JP2006211994A - Method for determining anticancer characteristic of anticancer agent against non small cell lung cancer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a judging method by which an anticancer agent usable for therapy of non small cell lung cancer and taking effects on a patient. <P>SOLUTION: The method for determining the characteristics of the anticancer agent comprises getting the interrelation between an expression amount of a ganglioside GM3 synthetase gene (SAT-I) of a cell of the non small cell lung cancer and anticancer effects of the anticancer agent. When the characteristics of the anticancer agent is determined thus, it is expected that the useless administration of the anticancer agent is evaded, the side effects are reduced and the treatment takes effects by determining the amount of SAT-I mRNA in the cancer cell of the patient suffering from the non small cell lung cancer, and administering the anticancer agent with inhibition activities, having negative interrelation when the expression rate is high, or administering the anticancer agent having positive interrelation when the expression rate is low. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for determining the characteristics of an anticancer agent against non-small cell lung cancer, and more specifically, from the correlation between the expression level of ganglioside GM3 synthase gene (SAT-I) in non-small cell lung cancer cells and the anticancer effect of the anticancer agent. It relates to a method for determining characteristics.

  Lung cancer is the number one cause of cancer death among men in Japan, and women rank second after stomach cancer. In the future, the number of lung cancer patients is expected to increase, and it is predicted that the number of patients who newly develop lung cancer in 2015 will be 110,000 men and 37,000 women in 2015.

  Lung cancer is systematically classified into small cell caricinoma and non-small cell caricinoma, and non-small cell cancer accounts for about 80 to 85% of all lung cancer in Japan. It is divided into adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Adenocarcinoma is the most common in Japan and is about half of primary lung cancer. It accounts for 40% of male lung cancer and more than 70% of female lung cancer.

  Lung cancer is increasingly being detected early due to the spread of CT (computer tomography) screening. However, there is a high probability that death will occur after recurrence and metastasis, even if it is found early and partly excised. The 5-year survival rate (the rate of survival for 5 years after surgery) is said to be about 65% in stage IB where the stage of lung cancer (stage of progression) stage I lung cancer is 3 cm or more.

  Diagnosis of lung cancer includes chest radiographs, CT, contrast radiographs, CTMRI, endoscopy, bronchial fiber scope, biopsy histology, sputum cytology, etc. by symptoms and examination. The degree of progression of lung cancer is determined by this screening. Specifically, each of the spread of the primary tumor of the lung (T), lymph node metastasis (N), and distant metastasis (M) is determined, thereby determining the stage from stage I to stage IV. In general, stage I to a part of stage III are the target of surgery, but those in subsequent stages are mainly treated with radiation therapy and chemotherapy using an anticancer agent.

  Cisplatin, vinorelbine, gefitinib, vinblastine, carboplatin, paclitaxel and the like are used as anticancer agents for lung cancer.

  Among these, chemotherapy for non-small cell lung cancer is a combination of cisplatin and other anticancer drugs irinotecan, vinorelbine, gemcitabine, paclitaxel, docetaxel, and a combination of calplatin and gemcitabine or paclitaxel. Many findings have been obtained, and it is listed as the most recommended treatment method in the 2003 Lung Cancer Clinical Practice Guidelines. Thus, platinum preparations are the main anticancer agents that are administered for the treatment of non-small cell lung cancer.

  Recently, anticancer agents that suppress the growth of cancer by acting on the target molecule epidermal growth factor receptor (EGFR), a protein in the epithelium of cancer cells, have been developed and attract attention (Onn A. et al., Br J. Cancer, 2004 Aug, 91 Suppl 2: S11-7 (Non-patent document 1); Tamura K. et al., Int. J. Clin. Oncol., 2003 Aug 8 (4): 207-11 (non-patent) Reference 2)). Such new anticancer drugs are expected to be fully predicted for the administration of side effects and effects, since patients with side effects and effects are clearly identified.

  Anti-cancer agents are usually cytotoxic to normal cells and may cause fatal side effects. Therefore, it is becoming increasingly important to select and use an appropriate anticancer drug for each patient in order to enhance the effect and reduce the side effects.

