Application of thioredoxin TXN1 and TRP14 as bidirectional switch for regulating and controlling cell death mode and application of anticancer drug
By regulating the activity or expression of TXN1 and TRP14, a novel anticancer drug composition was developed to modulate the sensitivity of cells to apoptosis, ferroptosis, and disulfide death. This solved the problem of the unclear role of thioredoxin in the disulfide death pathway, enhanced the cells' resistance to disulfide death, and provided a new drug development strategy.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-07
- Publication Date
- 2026-04-03
AI Technical Summary
In the current technology, the role of thioredoxin TXN1 and TRP14 in the disulfide death pathway is unclear, and there is a lack of effective drug development strategies for disulfide death-related diseases such as malignant tumors.
By regulating the activity or expression of thioredoxins TXN1 and TRP14 in cells, and using TXN1 and TRP14 as drug targets, the sensitivity of cells to apoptosis, ferroptosis, and disulfide death can be regulated. Drug compositions containing TXN1 and/or TRP14 inhibitors can be developed, combined with apoptosis and ferroptosis inducers, for the treatment of diseases related to excessive activation of disulfide death.
It significantly enhances cells' resistance to disulfide death, provides new anticancer drug targets and diagnostic biomarkers, helps overcome tumor resistance to existing therapies, and discovers effective drugs through high-throughput screening.
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Figure CN121784296A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the fields of molecular biology, pharmaceutical technology and drug development, and relates to the drug application of thioredoxin TXN1 and TRP14 in regulating disulfide death in cells. It relates to the use of thioredoxin TXN1 and TRP14 as bidirectional switches for regulating cell death and their application as anticancer drugs. Specifically, it relates to the functions of thioredoxin family members TXN1 and TRP14 in regulating disulfide death, and their novel uses in the diagnosis, development and screening of drugs for related diseases. Background Technology
[0002] Programmed cell death (PCD) is a core process for maintaining homeostasis and eliminating abnormal cells. In addition to classic apoptosis, the discovery of novel PCD mechanisms such as ferroptosis and disulfidptosis in recent years has greatly expanded our understanding of cell fate regulation. Disulfidptosis, in particular, is a programmed cell death mechanism triggered by intracellular disulfide stress and dependent on the abnormal accumulation of cytoskeletal proteins. Studies have shown that under glucose deprivation conditions, cells highly expressing solute carrier family 7 member 11 (SLC7A11) experience excessive cystine uptake and insufficient NADPH, leading to the accumulation of abnormal disulfides among actin cytoskeletal proteins, ultimately resulting in cell death (see reference 1: Gan B, et al. Disulfidptosis: a novel form of regulated cell death. Nat Cell Biol. 2023). This discovery provides a novel approach for targeting tumors with specific metabolic characteristics.
[0003] The thioredoxin system (including TXN, TXNR, and NADPH) is central to maintaining intracellular redox homeostasis. TXN1 is the major cytoplasmic / nuclear isoform, and TRP14 is a member of the TXN superfamily. Existing research has touched upon their roles in apoptosis or ferroptosis, but their mechanism of action in the novel disulfide death pathway remains entirely unknown. Specifically, the roles of TXN1 and TRP14 in disulfide death, and the molecular mechanisms by which they influence cytoskeletal protein function and cell fate, are crucial scientific questions that urgently need to be elucidated. Clarifying this issue is of great value for developing new strategies targeting disulfide death-related diseases, such as certain malignancies. Summary of the Invention
[0004] To address the aforementioned technological gaps, this invention aims to solve the following problems: elucidate the specific roles and molecular mechanisms of thioredoxin TXN1 and TRP14 in the disulfide death pathway, and based on this mechanism, provide new solutions for drug development, disease treatment, and diagnosis, and provide a drug application of thioredoxin TXN1 and TRP14 in regulating cellular disulfide death.
