Application of TFAM inhibitor in preparation of antitumor drugs

Abstract

The invention relates to the technical field of antitumor drugs, in particular to application of a TFAM inhibitor in preparation of antitumor drugs. It is found for the first time that all reagents which take TFAM as a target spot and can inhibit TFAM gene or protein expression or inhibit TFAM gene or protein functions can affect expression of mitochondrial genes in cells, the mitochondrial functions are remarkably reduced, then cancer cell apoptosis is selectively induced, and tumor growth and migration are effectively inhibited; meanwhile, the ethidium bromide targeting TFAM can effectively remove tumors (such as colorectal tumor tissues) in vivo, and also has a very good inhibition effect on cancer cell proliferation and migration (such as migration of pancreatic cancer cells in pancreas, liver and lung tissues); more importantly, the invention finds that the side effect of the ethidium bromide targeting the TFAM is very small. Therefore, TFAM serving as a target for development of antitumor drugs has extremely high clinical application value, and clinical experiments and application thereof are expected to be promoted.

Description

Technical Field

[0001] This invention relates to the field of antitumor drug technology, and in particular to the application of a TFAM inhibitor in the preparation of antitumor drugs. Background Technology

[0002] It is estimated that over 85% of glucose in tumor cells is produced through inefficient glycolysis to generate lactate for energy, with one molecule of glucose producing approximately four molecules of ATP. In contrast, most normal cells produce acetyl-CoA through glycolysis, which then enters the tricarboxylic acid cycle to produce NADH, primarily through mitochondrial-dependent oxidative phosphorylation, with one molecule of glucose producing approximately 36 molecules of ATP. Therefore, tumor cells must ingest significantly more glucose to meet their energy needs for rapid proliferation, which is the working principle of PET-CT. This process presents a paradox: why do tumor cells, which require more energy, choose the inefficient glycolysis pathway to consume glucose and obtain energy? Researchers currently have a very unclear understanding of the mechanism behind this significant clinical and scientific question. Some scholars believe it is mainly due to the higher activity of glycolysis-related enzymes in tumor cells, while others believe it is caused by mitochondrial dysfunction within tumor cells.

[0003] Mitochondria are the most important site of metabolism for substances and energy within the body and cells, and maintaining their normal physiological functions is crucial for the regulation of metabolism in the body and cells. From energy providers to the hub controlling apoptosis, from participating in innate immunity to being associated with various major diseases, mitochondria play a vital role in cellular life activities. Although it is believed that only 5% of glucose in cancer cells is converted into ATP via the mitochondrial pathway, it accounts for more than half of their energy source. Given that cancer cells have higher energy requirements and are more sensitive to changes in energy supply, they may be more sensitive to drugs that target mitochondrial function.

[0004] In recent years, research has gradually revealed the dual role of TFAM in tumorigenesis and development. On the one hand, in the early stages of inflammation-related cancers (such as colorectal cancer), downregulation of TFAM expression is closely related to impaired intestinal epithelial barrier function and exacerbated inflammatory responses, suggesting that it may play a protective role in the tumor initiation stage. Yang et al. pointed out that TFAM expression is significantly decreased in inflammatory bowel disease (IBD) tissues, and its absence can promote the development of colitis and increase susceptibility to colorectal cancer; while in tumor tissues, upregulation of TFAM expression promotes cancer cell proliferation and tumor progression by enhancing mitochondrial biosynthesis, showing that it has opposite functions at different stages (DOI: 10.1002 / cac2.12184).

[0005] On the other hand, TFAM interacts directly with the tumor suppressor p53. Wong et al. demonstrated through biophysical methods that TFAM specifically binds to the N-terminal transcriptional activation domains (TAD1 and TAD2) of p53, and both participate in the DNA damage response and repair process. This study also found that both TFAM and p53 preferentially bind to structurally distorted or damaged DNA, suggesting a possible synergistic effect in the mitochondrial DNA damage repair pathway, further supporting TFAM's potential as a key node in tumor metabolic regulation (DOI: 10.1093 / nar / gkp750).

[0006] Despite the growing interest in the role of TFAM in tumor biology, there is currently a lack of strategies for developing anti-tumor drugs that directly target TFAM. Existing technologies largely focus on the impact of TFAM expression regulation on mitochondrial function, and have not fully explored its clinical translational potential as a therapeutic target. Therefore, developing drugs that can specifically inhibit the function of TFAM genes or proteins, thereby inducing tumor cell apoptosis and inhibiting their proliferation and migration, has significant scientific importance and application prospects. Summary of the Invention

[0007] To address the aforementioned problems, the purpose of this invention is to provide an application of a TFAM inhibitor in the preparation of antitumor drugs.