  Therefore, various anticancer drug anticancer efficacy test methods have been developed. Specifically, the cancer tissue of a patient collected using an endoscope or the like is subjected to enzyme treatment and dispersed into cells. This cell and an anti-cancer agent are cultured on a microplate, and the MTT method for determining the survival rate by the mitochondrial metabolism of the cell, or the ATP method for measuring the amount of ATP, and recently isolated in a collagen gel. CD-DST (collagen gel droplet embedded culture-drug sensitivity test) method that can selectively measure only the effect on cancer cells by embedding the cultured cells (Kobayashi H., et al., Recent Results Cancer Res., 2003, 161: 48-61 (Non-Patent Document 3)). However, there is a drawback that it takes a period of time such as culturing cells and takes a long time for determination.

  In addition, gene markers involved in anticancer drug resistance have been selected and RT-PCR methods and methods for determining with DNA chips are being actively developed, but there are still established genetic markers for examining the anticancer effects of anticancer drugs. It has not been found (Suzuki T., et al., Lung Cancer, 2003 Oct., 42 (1): 35-41 (Non-patent Document 4)). In addition, no data on discrimination of anticancer effects for various anticancer agents has been reported.

  The present inventor has found that the expression level of the ganglioside GM3 synthetic gene is an effective index as a gene effective as a sensitivity diagnostic marker for cancer cells against anticancer agents, and has already filed a patent application as an anticancer agent sensitivity test method (WO 2004/099407, publication date: November 18, 2004 (Patent Document 1)).

  Regarding ganglioside GM3 and anticancer drug resistance, there is a report that the resistance of anticancer drug such as adriamycin and the expression of ganglioside GM3 is proportional to cancer hamster, mouse and human cancer cells (June L. , et al, Reverse transformation of multidrug-resistant cells. Cancer Metastasis Rev., 1994, Vol. 13, p. 191-207. In this report, anticancer drug resistance is increased in GM3 high-expressing cancer cell lines, but since the malignancy of these cancers in vivo is significantly reduced, the inventors' mouse lung cancer cells were used. The results of this study, that is, GM3, is greatly different from the fact that GM3 enhances cancer malignancy and anticancer drug resistance.

Furthermore, it has been reported that the expression level of GM3 is increased in the doxorubicin resistant strain of glioblastoma but decreased in the doxorubicin resistant strain of hepatoma (Benchekroun. Et al, Alteration of ganglioside composition in Cisplatin). -resitant Lung cancer cell line. Anticancer Res. 1998, Vol. 18, p.2957-2960.
This suggests that there is a difference in the correlation between the GM3 expression level and drug resistance in each cancer type, and it can be said that each cancer type must be examined. This report has not been studied in human non-small cell lung cancer.

In relation to this, it has been reported that GM3 expression level was increased in cisplatin-resistant strains in human small cell lung cancer cells but GM2 expression level was increased in adriamycin-resistant strains (Kiura k. Et al, Gnaglioside composition of Human Melanoma And Response To Antitumor Treatment, Cancer Invest, 1990, Vol. 8, p, 161-167 (Non-patent Document 7)). This report does not discuss non-small cell lung cancer.
Non-small cell lung cancers such as adenocarcinoma, squamous cell carcinoma, and large cell carcinoma are less sensitive to chemotherapy and radiation therapy and treatment methods are significantly different than small cell lung cancer.

There is also a report on melanoma (Kono k. Et al, ganglioside composition of human melanoma and response to antitumor treatment, Cancer Invest, 1990, Vol. 8, p. 161-167 (Non-patent Document 8)). This report has not been studied for human non-small cell lung cancer. In melanoma, in addition to GM3, GM2, GD3, and GD2 are expressed, and there is a positive correlation between the expression level of GD2 and the sensitivity to radiotherapy and anticancer drugs, and the negative expression level of GM3 and the sensitivity to radiotherapy and anticancer drugs The correlation is obtained. Therefore, in melanoma, both GM3 expression level and GD2 expression level affect the sensitivity to radiation therapy and anticancer agents, and it can be said that it is important to examine the expression level of GM3 and GD2 therapy. When the present inventor examined non-small cell lung cancer patient tissue, in non-small cell lung cancer, GM3 is the main ganglioside and the others are small, and that GM3 synthase gene expression level can be an indicator of GM3 expression. To the present invention.
International publication WO 2004/099407 Onn A. et al., Br J. Cancer, 2004 Aug, 91 Suppl 2: S11-7 Tamura K. et al., Int. J. Clin. Oncol., 2003 Aug 8 (4): 207-11 Kobayashi H., et al., Recent Results Cancer Res., 2003, 161: 48-61 Suzuki T., et al., Lung Cancer, 2003 Oct., 42 (1): 35-41 June L. et al., Cancer Metastasis Rev., 1994, Vol. 13, P. 191-207 Benchekroun. Et al., Anticancer Res. 1998, Vol. 18, p. 2957-2960. Kiura K. et al., Cancer Invest, 1990, Vol. 8, p, 161-167 Kono K. et al., Cancer Invest, 1990, Vol. 8, p.161-167

