Application of AMPK inhibitor and ferroptosis inducer combined drug in tumor treatment

Abstract

The invention relates to an application of an AMPK inhibitor and ferroptosis inducer combined drug in tumor treatment, in particular to an application of a ferroptosis inducer (RSL3) combined AMPK inhibitor (Compound C) in preparation of a drug for tumor treatment, and belongs to the technical field of biological medicine. In-vivo and in-vitro experiments prove that the combination of a ferroptosis inducer (RSL3) and an AMPK inhibitor (Compound C) drug can significantly inhibit the proliferation of non-small cell lung cancer cells with high expression of MORC2. The drug combination exerts significant synergistic interaction, and has more effective tumor treatment effect than a single drug. Therefore, the combination of the ferroptosis inducer and the cell metabolism inhibitor serves as a new potential clinical treatment strategy, and a new thought is provided for tumor treatment of high-expression MORC2.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-tumor drugs, and specifically relates to the use of a ferroptosis inducer (RSL3) in combination with an AMPK inhibitor (Compound C) in the preparation of drugs for tumor treatment. Background Art

[0002] Lung cancer is one of the cancers with the highest mortality rate in the world. In my country, the incidence and mortality of lung cancer also rank first. Lung cancer is divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) according to the purpose of treatment. Non-small cell lung cancer accounts for more than 85% of lung cancer cases. Lung cancer is highly malignant and has a poor prognosis. Liver kinase B1 (LKB1) deficiency often occurs in NSCLC, and NSCLC with LKB1 deficiency often shows drug resistance clinically. At present, there is still a lack of effective treatment options for this type of NSCLC. Therefore, finding potential therapeutic targets, especially for the treatment of LKB1-deficient NSCLC, is of great clinical significance.

[0003] MORC family CW-type zinc finger protein 2 (MORC2) is a member of the nuclear protein MORC superfamily and is involved in gene transcription regulation, chromatin remodeling and DNA damage response. Our studies and those of others have shown that MORC2 is highly expressed in a variety of human malignancies and is associated with poor prognosis. Overexpression of MORC2 can promote tumor growth. However, there are currently no ideal targeted MORC2 inhibitors in the clinic. Therefore, finding MORC2 drug targets is expected to provide new potential therapeutic strategies for the treatment of tumors that overexpress MORC2.

[0004] Adenylate-activated protein kinase (AMPK) is an intracellular energy metabolism sensor, consisting of a catalytic subunit α and two regulatory subunits β and γ. As research deepens, it is found that the key phosphorylation activation site is Thr172, which is located on the catalytic subunit of AMPK, and the kinase that phosphorylates this site is mainly LKB1, which leads people to believe that AMPK has tumor suppressor properties. In addition, studies have shown that tumor cells participate in the AMPK pathway to cope with various cell-intrinsic and extrinsic stresses. Compound C is a pyrazolopyrimidine derivative that has an inhibitory effect on AMPK activation and is currently widely used as an AMPK inhibitor in many studies. Therefore, AMPK inhibitors are expected to be used as a concept to disrupt stress-induced metabolic adaptability and provide prospects for new therapies.

[0005] Ferroptosis is a type of programmed cell death, and its fundamental mechanism is based on the imbalance of intracellular redox homeostasis, which triggers lipid peroxidation, ultimately destroying membrane integrity and leading to cell death. Studies have found that glutathione peroxidase 4 (GPX4) is a key regulator of ferroptosis. Ferroptosis inducer RSL3, as an inhibitor of GPX4, directly targets GPX4 to inactivate it, leading to the accumulation of intracellular peroxides and inducing ferroptosis. Therefore, the combination of ferroptosis inducers and cell metabolism inhibitors may be a new method for future cancer treatment and an important strategy to advance treatment development. Summary of the invention

[0006] In view of the problems existing in the prior art, the purpose of the present invention is to use a ferroptosis inducer (RSL3) in combination with an AMPK inhibitor (Compound C) in the preparation of a drug for tumor treatment. The present invention uses in vitro and in vivo experiments to prove that the combination of ferroptosis inducer (RSL3) and AMPK inhibitor (Compound C) significantly inhibits the proliferation of non-small cell lung cancer cells that highly express MORC2. Therefore, the combination of ferroptosis inducers and cell metabolism inhibitors, as a new potential clinical treatment strategy, provides a new idea for the treatment of tumors that highly express MORC2, especially those that lack LKB1.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0008] The invention discloses a pharmaceutical preparation for treating non-small cell lung cancer, which is characterized by comprising the following preparations in independent or mixed packages: an AMPK inhibitor; and a ferroptosis inducer.