[0008] The objective of this invention can be achieved through the following technical solutions: The first objective of this invention is to provide an application of a TFAM inhibitor in the preparation of antitumor drugs.

[0009] In one embodiment of the present invention, the drug is a drug that selectively induces apoptosis of cancer cells and / or inhibits tumor metastasis.

[0010] TFAM, or mitochondrial transcription factor, is a key transcription factor in mitochondrial gene transcription and an important participant in mitochondrial gene replication. TFAM binds to the promoter region of mitochondrial DNA to aid in mitochondrial gene replication and transcription. Studies in mice have shown that this gene product is essential for regulating mitochondrial genome copy number and is crucial for embryonic development. The Kearns-Sayre syndrome mouse model arises when targeted disruption of heart and muscle cells eliminates gene expression. Furthermore, the TFAM protein possesses two HMG regions, which are key regions for binding to mtDNA. This binding is important for mitochondrial transcription initiation in mammals, and TFAM may also be involved in mitochondrial genome packaging, but its binding activity is non-sequence specific.

[0011] This invention is the first to discover that, targeting TFAM, any reagent that can inhibit the expression of TFAM gene or protein, or inhibit the function of TFAM gene or protein, can affect the expression of mitochondrial genes in cells, leading to a significant decrease in mitochondrial function and thereby inducing apoptosis in cancer cells. This can be further used to prepare drugs for treating cancer.

[0012] In one embodiment of the present invention, the drug is a drug for treating gastric cancer, liver cancer, colorectal cancer, breast cancer, lung cancer, pancreatic cancer, or leukemia; Preferably, the drug is a drug for treating colorectal cancer or pancreatic cancer.

[0013] In one embodiment of the present invention, the TFAM inhibitor is a reagent that inhibits the expression of the TFAM gene or protein, or promotes the degradation of TFAM, thereby inducing a decline in mitochondrial function.

[0014] In one embodiment of the present invention, the TFAM inhibitor is a reagent that inhibits the binding of TFAM to DNA, thereby promoting the degradation of TFAM.

[0015] In one embodiment of the present invention, the TFAM inhibitor is ethidium bromide.

[0016] This invention is the first to discover that ethidium bromide specifically targets the mitochondrial gene transcription factor TFAM, inducing rapid degradation of TFAM, leading to a decrease in protein expression in mitochondrial genes and causing mitochondrial dysfunction, thereby rapidly activating the apoptosis pathway and ultimately selectively inducing tumor cell apoptosis (early events inducing tumor cell apoptosis can occur in vitro and be reproduced in vivo).

[0017] Specifically, this invention targets TFAM. First, a TSA screening method is used to preliminarily screen compounds that can bind to the TFAM fusion protein. Then, a quantitative real-time PCR instrument is used to screen whether the DIVERSet™ diverse compound library and a self-selected library of compounds affecting mitochondrial function bind to the TFAM protein. After obtaining potential binding compounds, their cellular functions (mainly mitochondrial-related functions), such as mitochondrial oxygen consumption and apoptosis, are further screened. Through other methods such as fluorescence colocalization, plasma resonance (SPR) analysis, microthermophoretic binding (MST) analysis, and in vitro protein degradation, a small molecule compound specifically targeting TFAM, ethidium bromide, is obtained. Unexpectedly, a strong interaction between ethidium bromide and TFAM was discovered. Further research revealed that it can specifically enter the mitochondria of cells, bind specifically to TFAM, and cause a sharp decrease in its protein level, thereby affecting the expression of mitochondrial genes in the cells, leading to a decline in mitochondrial function, and thus inducing apoptosis, especially apoptosis of tumor cells. On normal cells, it only inhibits cell growth. Therefore, it has excellent anti-tumor effects and extremely high clinical application value. This clarifies the characteristics and unique advantages of TFAM as a target anti-tumor drug.

[0018] A second objective of this invention is to provide an antitumor drug containing a TFAM inhibitor; The drug is a drug that selectively induces apoptosis in cancer cells and / or inhibits tumor metastasis.

[0019] In one embodiment of the present invention, the drug is a drug for treating gastric cancer, liver cancer, colorectal cancer, breast cancer, lung cancer, pancreatic cancer, or leukemia; Preferably, the drug is a drug for treating colorectal cancer or pancreatic cancer.

[0020] In one embodiment of the present invention, the dosage form of the drug is selected from either a gastrointestinal dosage form or a non-gastrointestinal dosage form.