  The present invention provides a discriminating method capable of clarifying the relationship between an anticancer agent used in non-small cell lung cancer treatment and a gene marker, determining the characteristics of the anticancer agent, and selecting an anticancer agent that is effective for the patient. Objective.

The present inventors examined in detail the correlation between the SAT-I gene expression level of the non-small cell lung cancer strain and the anticancer drug sensitivity. As a result, IC ₅₀ and SAT-I mRNA expression levels for various non-small cell lung cancer strains were positively correlated with certain anticancer agents, while IC ₅₀ and SAT- for various non-small cell lung cancer strains were positively correlated with other types of anticancer agents. I mRNA expression level was found to show a negative correlation. That is, certain types of anticancer drugs have high IC _{50 in} cell lines with high SAT-I mRNA expression levels, and anticancer drugs are difficult to work with, while other types of anticancer drugs have low IC ₅₀ levels in cell lines with high SAT-I mRNA expression levels. It was found that the cancer effect was high.
That is, the gist of the present invention is a method for determining the characteristics of an anticancer agent, including determining the correlation between the expression level of the ganglioside GM3 synthase gene (SAT-I) in non-small cell lung cancer cells and the anticancer effect of the anticancer agent.

  If the characteristics of the anticancer agent are determined in this manner, the amount of SAT-I mRNA in cancer cells of patients with non-small cell lung cancer is quantified, and if the expression is high, an anticancer agent having a negative correlation is administered, If it is low, an anticancer agent having a positive correlation can be administered, and thus an anticancer effect can be obtained with a minimum amount of anticancer agent, and administration of useless anticancer agent can be avoided, and side effects can be reduced and treatment can be expected to be effective.

Non-small cell cancer can be applied to any of adenocarcinoma, squamous cell carcinoma, and large cell carcinoma.
The expression level of the SAT-I gene can be determined by measuring SAT-I mRNA or SAT-I protein expressed from the gene of cancer cells. The detection method is not particularly limited as long as it can detect SAT-I mRNA or protein and can compare the expression level between anticancer drug sensitive and resistant cells.

  The expression level of the ganglioside GM3 synthetic gene can be measured by measuring SAT-I mRNA, typically by Northern blotting. Northern blotting is a technique well known to those skilled in the art. In Northern blotting, RNA (mRNA) is extracted from cells, the RNA (mRNA) is electrophoresed, the pattern is transferred to a filter, and hybridized with a specific labeled probe labeled with an isotope or the like. To analyze the presence and amount of mRNA in the specimen.

  As a probe for detection in the hybridization, double-stranded DNA, single-stranded DNA or RNA is used. When mRNA is specifically detected by Northern blotting, single-stranded DNA or RNA having a complementary sequence to the mRNA itself is used as a probe. The chain length of the probe used is relatively arbitrary, and synthetic oligonucleotides of about 20 to 40 bases and DNA or RNA probes having a length of several hundred to 1 kbp can be used. When the DNA sequence to be synthesized is a relatively short DNA probe, a specific sequence portion of the gene to be detected is used. By using a tool for confirming gene homology (http://www.ncbi.nlm.nih.gov/Blast), it is possible to search for a sequence unique to the gene to be detected.

  There are two types of probe labeling methods used for hybridization: radiolabeling and non-radiolabeling. Radiolabeled oligonucleotides can be prepared mainly by PCR based on 32P-labeled nucleotides. However, this method has a problem of radioactive waste disposal and is not simple. Non-radioactive labeling uses a probe molecule with a special modification group added, and the added modification group is detected by fluorescence, chemiluminescence, or color development. Since there is no significant difference in the detection sensitivity of the nucleic acid detection method using this non-radioactive labeling method and the sensitivity of the radiolabeling method, it is relatively safe, and this non-radioactive labeling method should be used. Is desirable. As examples of non-radioactive labels, digoxigenin (DIG) labeling, biotin labeling using biotin and (strept) avidin, FITC (fluorescene isothiocyanate) molecules that emit fluorescence, and the like can also be applied.

