Anti-tumor metastasis pharmaceutical composition based on ferroptosis and tumor-bacterium co-inhibition

Abstract

The invention discloses an anti-tumor metastasis pharmaceutical composition based on ferroptosis and tumor-bacteria co-inhibition, and belongs to the technical field of medicines. The pharmaceutical composition comprises a ferroptosis inducer and an antibacterial drug, the ferroptosis inducer comprises an active oxygen inducer, a glutathione peroxidase 4 (GPX4) inhibitor, a System xc-activity inhibitor, a targeting iron ion regulator and a lipid metabolism pathway regulator, and the antibacterial drug comprises an antibiotic, a broad-spectrum antibacterial agent and a photosensitizer. The pharmaceutical composition provided by the invention redistributes or adjusts lipid composition and functions through collaborative treatment of ferroptosis induction and tumor-bacteria co-inhibition, so that tumor fatty acid metabolism is remodeled, tumor metastasis is inhibited, and the problem of insufficient pertinence of an existing treatment mode is solved.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to an anti-tumor metastasis pharmaceutical composition based on ferroptosis and tumor-bacteria co-inhibition. Background Art

[0002] Tumor metastasis is the leading cause of cancer-related mortality, making the development of effective treatments crucial for anti-tumor therapy. Currently, intratumoral bacteria have been identified in various cancers, including breast, pancreatic, colorectal, and melanoma. Compared to adjacent healthy tissue, tumor tissues, through mechanisms such as a hypoxic microenvironment and secretion of specific metabolic substrates, form a unique microbial niche, selectively enriching pro-metastatic strains such as Staphylococcus xylosus and Fusobacterium nucleatum. Specifically, intratumoral bacteria not only modulate the tumor microenvironment through multiple mechanisms, including promoting the proliferation of regulatory T cells, inducing macrophage polarization toward the immunosuppressive M2 type, and impairing the activity of cytotoxic T cells, but also contribute to the establishment of a pre-metastatic niche. More importantly, intratumoral bacterial metabolites can remodel the tumor lipid metabolism network: on the one hand, bacterial-derived effector proteins regulate membrane phospholipid unsaturation by regulating SCD1 / ACSL4 expression; on the other hand, intratumoral bacteria can activate the PPARα-CPT1A pathway to enhance fatty acid β-oxidation in tumor cells, providing energy support for the metastatic cascade. This metabolic reprogramming significantly enhances the oxidative stress tolerance of circulating tumor cells. Based on this, the present invention proposes a new therapeutic strategy for targeted elimination of intratumoral bacteria and reversal of abnormal tumor metabolism, providing a new perspective for anti-tumor metastasis treatment by intervening in bacteria-tumor lipid metabolism.

[0003] Ferroptosis, a form of cell death characterized by the accumulation of iron-dependent lipid peroxides, can effectively inhibit tumor growth and metastasis by regulating fatty acid metabolism. Specifically, lipid peroxide accumulation interferes with the metabolic network of tumor cell-bacteria interactions, inducing redox stress in the pre-metastatic microenvironment. On the one hand, by continuously depleting glutathione and thereby inhibiting GPX4 activity, it disrupts the NRF2 / KEAP1 oxidative stress buffering system that metastatic cancer cells rely on. On the other hand, reactive oxygen species (ROS) induce oxidative stress in commensal bacteria within the tumor (such as Fusobacterium nucleatum and Staphylococcus xylosus), simultaneously blocking the metabolic support provided by bacterial-derived antioxidant molecules for metastatic colonization, thereby disrupting the survival mechanisms of metastatic tumor cells. Importantly, the bactericidal mechanism induced by ferroptosis may help address the issue of bacterial resistance. Similar to cancer cells, bacterial cell membranes, rich in unsaturated fatty acids, are susceptible to lipid peroxidation. The ROS generated during ferroptosis not only damage bacterial cell membranes but also DNA and proteins, ultimately leading to bacterial death. Therefore, this mechanism is emerging as a promising antibacterial and anti-tumor therapeutic approach.

[0004] Current anti-tumor therapies have yet to effectively integrate ferroptosis induction, intratumoral bacterial inhibition, and fatty acid metabolism intervention. This invention aims to provide a drug combination that, through ferroptosis induction and tumor-bacterial co-inhibition, redistributes or modulates lipid composition and function, thereby inhibiting tumor metastasis. This approach addresses the lack of dual-dimensional regulation of "tumor-bacterial co-inhibition and lipid metabolism reprogramming" in existing anti-tumor therapies. Summary of the Invention

[0005] One of the purposes of the present invention is to provide an application of a ferroptosis regulator in combination with an antibacterial agent in the preparation of an anti-tumor metastasis pharmaceutical composition.

[0006] In the present invention, the ferroptosis regulator includes at least one of a reactive oxygen species inducer, a glutathione peroxidase 4 (GPX4) inhibitor, a System xc-activity inhibitor, a regulator targeting iron ions, and a lipid metabolism pathway regulator; the reactive oxygen species inducer includes doxorubicin; the GPX4 inhibitor includes RSL3; the System xc-activity inhibitor includes Erastin; the regulator targeting iron ions includes ferric chloride and ferrous sulfate; the lipid metabolism pathway regulator includes arachidonic acid and docosahexaenoic acid.

