CN117778322B - A method for screening cell lines resistant to enzalutamide and its application - Google Patents
A method for screening cell lines resistant to enzalutamide and its application Download PDFInfo
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Abstract
The invention belongs to the field of biological medicines, and particularly discloses a screening method and application of a cell line resistant to enzalutamide, which comprises the following steps of 1) resuscitating Lncap cells, carrying out cell plating after Lncap cells grow to a normal state, 2) changing a culture medium into an incomplete culture medium after cell plating, carrying out starvation treatment on Lncap cells, 3) changing the culture medium into a complete culture medium after starvation treatment, adding testosterone and 0.1-1 mg/mL of acetyl nitrosourea ENU, treating for 8-24 hours to induce cell mutation, 4) washing the cells twice with the complete culture medium, changing the complete culture medium, continuously culturing for more than 24 hours, 5) adding the first concentration of enzalutamide, culturing for one week, 6) culturing for one week with the second concentration of the enzalutamide with the increased concentration, 7) culturing for two weeks with the third concentration of the increased concentration of the enzalutamide, 8) after the culture is finished, selecting and freezing and preserving the monoclonal cells, and 9) identifying the cell line resistant to the enzalutamide.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly discloses a screening method of a cell line resistant to enzalutamide and application thereof.
Background
Prostate cancer is the second most common cancer worldwide and is also the fifth leading cause of cancer-related death in men worldwide. Most prostate cancer patients are diagnosed with localized disease, suggesting active monitoring and localized treatment in combination with Androgen Deprivation Therapy (ADT) when needed. Although cure rates after topical treatment are high, with elevated levels of Prostate Specific Antigen (PSA), some patients relapse, which is defined as castration-resistant prostate cancer (CRPC). It is well known that the androgen receptor (Androgen Receptor, AR) pathway is an important component of the pathogenesis of prostate cancer and has therefore been a major target for prostate cancer treatment. AR signaling pathway inhibitors, such as Abiraterone Acetate (AA) and enzalutamide, have been recently batched and routinely used to treat mCRPC. However, as with the drug resistance problem faced by almost all targeted drugs, inhibitors of AR signaling pathways such as enzalutamide are also mostly resistant in the later stages of disease progression, and the mechanism of drug resistance is due to various factors, such as mutation of target proteins or activation of other compensatory signaling pathways. In the development process of the next generation of antitumor drugs, an in-vitro cytology model and an in-vivo animal drug resistance model need to be constructed.
Disclosure of Invention
Aiming at the problems, the invention discloses a screening method of a cell line resistant to enzalutamide and application thereof.
The technical scheme of the invention is as follows:
A method of screening for a cell line resistant to enzalutamide, comprising the steps of:
1) Resuscitating Lncap cells, and performing cell plating when the Lncap cells grow to a normal state;
2) After cell plating, changing the culture medium into an incomplete culture medium, and starving Lncap cells;
3) After starvation treatment, changing the culture medium into a complete culture medium, adding testosterone and 0.1-1mg/mL of acetyl nitrosourea ENU, and treating for 8-24 hours to induce cell mutation;
4) Washing the cells twice with the complete medium, and replacing the complete medium to continue culturing for more than 24 hours;
5) Adding the enzalutamide with the first concentration for culturing for one week;
6) Culturing for one week with an increased concentration of enzalutamide at a second concentration;
7) Culturing for two weeks with a third concentration of enzalutamide having a further increased concentration;
8) After the culture is finished, selecting monoclonal cells for culture and amplification, and then freezing and preserving;
9) Obtaining a cell line resistant to enzalutamide by drug resistance identification;
The first concentration is the proliferation inhibition activity IC50 value of enzalutamide on normal LnCap cells.
Further, according to the screening method of the cell line resistant to enzalutamide, the incomplete culture medium is RPMI-1640 culture medium without FBS.
Further, the method for screening the cell line resistant to the enzalutamide comprises the step of completely culturing the strain in RPMI-1640 medium containing 10% FBS.