As a simple method for preparing a non-radioactive labeled probe, a method of adding a labeled nucleotide to the 5 ′ end during PCR operation can also be used. Examples of the labeling substance include biotin and digoxigenation. Oligonucleotide probes have the advantage of being able to produce a uniform mass in a relatively large amount, but problems remain in terms of sensitivity. When a label is added to a portion other than the probe end, a method for synthesizing a labeled DNA by the nick translation method can also be used.
When RNA molecules such as mRNA are detected by Northern blotting, the detection probe needs to be single-stranded. The preparation of a single-stranded DNA probe includes the use of oligonucleotides and random PCR. As the single-stranded probe, a method using an RNA probe is preferable because of its high sensitivity. An RNA probe synthesizes an antisense RNA using a DNA sequence cloned upstream of a promoter sequence of an RNA polymerase such as T7 or Sp6 as a template. At this time, a labeled RNA probe can be prepared by using a labeled nucleotide as a substrate.

A medium for culturing cancer cells (RPMI-1640 or DMEM, manufactured by Sigma) contains 10% of calf serum and antibiotics such as penicillin and streptomycin. Cells are usually cultured in 10 cm dishes in an incubator in the presence of 5% CO ₂ . The culture temperature is usually 37 ° C.
Cells are cultured in a 10 cm dish until confluent and washed 3 times with PBS (phosphate-buffered saline). Add 1 ml of Trizol (phenolic reagent for RNA extraction, manufactured by Ghizogo Co., Ltd.), let stand at room temperature for 2 minutes, then scrape and collect in a 1.5 ml tube. The cells are completely lysed by pipetting and then incubated for 10 minutes at room temperature. After adding 200 μl of chloroform and suspending for 30 seconds, the mixture is allowed to stand for 2 minutes, and centrifuged at 4 ° C. and 12,000 rpm for 15 minutes to perform phase separation. Transfer the upper layer to a new 1.5 ml tube, add an equal volume (500 μl) of cold isopropanol, suspend for 10 seconds, let stand for 10 minutes, and centrifuge at 4 ° C., 12,000 rpm for 10 minutes to precipitate RNA. . The supernatant is decanted, 1 ml of 70% ethanol-DEPC (diethyl pyrocarbonate) -treated water is added, lightly suspended and washed, centrifuged at 12,000 rpm at 4 ° C. for 5 minutes, and then decanted to remove salts. The tube is dried with a desiccator to completely remove water, and then RNA is dissolved with 20 μl of DEPC-treated water. Storage is performed at -80 ° C.

  Although it is desirable to purify the total RNA to mRNA, it is possible to analyze the total RNA as it is. It is placed in a solution of formamide and formalin, denatured at 55 ° C., and then electrophoresed on an agarose gel containing formalin. The gel is then transferred to a nitrocellulose or nylon filter with a high salt solution of 15-20 × SSC. After the total RNA is attached to the filter, in the case of nitrocellulose, it is treated in a vacuum oven at 80 ° C. for about 2 hours to immobilize the total RNA. In the case of a nylon membrane, it is fixed by applying ultraviolet rays for a while to form a crosslink. Next, in order to identify SAT-I mRNA on this filter, a probe derived from SAT-I cDNA is prepared. When a single-stranded probe and a filter are brought into contact with each other under specific conditions, those complementary to each other bind. Here, if the probe is labeled with a radioactive substance, only the bound SAT-I mRNA can be detected. The hybridization conditions are 5-6 × SSC, temperature 65 ° C. (42 ° C. in the presence of formamide).

  mRNA can also be measured by the RT-PCR method. In RT-PCR, RNA is first reverse transcribed into cDNA using reverse transcriptase, then PCR is performed using this cDNA as a starting material and a specific primer set and a heat-resistant DNA polymerase, and the target RNA Is detected and quantified in the form of amplification of its cDNA.

  The expression level of the ganglioside GM3 synthetic gene can be measured by measuring the protein expressed from the gene, typically using Western blotting. Cells are cultured in a 6-well dish, extracted with a protein extraction buffer (10 mM Tris-HCl, 150 mM sodium chloride, 5 mM ethylenediaminetetraacetate (EDTA)) containing a surfactant (1% TritonX-100), and on ice Centrifuge for 10 minutes at 15,000 rpm and collect the supernatant by centrifugation at 15,000 rpm for 30 minutes.

