Application of IGF2BP2 inhibitor CWI1-2 in preparation of medicine for treating gastric cancer

Abstract

The invention discloses an application of an IGF2BP2 inhibitor CWI1-2 in preparation of a medicine for treating gastric cancer. Experimental evidence shows that the CWI1-2 induces gastric cancer cell ferroptosis through IGF2BP2-GPX4 axis specificity, and a theoretical basis and experimental data support are provided for clinical development of IGF2BP2 targeted drugs or gastric cancer treatment drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-cancer drug development, and particularly relates to the application of an IGF2BP2 inhibitor CWI1-2 in the preparation of a drug for treating gastric cancer. Background Art

[0002] Gastric cancer (GC) is a malignant tumor of the digestive system that seriously threatens human health. Despite significant progress in radical surgical resection and systemic radiotherapy and chemotherapy, the main aim is to induce regulatory cell death mechanisms such as apoptosis, pyroptosis, or necrosis, but the prognosis of advanced GC remains poor.

[0003] Epigenetics is a reversible gene regulation method with tissue, cell, and gene specificity. Among them, m6A (N6-methyladenosine) is the most common internal RNA modification form in eukaryotes. The m6A modification is dynamically regulated by "Writer", "Eraser", and "Reader" proteins, ultimately affecting RNA splicing, translation, stability, and the epigenetic regulation of certain non-coding RNAs. IGF2BP2 is a member of the IGF2BP family (Insulin-Like Growth Factor2mRNA-Binding Protein), which can mediate the post-transcriptional fine regulation of the expression of genes related to tumor cell proliferation, survival, chemoresistance, and metastasis. Moreover, IGF2BP2 is highly expressed in gastric cancer. However, there is no relevant report on the application of the IGF2BP2-specific inhibitor CWI1-2 in gastric cancer.

[0004] Ferroptosis, as a form of non-apoptotic cell death driven by iron-dependent lipid peroxidation, is molecularly characterized by the dual collapse of the glutathione (GSH) biosynthesis system and the antioxidant defense system: GSH depletion leads to the loss of glutathione peroxidase 4 (GPX4) activity, thereby blocking its ability to catalyze the glutathione reductase (GSH-GSSG) cycle to scavenge lipid peroxides (LPO). GPX4, as a key molecule maintaining redox homeostasis, specifically scavenges membrane phospholipid hydroperoxides through GSH-dependent catalytic reactions, constituting the core barrier of the ferroptosis defense system. The existing studies on IGF2BP2 in gastric cancer have the following defects: (1) high off-target risk: siRNA / shRNA technology is prone to experimental conclusion deviation due to the non-specificity of gene silencing; (2) application limitation: Whether the IGF2BP2 inhibitor CWI1-2 has an inhibitory effect on gastric cancer and whether it regulates ferroptosis of gastric cancer cells by targeting GPX4 are currently unknown. Summary of the Invention

[0005] To overcome the disadvantages and deficiencies of the prior art, the purpose of the present invention is to provide an application of an IGF2BP2 inhibitor CWI1-2 in the preparation of a medicament for treating gastric cancer.

[0006] The present invention is realized as follows: an application of an IGF2BP2 inhibitor CWI1-2 in the preparation of a medicament for treating gastric cancer.

[0007] Preferably, an application of an IGF2BP2 inhibitor CWI1-2 in the preparation of a medicament for inhibiting the proliferation of gastric cancer cells, enhancing the lipid peroxidation of gastric cancer cells and / or inducing ferroptosis of gastric cancer cells.

