SLC16A3 inhibitor based on ferroptosis regulation and application of SLC16A3 inhibitor in lung adenocarcinoma

Abstract

The invention provides an SLC16A3 inhibitor based on ferroptosis regulation and application of the SLC16A3 inhibitor in lung adenocarcinoma, and relates to the technical field of biological treatment drugs. The SLC16A3 inhibitor is a drug MSC-4381, the drug MSC-4381 is an effective SLC16A3 inhibitor, the SLC16A3 inhibitor can be used for inhibiting lactic acid transporter SLC16A3, breaking the redox steady state of tumor cells, inducing ferroptosis and remarkably enhancing the curative effect of the drug gefitinib, and in an in-vivo and in-vitro model, cell proliferation, migration and invasion can be inhibited through SLC16A3 knock-down or pharmacological inhibition, so that the SLC16A3 inhibitor can be used for preventing and treating the tumor cells. The MSC-4381 and the gefitinib are combined to promote lipid peroxidation and iron ion accumulation, the tumor growth can be obviously delayed through drug combination of the MSC-4381 and the gefitinib, the effect can be partially reversed through the Ferrostatin-1, the ferroptosis-dependent mechanism of the MSC-4381 and the gefitinib is verified, and transcriptional regulation and control of the SLC16A3 by the HIF1A and influence on ferroptosis resistance are clear. The invention provides a feasible combined medication strategy, verifies the significant tumor inhibition effect of the HIF1A-SLC16A3 axis in cell and animal models, reveals the key effect of the HIF1A-SLC16A3 axis in lung adenocarcinoma drug resistance formation, and provides a new combined treatment strategy and a new molecular target.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological therapy drugs, in particular to SLC16A3 inhibitor based on iron death regulation and its application in lung adenocarcinoma. BACKGROUND

[0002] Lung cancer is the most common malignant tumor in the world, ranking first in global cancer mortality in 2020, with a five-year survival rate of less than 20%. And the morbidity ranks second only to breast cancer in women, with about 2.2 million new cases and 1.8 million deaths each year. According to histopathology, lung cancer has two main types: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC accounts for about 80-85% of all cases, including adenocarcinoma, squamous cell carcinoma and large cell carcinoma. Lung adenocarcinoma (LUAD) is the most common type of NSCLC, accounting for about half of all malignant NSCLC.

[0003] Lung adenocarcinoma patients generally develop acquired drug resistance after receiving EGFR-TKI (gefitinib) treatment, and inducing iron death is considered an effective means to overcome such drug resistance. Excessive accumulation of lactate metabolites in lung adenocarcinoma can inhibit iron death by regulating the redox state. SLC16A3, as the main lactate transporter, is highly expressed in lung adenocarcinoma, but its mechanism of action in iron death inhibition and EGFR-TKI (gefitinib) resistance is unknown. Existing research has not systematically linked SLC16A3 to the mechanisms of iron death, drug resistance and metabolic reprogramming, and there is a lack of convertible drugs for combined intervention verification.

[0004] Therefore, the skilled person in the art provides SLC16A3 inhibitor based on iron death regulation and its application in lung adenocarcinoma to solve the problems raised in the background art. SUMMARY

[0005] In view of the deficiencies in the prior art, the present application provides SLC16A3 inhibitor based on iron death regulation and its application in lung adenocarcinoma, which solves the problem that SLC16A3, as the main lactate transporter, is highly expressed in lung adenocarcinoma, but its mechanism of action in iron death inhibition and EGFR-TKI resistance is unknown.

[0006] To achieve the above purpose, the present application is realized by the following technical scheme:

[0007] The SLC16A3 inhibitor based on iron death regulation is a drug MSC-4381, and the SLC16A3 knockdown is realized by lentivirus transfection, using three shRNAs, and the nucleotide sequence is:

[0008] shSLC16A3#1: 5'-GCGACTTGTTTATCCACTTTA-3';

[0009] shSLC16A3#2: 5'-GCACCATCTTTGTATTCATTA-3';

[0010] shSLC16A3#3: 5'-GCAGTGGATTTCTTGTTCTTT-3';

[0011] The above three shRNAs were cloned into pLKO.1 vector (Addgene) and packaged into virus using Lipofectamine 3000 (Invitrogen) in HEK293T cells.

[0012] Further, the SLC16A3 overexpression vector is full-length human SLC16A3 cDNA (NM_004207) cloned into pCDH vector (System Biosciences), and the stable cell line is obtained after screening for 7 days in 2 μg / mL puromycin (Sigma).

[0013] Further, a SLC16A3 inhibitor-containing overexpression PC9 cell line vector of claim 1.

