy, radiotherapy, and liver transplantation, but there are no anticancer drugs that can act specifically on liver cells (National Cancer Information Center). In order to develop anticancer drugs that can act specifically on liver cancer cells, the development of therapeutic methods based on the understanding of liver cancer mechanisms at the molecular biological level should be prioritized.

HBV, which causes chronic liver cancer, is a double-stranded DNA virus whose genome has four overlapping translation frames from which DNA polymerase, two structural proteins, and a regulatory protein, HBx (Hepatitis BX), are expressed. Tang, H., N. Oishi, et al. (2006). "Molecular functions and biological roles of hepatitis B virus x protein." Cancer Sci 97 (10): 977-83; Kim, H., YH Lee, et. al. (2001). "Through induction of juxtaposition and tyrosine kinase activity of Jak1, X-gene product of hepatitis B virus stimulates Ras and the transcriptional activation through AP-1, NF-kappaB, and SRE enhancers." Biochem Biophys Res Commun 286 (5): 886-94).

Among them, HBx is thought to play a major role in the development of liver cancer by activating several signaling pathways such as NF-kB, AP-1 and SRE, and interacting with proteins of host cells such as Jab1 through AP-1. Tanaka, Y., F. Kanai, et al. (2006). "The hepatitis B virus X protein enhances AP-1 activation through interaction with Jab1." Oncogene 25 (4): 633-42 ), The details of the mechanisms and the development of therapies based on them are unknown.

The present invention provides a system for screening a substance capable of inhibiting a signaling pathway based on a protein in a host cell that activates a signaling pathway that induces liver cancer through interaction with HBx.

In one aspect, the present invention provides a method for preparing an HBx protein comprising: providing HBx and APC proteins; Contacting the protein with a test substance; And comparing the interaction between HBx and APC in a control group not in contact with the protein in contact with the test substance.

In another aspect the invention provides a method wherein said HBx and APC are provided in the form of cells expressing it.

In another aspect the invention provides a method further comprising the step of selecting a substance that reduces the concentration of β-catenin compared to the control.

In another aspect, the invention provides a method wherein said HBx or APC is labeled with a detectable label.

In another embodiment the invention provides a method wherein said cells are HepG2, Huh7, Hep3B or PLC / PRF / 5.

In another aspect, the present invention provides a method of preparing a cell comprising the steps of providing a cell expressing HBx and APC proteins; Contacting said cell with a test substance; And it provides a liver cancer inhibitor screening method comprising the step of measuring and comparing the concentration of β-catenin in the control cell not in contact with the cell in contact with the test substance.

In another aspect, the present invention provides a method for screening a liver cancer suppressor further comprising the step of selecting a substance that reduces the concentration of β-catenin as a liver cancer inhibitor compared to the control group.

In another aspect, the present invention provides a method for screening a liver cancer inhibitor, wherein the liver cancer is caused by hepatitis B virus infection.

In another embodiment the invention provides a method wherein said cells are HepG2, Huh7, Hep3B or PLC / PRF / 5.

In another aspect, the present invention provides a method of controlling the concentration of β-catenin in cells infected with hepatitis B virus using APC gene or protein.

In another aspect, the present invention provides a method for preparing a protein comprising: providing a β-catenin protein; Contacting the protein with a test substance; And it provides a method for screening liver cancer inhibitor comprising the step of comparing the degree of β-catenin activity in the control group not in contact with the protein in contact with the test substance.

HBx, a protein of hepatitis B virus, is known to cause liver cancer by activating signaling pathways such as NF-kB, AP-1, and SRE through interactions with proteins present in host cells. One of these is that activation of the Wnt signaling pathway increases β-catenin concentrations and indeed β-catenin increases expression in 50-70% of hepatocellular carcinoma (Porfiri, E., B. Rubinfeld, et al. (1997). "Induction of a beta-catenin-LEF-1 complex by wnt-1 and transforming mutants of beta-catenin." Oncogene 15 (23): 2833-9; Suzuki, T., H. Yano, et al. (2002). "Beta-catenin expression in hepatocellular carcinoma: a possible participation of beta-catenin in the dedifferentiation process." J Gastroenterol Hepatol 17 (9): 994-1000; And Wong, CM, ST Fan, et al. (2001). "beta-Catenin mutation and overexpression in hepatocellular carcinoma: clinicopathologic and prognostic significance." Cancer 92 (1): 136-45). When the Wnt signaling pathway is not activated, β-catenin is typically phosphorylated by GSK3β and degraded by the ubiquitin complex, and APC plays an important scaffold role in phosphorylation by GSK3β with Axin (Morin, PJ, AB Sparks, et al. (1997). "Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC." Scienc e 275 (5307): 1787-90; and Satoh, S., Y. Daigo, et al. (2000). "AXIN1 mutations in hepatocellular carcinomas, and growth suppression in cancer cells by virus-mediated transfer of AXIN1." Nat Genet 24 (3): 245-50). However, no association with HBx, APC and β-catenin in the Wnt signaling pathway has been identified.

In the present invention, HBx, a regulatory protein of hepatitis virus, interacts with the host cell's Adenomatous Polyposis Coli (APC) protein as a binding partner, and as a result, interferes with the next step of APC and β-catenin, thereby preventing normal intracellular β- It is based on the discovery that it inhibits the breakdown of catenin and eventually increases its concentration. Increased β-catenin activates the Wnt signaling pathway and ultimately leads to liver cancer. HBx is a 17 KDa regulatory protein expressed by hepatitis B virus that activates the Wnt signaling pathway, but little is known about the mechanisms involving interaction with proteins present in host cells. Therefore, as shown in the present invention, by inhibiting the interaction of HBx and APC in the early stage of Wnt signaling, it is possible to suppress the increase of β-catenin concentration to inhibit the occurrence of liver cancer.

