KR20180110403A - Biomarker for predicting resistance or sensitivity to anticancer agent comprising ALDH3A1 - Google Patents

Abstract

The present invention relates to a biomarker for predicting resistance or sensitivity to an anticancer drug including aldehyde dehydrogenase 3 family member (A1ALDH3A1), a composition for predicting resistance or sensitivity to the anticancer drug containing a preparation for measuring a level of mRNA of an A1ALDH3A1 gene or a protein thereof, a kit, and a method for providing information. According to the present invention, the expression of the A1ALDH3A1 gene is remarkably reduced in a sensitive cell line showing high reactivity to the anticancer drug, thereby enabling effective prediction on resistance or sensitivity to the anticancer drug by using the same. In addition, by using the biomarker of the present invention, it is possible to carry out efficient anticancer therapy by selecting patients showing effectiveness to the anticancer agent. Thus, the biomarker of the present invention can be applied for developing drugs to increase sensitivity to the anticancer agent by targeting the gene.

Description

(Biomarker for predicting resistance or sensitivity to anticancer agent comprising ALDH3A1)

The present invention relates to a biomarker composition for predicting resistance or sensitivity to an anticancer agent comprising an aldehyde dehydrogenase 3 family member A1 (ALDH3A1), a resistance to an anticancer agent comprising an agent for measuring the mRNA of the ALDH3A1 gene or a protein level thereof, or Compositions for predicting susceptibility, kits and information providing methods.

Cancer is the major cause of death in Korea, and there have been many studies to conquer cancer, but it is still an incurable disease that has not been developed yet. Cancer can be categorized into surgery, radiotherapy, chemotherapy, and biological therapy, but there are limitations to each method. Specifically, surgery has a limitation in that it can not completely eliminate extensive metastasis. Radiation therapy has a limitation in that it can not selectively transmit radiation to target cancer cells, and immunotherapy has a limit that involves fatal toxicity to the patient. Considering these limitations, patients whose cancer or radiation therapy is not easy (about 50% of all cancer cases) and patients who have already metastasized are treated with chemotherapy.

Although many anticancer drugs have been used as effective treatments to date, the newly emerging problem is the resistance of cancer cells to anticancer drugs (Tsuruo et al., 1984). Resistance to anticancer drugs is caused by various mechanisms, such as long-term use of anticancer drugs, such that cells exposed to the drug decrease the intracellular accumulation of the drug, activate detoxification or excretion, or modify the target protein. This process is not only the biggest obstacle to cancer treatment, but also has a profound effect on treatment failure. In practice, chemotherapy is very limited in the treatment of cancer patients, and there is a limit to the chemotherapy regimens administered according to standard treatments. This is because the same chemotherapeutic agent can not be used for the recurrence after 1 ^st line chemotherapy and 2 ^nd line chemotherapy is administered. Repeated recurrence of cancer leads to a major problem in the resistance of previously used anticancer drugs, thus indicating the lack of available anticancer drugs. Recurrent cancer after chemotherapy resistance in cancer patients is very different from the type of early diagnosis cancer, and the characteristics of relapsed cancer lead to metastasis to other organs. In addition, despite the attempt of simultaneous administration of several kinds of anticancer drugs with different mechanism of action at the time of initial treatment, the phenomenon of no treatment effect can be observed frequently. Therefore, the limited range of available anticancer drugs is pointed out as an important problem in the chemotherapy of cancer, and predicting the susceptibility to anticancer drugs before chemotherapy is becoming an essential factor in determining the treatment direction of cancer patients . However, due to the complex action of bioreactors related to specific drugs, the diversity of therapeutic agents and methods of administration, and the difficulty of securing large samples, there is a limit to anticancer drug reactivity prediction.

Accordingly, the present inventors have made efforts to develop novel biomarkers for anticancer drug reactivity, and as a result, they have found that gene expression between cell lines exhibiting resistance or sensitivity to specific anticancer agents is comparatively analyzed and ALDH3A1 gene having a significantly decreased expression level in anticancer drug- Thereby completing the present invention.

