Application of fucoxanthin in the preparation of tumor cell pyroptosis inducers

Abstract

This invention discloses the application of fucoxanthin in the preparation of pyroptosis inducers for tumor cells. This invention innovatively discovers and confirms that fucoxanthin can induce strong GSDME-dependent pyroptosis in tumor cells, clearly elucidating the mechanism by which fucoxanthin exerts its pyroptosis-promoting effect dependent on the "ROS-apoptosis-GSDME" signaling cascade. Furthermore, through molecular docking technology, it is confirmed that fucoxanthin can bind with high affinity to the GSDME receptor pocket, forming a stable hydrogen bond network with key amino acid residues such as ILE250 and ASP256. This invention breaks the traditional understanding that natural carotenoids only induce apoptosis or autophagy, providing a highly promising natural drug candidate molecule and theoretical basis for the development of novel pyroptosis-promoting antitumor drugs and new combination drug strategies.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and natural drug extract technology, specifically relating to the application of fucoxanthin in the preparation of tumor cell pyroptosis inducers. Background Technology

[0002] Malignant tumors are characterized by high recurrence and metastasis rates, as well as a tendency to develop treatment resistance, seriously threatening human life, health, and quality of life. Current clinical treatments mainly include surgery, radiotherapy, chemotherapy, and targeted therapy, but these still face challenges such as limited efficacy, high recurrence rates due to drug resistance, and significant toxic side effects. Therefore, the search for highly effective, low-toxicity, and multi-target natural anti-tumor drugs is of significant clinical importance.

[0003] The regulation of cell death mechanisms is central to the development of anti-tumor drugs. Currently, the tumor-suppressing mechanisms of clinical anti-tumor drugs largely rely on inducing tumor cell apoptosis. However, tumor cells can activate apoptosis escape mechanisms through multiple pathways, leading to apoptosis resistance, treatment failure, and tumor recurrence and drug resistance, which greatly limits the therapeutic efficacy of traditional apoptosis-based anti-tumor drugs.

[0004] Pyroptosis is an inflammatory programmed cell death mechanism that has garnered significant attention in recent years. Its main characteristics include cell swelling, disruption of membrane integrity, and release of contents and inflammatory factors. Recent studies have revealed that pyroptosis not only participates in the body's inflammatory response but can also effectively induce tumor cell death and trigger a strong anti-tumor immune response, thus making it a promising new target for cancer therapy.

[0005] The gasdermin protein family, mainly composed of GSDMB, GSDMC, GSDMD, and GSDME, are key effector molecules in the pyroptosis process. These proteins, upon cleavage by specific proteases, release N-terminal domains with membrane-perforating activity, thereby inducing cell membrane perforation and pyroptosis. However, current research on the role of marine naturally occurring bioactive substances in mediating pyroptosis and intervening in cancer progression through GSDME remains limited.

[0006] Fucoxanthin is a natural carotenoid widely found in brown algae, possessing various biological activities such as antioxidant, anti-inflammatory, anti-obesity, and anti-tumor effects. Existing research focuses solely on fucoxanthin's anti-tumor effects through mechanisms such as inducing tumor cell apoptosis, arresting the cell cycle, and inhibiting tumor proliferation. However, whether it can exert its anti-cancer effects by activating Gasdermin protein-dependent pyroptosis has not yet been reported. Summary of the Invention

[0007] To address the problems of the prior art, the present invention aims to provide a novel application of fucoxanthin in the preparation of tumor cell pyroptosis inducers. Studies have confirmed that fucoxanthin can induce strong GSDME-dependent pyroptosis in tumor cells, providing a promising natural drug candidate molecule for new therapeutic strategies such as inducing pyroptosis and combination therapy.

[0008] This invention is achieved through the following technical solution:

[0009] On the one hand, the present invention provides the application of fucoxanthin in the preparation of tumor cell pyroptosis inducers.

[0010] The structural formula of fucoxanthin provided by the present invention is shown below. It can be prepared by conventional methods in the art or obtained commercially.

[0011]

[0012] Preferably, the tumor cell pyroptosis inducer is used to prepare an antitumor drug.

[0013] Preferably, the tumor is a tumor that highly expresses the GSDME protein.