  Thus, in one embodiment, the present invention is a method for screening a substance that inhibits the interaction of HBx and APC, wherein the substance selected by the method is useful as a liver cancer inhibitor substance. The method comprises providing HBx and APC proteins; Contacting the protein with a test substance; And it provides a method for screening a substance that inhibits the interaction of HBx and APC comprising the step of comparing the degree of interaction of HBx and APC in a control group not in contact with the protein in contact with the test substance.

The HBx of hepatitis B virus is a 17 KDa protein consisting of 154 amino acid residues, which may be derived from various hosts depending on the purpose. There may also be sequence variations, depending on the particular individual, region, environment, etc., even if they are derived from the same host, for example humans, and of course, some sequences have been modified (deleted, substituted, added), but functionally equivalent variants All can be used in the present invention. In one embodiment, hepatitis B virus is derived from a human host. In other embodiments, HBx derived from humans include, but are not limited to, CAA49453, BAD90001, and P69713 (above NCBI protein database accession no.), And the like.

Adenomatous Polyposis Coli (APC) is a protein consisting of 2843 amino acid residues expressed in a variety of animals including humans. These various derived APCs can be used in the present method, and although some sequences thereof have been modified (deleted, substituted, added), all functionally equivalent variants can be used in the present invention. Examples include, but are not limited to, those derived from humans, mice, and rats. In one embodiment, human derived APC (Accession No .: MP_000029, Version NP_000029.2, GI: 53759122) protein is used.

As used herein, HBx and APC can be prepared using methods known in the art. In particular, the method for producing the screening protein is to use a genetic recombinant technology. For example, the plasmid containing the corresponding gene encoding the protein can be delivered to prokaryotic or eukaryotic cells such as insect cells and mammalian cells, overexpressed and purified. The plasmid can be used, for example, after transfection of mammalian cell lines with pCMV-HA / HBx as used in exemplary embodiments herein, but not limited thereto.

Or by expressing the DNA or RNA sequence encoding the screening protein in a suitable host cell to make the cell lysate or by translating the mRNA of the screening protein in vitro and purifying the screening protein by protein isolation methods known in the art. Can be. Usually, in order to remove cell debris and the like, the cell lysate or the result of in vitro translation is centrifuged, followed by precipitation, dialysis, and various column chromatography. Ion exchange chromatography, gel-permeation chromatography, HPLC, reverse phase-HPLC, preparative SDS-PAGE, affinity columns and the like are examples of column chromatography. Affinity columns can be made, for example, using anti-screening protein antibodies.

The contact of the purified proteins can be used directly in vitro to confirm binding in the absence or presence of the test substance. In this case, the protein may be labeled using a variety of markers such as biotin, fluorescent material, acetylation, radioisotopes, or commercially available protein labeling kits for ease of detection, and labeled materials. Can be detected using a detector suitable for. Protein-protein interactions can be measured using various methods known in the art. For example, the East to Hybrid method and the Confocal Microscopy method for confirming the binding / interaction of intracellular proteins. Co-immunoprecipitation, surface plasma resonance (SPR) and spectroscopy methods include, but are not limited to, further references on comparisons and detailed test methods for these methods. Berggard et al., (2007) "Methods for the detection and analysis of protein-protein interactions ", PROTEOMICS Vol7: pp 2833-2842.

In one embodiment, HBx and APC proteins may be provided in the form of cells expressing it. For example mammalian cells expressing (transient or stable) HBx and APC, such as, but not limited to, HepG2, Huh7, Hep3B, PLC / PRF / 5, 293T cells incubated in a cell culture plate, and then After the test substance is added to the cells, after a certain time, the total protein is extracted from the cells, for example, by co-immunoprecipitation and immunofluorescence to check the interaction of HBx and APC. Compared to the control (untreated test material), inhibiting the interaction of HBx and APC can be selected as a candidate for liver cancer inhibitors.

The amount of protein used in this method, the type and concentration of cells and the amount and type of test substance will vary depending on the specific test method and the type of test substance used, and those skilled in the art will be able to select an appropriate amount. As a result of the experiment, a substance which inhibits the interaction between HBx and APC protein in the presence of the test substance is selected as compared with the control group which is not in contact with the test substance. Less than about 99%, less than about 95%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60% Reduction, about 55% reduction, about 50% or less, about 45% or less, but does not exclude the extent to which it is displaced.

In the present invention, interaction refers to contact / binding between proteins, and includes both interactions by covalent and non-covalent bonds. In the present invention, "test substance" means a substance which is expected to inhibit the interaction (binding) of HBx protein and APC, and thus, low molecular weight compounds, high molecular weight compounds, nucleic acid molecules (eg, DNA, RNA, PNA and aptamers). ), Proteins, sugars and lipids, and the like. The test substance may be a polypeptide having two or more amino acid residues, such as six, ten, twelve, twenty or less, or more than twenty such as fifty amino acid residues. The protein may comprise an antibody against HBx or APC.

For the purpose of screening drugs for inhibiting, treating and / or preventing the occurrence of liver cancer, the compound may have a low molecular weight therapeutic effect. For example, compounds of about 1000 Da in weight such as 400 Da, 600 Da or 800 Da can be used. Depending on the purpose, such compounds may form part of a compound library, and the number of compounds constituting the library may vary from tens to millions. Such compound libraries include peptides, peptoids and other cyclic or linear oligomeric compounds, and small molecule compounds based on templates such as benzodiazepines, hydantoin, biaryls, carbocycles and polycycle compounds (such as naphthalene, phenoty) Azine, acridine, steroids, and the like), carbohydrate and amino acid derivatives, dihydropyridine, benzhydryl and heterocycles (such as triazine, indole, thiazolidine, etc.), but these are merely illustrative. It is not limited to this.