[0007] In the present invention, the antibacterial drugs include antibiotics, broad-spectrum antibacterial agents, and photosensitizers; the antibiotics include sparfloxacin, vancomycin, and mitoxantrone; the broad-spectrum antibacterial agents include oleanolic acid and xanthohumol; and the photosensitizer includes indocyanine green.

[0008] In the present invention, the tumor is breast cancer, colon cancer, melanoma or pancreatic cancer.

[0009] Furthermore, the dosage ratio of the ferroptosis regulator to the antibacterial agent is w:w in the range of 0.25 to 5:1.

[0010] A second object of the present invention is to provide an anti-tumor metastasis pharmaceutical composition, comprising an effective amount of a ferroptosis regulator and an antibacterial drug; the ferroptosis regulator is selected from one or more of doxorubicin, RSL3, Erastin, ferric chloride, ferrous sulfate, arachidonic acid, and docosahexaenoic acid; the antibacterial drug is selected from one or more of sparfloxacin, vancomycin, mitoxantrone, oleanolic acid, xanthohumol, and indocyanine green.

[0011] Furthermore, the dosage ratio of the ferroptosis inducer and the antibacterial drug is 0.25 to 5:1 (w:w).

[0012] Compared with the prior art, the present invention has the following advantages:

[0013] (1) This invention emphasizes the combined effect of ferroptosis in inhibiting tumor cells and bacteria. This therapy simultaneously changes the lipid composition of tumor cells, thereby enhancing lipid peroxidation substrates and increasing the sensitivity of tumor cells and intratumoral bacteria to ferroptosis. Previous treatments often overlooked this critical metabolic vulnerability, which can actually significantly weaken the invasive ability of metastatic cancer cells.

[0014] (2) The present invention provides multiple types of ferroptosis regulators for the treatment of tumors and intratumoral bacterial killing, including reactive oxygen species inducers, glutathione peroxidase 4 (GPX4) inhibitors, System Xc-activity inhibitors, targeted iron ion regulators, and lipid metabolism pathway regulators. These agents combine bacterial clearance with tumor cell killing, simplifying the treatment regimen. The invention is applicable to a variety of solid tumors colonized by intratumoral bacteria, such as pancreatic cancer, colon cancer, melanoma, and breast cancer.

[0015] (3) The present invention innovatively targets intratumoral bacteria that are known to promote tumor metastasis, and uses ferroptosis regulators to specifically kill bacteria in metastatic tumors, thereby effectively inhibiting tumor metastasis.

[0016] (4) The synergistic effect of ferroptosis and antimicrobial agents takes advantage of the fact that bacterial membranes are extremely susceptible to the lipid peroxidation products produced during ferroptosis. Combined with antimicrobial agents, they further weaken the bacterial defense system and produce the strongest bactericidal effect.

[0017] Compared to existing treatments, this invention represents a significant leap forward in anti-metastatic therapy. By combining ferroptosis, fatty acid metabolism remodeling, and intratumoral bacterial clearance, it demonstrates superior efficacy, reduced side effects, and broader applicability, addressing key challenges in oncology and paving the way for innovative integrated cancer treatments. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 These are representative images of mouse tumor cell lysates plated after treatment in different groups in Example 1. DETAILED DESCRIPTION

[0019] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0021] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0022] Example 1

[0023] Ferric chloride, doxorubicin, and xanthohumol combined mediate ferroptosis and tumor-bacterial co-inhibition, and inhibit tumor metastasis by remodeling tumor fatty acid metabolism

[0024] First, a 4T1 breast cancer cell model colonized with Staphylococcus xylosus (MOI = 2) was established. Staphylococcus xylosus and 4T1 cells were co-cultured at a ratio of 2:1 for 4 hours, followed by treatment with 200 μg / mL gentamicin sulfate for 1 hour. Next, the 4T1 tumor cells infected with Staphylococcus xylosus were plated at 2.0×10 6 The density of cells / well was inoculated in 10 cm 2 After 24 hours of adherent growth in culture dishes, the serum-containing medium was removed. In each treatment group, ferric chloride concentrations of 3 μg / mL, doxorubicin concentrations of 1.5 μg / mL, and xanthohumol concentrations of 3 μg / mL were maintained for 24 hours. After trypsinization, the cell suspension was harvested (centrifuged at 1200 rpm for 5 minutes) and sample preparation was performed using a modified methyl tert-butyl ether (MTBE) lipid extraction method: 960 μL of a pre-chilled MTBE / methanol mixture (5:1, v / v) was added and lysed on ice for 10 minutes using sonication. After phase separation, 500 μL of the upper organic phase was transferred to a new EP tube. The remaining lower phase was extracted a second time with 500 μL of MTBE (vortexing for 10 minutes and sonicating on ice for 10 minutes). The two extracts were combined and evaporated to dryness after centrifugation at 14,000 × g for 10 minutes at 4°C. After fully evaporating, 1000 μL of liquid (isopropanol: acetonitrile: water = 75:20:5, v / v / v) was added for re-dissolution, and the cellular lipid composition was detected using liquid chromatography-mass spectrometry.