[0014] In a preferred embodiment of the present invention, the tumor is breast cancer.

[0015] Preferably, the fucoxanthin induces pyroptosis in tumor cells by specifically targeting and activating the GSDME protein.

[0016] In a preferred embodiment of the present invention, the drug further includes chemotherapeutic drugs that activate reactive oxygen species (ROS). Examples include ROT, doxorubicin, epirubicin, platinum-based antitumor drugs (cisplatin, carboplatin, oxaliplatin), taxanes (paclitaxel, docetaxel, albumin-bound paclitaxel), bleomycin, mitomycin C, etc.

[0017] Preferably, the drug further includes pharmaceutically acceptable excipients, which include any one or a combination of several of the following: carriers, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, antibacterial agents, or buffers.

[0018] The dosage form of the drug described in this invention can be any pharmaceutically acceptable dosage form, such as granules, capsules, tablets, powders, nano-formulations, liposome formulations, or microsphere targeted formulations.

[0019] On the other hand, the present invention provides the application of fucoxanthin in the preparation of GSDME protein-targeting activators for tumor cells.

[0020] Preferably, the tumor cells are breast cancer cells.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] This invention innovatively discovers and confirms that fucoxanthin can induce strong GSDME-dependent pyroptosis in tumor cells, precisely activate the cleavage activation of the N-terminal functional fragment of the GSDME protein, and has no significant activation effect on other pyroptosis family proteins, exhibiting extremely high target specificity. This discovery breaks the traditional understanding that natural carotenoids only have the ability to induce apoptosis or autophagy to fight tumors.

[0023] This invention, through ROS positive activation and reverse clearance experiments and Caspase-3 specific pharmacological inhibition experiments, fully elucidates the core upstream and downstream signaling pathways of fucoxanthin-induced pyroptosis in tumor cells: ROS oxidative stress is the key upstream initiation signal of this pyroptosis pathway. Fucoxanthin activates the ROS stress response, initiates the mitochondrial apoptosis pathway and activates Caspase-3, further cleaving and activating GSDME protein, ultimately mediating pyroptosis. This invention clarifies the upstream and downstream cascade regulatory mechanism of "ROS-apoptosis-GSDME", providing promising natural drug candidates and theoretical basis for the development of novel pyroptosis-promoting anti-tumor drugs and new combination drug strategies.

[0024] This invention, through molecular docking technology, reveals for the first time at the atomic level the direct physical binding mechanism between fucoxanthin and the GSDME protein. Fucoxanthin can stably bind to the GSDME receptor pocket with a high affinity of -7.8 kcal / mol, particularly forming a stable hydrogen bond network with ILE250 and ASP256 residues. This not only provides the most direct structural biological evidence for fucoxanthin activation of GSDME-mediated pyroptosis, but also provides a precise molecular template for designing novel targeted pyroptosis-promoting antitumor drugs based on this target binding pocket in the future. Attached Figure Description

[0025] Figure 1 Characterization of biological indicators related to pyroptosis induced by fucoxanthin in MDA-MB-231 breast cancer cells;

[0026] Figure 2 Characterization of biological indicators related to pyroptosis induced by fucoxanthin in MCF-7 breast cancer cells;

[0027] Figure 3 Fucoxanthin specifically activates GSDME expression and its N-terminal cleavage;

[0028] Figure 4 To regulate the effect of ROS-mediated oxidative stress on FX-induced pyroptosis;

[0029] Figure 5 The effects of apoptosis inhibitors on FX-mediated pyroptosis phenotype and GSDME cleavage;

[0030] Figure 6 The results show the molecular docking analysis of fucoxanthin binding to GSDME protein. Detailed Implementation

[0031] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. The described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] The fucoxanthin and reagents used in the embodiments of this invention are all commercially available.

[0033] Example 1: Fucoxanthin (FX) promotes the expression of biological markers of pyroptosis.

[0034] 1.1 Experimental Methods

[0035] PI staining: MCF-7 and MDA-MB-231 cells were stained at 1×10⁻⁶ cells per cell line. 6 Cells were seeded per well in 6-well plates. After cell adhesion, the treatment groups were treated with fucoxanthin (10, 20, 40 µM) for 48 h. Cell pellets were digested and collected. The pellets were washed twice with pre-cooled PBS, centrifuged at 1000 rpm for 5 min, and 1 mL of PI staining solution (3 µM, selected, 40710ES03) was added. The cells were incubated at room temperature for 15 min and then analyzed by flow cytometry.

