Use of helicobacter bacteria, metabolites of helicobacter bacteria or nocardamine in a medicament for the prevention and / or treatment of cancer

Abstract

The application discloses application of helicobacter bacteria, metabolites of helicobacter bacteria or Nocardamine in preparation of a medicine for preventing and / or treating cancer, and belongs to the front technology field of biological medicine targeted treatment. The application discloses that helicobacter bacteria, metabolites of helicobacter bacteria or Nocardamine can inhibit various malignant tumors including colorectal cancer, lung cancer, breast cancer, liver cancer, skin cancer, gastric cancer and head and neck cancer, and has broad-spectrum anticancer activity.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and microbiology, specifically the use of Helicobacter spp., Helicobacter spp. metabolites, or Nocardamine in drugs for the prevention and / or treatment of cancer. Background Technology

[0002] Clinical findings linking the human microbiome to cancer date back 4,000 years, with medical records observing how severe localized bacterial infections could occasionally lead to the miraculous eradication of malignant tumors in some individuals. However, it wasn't until the early 20th century that the scientific community gradually recognized, using modern immunology and molecular biology, the crucial double-edged sword role of the microbiome in both the pathogenesis and treatment of cancer. For example, attenuated Salmonella Typhimurium has been used to treat advanced pancreatic cancer, metastatic melanoma, and metastatic pancreatic ductal adenocarcinoma; attenuated or recombinant Listeria monocytogenes has been used to treat metastatic non-small cell lung cancer, liver metastases, advanced lung cancer, and ovarian cancer.

[0003] Among the many bacteria that colonize the human body, the relationship between Helicobacter and cancer has been extensively studied. Traditional research generally considers many Helicobacter species to be pathogenic or even carcinogenic microorganisms. Long-term host infection can trigger chronic mucosal inflammation, leading to impaired tissue repair and gene mutations. However, it is precisely this characteristic of long-term co-evolution with host epithelial cells and the mucosal immune system that gives Helicobacter bacteria an extremely strong ability to colonize host tissues, adapt to the microenvironment, and directly intervene in the molecular mechanisms of host cell signal transduction.

[0004] Currently, there is no clinically mature bacterial drug that can specifically target and inhibit tumor stem cells to eradicate cancer. Endogenous microorganisms, due to their natural compatibility with the host, controllable immunogenicity, ease of colonization in specific pathological microenvironments, and direct pathways of action, have advantages over exogenous or excessively genetically engineered microorganisms in tumor translational applications. Therefore, developing endogenous bacterial drugs that target tumor stem cells, are potent, safe, and low-cost to produce could potentially significantly prolong and improve the quality of life and overall survival of patients with intermediate and advanced cancer. Summary of the Invention

[0005] In view of the difficulties in eliminating tumor stem cells, the easy development of drug resistance in conventional anticancer drugs, and the lack of highly effective live bacterial drugs in the existing technology, the purpose of this invention is to provide a potential drug that can inhibit the self-renewal mechanism of tumor stem cells.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0007] The use of Helicobacter spp., Helicobacter spp. metabolites, or Nocardamine in the preparation of medicaments for the prevention and / or treatment of cancer.

[0008] Nocardamine, CAS number 26605-16-3, belongs to the class of cyclic siderophore compounds and has the function of an iron chelating agent.

[0009] Preferably, the Helicobacter genus bacteria is at least one of the following: Helicobacter mastomyrinus strain, Helicobacter ganmani strain, Helicobacter cinaedi strain, or Helicobacter canadensis strain.

[0010] Preferably, the metabolites of the Helicobacter spp. are prepared by the following method: immersing a culture medium containing Helicobacter spp. in an extraction solvent, then concentrating the extraction solvent to obtain an extract, and reconstitute the extract to obtain the metabolites of the Helicobacter spp.

[0011] Preferably, the nocardamine is an active ingredient of the metabolite of the Helicobacter genus bacteria.

[0012] Preferably, the drug specifically targets and inhibits the self-renewal of tumor stem cells, thereby inhibiting the formation of tumor cells into spheres.

[0013] Preferably, the cancer is at least one of colorectal cancer, lung cancer, breast cancer, liver cancer, skin cancer, stomach cancer, and head and neck cancer.