It is an object of the present invention to provide a biomarker composition for predicting resistance or sensitivity to an anticancer agent.

It is another object of the present invention to provide a composition for predicting resistance or sensitivity to an anticancer agent comprising an agent for measuring the level of the biomarker.

It is still another object of the present invention to provide a kit for predicting resistance or sensitivity to an anticancer agent comprising the composition.

It is still another object of the present invention to provide a method for providing information for predicting resistance or sensitivity to an anticancer drug using the biomarker.

Yet another object of the present invention is to provide a method for screening anticancer agents or anticancer agents using the biomarkers.

In order to solve the above problems, the present invention provides a biomarker composition for predicting resistance or sensitivity to an anticancer agent comprising ALDH3A1 (aldehyde dehydrogenase 3 family member A1).

In addition, the present invention provides a composition for predicting resistance or sensitivity to an anticancer agent comprising an agent for measuring mRNA of the ALDH3A1 gene or a protein level thereof.

In addition, the present invention provides a kit for estimating the resistance or sensitivity to an anticancer agent comprising the composition.

The present invention also relates to a method for detecting an ALDH3A1 gene, comprising the steps of: (a) measuring the mRNA of the ALDH3A1 gene or a protein level thereof in a biological sample; And (b) the mRNA or the protein level of the ALDH3A1 gene is higher than that of the control group, and the mRNA or protein level of the ALDH3A1 gene is lower than that of the control group. The method comprising the steps of: determining a resistance to an anticancer agent;

The present invention also provides a method for producing a biological sample, comprising the steps of: (a ') treating a candidate substance to a biological sample; And (b ') selecting a substance that reduces mRNA of the ALDH3A1 gene or a protein level thereof in the biological sample. The present invention also provides a method for screening an anticancer agent or a chemotherapeutic agent.

Since the expression of the ALDH3A1 gene of the present invention is significantly reduced in a sensitive cell line that is highly responsive to an anticancer agent, the resistance or sensitivity to the anticancer drug can be effectively predicted using the ALDH3A1 gene. In addition, by using the biomarker of the present invention, it is possible to efficiently treat chemotherapy by selecting patients showing an effect on the anticancer agent, and to develop a drug that targets the gene and increases the sensitivity of the anticancer agent.

FIG. 1 is a graph showing the anticancer effect of JQ1 through CCK8 ischemia in 9 types of gastric cancer cell lines. FIG.
Fig. 2 is a graph showing the results of confirming the IC50 of JQ1 in 9 kinds of gastric cancer cell lines. Fig.
Figure 3 shows the results of colony formation assays in highly sensitive gastric cancer cell lines for JQ1.
FIG. 4 is a graph showing the results of gene expression profile analysis between susceptible and resistant gastric cancer cell lines for JQ1.
FIG. 5 is a graph showing the results of analysis of gene expression profiles of JQ1 according to the treatment of JQ1 in a sensitive gastric cancer cell line. FIG.
FIG. 6 is a graph showing the results of analysis of ROS production level (left) and ALDH3A1 gene expression and ROS production in JR1 sensitive and resistant gastric cancer cell lines (right).
FIG. 7 is a graph showing the results of confirming the degree of ROS production according to the treatment of JQ1 in the sensitive and resistant gastric cancer cell lines for JQ1.

Hereinafter, the present invention will be described in more detail.

The present invention provides a biomarker composition for predicting resistance or sensitivity to an anticancer agent comprising ALDH3A1 (aldehyde dehydrogenase 3 family member A1).

In the present invention, "marker" is a substance that can predict reactivity to an anticancer drug, and includes polypeptides showing significant resistance to an anticancer agent and individuals showing sensitivity to an anticancer drug, nucleic acids (e.g., mRNA Etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.) and the like. For the purpose of the present invention, it is preferred that the marker comprises an ALDH3A1 (aldehyde dehydrogenase 3 family member A1) protein and a nucleic acid molecule (DNA or mRNA) encoding the same.