[0036] LDH content detection: MCF-7 cells were used at a concentration of 1.5 × 10⁻⁶. 4 MDA-MB-231 cells were seeded per well in 96-well plates, with a control group, a maximum enzyme activity control group, and fucoxanthin treatment groups (5, 10, and 20 µM) incubated for 24 h and 48 h, respectively. One h before the predetermined detection time point, 10 µL of LDH release reagent from the maximum enzyme activity control kit (Beyotime, C0017) was added to the control group, and after repeated pipetting, incubation continued for 1 h. After the predetermined time, 120 µL of supernatant from each group was added to a new 96-well plate, followed by 60 µL of LDH detection working solution (20 µL lactate solution + 20 µL 1×INT solution + 20 µL enzyme solution) to each well, mixed well, and incubated at room temperature for 30 min. The absorbance was measured at 490 nm, and the LDH release was calculated. MDA-MB-231 cells were seeded at 1.5 × 10⁻⁶ cells / well. 4Each well was seeded with one sample, and three control groups (20, 30, and 40 µM) were set up for incubation for 48 h. One h before the predetermined detection time, 10 µL of LDH release reagent from the LDH kit (Beyotime, C0017) was added to the control group, and the mixture was repeatedly pipetted and incubated for another h. After the predetermined time, 120 µL of the supernatant from each well was added to a new 96-well plate, and then 60 µL of LDH detection working solution (20 µL lactate solution + 20 µL 1×INT solution + 20 µL enzyme solution) was added to each well. The mixture was mixed, incubated at room temperature for 30 min, and the absorbance was measured at 490 nm to calculate the LDH release.

[0037] ATP content detection: MCF-7 and MDA-MB-231 cells were used at a concentration of 1×10⁻⁶. 6 Cells were seeded per well in 6-well plates. After cell adhesion, the treatment groups were incubated with fucoxanthin (10, 20, 40 µM) for 48 h, and the cell supernatant was collected for later use. Following the instructions of the ATP assay kit (Beyotime, S0026), the ATP standard solution was diluted with culture medium to create a concentration gradient (0, 1, 3, 9, 30, 90 nM) to prepare the ATP assay working solution. 100 µL of the ATP assay working solution was added to each well, incubated at room temperature for 3 min, and then 100 µL of sample or standard was added. RLU was measured using a chemiluminescence analyzer.

[0038] IL-1β level detection: MDA-MB-231 cells were cultured at 1.5 × 10⁻⁶ cells / cells. 4 Cells were seeded per well in 96-well plates. After cell adhesion, the treatment groups were incubated with fucoxanthin (40, 60 µM) for 48 h. Cell supernatant was collected and IL-1β concentration was detected according to the Human IL-1β Elsa Kit (Lianke Biotechnology, EK101B) instructions.

[0039] Transmission electron microscopy sample preparation: MDA-MB-231 and MCF-7 cells were prepared at 1×10⁻⁶ cells per cell. 6Cells were seeded per well in 6-well plates. After cell adhesion, the treatment group was cultured in medium containing fucoxanthin (60 µM) for 48 h. After washing with PBS, the cell pellet was collected and fixed in 1.5 ml centrifuge tubes containing 2.5% glutaraldehyde. The tubes were then placed in a 4 ℃ freezer for 12-24 h. The fixative was discarded, and the cells were washed three times with PBS buffer for 15 min each time. The samples were then fixed with 1% osmium tetroxide solution for 1-2 h. The osmium tetroxide waste was carefully removed, and the cells were washed three times with 0.1 M phosphate-buffered saline PBS (pH 7.4) for 15 min each time. The samples were then dehydrated sequentially in 30%, 50%, 70%, and 90% ethanol at 15 min intervals, twice with 100% ethanol for 20 min each time, and finally twice with 100% acetone for 20 min each time. Immediately afterward, the resin was infiltrated and embedded. Pure embedding medium was poured into an embedding plate, and the sample was inserted into the plate and baked in a 70 °C oven for 12-48 hours. The resin block was then removed for later use. The resin block containing the embedded sample was cut into 70-90 nm ultrathin sections using an ultramicrotome. The sections were retrieved using a copper mesh, stained with uranium acetate for 8-15 minutes, and then stained with lead citrate for 5-10 minutes. After drying, the sections were ready for examination. The morphology was observed under a transmission electron microscope, and images were acquired and analyzed.

