KR101075848B1 - Markers for Screening Inhibitors of HDAC - Google Patents

Abstract

The present invention uses genes that are up- or down-regulated in cancer cell lines treated with apicidin, which is a representative histone deacetylase (HDAC) inhibitor, as markers for discovering new HDAC inhibitors. The present invention relates to a kit for screening an HDAC inhibitor comprising an agent for measuring the expression level of the markers at the mRNA or protein level, and a method for screening the HDAC inhibitor using the same.

In recent years, the HDAC inhibitor has excellent anticancer effects and fewer side effects, so that the screening kit and method of the present invention can be advantageously used to discover a new HDAC inhibitor.

HDAC inhibitors, screening, biomarkers, apicidin

Description

Markers for Screening Inhibitors of HDAC}

The present invention relates to HDAC inhibitor screening, and more specifically, a kit for HDAC inhibitor screening comprising a marker for screening HDAC inhibitor, an agent measuring the expression level of the markers at the mRNA or protein level, and screening the HDAC inhibitor using the same. It is about a method.

Abnormal gene regulation plays an important role in tumor initiation and progression, and methods designed to correct this dysfunction will constitute a new anticancer strategy (Johnstone RW. Nat Rev Drug Discov 2002; 1: 287-99). Acetylation of lysine residues in histone proteins of nucleosomes is known to play an important role in chromatin structure and gene transcription (Cheung WL et al. Curr Opin Cell Biol 2000; 12: 326-33) . It is known that a family of histone deacetylases (HDACs) and histone acetyltransferases (HATs) is involved in the regulation of gene expression by determining histone acetylation (Johnstone RW et al. Cancer Cell 2003; 4: 13-8). Recently, HDAC inhibitors have emerged as excellent chemotherapeutic agents. The inhibitor can induce antitumor activity including induction of cell cycle arrest, promotion of differentiation and induction of apoptosis (Johnstone RW et al. Cancer Cell 2003; 4: 13-8; Marks PA et al. Curr Opin Oncol 2001; 13: 477-83). For example, suppression of tumor suppressor genes is known to promote carcinogenesis (Wolffe AP et al. Oncogene 2001; 20: 2988-90) and multi-proteins comprising DNA binding proteins The complex uses HDACs to inhibit transcription and block the function of tumor inhibitors. Furthermore, HDAC inhibitors can desuppress these genes, which cause anti-proliferative and in vivo anti-tumor effects in vitro (Johnstone RW et al. Nat Rev Drug Discov 2002; 1: 287-99, Kristeleit R et al. Expert Opin Emerg Drugs 2004; 9: 135-54, He LZ et al. J Clin Invest 2001; 108: 1321-30, Garcia-Manero G et al. Blood 2006; 108: 3271-9). However, the characteristic function of HDAC is that it is involved in the regulation of gene expression through transcriptional regulation.

HDACs are known to play an important role in the regulation of gene transcription and thereby participate in important biological processes during cell proliferation, differentiation and survival (Marks PA et al. Curr Opin Oncol 2001; 13 :: 477-83, Kouzarides T. Curr Opin Genet Dev 1999; 9: 40-8, Wade PA et al. Hum Mol Genet 2001; 10: 693-8). Despite no clear evidence that HDAC mutations are directly related to human type I and II cancers, it has been reported that HDAC is associated with several well-known oncogenic genes and tumor suppressors (Kristeleit R et. al. Expert Opin Emerg Drugs 2004; 9: 135-54. In conclusion, HDAC inhibitors have recently emerged as good chemotherapeutic agents, and some studies have shown antitumor including cell cycle arrest, differentiation promotion and apoptosis induction in a variety of transformed cells and tumors in which the inhibitor is cultured. It is proposed to induce activity (Johnstone RW et al. Cancer Cell 2003; 4: 13-8; Marks PA et al. Curr Opin Oncol 2001; 13 :: 477-83). In addition, unlike conventional chemotherapeutic agents that cause DNA damage in tumors and normal tissues, HDAC inhibitors are potent selectivity and less toxic to normal tissues (Johnstone RW et al. Cancer Cell 2003; 4: 13-8).

Therefore, as a new anticancer agent with excellent anticancer effect and few side effects, it is required to discover and develop HDAC inhibitors.