[0014] Preferably, the dosage form of the drug is at least one of the following: aerosol, spray, emulsion, suspension, enema, paste, ointment, gel, lotion, suppository, patch, film, capsule, tablet, pill, powder, granule, tincture, injection, syrup, or solution.

[0015] Preferably, the route of administration of the drug is at least one of the following: oral administration, sublingual administration, inhalation administration, mucosal administration, intravenous injection, arterial injection, intramuscular injection, intradermal injection, subcutaneous injection, intraperitoneal injection, rectal administration, vaginal administration, transdermal administration, or local administration.

[0016] Preferably, the drug further includes pharmaceutically acceptable additives or excipients.

[0017] More preferably, the pharmaceutically acceptable excipients include at least one of the following: diluents, binders, wetting agents, lubricants, disintegrants, solvents, emulsifiers, cosolvents, solubilizers, preservatives, pH adjusters, osmotic pressure adjusters, surfactants, coating materials, antioxidants, antibacterial agents, or buffers.

[0018] Compared with existing clinical conventional chemotherapy or targeted therapy techniques, the present invention has the following significant and unexpected beneficial effects:

[0019] 1) Broad-spectrum anti-tumor activity overcoming technological bias: For a long time, the industry has generally regarded many Helicobacter spp. bacteria as pathogenic or even carcinogenic factors. This invention overturns this inherent understanding. Through rigorous experiments, it has been proven that four specific types of Helicobacter spp. bacteria (Helicobacter mastomyrinus, Helicobacter ganmani, Helicobacter cinaedi, and Helicobacter canadensis) can inhibit various tumors, including colorectal cancer, lung cancer, breast cancer, liver cancer, skin cancer, gastric cancer, and head and neck cancer, and have broad-spectrum anti-cancer activity.

[0020] 2) Targeted inhibition of tumor stem cell self-renewal: The Helicobacter spp. bacteria, their metabolites, or Nocardamine of this invention exhibit concentration-dependent inhibitory effects, disrupting the in vitro self-renewal and clonal capacity of the aforementioned cancer stem cells. Since tumor stem cells are the cause of cancer recurrence, metastasis, and drug resistance, this invention achieves deep intervention in tumors from the most upstream level of the mechanism. Attached Figure Description

[0021] Figure 1 This is a diagram showing the results of H. mastomyrinus inhibiting liver metastasis of colorectal cancer in an embodiment of the present invention.

[0022] Figure 2 This is a diagram showing the results of H. cinaedi inhibiting colorectal cancer liver metastasis in an embodiment of the present invention.

[0023] Figure 3 This is a diagram showing the results of H. mastomyrinus inhibiting tumor cell spheroidization in an embodiment of the present invention.

[0024] Figure 4 This is a diagram showing the results of H. mastomyrinus metabolites inhibiting tumor cell spheroidization in an embodiment of the present invention.

[0025] Figure 5 This is a diagram showing the results of H. ganmani inhibiting tumor cell spheroidization in an embodiment of the present invention.

[0026] Figure 6This is a diagram showing the results of H. ganmani metabolites inhibiting tumor cell spheroidization in an embodiment of the present invention.

[0027] Figure 7 This is a diagram showing the results of H. cinaedi inhibiting tumor cell spheroidization in an embodiment of the present invention.

[0028] Figure 8 This is a diagram showing the results of H. cinaedi metabolites inhibiting tumor cell spheroidization in an embodiment of the present invention.

[0029] Figure 9 This is a diagram showing the results of H. canadensis inhibiting tumor cell spheroidization in an embodiment of the present invention.

[0030] Figure 10 This is a diagram showing the results of H. canadensis metabolites inhibiting tumor cell spheroidization in an embodiment of the present invention.

[0031] Figure 11 The figure shows the experimental results of detecting the presence of the siderophore Nocardamine in the metabolites of H. mastomyrinus, H. ganmani, H. cinaedi and H. canadensis in an embodiment of the present invention.

[0032] Figure 12 This is a diagram showing the results of Nocardamine inhibiting tumor cell spheroidization in an embodiment of the present invention. Detailed Implementation

[0033] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the technical solutions of this invention will be described in more detail below with reference to specific embodiments and accompanying drawings. It should be understood that the specific experimental steps and embodiments described herein are only for explaining and demonstrating the technical principles of this invention and are not intended to limit the legal protection scope of this invention.

