Use of trop2 inhibitors in the manufacture of ferroptosis sensitizing and / or inducing agents, and pharmaceutical compositions for enhancing ferroptosis

Abstract

The application provides an application of a TROP2 inhibitor in preparation of a ferroptosis sensitizer and / or inducer, and a pharmaceutical composition for enhancing ferroptosis. The pharmaceutical composition comprises a ferroptosis inducer and a TROP2 inhibitor. The application first discovers the role of TROP2 in regulating ferroptosis of lung cancer cells through experiments, and based on the discovery, the TROP2 inhibitor and the ferroptosis inducer (such as RSL3) are combined, and it is found that inhibiting TROP2 can enhance the sensitivity of lung cancer cells to ferroptosis induced by RSL3, so as to achieve the effect of jointly promoting cell ferroptosis and then inhibiting tumor development, and the application is expected to provide a new effective scheme for tumor treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to the use of a TROP2 inhibitor in the preparation of ferroptosis sensitizers and / or inducers, and pharmaceutical compositions that enhance ferroptosis. Background Technology

[0002] Lung cancer is one of the most common and deadliest malignant tumors worldwide, with a complex pathogenesis involving multiple genetic and environmental factors. Because early symptoms of lung cancer are often subtle, most patients are diagnosed at an advanced stage. Treatment options primarily include surgery, chemotherapy, radiotherapy, targeted therapy, and immunotherapy. Although molecularly targeted drugs and immunotherapy have significantly improved the prognosis of some patients in recent years, the overall five-year survival rate remains low, necessitating further research into new diagnostic technologies and treatment strategies to improve the early detection rate and treatment outcomes of lung cancer.

[0003] Ferroprelation, a form of programmed cell death mediated by iron ions and characterized by lipid peroxidation accumulation, was first proposed by Dixon et al. in 2012. This concept broadened our understanding of cell death mechanisms, differing significantly from traditional forms such as apoptosis, necrosis, and autophagy. Its occurrence is closely related to the inactivation of glutathione peroxidase 4 (GPX4) and the excessive accumulation of intracellular reactive oxygen species (ROS). However, studies have shown that insufficient cellular ROS levels and unsatisfactory drug accumulation in lung cancer lesions hinder the efficacy of ferroptosis therapy. Therefore, although the potential of ferroptosis in the treatment of some malignant tumors is gradually emerging, its specific molecular mechanisms are not yet fully elucidated, especially its role in the tumor microenvironment and heterogeneous tumors, which requires further investigation.

[0004] TROP2 (Trophoblast cell-surface antigen 2) is a transmembrane glycoprotein belonging to the EpCAM family. It was initially discovered in placental trophoblast cells and is widely expressed in various normal epithelial tissues and tumor tissues. A review of domestic and international literature revealed no reports on the application of TROP2 in ferroptosis. Summary of the Invention

[0005] The purpose of this invention is to provide the use of TROP2 inhibitors in the preparation of ferroptosis sensitizers and / or inducers, and pharmaceutical compositions that enhance ferroptosis, so as to improve the sensitivity of tumors (e.g., lung cancer) to ferroptosis therapy and improve antitumor effects.

[0006] Based on the above, the first objective of this invention is to provide the use of TROP2 inhibitors in the preparation of ferroptosis sensitizers and / or inducers.

[0007] Preferably, the TROP2 inhibitor comprises: siRNA or gene knockout vector of the TROP2 encoding gene, or a protein antibody of TROP2, or any one or more combinations of other inhibitors capable of inhibiting TROP2 expression.

[0008] Preferably, the TROP2 inhibitor is siRNA, and the nucleotide sequence of the siRNA is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0009] Preferably, when the TROP2 inhibitor is prepared as a ferroptosis sensitizer, it is used in combination with a ferroptosis inducer to enhance the ferroptosis effect, wherein the ferroptosis inducer includes at least one of RSL3, Erastin, or ML162.

[0010] A second object of the present invention is to provide a pharmaceutical composition for enhancing ferroptosis, the pharmaceutical composition comprising: a ferroptosis inducer and a TROP2 inhibitor.

[0011] Preferably, the TROP2 inhibitor is siRNA, and the nucleotide sequence of the siRNA is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0012] Preferably, the ferroptosis inducer includes at least one of RSL3, Erastin, or ML162.

[0013] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0014] A third object of the present invention is to provide the use of the aforementioned pharmaceutical composition in the preparation of antitumor drugs.

[0015] Preferably, the tumor includes lung cancer.