The present inventors identified genes that are up- or down-regulated as a result of treating breast cancer cell lines with apisidine, a representative HDAC inhibitor, and confirmed that new HDAC inhibitors can be identified by using the identified genes as markers. The invention has been completed.

One object of the present invention is to provide a marker for screening HDAC inhibitors at least one selected from the genes listed in Table 1.

Another object of the present invention is to provide a kit for HDAC inhibitor screening comprising an agent for measuring the expression level of one or more HDAC inhibitor screening markers selected from the genes listed in Table 1 at the mRNA or protein level.

Another object of the present invention, (1) treating the cancer cell line with the HDAC inhibitor candidate, (2) at least one selected from the genes listed in Table 1 between the cancer cell line treated with the candidate material and the untreated cancer cell line It is to provide an HDAC inhibitor screening method comprising the step of comparing the expression level of the marker for HDAC inhibitor screening.

The present invention relates to a marker capable of screening a histone deacetylase (HDAC) inhibitor consisting of the 625 genes listed in Table 1. In the present invention, 'markers' for screening HDAC inhibitors are 625 genes identified as being up- or down-regulated in breast cancer cell lines treated with the representative HDAC inhibitor 'apicidin', which is clearly described in Table 1.

Apicidine [cyclo (N-O-methyl-L-tryptophanyl-L-isoleucinyl-

L-2-amino-8-oxodecanoyl) is a potent and universal inhibitor of HDAC and has been reported to exhibit antitumor activity (eg, inhibit cell proliferation). In addition, this antitumor activity has been observed in vitro against other cancer cell lines (Darkin-Rattray SJ et al. Proc Natl Acad Sci USA 1996; 93: 13143-7; Han JW et al. Cancer Res 2000; 60: 6068-74). ^Apicidine , like other HDAC inhibitors, was first described as an anti-tumor agent that inhibits HeLa cell growth by inducing p21 ^{WAF1 / Cip1} (Han JW). et al. Cancer Res 2000; 60: 6068-74).

Genes that are up or down regulated in cancer cell lines treated with apisidine, established as representative HDAC inhibitors, will be similarly up or down regulated in cancer cell lines treated with another HADC inhibitor. Thus, the genes listed in Table 1 can be used to discover new HDAC inhibitors. In other words, between the cancer cell line treated with the HDAC inhibitor candidate and the untreated cancer cell line, the expression level of at least one of the genes listed in Table 1 was measured and compared at the mRNA or protein level to identify a significant change to identify a new HDAC inhibitor. Can be.

In the present invention, 'HDAC inhibitor candidate' or 'candidate' refers to any substance (eg, metal, compound, nucleic acid, protein, sugar, lipid, etc.) to be identified as having activity as an HDAC inhibitor. .

In the present invention, the 'cancer cell line' refers to cancer cells derived from breast cancer, cervical cancer, stomach cancer, liver cancer, lung cancer, pancreatic cancer, bronchial cancer, ovarian cancer, nasopharyngeal cancer, laryngeal cancer, bladder cancer, colon cancer, and the like. It is not limited.

The present invention does not specifically limit the number and combination of markers selected from Table 1 for HDAC inhibitor screening. That is, according to the purpose of discovering the HDAC inhibitor, a single gene may be used as a marker, or 2 to 625 plurality of genes may be selected in various combinations.

In one embodiment, the marker for screening HDAC inhibitors of the present invention may comprise genes that are upregulated among the genes listed in Table 1. Preferably, it may include genes that are up-regulated more than two times among the genes listed in Table 1. The 'up-regulated markers' are upregulated genes compared to untreated breast cancer cell lines in breast cancer cell lines treated with apisidine. In Table 1 upregulation is indicated with a (+) sign, corresponding to 433 genes. If the candidate is an HDAC inhibitor, it is expected to downregulate the expression of these genes in cancer cell lines, similarly to apisidine.

In another embodiment, the marker for screening HDAC inhibitors of the present invention may comprise genes that are down regulated among the genes listed in Table 1. Preferably, the genes listed in Table 1 may include two or more times down-regulated genes. The 'down-regulated genes' are genes that are down regulated compared to untreated breast cancer cell lines in breast cancer cell lines treated with apisidine. In Table 1 downregulation is indicated with a minus sign, corresponding to 198 genes. If the candidate is an HDAC inhibitor, it is expected that the expression of these genes is down-regulated in dark lines, similarly to apisidine.

