A method for screening cell lines resistant to enzalutamide and its application

Abstract

The invention belongs to the field of biomedicine, and specifically discloses a method for screening a cell line resistant to enzalutamide and an application thereof, comprising the following steps: 1) reviving Lncap cells, and plating cells after the Lncap cells grow to a normal state; 2) after plating the cells, replacing the culture medium with an incomplete culture medium, and starving the Lncap cells; 3) after the starvation treatment, replacing the culture medium with a complete culture medium, and adding testosterone and 0.1-1 mg / mL acetylnitrosourea ENU, and treating for 8-24 hours to induce cell mutation; 4) washing the cells twice with a complete culture medium, and replacing the complete culture medium to continue culturing for more than 24 hours; 5) adding a first concentration of enzalutamide and culturing for one week; 6) using a second concentration of enzalutamide with an increased concentration to culture for one week; 7) using a third concentration of enzalutamide with a further increased concentration to culture for two weeks; 8) after the culture is completed, selecting monoclonal cells for culture amplification, and freezing and preserving; 9) identifying drug resistance to obtain a cell line resistant to enzalutamide.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and specifically discloses a method for screening a cell line resistant to enzalutamide and an application thereof. Background Art

[0002] Prostate cancer is the second most common cancer and the fifth leading cause of cancer-related death in men worldwide. Most patients with prostate cancer are diagnosed with localized disease and are recommended to undergo active surveillance and local treatment in combination with androgen deprivation therapy (ADT) when needed. Although the cure rate after local treatment is high, some patients will relapse as prostate-specific antigen (PSA) levels increase, which is defined as castration-resistant prostate cancer (CRPC). It is well known that the androgen receptor (AR) pathway is an important component of the pathogenesis of prostate cancer and has therefore been the main target for prostate cancer treatment. AR signaling pathway inhibitors, such as abiraterone acetate (AA) and enzalutamide, are now approved and routinely used to treat mCRPC. However, like the drug resistance problem faced by almost all targeted drugs, AR signaling pathway inhibitors such as enzalutamide also mostly develop drug resistance in the late stage of disease progression. The resistance mechanism has multiple factors, such as mutations in the target protein or activation of other compensatory signaling pathways. In the development of the next generation of anti-tumor drugs, it is necessary to construct in vitro cellular models and in vivo animal resistance models. Summary of the invention

[0003] In view of the above problems, the present invention discloses a method for screening a cell line resistant to enzalutamide and application thereof.

[0004] The technical solution of the present invention is as follows:

[0005] A method for screening a cell line resistant to enzalutamide, comprising the following steps:

[0006] 1) Resuscitate Lncap cells and wait for them to grow to a normal state before plating the cells;

[0007] 2) After cell plating, the culture medium was replaced with incomplete culture medium to starve Lncap cells;

[0008] 3) After starvation treatment, the culture medium was replaced with complete culture medium, and testosterone and 0.1-1 mg / mL acetylnitrosourea ENU were added for 8-24 h to induce cell mutation;

[0009] 4) Wash the cells twice with complete medium, and replace the complete medium and continue culturing for more than 24 hours;

[0010] 5) adding the first concentration of enzalutamide and culturing for one week;

[0011] 6) culturing for one week with a second increasing concentration of enzalutamide;

[0012] 7) culturing for two weeks with a third concentration of enzalutamide at a further increased concentration;

[0013] 8) After the culture is completed, single clone cells are selected for culture, amplification, and cryopreservation;

[0014] 9) Drug resistance identification to obtain cell lines resistant to enzalutamide;

[0015] The first concentration is the IC50 value of the proliferation inhibition activity of enzalutamide on normal LnCap cells.

[0016] Furthermore, in the above method for screening a cell line resistant to enzalutamide, the incomplete culture medium is RPMI-1640 culture medium without FBS.

[0017] Furthermore, in the above method for screening a cell line resistant to enzalutamide, the complete culture medium is RPMI-1640 culture medium containing 10% FBS.