[0014] Further, the vector is human lung adenocarcinoma cell line PC9 or PC9GR, and normal lung epithelial cell line BEAS-2B, which is cultured in DMEM or RPMI-1640 medium containing 10% fetal bovine serum (FBS, Gibco) and maintained in a 37 ℃, 5% CO2 humidified environment.

[0015] Further, application of SLC16A3 inhibitor based on ferroptosis regulation in lung adenocarcinoma.

[0016] Further, application of SLC16A3 inhibitor based on ferroptosis regulation in lung adenocarcinoma to significantly inhibit PC9 cell proliferation and invasion.

[0017] Further, application of SLC16A3 inhibitor based on ferroptosis regulation in lung adenocarcinoma to enhance the expression of ferroptosis markers and significantly enhance the efficacy of gefitinib.

[0018] Further, the stable cell line vector is regulated and verified experimentally, as follows:

[0019] S1. Construction of stable cell line

[0020] SLC16A3 knockdown is achieved by lentiviral transfection using three shRNAs:

[0021] shSLC16A3#1: 5′-GCGACTTGTTTATCCACTTTA-3′;

[0022] shSLC16A3#2: 5′-GCACCATCTTTGTATTCATTA-3′;

[0023] shSLC16A3#3:5′-GCAGTGGATTTCTTGTTCTTT-3′ was cloned into the pLKO.1 vector (Addgene). The virus was packaged in HEK293T cells using Lipofectamine 3000 (Invitrogen). The SLC16A3 overexpression vector was then cloned into the pCDH vector (System Biosciences). After stabilizing the cells, they were screened for 7 days in 2 µg / mL puromycin (Sigma) for subsequent experiments.

[0024] S2. RNA extraction and real-time quantitative PCR

[0025] Total RNA was extracted using the RNA-easy™ Isolation Kit (Vazyme), and first-strand cDNA was synthesized using the HiScript® Ⅲ RTSuperMix for qPCR (Vazyme). Quantitative PCR was performed using the ChamQ Universal SYBRqPCR Master Mix (Vazyme) on a QuantStudio 6 Flex Real-Time PCR system (AppliedBiosystems). GAPDH was used as an internal control, and the relative expression level was calculated using the 2^–ΔΔCt method. Each group was tested in triplicate.

[0026] S3. Western blot

[0027] After transfection or treatment, tissue and cell samples were homogenized and sonicated. Total protein was extracted using RIPA lysis buffer (Beyotime) combined with protease and phosphatase inhibitors (Roche). Protein concentration was determined using a BCA kit (Thermo). 25 µg of total protein per sample was boiled for denaturation, followed by SDS-PAGE separation. Protein was transferred to PVDF membranes (Millipore), blocked with 5% skim milk powder TBST for 2 hours at room temperature, and then incubated overnight at 4 °C. Primary antibodies including SLC16A3 (Proteintech), HIF1A (Abcam), SLC7A11 (Abcam), GPX4 (Abcam), FSP1 (Abcam), TFRC (Abcam), DHODH (Proteintech), and β-actin (Proteintech) were used. After washing, secondary antibodies labeled with HRP (CellSignaling) were used. (Technology) Incubated at room temperature for 1 hour, developed using ECL reagent (Thermo), and images were acquired using an automated chemiluminescence imaging system. Band density was quantified using ImageJ software, and the expression level of the target protein was normalized to β-actin. All experiments were performed in triplicate.

[0028] S4. Cell proliferation and colony formation

[0029] For cell proliferation, 2 × 10³ cells were seeded per well in a 96-well plate and cultured to the specified time point. Cell counting was performed using a Cell Counting Kit-8 (CCK-8, Sigma-Aldrich). For colony formation, 500 cells were seeded per well in a 6-well plate and cultured for 10–14 days. Cells were then fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and counted using ImageJ software. All experiments were performed in triplicate.

[0030] S5. Migration and Invasion

[0031] The healing process was observed by puncturing the monolayer of cells with a pipette tip and taking pictures at 0h and 24h. Transwell migration and invasion were performed using uncoated and Matrigel-coated insertion cavities (Corning / BD), respectively. Migrating cells were stained with 0.1% crystal violet and quantified using ImageJ. All experiments were performed in triplicate.