[0034] The strains used in the embodiments of this invention are from the following sources:

[0035] The Helicobacter mastomyrinus strain (hereinafter referred to as H. mastomyrinus strain in this example) was purchased from the Culture Collection Center of the University of Gothenburg, Sweden, with accession number CCUG-52417T.

[0036] The Helicobacter ganmani strain (hereinafter referred to as H. ganmani strain in the examples) was purchased from the Culture Collection Center of the University of Gothenburg, Sweden, strain accession number: CCUG-43526.

[0037] The Helicobacter cinaedi strain (abbreviated as H. cinaedi strain in the examples) was purchased from the Culture Collection Center of the University of Gothenburg, Sweden, strain accession number: CCUG-15432.

[0038] The Helicobacter canadensis strain (hereinafter referred to as H. canadensis strain in the examples) was purchased from the Culture Collection Center of the University of Gothenburg, Sweden, strain accession number: CCUG-47163T.

[0039] The Non-toxigenic Bacteroides fragilis strain used as a control in the examples (hereinafter referred to as the NTBF strain in the examples) was purchased from the American Type Culture Collection, strain accession number: ATCC25285.

[0040] The Bacteroides vulgatus strain used as a control in the examples (hereinafter referred to as B. vulgatus strain in the examples) was purchased from the American Type Culture Collection, strain accession number: ATCC8482.

[0041] The metabolites of the above-mentioned strains in the embodiments of the present invention were prepared by the following methods:

[0042] Cut the agar plate containing the strain into 1cm pieces. 3 The samples were soaked in ethyl acetate solution for 3 days; then the ethyl acetate solution was removed by rotary evaporation to obtain a concentrate containing the strain's metabolites; the concentrate was redissolved in DMSO to finally obtain the metabolites dissolved in DMSO.

[0043] The Nocardamine used in this embodiment of the invention was purchased from MCE Company, CAS number 26605-16-3.

[0044] All in vitro cultured tumor cell lines used in the embodiments of this invention, including HCT116 (CCL-247), CT26 (CRL-2638), H1299 (CRL-5803), H460 (HTB-177), MDA-MB-231 (HTB-26), 4T1 (CRL-2539), HeP3B (HB-8064), A2058 (CRL-11147), SUN-1 (CRL-5971), and CAL-27 (CRL-2095), were purchased from the American Type Culture Collection Center (ATCC).

[0045] Example 1: Animal experiments on the inhibition of colorectal cancer liver metastasis by H. mastomyrinus and H. cinaedi strains.

[0046] Tumor metastasis is the leading cause of death in colorectal cancer patients, and the liver is the most common target organ for colorectal cancer metastasis. This embodiment aims to verify the inhibitory effects of H. mastomyrinus and H. cinaedi strains on liver metastasis of colorectal cancer by constructing a mouse model of colorectal cancer with orthotopic cecal injection.

[0047] (1) Animal experiments on the inhibition of colorectal cancer liver metastasis by H. mastomyrinus strain

[0048] BALB / c mice were administered a quadruple antibiotic regimen (1 g / L ampicillin, 1 g / L neomycin, 0.25 g / L metronidazole, 0.5 g / L vancomycin) via drinking water for 7 days to clear their intestinal flora. Afterward, they were given normal drinking water for 1 day to allow for the metabolism of drug residues. The experimental group received H. mastomyrinus-resistant antibiotics (0.5 g / L vancomycin, 2 g / L cefotaxime, 0.8 g / L trimethoprim) in their drinking water, while the control group received NTBF-resistant antibiotics (1 g / L kanamycin, 1 g / L streptomycin). This helped maintain the survival of the target bacterial strain administered via gavage and reduced interference from other strains. Starting from day 9, the mice were inoculated with the corresponding bacterial culture via gavage at a dose of 1 × 10⁻⁶ every 7 days. 8 CFU. Inject 1×10⁻⁶ CFU into the cecal wall on day 0. 5 CT26 cells were used. The tumor in situ was removed on day 10. Mice were sacrificed on day 35, and the fluorescence / bioluminescence signals of liver metastases were examined by dissection.