The method may further comprise the step of selecting a substance that reduces the concentration of β-catenin as compared to the control group untreated test substance when the interaction of HBx and APC in the cell. When HBx and APC interact, the next step, which is usually the case, is that the interaction of APC and β-catenin is interrupted, resulting in an increase in β-catenin concentration as described in the Examples herein. As mentioned above, β-catenin was increased in a large number of liver cancer tissues. Therefore, compounds that reduce the concentration of β-catenin can be selected as candidates for inhibiting liver cancer. The concentration of β-catenin is one that includes changes in mRNA and / or protein levels. Analytical methods for measuring mRNA levels are known in the art and include, for example, Reverse Transcription Polymerase Chain Reaction (RT-PCR), competitive RT-PCR, real-time RT-PCR, RNase protection assays, northern blotting, DNA chips Etc., but is not limited thereto. Such methods may be referred to as appropriate in the literature list described immediately preceding the examples herein.

Methods for measuring the concentration of proteins are also known, for example, reference may be made to the appropriate ones in the literature list immediately preceding the examples. Also, for example, a method using "antigen-antibody complex" formation between β-catenin protein extracted from cells and specific antibodies thereof, Western blot, Enzyme Linked ImmunoSorbant Assay (ELISA), radioimmunoassay, radioimmunoproliferation method, tissue immunostaining , Immunoprecipitation assay, complement fixation assay, FACS (Fluorescent Activated Cell Sorter), protein chips, and the like, but are not limited thereto. Through the above detection methods, the amount of β-catenin mRNA or protein expressed in the control group and the amount of expression expressed in the test group treated with the test substance were reduced and the candidates that can inhibit the interaction between HBx and APC. Selected by substance. Such materials may be useful as liver cancer inhibitors, in particular liver cancer inhibitors due to hepatitis B virus infection.

In another aspect, the present invention provides a method of preparing a cell comprising the steps of providing a cell expressing HBx and APC proteins; Contacting said cell with a test substance; And it provides a liver cancer inhibitor screening method comprising the step of measuring and comparing the concentration of β-catenin in a control group not in contact with the cell in contact with the test substance. As mentioned above, substances that inhibit the interaction of HBx and APC may be useful candidates for liver cancer inhibitors. Cell lines that can be used in the method are as described above, in particular, but not limited to animal cells, more particularly hepatocytes, even more particularly HepG2, Huh7, Hep3B, PLC / PRF / 5. The cell line was transfected with plasmids expressing HBx and plasmids expressing APC, or plasmids expressing both proteins, as described above, and then the total protein was extracted from the cells after a certain time, for example 48 hours. The concentration of β-catenin is measured and compared to a control not treated with the candidate. Compounds having a reduced β-catenin concentration compared to the control are substances capable of inhibiting liver cancer targeted in the present invention. The method of measuring the concentration of protein in the cell extract is as mentioned above. The amount of test substance depends on the specific test method used and the type of test substance, and a person skilled in the art can select an appropriate amount. As a result of the test, compared with the control group not in contact with the test substance, in the presence of the test substance, less than about 99% reduction, less than about 95% reduction, about 90% reduction, about 85% reduction, about 80% reduction, about 75% Decrease, about 70% decrease, about 65% decrease, about 60% decrease, about 55% decrease, about 50% decrease, about 45% decrease, but not beyond

In another aspect the present invention relates to a method for controlling the concentration of β-catenin in cells infected with hepatitis B virus using APC gene or protein. The method includes in vitro or in vivo concentration control. In one embodiment, in vitro regulation. In another embodiment, in vivo regulation. HBx inhibits the degradation of β-catenin by increasing the concentration of HBx by interfering with the interaction of APC with β-catenin at a lower level. Thus, overexpression of APCs in cells can inhibit this phenomenon as described in the Examples herein, resulting in a decrease in β-catenin concentrations compared to otherwise. As the APC gene or protein that can be used in the present method, those from various kinds as mentioned above can be used. In one embodiment, human derived genes or proteins can be used. In other embodiments, a gene is used, for example, a sequence such as human-derived NM_001127511.1, NM_001127510.1, or NM_000038.4 (above NCBI nucleic acid sequence database), which gene is amplified by a method such as PCR to eukaryotic cells. Can be cloned into an expression vector. Thereafter, known methods such as, but not limited to this, can be transfected into cells using liposomes and calcium phosphate methods to overexpress them. The concentration control refers to the regulation of the β-catenin gene transcription and / or translation level of the transcribed mRNA, in particular, the reduced β-catenin concentration compared to the negative control group (cells not overexpressing APC) it means.

In another aspect, the present invention also provides a method of preparing a β-catenin protein; Contacting the protein with a test substance; And it provides a method for screening liver cancer inhibitor comprising the step of comparing the degree of β-catenin activity in the control group not in contact with the protein in contact with the test substance. HBx activates the wnt signaling pathway to increase the concentration of β-catenin. This increase in β-catenin concentration and thus activation by HBx is inhibited by β-catenin inhibitors (see Example 4). Therefore, for the development of liver cancer inhibitors, in addition to hindering the interaction of HBx and APC in the previous stage, compounds that inhibit β-catenin in the later stage may also be usefully used as liver cancer inhibitors. The β-catenin gene or protein that may be used in the present method may be one derived from various animals. In one embodiment, a gene or protein derived from human can be used. In other embodiments, sequences derived from humans are used, such as CAA79497.1 (GenBank Accession) NP_001895.1 (NCBI reference no.). The method for purifying the expression of the protein and the test substance is as mentioned above. It is useful as a liver cancer inhibitor candidate to reduce β-catenin activity by comparing the degree of β-catenin activity in the control group not in contact with the protein in contact with the test substance. The degree of β-catenin activity may be determined by a plasmid comprising a promoter comprising a nucleic acid site to which β-catenin binds and a reporter gene (eg, luciferase, etc.) linked thereto, for example, as described in Examples 3 or 4 herein. It can be carried out by expressing it in eukaryotic cells using the method and measuring the activity of the reporter. Determination of the activity of a reporter can be assayed using known methods suitable for a particular reporter or methods such as those described in the Examples herein.