[0016] Compared with the prior art, the beneficial effects of the present invention include at least the following:

[0017] This invention is the first to experimentally discover the role of TROP2 in regulating ferroptosis in lung cancer cells. Based on this discovery, a TROP2 inhibitor and a ferroptosis inducer (such as RSL3) were combined. The results showed that inhibiting TROP2 could enhance the sensitivity of lung cancer cells to RSL3-induced ferroptosis, thereby achieving the combined effect of promoting cell ferroptosis and inhibiting tumor development, which is expected to provide a new and effective solution for tumor treatment. Attached Figure Description

[0018] Figure 1 This indicates that interfering with TROP2 inhibits the growth of lung cancer cells; among which:

[0019] A represents the results of TROP2 expression level detection in lung cancer cells after transfection with TROP2-si#1 and TROP2-si#2.

[0020] B represents the results of cell viability detection for lung cancer cells after transfection with TROP2-si#1 and TROP2-si#2.

[0021] C represents the clonal proliferation of lung cancer cells after transfection with TROP2-si#1 and TROP2-si#2.

[0022] Figure 2 This indicates the results of an investigation into the mechanism by which TROP2 inhibits lung cancer cell growth; among which:

[0023] A represents the transcriptome sequencing results of lung cancer cells with / without TROP2 interference;

[0024] B represents the MDA level detection results of lung cancer cells after transfection with TROP2-si#1 and TROP2-si#2.

[0025] C represents the GSH level detection result of lung cancer cells after transfection with TROP2-si#1 and TROP2-si#2;

[0026] D shows the results of cell morphology observation in each group using transmission electron microscopy.

[0027] Figure 3 This indicates that inhibiting TROP2 expression enhances RSL3-induced ferroptosis in lung cancer cells; where:

[0028] A represents the cell viability test results for each group of cells;

[0029] B represents the MDA level detection results for each group of cells;

[0030] C represents the GSH level detection results for each group of cells.

[0031] Figure 4 The results indicate that RSL3 and TROP2 interference significantly inhibited the growth of PC9 tumors in nude mice; among which:

[0032] A shows photographs of the final tumor tissue of mice in each group;

[0033] B represents the final tumor tissue weight of each group of mice;

[0034] C represents the growth curve of tumor volume in each group of mice. Detailed Implementation

[0035] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0036] Terminology Explanation

[0037] Malondialdehyde (MDA) is a product of lipid peroxidation, and elevated levels typically indicate intracellular lipid peroxidation, a key marker of ferroptosis. During ferroptosis, intracellular iron ions react with hydrogen peroxide to generate hydroxyl radicals (·OH), which in turn trigger lipid peroxidation, leading to the accumulation of oxidation products such as MDA. Therefore, detecting MDA levels can indirectly assess the extent of ferroptosis.

[0038] Glutathione (GSH) is an important antioxidant that neutralizes free radicals in cells, protecting them from oxidative stress damage. During ferroptosis, GSH levels change, typically manifesting as GSH depletion or impaired synthesis. Detecting GSH levels allows us to understand changes in intracellular antioxidant capacity, thereby assessing the extent of ferroptosis. Furthermore, changes in GSH synthesis are also a key marker of ferroptosis.

[0039] As described in the background section, although the potential of ferroptosis in the treatment of malignant tumors is gradually emerging, the efficacy of ferroptosis varies greatly among different types of tumors due to the significant differences in tumor microenvironment and tumor heterogeneity. The efficacy of ferroptosis in some malignant tumors (such as lung cancer) is not satisfactory.

[0040] To address the aforementioned technical problems, this invention conducted extensive experiments and research, unexpectedly discovering that when siRNA is used to interfere with TROP2 expression in lung cancer cells, it leads to a slowdown in lung cancer cell growth and an increase in ROS levels. Sequencing revealed a high correlation between TROP2 and ferroptosis. Further in vivo and in vitro experiments confirmed that TROP2 interference can increase the sensitivity of lung cancer cells to ferroptosis therapy, promote ferroptosis in lung cancer cells, and ultimately significantly improve the anti-tumor effect.

[0041] Based on the above, this invention first provides the application of TROP2 inhibitors in the preparation of ferroptosis sensitizers and / or ferroptosis inducers. When TROP2 inhibitors are prepared as ferroptosis sensitizers, they are used in combination with ferroptosis inducers to enhance the ferroptosis effect.

[0042] In some embodiments, the TROP2 inhibitor comprises: siRNA or gene knockout vector of the gene encoding TROP2, a protein antibody of TROP2, or any combination of one or more other inhibitors capable of inhibiting TROP2 expression. As an example, the TROP2 inhibitor is siRNA, and the nucleotide sequence of the siRNA is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0043] In some embodiments, the ferroptosis inducer includes at least one of RSL3, Erastin, or ML162.