[0049] Experimental results and statistical analysis: Figure 1 The study clearly presents representative in vitro liver imaging images of each group of mice, along with corresponding bar charts showing the quantitative statistical data of liver metastasis signals. Rigorous statistical analysis (using one-way ANOVA with Fisher's LSD test as a post-test to assess inter-group differences) clearly revealed that compared to the control group inoculated with NTBF strain, the experimental group mice regularly administered H. mastomyrinus strain showed significantly reduced liver metastasis bioluminescent signals. P<0.05; (P<0.01). The experimental results demonstrate that H. mastomyrinus has the function of inhibiting liver metastasis of colorectal cancer.

[0050] (2) Animal experiments on the inhibition of colorectal cancer liver metastasis by H. cinaedi strain

[0051] Experimental methods and procedures:

[0052] To verify the efficacy of the H. cinaedi strain and exclude interference from the host immune system, this example uses NCG severely immunodeficient mice (lacking functional T, B, and NK cells).

[0053] The procedure for clearing the intestinal background was the same as before: the patient freely drank the above-mentioned quadruple antibiotic solution for 7 days, followed by 1 day of normal drinking water. The experimental group received H. cinaedi-resistant antibiotics (0.5 g / L erythromycin, 0.3 g / L ciprofloxacin) in their drinking water, while the control group received B. vulgatus-resistant antibiotics (1 g / L ampicillin, 0.5 g / L cefotaxime). Simultaneously, starting from day 9, the corresponding bacterial culture was inoculated by gavage at a rate of 3 × 10⁶ cells / day every 3 days. 8 CFU. 1×10⁻⁶ Cephalexin injection on day 0. 6 HCT116 cells were used to remove the tumor in situ on day 10. Mice were sacrificed on day 30, and the fluorescence / bioluminescence signals of liver metastases were examined by dissection.

[0054] Experimental results and statistical analysis: Figure 2 The experimental results show representative mouse liver imaging images and statistical data on liver metastasis signals. One-way ANOVA analysis was used, with Fisher's LSD test as a post-test, to analyze the statistical differences in liver metastasis signals. P<0.05; (P<0.001). Experimental results demonstrate that H. cinaedi has the function of inhibiting liver metastasis of colorectal cancer.

[0055] Example 2: Experiment on the inhibition of spheroidization of various tumor cells by H. mastomyrinus strain and its metabolites.

[0056] As described in the background section, tumor stem cells possess extremely strong self-renewal capacity and differentiation potential, which are the main reasons for the persistent growth of solid tumors, their strong resistance to radiotherapy and chemotherapy, and their ability to escape the immune system. Based on the metastatic inhibition effect shown in Example 1, this strain cuts off the source of carcinogenesis by targeting and eliminating tumor stem cells in the microenvironment. In the field of cell biology, the tumor cell spheroidization assay is recognized as the "gold standard" for the in vitro quantitative detection of the self-renewal capacity of tumor stem cells. The medical community often uses this in vitro experiment to evaluate the killing effect of advanced anticancer drug candidates on the tumor stem cells of specific patients.

[0057] Table 1 Culture conditions for tumor cell line spheroidization assay

[0058]

[0059] (1) Experiment on H. mastomyrinus strain inhibiting the formation of multiple cancer cells into spheroids

[0060] According to the culture conditions in Table 1, the experiment also included negative control bacteria NTBF and H. mastomyrinus treatment groups. HCT116, CT26, MDA-MB-231, 4T1, and HeP3B were added at 3×10⁻⁶ mg / L. 6 1×10 7 Or 3×10 7 CFU bacteria, H1299, H460, A2058 and SUN-1, were added at 1×10⁻⁶ each. 7 Or 3×10 7 CFU bacteria, CAL-27 added 3×10 7 CFU bacteria were co-cultured for 7 days, and the tumor spheres were counted and photographed. Figure 3 ).

[0061] Figure 3 The experimental results show the number of tumor spheres and representative tumor sphere photographs from at least three independent experiments. One-way ANOVA was used, with Fisher's LSD test as a post-test, to analyze the statistical differences in the number of tumor spheres among the groups (ns, no significance). P<0.05; P<0.01; P < 0.001; (P<0.0001). The experimental results demonstrate that H. mastomyrinuss has the ability to inhibit the self-renewal of tumor stem cells in a concentration-dependent manner.