[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0006] In a first aspect, the present invention provides the use of thioredoxin TXN1 and TRP14 as a bidirectional switch for regulating cell death, specifically the use of thioredoxin TXN1 and TRP14 as drug targets in regulating cellular disulfide death sensitivity, which can regulate cellular sensitivity to programmed cell death, specifically by regulating the activity or expression level of thioredoxin-1 (TXN1) and / or thioredoxin-associated protein 14 (TRP14) in target cells; wherein, reducing the activity or expression of TXN1 and / or TRP14 can increase the cellular sensitivity to apoptosis and / or ferroptosis, while reducing the cellular sensitivity to disulfide death.
[0007] Furthermore, the apoptosis was induced by astrosporin (STS), the ferroptosis by RSL3, Erastin, ML210, or FIN56, and the disulfide death by glucose deprivation. Increased cellular sensitivity to apoptosis was achieved by activating the ASK1-JNK signaling pathway and upregulating the Bax / Bcl-2 ratio. Increased cellular sensitivity to ferroptosis was associated with decreased glutathione (GSH) and / or reduced thiol levels and downregulation of GPX4 protein. Decreased sensitivity to disulfide death was associated with the inhibition of abnormal aggregation of cytoskeletal proteins FLNA and / or Talin-1.
[0008] Experiments show that reducing the protein expression or activity of TXN1 or TRP14 in cells can significantly reduce cellular sensitivity to disulfide death and enhance cellular survival under disulfide death stress. This suggests that inhibiting the function of TXN1 or TRP14 could be a drug action strategy for treating diseases related to excessive activation of disulfide death (such as certain types of tumors). Disulfide death can be triggered by conditions such as glucose deprivation that induce intracellular disulfide bond stress. Its resistance mechanism is associated with the significant inhibition of abnormal aggregation of cytoskeletal proteins Filamin A (FLNA) and / or Talin-1. Furthermore, TXN1 and TRP14 have a functional compensatory relationship in this pathway; knocking down one leads to upregulation of the other, thereby jointly maintaining a resistant phenotype to disulfide death. This suggests that drug design should consider simultaneously or selectively inhibiting both to achieve optimal therapeutic efficacy.
[0009] Secondly, based on the anti-disulfide death effect discovered in the first aspect, this invention provides the application of thioredoxin TXN1 and TRP14 in anticancer drugs for the preparation of pharmaceutical compositions for treating cancer, specifically a pharmaceutical composition or formulation for protecting cells from disulfide death damage or treating diseases related to excessive activation of disulfide death. The composition comprises a first active ingredient and a second active ingredient; the first active ingredient is an inhibitor of TXN1 and / or TRP14; the second active ingredient is an apoptosis inducer and / or ferroptosis inducer; that is, the pharmaceutical composition is an active inhibitor containing an effective dose of TXN1 and / or TRP14.
[0010] Furthermore, the ferroptosis inducer is RSL3, Erastin, ML210, or FIN56. The apoptosis inducer is STS.
[0011] Furthermore, the inhibitors of TXN1 and / or TRP14 are selected from small molecule inhibitors, antisense oligonucleotides, siRNA, shRNA, or the CRISPR-Cas9 gene editing system. Such formulations can be used to prepare drugs for the prevention or treatment of diseases caused by disulfide metabolism disorders, such as cancer, neurodegenerative diseases, etc.
[0012] Thirdly, the present invention provides the use of thioredoxin TXN1 and TRP14 or their encoding genes as biomarkers to assess the susceptibility or resistance of cells, tissues or individuals to disulfide death, thereby guiding clinical drug selection, predicting patient response to disulfide death induction therapies (such as certain chemotherapy or targeted therapies) and prognosis.
[0013] Fourthly, this invention provides a method for screening anti-disulfide death candidate drugs, comprising the following steps: culturing cells in the presence of a test substance and applying disulfide death-inducing conditions (such as glucose deprivation); detecting cell viability or the aggregation status of FLNA / Talin-1; if the test substance can improve cell viability and inhibit abnormal aggregation of cytoskeletal proteins, and this effect depends on or is accompanied by a decrease in TXN1 / TRP14 activity, then the substance is a potential anti-disulfide death candidate drug. This method can be used in high-throughput drug screening platforms to rapidly discover disulfide death regulators targeting TXN1 / TRP14.
