KR20100099442A - Gemcitabine-resistant human bladder cancer cell line - Google Patents

Abstract

PURPOSE: A bladder cancer cell line is provided to ensure resistance to gemcitabine and to use for treating bladder cancer. CONSTITUTION: A human bladder cell line T24GR10 has a resistance to gemcitabine. The cell line has high Glutathione S transferase pi(GSTpi) gene expression level. The cell line is isolated by gradually treating gemcitabine to T24 cells from low concentration to high concentration and culturing.

Description

Gemcitabine-resistant bladder cancer cell line {GEMCITABINE-RESISTANT HUMAN BLADDER CANCER CELL LINE}

The present invention relates to bladder cancer cell lines, more particularly bladder cancer cell lines resistant to Gemcitabine.

Bladder cancer is the most common urinary cancer in Korea, ranking fifth among cancers in South Korea in 1997, and it occurs in over 200,000 men and 60,000 women annually worldwide (Glas AS, et. Tumor markders in the diagnosis of primary bladder cancer.A systematic review.J Urol 2003; 169 ”975-82). Among patients with bladder cancer, metastases developed at the time of initial diagnosis, 50% of patients with invasive bladder cancer, and 10-15% of patients with superficial bladder cancer eventually progress and eventually die of bladder cancer. In metastatic bladder cancer, cisplatin-based chemotherapy (MVAC, GC, etc.) has been mainly used, and the combination of gemcitabine-cisplatin is currently the standard of care (von der Maase H, et al. Gemcitabine and cisplatin versus methotrexate, vinblastine, doxorubicin, and cisplatin in advanced or metastatic bladder cancer: results of a large, randomized, multinational, multicenter, phase III study.J Clin Oncol 2000; 18: 3068-77)

Gemcitabine, a deoxycytidine analogue (2,2-difluorodeoxycytidine), is an anticancer drug approved by the Food and Drug Administration (FDA) for the treatment of advanced pancreatic cancer, lung cancer, ovarian cancer, breast cancer, and bladder cancer. (Moore et al. Gemcitabine. In: Ratain MJ, Tempero M, Skosey C, editors. Outline of oncology therapeutics. 1st ed. Philadelphia: Saunders; 2001; 51). In particular, the effect of increasing the action of cisplatin, one of the very effective anticancer agents used in the treatment of various types of cancer, the combination therapy of gemcitabine and cisplatin has been applied to cancer of many organs.

However, the therapeutic effect is limited due to the expression of cancer cells to anticancer drugs. Therefore, drug resistance to gemcitabine is a very important problem in treating various types of cancer including bladder cancer. The mechanisms of resistance to gemcitabine studied in several cancer cell lines have been reported (Bergman et al. Determinants of resistance to 2 ', 2'-difluorodeoxycytidine (gemcitabine). Drug Resist Updat 2002; 5: 19- 33).

Therefore, various studies have been conducted to treat cancers that have become resistant to gemcitabine using proteins and genes of cancer cells that induce resistance to gemcitabine.

However, to date, studies on which genes or proteins induce gemcitabine resistance in gemcitabine-resistant bladder cancer and studies on how to effectively treat bladder cancer resistant to gemcitabine are insufficient.

Therefore, in the present invention, to establish a cell line showing resistance to gemcitabine among bladder cancer cell lines, and to identify genes related to gemcitabine resistance by identifying the difference in gene expression after gemcitabine treatment in cells showing resistance to gemcitabine. It was.

The present invention is to provide a human bladder cancer cell line resistant to gemcitabine in order to solve the problems of the prior art as described above.

The present invention also aims to provide genes related to gemcitabine resistance by identifying gene expression differences after gemcitabine treatment in cells showing resistance to gemcitabine.

For this purpose, the present invention provides a human bladder cancer cell line resistant to gemcitabine.

Human bladder cancer cell line T24 introduced from the American Type Culture Collection, Rocksville, MD (ATCC) was used to induce gemcitabine resistant cell lines. T24 is a cell line established from bladder cancer tissue collected from 81-year-old white women, and is widely used in experiments because it shows various characteristics of bladder cancer.

By treating the bladder cancer cell line T24 cells with a concentration of gemcitabine gradually increased from a low concentration, the cell mutant strains that were resistant to gemcitabine were isolated and confirmed through a cytotoxicity test. January 30, 2009 It was deposited with the Institute of Biotechnology Research Institute Gene Bank (Accession Number KCTC 11461BP).