[0062] (2) Experiment on the inhibition of various cancer cell spheroidization by metabolites of H. mastomyrinus strain

[0063] According to the culture conditions in Table 1, the experiment also included a negative control group (NTBF metabolite treatment) and a H. mastomyrinus metabolite treatment group. HCT116, CT26, MDA-MB-231, 4T1, and HeP3B were added at 3×10⁻⁶ mg / L. 6 1×10 7 Or 3×10 7 CFU bacterial metabolites, H1299, H460, A2058 and SUN-1, were added at 1×10⁻⁶ each. 7 Or 3×10 7 CFU bacterial metabolites, CAL-27 added at 3×10 7 Bacterial metabolites of CFU were co-cultured for 7 days, and tumor spheres were counted and photographed. Figure 4 Mets, metabolic products.

[0064] Figure 4The experimental results show the number of tumor spheres and representative tumor sphere photographs from at least three independent experiments. One-way ANOVA was used, with Fisher's LSD test as a post-test, to analyze the statistical differences in the number of tumor spheres among the groups (ns, no significance). P<0.05; P < 0.001; (P<0.0001). The experimental results demonstrate that H. mastomyrinuss metabolites have the ability to inhibit the self-renewal of tumor stem cells in a concentration-dependent manner.

[0065] Example 3: Experiment on the inhibition of spheroidization of various tumor cells by H. ganmani strain and its metabolites

[0066] Through tumor spheroidization experiments, this example demonstrates that H. ganmani and its metabolites can inhibit the self-renewal capacity of tumor stem cells in colorectal cancer, lung cancer, breast cancer, liver cancer, melanoma, gastric cancer, and head and neck cancer. Figure 5-6 ).

[0067] (1) Experiment on H. ganmani strain inhibiting the formation of multiple cancer cells into spheroids

[0068] According to the culture conditions in Table 1, the experiment also included a negative control group NTBF and a H. ganmani treatment group. HCT116, CT26, MDA-MB-231, 4T1, and HeP3B were added at 3×10⁻⁶ mg / L. 6 1×10 7 Or 3×10 7 CFU bacteria, H1299, H460, A2058 and SUN-1, were added at 1×10⁻⁶ each. 7 Or 3×10 7 CFU bacteria, CAL-27 added 3×10 7 CFU bacteria were co-cultured for 7 days, and the tumor spheres were counted and photographed. Figure 5 ).

[0069] Figure 5 The experimental results show the number of tumor spheres and representative tumor sphere photographs from at least three independent experiments. One-way ANOVA was used, with Fisher's LSD test as a post-test, to analyze the statistical differences in the number of tumor spheres among the groups (ns, no significance). P<0.05; P<0.01; P < 0.001; (P<0.0001). The experimental results demonstrate that H. ganmani has the ability to inhibit the self-renewal of tumor stem cells in a concentration-dependent manner.

[0070] (2) Experiment on the inhibition of various cancer cell spheroidization by the metabolites of H. ganmani strain

[0071] According to the culture conditions in Table 1, the experiment also included a negative control group (NTBF metabolite treatment group) and a H. ganmani metabolite treatment group. HCT116, CT26, MDA-MB-231, 4T1, and HeP3B were added at 3×10⁻⁶ mg / L. 6 1×10 7 Or 3×10 7 CFU bacterial metabolites, H1299, H460, A2058 and SUN-1, were added at 1×10⁻⁶ each. 7 Or 3×10 7 CFU bacterial metabolites, CAL-27 added at 3×10 7 Bacterial metabolites of CFU were co-cultured for 7 days, and tumor spheres were counted and photographed. Figure 6 Mets, metabolic products.

[0072] Figure 6 The experimental results show the number of tumor spheres and representative tumor sphere photographs from at least three independent experiments. One-way ANOVA was used, with Fisher's LSD test as a post-test, to analyze the statistical differences in the number of tumor spheres among the groups (ns, no significance). P<0.01; P < 0.001; (P<0.0001). The experimental results demonstrate that H. ganmani metabolites have the ability to inhibit the self-renewal of tumor stem cells in a concentration-dependent manner.