  Western blotting is a method in which a protein is fractionated by polyacrylamide gel electrophoresis (SDS-PAGE) containing SDS, transferred to a nitrocellulose filter, and this is detected using an antibody. The protein is transferred from the polyacrylamide gel to a suitable filter such as a nitrocellulose film. The protein attached to the filter can be identified by using a SAT-I protein antibody as a probe. For example, a peroxidase-conjugated secondary antibody method is used for antibody detection. Transfer from gel to filter is often done by electroblotting. That is, a gel on which a protein is developed by electrophoresis and a filter are brought into close contact with each other, and a current is passed for a certain period of time with the filter as the anode side and the gel side as the cathode. This will transfer the protein from the gel to the filter. Several hours later, this membrane is taken out, reacted with an antibody that specifically binds to the SAT-I protein, and then the antibody is stained.

  Methods for producing antibodies are well known. The SAT-I protein or fragment thereof is used to induce antibodies. Animals such as rabbits, rats, and mice are immunized by intraperitoneal and / or intradermal injection of an emulsion containing, for example, 100 μg protein and Freund's adjuvant. For example, the antibody can be detected by an ELISA assay using a protein adsorbed on a solid surface. To obtain a high titer, booster injections are required several times, for example at intervals of about 2 weeks. As the antibody, a monoclonal antibody (Harlow, E and Lane, D. (1988), Antibodies: A LABORATORY MANUAL, Cold Spring Harbor Laboratory) can be used in addition to the polyclonal antibody obtained by the above-described method.

  As a protein detection method, a detection method using fluorescence microscope, observation with laser microscope and image processing, measurement by flow cytometry, evanescent light, etc. without pulverizing cells is possible.

  Fluorescene-activated cell sorter (FACS) is a device that fluorescently labels cells with antibodies and sorts the cells according to the fluorescence intensity. The experimental method using this device is called flow cytometry. . For example, an anti-GM3 synthase is allowed to act on the cells, and then an FITC (Fluorescein isothiocyanate) labeled antibody that reacts with the anti-GM3 synthase is allowed to act on the cells. Whether or not GM3 synthase is highly expressed can be determined by subjecting the cells to FACS and measuring the intensity of fluorescence intensity.

（式中、
Ｒ_１は、ＣＨ_３０，または

を表し、
Ｒ_２は水素、またはフルオロを表す）
で表される化合物である。
実施例 The anticancer agent is not limited at all. Examples of anticancer agents in which the correlation between the expression level of ganglioside GM3 synthase gene (SAT-I) in non-small cell lung cancer cells and the anticancer effect of the anticancer agent is positive are platinum preparations such as cisplatin and carboplatin, negative An example of a correlative anticancer agent is an EGFR (epidermal growth factor receptor) tyrosine kinase inhibitor. Examples of EGFR (epidermal growth factor receptor) tyrosine kinase inhibitors have the general formula

(Where
R ₁ is CH ₃ 0, or

Represents
R ₂ represents hydrogen or fluoro)
It is a compound represented by these.
Example

First, the experimental methods used in the following Examples 1 to 3 will be described.
-Cell culture The following non-vesicular cancer cells were used.
Human lung adenocarcinoma cell lines (PC3, LCSC # 1, LCSC # 2, NCI-H23, ABC-1)
Human lung squamous cell carcinoma cell lines (NCI-H226, LK-2, EBC-1)
Human lung cell carcinoma cell lines (OBA-LK1, Lu99, Lu99B, Lu65)
A 10% fetal bovine serum (FBS),
These cells were cultured in an RPMI1640 medium containing 100 units / mL penicillin and 100 ng / mL streptomycin (hereinafter, the medium is referred to as this medium unless otherwise specified).
PC3, NCI-H23, NCI-H226, and ABC-1 were kindly provided by Associate Professor Katsuichi Inoue, Hokkaido University. Others were obtained from Tohoku University Institute of Aging Medicine, Medical Cell Resource Center.