In addition, the present invention observed the ultrafine structure of the cancer cell line obtained the resistance to gemcitabine by electron microscopy. In addition, the present invention compared the gene expression in the cancer cell line obtained resistance to gemcitabine and the gene expression in the cancer cell line without obtaining resistance compared with the DNA microarray method, microarray method RT-PCR was performed on the basis of the increase in the expression of glutathione S transferase pi (GSTpi).

The present invention provides a gene associated with cancer cell lines and gemcitabine resistance that have obtained resistance to gemcitabine, and explores drug resistance mechanisms of gemcitabine in the future, and effectively uses the gene to bladder cancer that has become resistant to gemcitabine. It is expected to be used as a basis for treatment.

Hereinafter, the present invention will be described in more detail with reference to Examples.

The following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

Example 1 Induction and Culture of Gemcitabine Resistant Cell Lines

In order to induce a gemcitabine resistant cell line, a bladder cancer cell line T24 introduced from the American Type Culture Collection, Rocksville, MD (USA) was used. T24 is a cell line established from bladder cancer tissue collected from 81-year-old white women, and is widely used in experiments because it shows various characteristics of bladder cancer.

Medium for cell line culture was 10 mM HEPES-buffer (Gibco Laboratories), 100 units / ml penicillin and 100 μg / ml streptomycin in RPMI 1640 medium (Gibco Laboratories, Grand Island, NY). ) (penicillin-streptomycin solution, Gibco Laboratories) and 10% fetal bovine serum (Gibco Laboratories) boiled at 56 ° C. for 30 minutes were used. T24 cell lines were grown by incubating at 37 ° C. in a 5% CO ₂ -95% air thermohygrostat and adhering to a single cell layer.

Gemcitabine-resistant cell lines are treated with gemcitabine at low concentrations of 10 ng / ml and gradually grown in increasing concentrations to select resistant cell lines with a dose of 1 μg / ml, 5 μg of gemcitabine. T24 cells were cultured at / ml and 10 μg / ml to establish a cell line resistant to each concentration.

First, about 10 ml of phosphated buffered saline (PBS) or RPMI culture medium was dispensed into a 15 ml tube, and the T24 cell line frozen in cryovial was quickly dissolved in a 37 ° C. water bath. After 70% ethanol spray, the cells were transferred to a 15 ml tube containing PBS and centrifuged at 1500 rpm for 5 minutes in a centrifuge. After centrifugation, the supernatant was removed, suspended in about 10-12 ml of culture, transferred to a T75 flask, and cultured in a 37 ° C. incubator.

The culture medium of the T75 flask was removed, washed with about 10 ml of PBS, 1 ml of trypsin-EDTA was added, and then evenly spread and incubated at 37 ° C. for 5 minutes. The cells were collected in the culture medium and then transferred to a conical tube and centrifuged at 1500 rpm for 5 minutes. After centrifugation, the supernatant was removed, suspended in culture, and then moved to T75 flask for about 1/4 to incubate 10-12 ml of the culture solution. Longitudinal strains resistant to gemcitabine at 1 μg / ml, 5 μg / ml, and 10 μg / ml were named T24GR1, T24GR5, and T24GR10, respectively.

Example 2 Cytotoxicity Test with Increasing Gemcitabine Concentration in Gemcitabine Resistant Cell Line

Cell Counting Kit 8 (CCK-8 assay, Dojindo Molecular Technologies, Inc.) was used for cytotoxicity experiments on gemcitabine resistance of established gemcitabine resistant cell lines.

Dispense the cell line into a 96 well plate (1X10 ⁵ / well), incubate for 1 day in an incubator (5% CO ₂ , 37 ° C), add 10 μl of gemcitabine at various concentrations, and add CCK-8 to the medium. Incubated for 4 hours. After 4 hours of incubation, the absorbance was measured using a microplate reader (OD 450 nm). Gemcitabine resistant cell lines T24GR1, T24GR5, and T24GR10, including the parental cell line T24, were cultured in media containing gemcitabine at various concentrations, respectively, and subjected to cytotoxicity experiments using CCK-8 assay. It was.

As shown in FIG. 1, the T24GR1, T24GR5, and T24GR10 cultured in a medium containing various concentrations of gemcitabine showed a high absorbance in a constant pattern as the concentration of the drug increased, resulting in a decrease in cell viability. Compared with parental cell line T24, drug-resistant cell lines showed higher cell viability even at higher drug concentrations, resulting in higher survival rate of drug-resistant cells than parental cells. In addition, even among drug-resistant cell lines, the cell lines that were resistant to high concentrations of drugs showed higher survival at the same drug concentrations than the cell lines that established resistance at low concentrations of drugs.