[0008] The present invention overcomes the deficiencies of the prior art and provides an application of an IGF2BP2 inhibitor CWI1-2 in the preparation of a medicament for treating gastric cancer. In the present invention, the IGF2BP2 inhibitor CWI1-2 (Cas No.: 2408590-36-1, molecular formula: C 22 H 17 C l3 N6O3, molecular weight: 519.77, alias: N',N',2-tris(5-chloro-2-hydroxybenzylidene)hydrazine-1-carbohydrazide) inhibits the expression of IGF2BP2, down-regulates the stability of GPX4 mRNA through m6A modification. In addition, CWI1-2 induces the accumulation of lipid peroxides MDA and 4-HNE by targeting GPX4, and finally achieves the purpose of inhibiting the proliferation of gastric cancer cells, enhancing the lipid peroxidation of gastric cancer cells and inducing ferroptosis of gastric cancer cells.

[0009] Compared with the disadvantages and deficiencies of the prior art, the present invention has the following beneficial effects: Experimental evidence of the present invention shows that CWI1-2 specifically induces ferroptosis of gastric cancer cells through the IGF2BP2-GPX4 axis, which provides a theoretical basis and experimental data support for the clinical development of IGF2BP2-targeted drugs or medicaments for treating gastric cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1The m6A key "reader" protein IGF2BP2 is highly expressed in gastric cancer, and gastric cancer patients with high expression of IGF2BP2 have poor prognosis; among them, (A) TIMER (Tumor IMmune Estimation Resource) online data analysis of the expression levels of IGF2BP2 in gastric cancer tissues and adjacent tissues; (B) Kaplan-Meier online database analysis of the relationship between the expression of IGF2BP2 and the survival prognosis of gastric cancer; (C) Western Blot detection of the protein levels of IGF2BP2 in gastric cancer cells and GES-1 cells; (D) qRT-PCR detection of the mRNA levels of IGF2BP2 in gastric cancer cells and GES-1 cells; (E) IHC detection of the levels of IGF2BP2 in gastric cancer tissues and adjacent tissues.

[0011] Figure 2 The IGF2BP2 inhibitor CWI1-2 inhibits the proliferation of gastric cancer cells and induces lipid peroxidation in gastric cancer cells; among them, (A) CWI1-2 was used to treat gastric cancer cells HGC-27, MKN-45 and MGC-803, and CCK-8 was used to detect cell viability; (B) CWI1-2 was used to treat gastric cancer cells HGC-27, MKN-45 and MGC-803, and the H2DCFDA fluorescent probe was used to detect the intracellular ROS level; (C) CWI1-2 was used to treat gastric cancer cells HGC-27, MKN-45 and MGC-803, and the MDA kit was used to detect the intracellular MDA level; (D) CWI1-2 was used to treat gastric cancer cells HGC-27, MKN-45 and MGC-803, and Western Blot was used to detect the levels of IGF2BP2 and 4-HNE.

[0012] Figure 3 The IGF2BP2 inhibitor CWI1-2 downregulates its expression by reducing the mRNA stability of GPX4; among them, (A) CWI1-2 was used to treat gastric cancer cells HGC-27, MKN-45 and MGC-803, and qRT-PCR was used to detect the mRNA level of GPX4; (B) CWI1-2 was used to treat gastric cancer cells HGC-27, MKN-45 and MGC-803, and Western Blot was used to detect the protein level of GPX4; (C) After treating HGC-27, MKN-45 and MGC-803 cells with CWI1-2, actinomycin D (2 μg / mL) was used to treat for specified times (0, 2, 4 and 8 h), and qRT-PCR was used to detect the mRNA level of GPX4.

[0013] Figure 4CWI1-2 inhibits IGF2BP2 from inducing ferroptosis in gastric cancer cells by targeting GPX4; among them, (A) qRT-PCR was used to verify the overexpression effect of GPX4; (B) Western Blot was used to verify the effects of GPX4 overexpression lentivirus (upper) and overexpression plasmid (lower); (C-F) MGC-803 cells treated with CWI1-2 for control or GPX4-OE for 72 hours, and the cell viability was detected by CCK-8 assay (C), the intracellular MDA level was detected by colorimetry (D), the protein levels of GPX4 and 4-HNE were detected by Western Blot (E), and the intracellular ROS level was detected by flow cytometry (F). Detailed implementation manners