[0014] The principle of this invention is as follows:
[0015] This invention systematically investigated the effects of stable knockdown of TXN1 (shTXN1) or TRP14 (shTRP14) on disulfide death in human non-small cell lung cancer A549 cells. The results unexpectedly revealed that knockdown of TXN1 or TRP14, especially TXN1, significantly enhanced the resistance of cancer cells to disulfide death (glucose deprivation induced) through mechanisms such as mutually compensatory upregulation (TXN1 knockdown leads to TRP14 upregulation, and vice versa). This was manifested in increased cell survival and inhibition of abnormal aggregation of the cytoskeletal protein FLNA / Talin-1.
[0016] Based on the above findings, the present invention proposes the following drug-related applications:
[0017] Diagnostic / Prognostic Applications: Detecting the expression levels of TXN1 and / or TRP14 in tumor tissue can serve as biomarkers for predicting patient sensitivity to disulfide death-inducing therapy. Low expression may predict a better treatment response, thereby guiding the development of personalized treatment plans.
[0018] Drug Development Applications: TXN1 and TRP14 have become new targets for developing novel anti-tumor drugs. Developing inhibitors targeting these two targets could be used to treat cancers with disulfide death sensitivity or to overcome tumor resistance to existing therapies, such as certain drugs that induce disulfide death.
[0019] Drug screening applications: Establish cell or molecular models with TXN1 / TRP14 activity or expression as detection endpoints for high-throughput screening of candidate anticancer drugs that can inhibit TXN1 / TRP14 function, accelerating the discovery and optimization of lead compounds.
[0020] Compared with the prior art, the present invention has the following significant advantages:
[0021] First discovery and mechanism elucidation: For the first time internationally, the key negative regulatory roles of TXN1 and TRP14 in the disulfide death pathway have been systematically elucidated, filling the gap in the study of the function of downstream executive molecules of the thioredoxin system in disulfide death, and revealing a new mechanism by which they regulate by affecting the aggregation of cytoskeletal proteins, providing clear targets and mechanistic basis for drug design targeting this pathway.
[0022] Revealing complex interactions: The discovery of a compensatory relationship between TXN1 and TRP14 in regulating disulfide death deepens our understanding of the functional network within the thioredoxin family. This suggests that when developing targeted drugs, dual-target inhibition or the design of compounds that can disrupt their compensatory balance should be considered to improve efficacy and reduce drug resistance.
[0023] This research provides novel drug development strategies: Based on the discovery of "negative regulation," a series of translational directions with clear drug development prospects have emerged: a) It provides a new therapeutic strategy and drug target for treating disulfide death-resistant cancers by inhibiting TXN1 / TRP14; b) It establishes TXN1 / TRP14 as a novel companion diagnostic biomarker for predicting the efficacy of disulfide death therapy; c) It constructs a specific drug screening and evaluation model targeting this pathway. These applications provide entirely new ideas, targets, and tools for developing next-generation anti-tumor drugs and overcoming tumor treatment resistance. Attached Figure Description
[0024] Figure 1 The graph shows the changes in cell viability over time in the control group and the TXN1 / TRP14 knockdown group under glucose deprivation induction, demonstrating that knockdown cells acquire disulfide death resistance.
[0025] Figure 2 The images show the cell status of the control group and the TXN1 / TRP14 knockdown group under glucose deprivation induction, demonstrating that knockdown cells have better survival. Figure 2 (a) in the figure is the glucose state diagram of the control group; Figure 2 (b) shows the glycogenic state of TXN1 knockdown cells; Figure 2 (c) in the figure shows the glycogen state of TRP14 knockdown cells; Figure 2 (d) in the figure represents the sugar-free state of the control group; Figure 2 (e) in the figure shows the glucose-free state of TXN1 knockdown cells; Figure 2 (f) in the figure shows the glucose-free state of TRP14 knockdown cells;
[0026] Figure 3 The Western Blot validation plot shows that after glucose deprivation induction, there was significant abnormal aggregation of FLNA protein in the control group, while this aggregation was significantly reduced in the TXN1 knockdown group.