The invention may be practiced using conventional techniques that are within the skill of those skilled in the art of cell biology, cell culture, molecular biology, gene transformation techniques, microbiology, DNA recombination techniques, and immunology, unless otherwise noted. See also the following books and literature for a more detailed description of general techniques. For general methods of molecular biology and biochemistry, see Molecular Cloning: A Laboratory Manual, 3rd Ed. Sambrook et al., Cold Spring Harbor Laboratory Press 2001; Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. Eds., John Wiley & Sons 1999); DNA Cloning, Volumes I and II (Glover ed., 1985); Oligonucleotide Synthesis (Gait ed., 1984); Nucleic Acid Hybridization (Hames and Higgins eds. 1984); Transcription And Translation (Hames and Higgins eds. 1984); Culture Of Animal Cells (Freshney and Alan, Liss, Inc., 1987); Gene Transfer Vectors for Mammalian Cells (Miller and Calos, eds.); Current Protocols in Molecular Biology and Short Protocols in Molecular Biology, 3rd Edition (Ausubel et al., Eds.); And Recombinant DNA Methodology (Wu, ed., Academic Press). See also Gene Transfer Vectors For Mammalian Cells (Miller and Calos, eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al., Eds.); Immobilized Cells And Enzymes (IRL Press, 1986); Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (Weir and Blackwell, eds., 1986); Protein Methods (Bollag et al., John Wiley & Sons 1996); Non-viral Vectors for Gene Therapy (Wagner et al. Eds., Academic Press 1999); Viral Vectors (Kaplitt & Loewy eds., Academic Press 1995); Immunology Methods Manual (Lefkovits ed., Academic Press 1997); And Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998). For general techniques regarding cell culture and medium, see Large Scale Mammalian Cell Culture (Hu et al., Curr. Opin. Biotechnol. 8 (1997), 148); Serum-free Media (Kitano, Biotechnology 17 (1991), 73); Large Scale Mammalian Cell Culture (Curr. Opin. Biotechnol. 2 (1991), 375); And Suspension Culture of Mammalian Cells (Birch et al., Bioprocess Technol. 19 (1990), 251).

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example  One HBx Identification of protein of host cell that interacts with

(One) HBx Activation site determination

In order to identify proteins that interact with HBx and activate signaling pathways, a transactivation site of HBx was first determined. As shown in FIG.

Comparison of HBx sequences of woodchuck (WHBV), ground squirrel (GSHV), and human hepatitis B virus, derived from human sequences lacking the 12-142 amino acid residues at the C-terminal region where the conserved sequence was concentrated Mutant pCMV-HA / HBx Δ126-142, pCMV / HA / HBxΔ71-100 lacking amino acid residues 71-100, pCMV-HA / HBxC137T with 137 th cysteine substituted with threonine, wild type PCMV-HA / HBX [plasmids comprising human HBx sequences and specific PCR methods are described in Lee et al. (2005). "The hepatitis B virus X protein up-regulates lymphotoxin alpha expression in hepatocytes." PCR (Polymerase Chain Reaction, conditions 95 ° C 10sec, 55 ° C 30sec, 72 ° C 60sec, 30 cycles total), AmpliTaq Gold (Applied Biosystems, USA) PCMV-HA was purchased from Clonetech, Inc. Primers were as follows:

pCMV / HA / HBx Δ71-100 : Forward 5'-TCGGACGTTGACATTGCTGAAAGTGCACACGGTCCGGCAG-3 'and Reverse 5'-CTGCCGGACCGTGTGCACTTTCAGCAATGTCAACGTCCGA-3';

pCMV / HA / HBx Δ126-142 : Forward 5'-TTGCATGGTGCTGGTGAACACTCCTCCCCCAACTCCTCCC-3 'and Reverse 5'-GGGAGGAGTTGGGGGAGGAGTGTTCACCAGCACCATGCAA-3';

pCMV / HA / HBxC137T : Forward 5'-TCTTTGTACTAGGAGGCACCAGGCATAAATTGGTCTG-3 'and

Reverse 5'-CAGACCAATTTATGCCTGGTGCCTCCTAGTACAAAGA-3 '.

The function, expression level and intracellular location of the prepared mutants were analyzed as follows. First, 2 μg of the prepared plasmid was expressed using Fugene (Roche, USA), and the promoter having the NF-kappaB, SIE, and SRE sites expresses the plasmid and Renilla luciferase linked to the luciferase gene according to the manufacturer's method. Transfected into Huh7 cells containing the plasmid. The Huh7 cells are described in Kim et al., (2001). "Through induction of juxtaposition and tyrosine kinase activity of Jak1, X-gene product of hepatitis B virus stimulates Ras and the transcriptional activation through AP-1, NF-kappaB, and SRE enhancers. "Biochem Biophys Res Commun 286 (5): 886-94). For the analysis, 12-well plates were used for seeding at 0.3 × 10 ⁶ cells per well, and the medium was used by adding 10% fetal bovine serum (JBI, Daegu, Korea) to Dulbecco's modified eagle's medium (Life Technologies Inc. USA). . After incubation at 5% CO ₂ , 37 ° C., cells were disrupted after 48 hours and luciferase activity was analyzed using the Dual Luciferase Reporter Assay System (Promega, USA) as recommended by the manufacturer. Luciferase luminescence was read using a Victor2 luminometer (Millipore, Billerica, Mass.). Renilla was used to normalize luciferase activity.

In Western blots, after 48 hours, cells were disrupted with RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% DOC (deoxycholic acid), 0.1% SDS, 50 mM Tris pH 8.0) to form a total protein fraction. 100 μg of the solution was electrophoresed on an 8% SDS / PAGE gel, and then the protein in the gel was transferred to the PVDF membrane (Millipore, USA) as recommended by the manufacturer, followed by use of an anti-HA antibody (ab9110 Abcam, UK, 1: 500 dilution). ) And β-actin (AC-15 Sigma, USA, using 1: 500 dilution) for 48 hours, followed by Western blot using the Western Blot Coloring Kit (Thermo Scientific Pierce, ECL Substrate) as recommended by the manufacturer. Color development was performed. As shown in FIG. 2A, it can be seen that the HBx protein having a Δ126-142 region deficient has a function of activating a promoter at a negative control level. Western blot pictures directly below Figure 2a shows that the mutants are expressed at the wild type level in cells, indicating that the promoter activity is not due to decreased protein expression.