In another embodiment, the marker for screening the HDAC inhibitor of the present invention may include genes involved in the cell cycle among the genes listed in Table 1. Genes involved in the cell cycle may include SMADs, Cyclin-dependent kinases (CDKs), Cyclins, Cell division cycles (CDCs), Cyclin-dependent kinase inhibitors (CDKNs), E2F / DP complexes, and E2F / DP target genes. have. Specific genes belonging to each may refer to FIG. 4.

In another embodiment, the marker for screening the HDAC inhibitor of the present invention is a cell division cycle 2 (CDC2), minichromosome maintenance 5 (MCM5), proliferating cell nuclear antigen (PCNA), minichromosome (MCM7) selected from the genes listed in Table 1 maintenance 5), transcription factor Dp-1 (TFDP1), E2F1, cyclin B1 (CCNB1), cyclin-dependent kinase 4 (CDK4), histone deacetylase 5 (HDAC5), SMAD3 and origin recognition complex subunit 4-like It may include.

In the present invention, the method for measuring at least one expression level selected from the genes listed in Table 1 at the level of mRNA or protein in cancer cells treated with candidates and untreated cancer cell lines may include all methods in the art. In addition, HDAC inhibitors can be screened by comparing expression levels measured in cancer cell lines treated with candidates and cancer cell lines not treated with candidates.

The present invention relates to a kit for HDAC inhibitor screening comprising an agent for measuring the mRNA level of at least one HDAC inhibitor screening marker selected from the genes listed in Table 1.

Agents for measuring mRNA levels of genes are preferably primer pairs or probes that specifically bind to markers for HDAC inhibitor screening, and those skilled in the art will specifically amplify specific regions of the gene based on the known sequence of the stepped marker. Primers or probes can be designed.

An "primer", an example of an agent for measuring mRNA levels, is a nucleic acid sequence with a short free 3 'hydroxyl group that can form complementary templates and base pairs and is the starting point for template strand copying. It refers to a short nucleic acid sequence that functions as. It consists of primer pairs in the forward and reverse directions, and usually has a length of 7 to 50, more preferably 10 to 30 nucleic acids. Primers can initiate DNA synthesis in the presence of enzymes, reagents and four different nucleoside triphosphates for polymerization in the appropriate buffer and temperature. Primers can incorporate additional features that do not change the basic properties of the primers that serve as a starting point for DNA synthesis.

In addition, the primer nucleic acid sequences of the present invention may, if necessary, include a label that can be detected directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Examples of labels include enzymes (eg horseradish peroxidase, alkaline phosphatase), radioisotopes (eg ³³ P), fluorescent molecules, chemical groups (eg biotin), and the like. There is this.

Those skilled in the art can easily determine the combination of various primer pairs having high specificity and sensitivity without causing non-specific amplification by considering variables such as the degree of hybridization between sequences in target genes based on the gene sequences of known markers. As used herein, the term "nonspecific amplification" refers to nucleic acid amplification other than the target sequence caused by hybridization of a primer to a nucleic acid sequence other than the target sequence and acting as a substrate for primer extension.

Another example of an agent for measuring mRNA levels, "probe" refers to a nucleic acid fragment, such as RNA or DNA, which is short to several hundred bases that can achieve specific binding with mRNA, and is labeled to be specific. The presence of mRNA can be confirmed. The probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like.

Primers or probes of the invention can be chemically synthesized using phosphoramidite solid support methods, or other well known methods. Moreover, it can transform into methylation, capping, etc. by a well-known method.

Analytical methods for measuring mRNA levels include, but are not limited to, RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assays, northern blotting, DNA microarray chips, and the like. Kits for measuring mRNA levels of markers for screening HDAC inhibitors are specifically RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assays, northern blotting or diagnostic kits for DNA microarray chips, suitable for assay methods. One or more other component compositions, solutions or devices.

For example, kits for RT-PCR can contain test tubes or other appropriate containers, reaction buffers (variable pH and magnesium concentrations), deoxynucleotides (dNTPs), Taq-polymers, in addition to individual primer pairs specific for the marker gene. Enzymes such as enzymes and reverse transcriptases, DNAse, RNAse inhibitors, DEPC-water, sterile water, and the like. It may also include primer pairs specific for the gene used as the quantitative control.