[0021] In one embodiment of the present invention, the gastrointestinal dosage form is selected from one of tablets, granules, capsules, solutions, dry suspensions, powders, sustained-release agents, effervescent tablets, emulsions, suspensions, syrups, drops, or chewable tablets. The non-gastrointestinal drug delivery dosage forms include injection dosage forms, respiratory drug delivery dosage forms, cavity drug delivery dosage forms, mucosal drug delivery dosage forms, and skin drug delivery dosage forms.

[0022] In one embodiment of the invention, the antitumor drug is administered at a dose of 1 mg / kg to 100 mg / kg body weight per day, for example, at doses of about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg body weight per day.

[0023] In one embodiment of the invention, the application is carried out continuously for at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year. One or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) courses of treatment are administered, each course lasting at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks or at least 4 weeks; and the interval between each two courses is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days, two weeks, three weeks or four weeks.

[0024] In one embodiment of the invention, the drug comprises pharmaceutically acceptable salts and / or hydrates and / or carriers and / or excipients.

[0025] In one embodiment of the present invention, the carrier includes one or more of a solvent, a polymer, or a liposome; The excipients include one or more of diluents, excipients, or stabilizers.

[0026] A third objective of this invention is to provide the application of TFAM in the preparation of a kit for screening antitumor drugs, wherein the drugs are those that selectively induce apoptosis in cancer cells and / or inhibit tumor metastasis.

[0027] In one embodiment of the present invention, the drug is a drug for treating gastric cancer, liver cancer, colorectal cancer, breast cancer, lung cancer, pancreatic cancer, or leukemia; Preferably, the drug is a drug for treating colorectal cancer or pancreatic cancer.

[0028] Compared with the prior art, the present invention has the following beneficial effects: This invention is the first to discover that ethidium bromide targeting TFAM exhibits excellent selective killing effects on cancer cells, selectively inducing apoptosis and effectively inhibiting tumor growth and migration. This invention also reveals that ethidium bromide targeting TFAM can effectively eliminate tumors (e.g., colorectal tumor tissue) in vivo and has a very good inhibitory effect on cancer cell proliferation and migration (e.g., the migration of pancreatic cancer cells in the pancreas, liver, and lung tissues). Furthermore, this invention is the first to discover that ethidium bromide targeting TFAM has very few side effects, which is significantly different from existing understanding. Therefore, using TFAM as a target for anti-tumor drug development has extremely high clinical application value and is expected to advance clinical trials and applications. Attached Figure Description

[0029] Figure 1 In the diagram, (a) is a flowchart of the fluorescent reporter gene screening system, (b) is a schematic diagram of the absolute fluorescence ratio of each transfection group, and (c) is a schematic diagram of the absolute fluorescence ratio of each compound group.

[0030] Figure 2 Figure showing the results of an investigation into the intracellular localization of ethidium bromide (EtBr) and its effect on TFAM.

[0031] (a) Image obtained by super-resolution microscopy after treating cells with 1 μM ethidium bromide and staining with MitoTracker Green; (b) Image obtained by dividing AGS cells into two groups: i) treated with 1 μM ethidium bromide and MitoTracker Green, then fixed with 4% paraformaldehyde; ii) fixed first, then stained with ethidium bromide and MitoTracker Green; (c) Image obtained by confocal microscopy of cells transfected with GFP-labeled TFAM after ethidium bromide treatment, or after staining with PicoGreen and then ethidium bromide treatment; (d) Image of Western blot results; (e) Image obtained by super-resolution microscopy after treating cells with 1 μM ethidium bromide and MitoTracker Green; (f) Graph showing the decrease in expression of TFAM and mitochondrial-encoded proteins ATP8 and COX2 over time after treatment with μM ethidium bromide; (g) Graph showing the representative immunoblotting results of cell lysates from each group (control, ethidium bromide, doxorubicin, and 8-acetyl-ethidium bromide treatment groups): detection of TFAM, LonP1, HSP90, and GAPDH; (h) Graph showing the mRNA level of TFAM after ethidium bromide treatment by qRT-PCR; (h) Graph showing the expression changes of TFAM, Gapdh, Cyt b, Cox3, Atp8, and Cox2 after treatment with ethidium bromide, doxorubicin, or 8-acetyl-ethidium bromide.