[0009] Furthermore, the AMPK inhibitor includes Compound C or siRNA; and the ferroptosis inducer includes RSL3.

[0010] Furthermore, the non-small cell lung cancer also includes LKB1-deficient type.

[0011] Furthermore, the pharmaceutical product also includes pharmaceutically acceptable excipients.

[0012] The present invention also discloses the use of an active ingredient of an AMPK inhibitor and a ferroptosis inducer in combination in the preparation of a product for inhibiting the proliferation and invasion ability of tumor cells.

[0013] Furthermore, the AMPK inhibitor includes Compound C or siRNA; and the ferroptosis inducer includes RSL3.

[0014] Furthermore, the non-small cell lung cancer also includes LKB1-deficient type.

[0015] The invention discloses a pharmaceutical composition, characterized in that the pharmaceutical composition comprises an AMPK inhibitor and a ferroptosis inducer.

[0016] Furthermore, the AMPK inhibitor includes Compound C or siRNA; and the ferroptosis inducer includes RSL3.

[0017] Furthermore, the above-mentioned pharmaceutical composition is used in the preparation of a drug for treating non-small cell lung cancer.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0019] This invention uses LKB1-deficient non-small cell lung cancer as the research object, and detects the effects of AMPK inhibitor (Compound C) and ferroptosis inducer (RSL3) on non-small cell lung cancer through plate clone formation experiment, cell soft agar clone experiment and construction of tumor-bearing mice. It is the first time to propose and confirm that the combination of AMPK inhibitor (Compound C) and ferroptosis inducer (RSL3) can significantly inhibit the proliferation of non-small cell lung cancer with high expression of MORC2, especially LKB1 deficiency. This study provides ideas and basis for the diagnosis and treatment of non-small cell lung cancer with high expression of MORC2, and has certain theoretical and clinical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 MORC2 is highly expressed in human lung cancer tissues. A is the expression of MORC2 in non-matching tissue samples of lung adenocarcinoma and lung squamous cell carcinoma predicted by the TCGA database; B is the expression of MORC2 in matching tissue samples of lung adenocarcinoma and lung squamous cell carcinoma predicted by the TCGA database; C is the CCK-8 experiment confirming that overexpression of MORC2 promotes the proliferation of H838, A549 and H1650 cells. * P <0.05;** P <0.01;*** P <0.001.

[0021] Figure 2To predict the docking structure binding mode of MORC2 with AMPK inhibitor (Compound C) and ferroptosis inducer (RSL3) respectively using induced fit docking technology. Among them, A is the ranking of the top four conformations of RSL3 induced fit docking scores and the top three conformations of Compound C induced fit docking scores using metadynamics simulation. The collective variable (CV) is the root mean square deviation (RMSD) of the ligand. During the simulation, it is calculated by superimposing the binding site residues to consider the drift of the entire protein-ligand complex relative to the starting position; B is the 3D structure of MORC2 docked with RSL3 shown by induced fit docking technology, and the right picture is the 2D structure of MORC2 docked with RSL3; C is the 3D structure of MORC2 docked with Compound C shown by induced fit docking technology, and the right picture is the 2D structure of MORC2 docked with Compound C.

[0022] Figure 3 The combined use of AMPK activity inhibitor (Compound C) and ferroptosis inducer (RSL3) can significantly inhibit the proliferation of NSCLC with high expression of MORC2. AB are clone formation experiments to detect the clone formation ability of A549 and H838 cells overexpressing MORC2 and their control groups treated with Compound C / RSL3, and the right side is a statistical chart of the number of cell clones; C is a soft agar clone formation experiment to detect the sphering ability of A549 cells overexpressing MORC2 and their control groups treated with Compound C / RSL3 (scale: 500μm), and the right figure analyzes the clone area of ​​cells in soft agar. * represents P <0.05; ** represents P <0.01; *** represents P <0.001; ns means p >0.05 is not statistically significant.