Also, for example, a kit for a DNA microarray chip may include a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached with a probe, and the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof. The DNA microarray chip of the present invention can be produced by methods known to those skilled in the art. The method of manufacturing the microarray chip is as follows. In order to immobilize the searched biomarker as a probe DNA molecule on a substrate of a DNA microarray chip, a micropipetting method or a pin-shaped spotter using a piezoelectric method ), Etc. are used. The DNA microarray chip substrate may be coated with active groups such as amino-silane, poly-L-lysine, and aldehyde. Further, the substrate may be a slide glass, plastic, metal, silicon, nylon film, or nitrocellulose film. The kit may also consist of buffers used for hybridization, reverse transcriptase for synthesizing cDNA from RNA, cNTPs and rNTPs (premixed or separate feed), marker reagents such as chemical inducers of fluorescent dyes, wash buffers, and the like. Can be.

The present invention also relates to a kit for HDAC inhibitor screening comprising an agent for measuring the protein level of at least one HDAC inhibitor screening marker selected from the genes listed in Table 1.

Agents for measuring protein levels of genes are preferably antibodies to markers for screening HDAC inhibitors, which can be readily prepared using techniques well known in the art.

In the present invention, an antibody includes all polyclonal antibodies, monoclonal antibodies and recombinant antibodies. In addition, the antibody of the present invention is not only a complete form having two full-length light chains and two full-length heavy chains, but also antigen binding of antibody molecules such as Fab, F (ab '), F (ab') 2 and Fv. It may be a functional fragment that holds a function.

Methods for measuring protein levels include Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein Chips and the like, but is not limited thereto. Kits for measuring the protein level of the HDAC inhibitor screening markers specifically include the ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, a kit for protein chips.

For example, a kit for ELISA may comprise reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (eg conjugated with antibodies) and substrates thereof. have. It may also include antibodies specific for quantitative control proteins. ELISA is a direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, an indirect ELISA using a labeled antibody that recognizes a capture antibody in a complex of an antibody that recognizes an antigen attached to a solid support, and attaches to a solid support Direct sandwich ELISA using another labeled antibody that recognizes the antigen in a complex of antibodies and antigens, a label that recognizes the antibody after reacting with another antibody that recognizes the antigen in a complex of the antibody and the antigen attached to a solid support Kits for a variety of ELISAs, including indirect sandwich ELISAs using secondary antibodies.

Also, for example, the present invention relates to diagnostic markers for protein chips in which corresponding antibodies to one or more markers are arranged at fixed locations on a substrate and immobilized at high density. The protein chip kit of the present invention may be composed of a buffer solution, a marker reagent, a wash buffer solution, and the like used for hybridization.

Kits for screening HDAC inhibitors provided herein may require certain additional equipment, such as a PCR cycler, microarray reader, or FACS device.

In addition, the present invention (1) the treatment of cancer cell lines with HDAC inhibitor candidate, (2) at least one HDAC inhibitor screening selected from the genes listed in Table 1 between the cancer cell line treated with the candidate and untreated cancer cell line It relates to an HDAC inhibitor screening method comprising the step of comparing the expression level of the marker for.

Isolation of mRNA or protein from cancer cell lines can be carried out using known processes, and mRNA or protein levels can be measured by the various methods discussed above. mRNA or protein levels can be expressed as the absolute (eg μg / ml) or relative (eg relative intensity of signal) differences of the markers described above.

The expression levels of markers in cancer cell lines treated with HDAC inhibitor candidates and the expression levels of markers in untreated cancer cell lines can be compared at the mRNA or protein level. Can be screened.

The present invention identifies genes that are up- or down-regulated compared to breast cancer cell lines that are not treated with apisidine after treating breast cancer cell lines with apisidine, a representative HDAC inhibitor.

Since other HDAC inhibitors will show similar changes in gene expression patterns, similar to apidine, the identified genes can be used as markers for screening HDAC inhibitors.

Therefore, the marker for screening the HDAC inhibitor of the present invention will be usefully used to find a new HDAC inhibitor with excellent anticancer effect and fewer side effects.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. It will be obvious to you.