[0032] Figure 3Schematic diagram of the binding interaction between TFAM, ethidium bromide, and mtDNA; (a-e) Surface plasmon resonance (SPR) analysis results of binding affinity, where (a) is TFAM with ethidium bromide; (b) is double-stranded DNA with ethidium bromide; (c) is ethidium bromide and TFAM-double-stranded DNA complex (TFAM pre-incubated with double-stranded DNA); (d) is TFAM with double-stranded DNA; (e) is double-stranded DNA with TFAM pre-incubated with ethidium bromide; (f-h) Micro-thermophoretic (MST) binding analysis results, where (f) is TFAM with ethidium bromide; (g) is TFAM with double-stranded DNA; (h) is TFAM-double-stranded DNA binding in the presence of ethidium bromide (TFAM pre-incubated with ethidium bromide); (i) Schematic diagram of TFAM domains, showing the positions of FLAG and GFP tags; (j-l) Confocal microscopy observation of cells expressing TFAM deletion mutants (after 2 μM... The results of EtBr treatment for 15 minutes are shown in the figure; (j) is TFAM without double-stranded DNA binding domain; (k) is TFAM without mitochondrial transport signal; (l) is TFAM without non-DNA binding domain (showing colocalization of TFAM with ethidium bromide).

[0033] Figure 4 Schematic diagram of the interaction between TFAM, double-stranded DNA, ethidium bromide and the TFAM proteolytic enzyme LonP1; (a-d) are surface plasmon resonance (SPR) binding analysis diagrams, where (a) shows the binding affinity of recombinant TFAM to LonP1; (b) shows TFAM incubated with ethidium bromide before the addition of LonP1; (c) shows TFAM incubated with double-stranded DNA before exposure to LonP1; (d) shows TFAM incubated with both double-stranded DNA and ethidium bromide before the addition of LonP1; (e-h) are the results of LonP1-mediated in vitro proteolytic experiments of TFAM; where (e) shows the time-dependent degradation of TFAM by LonP1; (f) shows the time-dependent degradation of TFAM by LonP1 before the reaction of TFAM with double-stranded DNA. A) Incubation followed by LonP1 treatment; (g) Effect of ethidium bromide on LonP1 degradation of TFAM in the absence of DNA; (h) Sequence-dependent degradation of TFAM by LonP1 in the presence of DNA and ethidium bromide (protein levels were assessed by immunoblotting of TFAM, LonP1 and FLAG antibodies in all experiments); (i) Schematic diagram of the mechanism model of EtBr's selective antitumor activity: Under physiological conditions, TFAM binds to mtDNA to maintain mitochondrial gene transcription and evades LonP1 degradation; after ethidium bromide treatment, the drug intercalates into mtDNA and replaces TFAM, promoting LonP1 degradation of it, thereby disrupting mitochondrial function—this process preferentially affects tumor cells with high ATP demand.

[0034] Figure 5Figure 1 shows the effects of ethidium bromide on oxygen consumption and glycolysis levels in cells; (a) Effect of ethidium bromide on oxygen consumption in human gastric cancer cells AGS; (b) Effect of ethidium bromide on oxygen consumption in normal gastric fibroblast cells GES1; (c) Effect of ethidium bromide on oxygen consumption in pancreatic cancer cells Panc1; (d) Effect of ethidium bromide on oxygen consumption in hepatocytes Chang; (e) Effect of ethidium bromide on glycolysis levels in human gastric cancer cells AGS; (f) Effect of ethidium bromide on glycolysis levels in normal gastric fibroblast cells GES1; (g) Effect of ethidium bromide on glycolysis levels in pancreatic cancer cells Panc1; (h) Effect of ethidium bromide on glycolysis levels in hepatocytes Chang.

[0035] Figure 6 The following graphs were used to investigate the effectiveness of ethidium bromide (EtBr) in inhibiting colorectal cancer and pancreatic tumors: (a) a representative image of AOM / DSS-induced colorectal tumors in mice before ethidium bromide treatment; (b) a comparison of colon specimens from the control group and the ethidium bromide gavage treatment group (20 mg / kg, 4 weeks); (c) ex vivo tumor specimens from two representative mice in each group; (d) a graph showing the percentage of mice without tumors or with only a small amount of residual tumors in the ethidium bromide treatment group; (e) a graph comparing tumor weight between the control group and the ethidium bromide treatment group; (f~g) graphs showing physiological index analysis, where (f) is a graph showing changes in mouse body weight; (g) a graph showing a comparison of the weight of various organs; (h~j) graphs showing blood test indicators, where (h) is white blood cell count (WBC); (i) is alanine aminotransferase (ALT); and (j) is aspartic acid... Acid aminotransferase (AST); (k~o) are experimental data graphs of pancreatic tumors: (k) is an in situ map of pancreatic tumors as shown by open surgery; (l) is isolated pancreatic and liver tissues; (m) is the percentage of tumor-free mice (control vs. gavage vs. intravenous injection group); (n) is isolated tumor tissue; (o) is the tumor weight statistics of each group; (p~r) are dynamic magnetic resonance imaging (MRI) monitoring results: (p) is the tumor progression time series graph of the control group and the intravenous ethidium bromide injection group; (q) is the tumor volume quantitative analysis graph; (r) is the final tumor weight comparison graph.