[0014] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0015] Example 1 The m6A key "reader" protein IGF2BP2 is highly expressed in gastric cancer, and gastric cancer patients with high expression of IGF2BP2 have poor prognosis

[0016] 1. Online analysis of TIMER database and Kaplan MeierPlotter database

[0017] Using the TIMER (Tumor IMmune Estimation Resource) (https: / / cistrome.shinyapps.io / timer / ) online database, select the "DiffExp module" to study the differential expression of the IGF2BP2 gene between tumor and adjacent normal tissues in all tumors from the TCGA database. Box plots were used to show the distribution of gene expression levels, and the Wilcoxon test was used to evaluate the statistical significance of differential expression. The gastric cancer dataset was STAD, and the results showed that the IGF2BP2 gene was significantly more highly expressed in gastric cancer tumor tissues than in adjacent normal tissues.

[0018] Using the Kaplan MeierPlotter (https: / / kmplot.com / analysis / ) online database, select the "gastric cancer module" to analyze the overall survival rate (OS) of the IGF2BP2 (218847_at) gene in gastric cancer patients.

[0019] The results are as Figure 1As shown in A - B, the differential analysis results from the online databases TIMER (https: / / cistrome.shinyapps.io / timer / ) and KaplanMeierPlotter (https: / / kmplot.com / analysis / ) showed that IGF2BP2 was highly expressed in gastric cancer tissues, and gastric cancer patients with high expression of IGF2BP2 had poor prognosis.

[0020] 2. Western Blot (WB)

[0021] Cells or tissues were lysed on ice with RIPA lysis buffer (P0038 - 100ml, Beyotime, China) containing 0.1M PMSF (phenylmethanesulfonyl fluoride) (ST507 - 10ml, Beyotime, China). The supernatant was collected, and the protein concentration was detected using a BCA kit (P0012, Beyotime, China); boiled at 95°C for 10 min. Protein samples were separated by SDS - PAGE (sodium dodecyl sulfate - polyacrylamide gel electrophoresis), and then the separated proteins were transferred onto a PVDF membrane (ISEQ00010, Millipore, USA). Blocked with 5% skim milk at room temperature for 1 h, and the primary antibodies were incubated overnight at 4°C. The primary antibodies included IGF2BP2 (Proteintech, 11601 - 1 - AP, 1:2000), β - actin (ABclonal, AC026, 1:10000). The HRP - conjugated rabbit secondary antibody (ABclonal, AS014, 1:10000) was incubated at room temperature for 2 h. Finally, the ECL luminescence reagent (BMU102 - CN, Abbkine, China) generated chemiluminescence signals at the antibody - binding sites on the protein membrane, and was observed using a ChemiDoc imaging system (Bio - Rad, USA).

[0022] The results were as Figure 1 shown in C. As can be seen from the figure, compared with GES - 1 cells, the expression of IGF2BP2 was increased in gastric cancer cell lines.

[0023] 3. Real - Time Fluorescent Quantitative PCR (qRT - PCR)

[0024] Add 500 μL of RNA-easy Isolation Reagent (R701-01, Vazyme, China) to the cell samples, pipette thoroughly to mix well, and extract total RNA according to the instruction manual. Use HiScript II Q RT SuperMix for qPCR (+gDNAwiper) (R223, Vazyme, China) and ChamQ Universal SYBR qPCR Master Mix (Q711, Vazyme, China) for cDNA synthesis and PCR reaction. The primers are as follows:

[0025] IGF2BP2-Forward: 5’-AATCTCTTCATCCCAACCCAG-3’;

[0026] IGF2BP2-Reverse: 5’-ATGACCATCCTTTCGCTGAC-3’;

[0027] β-actin-Forward: 5’-AATCTCTTCATCCCAACCCAG-3’;

[0028] β-actin-Reverse: 5’-ATGACCATCCTTTCGCTGAC-3’.