Reagents and Methods

Example 1 Cell and Cell Culture

Human breast cell lines MDA-MB-435, MDA-MB-231, MCF-7, SCC-1395, SK-BR-3 and ZR-75-1 were purchased from the American Type Culture Collection (Manassas, VA) Incubated in appropriate culture medium supplemented with 10% FBS [fetal bovine serum (HyClone Laboratories, Logan, UT)] and 1% penicillin / streptomycin (Life Technologies). Incubation was at 37 ° C. in humid air of 5% CO ₂ .

Example 2: RT-PCR Analysis

Total cell RNA was extracted from six breast cancer cell lines using Trizol ^® reagent (Gibco, USA) according to the manufacturer's instructions. The quality of RNA was confirmed by checking the integrity of ribosomal RNAs (28s and 18s) on 1% agarose gels. 1 ㎍ of RNA extracted at 0, 1, 2, 4, 8, 24 and 48 hours after apisidine treatment was reverse transcribed using oligo-dT primers. CDNA was synthesized using Superscript II enzyme (Invitrogen, San Diego, CA) and HDAC specific primers listed in Table 2 below.

Example 3: Apicidine Treatment

Apicidine [cyclo (N-O-methyl-L-tryptophanyl-L-isoleucinyl-

D-pipecolinyl-L-2-amino-8-oxode canoyl] was purchased from CALBIOCHEM ^® (Merck KGaA, Darmstadt, Germany). MDA-MD-435 breast cancer cell lines were harvested and seeded at 1.5 × 10 ⁵ cells in 60 mm dishes and incubated overnight in 37% C. 5% CO ₂ wet incubator with complete growth medium (DMEM / 10% FBS). The cultured cells were then treated with the specified amount of apisidine for cell growth and apoptosis experiments. For comprehensive genomic analysis, apicidine (1 μg / ml) was added to MDA-MB-435 breast cancer cells at the designated time periods and the cells treated with apisidine (1 μg / ml) at the specified time periods. RNA was analyzed by DNA microarray.

Example 4: Antibody

Antibodies against PARP, p21, CDK2, CDK4 were purchased from Cell Signaling (Cell Signaling Technology, Inc. Danvers, MA). Antibodies against DP-1, E2F1, HDAC1, b-actin were purchased from Santa Cruz (Santa Cruz Biotechnology, Inc., CA), and antibodies against histone H4, a-tubulin and acetyl-histone H4 were upstate (Upstate Biotechnology , Inc., Lake Placid, NY).

Example 5: Apoptosis Measurement

MDA-MB-435 breast cancer cells were seeded at a density of 1.5 × 10 ⁵ cells / plate and apicidine was treated for 4 days at the concentrations described above. Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences, San Diego, Calif.) Was used to quantify the degree of apoptosis induced by apicidine. That is, the cells were trypsinized and centrifuged and then resuspended in binding buffer at 1 × 10 ⁶ cells / ml, and 5 μl of Annexin V-FITC and 5 μl of PI were added to 100 μl of suspension (˜1 × 10 ⁵ cells). The cells were left in dark at room temperature and measured by flow cytometry (FACS Vantage SE Flow Cytometric system; Becton Dickinson, NJ).

Example 6: Cell Growth Measurement

Cells were seeded at 1 × 10 ⁵ cells / sphere on 6 plate and 24 hours later a stock solution of acipidin dissolved in ethanol was added at a concentration of 0, 0.5, 1 or 2 μg / mL. Viable cells were counted using the trypan blue exclusion method. All experiments were performed in three replicates.

Example 7: Western Blot Analysis

After apisidine treatment, total cells were washed twice with ice-cold PBS and 200 μl of RIPA buffer [50 mM Tris-HCl, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 tablet / 50 mL of protease cocktail (Roche, Mannheim, Germany)]. Protein concentration in total cell lysate was determined using BCA protein assay reagent (Pierce, Rockford, IL 61105), 20 μg of protein was subjected to SDS-polyacrylamide gel electrophoresis, followed by polyvinylidene difluoride (PVDF) membrane (Amersham, US). Membranes were blocked with Tris buffer (TBS) containing 0.1% Tween-20 and 5% (w / v) dried skim milk powder and allowed to stand at room temperature for 1 hour with blocking solution containing appropriately diluted primary antibody. . The blot was then washed three times for 5 minutes with TBS-T and left at room temperature for 1 hour with secondary antibody conjugated with horseradish peroxidase diluted 1: 5000 (Pierce, Rockford, IL). , 3 washes for 5 minutes with TBS-T. Protein antibody responses were detected using an ECL detection kit (Amersham Biosciences, Piscataway, NJ).