[0027] Figure 4 The Western Blot validation plot shows that TRP14 expression is compensatorily upregulated in TXN1 knockdown cells.
[0028] Figure 5 Conversely, this shows that TXN1 expression is compensatorily upregulated in TRP14 knockdown cells.
[0029] Figure 6 The graphs showing the change in cell viability over time in the control group and the TXN1 / TRP14 knockdown group under the BAY-876 condition demonstrate that TXN1 / TRP14 does indeed affect disulfide death by influencing glucose transport. Detailed Implementation
[0030] The present invention will be further illustrated below with reference to specific implementation examples. The embodiments are intended to illustrate the technical solutions of the present invention and their application in drug development, but the scope of protection of the present invention is not limited thereto.
[0031] Example 1: Construction of gene knockdown cell lines targeting TXN1 and TRP14 and verification of their resistance to disulfide death.
[0032] TXN1 or TRP14 in human non-small cell lung cancer A549 cells were stably knocked down using shRNA lentiviral vectors to mimic the effect of drug inhibition of target gene expression. The knockdown efficiency was verified by Western blotting. Control and knockdown cells were cultured in glucose-free medium for different time periods, and cell viability was assessed using the MTT assay. The results showed that, compared with the control group, the survival rate of shTXN1 and shTRP14 cells was significantly increased under glucose deprivation conditions, demonstrating that inhibiting TXN1 or TRP14 function can enhance cellular resistance to disulfide death, suggesting that these two cells could serve as effective targets for drug intervention.
[0033] Example 2: Morphological observation of TXN1 / TRP14 knockdown cells under glucose deprivation conditions
[0034] A549 cells were cultured in both glucose-exposed and glucose-exposed conditions, and cell morphological changes were observed under a microscope. The results showed that under normal glucose conditions, there was no significant difference in cell morphology among the groups. However, under glucose deprivation conditions, control group cells exhibited significant shrinkage, detachment, and other death morphologies, while shTXN1 and shTRP14 cells maintained good adhesion and morphological integrity, directly demonstrating that knocking down TXN1 or TRP14 can enhance the morphological resistance of cells to disulfide death.
[0035] Example 3: Inhibitory effect of TXN1 knockdown on abnormal aggregation of FLNA protein in disulfide death
[0036] The expression and aggregation of FLNA protein in cells of different groups after glucose deprivation were detected by Western blotting. The results showed that control cells exhibited significant high-molecular-weight FLNA aggregation bands after glucose deprivation treatment, while this aggregation was significantly reduced in shTXN1 cells. This indicates that TXN1 knockdown can inhibit the abnormal aggregation of the cytoskeletal protein FLNA during disulfide death, suggesting that TXN1 plays a crucial role in regulating cytoskeletal stability.
[0037] Example 4: Verification of compensatory upregulation of TRP14 expression induced by TXN1 knockdown
[0038] In the A549 cell line with stable TXN1 knockdown, the expression level of TRP14 protein was detected by Western blotting. The results showed that TRP14 expression was significantly upregulated in shTXN1 cells compared to the control group. This result indicates a functional compensatory mechanism between TXN1 and TRP14; the loss of TXN1 triggers increased TRP14 expression to synergistically maintain the cell's resistance to disulfide death.
[0039] Example 5: Verification of compensatory upregulation of TXN1 expression induced by TRP14 knockdown
[0040] In the A549 cell line with stable TRP14 knockdown, the expression level of TXN1 protein was detected by Western blotting. The results showed that TXN1 expression was significantly upregulated in shTRP14 cells compared to the control group. This further confirms the bidirectional compensatory regulatory relationship between TXN1 and TRP14 in regulating disulfide death, providing a mechanistic basis for developing dual-target inhibition strategies.
[0041] Example 6: Effect of TXN1 / TRP14 knockdown on disulfide death resistance under glucose transport inhibition conditions
[0042] In the presence of the glucose transport inhibitor BAY-876, glucose deprivation was applied to cells in the control, shTXN1, and shTRP14 groups, and cell viability was assessed using the CCK-8 assay. The results showed that even with glucose uptake inhibition, shTXN1 and shTRP14 cells exhibited higher survival rates than the control group, indicating that the regulatory role of TXN1 / TRP14 in disulfide death is independent of the glucose transport pathway and may involve broader metabolic or signaling network regulation.