[0032] S6. Detection of Ferroptosis and Cell Death

[0033] Lipid peroxidation was detected using BODIPY 581 / 591 C11 (Thermo Fisher Scientific), mitochondrial membrane potential was assessed using JC-1 dye (Sigma-Aldrich), and intracellular Fe... 2+The activity of ferrostatin-1 (5 µM), Z-VAD-FMK (10 µM), chloroquine (10 µM), or glutathione (GSH, 5 mM) was detected using FerroOrange (MedChemExpress) according to the instructions. To evaluate the effects of different cell death pathways, cells were treated with ferrostatin-1 (5 µM), Z-VAD-FMK (10 µM), chloroquine (10 µM), or glutathione (GSH, 5 mM) for 24 hours, and their activity was detected using CCK-8 assay. All experiments were performed in triplicate.

[0034] S7. Detection of lactate, glucose uptake and ATP content

[0035] Intracellular and extracellular lactate levels were measured using a lactate assay kit (Beyotime), glucose uptake was measured using the Glucose Uptake-Glo Assay (Beyotime), and ATP content was measured using the ATP Determination Kit (Beyotime). All experiments were performed in triplicate.

[0036] S8. Subcutaneous xenograft model and in vivo drug treatment

[0037] PC9 cells (5 × 10) 6 (PBS and Matrigel 1:1 mixture) was injected subcutaneously into the axillary region. The genetic intervention group used PC9 cells that stably expressed shSLC16A3, OE-SLC16A3, shHIF1A or control.

[0038] In the pharmacodynamic study, mice were randomly assigned to receive DMSO (control), gefitinib (50 mg / kg), MSC-4381 (10 mg / kg), or a combination thereof, all administered via daily intraperitoneal injection. The ferroptosis remission group received additional Ferrostatin-1 (10 mg / kg). Tumor volume (length × width) was measured every 4 days using calipers. 2 / 2), mice were sacrificed on day 24, and tumors were collected for weighing, imaging, and subsequent analysis;

[0039] S9. Immunohistochemistry

[0040] Tumor tissue was fixed in formalin, embedded in paraffin and sectioned. After antigen retrieval, primary antibodies (such as SLC16A3 and Ki-67) were added, followed by HRP secondary antibody and DAB staining (ZSGB-Bio). The sections were counterstained with hematoxylin and imaged using a Leica DM6 microscope.

[0041] S10. Multiple immunohistochemistry

[0042] Tumor tissue sections were embedded in paraffin using animal models with a thickness of 4 μm. The sections were dewaxed, rehydrated, and heated in citrate buffer (pH 6.0) for antigen retrieval. Multiple rounds of staining were performed, including primary antibodies such as HIF1A, SLC16A3, GPX4, and SLC7A11. After each round, HRP secondary antibody and fluorescence development were added. Antibody stripping was performed between rounds to avoid cross-reaction. Cell nuclei were counterstained with DAPI. The sections were scanned using a multispectral imaging system, and quantitative analysis was performed using inForm software. Two tumor samples were used in each group for repeated experiments.

[0043] This invention provides an SLC16A3 inhibitor based on ferroptosis regulation and its application in lung adenocarcinoma. It possesses the following beneficial effects:

[0044] 1. This invention provides an SLC16A3 inhibitor based on ferroptosis regulation and its application in lung adenocarcinoma. By constructing SLC16A3 knockdown and PC9 overexpression cell lines, its promoting effects on proliferation, migration, and metabolism were verified. In the PC9GR model, SLC16A3 was found to participate in drug resistance formation, and KEGG enrichment suggested that it regulates the ferroptosis pathway. Knockdown of SLC16A3 enhances lipid peroxidation, iron accumulation, and mitochondrial depolarization.

[0045] 2. This invention provides an SLC16A3 inhibitor based on ferroptosis regulation and its application in lung adenocarcinoma. MSC-4381 is an effective SLC16A3 inhibitor that can enhance the efficacy of gefitinib and induce ferroptosis. Animal experiments have verified that the combination of MSC-4381 and gefitinib significantly inhibits tumor growth. Ferrostatin-1 can partially reverse the antitumor effect of MSC-4381. It is clear that HIF1A transcriptional regulation of SLC16A3 expression affects ferroptosis resistance and drug resistance mechanisms. HIF1A knockdown significantly inhibits cell invasion, migration, and colony formation. HIF1A knockdown leads to downregulation of SLC7A11 and GPX4. SLC16A3 overexpression can partially reverse the antitumor effect of HIF1A knockdown, and SLC16A3 overexpression can enhance cell resistance to erastin-induced ferroptosis.