[0073] Example 4: Experiment on the inhibition of spheroidization of various tumor cells by H. cinaedi strain and its metabolites

[0074] Through tumor spheroidization experiments, this example demonstrates that H. cinaedi and its metabolites can inhibit the self-renewal capacity of tumor stem cells in colorectal cancer, lung cancer, breast cancer, liver cancer, melanoma, gastric cancer, and head and neck cancer. Figure 7-8 ).

[0075] (1) Experiment on H. cinaedi strain inhibiting the formation of multiple cancer cells into spheroids

[0076] According to the culture conditions in Table 1, the experiment also included negative control bacteria NTBF and H. cinaedi treatment groups. HCT116, CT26, MDA-MB-231, 4T1, and HeP3B were added at 3×10⁻⁶ mg / L. 6 1×10 7 Or 3×10 7 CFU bacteria, H1299, H460, A2058 and SUN-1, were added at 1×10⁻⁶ each. 7 Or 3×10 7 CFU bacteria, CAL-27 added 3×10 7 CFU bacteria were co-cultured for 7 days, and the tumor spheres were counted and photographed. Figure 7 ).

[0077] Figure 7 The experimental results show the number of tumor spheres and representative tumor sphere photographs from at least three independent experiments. One-way ANOVA was used, with Fisher's LSD test as a post-test, to analyze the statistical differences in the number of tumor spheres among the groups (ns, no significance). P<0.05; P<0.01; P < 0.001; (P<0.0001). The experimental results demonstrate that H. cinaedi has the ability to inhibit the self-renewal of tumor stem cells in a concentration-dependent manner.

[0078] (2) Experiment on the inhibition of multiple cancer cell spheroidization by metabolites of H. cinaedi strain

[0079] According to the culture conditions in Table 1, the experiment also included a negative control group (NTBF metabolite treatment group) and a H. cinaedi metabolite treatment group. HCT116, CT26, MDA-MB-231, 4T1, and HeP3B were added at 3×10⁻⁶ mg / L. 6 1×10 7 Or 3×10 7 CFU bacterial metabolites, H1299, H460, A2058 and SUN-1, were added at 1×10⁻⁶ each. 7 Or 3×10 7 CFU bacterial metabolites, CAL-27 added at 3×10 7 Bacterial metabolites of CFU were co-cultured for 7 days, and tumor spheres were counted and photographed. Figure 8 Mets, metabolic products.

[0080] Figure 8The experimental results show the number of tumor spheres and representative tumor sphere photographs from at least three independent experiments. One-way ANOVA was used, with Fisher's LSD test as a post-test, to analyze the statistical differences in the number of tumor spheres among the groups (ns, no significance). P < 0.001; (P<0.0001). The experimental results demonstrate that H. cinaedi metabolites have the ability to inhibit the self-renewal of tumor stem cells in a concentration-dependent manner.

[0081] Example 5: Experiment on the inhibition of spheroidization of various tumor cells by H. canadensis strain and its metabolites.

[0082] Through tumor spheroidization experiments, this example demonstrates that H. canadensis and its metabolites can inhibit the self-renewal capacity of tumor stem cells in colorectal cancer, lung cancer, breast cancer, liver cancer, melanoma, gastric cancer, and head and neck cancer. Figure 9-10 ).

[0083] (1) Experiment on H. canadensis strain inhibiting the formation of multiple cancer cells into spheroids

[0084] According to the culture conditions in Table 1, the experiment also included negative control bacteria NTBF and H. canadensis treatment groups. HCT116, CT26, MDA-MB-231, 4T1, and HeP3B were added at 3×10⁻⁶ mg / L. 6 1×10 7 Or 3×10 7 CFU bacteria, H1299, H460, A2058 and SUN-1, were added at 1×10⁻⁶ each. 7 Or 3×10 7 CFU bacteria, CAL-27 added 3×10 7 CFU bacteria were co-cultured for 7 days, and the tumor spheres were counted and photographed. Figure 9 ).

[0085] Figure 9 The experimental results show the number of tumor spheres and representative tumor sphere photographs from at least three independent experiments. One-way ANOVA was used, with Fisher's LSD test as a post-test, to analyze the statistical differences in the number of tumor spheres among the groups (ns, no significance). P<0.05; P<0.01; P < 0.001; (P<0.0001). The experimental results demonstrate that H. canadensis has the ability to inhibit the self-renewal of tumor stem cells in a concentration-dependent manner.