[0018] Furthermore, in the above method for screening a cell line resistant to enzalutamide, the first concentration is determined by the following steps:

[0019] Lncap cells were revived and cultured in RPMI-1640 medium containing 10% FBS; first, the drug response curve of LnCap cells to enzalutamide was tested; Lncap cells were seeded in a 96-well plate at a density of 4000 cells / well, and after overnight culture, three-fold equal dilutions of enzalutamide were added, with a final concentration range of 50μM-7.6nM, a total of 9 concentrations, and 0.016% DMSO was added to the control wells; after incubation for 72 hours, the live cells in each well were detected using the Celltiter-Glo cell viability detection kit; the IC50 value of the proliferation inhibition activity of enzalutamide on LnCap cells was fitted and calculated using GraphPad Prism software.

[0020] Furthermore, in the above method for screening a cell line resistant to enzalutamide, the concentration of testosterone is preferably 50 nM; and the concentration of acetylnitrosourea ENU is preferably 0.5 or 1 mg / mL.

[0021] Furthermore, in the above method for screening a cell line resistant to enzalutamide, the first concentration of enzalutamide is 3.6 μM, the second concentration is 10 μM, and the third concentration is 20 μM.

[0022] Furthermore, in the above method for screening a cell line resistant to enzalutamide, the drug resistance identification comprises the following steps:

[0023] S1 Wild-type LnCap-WT and the drug-resistant strain LnCap-R to be identified were seeded in a 96-well plate at a density of 4000 cells / well. After overnight culture, three-fold equal dilutions of enzalutamide were added, with a final concentration range of 50 μM-7.6 nM, for a total of 9 concentrations. 0.016% DMSO was added to the control wells;

[0024] After incubation with S2 for 72 hours, the living cells in each well were detected using the 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 using GraphPad Prism software.

[0025] On the other hand, the present invention discloses a cell line resistant to enzalutamide, which is obtained by screening using the above screening method.

[0026] On the other hand, the present invention discloses the use of the above screening method or cell line in preparing an enzalutamide-resistant tumor-bearing mouse model.

[0027] On the other hand, the present invention also discloses the use of the above screening method or cell line in developing drugs for AR-resistant prostate cancer.

[0028] The present invention has the following beneficial effects:

[0029] The present invention discloses a method for screening a cell line resistant to enzalutamide and an application thereof, and independently creates a pre-treatment of LnCap cells and a combined application of ENU treatment + testosterone + starvation. Different numbers of clones are formed under different treatment conditions. Finally, an optimal treatment scheme is found, which can more effectively form drug-resistant clones.

[0030] The technical solution of the present invention can quickly discover LnCap cell clones resistant to enzalutamide, and can provide an in vitro cytological and in vivo pharmacodynamic evaluation platform for the development of next-generation AR inhibitors or related drugs for the treatment of prostate cancer that are resistant to enzalutamide. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 ; Schematic diagram of the construction process of the LnCap cell line resistant to enzalutamide;

[0032] Figure 2 : The number of drug-resistant clones formed under different treatment conditions;

[0033] Figure 3 : Comparison of the proliferation inhibitory activity of enzalutamide on WT-LnCap and Lncap-R cells;

[0034] Figure 4: Comparison of tumor volumes from WT-LnCap and Lncap-R under enzalutamide treatment. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] like Figure 1 Schematic diagram of the construction process of the LnCap cell line resistant to enzalutamide.

[0037] The reagents or instruments used in the examples of the present invention without indicating the manufacturer are all conventional reagent products that can be obtained through commercial purchase.

[0038] Example 1

[0039] Inducing mutations in LnCap cells using ENU mutagen

[0040] 1. IC50 determination of enzalutamide in normal Lncap cells

[0041] Lncap cells were revived and cultured in RPMI-1640 medium (complete medium) containing 10% FBS. First, the drug response curve of LnCap cells to enzalutamide was tested. Lncap cells were seeded in a 96-well plate at a density of 4000 cells / well. After overnight culture, three times equal dilutions of enzalutamide were added, with a final concentration range of 50μM-7.6nM, a total of 9 concentrations, and 0.016% DMSO was added to the control wells. After incubation for 72 hours, the live cells in each well were detected using the Celltiter-Glo cell viability assay kit. The proliferation inhibitory activity of enzalutamide on LnCap cells was fitted and calculated using GraphPad Prism software, and its IC50 value was 3.62±0.79μM and the IC80 value was 9.76±1.23μM.