In the present invention, "ALDH3A1 (aldehyde dehydrogenase 3 family member A1)" is one of the subfamilies of ALDH. ALDH is a group of enzymes that catalyze aldehyde oxidation (dehydrogenation). To date, 19 ALDH genes have been identified in the human genome, and these genes are involved in a variety of biological processes, including detoxification of exogenous and intrinsically generated aldehydes. The protein name of the ALDH3A1 gene is Aldehyde dehydrogenase, dimeric NADP-prefeffing. It is a type of aldehyde dehydrogenase which oxidizes various aldehydes to corresponding acids. It is also known to be involved in detoxification of alcohol-derived acetaldehyde and metabolism of corticosteroids, bio-amines, neurotransmitters and lipid peroxidation, and the gene is located on chromosome 17. The specific nucleotide sequence and protein information of the ALDH3A1 gene is known from NCBI (Gene ID: 218).

In the present invention, " resistant "refers to a condition that is not easily affected by an anticancer agent and does not significantly change cell proliferation despite treatment with an anticancer agent.

In the present invention, "sensitivity" refers to a state in which cell proliferation is effectively changed by an anticancer agent, that is, a state in which cancer cells die.

In the present invention, "prediction of resistance or sensitivity to an anticancer agent" means predicting whether a particular individual will respond favorably or non-favorably to the anticancer agent treatment, or predicting the risk of the anticancer agent resistance. For example, the predictions of the present invention can be used clinically to determine treatment by selecting the most appropriate treatment for a cancer patient. Prediction of the present invention is a useful tool in predicting whether a patient will respond favorably to a therapeutic treatment, such as a given therapeutic treatment, such as administration of a given therapeutic or combination, surgical intervention, chemotherapy, and the like.

In one embodiment of the present invention, the expression of JQ1, which is a kind of inhibitor of the BET family protein, is markedly decreased in a sensitive cell line as compared to a resistant cell line, and the expression level of JQ1 is further reduced by treatment with JQ1 The ALDH3A1 gene was identified and selected by biomarker.

Accordingly, in one aspect, the present invention provides a composition for predicting resistance or sensitivity to an anticancer agent comprising an agent for measuring mRNA of the ALDH3A1 gene or a protein level thereof.

In the present invention, the "anticancer agent" is preferably an inhibitor of the BET (Broodomain and Extra-Terminal domain) family protein, and the BET family includes BRD2 (bromodomain- containing protein 2), BRD3 , Bromodomain-containing protein 4 (BRD4) and bromodomain testis-specific protein (BRDT), and the like.

In the present invention, the term " inhibitor of BET family protein " refers collectively to the BET family protein itself or any substance capable of interfering with the interaction between the protein and chromatin, for example, JQ1, tetrahydroquinoline ), 3,5-dimethylisoxazole, 2-thiazolidinone or derivatives thereof, but is not limited thereto.

In the present invention, JQ1 or derivatives thereof may be represented by the following general formula (1).

[Chemical Formula 1]

X is N or CR < 5 >;

R5 is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted;

RB is H, alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or -COO-R3, each of which is optionally substituted;

Each RA is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; Any two of RA may form a fused aryl or heteroaryl group with each of the atoms to which they are attached;

R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted;

R'1 is H, -COO-R3, -CO-R3, optionally substituted aryl, or optionally substituted heteroaryl;

Each R3 is independently selected from the group consisting of:

(i) H, aryl, substituted aryl, heteroaryl, substituted heteroaryl;

(ii) heterocycloalkyl or substituted heterocycloalkyl;

(iii) -C1-C8 alkyl, -C2-C8 alkenyl or -C2-C8 alkynyl, each of which contains 0, 1, 2 or 3 heteroatoms selected from O, S or N; -C 3 -C 12 cycloalkyl, substituted -C 3 -C 12 cycloalkyl, -C 3 -C 12 cycloalkenyl, or substituted -C 3 -C 12 cycloalkenyl, each of which is optionally substituted;

m is 0, 1, 2 or 3;

Provided that R'1 is -COO-R3, X is N, R is substituted phenyl, and RB is methyl, in which case R3 is not methyl or ethyl.

Preferably, R is aryl or heteroaryl, each of which is optionally substituted.