[0086] (2) Experiment on the inhibition of various cancer cell spheroidization by metabolites of H. canadensis strain

[0087] According to the culture conditions in Table 1, the experiment also included a negative control group (NTBF metabolite treatment group) and a H. canadensis metabolite treatment group. HCT116, CT26, MDA-MB-231, 4T1, and Hep3B were added at 3×10⁻⁶ mg / L. 6 1×10 7 Or 3×10 7 CFU bacterial metabolites, H1299, H460, A2058 and SUN-1, were added at 1×10⁻⁶ each. 7 Or 3×10 7 CFU bacterial metabolites, CAL-27 added at 3×10 7 Bacterial metabolites of CFU were co-cultured for 7 days, and tumor spheres were counted and photographed. Figure 10 Mets, metabolic products.

[0088] Figure 10 The experimental results show the number of tumor spheres from at least three independent experiments and representative tumor sphere photographs. One-way ANOVA was used, with Fisher's LSD test as a post-test, to analyze the statistical differences in the number of tumor spheres among the groups. P<0.01; P < 0.001; (P<0.0001). The experimental results demonstrate that H. canadensis metabolites have the ability to inhibit the self-renewal of tumor stem cells in a concentration-dependent manner.

[0089] Example 6: Experiment on the inhibition of spheroidization of various tumor cells by nocardamine

[0090] Based on the experimental verification in Examples 2 to 5, it can be known that four members of the Helicobacter genus, namely H. mastomyrinus, H. ganmani, H. cinaedi, and H. canadensis, release a specific compound in the microenvironment to specifically disrupt the homeostasis of tumor stem cells.

[0091] In molecular microbiology, siderophores are a class of low-molecular-weight (typically between 200-1500 Daltons) high-affinity iron chelating compounds actively secreted by specific bacteria in iron-deficient environments, specifically designed to capture scarce ferric ions essential for survival in the surrounding environment. Through high-performance liquid chromatography-mass spectrometry (LC-MS) analysis, the inventors ultimately identified a core siderophore molecule with extremely high biological activity, Nocardamine, in the respective metabolites of *H. mastomyrinus*, *H. ganmani*, *H. cinaedi*, and *H. canadensis*. Figure 11 ).

[0092] Through tumor spheroidization experiments, this example demonstrates that Nocardamine can inhibit the self-renewal capacity of tumor stem cells in colorectal cancer, lung cancer, breast cancer, liver cancer, melanoma, gastric cancer, and head and neck cancer. Figure 12 ).

[0093] (1) Mass spectrometry qualitative detection of nocardamine in metabolites

[0094] The metabolite samples obtained from the previous extraction were scanned using a liquid chromatography-mass spectrometry system. The analysis system was set to multiple reaction monitoring (MRM) mode, specifically for monitoring the signal channel of ion pairs from m / z 599.1 to 399.0.

[0095] like Figure 11 The chromatographic output shows that a highly significant elution peak appeared at a precise retention time of 2.66 minutes in all four independent strains of metabolites tested. This peak was compared with a high-purity known nocardamine standard, and the retention times were consistent, thus confirming the stable presence of nocardamine in the metabolites at the physicochemical level.

[0096] (2) Experiment on the inhibition of multiple cancer cell spheroidization by Nocardamine

[0097] According to the culture conditions in Table 1, the following cell lines were cultured: colorectal cancer cell line HCT116, CT26; lung cancer cell lines H1299, H460; breast cancer cell lines MDA-MB-231, 4T1; liver cancer cell line Hep3B; melanoma cell line A2058; gastric cancer cell line SUN-1; and head and neck cancer cell line CAL-27. Figure 12Tumor spheroidization assays were performed. HCT116 cells were treated with 3 μM, 10 μM, 30 μM, and 100 μM Nocardamine, respectively, while the other nine cell lines were treated with 10 μM, 30 μM, and 100 μM Nocardamine, respectively. After 7 days, the tumor spheroids were counted and photographed.