[0040] 1.2 Experimental Results

[0041] like Figure 1 As shown in Figure AB, in MDA-MB-231 cells, compared with the control group, FX treatment significantly increased the LDH release rate and ATP release in the cell supernatant, suggesting cell membrane rupture and extracellular leakage of intracellular substances, which are one of the main characteristics of pyroptosis; Figure 1 The C-value indicates that FX treatment significantly increased the secretion of the pro-inflammatory factor IL-1β, reaching 18.05 ± 2.35 pg / mL, suggesting that FX can activate breast cancer cells to release inflammatory factors; Figure 1 As shown in the DE, the PI staining ratio of cells significantly increased after FX treatment, from 2.54±0.29% to 4.32±0.34%, indicating impaired cell membrane integrity, consistent with the results of LDH and ATP. Figure 1 As shown in Figure F, transmission electron microscopy revealed typical pyroptosis morphological features in breast cancer cells treated with FX, including cell swelling, cell membrane perforation, and accompanying bubble formation. These results indicate that FX can significantly induce pyroptosis in breast cancer cells.

[0042] like Figure 2 As shown in Figure AB, in MCF-7 cells, compared with the control group, the LDH release rate in the cell supernatant was significantly increased after FX treatment, indicating cell membrane rupture, with the activation effect at 48 h being particularly significant. Figure 2 The B group was significantly better than the 24-hour treatment group. Figure 2(A). In addition, such as Figure 2 The C-cell assay showed a significant increase in ATP release from the cell supernatant after FX treatment, indicating cell membrane rupture and extracellular leakage of intracellular substances, which is one of the main characteristics of pyroptosis; Figure 2 As shown in the DE diagram, FX treatment significantly increased the PI staining ratio in cells and impaired cell membrane integrity, consistent with the results of LDH and ATP treatment; Figure 2 As shown in Figure F, transmission electron microscopy revealed typical pyroptosis morphological features in breast cancer cells treated with FX, including cell swelling, cell membrane perforation, and accompanying bubble formation. These results indicate that FX can significantly induce pyroptosis in breast cancer cells.

[0043] Example 2: Fucoxanthin-specific activation of GSDME-mediated pyroptosis

[0044] 2.1 Experimental Methods

[0045] Protein expression detection: MDA-MB-231 and MCF-7 cells were expressed at a concentration of 1×10⁻⁶. 6 Cells were seeded per well in 6-well plates. After cell adhesion, the treatment groups were given medium containing fucoxanthin (40, 60 µM) and cultured for 24 h. PBS was added to collect the cells, and they were centrifuged at 12000 r / min at 4℃ for 3 min. The cell pellet was collected, and a certain amount of Loading Buffer (5×) was added and boiled at 99℃ for 15 min. Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins on the gel were transferred to a PVDF membrane (200 mA 60 min), blocked with 5% skim milk (Beyotime, P0216) at room temperature for 1.5 h, incubated with primary antibody at room temperature for 1.5 h, washed three times with TBST for 10 min each time, incubated with secondary antibody at room temperature for 1 h, washed three times with TBST for 10 min each time, developed with BeyoECLMoon hypersensitive chemiluminescence reagent (Beyotime, P0028FS), exposed using a Tanon-4600 chemiluminescence image analysis system, and the gray values ​​of the bands were analyzed using ImageJ software.