Preferably, R is phenyl or pyridyl, each of which is optionally substituted.

Preferably, R is p-Cl-phenyl, o-Cl-phenyl, m-Cl-phenyl, p-F-phenyl, o-F-phenyl, m-F-phenyl or pyridinyl.

Preferably, R'1 is -COO-R3, optionally substituted aryl, or optionally substituted heteroaryl; R3 is -C1-C8 alkyl comprising 0, 1, 2 or 3 heteroatoms selected from O, S or N, which may be optionally substituted.

Preferably, R'1 is -COO-R3 and R3 is methyl, ethyl, propyl, i-propyl, butyl, sec-butyl or t-butyl; Or R'1 is H or optionally substituted phenyl.

Preferably, the RB is methyl, ethyl, hydroxymethyl, methoxymethyl, trifluoromethyl, COOH, COOMe, COOEt, COOCH2OC (O) CH3.

Preferably, the RB is methyl, ethyl, hydroxymethyl, methoxymethyl, trifluoromethyl, COOH, COOMe, COOEt, or COOCH2OC (O) CH3.

Preferably, each of said RA's is independently optionally substituted alkyl, or any two of RA's may form fused aryl with each of these attached atoms.

Preferably, each of said RA's is methyl.

More specifically, the JQ1 may be represented by the following formula (2), and the derivative of the JQ1 may be represented by, for example, the following formula (3) or (4).

(2)

(3)

[Chemical Formula 4]

In the present invention, the tetrahydroquinoline derivative may be represented by, for example, the following formulas (5) to (11), but is not limited thereto.

[Chemical Formula 5]

[Chemical Formula 6]

(7)

[Chemical Formula 8]

[Chemical Formula 9]

[Chemical formula 10]

(11)

In the present invention, the 3,5-dimethylisoxazole derivative may be represented by, for example, the following formulas (12) to (26), but is not limited thereto.

[Chemical Formula 12]

[Chemical Formula 13]

[Chemical Formula 14]

[Chemical Formula 15]

[Chemical Formula 16]

[Chemical Formula 17]

[Chemical Formula 18]

[Chemical Formula 19]

[Chemical Formula 20]

[Chemical Formula 21]

[Chemical Formula 22]

(Wherein R is phenyl, p-F-phenyl, m-F-phenyl, p-Cl-phenyl or m-

(23)

(In the above formula (23), R is H, Ac, CH2CH2OH, methyl or CH2CH2OMe)

≪ EMI ID =

(25)

(26)

In the present invention, the 2-thiazolidinone derivative may be represented by, for example, the following formulas (27) to (33), but is not limited thereto.

(27)

(28)

[Chemical Formula 29]

(30)

(31)

(32)

(In the above formula (32), R < 1 > is Me, F or NHCOCH2OCH3)

(33)

(Wherein R < 1 > is H or methyl)

The composition according to the present invention can be a cancer, for example, a digestive cancer, and more specifically, can be used for prediction of resistance or sensitivity to an anticancer drug in gastric, colon, small intestine, rectal, esophageal, colon or pancreatic cancer And more preferably, it can be used for predicting resistance or sensitivity to an anticancer drug in gastric cancer, but it is not limited thereto.

In the present invention, the agent for measuring the mRNA level of the gene preferably comprises a primer pair or a probe that specifically binds to the ALDH3A1 gene. Since the nucleic acid sequence of the genes encoding ALDH3A1 is registered in the gene bank, Can design a primer pair or a probe for specifically amplifying a specific region of these genes based on the above sequence.

In the present invention, "primer" is a nucleic acid sequence having a short free 3 'hydroxyl group and can form a base pair with a complementary template, and a starting point for copying the template ≪ / RTI > The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. In the present invention, the sequence of the primer is not limited as long as the primer can be complementarily bound and amplified with the ALDH3A1 gene.

In the present invention, the term "probe" means a nucleic acid fragment such as RNA or DNA corresponding to a short period of a few nucleotides or several hundreds of nucleotides capable of specifically binding to a gene or mRNA, The presence or absence of the presence of the user can be confirmed. The probe may be prepared in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, or an RNA probe. In the present invention, the sequence of a probe as long as the probe can bind complementarily to the ALDH3A1 gene is not limited.