Further, the screening method of the cell line resistant to enzalutamide, wherein the first concentration is determined by the following steps:
Resuscitates LnCap cells, cultures in RPMI-1640 medium containing 10% FBS, tests the drug response curve of LnCap cells to enzalutamide first, inoculates Lncap cells in 96 well plates according to the density of 4000 cells/well, adds three times equal ratio of diluted enzalutamide with the final concentration range of 50 mu M-7.6nM after overnight culture, adds 9 total concentrations, adds DMSO with 0.016% in control wells, tests the living cells in each well with Celltiter-Glo cell viability test kit after 72 hours of incubation, and calculates the proliferation inhibition activity IC50 value of the enzalutamide to LnCap cells by GRAPHPAD PRISM software fitting.
Further, in the above method for screening a cell line resistant to enzalutamide, the concentration of testosterone is preferably 50nM, and the concentration of acetyl nitrosourea ENU is preferably 0.5 or 1mg/mL.
Further, in the method for screening the cell line resistant to the enzalutamide, the first concentration of the enzalutamide is 3.6 mu M, the second concentration of the enzalutamide is 10 mu M, and the third concentration of the enzalutamide is 20 mu M.
Further, the screening method of the cell line resistant to enzalutamide comprises the following steps:
S1, inoculating wild LnCap-WT and drug-resistant strain LnCap-R cells to be identified into a 96-well plate according to the density of 4000 cells/well respectively, culturing overnight, adding triple equal ratio diluted enzalutamide, wherein the final concentration range is 50 mu M-7.6nM, adding DMSO with concentration of 0.016% into a control hole;
After 72 hours of S2 incubation, the living cells in each well were detected with Celltiter-Glo cell viability assay kit, and the proliferation inhibitory activity of enzalutamide on WT-LnCap and Lncap-R cells to be identified was calculated by fitting GRAPHPAD PRISM software.
On the other hand, the invention discloses a cell line resistant to enzalutamide, which is obtained by screening by the screening method.
In another aspect, the invention discloses the use of the above screening method or cell line in the preparation of a tumor-bearing mouse model of enzalutamide resistance.
On the other hand, the invention also discloses the application of the screening method or the cell line in developing medicaments for AR resistant prostate cancer.
The invention has the following beneficial effects:
The invention discloses a screening method of a cell line resistant to enzalutamide and application thereof, which creates the combined application of pretreatment of LnCap cells, ENU treatment, testosterone and starvation, and finally finds an optimal treatment scheme to form drug-resistant clones more effectively.
According to the technical scheme, lnCap cell clone resistant to enzalutamide can be rapidly found, and an in vitro cytology and in vivo pharmacodynamics evaluation platform can be provided for development of the next generation AR inhibitor resistant to enzalutamide or related medicaments for treating prostate cancer.
Drawings
FIG. 1 is a schematic diagram of the construction process of LnCap cell line resistant to enzalutamide;
FIG. 2 number of drug resistant clones formed under different treatment conditions;
FIG. 3 comparison of proliferation inhibitory activity of enzalutamide on WT-LnCap and Lncap-R cells;
FIG. 4 comparison of tumor volumes of sources WT-LnCap and Lncap-R in the case of enzalutamide administration.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
FIG. 1 is a schematic representation of the construction process of LnCap cell line resistant to enzalutamide.
The reagents or instruments used in the examples of the present invention were not manufacturer-identified and were conventional reagent products commercially available.
Example 1
Induction of mutations in LnCap cells by means of an ENU mutagen or the like
1. IC50 assay of enzalutamide in normal Lncap cells
Lncap cells were recovered and cultured in RPMI-1640 medium (complete medium) containing 10% FBS. The drug response curve of LnCap cells to enzalutamide was first tested. Lncap cells were seeded at 4000 cells/well in 96-well plates and after overnight incubation, triple-diluted enzalutamide was added at a final concentration ranging from 50. Mu.M to 7.6nM for 9 total concentrations, and control wells were added with DMSO at 0.016%. After 72 hours of incubation, the viable cells in each well were detected using the Celltiter-Glo cell viability detection kit. Proliferation inhibitory activity of enzalutamide on LnCap cells was calculated by fitting with GRAPHPAD PRISM software, with an IC50 value of 3.62±0.79 μm and an IC80 value of 9.76±1.23 μm.