[0046] 2.2 Experimental Results

[0047] A comprehensive screening of Gasdermin family proteins was conducted using protein expression assays to identify key FX-mediated execution proteins. For example... Figure 3 As shown in the middle AE, Western blot results indicate that among numerous pyroptosis-related proteins, FX is the only one that significantly activates the N-terminal cleavage activation of the GSDME protein. Figure 3 In the GSDME-N protein family (GSDME-N), expression was significantly upregulated at the 35 kDa position, while no significant activation effect was observed on other family members. Figure 3As shown in the GJ, relative quantitative analysis was performed on the full-length and splice variant expression levels of pyroptosis proteins. The results showed that FX treatment did not cause widespread and consistent changes in the Gasdermin family proteins, but rather exhibited distinct GSDME-specific regulatory characteristics. For GSDMB, GSDMC, and GSDMD, only slight fluctuations in some isoforms or splice fragments were observed after FX treatment, and there was a lack of consistent dose-dependent changes between different concentrations, suggesting that FX did not universally activate Gasdermin family members. In contrast, GSDME showed the most significant and stable activation response pattern. Figure 3 (J). With increasing FX concentration, the amount of full-length GSDME protein (GSDME-FL) continuously decreased, while its active cleavage product GSDME-N significantly increased, showing a clear dose-dependent trend. These results indicate that FX exerts its pyroptotic activity by promoting the conversion of GSDME from its full-length form to its active N-terminal cleavage fragment, rather than affecting the expression of other Gasdermin family proteins, suggesting that FX induces pyroptosis in breast cancer cells by specifically targeting GSDME activation. Furthermore, the upstream protease Caspase-3, which induces GSDME cleavage, was also detected and relatively quantified. It was found that FX significantly activated the cleavage activity of Caspase-3, exhibiting a certain dose-dependent effect. Figure 3 (F, K)

[0048] Example 3: ROS-mediated oxidative stress regulates FX-mediated pyroptosis

[0049] 3.1 Experimental Methods

[0050] CCK-8 cell viability assay: MDA-MB-231 cells were sputtered at 1.5 × 10⁶ cells per well. 4 In 96-well plates, seeded at a density of 1000 μL each, the following groups were established: Control group, ROS activator Rotenone (ROT, 1 µM) group, ROS scavenger N-acetylcysteine ​​(NAC, 0.1 mM) group, fucoxanthin group (40 µM), ROT combined with fucoxanthin group, and NAC combined with fucoxanthin group. ROT / NAC was pretreated for 2 h, followed by fucoxanthin treatment for 48 h. Afterward, the drug-containing medium was removed, the plates were rinsed with PBS, and 100 µL of medium (containing 10% CCK-8 reagent) was added to each well. The plates were incubated at 37°C for 1 h, and absorbance was measured at 450 nm.

[0051] ATP content detection: MDA-MB-231 cells were used at a concentration of 1×10⁻⁶. 6Cells were seeded per well in 6-well plates, with groups including Control, Rotenone (ROT, 2.5 µM) activator, N-acetylcysteine ​​(NAC, 0.1 mM) scavenger, fucoxanthin (60 µM), ROT combined with fucoxanthin, and NAC combined with fucoxanthin. ROT / NAC pretreatment was performed for 2 h, followed by fucoxanthin treatment for 48 h. Cell supernatant was collected for later use. ATP standard solutions were diluted with culture medium to create a concentration gradient (0, 1, 3, 9, 30, 90 nM). ATP detection working solution was prepared according to the ATP assay kit (Beyotime, S0026) instructions. 100 µL of ATP detection working solution was added to each well, incubated at room temperature for 3 min, and then 100 µL of sample or standard was added. RLU was measured using a chemiluminescence analyzer.

[0052] LDH release assay: MDA-MB-231 cells were released at a concentration of 1.5 × 10⁻⁶. 4 Samples were seeded per well in a 96-well plate, with groups including Control, ROT (1 µM), NAC (0.2 mM), fucoxanthin (40 µM), ROT combined with fucoxanthin, and NAC combined with fucoxanthin. ROT / NAC pretreatment was performed for 2 h, followed by fucoxanthin treatment for 48 h. One hour before the predetermined detection time, 10 µL of LDH release reagent from the LDH kit (Beyotime, C0017) was added to the maximum enzyme activity control group. After repeated pipetting, incubation continued for 1 h. Upon reaching the predetermined time, 120 µL of supernatant from each group was added to a new 96-well plate. Immediately afterwards, 60 µL of LDH detection working solution (20 µL lactate solution + 20 µL 1×INT solution + 20 µL enzyme solution) was added to each well, mixed thoroughly, and incubated at room temperature for 30 min. The absorbance was measured at 490 nm, and the LDH release was calculated.