[0023] Figure 4 The combination of AMPK and ferroptosis inducers can significantly inhibit the proliferation of NSCLC with high MORC2 expression. AB shows the A549 and H838 cells overexpressing MORC2 and their control groups, which were transiently transfected with siAMPK for 24 hours, and then treated with RSL3 for 24 hours. The cell proliferation ability was detected by cloning assay, and the right side is a statistical chart of the number of cell clones; C shows the A549 cells overexpressing MORC2 and their control group, which were transiently transfected with siAMPK for 24 hours, and then treated with RSL3 for 24 hours. The soft agar cloning assay was used to detect the cell sphering ability (scale: 500μm). The right figure analyzes the clone area of ​​cells in soft agar. * represents P <0.05; ** represents P <0.01; *** representsP <0.001; ns represents p >0.05 is not statistically significant.

[0024] Figure 5 The experiment on tumor-bearing mice verified that the combined use of AMPK activity inhibitor (Compound C) and ferroptosis inducer (RSL3) can significantly inhibit the tumor growth of NSCLC caused by high expression of MORC2. Among them, A is a schematic diagram of subcutaneous tumor formation and dosing regimen; B is a tumor image of subcutaneous transplanted tumor after treatment with Compound C and RSL3; C is the tumor volume measurement result of subcutaneous transplanted tumor after treatment with Compound C and RSL3; D is the tumor weight measurement result of subcutaneous transplanted tumor after treatment with Compound C and RSL3; E is an immunohistochemical experiment to detect the expression of Ki67 in tumor tissue sections after treatment with Compound C and RSL3 (scale: 100μm), and the right picture is a statistical chart of Ki67 protein immunohistochemical staining results. * represents P <0.05; ** represents P <0.01; *** represents P <0.001; ns means p >0.05 is not statistically significant. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below with specific embodiments, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

[0026] Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0027] Experimental materials.

[0028] 1. Cell lines: The human lung cancer cell lines used in the experiment were all from the American Type Culture Collection (ATCC) cell bank. The H1650 cells used in the examples are LKB1 wild-type human lung cancer cells; H838 and A549 cells are LKB1-deficient human lung cancer cells.

[0029] 2. The transient interference AMPK (siAMPK) sequence was synthesized by Suzhou Jima Biotechnology Co., Ltd. .

[0030] 3. Experimental animals: 4-6 weeks old immunodeficient nude mice BALB / c Nude were purchased from Beijing Sibeifu Biotechnology Co., Ltd. The nude mice weighed 13-17 g. All experimental animals were kept in the Experimental Animal Department of China Medical University and approved by the Experimental Animal Ethics Committee with the ethics approval number CMU20240051.

[0031] 4. Main experimental reagents: Ferroptosis inducer RSL3 (catalog number HY10028) and Compound C (catalog number HY-13418A) were purchased from MCE, and RPMI-1640 culture medium was purchased from Gibco.

[0032] Related experimental operation methods.

[0033] 1. Bioinformatics analysis.

[0034] The TCGA database was used to analyze the mRNA levels of MORC2 between tumor tissues and normal tissues in lung adenocarcinoma and lung squamous cell carcinoma.

[0035] 2. Induced fit docking.

[0036] In this study, binding site analysis was performed on the MORC2 protein structure (PDB 5OF9) using Schrodinger Sitemap. Induced fit docking of AMPK inhibitor (Compound C) and ferroptosis inducer (RSL3) used the MORC2 protein structure (PDB 5OF9) as the receptor model. Induced fit docking and metadynamics simulations used induced fit and binding pose metadynamics programs as implemented in the Maestro suite and followed published procedures. Clustering of ligand binding poses used the StructuralInteraction Fingerprints program in the Maestro suite and followed published procedures. Simulation trajectories were analyzed using simulation interaction diagrams implemented in Schrodinger Desmond.

[0037] 3. Construction of cell lines stably expressing target proteins.

[0038] MORC2 overexpression lentivirus (pGC-Flag-MORC2-Lentivirus) and negative control lentivirus (pGC-Flag-vector-Lentivirus) were purchased from GeneCare Biotech. Cells infected with MORC2 overexpression lentivirus were the experimental group (oe-MORC2 group), and cells infected with negative control lentivirus were the control group (oe-NC group). The specific steps are as follows.