Co-treatment of LnCap cells with ENU and enzalutamide to establish an enzalutamide drug resistant cell strain
Acetyl nitrosourea (N-methyl-N-nitrosourea, ENU) transfers its ethyl group to the oxygen or nitrogen atom of the DNA base, resulting in a base mismatch or base substitution. We performed ENU mutagenesis to more rapidly generate drug resistant strains resistant to enzalutamide. After Lncap cells grew to normal, cells were seeded in 6-well plates, 1X 10 6 cells per well. After 24 hours of cell plating, the medium was replaced with RPMI-1640 medium without FBS and the culture was continued for 24 hours for starvation. After starvation treatment, the medium was changed to RPMI-1640 medium containing 10% FBS, and final concentrations of 50nM testosterone and 0.1,0.5,1mg/mL ENU were added and the treatment was performed for 8-24 hours to induce cell mutation.
After ENU treatment, the cells were washed twice with complete medium and the culture was continued for 24 hours with replacement of complete medium. 3.6. Mu.M enzalutamide (IC 50 value) was directly added to the cell culture media, and the cells were cultured for one week, and found to have significantly improved viability of 0.5,1mg/mL ENU treated cells, and then after one week of continued culture with complete medium containing 10. Mu.M enzalutamide, the culture was continued for two weeks with complete medium containing 20. Mu.M enzalutamide. During the culture, the complete medium containing the same concentration of enzalutamide was changed every three days. After the incubation was completed, the number of clone formations per well was counted and the monoclonal cells were manually picked into 96-well plates for further incubation. All monoclonal cells are frozen for preservation after culture and amplification, and then are identified later. The experiment was repeated three times, and the number of drug-resistant clones formed under different treatment conditions is shown in Table 1 and FIG. 2. It can be seen that in the case of cell starvation treatment, treatment with 0.5 or 1mg/mL ENU for 16-24 hours resulted in more colonies of cells resistant to enzalutamide.
TABLE 1 establishment of different treatment conditions for Enzalutamine resistant cell lines
| FBS starvation treatment | 50NM testosterone | ENU(mg/mL) | ENU processing time | |
| Con.0 | - | - | - | 0 |
| Con.1 | - | + | 0.1 | 8 Hours |
| Con.2 | - | + | 0.5 | 8 Hours |
| Con.3 | - | + | 1 | 8 Hours |
| Con.4 | + | + | 0.1 | 8 Hours |
| Con.5 | + | + | 0.5 | 8 Hours |
| Con.6 | + | + | 1 | 8 Hours |
| Con.7 | - | + | 0.1 | For 12 hours |
| Con.8 | - | + | 0.5 | For 12 hours |
| Con.9 | - | + | 1 | For 12 hours |
| Con.10 | + | + | 0.1 | For 12 hours |
| Con.11 | + | + | 0.5 | For 12 hours |
| Con.12 | + | + | 1 | For 12 hours |
| Con.13 | - | + | 0.1 | 16 Hours |
| Con.14 | - | + | 0.5 | 16 Hours |
| Con.15 | - | + | 1 | 16 Hours |
| Con.16 | + | + | 0.1 | 16 Hours |
| Con.17 | + | + | 0.5 | 16 Hours |
| Con.18 | + | + | 1 | 16 Hours |
| Con.19 | - | + | 0.1 | 24 Hours |
| Con.20 | - | + | 0.5 | 24 Hours |
| Con.21 | - | + | 1 | 24 Hours |
| Con.22 | + | + | 0.1 | 24 Hours |
| Con.23 | + | + | 0.5 | 24 Hours |
| Con.24 | + | + | 1 | 24 Hours |
Example 2
Identification of Enzalutamide drug-resistant cell lines
Drug resistant cell lines with similar doubling times to wild type LnCap cells were selected for identification. STRs of all cell clones that were successfully pooled were examined and SNP at 205 conserved sites was detected by the method of second generation sequencing identification (https:// doi. Org/10.1093/nargab/lqaa 060) and compared with wild type LnCap cells. Finally, 12 strains of cells were found to have more than 95% similarity to wild-type LnCap cells in second generation sequencing assays. And then tested for resistance to enzalutamide. Taking the drug resistant clone C24-9 as an example (namely LnCap-R). First, drug response curves of wild type LnCap (LnCap-WT) cells and LnCap drug resistant strain (LnCap-R) to enzalutamide were tested. LnCap-WT and LnCap-R cells were seeded in 96-well plates at 4000 cells/well density, respectively, and after overnight incubation, triple equi-diluted enzalutamide was added at a final concentration ranging from 50. Mu.M to 7.6nM for 9 total concentrations, and control wells were added with DMSO containing 0.016%. After 72 hours of incubation, the viable cells in each well were detected using the Celltiter-Glo cell viability detection kit. Proliferation inhibitory activity of enzalutamide on WT-LnCap and Lncap-R cells was calculated by fitting GRAPHPAD PRISM software, and as shown in FIG. 3, the IC50 value of WT-LnCap cells was 2.4. Mu.M, and the IC50 value of Lncap-R cells was 14.8. Mu.M. CTG results show that the inhibition rate of enzalutamide on LnCap-R cell proliferation is obviously reduced, and the construction success of the LnCap-R cell line is proved.