[0029] Quantitative analysis was performed using the 2 -ΔΔCt method.

[0030] The results are as Figure 1 shown in Figure D. As can be seen from the figure, compared with GES-1 cells, the expression of IGF2BP2 is increased in gastric cancer cell lines.

[0031] 4. Immunohistochemistry (IHC)

[0032] Gastric cancer tissue and corresponding adjacent normal tissue samples were fixed with formalin, embedded in paraffin, and sectioned. The sections were dewaxed, hydrated, and heat-induced antigen retrieval was performed. Then, 3% hydrogen peroxide was added dropwise to quench endogenous peroxidase activity, and the sections were blocked with 3% goat serum. The primary antibody was incubated overnight at 4°C. The primary antibody was IGF2BP2 (Proteintech, 11601-1-AP, 1:200). The HRP-conjugated rabbit secondary antibody (Servicebio, G1215, 1:200) was incubated with the sections at room temperature for 2 h, stained with 3,3-diaminobenzidine tetrahydrochloride, counterstained with hematoxylin, and sealed with neutral resin. The results were observed with an upright fluorescence microscopy imaging system (Olympus BX73, Tokyo, Japan) as Figure 1 shown in Figure E. As can be seen from the figure, the level of IGF2BP2 in gastric cancer tissue is significantly increased.

[0033] Example 2: The IGF2BP2 inhibitor CWI1-2 inhibits the proliferation of gastric cancer cells and induces lipid peroxidation in gastric cancer cells

[0034] 1. Cell viability and proliferation assay (CCK-8)

[0035] Cell viability and proliferation were measured using a Cell Counting Kit-8 (CCK-8, C6005, NCM Biotech, China). Gastric cancer cells HGC-27, MKN-45, and MGC-803 were seeded into 96-well plates at a density of 3000 cells per well and then treated with DMSO, 0.25 μM, 0.5 μM, or 1 μM CWI1-2 (HY-153274, MCE, China) for 72 h. 10 μL of CCK-8 reagent was added to each well and incubated at 37 °C for 2 h. The absorbance at 450 nm was measured using a microplate reader (Varioskan LUX, Thermo Fisher Scientific, USA).

[0036] The results were as Figure 2 shown in Fig. A. Treatment of gastric cancer cells HGC-27, MKN-45, and MGC-803 with the IGF2BP2 inhibitor CWI1-2 significantly inhibited cell proliferation.

[0037] 2. Detection of intracellular reactive oxygen species (ROS)

[0038] Gastric cancer cells HGC-27, MKN-45, and MGC-803 at 50000 cells per well were seeded into 6-well plates and cultured overnight in a 37 °C incubator. Each well was treated with DMSO, 1 μM, or 2 μM CWI1-2 (HY-153274, MCE, China) for 72 h. 5 μM 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA, D6883-50MG, Sigma-Aldrich, Germany) was added and incubated at 37 °C in the dark for 30 min, and then washed three times with PBS. The intracellular ROS fluorescence intensity was observed using an inverted fluorescence microscopy imaging system (Olympus IX73, Tokyo, Japan).

[0039] The results were as Figure 2 shown in Fig. B. Treatment of gastric cancer cells HGC-27, MKN-45, and MGC-803 with the IGF2BP2 inhibitor CWI1-2 significantly increased the level of ROS, a product related to intracellular lipid peroxidation.

[0040] 3. Detection of malondialdehyde (MDA)

[0041] Gastric cancer cells HGC-27, MKN-45, and MGC-803 at 50,000 cells / well were seeded into 6-well plates and cultured overnight in an incubator at 37°C. They were treated with DMSO and 2 μM CWI1-2 (HY-153274, MCE, China) for 72 h respectively. The determination of lipid peroxidation product MDA was based on the instruction manual (KTB1050, Abbkine, China). The protein lysates of gastric cancer cells in different treatment groups were mixed with the reaction mixture solution and incubated at 95°C for 30 min. The absorbance at 532 nm and 600 nm was read using an enzyme-linked immunosorbent assay (ELISA) reader (Varioskan LUX, Thermo Fisher Scientific, USA), and the MDA content was calculated according to the formula in the instruction manual.