[0039] (1) Cultivate the required cells. When the growth density reaches 70%-80%, digest and count 2×10 5The cells were plated in 6-well plates.

[0040] (2) On the second day, when the density reaches 40%-50% and the cells are in good condition, virus transfection can be performed. Calculate the required volume of virus solution in advance (virus volume = MOI × number of cells / virus titer). Preliminary experiments have shown that the MOI value of H838, H1650 and A549 cells is approximately 100. Discard the culture medium, wash once with PBS, and add 1 mL of fresh culture medium containing 10% FBS and the calculated volume of virus solution and virus infection reagent (40 μL) in turn, mix well and place in the incubator for continued culture.

[0041] (3) Within 24 hours of infection, replace the medium with 2 mL of fresh RPMI 1640 complete medium and continue culturing.

[0042] (4) After 48 hours of infection, the culture medium was replaced and puromycin (2 μg / mL) was added to screen the cells. Based on the cell screening results, the transfection effect of the target protein MORC2 was detected by RT-qPCR and Western blot.

[0043] In this example, the combined use of an AMPK activity inhibitor (Compound C) and a ferroptosis inducer (RSL3) can significantly inhibit the proliferation of NSCLC cells that highly express MORC2.

[0044] 4.CCK8 experiment.

[0045] (1) Cell treatment: Collect cells in good condition, digest them with trypsin, centrifuge at 800 rpm for 5 min, discard the supernatant, resuspend the cell pellet in 1 mL of culture medium, count the cells, and dilute the cells to a concentration of 2 × 10 4 / mL, aspirate 100 μL of cell suspension into a 96-well plate, set up 3 replicate wells for each group, and incubate in a cell culture incubator.

[0046] (2) Drug treatment: Add 10 μL of CCK8 reagent to each well of 100 μL culture medium. Add CCK8 reagent to the 96-well plate at 0 h (4 h after cell inoculation), 24 h, 48 h, and 72 h, mix well, and continue to culture in the cell culture incubator for 2 h.

[0047] (3) Measurement: Measure the absorbance at 450 nm using an ELISA reader, draw a curve and perform analysis.

[0048] 5. Plate colony formation experiment.

[0049] (1) Cell treatment: H838 and A549 cells (oe-NC and oe-MORC2 groups) in the logarithmic growth phase and in good condition were digested with trypsin and 3×105 Cells were plated in 6-well plates and divided into eight groups according to the needs: oe-NC+DMSO group, oe-NC+Compound C group, oe-NC+RSL3 group, oe-NC+CompoundC+RSL3 group, oe-MORC2+DMSO group, oe-MORC2+Compound C group, oe-MORC2+RSL3 group, oe-MORC2+Compound C+RSL3 group. The final concentration of Compound C was (10μM), and the final concentration of RSL was (2μM). After mixing, the cells were cultured for 24 hours. After treatment, 1000 cells were taken after trypsin digestion, inoculated in 6-well plates and incubated in a cell culture incubator. The complete culture medium was replaced every 2-3 days.

[0050] (2) Fixation: After the cells have been incubated in the incubator for 10 days, remove the 6-well plate, discard the culture medium, and wash twice with PBS. Add 1 mL of pre-cooled 75% ethanol to each well and fix at room temperature for 15 minutes.

[0051] (3) Staining: Remove 75% ethanol, wash twice with PBS, add 1 mL of crystal violet stain (1%) to each well, and stain for 15 min at room temperature. Then remove the stain and rinse the 6-well plate under running water along the edge of the plate to wash off excess stain.

[0052] (4) Observation: Place the plate upside down on the table, let it dry, then take a photo and analyze it.

[0053] 6. Cell soft agar cloning experiment.

[0054] (1) Preparation: Weigh 0.6g and 1.2g of agar powder respectively and add them to two glass bottles containing 100mL PBS to make agar concentrations of 0.6% and 1.2%. Stir and dissolve, then place in an autoclave for sterilization. When the temperature drops to about 50℃, take it out and place it in a 65℃ oven to keep it in a melted state for later use.

[0055] (2) Preparation of the lower layer of gel: prepare a 50 mL centrifuge tube, take out 1.2% agar and complete culture medium in equal proportions from the oven and add them to the centrifuge tube. Mix well with a pipette and add to a 6-well plate (2 mL per well). Cool and solidify at room temperature and set aside.