[0036] Where ns indicates not significant, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001. Detailed Implementation

[0037] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0038] Unless otherwise specified, all reagents used in the following embodiments are commercially available reagents, and all detection methods and techniques used are conventional detection methods and techniques in the art.

[0039] Example 1 This embodiment establishes a TFAM-based screening system to screen compounds targeting TFAM, as detailed below: To evaluate the effects of compounds or different growth conditions on TFAM transcriptional function, the human wild-type TFAM gene sequence was cloned into the pCMV vector using BamHI and XhoI to obtain the recombinant plasmid pCMV-TFAM; the LSP-DNA gene sequence was cloned into the pGL4 luciferase vector using XhoI and NheI to obtain the recombinant plasmid pGL4-LSP-DNA. Figure 1 a) HEK293T cells were transfected with Lipo-2000 (as control group), Lipo-2000 with pCMV-TFAM (plasmid concentration 2 μg), Lipo-2000 with pGL4-LSP-DNA (plasmid concentration 2 μg), and Lipo-2000 with pCMV-TFAM and pGL4-LSP-DNA (plasmid concentrations of 0.5 μg, 1 μg, and 2 μg, respectively) for 24 h. The absolute fluorescence ratio was then observed. Figure 1 (b) It was found that when the plasmid concentration was 2 μg and the number of cells seeded per well was 2... 10 4 At that time, compared with the untransfected group (Ctrl), its absolute fluorescence ratio reached more than 3 times, proving that the system has a better detection window.

[0040] To evaluate the effects of target compounds at varying concentrations on TFAM transcriptional function (with the untreated group (Control group) and DMSO group as control groups), transfected cells were treated with a series of compounds (ethidium bromide (purchased from Sigma (E7637)), Mdivi-1, rotenone, and antimycin A, at concentrations of 0.3 μM, 3 μM, 1 μM, and 10 μM, respectively) for 24 h under the above experimental conditions. The results showed that only etidium bromide could dose-dependently downregulate TFAM transcriptional function. Figure 1 c).

[0041] Example 2 This embodiment clarifies the intracellular localization of ethidium bromide (EtBr) and its effect on TFAM, as detailed below: Ethidium bromide itself contains a fluorescent group, and its excitation wavelength is approximately 580 nm, exhibiting orange-red fluorescence. GES1 and AGS cells were treated with 1 μM ethidium bromide and stained with MitoTracker Green, respectively. Imaging was performed using a super-resolution microscope. Figure 2 a) The results showed that ethidium bromide specifically entered the mitochondria and accumulated in certain locations within the mitochondria.

[0042] Because the conventional wisdom holds that ethidium bromide binds to DNA after entering cells, this study investigated AGS cells treated with 1 μM ethidium bromide and stained with MitoTracker Green, followed by fixation with 4% paraformaldehyde. The results showed that ethidium bromide lost its mitochondrial localization characteristics, instead becoming diffusely distributed in the cytoplasm after signaling weakening. If it were to bind to mtDNA within the mitochondria, this should not be affected by cell fixation. Furthermore, after staining the fixed cells with ethidium bromide again, it was found that the bromide specifically binds to the nuclear genome, indicating that its binding to DNA is unaffected by cell fixation. Figure 2 b).

[0043] Subsequently, the co-localization of ethidium bromide with mitochondrial DNA (mtDNA) binding proteins (such as TFAM and mtSSB single-stranded binding proteins) was further investigated. The results showed significant co-localization signals between ethidium bromide and TFAM and the double-stranded DNA dye PicoGreen, but no co-localization with mtSSB or PicoGreen-labeled nuclear DNA. Figure 2 c). In contrast, 8-acetyl-ethidium bromide did not exhibit similar co-localization, a result that confirms the functional importance of the ethidium bromide / mtDNA / TFAM interaction.

[0044] Example 3 This embodiment investigates the ethidium bromide-induced degradation of TFAM, as detailed below: To investigate the effect of ethidium bromide on TFAM, Western blot analysis was performed. GES1 and AGS cells were treated with ethidium bromide at concentrations of 0, 0.05 μM, 0.1 μM, 0.2 μM, 0.5 μM, and 1 μM, respectively (HSP90 and GAPDH were used as loading controls). Western blot results showed that after ethidium bromide treatment, TFAM protein levels decreased significantly in a dose-dependent manner with increasing ethidium bromide concentration. Figure 2 d).