The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, capping, substitution with one or more of the natural nucleotide analogs, and modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, Amidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).

In the present invention, the agent for measuring the protein level may preferably comprise an antibody specific for the ALDH3A1 protein.

In the present invention, "antibody" means a specific protein molecule directed against an antigenic site. Such an antibody can be prepared by cloning each gene into an expression vector according to a conventional method to obtain a protein encoded by the gene, and obtaining the protein from the obtained protein by a conventional method. Also included are partial peptides that can be made from the protein, for example, the partial peptides include at least 7 amino acids, preferably 9 amino acids, more preferably 12 amino acids. The form of the antibody of the present invention is not particularly limited, and any polyclonal antibody, monoclonal antibody or antigen-binding antibody thereof may be included in the antibody of the present invention and include all immunoglobulin antibodies. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies. Antibodies used in the present invention include functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

In addition, the present invention provides a kit for estimating the resistance or sensitivity to an anticancer agent comprising the composition.

The kit of the present invention can be used to predict the resistance or sensitivity to anticancer drugs by confirming the mRNA expression level or protein expression level of the ALDH3A1 gene corresponding to the marker. The kit of the present invention may include one or more other component compositions, solutions, or devices suitable for the assay, as well as primers, probes or antibodies for predicting resistance or sensitivity to anticancer agents.

As a specific example, the kit of the present invention may be a kit containing necessary elements necessary for performing RT-PCR. The RT-PCR kit can be used in combination with a test tube or other appropriate container, reaction buffer, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitor, DEPC- Water (DEPC-water), sterile water, and the like.

In addition, the kit of the present invention may be a kit including essential elements necessary for performing a microarray chip. The microarray chip kit may include a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and the substrate may include cDNA corresponding to a quantitative control gene or a fragment thereof, And can be easily produced by a production method conventionally used in the art. In order to fabricate a microarray chip, a micropipetting method using a piezo electric method or a pin-type method using a magnetized probe to immobilize the detected marker on a substrate of a DNA chip using the probe as a probe DNA molecule A method using a spotter of the above-mentioned, but the present invention is not limited thereto. The substrate of the microarray chip is preferably coated with an activator selected from the group consisting of amino-silane, poly-L-lysine and aldehyde, but is not limited thereto . In addition, the substrate is preferably selected from the group consisting of slide glass, plastic, metal, silicon, nylon film, and nitrocellulose membrane, but is not limited thereto.

In addition, the kit of the present invention may include a substrate, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, and a chromogenic substrate for immunological detection of the antibody. The substrate may be a nitrocellulose membrane, a plate synthesized with a polyvinyl resin, a plate synthesized with a polystyrene resin, or a slide glass made of glass. The chromogenic enzyme may be peroxidase, alkaline phosphatase, phosphatase and the like can be used, and fluorescent materials such as FITC and RITC can be used. The coloring substrate solution is ABTS (2,2'-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid) OPD (o-phenylenediamine), TMB (tetramethylbenzidine) can be used.

The present invention also relates to a method for detecting an ALDH3A1 gene, comprising the steps of: (a) measuring the mRNA of the ALDH3A1 gene or a protein level thereof in a biological sample; And (b) the mRNA or the protein level of the ALDH3A1 gene is higher than that of the control group, and the mRNA or protein level of the ALDH3A1 gene is lower than that of the control group. The method comprising the steps of: determining a resistance to an anticancer agent;

In the present invention, the biological sample of step (a) may be tissue, whole blood, serum or plasma, preferably cancer tissue, but is not limited thereto.

In the present invention, the measurement of the mRNA level in the step (a) is a step of confirming the presence and the degree of expression of the marker genes in the biological sample. The measurement of the mRNA level may be performed by PCR, reverse transcription polymerase chain reaction RT-PCR, real-time RT-PCR, RNase protection assay (RPA), northern blotting or DNA microarray analysis can be used. But is not limited thereto.