[0056] (3) Cell treatment: H838 and A549 cells (oe-NC and oe-MORC2 groups) in the logarithmic growth phase and in good condition were digested with trypsin and 3×10 5Cells were plated in 6-well plates and divided into eight groups according to the needs: oe-NC+DMSO group, oe-NC+Compound C group, oe-NC+RSL3 group, oe-NC+CompoundC+RSL3 group, oe-MORC2+DMSO group, oe-MORC2+Compound C group, oe-MORC2+RSL3 group, oe-MORC2+Compound C+RSL3 group. The final concentration of Compound C was (10μM), and the final concentration of RSL was (2μM). After mixing, the cells were cultured for 24h. After treatment, the cells were digested with trypsin and counted, and the cells were diluted to a concentration of 1×10 6 / mL.

[0057] (4) Preparation of the upper gel: Take one hole in a 6-well plate as an example, take 30 μL of cell suspension and add it to a new 15 mL centrifuge tube, then add 2 mL of complete culture medium and mix well, then add 1 mL of 0.6% agar solution, mix gently to avoid bubbles, take out 1 mL of the mixed solution and add it to the prepared lower gel. After cooling and solidifying at room temperature, put it in a cell culture incubator for incubation. The final number of cells in each well of the 6-well plate is 10 4 cells.

[0058] (5) Observation: After incubation for about 10 days, when cell spheres are observed under a microscope, take photos and count the number of cell clones and the size of the spheres.

[0059] 7. Construction of tumor-bearing mouse model and in vivo experiments.

[0060] (1) Animal husbandry: 5-week-old female immunodeficient nude mice were purchased from Beijing Sibeifu Biotechnology Co., Ltd. All experimental animals were kept in the Experimental Animal Department of China Medical University (SPF grade). All experiments were approved by the Experimental Animal Ethics Committee before they were carried out.

[0061] (2) Cell preparation: H1650 cells from the oe-MORC2 group and the control group (oe-NC) with good tumorigenicity and in the logarithmic growth phase were selected, digested with trypsin, resuspended in PBS, centrifuged at 800 rpm for 5 min, discarded the supernatant, and repeated three times. Resuspend the cells in PBS, count and adjust the cell concentration to 5×10 7 cells / mL and placed on ice for later use. Before injection, 100 μL of cell suspension was taken out from each tube and mixed evenly with an equal volume (100 μL) of matrix gel.

[0062] (3) Subcutaneous injection: 16 5-week-old female nude mice weighing 13-17 g were randomly divided into 4 groups. The injection site was disinfected with iodine before inoculation. 200 μL of the H1650 cell matrix gel suspension of the control group was subcutaneously inoculated in the left axilla, and 200 μL of the H1650 cell matrix gel suspension of the oe-MORC2 group was subcutaneously inoculated in the right axilla. The mice were kept in a sterile environment. The general condition and tumor growth of the nude mice were observed daily. When the subcutaneous tumor was visible to the naked eye, the longest and shortest parts of the tumor were measured with a vernier caliper to calculate the tumor volume.

[0063] Tumor volume = 1 / 2 × a × b 2 (a is the longest diameter of the tumor, b is the shortest diameter of the tumor).

[0064] (4) Drug experiment: When the subcutaneous tumor volume of nude mice reaches 50-100mm 3 Drug experiments were conducted at the same time. Nude mice were randomly divided into four groups, with 4 nude mice in each group, and treated with solvent control (PBS), Compound C, RSL3, and Compound C + RSL3 combined treatment. The specific administration methods are as follows: solvent control (PBS), Compound C, and RSL3 single administration groups were set up. The concentrations of RSL3 and Compound C were 100 mg / kg and 10 mg / kg, respectively. RSL3 was administered by intratumoral injection, and Compound C was administered by intraperitoneal injection. The combined medication group was first given RSL3 (twice a week, for 2 weeks), stopped for 1 day, and then given Compound C (once a day, for 1 week). The general vital signs of nude mice, such as breathing and vitality, were observed every day to be normal, and the weight and tumor size of nude mice were recorded. After the drug experiment was completed, the mice were killed by cervical dislocation, and the tumors of tumor-bearing mice were separated and photographed. The weight of each tumor was weighed separately. The tumor was stored in a liquid nitrogen tank for subsequent experiments.