[0053] PI staining: MDA-MB-231 cells were stained at a concentration of 1×10⁻⁶. 6 Cells were seeded per well in 6-well plates, with the following groups: Control group, ROS activator Rotenone (ROT, 1 µM) group, ROS scavenger N-acetylcysteine ​​(NAC, 2 mM) group, fucoxanthin group (60 µM), ROT combined with fucoxanthin group, and NAC combined with fucoxanthin group. ROT / NAC pretreatment was performed for 2 h, followed by fucoxanthin treatment for 48 h. Cell pellet was collected by digestion, washed twice with pre-cooled PBS, centrifuged at 1000 rpm for 5 min, and 1 mL of PI staining solution (3 µM, selected, 40710ES03) was added. The cells were incubated at room temperature for 15 min and then analyzed by flow cytometry.

[0054] Protein expression detection: MDA-MB-231 cells were expressed at a concentration of 1×10⁻⁶. 6 Cells were seeded per well in 6-well plates, with control, ROT (1µM), NAC (2 mM), fucoxanthin (60 µM), ROT combined with fucoxanthin, and NAC combined with fucoxanthin treatments. ROT / NAC pretreatment was performed for 2 h, followed by fucoxanthin treatment for 24 h. After adding PBS, the cells were centrifuged at 12000 r / min at 4 ℃ for 3 min, and the cell pellet was collected. A certain amount of Loading Buffer (5×) was added, and the cells were boiled at 99 ℃ for 15 min. Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins on the gel were transferred to a PVDF membrane (200 mA 60 min), blocked with 5% skim milk at room temperature for 1.5 h, incubated with primary antibody at room temperature for 1.5 h, washed three times with TBST for 10 min each time, incubated with secondary antibody at room temperature for 1 h, washed three times with TBST for 10 min each time, developed with BeyoECLMoon hypersensitive chemiluminescence reagent, exposed using a Tanon-4600 chemiluminescence image analysis system, and the gray values ​​of the bands were analyzed using ImageJ software.

[0055] 3.2 Experimental Results

[0056] To explore upstream triggering factors, the regulatory role of reactive oxygen species (ROS) was evaluated. Figure 4 As shown in AC, the ROS activator ROT significantly enhanced FX-mediated breast cancer cell death and the leakage and release of ATP and LDH (indicating enhanced membrane rupture), while also synergistically promoting FX-induced GSDME-N-terminal cleavage activation at the protein level. Figure 4 (Comparison of lanes G, 3, and 4) indicates that ROS activation exacerbates FX-mediated pyroptosis; such as Figure 4 As shown in the middle DF, the ROS scavenger NAC significantly reduced FX-mediated breast cancer cell death and the leakage and release of ATP and LDH (indicating reduced membrane rupture), while reversing FX-induced GSDME-N-terminal cleavage activation at the protein level. Figure 4 Comparison of lanes 3 and 4 (H, lanes 4 and 3) indicates that inhibiting ROS alleviates FX-mediated pyroptosis. Figure 4 As shown in Figure IK, flow cytometry results of PI staining indicated that the ROS activator ROT significantly enhanced the FX-mediated PI staining ratio in breast cancer cells. Figure 4 (J), while the ROS scavenger NAC significantly reduced the FX-mediated PI staining ratio in breast cancer cells (J). Figure 4 (Middle K). In summary, the above results fully demonstrate that ROS-mediated oxidative stress is a key upstream regulatory signal for FX-activated GSDME pyroptosis.

[0057] Example 4: Partial Regulation of Apoptosis in FX-Mediated Pyroptosis

[0058] 4.1 Experimental Methods

[0059] Cell viability CCK8 assay: MDA-MB-231 cells were cultured at 1.5 × 10⁶ cells per well. 4 The cells were seeded at a density of 100 μL in 96-well plates, with control group, Z-DEVD-FMK (ZDF, 20 µM, MCE, HY-12466) group, fucoxanthin group (40, 60 µM), and Z-DEVD-FMK combined with fucoxanthin group. Z-DEVD-FMK was pretreated for 2 h, followed by fucoxanthin treatment for 48 h. Finally, the drug-containing medium was removed and the cells were rinsed with PBS. 100 µL of medium (containing 10% CCK-8 reagent) was added to each well, and the cells were incubated at 37 °C for 1 h. The absorbance was measured at 450 nm.