(2) HBx Interact with In host cells  Protein Identification

For this purpose, proteins that bind to them differentially were selected using a Tandem Affinity Purification (TAP) system using HBx and HBxΔ126-142 proteins as described in FIG. 2B. Subsequently, mass spectrometry and database searches were performed to identify selected proteins. TAP is an experimental technique for analyzing protein-protein interactions. Puig, O., et al. (2001). "The Tandem Affinity Purification (TAP) Method: A General Procedure of Protein Complex Purification". It was performed according to the method described in Methods 24: 218-229. The experiment was repeated three times independently. In summary, for this purpose, each of the plasmids of HBx and HBxΔ126-142 of (1) was cloned into an EcoRI site of pIRES / EGFP (Clontech, USA), whereby a triple epitope or S-Flag-SBP- (S protein, Flag, Expression plasmids tagged with Streptavidin Binginding Protein) were constructed (pSFS-HBx and pSFS-HBxΔ126-142). After each plasmid was purified using a Qiagen plasmid purification kit, 2 μg of each was transfected into Huh7 cells as described in (1). After 48 hours, cells were treated with NTEN disruption buffer containing phosphatase (150 mM NaCl, 20 mM Tris-HCl, pH7.4, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM NaF, 1 mM Na).₃VO₄, And 50 μg / ml PMSF). The lysate was then reacted with 100 μl of S-protein and Streptavidin-Sepharose 6 Fast Flow resin (Amersham Biosciences) for 1 hour, washed three times with 1 ml TBS (Tris Buffered Saline), and then 500 μl of TEV buffer ( Invitrogen, USA). The tagged protein was isolated from the beads by reaction overnight at 4 ° C. using 50 U TEV buffer in a total volume of 200 μl. Proteins complexed with HBx and HBxΔ126-142 of the supernatant were then reduced and alkylated and then treated with trypsin overnight (Kirkpatrick, DS, SF Weldon, et al. (2005). "Proteomic identification of ubiquitinated proteins from human cells expressing His-tagged ubiquitin. "Proteomics 5 (8): 2104-11). Peptides were analyzed using MS / MS (Finnigan LTQ, Thermo Fisher Scientific, Inc. Waltham, Mass.) As recommended by the manufacturer. MS / MS results were searched for protein databases using SEQUEST Cluster (Thermo Fisher Scientific, Inc). As a result of the database search, a human APC (adenomous polyposis coli) protein (Accession No .: MP_000029, Version NP_000029.2, GI: 53759122) consisting of 2843 amino acid residues was identified. . All three experiments detected the same protein.

(3) HBx Wow APC Confirm interaction of

In order to confirm the interaction between the identified APC and HBx, a co-immunization experiment was performed to immunoprecipitate AH protein in Huh7 cells using HBx and HBxΔ126-142 proteins tagged with Flag. To this end, Huh7 cells were transfected with p3xFlag / CMV / HBx and P3xFlag / CMV / HBxΔ126-142, respectively, as in (1). Plasmids p3xFlag / CMV / HBx and p3xFlag / CMV / HBxΔ126-142 were constructed by cloning the HBx portion of the plasmid prepared in (1) with EcoRI and cloning into p3xFlag / CMV (Sigma, USA). Cells transfected with each plasmid were incubated for 48 hours as described in (1) and then washed twice with 4 ° C. PBS (pH 7.4) to collect the cells, and 300 μl into cells in one well of a 12 well plate. Lysis buffer NETN buffer (0.5% Nonidet P-40, 20mM Tris (pH 8.0), 50mM NaCl, 50mM NaF, 100 mM Na ₃ VO ₄ , 1mM DTT and 50μg / ml PMSF) Leave at 4 ° C. for 10 minutes. Then it was centrifuged for 5 minutes at 4 ℃ 14000 rpm. The supernatant was then used with anti-Flag (using M2, Sigma, 1: 500), anti-APC (using Ali12-28, Santa Cruz, USA, 1: 200) and mouse IgG (Santa Cruz) as negative control, FLAG-tagged protein immunoprecipitation kits were analyzed using the manufacturer's instructions. In summary, 100 μl of Flag M2 agarose beads (Sigma) and the prepared supernatant were mixed and incubated with gentle successive mixing at 4 ° C. for 2 hours. After centrifugation at 9000 rpm for 30 seconds, the beads were washed three times with the buffer contained in the kit, the protein complexes attached to the beads were separated, and then boiled for 5 minutes in a loading buffer (Protein 2x, Elpis Biotech), followed by SDS. Electrophoresis on / PAGE gel. Thereafter, the electrophoresis gel was transferred to the membrane as described in (1), and then Western blot was performed using the antibody described in FIG. 2C. The result is in FIG. 2C. As shown in the upper photo in FIG. 2C, the APC protein is immunoprecipitated by binding to HBx, but not immunoprecipitated by HBxΔ126-142 (photo below). This confirms the interaction of HBx and APC. In all the figures below, IP represents an immunoprecipitating antibody, and IB represents Immunoblot (an antibody used in western blot).

Subsequently, the two proteins were located at the same site in the cell using immunofluorescence. Experiments were performed as described in Park et al., Cancer Letters 282 (2009) 229-237. In summary, after transfecting Huh7 cells with each pCMV / HA / HBx and pEGFP / APC, 48 hours later, the cells (0.05 × 10 ⁶ cells / coverslip) were transferred to poly-L-lysine coated coverslips. Thereafter, 4% paraformaldehyde was fixed, followed by blocking with 1% BSA by increasing permeability with 0.2% triton-containing phosphate buffer. Then incubated with the anti-HA antibody (1: 100) and anti-APC (1: 100) used for 2 hours at 4 ° C, followed by washing twice with PBS, followed by FITC conjugates produced in rabbits as secondary antibodies. Stained with anti-mouse IgG antibody (Sigma, USA, 1: 100) (APC) or rhodamine conjugated anti-rabbit IgG antibody (Chemicon, USA, 1: 100) (HBx) produced in goats, and then Washed twice with PBS and then counterstained (blue) using DAPI (Invitrogen, USA) as instructed by the manufacturer. Observation was performed at fluorescence microscope (Olympus, USA) at x1000 magnification. The result is in FIG. 2D. As shown in FIG. 2D, APC (lime green) and HBX (red) are both located at the same site in the cell, and can be more clearly seen when merged. Furthermore, the sites deleted in HBxΔ126-142 are only involved in binding to APC and are not related to intracellular location.