-Extraction and purification of total RNA from cell line Each cell is cultured in a 10 cm dish until confluent, washed with physiological saline, 1 mL of Trizol (phenol reagent for RNA extraction, Gibco) is added, and the mixture is allowed to stand at room temperature for 2 minutes. After being placed, it was scraped and collected in a 1.5 mL tube. The cells were completely lysed by pipetting and then incubated at room temperature for 10 minutes. After adding 200 μL of chloroform and suspending for 30 seconds, the mixture was allowed to stand for 2 minutes and centrifuged at 12,000 rpm at 4 ° C. for 15 minutes to perform phase separation. The upper layer was transferred to a new 1.5 mL tube, 500 μL of cold isopropanol was added, suspended for 10 seconds, allowed to stand for 10 minutes, and centrifuged at 12,000 rpm at 4 ° C. for 10 minutes to precipitate RNA. The supernatant was discarded, 1 mL of 70% ethanol-DEPC (diethyl pyrocarbonate) -treated water was added, and the suspension was lightly suspended and washed. After centrifugation at 12,000 rpm for 5 minutes at 4 ° C., the supernatant was removed and the salt was removed. The tube was dried with a desiccator to completely remove ethanol, and then RNA was dissolved with 20 μL of DEPC-treated water.

・ Reverse transcription of extracted total RNA
1st Strand cDNA Synthesis kit for RT-PCR (AMV) was used.
Extracted and purified total RNA (2.5 μg) was diluted with RNase-free water to 23.2 μl. To this, 5 μl of 10 × reaction buffer, 10 μl of 25 mM MgCl ₂ , 5 μl of deoxynucleotide Mix, and 5 μl of random primer were added, stirred well, spun down, and incubated at 65 ° C. for 5 minutes. 1 μl of RNase inhibitor and 0.8 μl of AMV reverse transcriptase were added, and incubated at room temperature for 10 minutes, then at 42 ° C. for 60 minutes, and then at 95 ° C. for 5 minutes to synthesize cDNA. Real-time PCR
Real-time PCR was performed using the synthesized cDNA as a template and a specific probe / primer set for SAT-I and endogenous control (RPLP0; ribosomal protein Large P0). A 7500 real-time PCR system (ABI PRISM) was used for detection and quantification. Shaking due to experimental techniques was corrected by endogenous control, and the amount of SAT-I mRNA expression was quantified.
The probes and primers used are as follows.
Human SAT-I
hSAT-I probe 5'-FAM-gaaaccctgccattctgggtacgac-MGB-3 '(FAM represents fluorescein carboxamide, and MGB represents minor groove binder (Tm enhancer))
hSAT-I forward primer 5'-gcgcaccactgtctgacctt-3 '
hSAT-I reverse primer 5'-ttctgccacctgcttccaa-3 '

Human RPLP0 represents Pre-Developed TaqMan (registered trademark) Assay Reagents Human RPLP0 (Applied Biosystems).

（式中、ｙは阻害率（％）を表し、Ｅｍａｘは最大阻害率を表し、Ｅｍｉｎは最小阻害率を表し、γは定数を表し、Ｘは濃度を表す） Anticancer agent sensitivity test Cells were seeded in 96-well plates, 1 × 10 ^{4 for} PC3, which was very slow in growth, and 5 × 10 ^{3 for} other cells, and precultured in a medium for 24 hours. In the preparation of the drug solution, cisplatin was dissolved in DMSO (dimethyl sulfoxide) at 300 mM, carboplatin was dissolved in 3 mM in the medium, and AG1478 was dissolved in DMSO at 10 mM. These were used as stock solutions and diluted appropriately with each solvent. These chemical solutions were added to the medium, and a medium containing a chemical solution was prepared so that cisplatin had a final concentration of 1 nM to 3 mM, carboplatin had a final concentration of 1 nM to 3 mM, and AG1478 had a final concentration of 0.1 nM to 100 μM. The cell culture medium preliminarily cultured at this time was replaced with 100 μL of the medium containing these chemical solutions and a medium containing only the solvent in which the drug was diluted (no drug contact), and cultured for 70 hours. After 70 hours, 10 μL of each cell counting reagent Cell Counting Kit-8 was added, and after 2 hours, the absorbance of the medium at 450 nM was measured. From this absorbance value, the inhibition rate (%) = (1−drug contact group / drug non-contact group) × 100
The inhibition rate (%) was calculated according to the above formula, and using the analysis software Origin, the inhibition rate (%) was taken on the Y-axis and the logarithm of concentration was taken on the X-axis to create a concentration-inhibition effect curve. Further, IC ₅₀ was calculated by fitting to the following equation (sigmoid curve).