[0046] 3. This invention provides an SLC16A3 inhibitor based on ferroptosis regulation and its application in lung adenocarcinoma. Knocking down SLC16A3 significantly inhibits the proliferation and invasion of PC9 cells, and MSC-4381 enhances the expression of ferroptosis markers, significantly enhancing the efficacy of gefitinib. In the combined treatment group, tumor growth in animal models was significantly slowed, Ki-67 levels decreased, tumor proliferation was inhibited, and ferroptosis inhibition could restore Ki-67 expression, fully demonstrating that it works through the ferroptosis mechanism. Attached Figure Description

[0047] Figure 1The figure shows the SLC16A3 suppression results of this invention.

[0048] Figure 2 The figure shows the results of the SLC16A3 knockdown inhibition of lung adenocarcinoma cell proliferation, invasion and tumorigenesis in this invention;

[0049] Figure 3 This is a diagram showing the results of the present invention regarding the enhancement of lung adenocarcinoma cell migration and invasion ability through SLC16A3 overexpression.

[0050] Figure 4 The diagram shows the results of SLC16A3 knockdown in this invention enhancing ferroptosis in lung adenocarcinoma cells by disrupting iron and redox homeostasis, with lactate playing a partial regulatory role.

[0051] Figure 5 This is a diagram showing the results of HIF1A regulation of SLC16A3 transcription and ferroptosis sensitivity in this invention;

[0052] Figure 6 The figure shows the results of the present invention, which targets the HIF1A–SLC16A3 axis to enhance the sensitivity of gefitinib-resistant LUAD cells to ferroptosis.

[0053] Figure 7 The figure shows the results of the pharmacological inhibition of SLC16A3 by the present invention, which enhances the in vivo antitumor effect of gefitinib by inducing ferroptosis. Detailed Implementation

[0054] The technical solutions of the specific embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described specific embodiments are only a part of the specific embodiments of the present invention, and not all of them. Based on the specific embodiments of the present invention, all other specific embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0055] Example 1:

[0056] like Figure 1 As shown, the following are the specific experiments on the regulation and verification of stable cell line vectors:

[0057] S1. Construction of stable cell lines

[0058] SLC16A3 knockdown was achieved via lentiviral transfection using three shRNAs:

[0059] shSLC16A3#1: 5′-GCGACTTGTTTATCCACTTTA-3′;

[0060] shSLC16A3#2: 5′-GCACCATCTTTGTATTCATTA-3′;

[0061] shSLC16A3#3:5′-GCAGTGGATTTCTTGTTCTTT-3′ was cloned into the pLKO.1 vector (Addgene). The virus was packaged in HEK293T cells using Lipofectamine 3000 (Invitrogen). The SLC16A3 overexpression vector was then cloned into the pCDH vector (System Biosciences). After stabilizing the cells, they were screened for 7 days in 2 µg / mL puromycin (Sigma) for subsequent experiments.

[0062] S2. RNA extraction and real-time quantitative PCR

[0063] Total RNA was extracted using the RNA-easy™ Isolation Kit (Vazyme), and first-strand cDNA was synthesized using the HiScript® Ⅲ RTSuperMix for qPCR (Vazyme). Quantitative PCR was performed using the ChamQ Universal SYBRqPCR Master Mix (Vazyme) on a QuantStudio 6 Flex Real-Time PCR system (AppliedBiosystems). GAPDH was used as an internal control, and the relative expression level was calculated using the 2^–ΔΔCt method. Each group was tested in triplicate.

[0064] S3. Western blot

[0065] After transfection or treatment, tissue and cell samples were homogenized and sonicated. Total protein was extracted using RIPA lysis buffer (Beyotime) combined with protease and phosphatase inhibitors (Roche). Protein concentration was determined using a BCA kit (Thermo). 25 µg of total protein per sample was boiled for denaturation, followed by SDS-PAGE separation. Protein was transferred to PVDF membranes (Millipore), blocked with 5% skim milk powder TBST for 2 hours at room temperature, and then incubated overnight at 4 °C. Primary antibodies including SLC16A3 (Proteintech), HIF1A (Abcam), SLC7A11 (Abcam), GPX4 (Abcam), FSP1 (Abcam), TFRC (Abcam), DHODH (Proteintech), and β-actin (Proteintech) were used. After washing, secondary antibodies labeled with HRP (CellSignaling) were used. (Technology) Incubated at room temperature for 1 hour, developed using ECL reagent (Thermo), and images were acquired using an automated chemiluminescence imaging system. Band density was quantified using ImageJ software, and the expression level of the target protein was normalized to β-actin. All experiments were performed in triplicate.

[0066] S4. Cell proliferation and colony formation

[0067] For cell proliferation, 2 × 10³ cells were seeded per well in a 96-well plate and cultured to the specified time point. Cell counting was performed using a Cell Counting Kit-8 (CCK-8, Sigma-Aldrich). For colony formation, 500 cells were seeded per well in a 6-well plate and cultured for 10–14 days. Cells were then fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and counted using ImageJ software. All experiments were performed in triplicate.