[0042] The results are as Figure 2 shown in Figure C. Treatment of gastric cancer cells HGC-27, MKN-45, and MGC-803 with the IGF2BP2 inhibitor CWI1-2 significantly increased the level of the lipid peroxidation-related product MDA in the cells.

[0043] 4. Western blot assay (WB)

[0044] Gastric cancer cells HGC-27, MKN-45, and MGC-803 at 50,000 cells / well were seeded into 6-well plates and cultured overnight in an incubator at 37°C. Each well was treated with DMSO, 1 μM, and 10 μM CWI1-2 (HY-153274, MCE, China) for 72 h respectively. Cells were lysed on ice using RIPA lysis buffer (P0038-100 ml, Beyotime, China) containing 0.1 M PMSF (phenylmethanesulfonyl fluoride) (ST507-10 ml, Beyotime, China), and the supernatants were collected. The WB experimental procedure was the same as in Example 1C to detect the protein levels of IGF2BP2 and 4-HNE in gastric cancer cells after CWI1-2 treatment. Among them, the primary antibody for IGF2BP2 (Proteintech, 11601-1-AP, 1:2000), the primary antibody for 4-HNE (abcam, ab46545, 1:1000), the primary antibody for GAPDH (Proteintech, 10494-1-AP, 1:10,000); the HRP-conjugated rabbit secondary antibody (ABclonal, AS014, 1:10,000), the HRP-conjugated mouse secondary antibody (ABclonal, AS003, 1:10,000).

[0045] The results are as Figure 2As shown in D, treatment of gastric cancer cells HGC-27, MKN-45, and MGC-803 with the IGF2BP2 inhibitor CWI1-2 significantly increased the level of 4-HNE, a product related to intracellular lipid peroxidation. Moreover, the IGF2BP2 inhibitor CWI1-2 was able to inhibit the expression of IGF2BP2.

[0046] Example 3: The IGF2BP2 inhibitor CWI1-2 downregulates its expression by reducing the stability of GPX4 mRNA

[0047] 1. Real-time fluorescence quantitative PCR (qRT-PCR)

[0048] Gastric cancer cells HGC-27, MKN-45, and MGC-803 at 50,000 cells / well were seeded in 6-well plates and cultured overnight in a 37°C incubator. They were treated with DMSO and 2 μM CWI1-2 (HY-153274, MCE, China) for 72 h respectively. 500 μL of RNA-easy Isolation Reagent (R701-01, Vazyme, China) was added to each sample and thoroughly pipetted and mixed.

[0049] The qRT-PCR method was basically the same as the operation of real-time fluorescence quantitative PCR in Example 1. In this example, after treatment with CWI1-2, the mRNA level of GPX4 in gastric cancer cells was detected. Among them, the GPX4 primers were:

[0050] GPX4-Forward: 5’-GAGGCAAGACCGAAGTAAACTAC-3’;

[0051] GPX4-Reverse: 5’-CCGAACTGGTTACACGGGAA-3’.

[0052] The results were as Figure 3 shown in A. Treatment of gastric cancer cells HGC-27, MKN-45, and MGC-803 with the IGF2BP2 inhibitor CWI1-2 significantly downregulated the mRNA level of GPX4, a key molecule against ferroptosis.

[0053] 2. Western blotting (WB)

[0054] 50,000 gastric cancer cells HGC-27, MKN-45, and MGC-803 per well were seeded into 6-well plates and cultured overnight in an incubator at 37°C. Each well was treated with DMSO, 1 μM, or 10 μM CWI1-2 (HY-153274, MCE, China) for 72 h. Cells were lysed on ice using RIPA lysis buffer (P0038-100 ml, Beyotime, China) containing 0.1 M PMSF (phenylmethanesulfonyl fluoride) (ST507-10 ml, Beyotime, China), and the supernatants were collected.

