Application of non-small cell lung cancer RAS inhibitor drug-resistant cell strain

Abstract

The invention discloses an application of a non-small cell lung cancer RAS inhibitor drug-resistant cell strain in screening of a drug for treating RMC-7977-resistant non-small cell lung cancer, which is characterized in that a non-small cell lung cancer cell strain A549 is adopted as an induction object, and a continuous induction method is adopted to establish an RMC-7977 drug-resistant non-small cell lung cancer cell strain A549-R; experiments find that the constructed non-small cell lung cancer cell strain A549-R tolerates RMC-7977, in an RPMI 1640 culture medium containing the RMC-7977, the IC50 value of A549-R cells is greater than that of normal A549, and the clone formation ability is also higher than that of A549; the P-akt protein expression quantity is increased; the cell strain is used for screening drugs for treating RMC-7977-resistant non-small cell lung cancer, and can be used for revealing the drug resistance mechanism of the non-small cell lung cancer, reversing drug resistance and finding a better treatment method.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of drug-resistant cell strain preparation, and particularly relates to construction and application of a non-small cell lung cancer RAS inhibitor-resistant cell strain. BACKGROUND

[0002] Non-small cell lung cancer (NSCLC) is one of the most common types of cancer worldwide, accounting for more than 28% of all cancer-related deaths. It is estimated that more than 75% of lung cancer cases are NSCLC, and the rest are small cell lung cancer.

[0003] The KRAS gene is one of the most common oncogenes in human cancers, and plays a key role in NSCLC. KRAS mutations are mainly missense mutations, and common mutation types include G12D (29.19%), G12V (22.97%), and G12C (13.43%). These mutations cause KRAS protein to be continuously activated, promoting abnormal cell proliferation and transformation. KRAS mutations are particularly significant in the early stages of tumors, especially in adenocarcinoma (ADC), with a frequency of about 25%, and even higher in certain Asian populations. These mutations lead to dysregulation of cell signaling, promoting the occurrence and development of cancer. Therefore, KRAS mutations have important diagnostic and therapeutic significance in NSCLC.

[0004] KRAS protein was once considered an "undruggable" target, but scientists discovered that KRAS G12C mutants can be targeted by covalent small molecule drugs, leading to the development of sotorasib and adagrasib. However, these inhibitors only target inactive KRAS G12C, and patients may develop resistance. To overcome this problem, researchers developed RMC-7977, a new broad-spectrum RAS inhibitor. RMC-7977 first binds to cyclophilin A (CYPA) to form a binary complex, and then binds to activated RAS protein (including KRAS, NRAS, and HRAS) to form a ternary complex, thereby sterically blocking the interaction between RAS and effectors, inhibiting the RAS downstream signaling pathway. RMC-7977 is a broad-spectrum RAS-GTP inhibitor with potential for treating multiple cancers. Although preliminary experiments have shown its effectiveness against cancers such as NSCLC, tumors can develop resistance to RMC-7977 after continuous treatment, reducing the effectiveness of the drug, so it is important to establish a RMC-7977-resistant RAS mutant A549 cell strain to study its resistance mechanism.

[0005] Most of the current research on tumor drug resistance mechanism is carried out on tumor drug resistant cells, and a series of researches can be carried out on tumor drug resistant cell lines, such as revealing drug resistance mechanism, reversing drug resistance, developing new targeted drugs, and evaluating treatment response. There are two methods for screening drug resistant cell lines, namely continuous induction method and high dose impact method.

[0006] So far, there is no report on the establishment of non-small cell lung cancer cell line A549-R resistant to RMC-7977 by using human non-small cell lung cancer cell line A549 as the induction object. SUMMARY

[0007] The application provides a use of a non-small cell lung cancer RAS inhibitor drug resistant cell line in screening a drug for treating non-small cell lung cancer resistant to RMC-7977; the non-small cell lung cancer RAS inhibitor drug resistant cell line is prepared by using RMC-7977 to induce non-small cell lung cancer cell line A549 resistant to RMC-7977 by using a concentration gradient continuous induction method.