[0043] The above detailed embodiments further illustrate the purpose, technical solution, drug application prospects, and beneficial effects of the present invention. It should be understood that the above embodiments are merely examples of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made based on the technical solution and drug application concept of the present invention should be included within the scope of protection of the present invention.
Claims
1. The use of thioredoxins TXN1 and TRP14 as a bidirectional switch regulating cell death, characterized in that, The use of thioredoxins TXN1 and TRP14 as drug targets in regulating cellular disulfide death sensitivity can modulate cellular sensitivity to programmed cell death, specifically: This is achieved by regulating the activity or expression levels of thioredoxin-1 (TXN1) and / or thioredoxin-associated protein 14 (TRP14) in target cells; wherein, reducing the activity or expression of TXN1 and / or TRP14 can increase the sensitivity of cells to apoptosis and / or ferroptosis, while reducing the sensitivity of cells to disulfide death.
2. The use of thioredoxin TXN1 and TRP14 as a bidirectional switch regulating cell death according to claim 1, characterized in that, The apoptosis was induced by astrocytes (STS), the ferroptosis was induced by RSL3, Erastin, ML210, or FIN56, and the disulfide death was induced by glucose deprivation.
3. The use of thioredoxin TXN1 and TRP14 as a bidirectional switch regulating cell death according to claim 1, characterized in that, Increased cellular sensitivity to apoptosis is achieved by activating the ASK1-JNK signaling pathway and upregulating the Bax / Bcl-2 ratio; increased cellular sensitivity to ferroptosis is associated with decreased glutathione (GSH) and / or reduced thiol levels and downregulation of GPX4 protein; decreased sensitivity to disulfide death is associated with the inhibition of abnormal aggregation of cytoskeletal proteins FLNA and / or Talin-1.
4. The application of thioredoxins TXN1 and TRP14 in anticancer drugs, characterized in that, This is used in the preparation of pharmaceutical compositions for treating cancer, and is a pharmaceutical composition or formulation for protecting cells from disulfide death damage or treating diseases related to excessive activation of disulfide death.
5. The application of thioredoxin TXN1 and TRP14 in an anticancer drug according to claim 4, characterized in that, The composition comprises a first active ingredient and a second active ingredient; the first active ingredient is an inhibitor of TXN1 and / or TRP14; the second active ingredient is an apoptosis inducer and / or ferroptosis inducer; that is, the pharmaceutical composition is an active inhibitor containing an effective dose of TXN1 and / or TRP14.
6. The application of thioredoxin TXN1 and TRP14 in an anticancer drug according to claim 4, characterized in that, The ferroptosis inducer is RSL3, Erastin, ML210, or FIN56.
7. The application of thioredoxin TXN1 and TRP14 in an anticancer drug according to claim 4, characterized in that, The apoptosis inducer mentioned is STS.
8. The application of thioredoxin TXN1 and TRP14 in an anticancer drug according to claim 4, characterized in that, The inhibitors of TXN1 and / or TRP14 are selected from small molecule inhibitors, antisense oligonucleotides, siRNA, shRNA, or the CRISPR-Cas9 gene editing system.
9. The use of a thioredoxin TXN1 and TRP14 or their encoding gene as a biomarker, characterized in that, Used to assess the susceptibility or resistance of cells, tissues, or individuals to disulfide death.
10. A method for screening candidate drugs against disulfide death, characterized in that, Includes the following steps: Cells were cultured in the presence of the test substance and subjected to disulfide death-inducing conditions; Detect cell viability or FLNA / Talin-1 aggregation status; if the test substance can improve cell viability and inhibit abnormal aggregation of cytoskeletal proteins, and this effect depends on or is accompanied by a decrease in TXN1 / TRP14 activity, then the substance is a potential candidate drug for anti-disulfide death. The method is used in a high-throughput drug screening platform to rapidly discover disulfide death modulators targeting TXN1 / TRP14.