In the present invention, the measurement of the protein level in the step (a) is performed by confirming the presence and the degree of expression of the protein expressed from the marker gene in the biological sample, and may be determined by Western blotting, enzyme linked immunosorbent assay ), Radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, Rocket immunoelectrophoresis, tissue immuno staining, immunoprecipitation assay, complement fixation assay complete fixation assay, Fluorescence Activated Cell Sorter (FACS) or protein chip analysis, but the present invention is not limited thereto.

According to one embodiment of the present invention, the expression of ALDH3A1 was markedly reduced in a sensitive cell line that is highly reactive to JQ1, which is an inhibitor of the BET family protein, compared to the resistant cell line. In the biological sample, Or its protein level is compared with a control, i.e., a sample that does not show sensitivity to the anticancer agent, the resistance / sensitivity of the individual to the anticancer agent can be determined. In addition, it is possible to provide customized chemotherapy through prediction of resistance / sensitivity to the anticancer agent.

The present invention also provides a method for producing a biological sample, comprising the steps of: (a ') treating a candidate substance to a biological sample; And (b ') selecting a substance that reduces mRNA of the ALDH3A1 gene or a protein level thereof in the biological sample. The present invention also provides a method for screening an anticancer agent or a chemotherapeutic agent.

According to one embodiment of the present invention, the expression of ALDH3A1 gene is inversely proportional to the degree of ROS production in cancer cells, and it is confirmed that this is an important factor related to the anticancer mechanism of JQ1. Can be developed.

Hereinafter, the present invention will be described in detail with reference to Examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example  1. Anticancer effect of JQ1 in various gastric cancer cell lines

JQ1 is a representative compound belonging to the BET family protein inhibitor, including thiothalonaziadiazepines, BRD2, BRD3, BRD4 and BRDT. JQ1 ((Bromodomain Inhibitor) was applied to the gastric cancer cell lines of 10 different types (AGS, SNU16, SNU638, NUGC3, MKN45, MKN28, KATO3, NCI-N87, YCC1, YCC3) in order to confirm the anticancer effect of JQ1 in various gastric cancer cell lines. (Obtained from Dr. James E. Bradner of Dana-Farber Cancer Institute) was treated with concentration (0.01 to 5 μM), and CCK8 assay (Dojindo) was performed according to a conventionally known method. Each gastric cancer cell line was purchased from Korean Cell Line Bank and ATCC, and RPMI1640 and DMEM medium containing 10% FBS (Fetal bovin serum) and 1% antibiotic were used, and the cells were cultured at 37 ° C and 5% CO ₂ ) Were fed. More specifically, 10 kinds of gastric cancer cell lines were seeded in 96-well plates at 1 × 10 ^ 3 to 10 ^ 4 cells / well, respectively, and cultured for one day, and JQ1 was treated for 48 hours according to the predetermined concentration. After the reaction, CCK8 solution was added in an amount of 10 ul per well, followed by further incubation for 1 hour, and the absorbance at 450 nm was measured. The results are shown in FIG. 1, and the concentration (IC50: Half Maximal Inhibitory Concentration) at which JQ1 inhibits 50% cell growth of the gastric cancer cell line is shown in FIG.

As shown in FIG. 1, it was confirmed that JQ1 shows a difference in the cancer cell killing ability according to the gastric cancer cell line.

As shown in Fig. 2, the IC50 concentration of 8.0 was set as an intermediate reference value. IC50 of 10 kinds of gastric cancer cell lines was 8.0, sensitivity was lower than that of 8.0, IC50 8.0 was modest (black) Resistant (red) cells above 8.0 were used for the experiment.

Example  2. Anticancer effect on sensitive cell lines for JQ1

In order to re-examine the anticancer effect in the highly sensitive cell line NUGC3, SNU638 or AGS cell line which showed high reactivity to JQ1 in Example 1, a colony formation assay was performed according to a conventionally known method . More specifically, each gastric cancer cell line was seeded in a 6-well plate at 1 × 10 ^ 2 cells / well, cultured for one day, and treated with JQ1 at a concentration of 0.1, 0.5 or 1 μM. The culture was changed once every 3 days and cultured for 10-15 days. After confirming the formation of colonies, the number of colonies was counted through Crystal Violet staining. At this time, the composition of the crystal violet dyeing reagent is composed of 0.5 g of crystal violet, 1% formaldehyde and 1% methanol. The results are shown in Fig.