[0042] 2. ENU and Enzalutamide co-treatment of LnCap cells to establish Enzalutamide-resistant cell lines

[0043] Acetylnitrosourea (N-ethyl-N-nitrosourea, ENU) transfers its ethyl group to the oxygen or nitrogen atom of the DNA base, resulting in base mismatch or base substitution. We performed ENU mutagenesis to generate a strain resistant to enzalutamide more quickly. After Lncap cells grew to a normal state, the cells were seeded in a 6-well plate, with 1x 10 6After 24 hours of cell plating, the culture medium was replaced with RPMI-1640 medium without FBS, and the cells were starved for another 24 hours. After starvation, the culture medium was replaced with RPMI-1640 medium containing 10% FBS, and testosterone with a final concentration of 50 nM and ENU at 0.1, 0.5, and 1 mg / mL were added for 8-24 hours to induce cell mutation.

[0044] After ENU treatment, the cells were washed twice with complete medium, and the complete medium was replaced for a further 24 hours. 3.6 μM enzalutamide (IC50 value) was directly added to the cell culture medium and cultured for one week. It was found that the survival rate of cells treated with 0.5 and 1 mg / mL ENU was significantly improved. Then, the cells were cultured for a further week with complete medium containing 10 μM enzalutamide, and finally, the cells were cultured for a further two weeks with complete medium containing 20 μM enzalutamide. During the culture process, the complete medium containing the same concentration of enzalutamide was replaced every three days. After the culture was completed, the number of clones formed in each well was counted and monoclonal cells were manually picked and continued to be cultured in a 96-well plate. All monoclonal cell cultures were amplified, frozen, and subsequently identified. This experiment was repeated three times. The number of drug-resistant clones formed under different treatment conditions is shown in Tables 1 and Figure 2 It can be seen that under the condition of cell starvation, treatment with 0.5 or 1 mg / mL ENU for 16-24 hours can form more cell clones resistant to enzalutamide.

[0045] Table 1 Different treatment conditions for establishing enzalutamide-resistant cell lines

[0046] 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 12 hours Con.8 - + 0.5 12 hours Con.9 - + 1 12 hours Con.10 + + 0.1 12 hours Con.11 + + 0.5 12 hours Con.12 + + 1 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

[0047] Example 2

[0048] Identification of enzalutamide-resistant cell lines

[0049] Resistant cell lines with a similar doubling time to wild-type LnCap cells were selected for identification. The STRs of all successfully constructed cell clones were detected, and the SNPs of 205 conserved sites were detected by the second-generation sequencing identification method (https: / / doi.org / 10.1093 / nargab / lqaa060), and compared with wild-type LnCap cells. Finally, 12 cell lines were found to have more than 95% similarity with wild-type LnCap cells in the second-generation sequencing identification. Then their resistance to enzalutamide was tested. Take the resistant clone C24-9 as an example (i.e., LnCap-R). First, the drug response curves of wild-type LnCap (LnCap-WT) cells and LnCap resistant strains (LnCap-R) to enzalutamide were tested. LnCap-WT and LnCap-R cells were seeded in 96-well plates at a density of 4000 cells / well. After overnight culture, three-fold isocratic dilutions of enzalutamide were added, with a final concentration range of 50μM-7.6nM, a total of 9 concentrations, and 0.016% DMSO was added to the control wells. After 72 hours of incubation, the live cells in each well were detected using the Celltiter-Glo cell viability assay kit. The proliferation inhibitory activity of enzalutamide on WT-LnCap and Lncap-R cells was calculated using GraphPad Prism software. Figure 3 As shown, the IC50 value of WT-LnCap cells was 2.4 μM, and the IC50 value of Lncap-R cells was 14.8 μM. CTG results showed that the inhibition rate of enzalutamide on the proliferation of LnCap-R cells was significantly reduced, proving that the LnCap-R cell line was successfully constructed.

[0050] Example 3

[0051] In vivo drug resistance evaluation

[0052] LnCap-WT and LnCap-R cells were cultured in RPMI-1640 medium containing 10% FBS. Cells in the exponential growth phase were collected, resuspended in an appropriate volume of PBS, and an equal volume of Matrigel (v / v = 1:1) was added. 0.2 mL of the suspension (containing 1×10 7 The cells were subcutaneously inoculated in NOD SCID mice. The average tumor volume was about 150 mm 3 The animals were divided into two groups, one group was the control group, and the other group was given enzalutamide 10 mg / kg, QD*21Days. Before the start of administration, all animals were weighed and the tumor volume was measured with a vernier caliper. Using the Excel randomization software program, the animals were grouped according to tumor size using a stratified randomization method, with 6 animals in each group. The coefficient of variation (CV) of the tumor volume within each group was calculated using the formula CV = SD (standard deviation) / MTV (mean tumor volume) × 100%, which should be less than 20%.