[0008] The application uses non-small cell lung cancer cell line A549 as the induction object, and uses the continuous induction method to establish RMC-7977-resistant non-small cell lung cancer cell line A549-R; it is found through experiments that the constructed non-small cell lung cancer cell line A549-R tolerates RMC-7977, and the IC50 value of A549-R cells in the RPMI 1640 culture medium containing RMC-7977 is greater than that of normal A549, and the clonogenic ability is also stronger than that of A549; and the expression amount of P-akt protein is increased.

[0009] The non-small cell lung cancer RAS inhibitor drug resistant cell line constructed by the application can be used as a tumor drug resistance model for in vivo and in vitro experiments, can be used for screening a drug for treating non-small cell lung cancer resistant to RMC-7977, and can be used for revealing the non-small cell lung cancer drug resistance mechanism, reversing drug resistance, and finding a better treatment method. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 IC value diagram of A549 and A549-R cells after being treated with RMC-7977 for 48h; 50 Figure 2 Microscope observation of A549 (A) and A549-R (B) cell lines in 1640 culture medium, wherein the left graph is 4x, and the right graph is 10x; Figure 3 Clonogenic experiment results of A549 and A549-R cells after being treated with different concentrations of RMC-7977; Figure 4 ​The protein expression results of A549 and A549-R cells after treatment with 500 nM / mL RMC-7977 for 24 h; Figure 5 The volcano plot of differentially expressed genes of A549 and A549-R cells after treatment with 500 nM / mL RMC-7977 for 24 h. DETAILED DESCRIPTION

[0011] The application will be further described in conjunction with the following examples. However, the scope of the application is not limited to the following examples. Those skilled in the art can understand that various changes and modifications can be made to the application without departing from the spirit and scope of the application. The instruments, reagents, and materials involved in the following examples are conventional instruments, reagents, and materials available in the prior art, and can be obtained through regular commercial channels, unless otherwise specified. The experimental methods and detection methods involved in the following examples are conventional experimental methods and detection methods available in the prior art, unless otherwise specified.

[0012] Example 1: Establishment of RMC-7977-resistant non-small cell lung cancer cell line A549-R The non-small cell lung cancer cell line A549 was used as the induction object, and the RMC-7977-resistant non-small cell lung cancer cell line A549-R was established by using the continuous induction method. The continuous induction method is as follows: (1) Take the existing non-small cell lung cancer cell line A549 in the laboratory, and culture it in 1640 medium (containing 10% fetal bovine serum, 100 U / mL penicillin, and 10 μg / mL streptomycin) at 5% CO2 and 37°C to the logarithmic growth phase after resuscitation; (2) After two generations, take the logarithmic growth phase A549 cells and adhere them to a T25 culture flask. When the cell density reaches about 80%, add 1640 medium containing RMC-7977 to induce them. The initial concentration is 100 nM / mL, and the culture is carried out at 5% CO2 and 37°C. The medium is changed in time to maintain a drug concentration of 100 nM / mL until the cells can survive and grow at this concentration; (3) After the cells adapt to the initial concentration and restore the growth speed before drug administration, the cells are passaged, and the method in step (2) is repeated to induce them. The drug concentration is changed to 500 nM / mL, 1 µM / mL, 2 µM / mL, 5 µM / mL, and 10 µM / mL. This repeated induction, medium change, and passaging are carried out until the cells can grow and be passaged normally in the 10 µM / mL drug-containing medium. At this time, the cell line is the RMC-7977-resistant non-small cell lung cancer cell line A549-R; (4) The RPMI 1640 medium was configured to add 10% fetal bovine serum, 100 U / mL penicillin and 10 μg / mL streptomycin, and then the A549 and A549-R cell densities were adjusted to 3 x 10 4 cells / mL with RPMI 1640 medium, and the A549 and A549-R cell suspensions with adjusted cell densities were inoculated into 96-well plates at a volume of 100 μL per well, and the A549 and A549-R cells were cultured at 37°C in a humidified atmosphere of 5% CO2 for 24 h to adhere to the wall; the A549 and A549-R cells in the 96-well plates were incubated with RPMI 1640 medium containing RMC-7977 (0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 µM, 5 µM, 10 µM) at a volume of 100 μL per well for 48 h as an experimental group; the negative control group was incubated with only RPMI 1640 medium and A549 and A549-R cells for 48 h; the blank control group was only used with RPMI 1640 medium; then 10 μL of CCK8 reagent was added to each well, mixed gently, and incubated at 37°C for 2 h in the dark, and then the absorbance (OD) was detected at 450 nm using a microplate reader. The calculation method of cell viability is: ; The results are shown in Figure 1 Compared with normal A549, the IC 50 value of A549-R cells increased significantly, indicating that the drug-resistant cell line A549-R was successfully constructed.