[0025] The results are shown in Table 1. The triple treatment regimen of ferric chloride, doxorubicin and xanthohumol significantly changed the composition characteristics of glycerophospholipid molecular species by synergistically regulating the lipid metabolic network. The content of glycerophospholipids containing sn-1 saturated / monounsaturated and sn-2 long-chain polyunsaturated fatty acids (C20:4 / C22:4) was significantly increased, leading to fatty acid metabolic reprogramming and significantly enhancing the ferroptosis effect.

[0026] Table 1 Lipidomics of ferric chloride + doxorubicin + xanthohumol

[0027]

[0028] Note: Compared with the control group ** P<0.01, *** P<0.001, **** P<0.0001, compared with the ferric chloride + doxorubicin group # P<0.05, ## P<0.01,### P<0.001, #### P<0.0001, compared with the xanthohumol group & P<0.05, && P<0.01, &&& P<0.001, &&&& P<0.0001.

[0029] Next, the number of intracellular Staphylococcus xylosus in the control group, 3 μg / mL ferric chloride + 1.5 μg / mL doxorubicin group, 3 μg / mL xanthohumol group, and the physical mixture of the three groups were incubated with 4T1 cells colonized with Staphylococcus xylosus for 24 hours to evaluate the antibacterial effect of each group. Figure 1 As shown in Table 2, representative images of cell lysate plating and bacterial quantitative analysis results from each treatment group demonstrate that the number of intracellular bacteria in the ferric chloride + doxorubicin and xanthohumol treatment groups was significantly reduced compared to the control group. In contrast, the combined ferric chloride, doxorubicin, and xanthohumol treatment group demonstrated a stronger antibacterial effect, with statistically significant differences compared to the ferric chloride + doxorubicin and xanthohumol monotherapy groups.

[0030] Table 2 Evaluation of the antibacterial effect of ferric chloride + doxorubicin + xanthohumol

[0031]

[0032] Note: Compared with the control group **** P<0.001, compared with the ferric chloride + doxorubicin group, ### P<0.001, compared with the xanthohumol group, &&&& P<0.0001.

[0033] Furthermore, tumor-bearing Balb / c mice infected with Staphylococcus xylosus were used to evaluate the effect of ferric chloride, doxorubicin, and xanthohumol on mediating ferroptosis and tumor-bacteria co-inhibition, and inhibiting tumor metastasis by remodeling tumor fatty acid metabolism. 6 The bacterial colonization orthotopic breast tumor model was established by inoculating the mouse mammary fat pad with bacteria. 3At 4 pm, drug treatment was started, with 3 mice in each group. The experimental animals were divided into the following groups: control group, ferric chloride + doxorubicin group, xanthohumol group, and physical mixture ferric chloride + doxorubicin + xanthohumol group (with a dosage ratio of 2:1:2). On days 0, 2, and 4, ferric chloride, doxorubicin, and xanthohumol were injected through the tail vein at doses of 10 mg / kg, 5 mg / kg, and 10 mg / kg, respectively. The mice were killed on day 21. The lung metastasis of tumor-bearing mice was judged by H&E staining. As shown in Table 4, compared with the control group, the lung metastasis area of ​​the ferric chloride + doxorubicin group and the xanthohumol group was significantly reduced, and the ferric chloride + doxorubicin + xanthohumol combined treatment group had the most significant lung metastasis inhibition effect. This shows that the synergistic treatment of ferroptosis induction and tumor-bacteria co-inhibition can effectively inhibit the lung metastasis of tumors. This combined treatment provides a new idea for the design of related drug combinations.

[0034] Table 3 Evaluation of the anti-metastatic effect of anti-tumor combination drugs in bacterially infected tumor-bearing mice

[0035]

[0036] Note: Compared with the control group * P<0.05, ** P<0.01, *** P<0.001, compared with the ferric chloride + doxorubicin group ## P<0.01.

[0037] Example 2

[0038] Arachidonic acid, ferric chloride, and sparfloxacin combined mediate ferroptosis and tumor-bacterial co-inhibition and inhibit tumor metastasis by remodeling tumor fatty acid metabolism

[0039] First, a 4T1 breast cancer cell model colonized with Fusobacterium nucleatum (MOI = 100) was established. Fusobacterium nucleatum and 4T1 cells were co-cultured at a ratio of 100:1 for 24 hours, followed by treatment with 200 μg / mL gentamicin sulfate for 1 hour. Next, the pre-treated Fusobacterium nucleatum-infected 4T1 tumor cells were plated at 2.0×10 6 The density of cells / well was inoculated in 10 cm 2Cells were grown in culture dishes for 24 hours, after which the serum-containing medium was removed. In each treatment group, arachidonic acid concentrations were 3 μg / mL, ferric chloride concentrations were 5 μg / mL, and sparfloxacin concentrations were 3 μg / mL. All samples were incubated under these conditions for 24 hours. After trypsinization, the cell suspension was harvested (centrifuged at 1200 rpm for 5 minutes) and sample preparation was performed using a modified methyl tert-butyl ether (MTBE) lipid extraction method: 960 μL of pre-chilled MTBE / methanol mixture (5:1, v / v) was added and lysed on ice for 10 minutes using sonication. After phase separation, 500 μL of the upper organic phase was transferred to a new EP tube. The remaining lower phase was extracted a second time with 500 μL of MTBE (vortexing for 10 minutes and sonicating on ice for 10 minutes). The extracts were then centrifuged at 14,000 × g for 10 minutes at 4°C. The two extracts were combined and the solvent evaporated to dryness. After fully evaporating, 1000 μL of liquid (isopropanol: acetonitrile: water = 75:20:5, v / v / v) was added for re-dissolution, and the cellular lipid composition was detected using liquid chromatography-mass spectrometry.