[0065] Experimental results.

[0066] Example 1 MORC2 is highly expressed in human lung cancer tissues and promotes tumor proliferation.

[0067] To explore the differential expression of MORC2 in non-small cell lung cancer, we downloaded the relevant information of lung adenocarcinoma and lung squamous cell carcinoma from the TCGA database. Figure 1 As shown in A, the expression of MORC2 in lung adenocarcinoma and lung squamous cell carcinoma tissues was significantly higher than that in normal lung tissues ( P <0.01 is statistically significant). Figure 1 As shown in B, the differential analysis results of TCGA paired samples showed that MORC2 showed the same trend in lung adenocarcinoma and lung squamous cell carcinoma tissues ( P<0.01 was statistically significant). We then used CCK8 assay to detect the proliferation level of non-small cell lung cancer cells in the control group and the group with stable overexpression of MORC2. Figure 1 As shown in C, the proliferation rate of H838, A549, and H1650 cells stably overexpressing MORC2 increased compared with the control group ( P <0.05 was statistically significant). These results indicate that MORC2 is upregulated in non-small cell lung cancer cells and can promote the proliferation of non-small cell lung cancer.

[0068] Example 2 In this example, the docking structure binding mode of MORC2 with AMPK inhibitor (Compound C) and ferroptosis inducer (RSL3) was predicted by induced fit docking technology.

[0069] To explore whether MORC2 can be used as a drug target to provide a new potential therapeutic strategy for the treatment of tumors with high MORC2 expression. First, we performed molecular docking of RSL3 and Compound C with the protein structure of MORC2 by induced fit docking (IFD). The top four conformations of RSL3 induced fit docking scores and the top three conformations of Compound C induced fit docking scores were selected, and then metadynamics simulation was used to rank the possible conformations, such as Figure 2 As shown in Figure A, RSL3 conformation 4 and Compound C conformation 1 were selected as the most reasonable binding conformations for induced fit docking. The results showed that both RSL3 and Compound C could bind to the GHKL domain of MORC2 protein (e.g. Figure 2 (shown in BC).

[0070] Example 3 The combined use of an AMPK activity inhibitor (Compound C) and a ferroptosis inducer (RSL3) can significantly inhibit the proliferation of NSCLC cells that highly express MORC2.

[0071] Next, in order to verify that RSL3 and Compound C can provide a new method for the treatment of MORC2-overexpressing tumors. First, we treated A549 and H838 cells in the control group and MORC2-overexpressing group with RSL3 and Compound C or a combination of the two for 24 hours, and divided them into eight groups: oe-NC+DMSO group, oe-NC+Compound C group, oe-NC+RSL3 group, oe-NC+Compound C+RSL3 group, oe-MORC2+DMSO group, oe-MORC2+Compound C group, oe-MORC2+RSL3 group, oe-MORC2+Compound C+RSL3 group, and detected the cell proliferation ability through colony formation and soft agar colony formation experiments. Figure 3 As shown in Figures AB, compared with the oe-NC+DMSO group, the number of clones in the oe-MORC2+DMSO group increased significantly, and the oe-MORC2+Compound C group and the oe-MORC2+RSL3 group decreased to varying degrees compared with the oe-MORC2+DMSO group. After the combination of the two, the oe-MORC2+CompoundC+RSL3 group decreased significantly compared with the oe-MORC2+DMSO group. The results of the soft agar clone formation experiment were consistent with those of the clone formation experiment (e.g. Figure 3 This indicates that the combined use of AMPK activity inhibitor (Compound C) and ferroptosis inducer (RSL3) can significantly inhibit the proliferation of NSCLC cells with high expression of MORC2.

[0072] We then transiently transfected siAMPK based on overexpression of MORC2, and then added RSL3 for 24 hours. We also tested the cell proliferation ability by colony formation and soft agar colony formation experiments. Figure 4 As shown in Figures AB, compared with the oe-NC+DMSO group, the number of clones formed in the oe-MORC2+DMSO group increased significantly, and the oe-MORC2+siAMPK group and the oe-MORC2+RSL3 group decreased to varying degrees compared with the oe-MORC2+DMSO group. After the combination of the two, the oe-MORC2+siAMPK+RSL3 group decreased significantly compared with the oe-MORC2+DMSO group. The results of the soft agar clone formation experiment were consistent with those of the clone formation experiment (e.g. Figure 4 (as shown in C).