(In the formula, y represents the inhibition rate (%), Emax represents the maximum inhibition rate, Emin represents the minimum inhibition rate, γ represents a constant, and X represents the concentration)

Using the above experimental method, the relationship between cisplatin IC ₅₀ and SAT-I mRNA expression level for various non-small cell lung cancers was examined (FIG. 1). It was observed that the IC ₅₀ value was higher in the cell line with higher SAT-I mRNA expression level. That is, it was found that the SAT-I mRNA expression level was positively correlated with the IC ₅₀ value, and that the anticancer effect of cisplatin was low when the SAT-mRNA expression level was high. In other words, it turned out to be ineffective.

Using the above experimental method, the relationship between IC ₅₀ and SAT-I mRNA expression level of carboplatin for various non-small cell lung cancers was examined (FIG. 2). It was observed that the IC ₅₀ value was higher in the cell line with higher SAT-I mRNA expression level. That is, it was found that the SAT-I mRNA expression level and the IC50 value had a positive correlation, and the anticancer effect of carboplatin was low when the SAT-mRNA expression level was high.

Using the above experimental method, the IC ₅₀ and SAT of AG1478 (4- (3-chloroanilino) -6,7-dimethoxyquinazoline), an (epidermal growth factor receptor) tyrosine kinase inhibitor, against various non-small cell lung cancers. -I mRNA expression level was examined (FIG. 3). It was found that the IC ₅₀ value decreased as the SAT-I mRNA expression level increased. That is, it was found that SAT-I mRNA expression level and IC ₅₀ value had a negative correlation, and that non-small cell cancer with high SAT-I mRNA expression level had a high anticancer effect of AG1478.

  According to the results of Examples 1 to 3, the SAT-I mRNA expression level of cancer cells of patients with non-small cell cancer is measured, and when the expression level is low, a platinum preparation that inhibits DNA synthesis such as cisplatin and carboplatin is used. It is conceivable that a two-drug combination therapy of combining paclitaxel, gemcitabine, etc. may be performed, and if the expression level is high, molecular target therapy targeting EGFR may be performed.

The present invention provides knowledge about the properties and usage of anticancer agents.

Shows the relationship between the _{IC 50} and the SAT-I mRNA expression level to various non-small cell lung cancer cisplatin. Shows the relationship between the _{IC 50} and the SAT-I mRNA expression level to various non-small cell lung cancer carboplatin. Shows the relationship between the _{IC 50} and the SAT-I mRNA expression level to various non-small cell lung cancer AG1478 (4- (3- chloroanilino) 6,7-dimethoxyquinazoline).

Claims (12)

  A method for characterization of an anticancer agent, comprising determining a correlation between the expression level of a ganglioside GM3 synthase gene (SAT-I) in non-small cell lung cancer cells and the anticancer effect of the anticancer agent.   The method according to claim 1, wherein the non-small cell cancer is adenocarcinoma, squamous cell carcinoma, or large cell carcinoma.   The method according to claim 1 or 2, wherein the expression level of the ganglioside GM3 synthase gene (SAT-I) is measured by the amount of mRNA expressed from the gene.   The method according to claim 3, wherein the mRNA expression level is measured by Northern blotting.   A probe comprising a DNA having the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof, or a DNA corresponding thereto, which is used for measuring the mRNA expression level according to claim 3 or 4.   The method according to any one of claims 1 to 2, wherein the expression level of the ganglioside GM3 synthase gene is measured for an expressed protein comprising the gene.   The method according to claim 6, wherein the protein expression level is measured by Western blotting.   The antibody with respect to the protein which has the amino acid sequence of sequence number 2, or its fragment used in order to measure the protein expression level of Claims 6-8.   The method according to any one of claims 1 to 7, wherein the anticancer agent is a platinum preparation.   The method according to claim 9, wherein the platinum preparation is cisplatin or carboplatin.   8. The method according to any one of 1 to 7, wherein the anticancer agent is an EGFR (epidermal growth factor receptor) tyrosine kinase inhibitor.   12. The method according to claim 11, wherein the EGFR (epidermal growth factor receptor) tyrosine kinase inhibitor is AG1478 (4- (3-chloroanilino) -6,7-dimethoxyquinazoline) or a derivative thereof.

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