[0040] As shown in Table 4, after treatment with arachidonic acid + ferric chloride + sparfloxacin, arachidonic acid can combine with cell membrane phosphatidylethanolamine to provide more polyunsaturated bonds, resulting in a significant increase in the content of glycerophospholipids containing sn-1 saturated / monounsaturated and sn-2 long-chain polyunsaturated fatty acids (C20:4 / C22:4), thereby inducing fatty acid metabolic reprogramming and significantly enhancing the ferroptosis effect.

[0041] Table 4 Lipidomics analysis of the combination of arachidonic acid + ferric chloride + sparfloxacin

[0042]

[0043] Note: Compared with the control group **** P<0.0001, compared with the arachidonic acid + ferric chloride group # P<0.05, ## P<0.01, ### P<0.001, #### P < 0.0001, compared with the sparfloxacin group &&& P<0.001, &&&& P<0.0001.

[0044] Next, the number of intracellular F. nucleatum was counted using the spread plate method in the blank control group, the 3 μg / mL arachidonic acid and 5 μg / mL ferric chloride combined treatment group, the 3 μg / mL sparfloxacin monotherapy group, and the combined intervention group (arachidonic acid, ferric chloride, and sparfloxacin) after 24 hours of incubation with 4T1 cells colonized with F. nucleatum. Table 5 shows the quantitative bacterial counts in each treatment group. Compared with the arachidonic acid, ferric chloride, and sparfloxacin groups, the arachidonic acid, ferric chloride, and sparfloxacin group demonstrated a more potent bactericidal effect.

[0045] Table 5 Evaluation of the antibacterial effect of arachidonic acid + ferric chloride + sparfloxacin combined

[0046]

[0047] Note: Compared with the control group ** P<0.01, *** P<0.001, compared with the arachidonic acid + ferric chloride group # P<0.05.

[0048] Furthermore, the therapeutic effect of arachidonic acid, ferric chloride, and sparfloxacin on mediating ferroptosis and tumor-bacteria co-inhibition, thereby inhibiting tumor metastasis, was evaluated in bacterially infected tumor-bearing Balb / c mice. 6 The bacterial infection model of breast tumor was established by inoculating the mouse mammary fat pad with the bacterial infection virus. 3 Drug treatment was initiated at 4:00 p.m., with 3 mice in each group. The experimental animals were divided into the following groups: control group, arachidonic acid + ferric chloride group, sparfloxacin group, and physical mixture arachidonic acid + ferric chloride + sparfloxacin group (dosage ratio: 3:5:3). Arachidonic acid, ferric chloride, and sparfloxacin were injected intratumorally at doses of 3 mg / kg, 5 mg / kg, and 3 mg / kg on days 0, 2, and 4, respectively. Mice were sacrificed on day 21. Lung metastasis was further assessed by H&E staining. The lung metastasis area of ​​each treatment group is detailed in Table 6. Compared with the control group, the arachidonic acid + ferric chloride + sparfloxacin group had the most significant inhibitory effect on lung metastasis, indicating that the synergistic therapeutic strategy of inducing ferroptosis and tumor-bacteria co-inhibition significantly inhibited tumor lung metastasis.

[0049] Table 6 Evaluation of the anti-metastatic effect of anti-tumor combination drugs in bacterially infected tumor-bearing mice

[0050]

[0051] Note: Compared with the control group * P<0.05, ***P<0.001, compared with the arachidonic acid + ferric chloride group ## P < 0.01, compared with the sparfloxacin group @ P<0.05.

[0052] Example 3

[0053] Combination of docosahexaenoic acid, ferrous sulfate, and vancomycin mediates ferroptosis and tumor-bacterial co-inhibition, and inhibits tumor metastasis by remodeling tumor fatty acid metabolism

