Olaparib-resistant human ovarian cancer cell strain

Abstract

An olaparib-resistant human ovarian cancer cell strain, which is named A2780-olaparib and is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: C2022153. The cell strain A2780-olaparib can stably grow, passage and resuscitate in 100 nM of olaparib, has a resistance index to olaparib of 28.97, and has cross-drug resistance to cisplatin and paclitaxel in addition to olaparib resistance. In addition, after the drug-resistant cell strain is constructed, the cells are cultured for 3 months in the absence of the drug, then cryopreserved in liquid nitrogen for half a year, and then resuscitated to retest the drug resistance of the cells. The drug resistance of the drug-resistant cell strain is over 95% of the original level, indicating that the drug resistance stability of the drug-resistant cell strain is excellent. A drug-resistant tumor cell model is provided for studying the drug resistance mechanism of tumors, analyzing the sensitivity of chemotherapeutic drugs and screening and evaluating chemotherapeutic drugs, developing tumor resistance reversal drugs, studying more effective methods for treating tumors, etc.

Description

A human ovarian cancer cell line resistant to olaparib Technical Field

[0001] The present invention relates to a human ovarian cancer PARP inhibitor-resistant cell line induced by the PARP inhibitor Olaparib. Background Art

[0002] Malignant ovarian tumors have the highest mortality rate among all gynecological cancers. In the United States, the five-year relative survival rate for ovarian malignancies is approximately 47%, while in China it is approximately 41.8%. Due to its insidious symptoms, 70%-80% of ovarian cancer patients are diagnosed in the advanced stage. Despite improvements in diagnostic technology and treatment methods in recent decades, the five-year survival rate for advanced epithelial ovarian cancer has remained at 30%-40%. In 2005, an article published in Nature first proposed the use of PARP inhibitors and the synthetic lethality of homologous recombination deficiency (HRD) in tumors as a new targeted therapy for BRCA1 / 2 mutant tumors.

[0003] PARP (poly ADP-ribose polymerase) is a key regulator of DNA damage repair and plays a key role in repairing single-strand breaks (SSBs) in DNA through base excision. In clinical applications, PARP inhibitors not only have obvious benefits for people with BRCA mutations, but also have significant therapeutic effects in all people with high-grade serous ovarian cancer. However, from the current situation, resistance to PARP inhibitors has gradually become a major problem that needs to be faced in clinical practice. Studies on patients and mouse models have shown that the efficacy of PARP inhibitors is usually reduced due to the high resistance rate. Currently, PARP inhibitors approved by the US FDA for maintenance treatment of platinum-sensitive recurrent ovarian cancer include Olaparib, Niraparib, and Rucaparib. BRCA mutations or HRD are currently commonly used biomarkers in the application of PARP inhibitors. Based on the clinical research evidence already obtained, the US FDA approved olaparib and rucaparib for the first-line maintenance treatment of ovarian cancer patients who have achieved clinical complete remission and partial remission after initial chemotherapy and carry germline or systemic BRCA1 / 2 gene mutations.

[0004] Based on the results of several in vitro and in vivo studies, several resistance mechanisms have been identified, which can be divided into three main categories: 1. Affecting the intracellular content of PARP inhibitors; 2. Enhancing the efficiency of homologous recombination repair; and 3. Strengthening the stability of replication forks. Due to the relatively short time since the drugs were put into clinical use, our current research on acquired resistance to PARP inhibitors lacks sufficient materials and still has certain limitations. PARP inhibitors are important drugs in the maintenance treatment stage of ovarian cancer after chemotherapy and are now gradually being widely used in clinical practice. Studying the mechanism of resistance of human ovarian cancer cells to PARP inhibitors is of great significance for improving the treatment effect of ovarian cancer patients. Establishing human ovarian cancer PARP inhibitor-resistant cell lines is an important prerequisite and tool for this work.

[0005] Summary of the Invention

[0006] The purpose of this invention is to establish a human ovarian cancer PARP inhibitor-resistant cell line A2780-Olaparib, providing a drug-resistant tumor cell model for related research to study the morphology and biological characteristics of drug-resistant tumor cells, study the mechanism of tumor multidrug resistance, analyze the sensitivity of chemotherapy drugs and screen chemotherapy drugs, and study more effective tumor treatment methods.

[0007] The present invention adopts the following technical solution: Human ovarian cancer olaparib-resistant cell line A2780-Olaparib, whose deposit number is CCTCC NO: C2022153. Classification name: Human ovarian cancer drug-resistant cell line, deposited on May 18, 2022, with the China Center for Type Culture Collection, Wuhan University, Wuhan, China.