[0045] GES1 and AGS cells were treated with 1 μM ethidium bromide and 8-acetyl-ethidium bromide, respectively (HSP90 was used as a loading control); Western blot results showed that the expression of TFAM and mitochondrial-encoded proteins ATP8 and COX2 decreased over time 30 minutes after ethidium bromide administration. Figure 2 e). Because TFAM is crucial for mitochondrial gene transcription, the protein expression of mitochondrial-encoding genes (such as Atp8 and Cox2) also decreases over time.

[0046] GES1 and AGS cells were treated with 1 μM ethidium bromide, doxorubicin, and 8-acetyl-ethidium bromide, respectively (the blank group served as the control group, and HSP90 and GAPDH were used as loading controls). The results showed that LonP1 (a known mitochondrial protease that degrades free TFAM) expression was affected. Neither 8-acetyl-ethidium bromide nor the chemotherapy drug doxorubicin (DXR) affected TFAM expression, highlighting the unique role of ethidium bromide. Figure 2 f).

[0047] GES1 and AGS cells were treated with 1 μM ethidium bromide for 0 h, 2 h, 6 h, 12 h, and 24 h, respectively. The results showed that TFAM mRNA levels remained unchanged, indicating that TFAM degradation was not caused by altered gene expression or increased protease activity. Figure 2 g).

[0048] Cells were treated with ethidium bromide, doxorubicin, and 8-acetyl-ethidium bromide at concentrations of 1 μM, respectively, and the mRNA levels of multiple mitochondrial genes were observed. The results showed that ethidium bromide treatment significantly decreased the mRNA levels of multiple mitochondrial genes (including Cyt b, Cox3, Atp8, and Cox2). Figure 3 (h), further confirming mitochondrial dysfunction caused by TFAM degradation.

[0049] Example 4 This embodiment demonstrates, through affinity experiments, that ethidium bromide disrupts the interaction between TFAM and mtDNA, as detailed below: To verify the direct interaction between ethidium bromide, TFAM, and mtDNA, in vitro affinity analysis was performed using surface plasmon resonance (SPR) and micro-thermophoresis (MST) (results are shown in Figure 1). Figure 3 (As shown).

[0050] First, SPR assay combined with affinity analysis data showed that ethidium bromide had a weak binding to TFAM (KD = 1592 μM). Figure 3a), but ethidium bromide reacts with double-stranded DNA (LSP-DNA) (KD = 68.9 μM; Figure 3 b) and TFAM with double-stranded DNA (KD = 102.5 μM; Figure 3 c) shows stronger binding. Meanwhile, TFAM alone exhibits high affinity for double-stranded DNA (KD = 546 nM). Figure 3 d), but the effect was significantly reduced after the addition of ethidium bromide (KD = 1152 nM; Figure 3 e).

[0051] Furthermore, to further verify the interaction between ethidium bromide, TFAM, and mtDNA, MST analysis was used to further verify whether ethidium bromide disrupted TFAM / DNA binding. The results showed that after treatment with 100 μM ethidium bromide, the Kd value of the TFAM–LSP-DNA complex increased from 869 nM to 2506 nM. Figure 3 (f–h). However, 8-acetyl-ethidium bromide had almost no effect on TFAM–DNA binding, indicating that acetylation would disrupt its DNA-binding ability. Although ethidium bromide altered the TFAM–DNA interaction, its high KD value with TFAM suggests that at function-related concentrations it does not directly bind TFAM, but rather functions through DNA-mediated binding.

[0052] To confirm whether ethidium bromide / TFAM colocalization depends on mtDNA, a series of TFAM deletion mutants (targeting DNA binding domain and non-binding domain) were constructed. Figure 3 i) Treatment of the TFAM deletion mutant with 2 μM ethidium bromide for 15 minutes revealed that the co-localization of TFAM with the mtDNA binding domain was completely lost. Figure 3 j), while TFAM lacking mitochondrial targeting signals localizes to the cell nucleus (j ...). Figure 3 k), TFAM lacking only the non-mtDNA binding domain still co-localizes with ethidium bromide in mitochondria. Figure 3 l).

[0053] These results indicate that ethidium bromide indirectly targets TFAM by binding to mtDNA, and its mitochondrial localization and functional disruption depend on the presence of mtDNA.