Example 3
Evaluation of in vivo drug resistance
LnCap-WT and LnCap-R cells were cultured in RPMI-1640 medium containing 10% FBS. Cells in exponential growth phase were collected, resuspended in the appropriate volume of PBS, and an equal volume of matrigel was added (v/v=1:1). 0.2mL of the suspension (containing 1X 10 7 cells) was inoculated subcutaneously into NOD SCID mice. The average tumor volume was up to about 150mm 3 divided into two groups, one group being the control group and one group being given 10mg/kg of enzalutamide, QD x 21Days. All animals were weighed before dosing began and tumor volumes were measured with vernier calipers. A hierarchical randomization method was used to group 6 tumors according to tumor size using an Excel random software program. The Coefficient of Variation (CV) of tumor volumes within each group, calculated as formula cv=sd (standard deviation)/MTV (mean tumor volume) ×100%, should be less than 20%.
Growth curves for LnCap-WT and LnCap-R subcutaneous transplantation tumor models are shown in FIG. 4. At the end of the experiment, the average tumor volume of Vehicle was 1091.2mm 3, the average tumor volume of enzalutamide-administered group was 448.8mm 3, the tumor volume inhibition rate TGI TV was 65%, and statistical analysis showed that the tumor volume of the enzalutamide-administered group was significantly reduced compared to Vehicle group (p=0.0147, t-test). Growth curves of LnCap-R subcutaneous engraftment tumor model showed that average tumor volume of veccle was 1701.1mm 3, average tumor volume of enzalutamide-administered group was 1615.2mm 3, tumor volume inhibition rate TGI TV was 5%, and statistical analysis showed no significant change in tumor volume of enzalutamide-administered group compared to veccle group (p=0.8396, t-test).
The data show that enzalutamide has remarkable effect of inhibiting proliferation of LnCap-WT cell subcutaneous transplantation tumor model in mice, but has no remarkable pharmacodynamic activity in LnCap-R cells.
In summary, according to the embodiment of the invention, lnCap cell clone resistant to enzalutamide can be rapidly found by the method of the invention, and an in vitro cytology and in vivo pharmacodynamics evaluation platform can be provided for the development of the next generation AR inhibitor resistant to enzalutamide or related medicaments for treating prostate cancer.
Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Numerous modifications and substitutions of details are possible in light of all the teachings disclosed, and such modifications are contemplated as falling within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.
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| CN117587111A (en) * | 2023-11-30 | 2024-02-23 | 苏州拓维生物技术有限公司 | A method for rapid batch screening of compound activity in kinase cell lines |
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| RUVBL1通过调控PLXNA1-CRAF-MAPK信号通路促进前列腺癌恩杂鲁胺耐药的研究;孙菲菲;中国博士学位论文全文数据库 医药卫生科技辑;20240215(第02期);E067-25 * |
| 靶向KDM4A-AS1抑制AR/AR-Vs去泛素化并增强CRPC恩杂鲁胺响应;张博雅;中国博士学位论文全文数据库 医药卫生科技辑;20240615(第06期);E067-30 * |
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