[0060] ATP content detection: MDA-MB-231 cells were used at a concentration of 1×10⁻⁶. 6 Cells were seeded per well in 6-well plates, with options including a Control group, a Z-DEVD-FMK (20 µM) group, a fucoxanthin group (40, 60 µM), and a Z-DEVD-FMK combined with fucoxanthin group. Z-DEVD-FMK pretreatment was performed for 2 h, followed by fucoxanthin treatment for 48 h. Cell supernatant was collected for later use. ATP standard solutions were diluted with culture medium to create a concentration gradient (0, 1, 3, 9, 30, 90 nM). Following the instructions of the ATP assay kit (Beyotime, S0026), 100 µL of ATP assay working solution was added to each well, incubated at room temperature for 3 min, and then 100 µL of sample or standard was added. RLU was measured using a chemiluminescence analyzer.

[0061] LDH content detection: MDA-MB-231 cells were used at a concentration of 1×10⁻⁶ 4 Each well was seeded with one sample of the following: Control group, Z-DEVD-FMK (20 µM) group, fucoxanthin group (40, 60 µM), and Z-DEVD-FMK combined with fucoxanthin group. Z-DEVD-FMK pretreatment was performed for 2 h, followed by fucoxanthin treatment for 48 h. One h before the predetermined detection time, 10 µL of LDH release reagent from the LDH kit (Beyotime, C0017) was added to the maximum enzyme activity control group. After repeated pipetting, incubation continued for 1 h. Upon reaching the predetermined time, 120 µL of supernatant from each group was added to a new 96-well plate. Immediately afterwards, 60 µL of LDH detection working solution (20 µL lactate solution + 20 µL 1×INT solution + 20 µL enzyme solution) was added to each well, mixed thoroughly, and incubated at room temperature for 30 min. The absorbance was measured at 490 nM, and the LDH release was calculated.

[0062] Protein expression detection and analysis statistics: MDA-MB-231 cells were expressed at a concentration of 1×10⁻⁶. 6 Cells were seeded per well in 6-well plates, with control group, Z-DEVD-FMK (20 µM) group, fucoxanthin group (40, 60 µM), and Z-DEVD-FMK combined with fucoxanthin group. Z-DEVD-FMK pretreatment was performed for 2 h, followed by fucoxanthin treatment for 48 h. PBS was added, and the cells were centrifuged at 12000 r / min at 4 ℃ for 3 min. The cell pellet was collected, and a certain amount of Loading Buffer (5×) was added and boiled at 99 ℃ for 15 min. Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins on the gel were transferred to a PVDF membrane (200 mA 60 min), blocked with 5% skim milk at room temperature for 1.5 h, incubated with primary antibody at room temperature for 1.5 h, washed three times with TBST for 10 min each time, incubated with secondary antibody at room temperature for 1 h, washed three times with TBST for 10 min each time, developed with BeyoECLMoon hypersensitive chemiluminescence reagent, exposed using a Tanon-4600 chemiluminescence image analysis system, and the gray values ​​of the bands were analyzed using ImageJ software.

[0063] 4.2 Experimental Results

[0064] GSDME can be cleaved by the apoptosis-executing protein Caspase-3. This example verifies the potential impact of apoptosis on FX-induced pyroptosis. Figure 5 As shown in Figure A, when breast cancer cells were pretreated with the Caspase-3 specific inhibitor Z-DEVD-FMK, FX-induced GSDME cleavage activation was significantly blocked, as evidenced by reduced GSDME-N-terminal expression (comparison between lanes 3 and 6), and Caspase-3 cleavage expression was also significantly inhibited (comparison between lanes 3 and 6). Furthermore, as... Figure 5 As shown in the middle BD, Z-DEVD-FMK pretreatment significantly reduced FX-mediated cell death and various pyroptosis biological indicators such as PI positivity rate. Figure 5 (B), LDH ( Figure 5 (C) and ATP release ( Figure 5 (D). This result confirms that FX-mediated pyroptosis partially depends on the initiation of apoptotic signals, representing a classic new mechanism for the transition from apoptosis to pyroptosis.