[0013] Example 2: Observation of the morphology of A549 and A549-R cell lines under a microscope After logarithmic growth of A549 and A549-R cells was cultured in 1640 medium for 48 h, the morphology of the two cells was observed under a 4x and 10x microscope, and the results are shown in Figure 2 Compared with normal A549, A549-R cells were spindle-shaped, some were linear, and were elongated.

[0014] Example 3: Cloning formation experiment of A549 and A549-R cells after treatment with different concentrations of RMC-7977 A549, A549-R cells were inoculated in six-well plates (1000 per well), and after 2-3 days of culture, the culture medium was replaced with a culture medium containing different concentrations of RMC-7977 (0, 1 nM, 10 nM, 100 nM, 200 nM, 500 nM, ), and the liquid was replaced every 3 days until the cell colonies were about 50 cells, and then the drug administration was stopped. The culture medium in the hole was aspirated, washed twice with PBS, 4% paraformaldehyde was added for fixation for 30 minutes, and then washed twice with PBS, 0.1% crystal violet solution was added, and stained for 15-20 minutes. After staining, pure water was washed until the six-well plate background was clear, and then dried, photographed.

[0015] As shown in Figure 3 compared with normal A549, A549-R cells can grow and complete cloning after RMC-7977 treatment at different concentrations.

[0016] Example 4: Protein expression of A549, A549-R cells after 24h treatment of 500nM / mL RMC-7977 A549, A549-R cells were inoculated in six-well plates (200000 per well), and after 2-3 days of culture, the culture medium was replaced with a culture medium containing different concentrations of RMC-7977 (0, 1 nM, 10 nM, 100 nM, 200 nM, 500 nM, ), and the liquid was replaced every 3 days until the cell colonies were about 50 cells, and then the drug administration was stopped. The culture medium in the hole was aspirated, washed twice with PBS, 4% paraformaldehyde was added for fixation for 30 minutes, and then washed twice with PBS, 0.1% crystal violet solution was added, and stained for 15-20 minutes. After staining, pure water was washed until the six-well plate background was clear, and then dried, photographed.

[0017] As shown in Figure 4 compared with normal A549, A549-R cells can grow and complete cloning after RMC-7977 treatment at different concentrations.

[0018] Example 5: Differential gene expression of A549, A549-R cells after 24h treatment of 500nM / mL RMC-7977 A549, A549-R cells were inoculated in six-well plates (200000 per well), and after 2-3 days of culture, the culture medium was replaced with a culture medium containing different concentrations of RMC-7977 (0, 1 nM, 10 nM, 100 nM, 200 nM, 500 nM, ), and the liquid was replaced every 3 days until the cell colonies were about 50 cells, and then the drug administration was stopped. The culture medium in the hole was aspirated, washed twice with PBS, 4% paraformaldehyde was added for fixation for 30 minutes, and then washed twice with PBS, 0.1% crystal violet solution was added, and stained for 15-20 minutes. After staining, pure water was washed until the six-well plate background was clear, and then dried, photographed. As shown in Figure 5As shown, after RMC-7977 treatment, there were 2130 genes significantly increased and decreased in A549-R cells compared with normal A549. Among them, 1138 genes were up-regulated and 992 genes were down-regulated. The genes related to drug resistance were analyzed in detail, which were mainly enriched in PI3K-Akt, MAPK, Hippo and other signaling pathways. Among them, the PI3K-Akt signaling pathway related genes changed significantly, which proved the important mechanism of RMC-7977 killing drug-resistant tumor cells.

Claims

1. Application of a non-small cell lung cancer RAS inhibitor-resistant cell line in screening drugs for the treatment of RMC-7977-resistant non-small cell lung cancer.

2. The application according to claim 1, characterized in that: The RAS inhibitor-resistant cell line for non-small cell lung cancer was obtained by inducing resistance in the A549 non-small cell lung cancer cell line using the concentration gradient continuous induction method with RMC-7977.

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