Example 3 Analysis of Morphological Changes of Ultrafine Structures

Bladder cancer cell lines and all T24GR1, T24GR5 and T24GR10 cells were ultrafine cut at pre-fixed and post-fixed 60nm and observed from 2,700 times to 14,000 times with H-7000 transmission electron microscope. At low magnification, the cell shape, cytoplasm, and nucleic acid were observed. At high magnification, intracellular organelles were observed and compared with the parent cell line.

Figure 2 shows the chromatin shape and overall morphology of the bladder cancer cell line (T24) and gemcitabine obtained resistance to 224 times electron microscope (T24GR1, T24GR5, T24GR10). The T24GR1, T24GR5, and T24GR10 cell lines obtained resistance to gemcitabine were found to be more abundant in cytoplasm than T24. In contrast to the heterochromatin form of T24, the chromatin of the T24GR family cells, which are resistant, is small and has euchromatin form. Cell processes were observed more in T24GR than in T24, and with increasing resistance levels, the cell processes were shorter and rounder.

Figure 3 shows the mitochondria observed with bladder cancer cell line (T24) and cell lines (T24GR1, T24GR5, T24GR10) obtained resistance to gemcitabine with a 14000 magnification electron microscope. Mitochondria of intracellular organelles were observed in various sizes of high-density densities in which cristase was lost compared to parental cells in T24GR.

Intracellular organelles of T24GR had increased golgi complex and rough endoplasmic reticulum compared to parental T24 (FIG. 4).

Example 4. DNA microarray analysis

The DNA chip (AB 1700 Full Genome Expression Human Microarray) was used to determine the difference in gene expression of bladder cancer cell line T24 and the cell line T24GR1 that acquired resistance to gemcitabine.The procedure was repeated three times to obtain an average value for analysis. It was. Gene path analysis and functional classification were performed using the PANTHER classification system (Applied Biosystems). Data normalization was performed with 253 non-human control genes mounted on the chip and control RNA included in the labeling kit. The microarray slides were scanned with a custom-built laser scanner and the image files were analyzed with a Dchip software package.

When the path distribution of the expressed genes is statistically compared with the known human (H. sapiens) genes, the genes expressed by more than two times increase in resistant cell lines are shown in Table 1 below.

Biological pathway Normal human expression gene count (NCBI: H. sapiens) Number of genes expressing resistant cell line (Gene Hits; T24GR1 / 5) P value (T24GR1 / 5) Phosphatidylinositide 3 kinase (PI3kinase) pathway 90 19/18 4.85E-03 / 1.27E-02 P53 path 139 21/25 7.73E-02 / 1.33E-02 Apoptosis Signaling Pathway 131 23/24 2.24E-03 / 9.97E-04

There are 14 (p <0.05) genes expressed by more than twofold increase in T24GR1 relative to the phosphatidylinositide 3 kinase pathway, a significant increase in cyclin D1 (4.50-fold increase) associated with activation of this pathway. An increase was seen.

Meanwhile, genes involved in the p53 pathway increased in both T24GR1 and T24GR5, but there was no statistical significance in T24GR1 (p> 0.05) (Table 1).

Among genes expressed by more than twofold reduction, the frequency of genes involved in the apoptosis signal pathway was significantly higher in both T24GR1 and T24GR5 (Table 1). In T24GR1, 10 genes such as BAD, protein kinase C, DAXX, ATF4, and FOSL1 showed decreased expression (p <0.05).

The results of analyzing genes involved in detoxification are shown in Table 2 below. As shown in Table 2, expression levels of genes related to three translational processes of glutathione S transferase pi (GSTpi), MDR1 and GAPDH were significantly increased (p <0.05).

Region Total Density (Gray Lev) Sample / GAPDH BLANK 0 T24-GSTpi 18463.5 0.383874905 T24GR1-GSTpi 19405.9 0.799458676 T24GR5-GSTpi 19698.8 0.995628067 T24GR10-GSTpi 40915.2 2.525239932 T24-MDR1 0 0 T24GR1-MDR1 48.1667 0.001984308 T24GR5-MDR1 449.583 0.022723082 T24GR10-MDR1 104 0.006418763 T24-GAPDH 48097.7 T24GR1-GAPDH 24273.8 T24GR5-GAPDH 19785.3 T24GR10-GAPDH 16202.5

Example 5. Investigation of Gene Expression by RT-PCR

In Table 2, after confirming the numbers of the genes GSTpi, MDR1, and GAPDH whose changes in expression were observed in the microarray technique analysis, a primer was prepared from the cDNA sequence of each gene.