[0054] First, a CT26 colon cancer cell model colonized with Fusobacterium nucleatum (MOI = 100) was established. Fusobacterium nucleatum and 4T1 cells were co-cultured at a ratio of 100:1 for 24 hours, followed by treatment with 200 μg / mL gentamicin sulfate for 1 hour. Next, the pretreated CT26 tumor cells were incubated at 2.0×10 6 The density of cells / well was inoculated in 10 cm 2 Cells were grown in culture dishes for 24 hours, after which the serum-containing medium was removed. Within each treatment group, the concentration of docosahexaenoic acid (DHA), ferrous sulfate (FeSO), and vancomycin (VCM) was set at 2 μg / mL, 8 μg / mL, and 2 μg / mL, and all samples were incubated under these conditions for 24 hours. After trypsinization, the cell suspension was harvested (centrifuged at 1200 rpm for 5 minutes) and sample preparation was performed using a modified methyl tert-butyl ether (MTBE) lipid extraction method: 960 μL of pre-chilled MTBE / methanol (5:1, v / v) was added and lysed on ice for 10 minutes using sonication. After phase separation, 500 μL of the upper organic phase was transferred to a new EP tube, and the remaining lower phase was extracted a second time with 500 μL of MTBE (vortexing for 10 minutes and sonicating on ice for 10 minutes). The two extracts were combined and evaporated to dryness after centrifugation at 14,000 × g for 10 minutes at 4°C. After fully evaporating, 1000 μL of liquid (isopropanol: acetonitrile: water = 75:20:5, v / v / v) was added for re-dissolution, and the cellular lipid composition was detected using liquid chromatography-mass spectrometry.

[0055] As shown in Table 7, after combined treatment with docosahexaenoic acid + ferrous sulfate + vancomycin, the content of glycerophospholipids containing sn-1 saturated / monounsaturated and sn-2 long-chain polyunsaturated fatty acids (C20:4 / C22:4) increased significantly, leading to reprogramming of the fatty acid metabolic profile and significantly enhancing the ferroptosis effect.

[0056] Table 7 Lipidomics analysis of docosahexaenoic acid + ferrous sulfate + vancomycin combination

[0057]

[0058] Note: Compared with the control group ** P<0.01, ***P<0.001, **** P<0.0001, compared with the docosahexaenoic acid + ferrous sulfate group ### P<0.001, #### P < 0.0001, compared with the sparfloxacin group &&&& P<0.0001.

[0059] Next, the antibacterial efficacy of each group was assessed by counting the number of intracellular Fusobacterium nucleatum in the control group, the 2 μg / mL docosahexaenoic acid group + 8 μg / mL ferrous sulfate group, the 2 μg / mL vancomycin group, and the docosahexaenoic acid, ferrous sulfate, and vancomycin combination group after 24 hours of incubation with CT26 cells colonized with Fusobacterium nucleatum. Table 8 shows the quantification of bacterial counts. Compared to the monotherapy groups, the docosahexaenoic acid, ferrous sulfate, and vancomycin group demonstrated superior bactericidal efficacy.

[0060] Table 8 Evaluation of the antibacterial effect of docosahexaenoic acid + ferrous sulfate vancomycin combination

[0061]

[0062] Note: Compared with the control group * P<0.05, **** P<0.0001.

[0063] Furthermore, the effect of docosahexaenoic acid, ferrous sulfate, and vancomycin combined to mediate ferroptosis and tumor-bacteria co-inhibition, thereby inhibiting tumor metastasis, was evaluated. CT26 tumor cells (1.5×10 6 ) were inoculated into the colon to construct a bacterial infection colon tumor model. When the tumor volume of the mouse grew to 50mm 3 At 4 pm, drug treatment was started, with 3 animals in each group. The experimental animals were divided into the following groups: control group, ferroptosis inducer docosahexaenoic acid group, antibacterial drug vancomycin group, and physical mixture docosahexaenoic acid + ferrous sulfate + vancomycin group (the dosage ratio was 1:4:1). Drugs were injected through the tail vein on days 0, 2, and 4. The dosages of docosahexaenoic acid, ferrous sulfate, and vancomycin were 2.5 mg / kg, 10 mg / kg, and 2.5 mg / kg, respectively. The mice were killed on day 21, and the status of lung metastasis was determined by H&E staining. As shown in Table 9, compared with the control group, the area of ​​lung metastasis in each treatment group was significantly reduced. It is worth noting that the docosahexaenoic acid + ferrous sulfate + vancomycin group had the most significant reduction in lung metastasis area, indicating that the synergistic treatment of ferroptosis induction and tumor-bacteria co-inhibition significantly inhibited the lung metastasis symptoms of the tumor.

[0064] Table 9 Evaluation of the anti-metastatic effect of anti-tumor combination drugs in bacterially infected tumor-bearing mice

[0065]

[0066] Note: Compared with the control group ** P<0.01, *** P<0.001, compared with the docosahexaenoic acid + ferrous sulfate group # P<0.05, compared with the vancomycin group @@ P<0.01.