As shown in FIG. 3, in the NUGC3, SNU638, or AGS cell lines, JQ1 has an excellent anticancer effect that reduces colony formation from a low concentration of 100 nM.

Example  3. Identification of genes affecting responsiveness to JQ1

In order to identify the genes affecting the responsiveness to JQ1, the gene expression profile constructed from the AGS, NUGC3 and SNU16 cell lines, which are sensitive gastric cancer cells, and the YCC1, YCC3 and NCI-N87 cell lines, which are resistant gastric cancer cells, , We identified genes that showed differences in expression between the cells of the sensitive and resistant groups for JQ1. The results are shown in Fig.

As shown in Fig. 4, genes showing a statistically significant difference (p < 0.001) between the cells of the sensitive and resistant group to JQ1 were firstly screened.

Next, total RNA was isolated from JQ1 after treating SNU638 or AGS cell line, which is a sensitive gastric cancer cell for JQ1. mRNA was amplified and gene expression profile was analyzed after microarray. The results are shown in Fig.

As shown in FIG. 5, genes showing a statistically significant difference in expression depending on treatment of JQ1 in the sensitive gastric cancer cell lines for JQ1 were secondly selected.

Based on the results of the above two experiments, it was confirmed that the expression of ALDH3A1 gene is significantly decreased in the sensitive cell line which is highly responsive to JQ1, and that the expression level of JQ1 is further reduced by JQ1 treatment, The ALDH3A1 gene was finally selected by a biomarker that affects the reactivity of the ALDH3A1 gene.

Example  4. ALDH3A1  Sensitivity of JQ1 through Mechanism  analysis

Based on the existing role of the ALDH3A1 gene identified as a biomarker for determining the reactivity to JQ1 in Example 3, it was confirmed whether there is a difference in the degree of ROS generation among the cells of the susceptible and resistant group to JQ1. More specifically, in order to determine the degree of endogenous ROS generation in each cell line of six gastric cancer cell lines, DCFH-DA, a ROS detection reagent, was treated with 20 μM of the ROS detection reagent for 1 hour and then luminescence intensity Respectively. The results are shown in Fig.

As shown in FIG. 6, it was confirmed that the ROS production level was significantly increased in the sensitive cell line for JQ1 as compared to the resistant cell line for JQ1, and the degree of ROS production was inversely proportional to the expression level of the ALDH3A1 gene.

Based on the above experimental results, in order to confirm whether the production of ROS directly affects the reactivity to JQ1, the degree of ROS generation was measured by the same method after treating JQ1 with the gastric cancer cell line. For negative control, DMSO was treated. The results are shown in Fig.

As shown in FIG. 7, the production of ROS by JQ1 was increased in the sensitive gastric cancer cell line for JQ1, but it was confirmed that there was no significant difference in the production of ROS by JQ1 in the JQ1-resistant gastric cancer cell line.

These results suggest that the expression of ALDH3A1 gene plays an important role in the production of ROS in cancer cells and this is an important factor related to the anticancer mechanism of JQ1.

Overall, it is possible to use the ALDH3A1 gene as a biomarker for predicting sensitivity to anticancer drugs, thereby maximizing the effect of chemotherapeutic treatment, such as predicting the patient's response to chemotherapy and the risk of anticancer drug resistance. And thus it can be used for screening new anticancer substances.