Example 8: Extensive Analysis of Gene Expression Profiles Using Oligonucleotide Arrays

Microarray analysis was performed using human DNA microarrays prepared by the Microdissection Genomics Research Center, College of Medicine, The Catholic University of Korea. Microarrays contain 19,000 genetic elements (Noh JH, et al. Mol Cell Biochem 2006; 288: 91-106). Total cell RNA was extracted from cells treated with apacidine using TRIzol ^® reagent (Gibco, US). Total RNA NanoDrop (Nanodrop Technologies, Wilmington, DE, USA). RNA quality was checked using Bioanalyzer 2100 (Agilent Technologies), and intact RNA samples (260/280 ratio> 1.8) were used for hybridization. Human universal reference RNA (Stratagene, La Jolla, Calif.) Was used as reference RNA. For each RNA sample, a total of 20 μg of RNA was primed with oligo-dT and labeled with Cy3-dUTP or Cy5-dUTP (NEN) during reverse transcription. Labeled cDNA targets were adjusted to ˜15 μL using Microcon-YM30 (Amicon, Bedford, Mass.). Hybridization was performed at 42 ° C. for 16 hours using the MAUI Hybridization System (BioMicro Systems, Salt Lake City, UT). After hybridization, the microarray was twice with 2 × standard sodium citrate and 0.1% SDS for 2 minutes, 1 × SSC and 0.1% SDS for 3 minutes, 0.2 × SSC for 3 minutes, 0.05 × SSC for 2 minutes Washed with distilled water for 2 minutes.

Example 9: Scanning and Data Analysis

Arrays with hybridized targets were scanned with an Axon scanner, imaged and analyzed with GenePix® Pro 4.1 software (Axon Instruments). Spots of poor quality in visual observation were excluded from further analysis. The resulting array was submitted to the BioArray Software Environment (BASE) database at the microarray core facility (http://genomics.catholic.ac.kr/) of the Catholic University College of Medicine. Data was standardized using the R-packages of the LIMMA package (Linear Models for Microarray Data) and SMA (Statistics for Microarray Analysis). Spots of 50 μm or less were excluded from the analysis unless otherwise specified. Pearson's correlation coefficient was calculated using S-PLU and Cluster and TreeView were used to visualize the data (Park JY, et al. Biochem Biophys Res Commun 2004; 325: 1346-52). Path analysis was performed with ArrayXPath (http: www.snubi.org/software/ArrayXPath/).

result

Anti-proliferative Effect of Apisidine on MDA-MB-435 Human Breast Cancer Cell Line

Apicidines are known to strongly inhibit HDAC activity in various tissue-derived cell lines and induce accumulation of acetylated histones in vivo (Noh JH, et al. Mol Cell Biochem 2006; 288: 91-106; Kwon SH et al. J Biol Chem 2002; 277: 2073-80; Davie JR et al. Prog Nucleic Acid Res Mol Biol 2001; 65: 299-340). First, to determine whether apisidine had a similar effect on breast cancer cells, the amount of intracellular HDAC in breast cancer cells was first measured. As shown in FIG. 1, it was confirmed that type 8 HDACs are expressed in various breast cancer cell lines. In particular, the MDA-MB-435 cell line was established from a classical metastatic breast cancer cell line and showed relatively high HDAC expression levels in all cell lines.

On the other hand, morphological changes induced by apisidine in vitro show antitumor effects on cancer cells (Han JW, et al. Cancer Res 2000; 60: 6068-74). As shown in FIG. 2A, MDA-MB-435 cells extended to linear lobes when treated with increased concentrations of apisidine (up to 2 μg / mL). As a result of measuring the cell growth rate at the same concentration of apicidine, the growth rate was markedly reduced by increasing concentrations of apicidine treatment (FIG. 2B), and this growth inhibition was partially due to apoptosis induced by apicidin. It can be explained by (Fig. 2d). This cell growth inhibition was confirmed by FACS analysis after staining cells treated with apisidine with Annexin V-FITC. As shown in FIG. 2C, Annexin V-FITC staining increased the percentage of cells in the M2 stage compared to the untreated control (48% to 19% at 2 mg / mL apicidine) after 48 hours of apisidine treatment. ) Previous studies have suggested that apicidin induces apoptosis through cytochrome C release of cytoplasm by selectively inducing Fas / Fas ligands and activating caspase-9 and caspase-3 (Han JW, et al. Cancer Res 2000; 60: 6068-74; Kwon SH, et al. J Biol Chem 2002; 277: 2073-80. To identify the mechanism of apoptosis by apicidin, PARP (PARP is a known substrate of caspase-3) cleavage, which is characteristic of cellular apoptosis, was analyzed. Endogenous PARP truncated fragments (85 kDa) were detected after treatment with <1.0 mM of apicid for 48 hours, and the fragments accumulated in a dose dependent manner as shown in FIG. 2D.