[0044] In another aspect, the present invention provides a pharmaceutical composition for enhancing ferroptosis, the pharmaceutical composition comprising: a ferroptosis inducer and a TROP2 inhibitor.

[0045] In some embodiments, the TROP2 inhibitor is siRNA, and the nucleotide sequence of the siRNA is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0046] In some embodiments, the ferroptosis inducer includes at least one of RSL3, Erastin, or ML162. RSL3 (RAS Selective Lethal 3) is a commonly used ferroptosis inducer that triggers lipid peroxidation and ultimately leads to ferroptosis by directly inhibiting GPX4 activity. When RSL3 inhibits GPX4, intracellular antioxidant defenses are weakened, lipid peroxide levels increase, leading to impaired cell membrane integrity and inducing ferroptosis.

[0047] In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" refers to a carrier used for the administration of a therapeutic agent, including various excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity after administration. Suitable pharmaceutically acceptable carriers are well known to those skilled in the art. A thorough description of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences. Pharmaceutically acceptable carriers in a composition may contain liquids such as water, phosphate buffer, Ringer's solution, physiological saline, balanced salt solution, glycerol, or sorbitol. Additionally, these carriers may contain auxiliary substances such as lubricants, flow aids, wetting agents or emulsifiers, pH buffers, and stabilizers such as albumin.

[0048] In another aspect, the present invention provides the use of the aforementioned pharmaceutical composition in the preparation of an antitumor drug. In some embodiments, the tumor includes lung cancer.

[0049] The following will be combined with the appendix Figures 1 to 4 The experimental investigation process of the present invention is described in detail, along with specific embodiments.

[0050] (I) Investigation into the mechanism by which TROP2 interferes with the growth of lung cancer cells

[0051] 1. Interfering with TROP2 expression can inhibit the growth of lung cancer cells.

[0052] In this invention, lung cancer cells PC9 and H292 purchased from ATCC were co-cultured with siRNA that interferes with TROP2 to observe the effect of TROP2 interference on the growth of lung cancer cells. si-CON was also included as an experimental control.

[0053] Two siRNAs were designed to interfere with TROP2, denoted as TROP2-si#1 and TROP2-si#2, with the following sequences:

[0054] The nucleotide sequence of TROP2-si#1 is: 5'-CGCUCAUCUAUUACCUGGA-3' (SEQ ID NO: 1);

[0055] The nucleotide sequence of TROP2-si#2 is: 5'-CGUGGACAACGAUGGCCUCUA-3' (SEQ ID NO: 2).

[0056] The experimental procedure is as follows:

[0057] (1) 18-24 hours before lentivirus transfection, lung cancer cells are seeded into 96-well plates to achieve a cell confluence of 30%-50% at the time of transfection.

[0058] (2) Dilute siRNA (i.e. TROP2-si#1 or TROP2-si#2) in an EP tube containing 250 μL of Opti-MEM medium and mix gently. In another EP tube, dilute Lipofectamine 2000 in 250 μL of Opti-MEM medium. The concentration ratio of Lipofectamine 2000 to siRNA (pmol) after dilution is 1:1. Incubate at room temperature for 5 min after dilution. After incubation, gently mix the diluted siRNA with the diluted Lipofectamine 2000 and incubate the mixture at room temperature for 20 min.

[0059] (3) Add 500 μL of the incubated mixture to the wells containing cells and culture medium, gently mix by cross-shading, and then incubate at 37°C in a CO2 incubator for 24 h. The medium can be replaced with complete culture medium 4-6 h after the start of transfection. During replacement, some cells can be collected, lysed using RIPA lysis buffer, and after BCA quantification, the effect of siRNA interfering with TROP2 can be detected by Western blotting. Results are as follows: Figure 1 As shown in Figure A, both TROP2-si#1 and TROP2-si#2 can significantly reduce the expression level of TROP2 in both types of lung cancer cells, with a particularly significant interference effect on H292 cells.

[0060] The remaining cells were seeded in 96-well plates at a density of 3000 cells per well. Six replicates were set up for the control group and the two experimental groups. CCK8 solution was added on days 1, 2, and 3, and absorbance at 450 nm was measured to evaluate the effect of siRNA interference with TROP2 on cell growth. Results are as follows: Figure 1 As shown in Figure B, compared with the control group (si-CON), the growth of cells in the experimental group was significantly inhibited.