[0055] The WB procedure was basically the same as that of the immunoblotting experiment in Example 1. In this example, the protein level of GPX4 in gastric cancer cells was detected after treatment with CWI1-2. Among them, the primary antibody against GPX4 was from HUABIO (ET1706-45, 1:2000).

[0056] The results were as Figure 3 shown in Figure B. Treatment of gastric cancer cells HGC-27, MKN-45, and MGC-803 with the IGF2BP2 inhibitor CWI1-2 significantly downregulated the protein level of the ferroptosis-resistant key molecule GPX4.

[0057] 3. RNA Stability Assay

[0058] 50,000 gastric cancer cells HGC-27, MKN-45, and MGC-803 per well were seeded into 6-well plates and cultured overnight in an incubator at 37°C. Cells were treated with DMSO and 2 μM CWI1-2 (HY-153274, MCE, China) for 72 h, respectively. To determine the stability of GPX4 mRNA, gastric cancer cells in different treatment groups were treated with 5 μg / mL actinomycin D (ActD, HY-17559, MCE, China) to stop transcription. RNA was collected at 0, 2, 4, and 8 h after transcription termination. The GPX4 mRNA level was evaluated using qRT-PCR. The qRT-PCR method in this example was the same as the operation of real-time fluorescence quantitative PCR in Example 1.

[0059] The results were as Figure 3 shown in Figure C. Treatment of gastric cancer cells with CWI1-2 inhibited the stability of GPX4 mRNA.

[0060] In summary Figure 3 The results in Figures A - C indicate that inhibition of IGF2BP2 by CWI1-2 can downregulate the expression of GPX4 by reducing the stability of GPX4 mRNA.

[0061] Example 4 CWI1-2 Inhibits IGF2BP2 to Induce Ferroptosis in Gastric Cancer Cells by Targeting GPX4

[0062] 1. Verification of plasmid transfection and lentivirus transfection

[0063] The overexpressed GPX4 (GPX4-OE) and control (NC) plasmids were purchased from GeneChem Co (Shanghai, China). Cells were cultured overnight in a 6-well plate at 37 °C. The GPX4-OE or NC plasmids at a concentration of 2000 ng / well were transfected using the Lipofectamine 3000 transfection kit (L3000-015, invitrogen, USA). Cells were collected at 48 or 72 h, and the transfection efficiency was detected by qRT-PCR and WB.

[0064] The GPX4-OE lentivirus was constructed using the Ubi MCS CBh-gcGFP IRES-puromycin vector (GeneChem Co, China). According to the manufacturer's instructions, cells were cultured overnight in a 6-well plate at 37 °C, and the multiplicity of infection (MOI) for gastric cancer cells was 10. After 72 h, stably transfected cells were selected and screened with 2 μg / mL puromycin (ST551, Beyotime, China) for stable cell lines, as shown in Figure 4 Figures A to 4B.

[0065] 2. Cell viability and proliferation assay (CCK-8)

[0066] Gastric cancer cells MGC-803 were cultured overnight in a 6-well plate at 37 °C. The GPX4-OE or NC plasmids at a concentration of 2000 ng / well were transfected using the Lipofectamine 3000 transfection kit (L3000-015, invitrogen, USA). After 24 h, the transfected cells were further treated with DMSO and 0.25 μM, 1 μM CWI1-2 (HY-153274, MCE, China) for 72 h. 10 μL of CCK-8 reagent was added to each well and incubated at 37 °C for 2 h, and the absorbance at 450 nm was detected using a microplate reader (Varioskan LUX, Thermo Fisher Scientific, USA).