2. Identification of characteristic molecular symbols of apicidin

To characterize transcript control by apicidin in breast cancer cells, MDA-MB-435 cells were treated with apicidine (1 μg / mL) for a specified time and the cell lysate was approximately 1,9000 human genetics. DNA microarrays containing urea were examined. We then designed an extensive gene expression analysis algorithm that recapitulates comprehensive gene expression changes (specific genes) induced by apisidine. The algorithm subtracted the expression level in untreated cells (0 hours) from the expression level at each time point. Due to down- or up-regulation, genes with more than two-fold changes compared to control expression were selected. All selected genes were expressed as heatmaps to provide molecular signals for apicidin (FIG. 3A). Heatmaps of 631 selected genes showed that expression of 198 genes increased continuously over time after apicidin treatment (Table 1). In addition, the expression of some genes was found to change rapidly after exposure to apicidin (FIG. 3A).

In order to analyze the functional properties of the 631 genes previously selected using ArrayXPath (http: www.snubi.org/software/ArrayXPath/), a path mimicking tool, selected 631 specific genes were assigned to known pathways. Application of this software has shown that apicidin primarily affects genes involved in cell cycle signaling pathways (data not shown). Eleven of 631 genes were assigned to the cell cycle pathway, and their expression changes over time are shown in FIGS. 3B and 3C as heat maps and graphs, respectively. Apisidine induced cell cycle arrest by activating negative cell cycle regulators and / or cell cycle promoter molecules at the transcript level. SMAD3 , a downstream effector of the TGF-β receptor, was highly activated by apisidine, but genes involved in G1 / S cell cycle progression and DNA replication ( CDC2, MCM5, MCM7, PCNA, E2F1 and CDK4 ) were inhibited (FIG. 3b and 3c).

3. Molecular Ablation of Cell Cycle Regulation Through Apisidine by Extensive Data Analysis

The aim of this study was to determine whether the regulation of genes in these cell cycle signaling pathways is characteristically mediated by apisidine or is a consequence of the cytotoxicity of apisidine. Accordingly, the amount of expression of all selected genes associated with the regulation of G1 / S cell cycle progression was shown as a heat map (FIG. 4A). For example, SMADs are a type of protein that alters the activity of transforming growth factor ligands. Cytoplasmic SMADs form complexes, which then transfer to the nucleus to act as transcription factors. As shown in FIG. 4A, the expression level of SMAD3 was immediately induced by apisidine, while SMAD4 was upregulated after 24 hours of apisidine treatment. Inhibitors of cyclin-dependent kinases (CDKs) and p21 ^Cip1 , p27 ^Kip1 , and p57 ^Kip2 are important G1 / S phase regulators and negatively regulate cell cycle progression. Western blotting results showed that p21 ^Cip1 was specifically induced by apicidin (FIGS. 4A and 4B), and CDK2 and CDK4 (but not other CDKs ) were characteristicly downregulated by apicidin (FIG. 4A And 4b). In addition, cyclin A2, B1 , and B2 and cell division cycle 2 ( CDC2 ) were characteristicly downregulated by apicidin, while other cyclins and CDCs were not. Indeed cyclins A and B and CDC2 are the major regulators of G2 / M rather than the G1 / S phase. These results show that apicidin affects cell cycle progression by modulating the expression of key components of the cell cycle clock.