[0008] Furthermore, the PARP inhibitor resistance index of A2780-Olaparib was 28.97.

[0009] Furthermore: After the A2780-Olaparib strain was established, the drug was withdrawn and cultured for 3 months. After being frozen in liquid nitrogen for half a year, the cells were revived and the drug resistance was 96.85% of the original.

[0010] The present invention also provides a use of a human ovarian cancer olaparib-resistant cell line A2780-Olaparib, comprising one or more of the following:

[0011] (1) Study the mechanism of tumor resistance in vitro;

[0012] (2) in vitro analysis of chemotherapy drug sensitivity;

[0013] (3) Preparation of tumor cell models, cell line organoid models, and animal tumor models; wherein the tumor cell models include progeny cells established from the present cell line or cells established from progeny cells established from the present cell line by transfection with fluorescently labeled genes. Animal tumor models include animal models of human ovarian cancer established by subcutaneous tumor loading or tail vein injection.

[0014] (4) In vitro screening and evaluation of chemotherapy drugs; the method for screening tumor chemotherapy drugs can be: adding different chemotherapy drugs to the culture medium of the human ovarian cancer olaparib-resistant cell line A2780-Olaparib, observing the drug cytotoxicity, and obtaining preliminary effective candidate drugs. Then, the candidate drugs are administered to the cells, and the half-maximal inhibitory concentration (IC50) of the screened effective drugs is calculated. The drug with the lowest IC50 is selected for further application to an animal model, and the survival, tumor size, metastasis, etc. of the animals in the non-drug group are compared to obtain potential drugs for the treatment of human ovarian cancer.

[0015] (5) In vitro development of tumor resistance reversal drugs. The method for developing tumor resistance reversal drugs can be: adding resistance reversal drugs and olaparib / paclitaxel / cisplatin to the culture medium of the human ovarian cancer olaparib-resistant cell line A2780-Olaparib, observing the drug cytotoxicity, and obtaining preliminary effective candidate drugs. Then, the candidate drugs are administered to the cells, the IC50 of the screened effective drugs is calculated, and the drug with the lowest IC50 is selected for further application to an animal model. The survival period, tumor size, metastasis, etc. of the animals in the non-drug group are compared to screen for potential resistance reversal drugs.

[0016] The present invention has the following beneficial effects: A2780-Olaparib can stably grow, passage, and recover in the presence of 100 nM Olaparib, with a resistance index to Olaparib of 28.97. In addition to resistance to Olaparib, the cell also exhibits cross-resistance to cisplatin and paclitaxel. Furthermore, after establishing the resistant cell line, the drug resistance was increased to over 95% of the original value after three months of culture with withdrawal of the drug and six months of cryopreservation in liquid nitrogen. The stability of the drug resistance of the resistant cell line is excellent. The present invention provides a drug-resistant tumor cell model for studying tumor resistance mechanisms, analyzing chemotherapy drug sensitivity, screening and evaluating chemotherapy drugs, developing drugs to reverse tumor resistance, and researching more effective tumor treatments. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 shows the morphology of A2780-Olaparib cells in the logarithmic growth phase, with a scale bar of 20 μm;

[0018] FIG2 shows the proliferation curves of A2780 and A2780-Olaparib under normal culture conditions and drug-added culture conditions;

[0019] Figure 3 shows the cell resistance index detected by the CCK-8 method;

[0020] FIG4 shows the colony formation assay of A2780 and A2780-Olaparib;

[0021] Figure 5 shows the cross-resistance of A2780 and A2780-Olaparib to cisplatin and paclitaxel. DETAILED DESCRIPTION

[0022] The steps for establishing the drug-resistant cell line of the present invention are as follows:

[0023] (1) Human ovarian cancer A2780 cell line was revived and cultured in RPMI-1640 medium (containing 10 wt% fetal bovine serum) in a 5 vol% CO2, 37°C incubator.

[0024] (2) A2780 cells in the logarithmic growth phase were taken, and fresh culture medium was replaced. Olaparib was added at a concentration of 0.05 μM. The cells were cultured in a 5 vol% CO2, 37°C incubator. When the surviving cells in the culture flask grew to about 80%, the cells were passaged and the culture medium containing 0.05 μM Olaparib was used for subculture.

[0025] (3) After culturing in a medium containing 0.05 μM olaparib for about 4 weeks, the cells are in good condition and are induced with the next concentration of olaparib, and the drug concentration is increased, with the olaparib concentration increasing by 1 fold each time;

[0026] (4) The human ovarian cancer olaparib-resistant cell line A2780-Olaparib was cultured for 6 months until the cell line A2780 could stably grow, passage, and recover in the presence of 1.6 μM Olaparib.