[0068] S5. Migration and Invasion

[0069] The healing process was observed by puncturing the monolayer of cells with a pipette tip and taking pictures at 0h and 24h. Transwell migration and invasion were performed using uncoated and Matrigel-coated insertion cavities (Corning / BD), respectively. Migrating cells were stained with 0.1% crystal violet and quantified using ImageJ. All experiments were performed in triplicate.

[0070] S6. Detection of Ferroptosis and Cell Death

[0071] Lipid peroxidation was detected using BODIPY 581 / 591 C11 (Thermo Fisher Scientific), mitochondrial membrane potential was assessed using JC-1 dye (Sigma-Aldrich), and intracellular Fe... 2+The activity of ferrostatin-1 (5 µM), Z-VAD-FMK (10 µM), chloroquine (10 µM), or glutathione (GSH, 5 mM) was detected using FerroOrange (MedChemExpress) according to the instructions. To evaluate the effects of different cell death pathways, cells were treated with ferrostatin-1 (5 µM), Z-VAD-FMK (10 µM), chloroquine (10 µM), or glutathione (GSH, 5 mM) for 24 hours, and their activity was detected using CCK-8 assay. All experiments were performed in triplicate.

[0072] S7. Detection of lactate, glucose uptake and ATP content

[0073] Intracellular and extracellular lactate levels were measured using a lactate assay kit (Beyotime), glucose uptake was measured using the Glucose Uptake-Glo Assay (Beyotime), and ATP content was measured using the ATP Determination Kit (Beyotime). All experiments were performed in triplicate.

[0074] S8. Subcutaneous xenograft model and in vivo drug treatment

[0075] PC9 cells (5 × 10) 6 (PBS and Matrigel 1:1 mixture) was injected subcutaneously into the axillary region. The genetic intervention group used PC9 cells that stably expressed shSLC16A3, OE-SLC16A3, shHIF1A or control.

[0076] In the pharmacodynamic study, mice were randomly assigned to receive DMSO (control), gefitinib (50 mg / kg), MSC-4381 (10 mg / kg), or a combination thereof, all administered via daily intraperitoneal injection. The ferroptosis remission group received additional Ferrostatin-1 (10 mg / kg). Tumor volume (length × width) was measured every 4 days using calipers. 2 / 2), mice were sacrificed on day 24, and tumors were collected for weighing, imaging, and subsequent analysis;

[0077] S9. Immunohistochemistry

[0078] Tumor tissue was fixed in formalin, embedded in paraffin and sectioned. After antigen retrieval, primary antibodies (such as SLC16A3 and Ki-67) were added, followed by HRP secondary antibody and DAB staining (ZSGB-Bio). The sections were counterstained with hematoxylin and imaged using a Leica DM6 microscope.

[0079] S10. Multiple immunohistochemistry

[0080] Tumor tissue sections were embedded in paraffin using animal models with a thickness of 4 μm. The sections were dewaxed, rehydrated, and heated in citrate buffer (pH 6.0) for antigen retrieval. Multiple rounds of staining were performed, including primary antibodies such as HIF1A, SLC16A3, GPX4, and SLC7A11. After each round, HRP secondary antibody and fluorescence development were added. Antibody stripping was performed between rounds to avoid cross-reaction. Cell nuclei were counterstained with DAPI. The sections were scanned using a multispectral imaging system, and quantitative analysis was performed using inForm software. Two tumor samples were used in each group for repeated experiments.

[0081] Example 2:

[0082] like Figure 2 As shown, SLC16A3 promotes the proliferation, migration, invasion, and tumor growth of LUAD cells.

[0083] AB, Western blot and qRT-PCR were used to verify the knockdown efficiency of three independent SLC16A3 shRNAs in PC9 cells;

[0084] C. Colony formation assays showed that SLC16A3 knockdown reduced cell proliferation.

[0085] D. Scratch assay to assess cell migration ability after SLC16A3 knockdown;

[0086] E, Transwell invasion assays showed that knocking down SLC16A3 significantly reduced cell invasion ability;

[0087] FH, in vivo tumorigenesis experiment: PC9 cells expressing control or shSLC16A3 were subcutaneously injected into BALB / c nude mice (n=4 per group) to assess tumor growth.

[0088] I. IHC staining of SLC16A3 in xenograft tumor tissue;

[0089] Changes in metabolic parameters after J,SLC16A3 knockdown: intracellular ATP level, intracellular and extracellular lactate content, and intracellular glucose uptake.