Example  2: HBx Wow APC  By interaction APC And β- With catenin  Coupling interference

Example 1 using anti-APC and anti-β-catenin antibodies in the presence of HBx or HBxΔ126-142 to determine whether the binding of APC and β-catenin is inhibited by HBx and APC interactions Coimmunoprecipitation experiments were performed as described. In summary, 293T cells (CRL-11268, ATCC) were treated with 37% 5% CO by adding 10% fetal bovine serum (JBI, Daegu, Korea) to RPMI medium with 10% fetal bovine serum (Life Technologies Inc. USA). Incubated at ₂ . After transfection with pCMV / HA / HBx and pCMV / HA / HBx Δ126-142 (see Example 1), lysate was prepared from the cells 48 hours later to prepare anti-APC and anti-β-catenin antibodies ( C-18, Santa Cruz), followed by treatment with goat anti-mouse IgG (using Santa Cruz, 1: 200) conjugated to agarose beads for 1 hour, followed by centrifugation of the beads. After collection, add SDS / PAGE sample buffer (100 mM Tris, pH 6.8, 2% SDS, 5% ß- mercaptoethanol, 15% glycerol), boil for 5 minutes, take supernatant and separate with 8% SDS / PAGE gel Western blots were then performed as described in Example 1 with the antibodies described in FIG. 3A. The results are described in FIG. 3A. As shown in Figure 3a, it can be seen that when HBx is present in cells, immunoprecipitates APC, thereby reducing the amount of β-catenin that binds to APC, and conversely, when immunoprecipitating β-catenin, β-catenin It can be seen that the amount of APC bound to is also reduced. HBxΔ126-142, on the other hand, had no effect on the interaction of APC with β-catenin. This demonstrates that the specific interaction between HBx and APC prevents β-catenin from binding to the APC / Axin / GSK3 disruption complex.

In order to determine the site where APC binds to HBx, the following experiment was performed. See previously described (Seo, E. and EH Jho (2007). "Axin-independent phosphorylation of APC controls beta-catenin signaling via cytoplasmic retention of beta-catenin." Biochem Biophys Res Commun 357 (1): 81-6. A full length APC or plasmid lacking some or all of the β-catenin binding site of APC was used to determine whether it was identical to the HBx binding site. APC plasmid with a portion of the myc tagged full length cut as described in Seo et al, and p3xFlag / CMV / HBx as described in Example 1. The plasmid was transfected into 293T cells as described above and after 24 hours the cell lysates were extracted using NTEN buffer followed by immunoprecipitation with anti-Flag (HBx) antibody followed by electrophoresis as described above. , Western blot was performed with the antibody described in Figure 3b (anti-Myc antibody is 9E10, manufactured by Santa Cruz). As shown in FIG. 3B, HBx was able to bind to APC only when it included at least two of at least three 15-amino acid residue repeats (APC 21 and APC 2). This indicates that the binding site of HBx and APC overlaps with the binding site of APC and β-catenin, which indicates that HBx structurally interferes with APC / β-catenin interaction (see also Example 3). In addition, there was no change in the amount of protein expression of APC (Data not shown).

Example  3: HBx Wow APC By the interaction of In liver cancer cell line  β- Catenin  Increase in concentration

Hepatocarcinoma cell lines Hep3B (ATCC HB 8064) and PLC / PRF / 5 (ATCC CRL-8024) were cultured identically to Huh7, and each of these cells was treated with pCMV / HA / HBx and pCMV / HA / HBx Δ126-142 (Example 1) After 48 hours of transfection in each of the methods of Example 1, immunofluorescence staining was performed as described in Example 1 (3). In summary, the cells were washed twice with PBS and then fixed with 4% paraformaldehyde. Cells were then stained using anti-HA (HBx) and anti-β-catenin antibodies and the secondary antibodies described in Example 1 and DAPI (nuclear staining). The result is in FIG. 3C. The number of cells expressing HBx (cytoplasmic) (green) and β-catenin (nucleus) (red) simultaneously was measured under a fluorescence microscope (Olympus, USA) at x1000 magnification. In one experiment, 300 cells were observed, and as a result of three independent experiments, the number of simultaneously expressed cells was measured as a percentage, and the results are shown as their average and standard deviation (Table 3C). pCMV / HA / HBx Δ126-142 both expressed HBx and β-catenin in less than 10% of cells, whereas pCMV / HA / HBx expressed HBx and HBx in 45%, 56% and 61% of cells. It was shown to express both β-catenin. From the results, it can be seen that the expression of HBx in liver cancer cell lines leads to an increase in the concentration of β-catenin.

In order to confirm this again, after transfection in the same manner as described above, after 48 hours, the nucleus, cytoplasm and whole cell fractions were extracted from the cells according to the manufacturer's recommendation using the Nuclear Extration kit (Active Motif, USA), Western blot was performed as described in Example 1 using the antibody described in FIG. 3D. Expression of HBx resulted in an increase in β-catenin concentration only in the nuclear extract in which β-catenin is located in all the liver cancer cell lines used, and HBx Δ126-142 did not result in an increase in β-catenin concentration as predicted. Anti-nucleophosmin, anti-β-actin, anti-HA did not change in any fraction, in any cell, as expected for loading control. This is a result consistent with FIG. 3C.