Quantitative analysis of GST-pi mRNA from the obtained purified total mRNA was performed using the reverse transcriptase polymerase chain reaction (RT-PCR) method, which is a common method in the art.

Total ribonucleic acid was first extracted as described above. cDNA production was reverse transcription using a superscript choice system (Gibco-BRL, Rockville, MD) using a random hexamer (Gibco-BRL, Rockville, MD). In amplifying the polymerase chain reaction product, the positive control group used a primer specific for the gene of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (forward: TTT GGT ATT GAA GAG GGC CT; reverse: ATT AAA GCC AAA GTA AAA GC) Amplification was performed by removing the reverse transcriptase.

The polymerase chain reaction conditions were 94 ℃ for 30 seconds for 30 cycles, 52-55 ℃ for 30 seconds, 72 ℃ for 30 seconds. The polymerase chain reaction product was digested in a 2% agarose gel containing ethidium bromide and analyzed for the intensity of the band generated by the image analyzer (Bio-Rad, Burlington, MA) under ultraviolet light. The results from the cell lines are compared and shown in FIG. 6.

As shown in FIG. 4, the results of the reverse transcriptase polymerase reaction, which is a semi-quantitative study conducted to identify a part of genes related to drug resistance, indicate that glutathione S transferase pi (GSTpi) gene expression is T24GR1 compared to bladder cancer cell line T24. , 1.1x and 1.1x respectively in T24GR5 and T24GR10. 2.3 times higher. However, multidrug resistant gene 1 (MDR1) was not expressed in bladder cancer cell lines T24 and gemcitabine, all of which obtained cell lines T24GR1, T24GR5, and T24GR10.

Glutathione-S-transferase (GST) -based proteins are involved in the translation of cytotoxic drugs. By catalyzing the conjugation of toxic and carcinogenic electrophilic molecules with glutathione, the GST enzyme protects cellular macromolecules from damage [Boyer et al., Preparation, characterization and properties of glutathione S-transferases. In: Zakim D, Vessey D (eds.) Biochemical Pharmacology and Toxicology. New York, NY: John Wiley and Sons, 1985]. Glutathione S-transferase Pi (GST-pi), a homogeneous isoform of these proteins, is widely expressed in human epithelial tissue and has been demonstrated to be overexpressed in some tumors (Terrier et al., Am J Pathol 1990; 137: 845 -853; Moscow et al., Cancer Res 1989; 49: 1422-1428).

As the expression of glutathione S-transferase Pi (GST-pi) is increased in the human bladder cancer cell line of the present invention, it is a new cell line that differs in genetic properties from the conventional human bladder cancer cell line, thereby improving gemcitabine resistance in bladder cancer. Not only is it an important material to be identified, it will be very beneficial for the development of new bladder cancer and / or bladder cancer therapies.

Figure 1 shows the IC50 changes through the cytotoxicity test for gemcitabine for bladder cancer cell line (T24) and the cell lines (T24GR1, T24GR5, T24GR10) obtained resistance to gemcitabine.

Figure 2 shows the chromatin shape and overall morphology of the bladder cancer cell line (T24) and gemcitabine obtained resistance to 224 times electron microscope (T24GR1, T24GR5, T24GR10).

Figure 3 shows the mitochondria observed with bladder cancer cell line (T24) and cell lines (T24GR1, T24GR5, T24GR10) obtained resistance to gemcitabine with a 14000 magnification electron microscope.

FIG. 4 shows three translational processes of glutathione S transferase pi, GSTpi, MDR1, and GAPDH against bladder cancer cell line (T24) and gemcitabine-resistant cell lines (T24GR1, T24GR5, T24GR10). The expression level of related genes was confirmed by RT-PCR (reverse transcriptase polymerase chain reaction).

Claims (2)

Human bladder cancer cell line T24GR10. The method of claim 1, The human bladder cancer cell line T24GR10 is characterized by high expression of glutathione S transferase pi (GSTpi) genes of human bladder cancer cell line T24GR10.

Images