[0027] The biological characteristics of the olaparib-resistant human ovarian cancer cell line A2780-Olaparib were characterized by flow cytometry to detect changes in cell cycle distribution, CCK-8 assay to plot cell growth curves, cell resistance index and stability of the resistant cell line, and clonogenic assay to assess single cell proliferation in the presence of drug. The cell line A2780-Olaparib showed rapid growth and an olaparib resistance index of 28.97. In addition to olaparib resistance, it also exhibited cross-resistance to cisplatin and paclitaxel. Furthermore, after establishing the resistant cell line, 3 months after drug withdrawal and 6 months after cryopreservation in liquid nitrogen, retesting of the cells revealed resistance levels exceeding 95% of the original level, demonstrating excellent stability of resistance. This approach provides new research methods for studying the mechanisms of resistance to PARP inhibitors in human ovarian cancer, analyzing PARP inhibitor sensitivity, screening targeted therapeutics, and developing more effective tumor treatments.

[0028] Specifically, the established A2780-Olaparib cells were subjected to morphological observation and biological characteristics identification:

[0029] 1. Morphological Observation

[0030] One bottle of A2780 and one bottle of A2780-Olaparib cells in the logarithmic growth phase were collected. After changing the medium, cell morphology was observed under an inverted microscope and photographed. As shown in Figure 1, the parental A2780 cells were relatively uniform in size and morphology, with a uniform intracellular structure. However, the drug-resistant A2780-Olaparib cells exhibited a different morphology, with a large accumulation of dark material, especially near the cell envelope. Their cell size and nuclear size were larger than those of A2780 cells.

[0031] 2. CCK-8 method to determine cell growth curve

[0032] Cell proliferation experiments were performed using the CCK-8 kit from Shanghai Biotech Co., Ltd. Human ovarian cancer cell line A2780-Olaparib-resistant and human ovarian cancer cell line A2780 in the logarithmic phase were collected and the cell suspension concentration was adjusted to 2×10 4Cells were plated at a density of 2000 cells / ml in a 96-well plate. Two groups were set up for the olaparib-resistant human ovarian cancer cell lines A2780-olaparib and A2780: A2780-olaparib group, A2780-olaparib plus treatment group, A2780 group, and A2780 plus treatment group, with five replicates per group. Cells were cultured overnight at 37°C in a 5 vol% CO2 incubator until the cell monolayer adhered. For the A2780-olaparib plus treatment group and the A2780 plus treatment group, 10 μM olaparib was added at 24 hours. OD values ​​were measured at 24, 48, 72, and 96 hours after treatment. 10 μl of CCK-8 solution was added to each well. The 96-well plate was placed in a cell culture incubator and incubated in the dark for 2 hours. The absorbance was then measured at 450 nm using a microplate reader. Each experiment was repeated three times, and a cell growth curve was plotted. As shown in Figure 1, it can be seen that the proliferation of the sensitive human ovarian cancer A2780 cell line was inhibited after drug addition, while the proliferation of the human ovarian cancer resistant cell line A2780-Olaparib was not significantly inhibited under drug addition conditions, showing obvious characteristics of a drug-resistant tumor cell line.

[0033] 3. Determination of Cell Resistance Index and Stability of Resistant Cells

[0034] Cytotoxicity experiments were performed using the Cell Count Kit-8 (CCK-8) kit (C0038) from Shanghai Biotech Co., Ltd. Cells were collected in the logarithmic phase and the cell suspension concentration was adjusted to 5×10 4 Cells were cultured at a density of 5000 cells / ml in a 96-well plate. 100 μl of cell suspension was added to each well of the plate and the cells were plated to a density of 5000 cells / well. The cells were cultured overnight in a 37°C, 5 vol% CO2 incubator until the cell monolayer adhered. Olaparib was then added at concentrations of 1 to 256 μM, with 5 replicates for each concentration. OD values ​​were measured 72 hours after drug treatment. 10 μl of CCK-8 solution was added to each well. After incubation in the cell culture incubator for another 2 hours, the absorbance was measured at 450 nm using a microplate reader (Bio-Rad, Model 680), as shown in Figure 3. The IC50 of the drug was calculated as shown in Table 1.