Claims (14)

A biomarker composition for predicting resistance or sensitivity to an anticancer agent comprising ALDH3A1 (aldehyde dehydrogenase 3 family member A1).
A composition for predicting resistance or sensitivity to an anticancer agent comprising an agent for measuring mRNA of ALDH3A1 (aldehyde dehydrogenase 3 family member A1) gene or a protein level thereof.
3. The composition according to claim 2, wherein the anticancer agent is an inhibitor of a BET (Brobodomain and Extra-Terminal domain) family protein.
4. The method of claim 3, wherein the inhibitor of the BET family protein is selected from the group consisting of JQ1, tetrahydroquinoline, 3,5-dimethylisoxazole, 2-thiazolidinone, Or a derivative thereof, wherein the composition is predictive of resistance or sensitivity to an anticancer agent.
5. The composition according to claim 4, wherein the JQ1 or derivative thereof is represented by the following formula (1).
[Chemical Formula 1]

X is N or CR &lt; 5 &gt;;
R5 is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted;
RB is H, alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or -COO-R3, each of which is optionally substituted;
Each RA is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; Any two of RA may form a fused aryl or heteroaryl group with each of the atoms to which they are attached;
R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted;
R'1 is H, -COO-R3, -CO-R3, optionally substituted aryl, or optionally substituted heteroaryl;
Each R3 is independently selected from the group consisting of:
(i) H, aryl, substituted aryl, heteroaryl or substituted heteroaryl;
(ii) heterocycloalkyl or substituted heterocycloalkyl;
(iii) -C1-C8 alkyl, -C2-C8 alkenyl or -C2-C8 alkynyl, each of which contains 0, 1, 2 or 3 heteroatoms selected from O, S or N; -C 3 -C 12 cycloalkyl, substituted -C 3 -C 12 cycloalkyl, -C 3 -C 12 cycloalkenyl, or substituted -C 3 -C 12 cycloalkenyl, each of which is optionally substituted;
m is 0, 1, 2 or 3;
Provided that R'1 is -COO-R3, X is N, R is substituted phenyl, and RB is methyl, in which case R3 is not methyl or ethyl. The composition according to claim 2, wherein the composition is for predicting resistance or sensitivity to an anticancer agent in at least one cancer selected from the group consisting of gastric cancer, colon cancer, small intestine cancer, rectal cancer, esophageal cancer, colon cancer and pancreatic cancer. 0.0 &gt; resistance &lt; / RTI &gt; or sensitivity. 3. The composition according to claim 2, wherein the agent for measuring the mRNA level of the ALDH3A1 gene comprises a primer pair or a probe specifically binding to the gene, for predicting the resistance or sensitivity to an anticancer agent. 3. The composition according to claim 2, wherein the agent for measuring the ALDH3A1 protein level comprises an antibody specific for the protein. A kit for estimating the resistance or sensitivity to an anticancer agent comprising the composition of any one of claims 2 to 8. (a) measuring mRNA or its protein level of ALDH3A1 (aldehyde dehydrogenase 3 family member A1) gene in a biological sample; And
(b) If the mRNA or protein level of the ALDH3A1 gene is higher than that of the control, it is determined that the ALDH3A1 gene is resistant to the anticancer agent. If the mRNA or the protein level of the ALDH3A1 gene is lower than that of the control, And determining a resistance or sensitivity to an anticancer agent. 11. The method according to claim 10, wherein the biological sample in step (a) is tissue, whole blood, serum or plasma. The method according to claim 10, wherein the mRNA level of step (a) is selected from the group consisting of a polymerase chain reaction (PCR), a reverse transcription polymerase chain reaction (RT-PCR), a competitive reverse transcription polymerase chain reaction Characterized in that the anticancer agent is measured by at least one method selected from the group consisting of Realtime RT-PCR, RNase protection assay (RPA), Northern blotting and DNA microarray analysis. A method for providing information for predicting resistance or sensitivity. [11] The method of claim 10, wherein the protein level of step (a) is selected from the group consisting of western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay, radioimmunodiffusion, Immunoprecipitation assays, complete fixation assays, Fluorescence Activated Cell Sorter (FACS) and protein chips (immunoassays) were performed using immunoassay, Rocket immunoelectrophoresis, tissue immuno staining, ) Analysis method according to any one of claims 1 to 5, wherein the method comprises the steps of: (a ') treating the candidate material with a biological sample; And
(b ') selecting a substance that reduces the mRNA of the ALDH3A1 (aldehyde dehydrogenase 3 family member A1) gene or its protein level in the biological sample.

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