[0061] In addition, transfected cells were seeded into six-well plates at a density of 1000 cells per well, with the culture medium changed regularly. After 10-14 days of culture, clones formed. The plates were gently washed with 1×PBS, fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and photographed. The results are as follows: Figure 1 As shown in Figure C, compared with the control group, inhibiting TROP2 expression significantly suppressed cell proliferation.

[0062] 2. Interference with TROP2 expression can promote ferroptosis in lung cancer cells.

[0063] Based on the above research, further investigation through cell collection and RNA extraction for transcriptome sequencing revealed a high correlation between slowed cell growth and ferroptosis after TROP2 reduction. This led to the hypothesis that TROP2 interference is associated with ferroptosis, and this hypothesis was validated through experiments. Changes in GSH and MDA levels in PC9 and H292 cells after TROP2 interference were detected using a kit, and cell structural changes were observed using electron microscopy to confirm this hypothesis. In this example, the TROP2-si#1 sequence was used to package a lentivirus (shTROP2) to achieve long-term interference with TROP2 in PC9 and H292 cells. The experimental procedure and results are as follows:

[0064] (1) Cells from the control group (shCON treatment) and the TROP2 interference treatment group (shTROP2 treatment) were collected. Each 10 million cells were resuspended in 100 μL of PBS, sonicated, and centrifuged at 8000g for 15 minutes. The supernatant was used for subsequent MDA detection. Samples were processed according to the kit instructions. The supernatant was incubated in a 95℃ metal bath for 1 hour, then cooled on ice, and centrifuged at 8000g for 10 minutes. 200 μL of the supernatant was placed in a clear 96-well plate, and the absorbance at 532 nm was measured using a microplate reader. For data analysis, the protein concentration of the samples was measured, and the MDA concentration was calculated using the standard curve method.

[0065] (2) Collect cells from the control and TROP2 interference treatments. Resuspend 1 million cells in 300 μL PBS and sonicate at 4°C for 30 seconds at a frequency of 25 kHz, with a 20-second interval, for a total duration of 30 minutes. Then, centrifuge the cells at 8000g for 10 minutes. Collect the supernatant, mix it with protein removal reagent, and centrifuge again. Obtain the supernatant for subsequent reduced glutathione detection. Mix the sample with the working solution of the detection probe and add it to a 96-well plate. After incubating at room temperature for 5 minutes, measure the absorbance at 412 nm using a microplate reader. Calculate the GSH concentration using a standard curve.

[0066] (3) Collect cells from the control group and those treated with TROP2 interference. Fix them with electron microscopy fixative for at least 24 hours, then fix them with 1% osmium tetroxide prepared in 0.1M phosphate buffer PB (pH 7.4) at room temperature in the dark for 2 hours. After graded dehydration with ethanol, embed them in resin blocks. Cut the resin blocks into 1.5μm semi-thin sections using a microtome, stain with toluidine blue, and locate them under an optical microscope. Cut the resin blocks into 60 80nm ultrathin sections using an ultramicrotome, stain them, and observe and photograph them under a transmission electron microscope.

[0067] The results are as follows Figure 2 As shown, transcriptome sequencing analysis revealed that reduced cell growth after TROP2 reduction was significantly associated with the ferroptosis pathway. Figure 2 (A). Compared to the control group, the MDA concentration in the experimental group cells was significantly increased ( Figure 2 B), GSH concentration decreased significantly ( Figure 2 (C), and TEM showed that after TROP2 inhibition, mitochondria in cells became smaller, membrane density increased, and cristae decreased or even disappeared. Figure 2 These results indicate that inhibiting TROP2 expression can promote ferroptosis in lung cancer cells and exacerbate the degree of ferroptosis.

[0068] 3. Inhibition of TROP2 expression can enhance RSL3-induced ferroptosis in lung cancer cells.

[0069] Based on the above research, this invention designs an experiment to combine a TROP2 inhibitor (in this embodiment, a lentivirus packaged with the TROP2-si#1 sequence, i.e., shTROP2) with a ferroptosis inducer (in this embodiment, RSL3) to achieve a synergistic effect, enhance ferroptosis in tumor cells, and thus improve the anti-tumor effect. The specific experimental procedure includes:

[0070] Lung cancer cells (PC9 or H292) were subjected to TROP2 interference (MOI = 10) using lentiviruses and divided into three groups, each receiving the following treatments: RSL3 group (5 μM RSL3), RSL3+Fer group (5 μM RSL3 and ferroptosis inhibitor Fer1), and DMSO group (1 volume of DMSO). Notably, untreated lung cancer cells were also included in these three treatments. After 24 hours, cells from each group were seeded into 96-well plates with 8000 cells per well and 6 replicates.