[0035] Resistance index (RI) = IC50 of resistant cells / IC50 of parental cells

[0036] Analysis showed that the resistance index (RI) of A2780-Olaparib cells to olaparib was 28.97, indicating high resistance. After establishing the A2780-Olaparib cell line, the cells were cultured with the drug withdrawn for three months and frozen in liquid nitrogen for six months before being revived. Resistance was then retested and the IC50 values ​​calculated using the same method as above. The results are shown in Table 1. Analysis showed that the resistance index (RI) of A2780-OlaR cells to olaparib was 28.06, indicating high resistance, with a resistance rate of 96.85% of the original resistance.

[0037] Table 1

[0038] 4. Colony Formation Assay

[0039] The clone formation test was stained with crystal violet powder (Sigma-C3886) of Shanghai Sigma High-Tech Co., Ltd. The cells in the logarithmic phase were collected, the concentration of the cell suspension was adjusted to 250 cells / ml, 2 ml of cell suspension was added to each well of a 6-well plate, and the cells were plated so that the density of the cells to be tested was 500 cells / well. The cells were cultured overnight in a 37°C, 5 vol% CO2 incubator until the cell monolayer adhered to the wall, and then olaparib was added at concentrations of 0.1 μM and 0.5 μM, respectively. The medium was changed every 2 days. After 14 days of culture, the cells were stained with crystal violet staining solution (0.05 g crystal violet, 2 ml methanol, 8 ml PBS) and photographed and recorded. The results are shown in Figure 4. Under the condition of drug addition, the clone formation ability of the human ovarian cancer olaparib-resistant cell line A2780-Olaparib was stronger, which further illustrates the obvious characteristics of the drug-resistant tumor cell line.

[0040] 5. Drug resistance spectrum analysis

[0041] Cytotoxicity experiments were performed using the Cell Count Kit-8 (CCK-8) kit (C0038) from Shanghai Biotech Co., Ltd. Cells were collected in the logarithmic phase and the cell suspension concentration was adjusted to 5×10 4 Cells were cultured at a density of 5000 cells / ml in a 96-well plate and 100 μl of cell suspension was added to each well. The cells were plated to a density of 5000 cells / well. The cells were cultured overnight in a 37°C, 5 vol% CO2 incubator. After the cell monolayer adhered, different concentrations of cisplatin and paclitaxel were added, with 5 replicates for each concentration. The OD value was measured 72 hours after drug treatment. 10 μl of CCK-8 solution was added to each well. After further incubation for 2 hours in a cell culture incubator, the absorbance was measured at 450 nm using a microplate reader (Bio-Rad, Model 680), as shown in Figure 5. Analysis showed that A2780-Olaparib cells were cross-resistant to cisplatin and paclitaxel in addition to being resistant to olaparib. See Table 2 for details.

[0042] Table 2

[0043] In summary, the A2780-Olaparib cell line of the present invention significantly slowed its proliferation rate and significantly increased its doubling time; its resistance index to olaparib was 28.97; and its clonogenicity was enhanced under olaparib treatment. In addition to resistance to olaparib, it also exhibited cross-resistance to cisplatin and paclitaxel. Furthermore, after establishing the A2780-Olaparib cell line, the drug resistance was maintained at over 90% of its original value after three months of culture with withdrawal of the drug and six months of cryopreservation in liquid nitrogen, demonstrating the excellent drug resistance stability of this cell line. This cell model can be used to study the mechanism of olaparib resistance in ovarian cancer, analyze chemotherapy sensitivity and screening, and develop more effective tumor treatments.

[0044] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications derived therefrom remain within the scope of protection of the present invention.

Claims

1. A human ovarian cancer olaparib-resistant cell line, named A2780-Olaparib, deposited in China Center for Type Culture Collection with the deposit number: CCTCC NO: C2022153.

2. The cell line according to claim 1, characterized in that The resistance index of human ovarian cancer olaparib-resistant cell line to olaparib is 28.

97.

3. The cell line according to claim 1, characterized in that After the establishment of the human ovarian cancer olaparib-resistant cell line, the drug was withdrawn and cultured for 3 months. The cells were revived after being frozen in liquid nitrogen for half a year, and the drug resistance was 96.85% of the original.

4. Progeny cells of the human ovarian cancer olaparib-resistant cell line according to any one of claims 1 to 3.

5. Use of the human ovarian cancer olaparib-resistant cell line according to any one of claims 1 to 3, characterized in that: include: (1) Study the mechanism of tumor resistance in vitro; (2) in vitro analysis of chemotherapy drug sensitivity; (3) Preparation of tumor cell models, cell line organoid models, or tumor animal models; (4) In vitro screening and evaluation of chemotherapeutic drugs; (5) Develop drugs to reverse tumor resistance in vitro.

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