[0053] Growth curves of LnCap-WT and LnCap-R subcutaneous transplant tumor models are shown in Figure 4 At the end of the experiment, the average tumor volume of Vehicle was 1091.2mm 3 The mean tumor volume in the enzalutamide group was 448.8 mm 3 , tumor volume inhibition rate TGI TV The statistical analysis showed that the tumor volume in the enzalutamide group was significantly reduced compared with the Vehicle group (p = 0.0147, t-test). The growth curve of the LnCap-R subcutaneous transplanted tumor model showed that the average tumor volume in the Vehicle group was 1701.1 mm 3 The mean tumor volume in the enzalutamide group was 1615.2 mm 3 , tumor volume inhibition rate TGI TV 5%. Statistical analysis showed that there was no significant change in tumor volume in the enzalutamide group compared with the Vehicle group (p=0.8396, t-test).

[0054] The above data show that enzalutamide has a significant inhibitory effect on the proliferation of LnCap-WT cell subcutaneous transplanted tumor model in mice, but has no obvious pharmacodynamic activity in LnCap-R cells.

[0055] From the above examples, it can be seen that the method of the present invention can quickly discover LnCap cell clones resistant to enzalutamide, and can provide an in vitro cytological and in vivo pharmacodynamic evaluation platform for the development of next-generation AR inhibitors or related drugs for the treatment of prostate cancer that are resistant to enzalutamide.

[0056] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and substitutions may be made to those details according to all the teachings disclosed, and these changes are within the scope of protection of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. A method for screening a cell line resistant to enzalutamide, characterized in that: The following steps are involved: 1) Resuscitate Lncap cells and wait for them to grow to a normal state before plating the cells; 2) After cell plating, the culture medium was replaced with incomplete culture medium to starve Lncap cells; 3) After starvation treatment, the culture medium was replaced with complete culture medium, and testosterone and acetylnitrosourea ENU were added for 8-24h to induce cell mutation; 4) Wash the cells twice with complete medium, and replace the complete medium and continue culturing for more than 24 hours; 5) Add the first concentration of enzalutamide and culture for one week; 6) culture for one week with a second increasing concentration of enzalutamide; 7) Incubate for two weeks with a third increasing concentration of enzalutamide; 8) After the culture is completed, single clone cells are selected for culture, amplification, and cryopreservation; 9) Drug resistance identification to obtain cell lines resistant to enzalutamide; The incomplete culture medium is RPMI-1640 culture medium without FBS; The complete culture medium is RPMI-1640 culture medium containing 10% FBS; The concentration of the testosterone is 50 nM; the concentration of the acetylnitrosourea ENU is 0.5 or 1 mg / mL; The first concentration of Enzalutamide is 3.6 μM, the second concentration is 10 μM, and the third concentration is 20 μM; The drug resistance identification comprises the following steps: S1 Wild-type LnCap-WT and the drug-resistant strain LnCap-R to be identified were seeded in a 96-well plate at a density of 4000 cells / well. After overnight culture, three-fold equal dilutions of enzalutamide were added, with a final concentration range of 50 μM-7.6 nM, for a total of 9 concentrations. 0.016% DMSO was added to the control wells; After incubation with S2 for 72 hours, the living cells in each well were detected using the Celltiter-Glo cell viability assay kit, and the proliferation inhibitory activity of enzalutamide on WT-LnCap and the Lncap-R cells to be identified was calculated using GraphPad Prism software.

2. A cell line resistant to enzalutamide, characterized in that: The method is obtained by screening according to claim 1.

3. Use of the screening method according to claim 1 or the cell line according to claim 2 in preparing an enzalutamide-resistant tumor-bearing mouse model.

4. Use of the screening method according to claim 1 or the cell line according to claim 2 in developing a drug for enzalutamide-resistant prostate cancer.

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