[0098] Figure 12 The experimental results show the number of tumor spheres and representative tumor sphere photographs from at least three independent experiments. One-way ANOVA was used, with Fisher's LSD test as a post-test, to analyze the statistical differences in the number of tumor spheres among the groups (ns, no significance). P<0.05; P<0.01; P < 0.001; (P<0.0001). Experimental results demonstrate that Nocardamine has the ability to inhibit the self-renewal of tumor stem cells in a concentration-dependent manner.

[0099] Theoretical description and conclusions:

[0100] The core experiments in this section demonstrate that the siderophore Nocardamine, independently identified in this invention, possesses the ability to directly deprive a broad spectrum of tumor stem cells of their core survival right to self-renewal at extremely low micromolar concentrations, and its inhibitory efficacy exhibits a highly stable concentration-dependent effect. It reconstructs the macroscopic anticancer manifestations exhibited in the aforementioned animal in vivo metastasis inhibition (Example 1) and the inhibition of tumor cell stemness by various strains and their metabolites (Examples 2 to 5), revealing the core secrets and material basis at the most fundamental molecular level of how specific Helicobacter spp. bacteria intervene in and reverse the process of malignant tumor development in their hosts.

[0101] Finally, it should be specifically noted that the detailed content and numerous parameter experiments described above are only intended to provide a detailed explanation and illustration of the core technical solution and inventive principle of this invention as optimal examples, and are not intended to reduce or limit the legal protection scope defined by the claims of this application in any way. Any simple conventional modifications or substitutions of conventional equivalent technical features made by those skilled in the art of medicine and microbiology, under the guidance of a thorough reading of the technical spirit conveyed in this patent specification, to the technical solution and drug implementation of this invention (e.g., simple expansion testing with other equivalent cancer types, or the use of other conventional organic extraction and purification methods, or the simple derivation of Nocardamine into other pharmaceutically equivalent and acceptable salt forms), should not depart from the essential core and overall spirit of the technical contribution made by this invention. All such equivalent substitution schemes should be legally covered within the broad protection scope of the appended claims of this application.

Claims

1. The use of Helicobacter spp., Helicobacter spp. metabolites, or Nocardamine in the preparation of medicaments for the prevention and / or treatment of cancer.

2. The application according to claim 1, characterized in that, The Helicobacter genus bacteria mentioned are at least one of the following: Helicobacter mastomyrinus, Helicobacter ganmani, Helicobacter cinaedi, and Helicobacter canadensis.

3. The application according to claim 1, characterized in that, The metabolites of the Helicobacter spp. are prepared by the following method: immersing a culture medium containing Helicobacter spp. in an extraction solvent, then concentrating the extraction solvent to obtain an extract, and then reconstituted the extract to obtain the metabolites of the Helicobacter spp.

4. The application according to claim 1, characterized in that, Nocardamine is the active ingredient in the metabolic products of the Helicobacter genus bacteria.

5. The application according to claim 1, characterized in that, The drug specifically targets and inhibits the self-renewal of tumor stem cells, thereby inhibiting the formation of tumor cells into spheres.

6. The application according to claim 1, characterized in that, The cancer mentioned is at least one of the following: colorectal cancer, lung cancer, breast cancer, liver cancer, skin cancer, stomach cancer, and head and neck cancer.

7. The application according to claim 1, characterized in that, The dosage form of the drug is at least one of the following: aerosol, spray, emulsion, suspension, enema, paste, ointment, gel, lotion, suppository, patch, film, capsule, tablet, pill, powder, granule, tincture, injection, syrup, and solution.

8. The application according to claim 1, characterized in that, The drug is administered via at least one of the following routes: oral administration, sublingual administration, inhalation administration, mucosal administration, intravenous injection, arterial injection, intramuscular injection, intradermal injection, subcutaneous injection, intraperitoneal injection, rectal administration, vaginal administration, transdermal administration, and local administration.

9. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable additives or excipients.

10. The application according to claim 9, characterized in that, The pharmaceutically acceptable excipients include at least one of the following: diluents, binders, wetting agents, lubricants, disintegrants, solvents, emulsifiers, cosolvents, solubilizers, preservatives, pH adjusters, osmotic pressure adjusters, surfactants, coating materials, antioxidants, antibacterial agents, and buffers.

Images