[0090] To investigate the functional role of SLC16A3, we performed knockdown experiments using three independent shRNAs in PC9 cells. Western blot and qRT-PCR confirmed successful knockdown of SLC16A3. Figure 2 A, B;

[0091] Clonogenesis assays showed that SLC16A3 knockdown significantly inhibited cell proliferation, such as Figure 2 C;

[0092] Scratch and Transwell tests showed a significant reduction in its migration and invasion capabilities, such asFigure 2 D, E;

[0093] In vivo, using a BALB / c nude mouse xenograft model, SLC16A3 knockdown significantly inhibited tumor growth, specifically manifested as a decrease in tumor weight and volume. Figure 2 F–H;

[0094] IHC staining results showed decreased SLC16A3 expression in tumors of the shRNA-treated group, such as Figure 2 I;

[0095] Furthermore, metabolic analysis revealed that SLC16A3 knockdown significantly reduced intracellular ATP levels, lactate production, and glucose uptake, suggesting a promoting effect on glycolysis. Figure 2 J.

[0096] Example 3:

[0097] like Figure 3 As shown, SLC16A3 overexpression enhances LUAD cell migration and invasion.

[0098] AB, Validation of SLC16A3 overexpression in PC9 cells. Western blot (A) and qRT-PCR (B) results showed that the protein and mRNA levels of SLC16A3 in the OE-SLC16A3 vector transfection group were significantly increased.

[0099] C. Scratch assay showed that OE-SLC16A3 cells had enhanced migration ability;

[0100] D. Transwell invasion assays showed that SLC16A3 overexpression enhanced cell invasiveness;

[0101] Overexpression experiments were performed in PC9 cells to further verify the pro-invasive function of SLC16A3. Western blot and qRT-PCR confirmed successful overexpression of SLC16A3. Figure 3 A, B;

[0102] Compared to the empty vector control, SLC16A3 overexpressing cells exhibited significantly enhanced migration, such as Figure 3 C, and the ability to invade, such as Figure 3 D supports its role in promoting malignant phenotypes.

[0103] Example 4:

[0104] like Figure 4 As shown, SLC16A3 knockdown promotes ferroptosis by disrupting iron and redox homeostasis, with lactate partially regulating this process.

[0105] A. KEGG pathway enrichment analysis of differentially expressed genes after SLC16A3 knockdown in PC9 cells: ferroptosis was the most significantly enriched pathway.

[0106] B. Western blot analysis showed changes in the expression of ferroptosis-related proteins (SLC7A11, GPX4, TFRC, FSP1, DHODH) after SLC16A3 knockdown;

[0107] Representative immunofluorescence images obtained by C, FerroOrange staining and quantitative results of intracellular Fe²⁺ levels;

[0108] D, BODIPY 581 / 591 C11 staining was used to detect lipid peroxidation and to quantify the oxidation / reduction fluorescence intensity ratio;

[0109] E,JC-1 staining assesses mitochondrial membrane potential; the red (aggregated state) / green (monomerized state) fluorescence ratio indicates the degree of mitochondrial depolarization.

[0110] F. Exogenous lactic acid treatment can reduce lipid peroxidation. BODIPY staining results showed that the oxidation level decreased after adding lactic acid.

[0111] Cell viability rescue experiments were conducted in G,shSLC16A3 cells using ferroptosis inhibitor (Fer-1), caspase inhibitor (Z-VAD-FMK), autophagy inhibitor (chloroquine), and antioxidant (GSH).

[0112] To investigate the mechanism by which SLC16A3 promotes tumor progression, KEGG pathway enrichment analysis was performed on differentially expressed genes after SLC16A3 knockdown. Ferropreservation was found to be the most significantly enriched pathway, such as... Figure 4 A;

[0113] Western blot analysis showed that SLC16A3 knockdown led to downregulation of the ferroptosis protection proteins SLC7A11 and GPX4, such as Figure 4 B, while the expression of FSP1, TFRC and DHODH showed no significant change;

[0114] FerroOrange staining showed intracellular Fe... 2+ Accumulation and increase, such as Figure 4 C, BODIPY C11 staining suggests increased lipid peroxidation, such as Figure 4 D,JC-1 staining shows mitochondrial depolarization, such as Figure 4 E;

[0115] Exogenous lactate supplementation can partially reverse lipid peroxidation, suggesting that lactate participates in ferroptosis resistance, such as Figure 4 F;

[0116] Further investigation using inhibitors of multiple cell death pathways (ferroptosis, apoptosis, autophagy, and oxidative stress) revealed that the decreased cell viability following SLC16A3 knockdown could be partially rescued, with ferrostatin-1 showing the most significant effect. Figure 4 G indicates that ferroptosis is the primary mode of cell death.