[0054] Example 5 This embodiment studies the TFAM degradation induced by ethidium bromide through LonP1-mediated proteolysis, as detailed below: LonP1 is a mitochondrial ATP-dependent protease belonging to the AAA+ protein family, capable of selectively degrading damaged or misfolded proteins (including TFAM). To investigate whether ethidium bromide promotes TFAM degradation through LonP1, the interactions between TFAM, ethidium bromide, LSP-DNA, and LonP1 were examined (e.g., Figure 4 (As shown).

[0055] First, in vitro binding experiments were conducted to reveal whether ethidium bromide regulates the TFAM-LonP1 interaction. The results showed that TFAM and LonP1 have a strong binding force (Kd = 20.8 nM). Figure 4 a) The binding was only slightly weakened after the addition of ethidium bromide (Kd = 30.9 nM); Figure 4 b). However, when TFAM was pre-bound to LSP-DNA, its affinity for LonP1 was significantly reduced (Kd = 217.8 nM); Figure 4 c) indicates that DNA-bound TFAM is protected and not easily degraded. The addition of ethidium bromide to the TFAM–LSP-DNA complex restored the high-affinity binding of TFAM to LonP1 (Kd = 58.6 nM). Figure 4 d) suggests that ethidium bromide makes mtDNA more susceptible to degradation by LonP1 by replacing TFAM with TFAM.

[0056] Subsequently, in vitro protein hydrolysis experiments were used to further verify the mechanism of action of ethidium bromide on TFAM. The results showed that LonP1 can progressively degrade free TFAM over time. Figure 4 e), but if TFAM is pre-bound to LSP-DNA, degradation is completely inhibited ( Figure 4 f), confirming the protective effect against DNA binding; however, when ethidium bromide acts alone, it does not enhance the degradation of free TFAM by LonP1 (f). Figure 4 (g) indicates that ethidium bromide does not directly activate LonP1. Therefore, it can be concluded that ethidium bromide effectively promotes TFAM degradation in the presence of DNA, regardless of the addition order. Figure 4 (h), the mechanism may be through embedding in DNA and replacing TFAM.

[0057] Based on these findings, a model is proposed regarding the mechanism by which ethidium bromide achieves selective antitumor activity by targeting mitochondria: Under normal conditions, TFAM binds to mtDNA, maintaining mitochondrial gene transcription and preventing degradation. After ethidium bromide treatment, the drug accumulates in the mitochondria and intercalates into mtDNA, replacing TFAM; free TFAM becomes a substrate for LonP1, leading to decreased TFAM levels, reduced mitochondrial transcription, and functional disorders. Figure 4 i).

[0058] Example 6 This embodiment studies the selective induction of tumor cell death by ethidium bromide, as detailed below: (1) Ethidium bromide selectively induces tumor cell apoptosis. To verify the important role of mitochondrial function in tumor treatment and development, this embodiment uses ethidium bromide to treat cancerous and non-cancer cells.

[0059] Cancer cells and normal cells from various tissue sources with a growth abundance of approximately 40% (as shown in Table 1) were treated with 1 μM, 2 μM, and 5 μM ethidium bromide for 48 hours, respectively, followed by MTT assay to detect apoptosis. Table 1 summarizes the median lethal dose (LD50) of ethidium bromide for various tumor cells and normal cells. The results show that the LD50 of ethidium bromide for most of these tumor cells was below 10 μM, while that for normal cells was mostly above 50 μM.

[0060] Table 1. Median lethal dose (IC50) of ethidium bromide for various tumor cell lines and normal cell lines 50 ) In summary, ethidium bromide can specifically induce the death of cancer cells; however, its effect on non-cancerous cells (normal cells) is merely to inhibit growth, without causing cell death. This may be related to the mechanism by which ethidium bromide induces mitochondrial damage and alters energy metabolism in tumor tissues.

[0061] (2) Ethidium bromide selectively inhibits mitochondrial function in cancer cells. To further verify the effect of ethidium bromide on mitochondrial function, various cell lines were treated with ethidium bromide (concentrations of 1 μM, 2 μM, and 5 μM), and mitochondrial function was assessed using Seahorse after 6 hours. The results showed that ethidium bromide treatment significantly reduced parameters reflecting mitochondrial function, such as oxygen consumption, glycolysis level, and glycolysis rate, in pancreatic cancer cells (Panc1) and gastric cancer cells (AGS), exhibiting a concentration-dependent effect (the numbers represent concentration values ​​in μM). In contrast, normal cell lines GES1 and Chang only showed a decrease at higher concentrations, such as 5 μM. The effective concentration of ethidium bromide for toxicity was significantly higher than that for cancer cells. Figure 5 ).