In G1 / S cell cycle metastasis, activation of the CDKs / cyclin complex phosphorylates the retinoblastoma (Rb) protein and no longer inhibits the G1 / S transcription factor E2F / DP-1, resulting in transcriptional activation of downstream target molecules. In addition, the present invention showed that apicidin downregulates E2F / DP-1 transcription factor expression. As shown in FIG. 4A, only E2F1 and DP-1 of the E2F family members were inhibited by apisidine . Interestingly, some HDAC genes were characteristically induced by apicidine, and HDAC is a co-regulator in the Rb / E2F / DP-1 complex. In addition, E2F / DP-1 downstream target target genes such as MCMs, PCNA and DHFR were also downregulated by apicidin (FIG. 4A).

1 is the result of RT-PCR showing the expression of HDAC of type 8 in various breast cancer cell lines.

Figure 2a shows the morphological changes of breast cancer cell line MDA-MB-435 treated with apacidine by concentration.

2B shows changes in cell viability of breast cancer cell line MDA-MB-435 treated with apacidine.

2C is a FACS result showing that apoptosis is induced in breast cancer cell line MDA-MB-435 by apisidine treatment.

FIG. 2D is a Western blot showing that apoptosis is induced in breast cancer cell line MDA-MB-435 by apisidine treatment.

FIG. 3A shows a heat map of genes whose expression levels are down- or up-regulated in the apisidine-treated breast cancer cell line MDA-MB-435 compared to the apisidine-treated breast cancer cell line MDA-MB-435.

Figure 3b is a heat map of 11 genes involved in the cell cycle among genes whose expression level is changed by apisidine treatment for breast cancer cell line MDA-MB-435.

Figure 3c is a graph showing the increase and decrease of the expression level of the 11 genes involved in the cell cycle among genes whose expression level is changed by apisidine treatment for breast cancer cell line MDA-MB-435.

4 is a heat map of all genes involved in the cell cycle among genes whose expression level is changed by apisidine treatment for breast cancer cell line MDA-MB-435.

Figure 4b shows the results of Western blot analysis for breast cancer cell line MDA-MB-435 treated with apicidine.

5 shows a schematic diagram of cell cycle regulation by apisidine.

<110> Catholic University Industry Academic Cooperation Foundation <120> Markers for Screening Inhibitors of HDAC <160> 16 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 ccagtattcg atggcctgtt 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ggcttgaaaa tggcctcata 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ggtgctggaa aaggcaaata 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tgcgcaaatt ttcaaacaaa 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gcaaggcttc accaagagtc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 cttttccagc cggttatcaa 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 ggagctgaag aatggctttg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 gatgacacca gcaccacatc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 cagcaggcgt tctacaatga 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gttgatgttg ggcttttgct 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 aaccaggcag cgaagaagta 20 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 tgaaccaaca tcagctcttc c 21 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 atgggcttct gcttcttcaa 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 tccaccctgt tacccagaag 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 ggccagtatg gtgcattctt 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 tgcagatcca aatccacgta 20  

Claims (12)

delete A kit for HDAC inhibitor screening comprising an agent measuring the expression level of a marker for screening HDAC inhibitors consisting of the genes listed in Table 1 at the mRNA or protein level. 3. The method of claim 2, The agent measuring at the mRNA level is a kit, characterized in that the primer pair or probe specifically binding to the marker for HDAC inhibitor screening. 3. The method of claim 2, The agent measuring at the protein level is a kit, characterized in that the antibody against the marker for HDAC inhibitor screening. 3. The method of claim 2, Among the genes described in Table 1, 428 to 625 gene is characterized in that the expression level is down-regulated. The method of claim 5, Among the genes listed in Table 1, the kits 428 to 463 are characterized in that the expression level is more than two times down-regulated. 3. The method of claim 2, 1 to 427 of the genes listed in Table 1, the kit characterized in that the expression level is up-regulated. The method of claim 7, wherein Among the genes listed in Table 1, the kits 325 to 427 are characterized in that the expression level is more than two times up-regulated. delete 3. The method of claim 2, Among the genes listed in Table 1, SMADs, CDKs (Cyclin-dependent kinases), CCNs (Cyclins), CDCs (Cell division cycles), CDKNs (Cyclin-dependent kinase inhibitors), E2F / DP complexes and E2F / DP target genes Kits, characterized in that the gene involved in the cell cycle. delete (1) treating cancer cell lines with HDAC inhibitor candidates; (2) HDAC inhibitor screening method comprising the step of comparing the expression level of the HDAC inhibitor screening marker consisting of the genes listed in Table 1 between the candidate cell treated cancer cell line and the untreated cancer cell line.

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