[0117] Example 5:

[0118] like Figure 5 As shown, HIF1A transcriptionally regulates SLC16A3 and affects ferroptosis sensitivity.

[0119] A, Predicted transcription factor binding motifs in the SLC16A3 promoter region;

[0120] B. Knock down candidate transcription factors and perform qRT-PCR analysis to verify that HIF1A is the main upregulator of SLC16A3 expression.

[0121] CD, based on the TCGA database (C, n=516) and clinical LUAD samples (D, n=24), analyzed the correlation between HIF1A and SLC16A3 mRNA expression;

[0122] E. Western blot and qRT-PCR validation showed that HIF1A knockdown reduced SLC16A3 protein and mRNA levels;

[0123] Functional experiments after knocking down HIF1A showed decreased invasion (F), migration (G), and colony formation (H) of LUAD cells;

[0124] I. Salvage experiment in BALB / c nude mice: shHIF1A-induced tumor growth inhibition can be partially reversed by SLC16A3 overexpression;

[0125] J. Western blot results showed that HIF1A knockdown led to downregulation of SLC16A3, SLC7A11 and GPX4 expression;

[0126] K,SLC16A3 overexpression can partially restore ferroptosis resistance after treatment with the ferroptosis inducer erastin;

[0127] L, cell viability assay showed that SLC16A3 overexpression could resist erastin-induced ferroptosis;

[0128] Transcription factor binding motif analysis from the JASPAR database predicts that HIF1A may regulate SLC16A3 promoter activity, such as Figure 5 A;

[0129] Following siRNA-mediated knockdown of HIF1A and other candidate transcription factors, SLC16A3 mRNA expression was downregulated, with HIF1A showing the most significant inhibitory effect. Figure 5 B;

[0130] Correlation analysis of expression in the TCGA database and clinical LUAD samples both showed a strong correlation between HIF1A and SLC16A3 expression, such as Figure 5 C, D;

[0131] Western blot further confirmed that HIF1A knockdown reduced SLC16A3 protein levels, such as Figure 5 E;

[0132] Functionally, HIF1A knockdown significantly inhibits cell invasion, such as Figure 5 F. Migration, such as Figure 5 G and clone-forming ability, such as Figure 5 H;

[0133] In vivo experiments showed that SLC16A3 overexpression could partially reverse the tumor-suppressive effect of HIF1A knockdown, such as Figure 5 I;

[0134] Mechanistically, HIF1A knockdown leads to downregulation of SLC7A11 and GPX4, while SLC16A3 salvage expression can reverse this effect, such as... Figure 5 J–K, in addition, SLC16A3 overexpression can enhance cellular resistance to erastin-induced ferroptosis, such as Figure 5 L.

[0135] Example 6:

[0136] like Figure 6 As shown, targeting the HIF1A–SLC16A3 axis can enhance the ferroptosis sensitivity of gefitinib-resistant LUAD cells.

[0137] A. Western blot and qRT-PCR showed that, compared with parental PC9 cells, the expression of HIF1A and SLC16A3 was significantly upregulated in gefitinib-resistant PC9GR cells;

[0138] B. In PC9 cells, gefitinib treatment further induced the expression of HIF1A and SLC16A3;

[0139] C, Knockdown of SLC16A3 in PC9GR cells led to decreased expression of GPX4 and SLC7A11;

[0140] D. Knocking down HIF1A also downregulated the expression of SLC16A3 and ferroptosis-related proteins.

[0141] E. Restoring SLC16A3 expression in the shHIF1A background can restore GPX4 and SLC7A11 expression, confirming the regulatory role of the HIF1A–SLC16A3 axis.

[0142] F, colony formation, scratch and Transwell invasion assays showed that the SLC16A3 inhibitor MSC-4381 could enhance the sensitivity of PC9GR cells to gefitinib and inhibit cell proliferation and migration.

[0143] G, MSC-4381 treatment reduced the expression of SLC7A11 and GPX4 in PC9GR cells;

[0144] H, BODIPY-C11 staining showed that lipid peroxidation was enhanced after treatment with gefitinib combined with MSC-4381.

[0145] I,JC-1 staining showed that combined treatment with MSC-4381 and gefitinib induced mitochondrial depolarization, suggesting ferroptosis-related mitochondrial damage.