[0067] Example 4

[0068] RSL3 and indocyanine green combine to mediate ferroptosis and tumor-bacteria co-inhibition and inhibit tumor metastasis by remodeling tumor fatty acid metabolism

[0069] First, a 4T1 breast cancer cell model colonized with E. coli (MOI = 20) was established. The specific experimental process is as follows: E. coli and 4T1 cells were co-cultured at a ratio of 20:1 for 1 hour, followed by treatment with 200 μg / mL gentamicin sulfate for 1 hour. Next, the pretreated tumor cells were incubated at 2.0×10 6 The cells were seeded at a density of 100 cells / well in a six-well culture plate. After the cells attached to the wall, 40 μg / mL RSL3 and 8 μg / mL indocyanine green were added and incubated for 12 h. The cells were then illuminated with an 808 nm laser at 1.0 W / cm 2 Cells were laser-irradiated for 5 minutes. The combined interventions were cultured for 24 hours at 37°C and 5% CO₂, and then harvested by centrifugation. Samples were prepared using a modified methyl tert-butyl ether (MTBE) lipid extraction method: 960 μL of a pre-chilled MTBE / methanol mixture (5:1, v / v) was added and sonicated on ice for 10 minutes. After phase separation, 500 μL of the upper organic phase was transferred to a new EP tube. The remaining lower phase was extracted again with 500 μL of MTBE (vortexing for 10 minutes and sonicating on ice for 10 minutes). The extracts were centrifuged at 14,000 × g for 10 minutes at 4°C. The two extracts were combined and the solvent was evaporated to dryness. After complete evaporation, the extracts were reconstituted with 1000 μL of a mixture (isopropanol:acetonitrile:water = 75:20:5, v / v / v). Cellular lipid composition was analyzed by liquid chromatography-mass spectrometry.

[0070] As shown in Table 10, levels of polyunsaturated fatty acid-containing phosphatidylethanolamine and phosphatidylcholine increased in 4T1 cells treated with RSL3 and indocyanine green. Furthermore, studies examining the degree of unsaturation in the polyunsaturated fatty acids phosphatidylethanolamine and phosphatidylcholine revealed a role for RSL3 in upregulating the synthesis of polyunsaturated fatty acid-containing lipids. This may contribute to increased oxidative sensitivity to anti-tumor therapies and promote lipid peroxidation, thereby enhancing the therapeutic effects of ferroptosis.

[0071] Table 10 Lipidomics analysis of RSL3 combined with indocyanine green

[0072]

[0073] Note: Compared with the control group *** P<0.001, **** P < 0.0001, compared with RSL3 group ### P<0.001, #### P < 0.0001, compared with the indocyanine green group &&&& P<0.0001.

[0074] Next, the number of intracellular E. coli after 24 hours of co-incubation of different treatment groups with 4T1 cells colonized with E. coli was quantitatively evaluated by the spread plate method to evaluate the antibacterial effect of each group. The experimental groups were set as blank control group, 40μg / mL RSL3 group, 8μg / mL indocyanine green group and its physical mixture group. The experimental data in Table 11 show that compared with the control group, the single drug and the combination of the two drugs showed significant antibacterial effects. It is worth noting that the physical mixture group achieved a bactericidal efficiency of >99.9%, almost completely killing the intracellular colonized E. coli.

[0075] Table 11 Evaluation of the antibacterial effect of RSL3 and indocyanine green combined

[0076]

[0077] Note: Compared with the control group *** P < 0.001, compared with RSL3 group ## P < 0.01, compared with the indocyanine green group @ P<0.05.

[0078] Furthermore, the therapeutic effect of RSL3 and indocyanine green in mediating ferroptosis and tumor-bacteria co-inhibition, and inhibiting tumor metastasis by remodeling tumor fatty acid metabolism, was evaluated in bacterially infected tumor-bearing Balb / c mice. 6 A breast cancer tumor model with bacterial infection was established. When the tumor volume of the mouse grew to 50 mm3 At 3 pm, drug treatment was started, with 3 mice in each group. The experimental animals were divided into the following groups: control group, RSL3, indocyanine green and physical mixture RSL3+indocyanine green group (with a dosage ratio of 5:1). The drugs were injected through the tail vein on days 0, 2 and 4, with doses of RSL3 and indocyanine green of 10 mg / kg and 2 mg / kg, respectively. The mice were killed on day 21. The lung metastasis of each group was judged by H&E staining of lung tissue. The metastasis area of ​​each group is shown in Table 12. Compared with the control group, there was almost no lung metastasis in the RSL3 and indocyanine green combination group, indicating that the synergistic treatment of ferroptosis induction and tumor-bacteria co-inhibition significantly inhibited the symptoms of tumor lung metastasis.

[0079] Table 12 Evaluation of the anti-metastatic effect of anti-tumor combination drugs in bacterially infected tumor-bearing mice

[0080]

[0081] Note: Compared with the control group *** P<0.001, **** P < 0.0001, compared with RSL3 group # P<0.05, #### P < 0.0001, compared with the indocyanine green group &&&& P<0.001.