[0062] This indicates that ethidium bromide can selectively inhibit mitochondrial function in cancer cells, and also demonstrates that ethidium bromide targets TFAM and then selectively induces apoptosis in cancer cells by inhibiting mitochondrial function. This further proves that mitochondrial dysfunction in cancer cells plays a crucial role in the selective induction of cancer cell death by ethidium bromide, and also suggests that mitochondrial TFAM may play a unique and important role in cancer treatment.

[0063] Example 7 This embodiment demonstrates that ethidium bromide, a small molecule targeting TFAM, eliminated AOM / DSS-induced colorectal tumors via gavage treatment, as detailed below: Colorectal cancer (CRC) is one of the most common and deadliest malignant tumors worldwide, urgently requiring more effective treatment strategies. To evaluate the antitumor effect of EtBr, an AOM / DSS-induced tumor model was established. Pre-treatment tumor presence was confirmed by imaging. Figure 6 a). After 4 weeks of oral ethidium bromide treatment (20 mg / kg) (control group was blank), most mice showed complete tumor regression or only a small amount of tumor remaining. Figure 6 bd), tumor quality significantly decreased ( Figure 6 e). More importantly, ethidium bromide treatment in mice did not significantly affect body weight or the weight of major organs (e). Figure 6 FG, hematological (white blood cell) and biochemical (liver and kidney function) tests showed no systemic toxicity. Figure 6 hj).

[0064] Example 8 This embodiment demonstrates that intravenous injection treatment completely eliminates pancreatic tumors, as detailed below: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers, characterized by its aggressive nature and dense stromal microenvironment, which severely impacts drug delivery. Currently, effective treatments are still lacking. Therefore, ethidium bromide was further evaluated in a PDAC xenograft mouse model. The study found that, compared to the control group (blank), both oral and intravenous administration of ethidium bromide significantly reduced the tumor burden in the peritoneum, pancreas, and liver. Figure 6 kl). It was even found that all mice in the intravenous injection group achieved complete tumor clearance ( Figure 6 Although approximately 18% of mice in the oral administration group achieved complete remission, significant tumor suppression was also observed. Figure 6 Meanwhile, safety observations revealed that, similar to the above, no significant toxicity was observed in either treatment group.

[0065] In addition, tumor progression is monitored in real time via weekly magnetic resonance imaging (MRI): It was found that ethidium bromide treatment led to a sustained reduction in tumor size. Figure 6 The control group showed significant ascites (indicated by arrows), while the treatment group did not exhibit this phenomenon. Figure 6 (p) The residual pancreatic tumors in some treated mice may be related to the non-SPF feeding conditions required for repeated MRI examinations.

[0066] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the interpretation of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. Use of a TFAM inhibitor in the preparation of an antitumor medicament. 2.The use of a TFAM inhibitor according to claim 1 in the preparation of an antitumor drug, characterized in that, The medicament is a medicament that selectively induces apoptosis of cancer cells and / or inhibits tumor metastasis. 3.The use of a TFAM inhibitor according to claim 1 in the preparation of an antitumor drug, characterized in that, The medicament is a medicament for treating gastric cancer, liver cancer, colorectal cancer, breast cancer, lung cancer, pancreatic cancer or leukemia. 4.The use of a TFAM inhibitor according to claim 1 in the preparation of an antitumor drug, characterized in that, The TFAM inhibitor is an agent that inhibits the expression of a TFAM gene or protein, or promotes the degradation of TFAM. 5.The use of a TFAM inhibitor according to claim 4 in the preparation of an antitumor drug, characterized in that, The TFAM inhibitor is ethidium bromide.

6. An antitumor agent, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The medicament contains a TFAM inhibitor. The medicament is a medicament that selectively induces apoptosis of cancer cells and / or inhibits tumor metastasis.

7. The antitumor drug according to claim 6, wherein The dosage form of the medicament is selected from one of a gastrointestinal administration dosage form or a parenteral administration dosage form.

8. The antitumor drug according to claim 6, wherein The antitumor medicament is administered at a dose of 1 mg / Kg to 100 mg / Kg of body weight per day.

9. The antitumor drug according to claim 6, wherein The medicament includes a pharmaceutically acceptable salt and / or hydrate and / or carrier and / or excipient.

10. Use of TFAM in the preparation of a kit for screening anti-tumor drugs, characterized in that, The medicament is a medicament that selectively induces apoptosis of cancer cells and / or inhibits tumor metastasis.

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