Example  4: HBx  And APC By the interaction of Wnt  Activation of the signaling pathway

Evidence of Wnt / β-catenin signaling pathway activation is the migration of β-catenin from the cytoplasm to the nucleus. Catenin interacts with TCF / LEF in the nucleus to promote expression of target proteins of Wnt signaling such as c-MYC (He, TC, AB Sparks, et al. (1998). "Identification of c-MYC as a target of the APC pathway. "Science 281 (5382): 1509-12; and Segditsas, S., and Tomlinson, I. (2006). Colorectal cancer and genetic alterations in the Wnt pathway. Oncogene 25, 7531-7537). Therefore, we investigated the activity of β-catenin promoter by HBx expression to see whether HBx binding to APC interacting with β-catenin is involved in gene activation of Wnt signaling pathway.

First, for this purpose, a plasmid containing a reporter regulated under a promoter regulated by TCF / β-catenin (pTOPFLASH) and a pFOPFLASH containing a mutation in the promoter were used. The plasmid used was described in Chen et al., (2201) The Journal of Cell Biology, Volume 152, Number 1, 87-96. Each of the plasmids was transfected with pTOPFLASH, or pFOPFLASH, and a Renilla plasmid (Example 1) in each of the Huh7, Hep3B and PLC / PRF / 5 cell lines. After 48 hours the cells were disrupted and the activity of the promoter was determined by assaying luciferase activity using the Dual Luciferase Reporter Assay System (Promega, USA) as recommended by the manufacturer. The results are described in FIG. 4A. pCMV / S37A β-catenin expresses the activated form of β-catenin and is a positive control. As shown in Figure 4a, HBx was found to increase the expression of genes involved in the Wnt signaling pathway (from the activity of the promoter) to the positive control level. HBxΔ126-142, on the other hand, did not, indicating that the binding of HBx with APC is important for wnt signaling activity.

Furthermore, we investigated whether signaling pathway activity was increased by increasing the amount of HBx expression. To this end, the cell lines described in FIG. 4B were each transfected with pTOPFLASH and Renilla plasmid with 0 μg, 0.5 μg, 1 μg, 1.5 μg of pCMV / HA / HBX or pCMV / HA / HBXΔ126-142 plasmid, respectively. Example 3) Luciferase activity was measured in the same manner as in FIG. 4A. The results are shown in Figure 4b, it can be seen that as the amount of HBx increases luciferase activity, it was not observed in HBxΔ126-142. This is consistent with the result of FIG. 4A.

In addition, in the case of expressing APC simultaneously with HBx in liver cancer cell lines, the Wnt signaling pathway was not activated. PCMV / HA / HBX or pCMV / HA / HBXΔ126-142 was combined with the full-length APC (FL-APC) or APC mutant construct (see Example 2) in the cell line described in FIG. 4C in the same manner as above. Luciferase activity was measured after transfection. In FIG. 4D, luciferase activity was measured when transfected with full-length APC. Unlike the expression of mutant APCs as shown in FIGS. 4C and 4D, it can be seen from the reduced promoter activity that the Wnt signaling pathway is not activated only when wild-type full-length APCs are expressed with HBx, which is the APC used. It was dependent on the amount of (Fig. 4d).

We also investigated whether β-catenin inhibitors could block HBx activity. pCMV / HA / HBX was applied to Huh7 cells as in Example 1 either transient transfection (FIG. 5A-left) or permanent transfection (FIG. 5A-right) (Gu, JM, SO Lim). , et al. (2008), "HBx modulates iron regulatory protein 1-mediated iron metabolism via reactive oxygen species." Virus Res 133 (2): performed as described in 167-77), followed by a β-catenin inhibitor After addition of 10 μM curcumin (Sigma) and 50 μM quercetin (Sigma), the relative promoter activity for pTOPFLASH was investigated as above. As shown in FIG. 5A, the presence of the two inhibitors showed that the activity of the promoters by HBx was remarkably separated as compared with the case without treatment with the two inhibitors. This shows that the Wnt signaling pathway activity caused by the expression of HBx is reduced by β-catenin inhibitor treatment, indicating that the Wnt signaling pathway triggered by HBx is transmitted through β-catenin. In addition, the inhibition of β-catenin alone can inhibit HBx-induced liver cancer.

Subsequently, the amount of transcription of c-Myc known to increase expression and E-cadherin known to decrease in the Wnt signaling pathway was confirmed by real-time PCR. RNA was extracted from the permanently transfected cells using Trizol (Invitrogen, USA) according to the manufacturer's instructions, and then 1 μg of RNA was converted to AMV reverse transcriptase (Promega, USA) using oligo-dT (Promega, USA). After reverse transcription as directed by the manufacturer, real-time quantitative PCR (ABI 7300, Applied Biosystems, USA) analysis was performed. The primers used were: 5'GGACGACGAGACCTTCATCAA-3'sense) and 5'CCAGCTTCTCTGAGACGAGCTT-3 '(anti-sense) for c-MYC; 5'GTCACTGACACCAACGATAATCCT-3 '(sense) and 5'TTTCAGTGTGGTGATTACGACGTTA-3' (anti-sense) for E-cadherin; 5'ATCATCCCTGCCTCTACTGG-3 '(sense) and 5'TGGGTGTCGCTGTTGAAGTCAG-3' (anti-sense) for GAPDH. The results are shown in Figure 5b, it can be seen that their transcription is inhibited in the presence of curcumin and quercetin. This indicates that the signal triggered by HBx is transferred to β-catenin, c-Myc and E-cadherin through the Wnt signaling pathway.

The c-Myc and E-cadherin RNA concentration changes were consistent with protein concentrations. The cells used in the real-time quantitative PCR analysis were treated with RIPA buffer to extract the total protein, and then c-Myc (9E10, Santa Cruz) and E-cadherin (36 / E-cadherin, BD Biosciences, UK) as in Example 1 Western blot was performed using antibodies against. The result is in FIG. 5C.