[0082] Example 5

[0083] Therapeutic approach and evaluation of erastin and mitoxantrone combined to mediate ferroptosis and tumor-bacteria co-inhibition, and to inhibit tumor metastasis by remodeling tumor fatty acid metabolism

[0084] First, a B16-F10 melanoma cell model colonized with Staphylococcus aureus (MOI = 20) was established. The specific experimental process is as follows: Staphylococcus aureus and B16-F10 cells were co-cultured at a ratio of 20:1 for 1 hour, followed by treatment with 200 μg / mL gentamicin sulfate for 1 hour. Next, the pretreated B16-F10 tumor cells were incubated at 2.0×10 6Cells were seeded at a density of 100 cells / well in a cell culture dish. Each group was treated with 2 μg / mL erastin, 8 μg / mL mitoxantrone, or a physical mixture of the two, and incubated for 24 hours. Cells were harvested by centrifugation. Samples were prepared using a modified methyl tert-butyl ether (MTBE) lipid extraction method: 960 μL of a pre-chilled MTBE / methanol mixture (5:1, v / v) was added and sonicated on ice for 10 minutes. After phase separation, 500 μL of the upper organic phase was transferred to a new EP tube. The remaining lower phase was extracted again with 500 μL of MTBE (vortexing for 10 minutes and sonicating on ice for 10 minutes). The extracts were centrifuged at 14,000 × g for 10 minutes at 4°C. The two extracts were combined and the solvent was evaporated to dryness. After complete evaporation, the cells were reconstituted with 1000 μL of a solution (isopropanol:acetonitrile:water = 75:20:5, v / v / v). Cell lipid composition was analyzed by liquid chromatography-mass spectrometry.

[0085] As shown in Table 13, compared with the control group, the combined treatment of Erastin and Mitoxantrone significantly increased the levels of PE (38:4) + 2O, PE (40:4) + 2O and PE (38:4) + 3O.

[0086] Table 13 Lipidomics analysis of Erastin and Mitoxantrone combined

[0087]

[0088] Note: Compared with the control group * P<0.05, **** P < 0.0001, compared with the Erastin group #### P < 0.0001, compared with the mitoxantrone group && P<0.01.

[0089] Next, the antibacterial effect of each group was evaluated by quantitatively counting the number of intracellular Staphylococcus aureus in different treatment groups after 24 hours of incubation with B16-F10 cells colonized with Staphylococcus aureus. The experimental setup included: a blank control group, a 2μg / mL Erastin group, an 8μg / mL mitoxantrone group, and a physical mixture group. Table 14 shows the results of the antibacterial quantitative analysis. Compared with the control group, the Erastin and mitoxantrone groups showed excellent bactericidal effects. Furthermore, the bactericidal effect of the Erastin + mitoxantrone group reached 99%, with almost no intracellular bacteria surviving.

[0090] Table 14 Evaluation of the antibacterial effect of Erastin and mitoxantrone combined

[0091]

[0092] Note: Compared with the control group * P<0.05,*** P < 0.001, compared with the Erastin group ## P < 0.01, compared with mitoxantrone @ P<0.05.

[0093] Furthermore, the combination of erastin and mitoxantrone was used to evaluate the therapeutic effect of ferroptosis and tumor-bacteria co-inhibition mediated by erastin and mitoxantrone in bacterially infected tumor-bearing Balb / c mice, and to inhibit tumor metastasis by remodeling tumor fatty acid metabolism. 6 A bacterial infection melanoma tumor model was established. When the tumor volume of the mouse grew to 50 mm 3 At , drug treatment was started, with 3 animals in each group. The experimental animals were divided into the following groups: control group, Erastin group, mitoxantrone group, physical mixture Erastin and mitoxantrone group (the dosage ratio was 1:4). The drugs were injected through the tail vein on days 0, 2, and 4, with Erastin at a dose of 1 mg / kg and mitoxantrone at a dose of 4 mg / kg. The mice were killed on day 21. The lung metastasis of each treatment group was observed by H&E staining of lung tissue. As shown in Table 15, compared with the control group, the Erastin group and the mitoxantrone group significantly reduced the area of ​​lung metastasis. It is worth noting that lung metastasis in the Erastin group and the mitoxantrone group was almost undetectable. This result shows that the synergistic treatment strategy of inducing ferroptosis and tumor-bacteria co-inhibition can effectively reshape tumor fatty acid metabolism and significantly inhibit the occurrence and development of tumor metastasis.

[0094] Table 15 Evaluation of the anti-metastatic effect of anti-tumor combination drugs in bacterially infected tumor-bearing mice

[0095]

[0096] Note: Compared with the control group ** P<0.01, *** P < 0.001, compared with the Erastin group ## P < 0.01, compared with mitoxantrone @@ P<0.01.

[0097] Example 6

[0098] RSL3 and oleanolic acid combine to mediate ferroptosis and tumor-bacteria co-inhibition and inhibit tumor metastasis by remodeling tumor fatty acid metabolism

[0099] First, a Pan02 cell model colonized with E. coli (MOI = 10) was established. The specific experimental process is as follows: E. coli and Pan02 cells were co-cultured at a ratio of 10:1 for 1 hour, followed by treatment with 200 μg / mL gentamicin sulfate for 1 hour. Next, the pretreated 4T1 tumor cells were cultured at 2.0×10 6 Cells were seeded at a density of 100 cells / well in a cell culture dish. Oleanolic acid concentrations were set at 5 μg / mL in each group, and RSL3 concentrations were set at 5 μg / mL. Samples were incubated under the above conditions for 24 hours. Cells were harvested by centrifugation. Samples were prepared using a modified methyl tert-butyl ether (MTBE) lipid extraction method: 960 μL of pre-chilled MTBE / methanol mixture (5:1, v / v) was added and lysed on ice for 10 minutes by sonication. After phase separation, 500 μL of the upper organic phase was transferred to a new EP tube. The remaining lower phase was extracted again with 500 μL of MTBE (vortexing for 10 minutes and sonicating on ice for 10 minutes). The extracts were centrifuged at 14,000 × g for 10 minutes at 4°C. The two extracts were combined and the solvent was evaporated to dryness. After complete evaporation, the cells were reconstituted with 1000 μL of a mixture (isopropanol:acetonitrile:water = 75:20:5, v / v / v). Cell lipid composition was analyzed by liquid chromatography-mass spectrometry.