[0146] In gefitinib-resistant PC9GR cells, the expression levels of HIF1A and SLC16A3 were significantly higher than those in parental PC9 cells. Figure 6 A, and further upregulated after treatment with gefitinib, such as Figure 6 B;

[0147] Knock down SLC16A3 in PC9GR cells (e.g.) Figure 6 C) or HIF1A (such as Figure 6 D) Both can downregulate GPX4 and SLC7A11 expression, while salvage expression of SLC16A3 can restore the above effects, such as Figure 6 E, further verifying that HIF1A regulates ferroptosis-related pathways through SLC16A3;

[0148] Functional experiments showed that treatment of PC9GR cells with the SLC16A3 inhibitor MSC-4381 in combination with gefitinib significantly reduced colony formation, migration, and invasion, demonstrating advantages over single-agent therapy. Figure 6 F;

[0149] Western blot showed that MSC-4381 reduced GPX4 and SLC7A11 expression, such as Figure 6 G, BODIPY staining confirmed enhanced lipid peroxidation, such as Figure 6 H,JC-1 staining showed mitochondrial damage, suggesting that ferroptosis was activated, such as Figure 7 I.

[0150] Example 7:

[0151] like Figure 7As shown, pharmacological inhibition of SLC16A3 enhances the in vivo efficacy of gefitinib by inducing ferroptosis.

[0152] A. A subcutaneous xenograft tumor model of BALB / c nude mice was constructed using PC9 cells. Mice were treated with DMSO, gefitinib, MSC-4381 (SLC16A3 inhibitor) or combination therapy (G+M). Representative tumor images, final tumor weight and tumor volume change curves are shown (n=4 per group). Combination therapy significantly inhibited tumor growth.

[0153] B. The ferroptosis inhibitor ferrostatin-1 can reverse the MSC-4381-mediated antitumor effect, confirming that it depends on the ferroptosis mechanism.

[0154] C, Ki-67 immunohistochemical staining of tumor tissue from group (A) showed that combined treatment could reduce the proliferation index;

[0155] D, Ki-67 staining of tumor tissue from (B) ferroptosis relief experiment showed that ferroptosis inhibition could restore the proliferation capacity inhibited by MSC-4381;

[0156] To verify the in vivo therapeutic potential, mice in the PC9 subcutaneous xenograft model were treated with MSC-4381, gefitinib, or a combination of these drugs.

[0157] The combined treatment group showed significant inhibition of tumor growth, such as Figure 7 A;

[0158] Ferrostatin-1 can reverse the antitumor effect of MSC-4381, suggesting that ferroptosis is the main mechanism. Figure 7 B;

[0159] IHC staining showed decreased Ki-67 expression in the combined treatment group, suggesting that proliferation was inhibited. Figure 7 C, while ferroptosis inhibition can restore Ki-67 expression, such as ​ D.

[0160] Although specific embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these specific embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An SLC16A3 inhibitor based on regulation of ferroptosis characterized in that, The SLC16A3 inhibitor is drug MSC-4381, and the SLC16A3 knockdown is achieved by lentiviral transfection using three shRNAs with the nucleotide sequences of: shSLC16A3#1: 5'-GCGACTTGTTTATCCACTTTA-3'; shSLC16A3#2: 5'-GCACCATCTTTGTATTCATTA-3'; shSLC16A3#3: 5'-GCAGTGGATTTCTTGTTCTTT-3'; The above three shRNAs are cloned into the pLKO.1 vector, and virus packaging is performed using Lipofectamine 3000 in HEK293T cells.

2. The SLC16A3 inhibitor according to claim 1, characterized in that, The SLC16A3 overexpression vector is a full-length human SLC16A3 cDNA cloned into the pCDH vector, and stable cells are obtained after 7 days of screening in 2 μg / mL puromycin.

3. An overexpression PC9 cell line vector comprising the SLC16A3 inhibitor of claim 1.

4. The carrier of claim 3, wherein, The vector is a human lung adenocarcinoma cell line PC9 or PC9GR, and a normal lung epithelial cell line BEAS-2B, and the cells are cultured in DMEM or RPMI-1640 medium containing 10% fetal bovine serum and maintained in a 37 ℃, 5% CO2 humidified environment for growth.

5. The SLC16A3 inhibitor based on ferroptosis regulation according to claim 1 in lung adenocarcinoma.

6. The SLC16A3 inhibitor based on ferroptosis regulation according to claim 1 in lung adenocarcinoma for significantly inhibiting PC9 cell proliferation and invasion.

7. The SLC16A3 inhibitor based on ferroptosis regulation according to claim 1 in lung adenocarcinoma for enhancing the expression of ferroptosis markers and significantly enhancing the efficacy of gefitinib.

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