Furthermore, Wnt / β-catenin signaling pathway activity triggered by HBx was confirmed in vivo using mice. Homozygous HBx transgenic mice (Yoo, YG, SH Oh, et al. (2003). "Hepatitis B virus X protein enhances transcriptional activity of hypoxia-inducible factor-1 alpha through activation of mitogen-activated protein kinase pathway. '' J Biol Chem 278 (40): 39076-84; Yoo, YG, TY Na, et al. (2008). "Hepatitis B virus X protein induces the expression of MTA1 and HDAC1, which enhances hypoxia signaling in hepatocellular carcinoma cells. "Oncogene 27 (24): 3405-13; and Yu, DY, HB Moon, et al. (1999)." Incidence of hepatocellular carcinoma in transgenic mice expressing the hepatitis B virus X-protein. "J Hepatol 31 (1) : 123-32), the liver tissue was extracted after the time indicated in FIG. 5, and the protein was extracted therefrom (Yoo et al., 2008). Western blot was performed. Expression of c-MYC in the Wnt signaling pathway is increased and expression of E-cadherin is known to be inhibited (Jung, JK, HJ Kwun, et al. (2007). "Hepatitis B virus X protein differentially affects the ubiquitin-mediated proteasomal degradation of beta-catenin depending on the status of cellular p53. "J Gen Virol 88 (Pt 8): 2144-54). Where to buy antibodies is as follows: MYC (9E10, Santa Cruz), E-cadherin (36 / E-cadherin, BD Biosciences, Oxford, UK), β-catenin (E-5, Santa Cruz), p (phosphorylated) β-catenin (9561, Cell Signaling, USA), GSK3β (27C10, Cell Signaling), and pGSK3β (9336, Cell Signaling). X represents transgenic mouse and WT represents wild type. As shown in FIG. 5, the expression of HBx results in an increase in the total amount of β-catenin protein, thereby reducing the phosphorylated degraded form of β-catenin, which activates the wnt signaling pathway to activate c- This led to an increase in MYC and a decrease in E-cadherin. β-actin is used for the titration of protein amount.

From the above results, HBx, a protein of hepatitis B virus, increases concentration by inhibiting β-catenin degradation through interaction with APC in host cells, and increased β-catenin increases liver cancer through Wnt signaling pathway activity. Generates. Therefore, based on the mechanism disclosed in the present invention, a compound capable of screening a compound that inhibits / inhibits the interaction between HBx and APC, or directly modulates the activity of β-catenin or the regulation of the wnt signaling pathway activated by HBx May be effectively used as a treatment for liver cancer.

Figure 1 shows the activation site of HBx protein and the conserved sequence HBx amino acids 71-100 and 126-142 site in three types of hepatitis B virus. Conserved 137th amino acid residue is indicated in yellow. Abbreviations are: SIE: stat inducible enhancer, RSV: Rous sarcoma virus-long terminal repeat, NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells, AP1: AP1 enhancer, FR: Gal4 Binding element , Cp: HBV core promoter, Xp: HBV X gene promoter. GSHV: ground squirrel hepatitis virus, WHBV: woodchuck hepatitis B virus, *: conserved amino acid.

Figure 2a is the result of measuring the transcriptional activity of the HBx protein missing a specific site shown in Figure 1.

2B schematically illustrates a tandem affinity tablet analysis.

Figure 2c is a result of analyzing the binding of the proteins APC and HBx selected by the tandem affinity analysis by immunoprecipitation method.

2D is the result of immunofluorescence assay showing intracellular locations of APCs and HBx.

Figure 3a is the result of immunoprecipitation analysis showing that the interaction of HBx and APC interferes with the binding of APC and β-catenin.

Figure 3b is the result of immunoprecipitation analysis showing that the site of APC interacting with HBx overlaps with the binding site with β-catenin.

Figure 3c is the result of immunofluorescence showing the increase in the concentration of β-catenin by the expression of HBx.

Figure 3d is a result of Western blot by cell fraction showing the increase of β-catenin concentration by the expression of HBx.

4A is a result showing that the expression of HBx leads to the activity of genes involved in the Wnt signaling pathway.

4B is a result showing that the phenomenon of FIG. 4A is dependent on the amount of HBx.

4C and 4D show that overexpression of wild-type full-length APC inhibits gene activity of the Wnt signaling pathway by HBx (4d) but not mutant APC.

5A shows that HBx acts through β-catenin as a result showing that the Wnt signaling pathway activity by HBx expression is reduced by β-catenin inhibitor treatment.

5B is a real-time PCR result showing that expression at the RNA level of genes involved in the Wnt signaling pathway by HBx expression is affected by β-catenin inhibitor treatment.

5C is a Western blot showing that expression at the protein level of genes involved in the Wnt signaling pathway by HBx expression is affected by β-catenin inhibitor treatment.

5D is a Western blot result of observing the activity of genes involved in the Wnt signaling pathway by HBx expression in liver tissue of HBx transgenic mice.

Claims (10)

A method for screening a substance that inhibits the interaction of HBx with APC, comprising the following steps: Providing HBx and APC proteins; Contacting the protein with a test substance; And  Comparing the interaction of HBx with APC in a control group not in contact with the protein in contact with the test substance. The method of claim 1, wherein the HBx and APC are provided in the form of cells expressing it. The method of claim 2, further comprising selecting a substance that reduces the concentration of β-catenin compared to the control. The method of claim 2, wherein the cells are HepG2, Huh7, Hep3B or PLC / PRF / 5. Providing a cell expressing HBx and APC proteins; Contacting said cell with a test substance; And  A method for screening liver cancer inhibitors comprising measuring and comparing the concentrations of β-catenin in control cells not in contact with cells in contact with the test substance.  The method according to claim 5, wherein the method further comprises the step of selecting a substance that reduces the concentration of β-catenin as a liver cancer inhibitor compared to the control group. The method of claim 5, wherein the liver cancer is caused by hepatitis B virus infection. The method of claim 5, wherein the cells are HepG2, Huh7, Hep3B or PLC / PRF / 5. delete providing a β-catenin protein; Contacting the protein with a test substance; And  Liver cancer inhibitor screening method comprising comparing the degree of β-catenin activity in the control group not in contact with the protein in contact with the test substance.

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