[0073] Example 4 Experiments on tumor-bearing mice verified that the combined use of an AMPK activity inhibitor (Compound C) and a ferroptosis inducer (RSL3) can significantly inhibit the tumor growth of NSCLC caused by overexpression of MORC2.

[0074] According to the above results, AMPK inhibitors combined with ferroptosis inducers can significantly inhibit the proliferation of non-small cell lung cancer cells caused by high expression of MORC2 in vitro, suggesting that AMPK inhibitors combined with ferroptosis inducers may also have a good killing effect on non-small cell lung cancer cells with high expression of MORC2 in vivo. To this end, we verified this through tumor-bearing mouse experiments. First, H1650 cells of the control group (oe-NC) and the oe-MORC2 group were inoculated subcutaneously in the axillae on both sides of nude mice. After tumor formation, the nude mice were randomly divided into four groups, with 4 nude mice in each group, and were given solvent control (PBS), Compound C, RSL3, and Compound C+RSL3 combined treatment (such as Figure 5 The breathing, vitality and other general vital signs of the nude mice were observed every day to see if they were normal, and the weight and tumor size of the nude mice were recorded. One week after the end of the drug experiment, the mice were killed by cervical dislocation, and the tumors of the tumor-bearing mice were isolated and photographed. The weight of each tumor was weighed separately. Consistent with the results of the in vitro experiment, we found that the tumor growth and tumor volume of the oe-MORC2+PBS group were significantly increased compared with the oe-NC+PBS group (as shown in Figure 2A). Figure 5 Meanwhile, the combined treatment of Compound C and RSL3 significantly inhibited the tumor growth of the oe-MORC2 group (as shown in Figure 5 As shown in B, the tumor volume of tumor-bearing mice decreased (as shown in Figure 5 C), decreased tumor weight (as shown in Figure 5 Next, we fixed and sliced ​​these tumor tissues and performed immunohistochemical staining of the proliferation-related antigen Ki67. The experimental results showed that the expression of Ki67 in the oe-MORC2+PBS group was higher than that in the control group, indicating that overexpression of MORC2 promoted the proliferation of non-small cell lung cancer cells in vivo. After the combined treatment of CompoundC+RSL3, the expression of Ki67 in the MORC2 overexpression group was significantly reduced (as shown in Figure 2A). Figure 5 (shown in E).

[0075] In summary, at the cellular level and in animal experiments, we found that the combined use of AMPK inhibitor (Compound C) and ferroptosis inducer (RSL3) can significantly inhibit the proliferation of NSCLC with high MORC2 expression and the growth of tumors in tumor-bearing mice. This study provides ideas and basis for the diagnosis and treatment of NSCLC with high MORC2 expression, especially LKB1 deficiency, and has certain theoretical and clinical significance.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A pharmaceutical preparation for treating non-small cell lung cancer, characterized in that: The invention comprises the following preparations in separate or mixed packages: containing an AMPK inhibitor; containing a ferroptosis inducer.

2. The pharmaceutical product according to claim 1, characterized in that The AMPK inhibitor includes Compound C or siRNA; the ferroptosis inducer includes RSL3.

3. The pharmaceutical product according to claim 1, characterized in that The non-small cell lung cancer also includes LKB1-deficient type.

4. The pharmaceutical product according to claim 1, characterized in that The pharmaceutical product also includes pharmaceutically acceptable excipients.

5. The application of the active ingredients of AMPK inhibitors and ferroptosis inducers in the preparation of products that inhibit the proliferation and invasion ability of tumor cells.

6. The use according to claim 5, characterized in that: The AMPK inhibitor includes Compound C or siRNA; the ferroptosis inducer includes RSL3.

7. The use according to claim 5, characterized in that: The non-small cell lung cancer also includes LKB1-deficient type.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises an AMPK inhibitor and a ferroptosis inducer.

9. The pharmaceutical composition according to claim 8, characterized in that The AMPK inhibitor includes Compound C or siRNA; the ferroptosis inducer includes RSL3.

10. Use of the pharmaceutical composition according to claim 8 in the preparation of a medicament for treating non-small cell lung cancer.

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