[0067] The results are shown in Figure 4 Figure C. Overexpression of GPX4 reduced gastric cancer cell death caused by IGF2BP2 inhibition.

[0068] 3. Malondialdehyde detection (MDA)

[0069] Gastric cancer cells MGC-803 were cultured overnight in a 6-well plate in an incubator at 37°C. GPX4-OE or NC plasmids at a concentration of 2000 ng / well were transfected using the Lipofectamine 3000 transfection kit (L3000-015, Invitrogen, USA). After 24 h, the transfected cells were further treated with DMSO and 1 μM CWI1-2 (HY-153274, MCE, China) for 72 h. The determination of lipid peroxidation product MDA was based on the instruction manual (KTB1050, Abbkine, China). The protein lysates of gastric cancer cells in different treatment groups were mixed with the reaction mixture solution and incubated at 95°C for 30 min. The absorbance at 532 nm and 600 nm was read using a microplate reader (Varioskan LUX, Thermo Fisher Scientific, USA), and the MDA content was calculated according to the formula in the instruction manual.

[0070] The results were as Figure 4 shown in D. Overexpression of GPX4 reduced the increase in MDA in gastric cancer cells caused by IGF2BP2 inhibition.

[0071] 4. Western blot assay (WB)

[0072] Gastric cancer cells MGC-803 were cultured overnight in a 6-well plate in an incubator at 37°C. GPX4-OE or NC plasmids at a concentration of 2000 ng / well were transfected using the Lipofectamine 3000 transfection kit (L3000-015, Invitrogen, USA). After 24 h, the transfected cells were further treated with DMSO and 10 μM CWI1-2 (HY-153274, MCE, China) for 72 h. Cells were lysed on ice using RIPA lysis buffer (P0038-100 ml, Beyotime, China) containing 0.1 M PMSF (phenylmethanesulfonyl fluoride) (ST507-10 ml, Beyotime, China), and the supernatant was collected. The WB experimental procedure in this example was the same as the immunoblotting experiment operation in Example 1.

[0073] The results were as Figure 4 shown in E. Overexpression of GPX4 reduced the increase in 4-HNE in gastric cancer cells caused by IGF2BP2 inhibition.

[0074] 5. Intracellular reactive oxygen species detection (ROS)

[0075] Gastric cancer cells MGC-803 were cultured overnight in a 6-well plate in an incubator at 37°C. GPX4-OE or NC plasmids at a concentration of 2000 ng / well were transfected using the Lipofectamine 3000 transfection kit (L3000-015, Invitrogen, USA). After 24 h, the transfected cells were further treated with DMSO and 2 μM CWI1-2 (HY-153274, MCE, China) for 72 h. Gastric cancer cells in different treatment groups were digested and centrifuged. The cell pellet was washed twice with PBS, incubated with 5 μM 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA, D6883-50MG, Sigma-Aldrich, Germany) in the dark at 37°C for 30 min, and then washed twice with PBS. Then, the intracellular ROS level was detected by flow cytometry (RaiseCyte-2L6C, China).

[0076] The results are as Figure 4 shown in Figure F. Overexpression of GPX4 reduced the increase in intracellular ROS in gastric cancer cells caused by IGF2BP2 inhibition. The above results indicate that overexpression of GPX4 can partially reverse the enhancement of lipid peroxidation level and cell death in gastric cancer cells caused by IGF2BP2 inhibition. The IGF2BP2 inhibitor CWI1-2 induces ferroptosis in gastric cancer cells by targeting GPX4.

[0077] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of an IGF2BP2 inhibitor CWI1-2 in the preparation of a medicament for treating gastric cancer.

2. The application according to claim 1, characterized in that, Use of an IGF2BP2 inhibitor CWI1-2 in the preparation of a medicament for inhibiting the proliferation of gastric cancer cells, enhancing lipid peroxidation of gastric cancer cells and / or inducing ferroptosis of gastric cancer cells.

Images