[0065] Example 5: Molecular docking analysis of fucoxanthin binding to GSDME protein

[0066] 5.1 Experimental Methods

[0067] Molecular docking: The 2D structures of small molecule ligands were obtained from the PubChem database, imported into Chem3D 23.1.1 software to construct and optimize 3D structures, and saved as mol2 format files. Protein target crystal structures were obtained by screening high-resolution structures from the RCSB PDB database. Redundant groups such as water molecules and phosphate groups were removed using PyMOL 2.6 software, and the preprocessed structures were saved as PDB format files. The AutoDock tool was used to perform hydrogenation, dehydration, charge distribution, and rotatable bond definition between the protein and ligands. Molecular docking simulations were performed using AutoDock Vina 1.2.5 software. The docking box coordinates and dimensions were set according to the protein's active pocket, and the optimal binding conformation was screened based on binding free energy scoring. Interaction visualization was performed using Discovery Studio 2019 and PyMOL 2.6 software, generating 2D interaction diagrams and 3D binding pattern diagrams.

[0068] 5.2 Experimental Results

[0069] like Figure 6 As shown in Figure A, molecular docking results demonstrate that FX can precisely embed itself in the active pocket of the GSDME protein, exhibiting extremely high affinity. Figure 6As shown in Figure B, its binding energy (BE) reaches -7.8 kcal / mol, indicating that the two can bind spontaneously and stably. Further amino acid residue interaction analysis showed that FX and the GSDME receptor formed a multi-dimensional binding network, specifically including: (1) Hydrogen bond interaction: FX forms strong hydrogen bonds with ILE250 and ASP256 residues on GSDME, with bond lengths of approximately 2.3 Å, which is the core force stabilizing the protein-ligand complex. (2) Hydrophobic interaction: It forms hydrophobic interactions with ARG151, LYS41, LYS380, LEU255, VAL253, and TYR254 residues on the GSDME receptor, which helps anchor Fucoxanthin in the hydrophobic cavity inside the protein. (3) Van der Waals forces: It forms extensive van der Waals interactions with residues MET384, GLN381, ALA377, PRO257, LEU258, ARG261, ASN152, ASP251, and SER252. (4) Pi-Sigma interactions: It forms Pi-Sigma interactions with residues PHE265 and TYR254. Notably, the binding sites of FX are concentrated in the 250-265 aa region of GSDME, which is adjacent to the Caspase-3-mediated GSDME cleavage site Asp270. This suggests that after FX binds to the region adjacent to the GSDME cleavage site, it may induce local conformational changes, promote the exposure of the Asp270 site, enhance the recognition and cleavage efficiency of Caspase-3 at the Asp270 site, thereby promoting the release of GSDME-N and triggering pyroptosis. In summary, the above results provide strong structural evidence that FX has a high probability of binding to GSDME. This stable spatial physical binding alters the conformation of GSDME, thereby promoting its N-terminal cleavage activation and ultimately triggering pyroptosis in breast cancer cells.

[0070] In summary, this invention clearly proposes and verifies a novel phenotype of fucoxanthin that effectively induces pyroptosis in breast cancer cells through the "ROS-apoptosis-GSDME" mechanism axis, providing a highly promising natural drug candidate molecule for new therapeutic strategies such as inducing pyroptotic death of breast cancer cells and combination therapy.

Claims

1. Application of fucoxanthin in the preparation of tumor cell pyroptosis inducers.

2. The application according to claim 1, characterized in that, The tumor cell pyroptosis inducer is used to prepare anti-tumor drugs. Tumor drugs.

3. The application according to claim 2, characterized in that, The tumor is a tumor that highly expresses the GSDME protein.

4. The application according to claim 3, characterized in that, The tumor is breast cancer.

5. The application according to claim 4, characterized in that, Fucoxanthin induces pyroptosis in tumor cells by specifically targeting and activating the GSDME protein.

6. The application according to claim 2, characterized in that, The drugs also include chemotherapeutic drugs that activate reactive oxygen species (ROS).

7. The application according to claim 6, characterized in that, The drug also includes pharmaceutically acceptable excipients.

8. Application of fucoxanthin in the preparation of GSDME protein-targeting activators for tumor cells.

9. The application according to claim 8, characterized in that, The tumor cells are breast cancer cells.

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