[0071] 10 μL of CCK8 reagent was added 24 hours after plating, and the cells were incubated in an incubator for two hours. The absorbance at 450 μm was then measured to evaluate cell viability in each group. Results are as follows: Figure 3 As shown in A, TROP2 interference increases the sensitivity of tumor cells to RSL3, and the use of Fer1 can partially restore cell viability.

[0072] In addition, the levels of GSH and MDA in each group of cells were measured, using methods described in the relevant sections above. The results are as follows: Figure 3 As shown in Figures B and C, compared to the control group (shCON group), treatment with RSL3 alone (shCON+RSL3 group) or treatment with only TROP2 interference (shTROP2 group) resulted in increased MDA levels and decreased GSH levels in cells. This indicates that treatment with either ingredient alone can induce ferroptosis, but the combined treatment (shTROP2+RSL3 group) is more effective, exhibiting a significant synergistic effect and significantly enhancing ferroptosis in lung cancer cells. These results demonstrate that inhibiting TROP2 expression enhances RSL3-induced ferroptosis.

[0073] 4. Interference with RSL3 and TROP2 significantly inhibited the growth of PC9 tumors in nude mice.

[0074] To further demonstrate the regulatory effects of RSL3 and TROP2 on tumors, relevant evaluations were conducted through animal experiments. Specifically, these included:

[0075] The experiment used PC9 cells at 60% density, infected with either a control lentivirus (shCON) with an MOI (Multiplicity of Infection) of 10 or an interfering lentivirus (shTROP2), with 5 μg / mL polybrene added to enhance infection efficiency. After infection, the cells were expanded to a density of 80-90%, digested with trypsin, washed with pre-cooled PBS, and resuspended in PBS to a suspension of 6 × 10⁶ cells / 100 μL. Twenty-four 6-8 week old nude mice were randomly divided into two groups (n = 12 / group), and either shCON or shTROP2-infected cells were injected subcutaneously into the back (100 μL / mouse). When tumors became visible to the naked eye, the nude mice treated with shCON were further divided into a control group (shCON) and an RSL3 treatment group (shCON + RSL3), and the nude mice treated with shTROP2 were divided into a TROP2 interference group (shTROP2) and a TROP2 interference + RSL3 group (n = 6 / group) for subsequent intervention. RSL3 was dissolved in DMSO. The RSL3-treated group and the TROP2 interference + RSL3-treated group were intraperitoneally injected with 5 mg / kg RSL3 solution every other day for 13 days. The body weight and tumor volume of the mice were measured regularly. After 13 days, the mice were sacrificed, and the tumors were dissected and photographed.

[0076] The results are as follows Figure 4 As shown in the AC, compared with RSL3 treatment alone or TROP2 interference mice, the final tumor volume and weight of the TROP2 interference + RSL3 treatment group were significantly smaller than the other three groups, indicating that inhibiting TROP2 expression has an enhancing effect on RSL3-induced ferroptosis.

[0077] In summary, this invention has for the first time discovered the role of TROP2 in regulating ferroptosis in lung cancer cells through experiments. Based on this discovery, a TROP2 inhibitor and a ferroptosis inducer (such as RSL3) were combined. The results showed that inhibiting TROP2 can enhance the sensitivity of lung cancer cells to RSL3-induced ferroptosis, thereby achieving the combined effect of promoting ferroptosis and inhibiting tumor development, which is expected to provide a new and effective solution for tumor treatment.

[0078] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. The application of TROP2 inhibitors in the preparation of lung cancer ferroptosis sensitizers and / or inducers, characterized in that, The TROP2 inhibitor is siRNA, and the nucleotide sequence of the siRNA is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. The application as described in claim 1, characterized in that, When the TROP2 inhibitor is prepared as a ferroptosis sensitizer, it is used in combination with a ferroptosis inducer to enhance the ferroptosis effect. The ferroptosis inducer includes at least one of RSL3, Erastin, or ML162.

3. A pharmaceutical composition for enhancing ferroptosis, characterized in that, The pharmaceutical composition comprises: a ferroptosis inducer and a TROP2 inhibitor, wherein the ferroptosis inducer is RSL3 and the TROP2 inhibitor is siRNA, and the nucleotide sequence of the siRNA is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

4. The pharmaceutical composition according to claim 3, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

5. The use of the pharmaceutical composition according to claim 3 or 4 in the preparation of an antitumor drug, characterized in that, The tumor is lung cancer.

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