[0100] As shown in Table 16, compared with the control group, the content of polyunsaturated fatty acids PE(16:0_20:4), PE(16:0_22:4), PE(18:0_20:4) and PE(18:0_22:4) were significantly increased after treatment with oleanolic acid and RSL3.

[0101] Table 16 Lipidomics analysis of oleanolic acid combined with RSL3

[0102]

[0103] Note: Compared with the control group ** P<0.01, *** P<0.001, **** P<0.0001, compared with the oleanolic acid group ## P<0.01, ### P<0.001, #### P < 0.0001, compared with RSL3 group &&&& P<0.0001.

[0104] Next, the antibacterial efficacy of each treatment group was assessed by quantitatively counting the number of intracellular E. coli bacteria after 24 hours of incubation with Pan02 cells colonized with E. coli using a spread plate method. The experimental setup included a blank control group, a 5 μg / mL oleanolic acid group, a 5 μg / mL RSL3 group, and a physical mixture of these groups. Table 17 shows the bacterial count quantification results. Compared to the control group, the physical mixture of oleanolic acid and RSL3 demonstrated superior bactericidal efficacy, with virtually no intracellular bacteria surviving.

[0105] Table 17 Evaluation of the antibacterial effect of oleanolic acid and RSL3 combined

[0106]

[0107] Note: Compared with the control group **** P<0.0001, compared with the oleanolic acid group #### P<0.0001 compared with RSL3 group &&&& P<0.0001.

[0108] Furthermore, bacterially infected tumor-bearing Balb / c mice were used to evaluate the effect of oleanolic acid and RSL3 on mediating ferroptosis and tumor-bacteria co-inhibition, and on inhibiting tumor metastasis by remodeling tumor fatty acid metabolism. Pan02 tumor cells (1.5×10 6 A pancreatic cancer tumor model with bacterial infection was established. When the tumor volume of the mouse grew to 50 mm 3 At 3 pm, drug treatment was started, with 3 mice in each group. The experimental animals were divided into the following groups: control group, oleanolic acid group, RSL3 group, and physical mixture oleanolic acid + RSL3 group (with a dosage ratio of 1: 1). The drugs were injected through the tail vein on days 0, 2, and 4, with doses of oleanolic acid and RSL3 of 2 mg / kg and 2 mg / kg, respectively. The mice were killed on day 21. Lung metastasis was judged by H&E staining of lung tissue. As shown in Table 18, compared with the control group, the single drug oleanolic acid and RSL3 groups significantly reduced the area of ​​lung metastasis, and the oleanolic acid + RSL3 group had almost no lung metastasis, indicating that the synergistic treatment of ferroptosis induction and tumor-bacteria co-inhibition significantly inhibited the occurrence and development of tumor metastasis.

[0109] Table 18 Evaluation of the anti-metastatic effect of anti-tumor combination drugs in bacterially infected tumor-bearing mice

[0110]

[0111] Note: Compared with the control group ** P<0.01, *** P<0.001, compared with the oleanolic acid group ### P < 0.001, compared with RSL3 group@@ P<0.01。

Claims

1. Application of ferroptosis regulators and antibacterial drugs in the preparation of anti-tumor metastasis drugs.

2. The use according to claim 1, characterized in that The ferroptosis inducer is selected from one or more of arachidonic acid, docosahexaenoic acid, doxorubicin, RSL3, Erastin, ferrous sulfate, and ferric chloride.

3. The use according to claim 1, characterized in that The antibacterial drug is selected from one or more of sparfloxacin, vancomycin, mitoxantrone, oleanolic acid, indocyanine green and xanthohumol.

4. The use according to claim 1, characterized in that The tumor is breast cancer, colon cancer, pancreatic cancer or melanoma.

5. The use according to any one of claims 1 to 4, characterized in that The dosage ratio of the ferroptosis inducer to the antibacterial agent is 0.25 to 5:

1.

6. An anti-tumor metastasis pharmaceutical composition, characterized in that: The invention comprises an effective amount of an iron death inducer and an antibacterial drug; the iron death inducer is selected from one or more of arachidonic acid, docosahexaenoic acid, doxorubicin, RSL3, Erastin, ferrous sulfate, and ferric chloride; the antibacterial drug is selected from one or more of sparfloxacin, vancomycin, oleanolic acid, mitoxantrone, indocyanine green, and xanthohumol.

7. The anti-tumor metastasis pharmaceutical composition according to claim 6, characterized in that The dosage ratio of the ferroptosis inducer to the antibacterial agent is 0.25 to 5:1.

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